WO2025128343A1 - Protein expression using trans-splicing and split selectable markers - Google Patents

Abstract

Disclosed are a multiple-vector expression system encoding a pre-mRNA molecule and a pre-mRNA trans-splicing molecule (PTM), and a cultured eukaryotic recombinant host cell containing the system. Also disclosed are methods of recombinantly expressing one or more proteins of interest in a cultured eukaryotic host cell line and of selecting eukaryotic host cells that recombinantly express a protein of interest, in vitro, which methods employ the multiple-vector expression system of the invention.

Description

JUST1781 PCT International Patent Application PROTEIN EXPRESSION USING TRANS-SPLICING AND SPLIT SELECTABLE MARKERS [0001] Sequence Listing [0002] The instant application contains a Sequence Listing which has been submitted elec- tronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 25, 2024, is named JUST1781_SL.xml and is 21,202 bytes in size. BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] This invention relates to recombinant production of proteins in cultured eukaryotic cells, such as mammalian cell lines. [0005] 2. Discussion of the Related Art [0006] The biopharmaceutical industry is undergoing major changes, prompted in part by the surge in approvals of new biotherapeutic proteins, including a variety of antibodies and fusion proteins, ongoing developments in cell science facilitating higher protein expression rates, and increased pressure from the biosimilars market. (Levine et al., Efficient,flexible facilities for the 21st century, BioProcess International 10(11):20–30 (2012)). [0007] More recently heterodimeric immunoglobulins or bispecific antibodies have been seen as a promising approach to improving target specificity by binding two different target antigens, and such bispecific antibodies have tremendous potential to improve therapy for a variety of diseases. (See, e.g., Rajendra et al., “Transient and stable CHO expression, purification and characterization of novel hetero-dimeric bispecific IgG antibodies.” Biotechol. Progress 33(2) (2016); Gong and Wu, Gong and Wu (2023) “Efficient production of bispecific antibody – optimization of transfection strategy leads to high-level stable cell line generation of a Fabs-in-tandem immunoglobin,” Antibody Therapeutics, 6(3):170-179 (2023)). [0008] Stable eukaryotic cell lines (e.g., mammalian cell lines) are commonly employed to produce protein drugs with appropriate post-translational glycosylation. However developing these stable cell lines, particularly those that express heterodimeric immunoglobulins, has typicallly involved the use of multiple cell selection markers, often including the expression JUST1781 PCT International Patent Application of antibiotic resistance genes and the addition of one or more antibiotics that must later be purified from the culture medium. Also, the expression of antibiotic resistance genes, may have undesirable effects on cell viability, purification, or production efficiency. Accordingly, industrial advantages have been proposed by using DHFR-knockouts and/or GS-knockouts as selectable producer clones with DHFR and/or GS as selection markers. (see, e.g., Zhou, Jingmin et al., “Cell Line for Recombinant Protein and/or Viral Vector Production,” US2019/0078099A1). [0009] Ketchem et al. broadly disclosed a concept for expressing a heteromultimeric antibody (e.g., a bispecific antibody) by using a first selectable marker that is capable of intragenic complementation (for example, GS) to express the heavy chain and light chain from a first antibody, and a different selectable marker such as a split metabolic enzyme or resistance marker, or a second selectable marker that is different than the first selectable marker and is capable of intragenic complementation. (Ketchem, Randal et al., “Direct Selection of Cells Expressing High Levels of Heteromeric Proteins Using Glutamine Synthetase Intragenic Complementation Vectors,” US2019/127452A1). [00010] In eukaryotic cells, a gene expression involves transcription of DNA into RNA. Chromosomal DNA contains coding regions (exons) and is generally transcribed into a pre- mRNA containing intervening noncoding regions (introns). Such introns are removed from the pre-mRNA via a fine process referred to as splicing. Splicing is known to take place as an interaction coordinated by several small ribonucleoproteins (snRNPs) and many protein factors that assemble to form an enzyme complex known as a spliceosome. (See, e.g., Will, Cindy L. and Luehrmann, Reinhard, “Spliceosome Structure and Function,” Cold Spring Harb. Perspect. Biol. (2011);3:a003707; Padgett, Richard A. et al., “Splicing of messenger RNA precursors,” Ann. Rev. Biochem.55:1119-150 (1986)). [00011] Pre-mRNA splicing proceeds by a 2-step mechanism. The first step involves cleavage of 5'splice site so as to generate a "free" 5' exon and a lariat intermediate. The second step involves freeing introns in the form of lariat products, as the 5' exon is ligated to 3'exon. These steps proceed via catalysis involving small nuclear ribonucleoproteins and a protein complex referred to as spliceosome. These splicing reaction sites are defined by consensus sequences in the peripheries of the 5' and 3'splice sites. The 5' splice site consensus sequence is AG/GURAGU (here, A=adenosine, U=uracil, G=guanine, C=cytosine, R=purine, and /=splice site). The 3' splice region is composed of three individual sequential elements: a JUST1781 PCT International Patent Application branch point or branch site, a polypyrimidine tract, and the 3' consensus splice sequence (YAG). These elements roughly define the 3' splice region. The 3' splice region can contain a 100-nucleotide intron upstream of the 3'splice site. A consensus sequence of a mammalian branch point is CURAY (here, Y=pyrimidine). Underlined A is a branch formation site (BPA=branch point adenosine). The 3' consensus splice sequence is YAG/G. A polypyrimidine tract is generally observed between a branch point and a splice site, which is important in a mammalian system for effective use of a branch point and recognition of the 3' splice site. The first Y, A, and G (trinucleotides) located downstream of a branch point and a polypyrimidine tract forms a most-frequently-used 3' splice site (Smith, C. W. et al., 1989, Nature 342: 243-247). [00012] Typically, the splicing reaction takes place within the same pre-mRNA molecule, which is referred to as “cis-splicing.” Splicing that takes place between two independently- transcribed pre-mRNAs is referred to as “trans-splicing.” In trans-splicing, exons from separately transcribed precursor RNA molecules are joined when a mature messenger RNA is generated. Trans-splicing was discovered for the first time in Trypanosoma (Sutton & Boothroyd, 1986, Cell 47: 527; Murphy et al., 1986, Cell 47: 517) and then discovered in nematode, flatworm (Rajkovic et al., 1990, Proc. Natl. Acad. Sci. U.S.A., 87: 8879; and Davis et al., 1995, J. Biol. Chem.270: 21813), and plant mitochondria (Malek et al., 1997, Proc. Natl. Acad. Sci. U.S.A.94: 553). [00013] The construction and genetic engineering of dual vector expression systems have been described, which are useful in other eukaryotic host cell types. These systems involve pre-mRNA molecules and pre-mRNA trans-splicing molecules (PTMs) to induce spliceosome-mediated trans-splicing reactions within the eukaryotic host cells. (See, e.g., U.S. Pat. Nos.6,083,702; 6,013,487; 6,280,978; 7,399,753; 7,879,321; 8,735,366; and 9,655,979). [00014] It is a desideratum to simplify and streamline the selection process in developing stable eukaryotic cell lines for the production of proteins of interest, particularly heterodimeric immunoglobulin biotherapeutics, such as bispecific antibodies. This and other benefits the present invention provides. SUMMARY OF THE INVENTION [00015] The present invention employs trans-splicing, facilitated by the host cell spliceosome, in an expression system which uses the 5′ splice site from one molecule (pre- JUST1781 PCT International Patent Application mRNA molecule) and the branch point, together with the 3′ splice site from another molecule (pre-mRNA trans-splicing molecule, or “PTM”), to ligate two exonic fragments of a selectable marker (e.g., glutamine synthetase or DHFR), and is capable of confirming expression of proteins of interest by the expression of the complete coding sequence for the marker from joined together from the two different molecules. One of the benefits of using the invention, e.g., with a split GS selection or split DHFR selection, for expressing bispecific or multi-chain antibodies is that only one selection marker is used for stable expression, instead of using multiple selection markers. [00016] In one aspect the present invention relates to a multiple-vector expression system encoding a pre-mRNA molecule and a pre-mRNA trans-splicing molecule (PTM). The inventive expression system involves: [00017] (A) a first vector encodes the pre-mRNA molecule, which comprises 5' to 3': [00018] (i) a promoter; [00019] (ii) a first coding sequence comprising a 5'-exonic coding fragment of a gene encoding a predetermined selectable protein marker; [00020] (iii) an intron, comprising a first splicing domain comprising: [00021] (a) a 5' donor acceptor splice site sequence; [00022] (b) a target binding sequence; [00023] (c) a branch point (BP) sequence; [00024] (d) a polypyrimidine-rich sequence (also known as a “polypyrimidine tract” or “PPT”); and [00025] (e) a 3' acceptor splice site sequence; and [00026] (B) a second vector encoding the PTM, wherein the PTM comprises 5' to 3': [00027] (i) a binding sequence complementary to the target binding sequence in the pre-mRNA molecule; [00028] (ii) a second splicing domain comprising: [00029] (a) a binding domain sequence complementary to a portion of the target binding sequence in (A)(iii)(b); JUST1781 PCT International Patent Application [00030] (b) a branch point (BP) sequence; [00031] (c)) a polypyrimidine-rich sequence (PPT); and [00032] (d) a 3' acceptor splice site sequence; and [00033] (iii) a second coding sequence comprising a 3'-exonic coding fragment of the gene encoding the predetermined selectable protein marker, sequential to the 5'-exonic coding fragment of the gene in (A)(ii), whereby in combination therewith, after a trans-splicing event, an operable coding sequence of the protein marker is obtained that is selectable in a eukaryotic cell. [00034] In another aspect the present invention relates to a cultured eukaryotic recombinant host cell (e.g., a mammalian cell) that does not endogenously express a predetermined functional selectable marker, wherein the eukaryotic host cell comprises the multiple-vector expression system. [00035] In still another aspect, the present invention is directed to a method of recombinantly expressing one or more proteins of interest in a cultured eukaryotic host cell line, which includes: [00036] (a) transfecting a plurality of cultured eukaryotic cells of a parent cell line, with the inventive multiple-vector expression system, wherein the parent cell line lacks a functional gene encoding the predetermined selectable protein marker (i.e., that a biologically functional or detectable version of the selectable protein marker is not expressed by the parent cell line); [00037] (b) culturing, in vitro, under physiological conditions that allows expression of proteins, in a culture medium not permissive to growth of the parent cell line; [00038] (c) selecting viable transfected cells from (b); and [00039] (d) harvesting the one or more proteins of interest. [00040] The present invention also relates to a method of selecting cultured eukaryotic host cells that recombinantly express a protein of interest, in vitro, which includes the steps of: [00041] (a) transfecting a plurality of cultured eukaryotic host cells with the inventive multiple-vector expression system, wherein the host cells lack a functional gene encoding the JUST1781 PCT International Patent Application predetermined selectable protein marker (i.e., that a biologically functional or detectable version of the selectable protein marker is not endogenously expressed by the host cells); [00042] (b) culturing, in vitro, under physiological conditions that allows expression of proteins, in a culture medium not permissive to growth of eukaryotic host cells that do not recombinantly express the selectable protein marker; and [00043] (c) selecting viable transfected cells from (b). [00044] The foregoing summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description of Embodiments. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. [00045] In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations defined by specific paragraphs above. For example, certain aspects of the invention that are described as a genus, and it should be understood that every member of a genus is, individually, an aspect of the invention. Also, aspects described as a genus or selecting a member of a genus, should be understood to embrace combinations of two or more members of the genus. Although the applicant(s) invented the full scope of the invention described herein, the applicants do not intend to claim subject matter described in the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention. BRIEF DESCRIPTION OF THE DRAWINGS [00046] Figure 1A-B shows a schematic diagram of a split glutamine synthetase (GS) mechanism for stable expression and DNA constructs. Figure 1A shows both the target and PTM plasmids, which were transfected into GS knock-out (KO) CHO-K1 cells. After nascent mRNA transcription, the binding domain on the PTM molecule recognizes a complementary sequence on the intron of the rhodopsin pre-mRNA, which facilitates trans-splicing, leading to the production of a full-length GS JUST1781 PCT International Patent Application mRNA. Branch point (BP), polypyrimidine tract (PPT) sequences and the 5’ and 3’ splice sites (SS) help facilitate splicing. Following transfection, cells were cultured in medium without glutamine, thereby allowing only cells that had successful expression of full-length GS, via trans-splicing, to survive selection. Forward and reverse PCR primers were designed to detect successful trans-splicing or cis-splicing. Figure 1B illustrates details of key elements on the target and PTM plasmid. The expression of the immunoglobulin heavy chain coding sequence (“HC”) and immunoglobulin light chain coding sequence (“LC”) on each plasmid are driven by a mouse CMV promoter (“mCMV”); expression is terminated by the 3’ polyadenylation signal (“PA”). The human CMV promoter (“hCMV”) drives expression of GS exons 1-3, followed by the intron containing the 5’ splice site, branch point, polypyrimidine tract, 3’ splice site, and rhodopsin exon on the target plasmid. On the PTM plasmid, the human CMV promoter drives the expression of the intron containing the binding domain, branch point, polypyrimidine tract, and GS exons 4-7, including the artificial intron (SEQ ID NO:2) between exons 4 and 5. These expression cassettes are flanked by inverted terminal repeat (“ITR”) sequences necessary for integration into TTAA chromosomal sites by the PiggyBac transposase. [00047] Figure 2A-E represents The use of split GS to express monoclonal antibody 1 (mAb1) in a GS KO CHO-K1 cell line. Figure 2A shows a schematic design of a proof-of-concept experiment for the use of split GS in stable expression of a monoclonal antibody (mAb1). In this embodiment, the control plasmid had both heavy and light chains of mAb1 on the same vector, as well as a full-length GS coding region. The target plasmid had GS exons 1-3 and the coding sequence for the immunoglobulin heavy chain, while the PTM plasmid had GS exons 4-7 and the coding region for the immunoglobulin light chain. The heavy and light chains coding sequences were on separate plasmids with different fragments of the GS gene. Forward (For) and reverse (Rev) PCR primers for GS were designed to detect successful trans-splicing. A GS-KO CHO-K1 cell line (CL-72) was transfected with the corresponding plasmids. Upon successful trans-splicing and minus glutamine selection, cells were put into a 10-day fed-batch production assay to obtain antibody titer. Figure 2B shows an agarose gel image of PCR products from two replicates of trans-spliced GS samples, control sample, and a mock transfected sample. Cell pellets were collected when the cells recovered fully from selection (>90% viability), and DNA and RNA were extracted. A PCR product seen only in the cDNA of the trans-spliced samples, but not the DNA indicated successful expression of the full-length GS upon trans-splicing. Figure 2C shows the percentage cell viability, and Figure 2D shows the viable cell density during selection. Cells transfected with both trans-splicing vectors showed complete recovery from minus glutamine selection, albeit much later than the control. Samples with the artificial intron between GS exons 4 and 5 showed no improvement in recovery during selection. Figure 2E illustrates that compared to the pJV145 control, titers from trans-splicing samples were low, about 20% of the control. JUST1781 PCT International Patent Application [00048] Figure 3A-C demonstrates improved selection recovery and mAb1 titers with artificial intron in an inducible GS-KO CHO-K1 cell line (CL-130). Figure 3A shows the percentage cell viability as measured by trypan blue exclusion during minus glutamine selection. Samples with the artificial intron between GS exons 4 and 5 (squares) showed improved recovery during selection relative to trans-spliced samples without the intron (circles). Figure 3B shows viable cell density as measured by trypan blue exclusion during minus glutamine selection. Figure 3C shows titer measurements at Day 10 of a fed-batch production assay. To induce expression of mAb1, 0.0125 µg/mL of doxycycline was added to the production media at days 0, 3, 6, and 8. Samples with the artificial intron showed significantly higher titers relative to trans-spliced samples without the artificial intron. This experiment was repeated two more times and showed reproducible results. [00049]_{Figure 4A-D shows} N⁶-methyladenosine (m6A) sequences within the 5’UTR of the heavy chain improves recovery from selection in the inducible GS KO cell line (CL-130). Figure 4A shows a schematic diagram of the location of putative m6A motif sequences (GGACT) on the target plasmid. This m6A motif sequence (SEQ ID NO:5) was inserted into the 5’UTR of the heavy chain in the target plasmid, upstream of the kozak sequence. Figure 4B shows the percentage cell viability, and Figure 4C shows viable cell density during, minus glutamine selection, as measured by a trypan blue exclusion assay. Samples transfected with pJV350_m6A (diamonds) had improved recovery during selection compared to trans-splicing samples without the m6A sequence (squares). Figure 4D shows titers from day 10 of a fed-batch production assay showed similar titers between trans-splicing samples with the m6A motif and those without. [00050] Figure 5A-C illustrates that N⁶-methyladenosine (m6A) sequence within the 5’UTR of the heavy chain improves recovery from selection in the GS-KO cell line CL-72. Figure 5A shows the percentage cell viability, and Figure 5B shows viable cell density during minus glutamine selection measured by a trypan blue exclusion assay. Samples transfected with pJV350_m6A (diamonds) had improved recovery during selection compared to trans-splicing samples without the m6A sequence (squares). Figure 5C) shows titers from day 10 of a fed-batch production assay, which demonstrated a trend of improved titers between trans-splicing samples with the m6A motif, compared to those without. [00051] Figure 6A-D illustrates an embodiment in which the N⁶-methyladenosine (m6A) sequence is in a location upstream of the intron containing the binding domain; this embodiment did not markedly improve recovery from selection in the CL-130 inducible GS KO cell line expressing another molecule M-2865. Figure 6A shows a schematic diagram of the location of putative m6A motif sequences (GGACT, appearing twice within a nucleotide sequence insert we employed: GGACTAAAGCGGACTTGT//SEQ ID NO:5) on the PTM plasmid. Figure 6B Percentage cell viability (Figure 6B) and viable cell density (Figure 6C) are shown during minus-glutamine selection, JUST1781 PCT International Patent Application measured by a trypan blue exclusion assay. Samples transfected with pJV351_BD1intron_M- 2865_m6A (squares) had similar recovery during selection compared to trans-splicing samples without the m6A sequence (circles; Figure 6D). Titers from day 10 of a fed-batch production assay showed similar titers between trans-splicing samples with the m6A motif and those lacking the m6A motif. M-2865 is an artificial humanoid IgG1 antibody generated in silico using a Generative Adversarial Network (GAN; see, Amimeur et al., “Designing feature-controlled humanoid antibody discovery libraries using Generative Adversarial Networks,” BioRxiv 2020.04.12.024844 (2020)). Titers of M-2865 generated by the trans-splicing vectors were about 67% of the control, wherein both heavy and light chains of M-2865 were on the same vector with intact GS, implying the successful use of the invcentive method for a second, different monoclonal antibody. [00052] Figure 7A-E illustrates the expression of a novel bispecific antibody using trans- splicing of GS. Figure 7A shows a schematic diagram of a bispecific antibody made from two different SARS-CoV2 antibodies generated in silico using a Generative Adversarial Network (GAN). Knob-into-hole mutations were engineered into the CH3 region of the heavy chains of the antibodies. These individual antibodies were determined to have good binding activity and can neutralize multiple SARS strains. Figure 7B shows a schematic representation of the design of trans-splicing constructs used in some experiments. The CH3 domain of the heavy chain of the M-3406 antibody had “hole” mutations, while the CH3 domain of the heavy chain of the M-3376 antibody had “knob” mutations. Both plasmids were transfected into the GS KO inducible cell line CL-130 and CL-165 in a 1:1 ratio. Figure 7C shows the titer of the M-3406/M-3376 IgG1 bispecific antibody from a 10-day fed batch production run. Starting at day 0 of production, 0.0125 micrograms per mL of doxycycline was added to the culture medium and gave a titer of about 1 mg/mL. Data are representative of two independent experiments. Figure 7D shows the results of size exclusion chromatography (SEC) with a main peak of greater than 85%. Figure 7E shows results of reduced capillary electrophoresis (rCE) that was also performed, and which detected both the light and heavy immunoglobulin chains. Samples were reduced with 5.5% 2-mercaptoethanol (BME) in 100 mM Tris, pH 9.0, 1% sodium dodecyl sulphate (SDS) and heated at 70ºC for 10 minutes. DETAILED DESCRIPTION OF EMBODIMENTS [00053] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [00054] Definitions JUST1781 PCT International Patent Application [00055] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms "a," "an," and "the," include plural referents unless the context clearly indicates otherwise. For example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes populations of a plurality of cells. [00056] A “pre-mRNA molecule” or “pre-mRNA” or “precursor mRNA” (used herein interchangeably), refers to a target pre-mRNA molecule having a targeted nucleotide sequence to which the target binding sequence of a pre-mRNA trans-splicing molecule is able to hybridize and specifically bind. The target binding sequence (or domain) of the PTM endows the PTM with a binding affinity for the target pre-mRNA molecule. As used herein, a target binding sequence or domain is defined as any molecule, i.e., nucleotide, protein, chemical compound, etc., that confers specificity of binding and anchors the pre-mRNA molecule closely in space to the PTM, so that the spliceosome processing machinery of the host cell nucleus can trans-splice a portion of the PTM to a portion of the pre-mRNA molecule. Within the scope of the present invention, the pre-mRNA contains at least one intron and one or more 5'-exonic coding fragments of a selectable marker, and it may also contain one or more coding sequences for a protein or protein subunit of interest. [00057] A “pre-mRNA trans-splicing molecule” (PTM) refers to a RNA molecule having: (a) at least one binding domain (i.e., a “target binding sequence”) that targets binding of the nucleic acid molecule to a pre-mRNA expressed within a cell; (b) at least one splicing domain containing motifs necessary for the trans-splicing reaction to occur, and (c) at least one coding domain, wherein said coding domain. The general design, construction and genetic engineering of PTMs and demonstration of their ability to successful induce spliceosome mediated trans-splicing reactions within the cell are described in detail in, e.g., U.S. Pat. Nos.6,083,702, 6,013,487, 6,280,978, 7,399,753, 6,280,978 , 8,735,366 and 9,655,979. In brief, the PTM molecule is designed to carry a nucleic acid target binding domain (“BD”), or sequence, complementary to and in antisense orientation to an intron sequence of the target pre-mRNA molecule, to suppress target cis-splicing while enhancing trans-splicing between the PTM and the target. A PTM further includes a splicing domain, comprising a strong conserved branch point (BP) sequence, a polypyrimidine-rich sequence JUST1781 PCT International Patent Application (also known as a “polypyrimidine tract” or “PPT”), and a 3′ acceptor splice site (ss). A spacer sequence separates the splicing domain from the target binding domain. And finally an PTM comprises at least one coding domain coding sequence to be trans-spliced to the target pre-mRNA. The coding domain can be a single exon, multiple exons or an entire coding sequence. In the context of the present invention. [00058] The “branch point” (BP) is usually an adenosine residue (but it may be a different residue), within a 5-residue BP sequence, which is a cis-acting intronic motif required for mRNA splicing. Within the context of the present invention, the BP sequence that is part of an intronic sequence or a splicing domain sequence can be the same or different branch point sequence. The BP forms-- through an enzymatically catalyzed reaction-- a covalent linkage to the 5’ splice junction, which then initiates the splicing reaction. For example, a consensus sequence for a useful 5-residue BP sequence is YunAY, where “Y” is a pyrimidine residue and “n” (or “N”) can be any residue (Gao et al., “Human branch point consensus sequence is yUnAy,” Nucleic Acids Research, 2008, Vol.36, No.72257–2267, doi:10.1093/nar/gkn073). For example, the nucleotide sequence surrounding the BP is typically: 5' (exon...AG)'GURAGU...intron...YunAY...intron...YYYYYYYYYYYYYYYNCAG'(G...exo n) (“YYYYYYYYYYYYYYYNCAG” disclosed as SEQ ID NO:19), with an intronic sequence being inserted at a residue between the “'” marks (apostrophes), as shown. A “polypyrimidine-rich sequence,” also known as a “polypyrimidine tract” or “PPT” (all these terms are used herein interchangeably), is of variable length, but typically a sequence of about 15 pyrimidine residues at the 3' end of the intronic sequence, which is upstream of the 3’ splice junction. Other useful functional variants of BP sequences that can be included in an intron or in a splicing domain, instead of the consensus sequence above, are known in the art. (See, e.g., Gao et al., “Human branch point consensus sequence is yUnAy,” Nucleic Acids Research, 2008, Vol.36, No.72257–2267, doi:10.1093/nar/gkn073; Kadri, N.K., Mapel, X.M. & Pausch, H. “The intronic branch point sequence is under strong evolutionary constraint in the bovine and human genome.” Commun Biol 4, 1206 (2021), doi.org/10.1038/s42003-021-02725-7). [00059] In general, the nucleic acid target binding domain (BD) brings specificity to trans- splicing by binding specifically to the target pre-mRNA, whereas the splicing and coding domains provide essential consensus motifs that are recognized by the spliceosome and make the trans-splicing reaction actually happen. The use of BP and PPT follows consensus JUST1781 PCT International Patent Application sequences which are needed for performance of the two phosphoryl transfer reaction involved in cis-splicing and, presumably, also in trans-splicing. These reactions, catalyzed by the spliceosome, must excise the introns precisely in order to produce functional mRNAs. In a manner similar to the RNA cis-splicing processes, the binding domain and splicing domain sequences of the PTM RNA are excised after trans-splicing and are not retained in the reprogrammed final mRNA products. Within the scope of the present invention, the pre- mRNA conatins one or more 3'-exonic coding fragments of a selectable marker, and it may also contain one or more coding sequences for a protein or protein subunit of interest. [00060] The methods of the invention involve contacting the PTMs of the invention with a target pre-mRNA, under physiological conditions within a cultured eukaryotic cell, in which a portion of the PTM is trans-spliced to a portion of the target pre-mRNA to form a novel RNA molecule that is further processed and expressed in the cultured host cell to produce a protein(s) of interest, such as, but not limited to an immunoglobulin of interest. [00061] The term "immunoglobulin" encompasses a gamut of antibody molecules, including full antibodies comprising two dimerized heavy chains (HC), each covalently linked to a light chain (LC), in which the monomers may be selected to specifically bind to the same or different antigen targets; a single undimerized immunoglobulin heavy chain, or a light chain covalently linked to a heavy chain (HC+LC; i.e., monomeric Ab), a Fc-fusion protein, or a chimeric immunoglobulin (light chain+heavy chain)-Fc heterotrimer (a so-called "hemibody"), or a single domain or single variable-domain antibody (i.e., “VHH domain” antibody, “sdAb,” or “nanobody,” or “Nb,” terms used interchangeably). An immunoglobulin molecule can be designed to be mono-specific, bi-specific, or poly-specific, with respect to the antigen target(s) to which it is capable of specifically binding. Although an "immunoglobulin" is a protein, it is not necessarily an antigen-binding protein, for it may optionally also be engineered not to have a known target or may naturally not specifically bind to a known target (e.g., a so-called “carrier antibody” for delivering a therapeutic peptide or other chemical moiety). [00062] An "isolated" protein, e.g., an immunoglobulin protein (such as an anrtibody), is one that has been identified and separated from one or more components of its natural environment or of a culture medium in which it has been secreted by a producing cell. In some embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural or culture medium environment that would JUST1781 PCT International Patent Application interfere with its therapeutic, diagnostic, prophylactic, research or other use. "Contaminant" components of its natural environment or medium are materials that would interfere with diagnostic or therapeutic uses for the protein, e.g., an antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous (e.g., polynucleotides, lipids, carbohydrates) solutes. Typically, an "isolated protein" constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. In some embodiments, the protein of interest, e.g., an antibody, will be purified (1) to greater than 95% by weight of protein, and most preferably more than 99% by weight, or (2) to homogeneity by SDS-PAGE, or other suitable technique, under reducing or nonreducing conditions, optionally using a stain, e.g., Coomassie blue or silver stain. Isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the protein's natural environment will not be present. Typically, however, the isolated protein of interest (e.g., an antibody) will be prepared by at least one purification step. The term "naturally occurring," where it occurs in the specification in connection with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to materials which are found in nature. [00063] A "domain" or "region" (used interchangeably herein) of a polynucleotide is any portion of the entire polynucleotide, up to and including the complete polynucleotide, but typically comprising less than the complete polynucleotide. A domain can, but need not, fold independently (e.g., DNA hairpin folding) of the rest of the polynucleotide chain and/or be correlated with a particular biological, biochemical, or structural function or location, such as a coding region or a regulatory region. [00064] A "domain" or "region" (used interchangeably herein) of a protein is any portion of the entire protein, up to and including the complete protein, but typically comprising less than the complete protein. A domain can, but need not, fold independently of the rest of the protein chain and/or be correlated with a particular biological, biochemical, or structural function or location (e.g., a ligand binding domain, or a cytosolic, transmembrane or extracellular domain). [00065] "Antigen binding region" or "antigen binding site" means a portion of a protein that specifically binds a specified target ligand or antigen, e.g., fentanyl and/or carfentanil. For example, that portion of an antigen binding protein that contains the amino acid residues that interact with a target ligand or an antigen and confer on the antigen binding JUST1781 PCT International Patent Application protein its specificity and affinity for the antigen is referred to as "antigen binding region." In an antibody, an antigen binding region typically includes one or more "complementary binding regions" ("CDRs"). Certain antigen binding regions also include one or more "framework" regions ("FRs"). A "CDR" is an amino acid sequence that contributes to antigen binding specificity and affinity. "Framework" regions can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen binding region and an antigen. In a traditional antibody, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions responsible for antigen binding and recognition. A variable region of an immunoglobulin antigen binding protein comprises at least three heavy or light chain CDRs, see, supra (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342: 877- 883), within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991, supra; see also Chothia and Lesk, 1987, supra). [00066] The term “target” or "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen binding protein (including, e.g., an antibody or immunologically functional fragment of an antibody), and additionally capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen may possess one or more epitopes that are capable of interacting with different antigen binding proteins, e.g., antibodies. [00067] The term "epitope" is the portion of a target molecule that is bound by an antigen binding protein (for example, an antibody or antibody fragment). The term includes any determinant capable of specifically binding to an antigen binding protein, such as an antibody or to a T-cell receptor. An epitope can be contiguous or non-contiguous (e.g., in a single-chain polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within the context of the molecule are bound by the antigen binding protein). In certain embodiments, epitopes may be mimetic in that they comprise a three- dimensional structure that is similar to an epitope used to generate the antigen binding protein, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antigen binding protein. Most often, epitopes reside on proteins, but in some instances they may reside on other kinds of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and they may have specific three- JUST1781 PCT International Patent Application dimensional structural characteristics, and/or specific charge characteristics. Generally, antigen binding proteins specific for a particular target will preferentially recognize an epitope on the target in a complex mixture of proteins and/or macromolecules. [00068] A protein of interest, such as an antigen-binding protein (e.g., an immunoglobulin, such as an antibody or antibody fragment or subunit) used in the practice of the invention, whether a variant or parent protein, is typically produced by recombinant expression technology. The term "recombinant" indicates that the material (e.g., a nucleic acid or a polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other well-known molecular biological procedures. Examples of such molecular biological procedures are found in Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). A "recombinant DNA molecule," is comprised of segments of DNA joined together by means of such molecular biological techniques. [00069] The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule, e.g., an antibody, which is expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and/or expresses a recombinant nucleic acid. [00070] The term "control sequence" or "control signal" refers to a polynucleotide sequence that can, in a particular host cell, affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences may depend upon the host organism. In particular embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding site, and a transcription termination sequence. Control sequences for eukaryotes may include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences or elements, polyadenylation sites, and transcription termination sequences. Control sequences can include leader sequences and/or fusion partner sequences. Promoters and enhancers consist of short arrays of DNA that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The JUST1781 PCT International Patent Application selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis, et al., Science 236:1237 (1987)). [00071] A “promoter” is a region of DNA including a site at which RNA polymerase binds to initiate transcription of messenger RNA by one or more downstream structural genes. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand). Promoters are typically about 100-1000 bp in length. [00072] An “enhancer” is a short (50-1500 bp) region of DNA that can be bound with one or more activator proteins (transcription factors) to activate transcription of a gene. [00073] The terms "in operable combination", "in operable order" and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences. [00074] A protein of interest for purposes of the present invention, whether it includes a variant or parental amino acid sequence, is typically produced by recombinant expression technology, although it can also be a naturally occurring protein. [00075] "Polypeptide" and "protein" are used interchangeably herein and include a molecular chain of two or more amino acids linked covalently through peptide bonds. The terms do not refer to a specific length of the product. Thus, "peptides," and "oligopeptides," are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide. The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be expressed recombinantly using known protein engineering JUST1781 PCT International Patent Application techniques. In addition, proteins can be derivatized as described herein and by other well- known organic chemistry techniques. [00076] The term "purify" or "purifying" a protein means increasing the degree of purity of the desired protein from a composition or solution comprising the protein of interest (i.e., the “POI,” and one or more contaminants by removing (completely or partially) at least one contaminant from the composition or solution. An "isolated" protein is one that has been identified and separated from one or more components of its natural environment or of a culture medium in which it has been secreted by a producing cell. In some embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural or culture medium environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. "Contaminant" components of its natural environment or medium are materials that would interfere with industrial, research, therapeutic, prophylactic, or diagnostic or uses for the protein of interest, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous (e.g., polynucleotides, lipids, carbohydrates) solutes. Typically, an "isolated protein" or, interchangeably, “protein isolate,” constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. In some embodiments, the isolated protein of interest will be “purified”: (1) to greater than 95% by weight of protein, and most preferably, more than 99% by weight, or (2) to homogeneity by SDS-PAGE, or other suitable technique, under reducing or nonreducing conditions, optionally using a stain, e.g., Coomassie blue or silver stain. An isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the protein's natural environment will not be present. Typically, however, the isolated or purified protein of interest (e.g., a purified protein drug substance) will be prepared by at least one purification step, which can include cell lysis (with or without centrifugation), filtration, and/or affinity chromatography. [00077] A "variant" of a polypeptide (e.g., an immunoglobulin, or an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants can include fusion proteins. [00078] The term peptide or protein “analog" refers to a polypeptide having a sequence that differs from a peptide sequence existing in nature by at least one amino acid residue substitution, internal addition, or internal deletion of at least one amino acid, and/or amino- or JUST1781 PCT International Patent Application carboxy-terminal end truncations, or additions). An "internal deletion" refers to absence of an amino acid from a sequence existing in nature at a position other than the N- or C-terminus. Likewise, an "internal addition" refers to presence of an amino acid in a sequence existing in nature at a position other than the N- or C-terminus. [00079] The term "fusion protein" indicates that the protein includes polypeptide components derived from more than one parental protein or polypeptide. Typically, a fusion protein is expressed from a “fusion gene” in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence from a different protein. The fusion gene can then be expressed by a recombinant host cell as a single protein. Fusion proteins incorporating an antibody or an antigen-binding portion thereof are known. [00080] A "secreted" protein refers to those proteins capable of being directed to the endoplasmic reticulum (ER), secretory vesicles, or the extracellular space as a result of a secretory signal peptide sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage. In some other embodiments, the antibody protein of interest can be synthesized by the host cell as a secreted protein, which can then be further purified from the extracellular space and/or medium. [00081] As used herein "soluble" when in reference to a protein produced by recombinant DNA technology in a host cell is a protein that exists in aqueous solution; if the protein contains a twin-arginine signal amino acid sequence the soluble protein is exported to the periplasmic space in gram negative bacterial hosts, or is secreted into the culture medium by eukaryotic host cells capable of secretion, or by bacterial host possessing the appropriate genes (e.g., the kil gene). Thus, a soluble protein is a protein which is not found in an inclusion body inside the host cell. Alternatively, depending on the context, a soluble protein is a protein which is not found integrated in cellular membranes, or, in vitro, is dissolved, or is capable of being dissolved in an aqueous buffer under physiological conditions without forming significant amounts of insoluble aggregates (i.e., forms aggregates less than 10%, and typically less than about 5%, of total protein) when it is suspended without other proteins in an aqueous buffer of interest under physiological conditions, such buffer not containing an JUST1781 PCT International Patent Application ionic detergent or chaotropic agent, such as sodium dodecyl sulfate (SDS), urea, guanidinium hydrochloride, or lithium perchlorate. In contrast, an insoluble protein is one which exists in denatured form inside cytoplasmic granules (called an inclusion body) in the host cell, or again depending on the context, an insoluble protein is one which is present in cell membranes, including but not limited to, cytoplasmic membranes, mitochondrial membranes, chloroplast membranes, endoplasmic reticulum membranes, etc., or in an in vitro aqueous buffer under physiological conditions forms significant amounts of insoluble aggregates (i.e., forms aggregates equal to or more than about 10% of total protein) when it is suspended without other proteins (at physiologically compatible temperature) in an aqueous buffer of interest under physiological conditions, such buffer not containing an ionic detergent or chaotropic agent, such as sodium dodecyl sulfate (SDS), urea, guanidinium hydrochloride, or lithium perchlorate. [00082] The term "polynucleotide" or "nucleic acid," used interchangeably herein, includes both single-stranded and double-stranded nucleotide polymers containing two or more nucleotide residues. The nucleotide residues comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate and phosphoroamidate. [00083] The term "oligonucleotide" means a polynucleotide comprising 200 or fewer nucleotide residues. In some embodiments, oligonucleotides are 10 to 60 bases in length. In other embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotides may be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides may be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides may be used, for example, as PCR primers, cloning primers or hybridization probes. [00084] A "polynucleotide sequence" or "nucleotide sequence" or "nucleic acid sequence," as used interchangeably herein, is the primary sequence of nucleotide residues in a polynucleotide, including of an oligonucleotide, a DNA, and RNA, a nucleic acid, or a character string representing the primary sequence of nucleotide residues, depending on JUST1781 PCT International Patent Application context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence can be determined. Included are DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end; the left-hand direction of double- stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences;" sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences." [00085] As used herein, an "isolated nucleic acid molecule" or "isolated nucleic acid sequence" is a nucleic acid molecule that is either (1) identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid or (2) cloned, amplified, tagged, or otherwise distinguished from background nucleic acids such that the sequence of the nucleic acid of interest can be determined. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the immunoglobulin (e.g., antibody) where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells. [00086] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of ribonucleotides along the mRNA chain, and also determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the RNA sequence and for the amino acid sequence. [00087] The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. Genes typically include coding sequences and/or the regulatory sequences required for expression of such coding sequences. The term "gene" applies to a specific genomic or recombinant sequence, as well as to a cDNA or mRNA encoded by that sequence. Genes also include non-expressed nucleic acid segments that, for example, form JUST1781 PCT International Patent Application recognition sequences for other proteins. Non-expressed regulatory sequences including transcriptional control elements to which regulatory proteins, such as transcription factors, bind, resulting in transcription of adjacent or nearby sequences. [00088] "Expression of a gene" or "expression of a nucleic acid" means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), translation of RNA into a polypeptide (possibly including subsequent post-translational modification of the polypeptide), or both transcription and translation, as indicated by the context. [00089] An expression cassette is a typical feature of recombinant expression technology. The expression cassette includes a gene encoding a protein of interest, e.g., a gene encoding an antibody sequence, such as an immunoglobulin light chain and/or heavy chain sequence. A eukaryotic “expression cassette" refers to the part of an expression vector that enables production of protein in a eukaryotic cell, such as a mammalian cell. It includes a promoter, operable in a eukaryotic cell, for mRNA transcription, one or more gene(s) encoding protein(s) of interest and a mRNA termination and processing signal. An expression cassette can usefully include among the coding sequences, a gene useful as a selective marker or reporter. In the expression cassette promoter is operably linked 5' to an open reading frame encoding an exogenous protein of interest; and a polyadenylation site is operably linked 3' to the open reading frame. Other suitable control sequences can also be included as long as the expression cassette remains operable. The open reading frame can optionally include a coding sequence for more than one protein of interest. A “selectable marker” or “selectable protein marker” (used interchangeably herein) is a gene product or protein, the recombinant expression of which confers a phenotypic trait or function upon a host cell that enables the survival, growth, and selection of the cell under restrictive environmental and/or nutritional conditions. Useful selectable markers and their coding sequences are well known in the art. They can confer traits such as, but not limited to, resistance to a toxin, heavy metal, antibiotic, or other agent, prototrophy in an auxotrophic host, the ability to grow in a medium free of an essential nutrient, the ability to synthesize an essential metabolite. Selectable markers commonly used in transfecting mammalian cells, such as CHO cells, include, but are not limited to an endogenously expressed selectable protein marker (i.e., a gene that is typically expressed in a non-auxotrophic parental cell line), which in the host cell employed in the invention has the gene encoding the selectable marker “knocked-out” (KO) in a “null” mutation of the gene encoding the marker, e.g., glutamine JUST1781 PCT International Patent Application synthetase (GS) or dihydrofolate reductase (DHFR), so that successful recombinant expression of the selectable protein marker allows the useful selection of transfected cells in a population of KO cells; or in some embodiments of the invention, the selectable marker can be an antibiotic resistance marker, such as puromycin-N acetyl transferase (PAC or PurR), neomycin resistance (NeoR), zeomycin resistance (ZeoR), blasticidin-S deaminase, hygromycin phosphotransferase (hpt), aminoglycoside phosphotransferase, nourseothircin N- acetyl transferase, or a protein that binds to zeocin. A useful example of a glutamine synthetase coding sequence for a functional GS enzyme has the nucleotide sequence of SEQ ID NO:14, or a degenerate DNA sequence: ATGGCCACCTCAGCAAGTTCCCACTTGAACAAAAACATCAAGCAAATGTACTTGT GCCTGCCCCAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACTGG AGAAGGACTGCGCTGCAAAACCCGCACCCTGGACTGTGAGCCCAAGTGTGTAGA AGAGTTACCTGAGTGGAATTTTGATGGCTCTAGTACCTTTCAGTCTGAGGGCTCCA ACAGTGACATGTATCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAGAGAT CCCAACAAGCTGGTGTTCTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAGAGA CCAATTTAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAACCAGCACCC CTGGTTTGGAATGGAACAGGAGTATACTCTGATGGGAACAGATGGGCACCCTTTT GGTTGGCCTTCCAATGGCTTTCCTGGGCCCCAAGGTCCGTATTACTGTGGTGTGGG CGCAGACAAAGCCTATGGCAGGGATATCGTGGAGGCTCACTACCGCGCCTGCTTG TATGCTGGGGTCAAGATTACAGGAACAAATGCTGAGGTCATGCCTGCCCAGTGGG AATTTCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCTCTGGGTGGCC CGTTTCATCTTGCATCGAGTATGTGAAGACTTTGGGGTAATAGCAACCTTTGACCC CAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACC AAGGCCATGCGGGAGGAGAATGGTCTGAAGCACATCGAGGAGGCCATCGAGAAA CTAAGCAAGCGGCACCGGTACCACATTCGAGCCTACGATCCCAAGGGGGGCCTGG ACAATGCCCGTCGTCTGACTGGGTTCCACGAAACGTCCAACATCAACGACTTTTC TGCTGGTGTCGCCAATCGCAGTGCCAGCATCCGCATTCCCCGGACTGTCGGCCAG GAGAAGAAAGGTTACTTTGAAGACCGCCGCCCCTCTGCCAATTGTGACCCCTTTG CAGTGACAGAAGCCATCGTCCGCACATGCCTTCTCAATGAGACTGGCGACGAGCC CTTCCAATACAAAAACTAA//SEQ ID NO:14. [00090] Typically, selectable markers are useful for choosing a particular cellular clone for its utility in producing a particular protein(s) of interest. JUST1781 PCT International Patent Application [00091] As used herein the term "coding region" or "coding sequence" when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA). [00092] Recombinant expression technology typically involves the use of a recombinant expression vector comprising an expression cassette and a mammalian host cell comprising the recombinant expression vector with the expression cassette or at least the expression cassette, which may for example, be integrated into the host cell genome. [00093] The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell. [00094] The term "expression vector" or "expression construct" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of the operably linked coding sequence in a particular host cell. An expression vector can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto. [00095] “Exons” are nucleic acid sequences in DNA or RNA that encode the amino acid sequence of a protein or proprotein; exons are present in mature mRNA after post- transcriptional modification. In eukaryotic cells, “introns” are intervening sequences between two exons; introns do not directly encode for protein sequences. In the eukaryotic nucleus, a transcribed, unprocessed mRNA with the introns present, is referred to as a “pre-mRNA” molecule. Introns are removed by RNA splicing before translation. (See, e.g., Calvet and Pederson, “Secondary structure of heterogeneous nuclear RNA: Two classes of double- stranded RNA in native ribonucleoprotein,” Proc. Natl. Acad. Sci. USA Vol.74, No.9, pp. 3705-3709 (1977)). [00096] N⁶-methyladenosine (“m6A”) is the most abundant and conserved ribonucleotide modification among eukaryotic messenger RNAs and is dynamically regulated through distinct protein complexes that methylate, demethylate, and/or interpret the m6A modification. Deposition of m6A onto RNAs is achieved by the m6A methyltransferase complex (used interchangeably herein with “m6A writer” or “m6A methyltransferase writer complex”), a large holocomplex of about 1000 JUST1781 PCT International Patent Application kDa in size that contains methyltransferase-like 3 and 14 (METTL3/14), Wilms’ tumor 1-associated protein (WTAP), KIAA1429 (VIRMA), Zinc finger CCCH domain-containing protein 13 (ZC3H13), RNA binding motif protein 15/15 paralog (RBM15/15B), and E3 ubiquitin ligase CBLL1 (HAKAI). (See, e.g., Shichen Su et al., “Cryo-EM structures of human m6A writer complexes,” Cell Research 32:982–994 (2022), doi.org/10.1038/s41422-022-00725-8). These m6A writer complex proteins, and the m6A modification, are involved in the regulation of gene expression, RNA stability, splicing and translation. Although the precise contribution of m6A to alternative splicing, exon selection and transcript stability has been debated there is substantial evidence to support a role for m6A in regulating context-dependent splicing in vivo. (see, e.g., Lothion-Roy, Jennifer et al., “Clinical and molecular significance of the RNA m⁶A methyltransferase complex in prostate cancer,” Front. Genet., 12 January 2023, Sec. Epigenomics and Epigenetics Vol.13 – 2022, doi.org/10.3389/fgene.2022.1096071; Ke, S., Alemu, E. A., Mertens, C., Gantman, E. C., Fak, J. J., Mele, A., et al. (2015). “A majority of m6A residues are in the last exons, allowing the potential for 3’ UTR regulation,” Genes Dev.29, 2037–2053 (2015), doi:10.1101/gad.269415.115; Haussmann, I. U., Bodi, Z., Sanchez-Moran, E., Mongan, N. P., Archer, N., Fray, R. G., et al., “m(6)A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination,” Nature 540, 301–304 (2016). doi:10.1038/nature205772016; Lence, T., Akhtar, J., Bayer, M., Schmid, K., Spindler, L., Ho, C. H., et al. (2016). “m6A modulates neuronal functions and sex determination in Drosophila.” Nature 540, 242–247 (2016), doi:10.1038/nature205682016; Ke, S., Pandya-Jones, A., Saito, Y., Fak, J. J., Vagbo, C. B., Geula, S., et al. (2017), “m(6)A mRNA modifications are deposited in nascent pre- mRNA and are not required for splicing but do specify cytoplasmic turnover,” Genes Dev.31, 990– 1006 (2017), doi:10.1101/gad.301036.117; Darnell, R. B., Ke, S., and Darnell, J. E., JR. (2018), “Pre- mRNA processing includes N(6)methylation of adenosine residues that are retained in mRNA exons and the fallacy of RNA epigenetics,” RNA 24, 262–267 (2018), doi:10.1261/rna.065219.117; Zhao, B. S., Nachtergaele, S., Roundtree, I. A., and He, C., “Our views of dynamic N(6)-methyladenosine RNA methylation,” RNA 24, 268–272 (2018), doi:10.1261/rna.064295.117). [00097] A “motif that is recognizable by a m⁶A methyltransferase writer complex” is a nucleotide sequence in a DNA or RNA molecule that is able to encode and/or, when transcibed into an RNA molecule, specifically bind, a m6A methyltransferase writer complex under physiological conditions to enable function of the m6A writer complex. For example, a putative m6A motif sequence is GGACT, i.e., an example of the so-called “DRACH” motif, which appears twice within one such nucleotide sequence insert we employed: GGACTAAAGCGGACTTGT//SEQ ID NO:5); the broader consensus sequence is best reflected by the motif “DRACH” (D=A, G or U; R=A or G; H=A, C or U). (See, e.g., Linder et al., “Single-nucleotide resolution mapping of m6A and m6Am throughout the transcriptome,” Nat. Methods 12(8):767-772 (2015); Schwartz et al., “Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites,” Cell rep 10;8(1): 284-296 JUST1781 PCT International Patent Application (2014); Meyer et al., “Comprehensive analysis of mRNA methylation reveals enrichment in 3'UTRs and near stop codons,” Cell 149:1635-1646 (2012)). However, the person of skill in the art can use other examples of such motifs that are known in the art. [00098] Nucleic acid sequences necessary for expression can include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, introns, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence of interest, so that the expressed polypeptide can be secreted by the recombinant host cell, for more facile isolation of the polypeptide of interest from the cell, if desired. Such techniques are well known in the art. (See, e.g., Goodey, Andrew R.; et al., Peptide and DNA sequences, U.S. Pat. No.5,302,697; Weiner et al., Compositions and methods for protein secretion, U.S. Pat. No.6,022,952 and U.S. Pat. No. 6,335,178; Uemura et al., Protein expression vector and utilization thereof, U.S. Pat. No. 7,029,909; Ruben et al., 27 human secreted proteins, US 2003/0104400 A1). For expression of multi-subunit proteins of interest, separate expression vectors in suitable numbers and proportions, each containing a coding sequence for each of the different subunit monomers, can be used to transform a host cell. In other embodiments, a single expression vector can be used to express several, or all of, the different subunit monomers of the protein of interest. [00099] The term "host cell" means a cell that has been transformed, or is capable of being transformed, with a nucleic acid and thereby expresses a gene or coding sequence of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present. Any of a large number of available and well-known host cells may be used in the practice of this invention to obtain the antigen-binding proteins of the invention, including mammalian cells, insect cells, microbial cells, or plant cells. For some embodiments mammalian host cells capable of post-translationally glycosylating antibodies may be preferred by the skilled artisan. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Modifications can be made at the DNA level, as well. The peptide-encoding DNA sequence may be changed to JUST1781 PCT International Patent Application codons more compatible with the chosen host cell. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. [000100] Within these general guidelines, eukaryotic host cells in culture, such as yeast cell lines (e.g., Saccharomyces, Pichia, Schizosaccharomyces, Kluyveromyces) and other fungal cells, algal or algal-like cells, insect cells, plant cells, that have been modified to incorporate humanized glycosylation pathways, can be used to produce fully functional glycosylated proteins of interest (e.g., antibody). However, mammalian (including human) host cells, e.g., CHO cells and HEK-293 cells, are especially useful. [000101] Examples of useful mammalian host cell lines are Chinese hamster ovary cells, including CHO-K1 cells (e.g., ATCC CCL61), CHO-S, DXB-11, DG-44, and Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture (Graham et al, J. Gen Virol.36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod.23: 243-251 (1980)); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y Acad. Sci.383: 44-68 (1982)); MRC 5 cells or FS4 cells; or mammalian myeloma cells, e.g., NS0 or sp2/0 mouse myeloma cells. “Cell,” “cell line,” and “cell culture” are often used interchangeably and all such designations herein include cellular progeny. For example, a cell “derived” from a CHO cell is a cellular progeny of a Chinese Hamster Ovary cell, which may be removed from the original primary cell parent by any number of generations, and which can also include a transformant progeny cell. Transformants and transformed cells include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Host cells are transformed or transfected with the JUST1781 PCT International Patent Application above-described nucleic acids or vectors for production of polypeptides (including antigen binding proteins, such as antibodies) and are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In addition, novel vectors and transfected cell lines with multiple copies of transcription units separated by a selective marker or reporter are particularly useful for the expression of polypeptides, such as antibodies. [000102] The inventive method of recombinantly expressing one or more proteins of interest in a cultured eukaryotic host cell line involves transfecting a plurality of cultured eukaryotic cells of a parent cell line, with the inventive multiple-vector expression system. [000103] The term "transfection" means the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. [000104] The term "transformation" refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is considered to have been "stably transformed" when the transforming DNA is replicated with the division of the cell. [000105] “Inducible expression” refers to non-constitutive expression from a promoter, in response to derepression of the promoter sequence. For example, in cells expressing the Tet repressor (TetR), tetracycline can be used to regulate expression from promoters containing the Tet operator sequence. Introduction of the Tet operator (TetO) sequence just 3’ of the TATA box prevents transcription from this promoter in the presence of the TetR. Presumably, TetR binds to TetO and prevents transcription factors from interacting with the transcription start site or interfere with the transcription initiation complex formation. JUST1781 PCT International Patent Application Notably, positioning of the TetO sequences is critical in determining if TetR can effectively modulate transcription. (See, Yao et al., Tetracycline repressor, tetR, rather than the tetR- mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells, Hum. Gene Ther.9(13):1939-50 (1998)). [000106] Yao et al. hypothesized that positioning the TetO sequence 10bp downstream of the TATATAA sequence allows binding of the TetR to the same surface as the TATA binding protein. Consistent with this hypothesis, Kim et al. found that insertion of TetO sequences at position 0 or 15 downstream of the hCMV TATAAG sequence failed to preserve repression in the presence of TetR. (See, Kim et al., "Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus", J. Virol.69: 2565-257 (1995)). Tetracycline (Tet), when added to the culture, will bind TetR and inhibit binding to the TetO sequence, thus allowing transcription. [000107] In some useful embodiments, in which tetracycline-inducible expression is desired, a promoter comprises one or more TetO sequences operably linked 3’ to other regulatory elements within the promoter of choice. A “TetO” sequence means a nucleotide sequence, which maintains the ability to bind tetracycline repressor protein (TetR). The TetO sequence is placed so that, in the presence of TetR protein, there is binding of TetR to the TetO sequence, thereby disrupting transcription to a detectable extent, compared to a control not having TetO in the promoter driving transcription of a gene of interest, or to a control not having TetR. Examples of the TetO sequence include a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:12 (TCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGA//SEQ ID NO:12) or having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:13 (CTCCCTATCAGTGATCAGTTCCTCCCTATCAGTGATAGAGA//SEQ ID NO:13). In some useful embodiments, there can be additional nucleotide residues 5’ or 3’ to the TetO sequence(s), or there can be intervening nucleotide linker sequences, as long as the ability to bind TetR protein is not eliminated. [000108] “Repression,” or “repressed,” within the context of the invention, refers to the interference of transcription of a gene of interest (encoding a protein of interest), occurring when TetR protein binds to a TetO binding site in the promoter that drives expression of the gene of interest, resulting in decreased expression of the protein of interest by the cell(s) JUST1781 PCT International Patent Application (which are cell(s) that express TetR). Expression of a gene of interest or of a protein of interest is said to be “derepressed,” when, in the presence of tetracycline in the medium, expression of the protein of interest is at least 1.5-fold over the basal levels of expression by the cell(s) in the absence of tetracycline in the medium. [000109] “Tetracycline” means tetracycline or an analog of tetracycline, such as doxycycline, anhydrotetracycline, minocycline, oxytetracycline, methacycline, chlortetracycline, or COL-3 (Chemically modified tetracycline-3). [000110] The host cells can be usefully grown in batch culture, fed-batch culture, intensified fed-batch culture (product retention perfusion), or in continuous culture systems employing liquid aqueous medium. Mammalian cells, such as CHO and BHK cells, are generally cultured as suspension cultures. That is to say, the cells are suspended in a liquid cell culture medium, rather than adhering to a solid support. In other embodiments, the mammalian host cells can be cultured on solid or semi-solid aqueous culture medium, for example, containing agar or agarose, to form a medium, carrier (or microcarrier) or substrate surface to which the cells adhere and form an adhesion layer. Another useful mode of production is a hollow fiber bioreactor with an adherent cell line. Porous microcarriers can be suitable and are available commercially, sold under brands, such as Cytoline^®, Cytopore^® or Cytodex^® (GE Healthcare Biosciences). [000111] "Cell culture medium" or “culture medium,” used interchangeably, is defined, for purposes of the invention, as a sterile medium suitable for growth of cells, and preferably animal cells, more preferably mammalian cells (e.g., CHO cells), in in vitro cell culture. Any medium capable of supporting growth of the appropriate cells in cell culture can be used. Suitably, the culture medium has an osmolality of between 210 and 650 mOsm, preferably 270 to 450 mOsm, more preferably 310 to 350 mOsm and most preferably 320 mOsm. Preferably, the osmolality of the cell culture supernatant is maintained within one or more of these ranges throughout the culturing of host cells. The cell culture medium can be based on any basal medium such as DMEM, or RPMI generally known to the skilled worker. Commercially available media such as ExpiCHO^TM Expression Medium (ThermoFisher Scientific), Ham's F10 (Sigma), Ham's F12, Medium 199, McCoy, Minimal Essential Medium ((MEM), (Sigma-Aldrich), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich) are suitable for culturing various host cells. In addition, any of the media described in Ham et al., Meth. Enz.58: 44 (1979), Barnes et al., Anal. JUST1781 PCT International Patent Application Biochem.102: 255 (1980), U.S. Patent Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Patent Re. No.30,985 may be used as culture media for the host cells, or modified appropriately to suit the cell line employed. Other examples include HyClone ActiPro™ and Lonza PowerCHO-2™. The basal medium can comprise a number of ingredients, including amino acids, vitamins, organic and inorganic salts, and sources of carbohydrate, each ingredient being present in an amount which supports the cultivation of a cell which is generally known to the person skilled in the art. The medium can contain auxiliary substances, such as buffer substances like sodium bicarbonate, antioxidants, stabilizers to counteract mechanical stress, or protease inhibitors. Any of these media may be supplemented as necessary with hormones and/or other growth factors (preferably recombinantly produced), such as insulin, insulin-like growth factor (IGF)-1, transferrin, or epidermal growth factor; salts, such as sodium chloride, calcium, magnesium, and phosphate; buffers, such as HEPES and/or sodium bicarbonate; nucleotides, such as adenosine and thymidine; antibiotics, such as gentamicin, neomycin, tetracycline, puromycin, or kanamycin; trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range); and glucose or an equivalent carbon and/or energy source, such that the physiological conditions of the cell in, or on, the medium promote expression of the protein of interest by the host cell; any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. [000112] Historically, mammalian cells have been cultured in media containing mammalian serum. The culture medium can include a suitable amount of serum such a fetal bovine serum (FBS). The term "serum-comprising" as applied to cell culture medium includes any cell culture medium that does contain serum. However, such media are incompletely defined and carry the risk of infection, therefore, preferably, the host cells can be adapted for culture in serum-free medium. The term "serum-free" as applied to medium includes any cell culture medium that does not contain serum. By "serum- free", it is understood that the medium has preferably less than 0.1% (v/v) serum and more preferably less than 0.01% (v/v) serum. The term "serum" refers to the fluid portion of the blood obtained after removal of the fibrin clot and blood cells. [000113] Those in the art have devised "protein-free" media that are either completely free of any protein or at least are free of any protein that is not recombinantly produced. For example, due to the labile nature of Factor VIII (FVIII), the productivity of the engineered host cells is severely reduced under protein-free conditions. Human serum albumin is JUST1781 PCT International Patent Application commonly used as a serum-free culture supplement for the production of recombinant proteins. The albumin itself stabilizes the FVIII, and the impurities present in serum-derived albumin preparations may also contribute to the stabilizing effect of albumin. Factors such as lipoprotein have been identified as a replacement for human serum albumin for the production of recombinant Factor VIII (FVIII), under serum-free conditions. Useful cell culture media include those disclosed in U.S. Pat. No.6,171,825 (Chan et al., Preparation of recombinant factor VIII in a protein free medium, Bayer, Inc.) and U.S. Pat. No.6,936,441 (Reiter et al., Recombinant cell clones having increased stability and methods of making and using the same, Baxter AG). The medium of U.S. Pat. No.6,171,825 (Chan et al.) comprises modified Dulbecco's Minimum Essential Medium and Ham's F-12 Medium (50:50, by weight) supplemented with recombinant insulin, iron, a polyol, copper and optionally other trace metals. [000114] If insulin is used, it should be recombinant and can be obtained commercially as “Nucellin” insulin (Eli Lilly. It can be added at 0.1 to 20 µg/mL (preferably 5-15 µg/mL, or about 10 µg/mL). The iron is preferably in the form of Fe²⁺ ions, for example provided as FeSO₄EDTA, and can be present at 5-100 µM (preferably about 50 µM). Suitable polyols include non-ionic block copolymers of poly(oxyethylene) and poly(oxypropylene) having molecular weights ranging from about 1000 to about 16,000 Da. A particularly preferred polyol is Pluronic F-68 (BASF Wyandotte), which has an average molecular weight of 8400 Da and consists of a center block of poly(oxypropylene) (20% by weight) and blocks of poly(oxyethylene) at both ends. It is also available as Synperonic F-68 from Unichema Chemie BV. Others include Pluronics F-61, F-71 and F-108. Copper (Cu²⁺) may be added in an amount equivalent to 50-800 nM CuSO4, preferably 100-400 nM, conveniently about 250 nM. The inclusion of a panel of trace metals such as manganese, molybdenum, silicon, lithium and chromium can lead to further increases in Factor VIII production. BHK cells grow well in this protein-free basal medium. [000115] The medium of U.S. Pat. No.6,936,441 (Reiter et al.) is particularly well suited to the culturing of CHO cells but may be used with other cells as well. The medium of U.S. Pat. No.6,936,441 is also based on a 50/50 mixture of DMEM and Ham's F12 but includes soybean peptone hydrolysate or yeast extract at between 0.1 and 100 g/L, preferably between 1 and 5 g/L. As a particularly preferred embodiment, soybean extract, e.g. soybean peptone, may be used. The molecular weight of the soybean peptone can be less than 50 kDa, preferably less than 10 kDa. The addition of ultrafiltered soybean peptone having an average JUST1781 PCT International Patent Application molecular weight of 350 Da has proven particularly advantageous for the productivity of the recombinant cell lines. It is a soybean isolate having a total nitrogen content of about 9.5% and a free amino acid content of about 13%, or about 7-10%. [000116] Another useful embodiment of a cell culture medium has the following composition: synthetic minimum medium (e.g.50/50 DMEM/Ham's F12) 1 to 25 g/L; soybean peptone 0.5 to 50 g/L; L-glutamine 0.05 to 1 g/L; NaHCO30.1 to 10 g/L; ascorbic acid 0.0005 to 0.05 g/L; ethanolamine 0.0005 to 0.05; and sodium selenite 1 to 15 µg/L. Optionally, a “defoaming” or “anti-foaming” agent can be added to the culture medium. Examples include, a silicone antifoam agent, or a non-ionic surface-active agent such as a polypropylene glycol (e.g. Pluronic F-61, Pluronic F-68, Pluronic F-71 or Pluronic F-108). Another example of a useful commercially available anti-foaming agent is Ex-Cell^® Antifoam (Sigma-Aldrich, Inc., St. Louis, MO; Product No.59920C). The anti-foam agent is generally applied to protect the cells from the negative effects of aeration ("sparging"), since without the addition of a surface-active agent the rising and bursting air bubbles may damage those cells that are at the surface of the air bubbles. [000117] The amount of non-ionic surface-active agent can range between 0.05 and 10 g/L, preferably between 0.1 and 5 g/L. Furthermore, the medium can also contain cyclodextrin or a derivative thereof. The serum- and protein-free medium can also contain a protease inhibitor, such as a serine protease inhibitor, which is suitable for tissue culture and which is of synthetic or vegetable origin. Non-ionic surfactants or antifoaming agents, if present in the cell culture medium, are preferably removed from the buffer in which the antibodies are dissolved before any affinity chromatography steps, lest they interfere. [000118] In another embodiment of a cell culture medium, the following amino acid mixture is can be added to the above-mentioned medium: L-asparagine (0.001 to 1 g/L; preferably 0.01 to 0.05 g/L; particularly preferably 0.015 to 0.03 g/1), L-cysteine (0.001 to 1 g/L; preferably 0.01 to 0.05 g/L; particularly preferably 0.015 to 0.03 g/L), L-proline (0.001 to 1.5 g/L; preferably 0.01 to 0.07 g/L; particularly preferably 0.02 to 0.05 g/L), L-tryptophan (0.001 to 1 g/L; preferably 0.01 to 0.05 g/L; particularly preferably 0.015 to 0.03 g/L) and L- glutamine (0.05 to 10 g/L; preferably 0.1 to 1 g/L). These amino acids may be added to the medium individually or in combination. The combined addition of the amino acid mixture containing all of the above-mentioned amino acids is particularly preferred. JUST1781 PCT International Patent Application [000119] In one embodiment, a serum- and protein-free medium is used additionally containing a combination of the above-mentioned amino acid mixtures and purified, ultrafiltered soybean peptone hydrolysate. [000120] Nutrient supplements such as yeast hydrolysate or various plant-based hydrolysates can be included in the medium, if desired. In some embodiments, the aqueous medium is liquid, such that the host cells are cultured in a cell suspension within the liquid medium. Alternate media capable of supporting CHO cell growth and productivity of antibody can be used interchangeably with the media used in the working example described herein. The possibilities are numerous and could include commercial media made by Sigma- Aldrich, Sartorius or Irvine Scientific, as well as, media especially formulated for a variety of suitable host cell types. [000121] The term "hydrolysate" includes any digest of an animal derived or plant derived source material, or extracts derived from yeast, bacteria, or plants, e.g.,"soy hydrolysate," which can be a highly purified soy hydrolysate, a purified soy hydrolysate or crude soy hydrolysate. [000122] A further suitable cell culture medium is the oligopeptide-free medium disclosed in US 2007/0212770 A1 (Grillberger et al., Oligopeptide-free cell culture media; Baxter International Inc., Baxter Healthcare S.A.), but any suitable cell culture medium that provides physiological conditions permitting the expression of antibody proteins by the host cells can be employed, including other media described in the Examples herein. [000123] The term "inoculation of the cells into the cell culture medium" refers to the step of contacting the cells with the cell culture medium under conditions which are suitable for growth and proliferation of the cells. [000124] The cell culture contemplated herein may be any cell culture independently of the kind and nature of the cultured cells and the growth phase of the cultured cells, e.g. adherent or non-adherent cells; growing, or growth-arrested cells. [000125] The term "sterile," as used herein, refers to a substance that is free, or essentially free, of microbial and/or viral contamination. In this respect the "contaminant" means a material that is different from the desired components in a preparation being a cell culture medium or at least a component of a cell culture medium. In the context of "sterile filtration", the term sterile filtration is a functional description that a preparation is filtered JUST1781 PCT International Patent Application through a sterile filter (with a pore size of 0.2 µm or less) to remove bacterial and/or mycoplasma contaminants. [000126] The "cell culture supernatant" is the extracellular medium in which the mammalian cells are cultured. This medium is not to be confused with feed medium that may be added to the culture after inoculation of the cells into the cell culture medium and cell growth has been commenced. A "cell culture" means the cell culture supernatant and the mammalian cells cultured therein. Conventionally, mammalian cells are cultured at 37°C ± 1°C. [000127] "Culturing at" or "maintaining at" a set point of a particular desired temperature, means that the process control systems are set to that desired temperature, in other words that the set point of temperature is the intended target temperature. The culture conditions, such as temperature (typically, but not necessarily, about 37°C), pH (typically, but not necessarily, a cell culture medium is maintained within the range of about pH 6.5-7.5, as modified consistent with the present invention), oxygenation, and the like, will be apparent to the ordinarily skilled artisan. Clearly, there will be small variations of the temperature of a culture over time, and from location to location through the culture vessel. Digital control units and sensory monitors are available commercially or can be constructed by the skilled artisan. Alternative digital control units (DCU) control and monitor the cell culture process are available commercially, made by companies such as B. Braun, New Brunswick, or Sartorius. For in-flask batch culture with shaker, numerous models of suitable cell culture incubators with built-in environmental controls (e.g., CO₂ and Multigas CO₂/O₂ controls) are commercially available, e.g., by Thermo Fisher Scientific. [000128] "Culturing at" or "maintaining at" a temperature that is set at X±Y°C, means that the set point is at a value of from X+Y°C to X-Y°C. For example, where X and Y are 37.0 and 0.9, respectively, the set point is set at a value of from 37.9 to 36.1°C. For each of the preferred values of X, e.g., X=31, X=32, X=33, X=34, X=35, X=36, or X=37, the set- point is at a value within the range X±0.9°C, ±0.8°C, ±0.7°C, ±0.6°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, or ±0.1°C. (See, e.g., Oguchi et al., pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture, Cytotechnology.52(3):199–207 (2006); Al-Fageeh et al., The cold-shock response in cultured mammalian cells: Harnessing the response for the improvement of recombinant protein production, Biotechnol. Bioeng.93:829–835 (2006); Marchant, R.J. et al., Metabolic rates, JUST1781 PCT International Patent Application growth phase, and mRNA levels influence cell-specific antibody production levels from in vitro cultured mammalian cells at sub-physiological temperatures, Mol. Biotechnol.39:69–77 (2008)). [000129] For any given set-point, slight variations in temperature may occur. Typically, such variation may occur because heating and cooling elements are only activated after the temperature has deviated somewhat from the set-point. In that case, the set-point is X±Y and the heating or cooling element is activated when the temperature varies by ±Z°C, as appropriate. Typically, the permissible degree of deviation of the temperature from the set- point before heating or cooling elements are activated may be programmed in the process control system. Temperature may be controlled to the nearest ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, or even ±0.1°C by heating and cooling elements controlled by thermostats. Larger differentials in temperature may also be programmed, such as ±1.0°C, ±0.9°C, ±0.8°C, ±0.7°C, or ±0.6°C. The temperature may also be controlled by immersion of the culture vessel in a heating bath at a particular temperature. Conceivably, there is no variation from the set-point because the heating is applied continually, which can also involve variable heating output in the case of variable ambient temperature. Another source of variation arises due to measurement error in the temperature of the cell culture supernatant. Typical thermometers used in cell culture equipment may have a variability of ±0.3°C, or ±0.2°C, or even ±0.1°C. [000130] Where the temperature set-point is set at a value within the range X±Y°C, and the tolerance of the temperature is ±Z°C (i.e. a heater or cooler is activated when the temperature deviates by ±Z°C, as appropriate) this can also be expressed as a set-point of (X- Y to X+Y)±Z°C. For each possible value of X, all combinations of ±Y°C. and ±Z°C, as indicated above, are envisaged. [000131] "Culturing at" or "maintaining at" a set point of a particular desired pH value, means that the process control systems are set to that desired pH value, in other words that the set point of pH is the intended target pH. "Culturing at" or "maintaining at" a pH that is set at X±Y, means that the set point is at a value of from X+Y to X-Y pH units. For each of the preferred values of X, the set-point is at a value within the range pH X±0.05, ±0.04, ±0.03, ±0.02 or ±0.01. JUST1781 PCT International Patent Application [000132] Where the pH set-point is set at a value within the range X±Y, and the tolerance is ±Z, this can also be expressed as a set-point of (X-Y to X+Y)±Z. For each possible value of X, all combinations of ±Y and ±Z, as indicated above. [000133] For any given pH set-point, slight variations in pH may occur. Typically, such variation can occur because means which control pH are only activated after the pH has deviated somewhat from the set-point. Typically, the pH is controlled to the nearest ±0.05, ±0.04, ±0.03, ±0.02, or ±0.01. Typically, sparging with CO₂ provides additional acid in mammalian cell culture. Liquid acids, e.g., HCl or H3PO4, are commonly used in microbial cultures. Sodium carbonate is usually the source of added alkali used to maintain pH for mammalian cell culture, and NH4OH is often selected to add alkali in microbial culture. [000134] The cell culture supernatant typically has a CO2 concentration of 1 to 10% (v/v), for example 4.0-9.0% (v/v), 5.5-8.5% (v/v) or about 6-8% (v/v). Conventionally, CO₂ concentration is higher than this due to the CO2 produced by the cells not being removed from the cell culture supernatant. Maintaining the CO2 concentration at 10% or lower is reported to increase the yield of recombinant protein; it helps the dCO2 (or pCO2) to be kept low if the feed medium is degassed (for example by bubbling air through it) as well as the cell culture supernatant in the bioreactor being sparged. (See, Giovagnoli et al., Cell Culture Processes, US2009/0176269, US2016/0244506, US9359629, EP2235197, EP2574676). [000135] Ways of monitoring culture parameters of temperature, pH and CO2 concentration are well known in this art and generally rely on probes that are inserted into the bioreactor, or included in loops through which the culture medium is circulated, or inserted into extracted samples of culture medium. Suitable monitoring equipment and appropriate alternatives are commercially available or can be constructed by the skilled artisan. Alternative gas analyzers are commercially available, such as RapidLab^® 248 (Siemens) and others made by Nova® Biomedical, Radiometer America and Roche Diagnostics. Mass flow controllers can also be used to control gas and liquid additions in labs that are properly equipped. A suitable in-line dCO2 (or pCO2) sensor and its use are described in Pattison et al (2000) Biotechnol. Prog.16:769-774. A suitable in-line pH sensor is Mettler Toledo InPro 3100/125/Pt100 (Mettler-Toledo Ingold, Inc., Bedford, Mass.). A suitable off-line system for measuring dCO2 (or pCO2), in addition to pH and pO2 is the BioProfile pHOx (Nova Biomedical Corporation, Waltham MA). In this system, or dCO₂ (or pCO₂) is measured by potentiometric electrodes within the range 3-200 mmHg with an imprecision resolution of JUST1781 PCT International Patent Application 5%. The pH may be measured in this system at a temperature of 37°C, which is close to the temperature of the cell culture supernatant in the bioreactor. Ways of altering the specified parameter in order to keep it at the predefined level are also well known. For example, keeping the temperature constant usually involves heating or cooling the bioreactor or the feed medium (if it is a fed-batch or continuous process); keeping the pH constant usually involves choosing and supplying enough of an appropriate buffer (typically bicarbonate) and adding acid, such as hydrochloric acid, or alkali, such as sodium hydroxide, sodium carbonate or a mixture thereof, to the feed medium as necessary; and keeping the CO₂ concentration constant usually involves adjusting the sparging rate (see further below), or regulating the flow of CO₂ in the head space. It is possible that the calibration of an in-line pH probe may drift over time, such as over periods of days or weeks, during which the cells are cultured. In that event, it may be beneficial to reset the in-line probe by using measurements obtained from a recently calibrated off-line probe. A suitable off-line probe is the BioProfile pHOx (Nova Biomedical Corporation, Waltham MA). [000136] Mammalian cell cultures, and many other kinds of microbial cells, need oxygen for the cells to grow, or can grow fastest under aerobic conditions. Normally, this is provided by forcing oxygen into the culture through injection ports. It is also necessary to remove the CO₂ that accumulates due to the respiration of the cells. This is achieved by “sparging,” i.e., passing a gas through the bioreactor in order to entrain and flush out the CO2. Conventionally, this can also be done using oxygen. However, the inventors have found that it is advantageous to use air instead. It has been found that usually a conventional inert gas such as nitrogen is less effective at sparging CO₂ than using air. Given that air is about 20% (v/v) oxygen, one might have thought that five times as much air would be used. However, this has been found to be inadequate in large scale cultures, particularly in cultures at 2500 L scale. In a 2500-L bioreactor, 7 to 10 times as much air, preferably about 9 times as much air, is used. For example, under standard conditions, the 2500-L bioreactor is sparged with O2 at a 10-µm bubble size at a rate of 0.02 VVH (volume O₂ per volume of culture per hour). The same 2500-L bioreactor used according to the methods of the invention would be sparged with air at a 10-µm bubble size at a rate of 0.18 VVH. [000137] Hence, the use of surprisingly high volumes of air has been found to provide adequate oxygen supply and to remove the unwanted CO2. Flushing the bioreactor head space with air is also a useful mechanism for removing excess CO₂. JUST1781 PCT International Patent Application [000138] During production phase, it is preferred to remove CO₂ by air sparging, as described above. This is especially the case when using bioreactors of large capacity, in which the cell culture supernatant would otherwise accumulate CO2 to deleteriously high levels. However, at the beginning of culture, or in small scale culture, such as at 1-L or 2.5-L scale, the head space may be overlayed with CO2. Under such conditions, low levels of dCO2 (or pCO₂) can still be achieved. Overlaying the headspace with CO₂ may also be used to reduce the pH to the set-point, if the pH is too basic. [000139] In accordance with inventive methods, the culturing of a plurality of mammalian host cells can be any conventional type of culture, such as batch, fed-batch, intensified fed-batch, or continuous. Suitable continuous cultures included repeated batch, chemostat, turbidostat or perfusion culture. For purposes of the present invention, the desired scale of the recombinant expression will be dependent on the type of expression system and the quantity of different theoretical antibody variants to be studied. As noted herein, typically, 100 milligrams of total antibody protein will suffice, requiring only a batch cell culture of 20 mL to 500 mL; while larger scale culture batches or continuous cell culture methods can be employed, larger volumes are typically not cost-effective. [000140] A batch culture starts with all the nutrients and cells that are needed, and the culture proceeds to completion, i.e. until the nutrients are exhausted or the culture is stopped for some reason. [000141] A fed-batch culture is a batch process in the sense that it starts with the cells and nutrients but it is then fed with further nutrients in a controlled way. The fed-batch strategy is typically used in bio-industrial processes to reach a high cell density in the bioreactor. The feed solution is usually highly concentrated to avoid dilution of the bioreactor. The controlled addition of the nutrient directly affects the growth rate of the culture and allows one to avoid overflow metabolism (formation of metabolic by-products) and oxygen limitation (anaerobiosis). In most cases the growth-limiting nutrient is glucose which is fed to the culture as a highly concentrated glucose syrup (for example 500-850 g/L). [000142] Different strategies can be used to control the growth in a fed-batch process. For example, any of dissolved oxygen tension (DOT, pO2), oxygen uptake rate (OUR), glucose concentration, lactate concentration, pH and ammonia concentration can be used to monitor and control the culture growth by keeping that parameter constant. In a continuous culture, nutrients are added and, typically, medium is extracted in order to remove unwanted JUST1781 PCT International Patent Application by-products and maintain a steady state. Suitable continuous culture methods are repeated batch culture, chemostat, turbidostat and perfusion culture. [000143] CHO cells, for example, may be cultured in a stirred tank or an airlift tank that is perfused with a suitable medium at a perfusion rate of from 1 to 10 volume exchanges per day and at an oxygen concentration of between 40% and 60%, preferably about 50%. Moreover, the cells may be cultured by means of the chemostat method, using the preferred pH value given above, an oxygen concentration of between 10% and 60% (preferably about 20%) and a dilution rate D of 0.25 to 1.0, preferably about 0.5. [000144] In a repeated batch culture, also known as serial subculture, the cells are placed in a culture medium and grown to a desired cell density. To avoid the onset of a decline phase and cell death, the culture is diluted with complete growth medium before the cells reach their maximum concentration. The amount and frequency of dilution varies widely and depends on the growth characteristics of the cell line and convenience of the culture process. The process can be repeated as many times as required and, unless cells and medium are discarded at subculture, the volume of culture will increase stepwise as each dilution is made. The increasing volume may be handled by having a reactor of sufficient size to allow dilutions within the vessel or by dividing the diluted culture into several vessels. The rationale of this type of culture is to maintain the cells in an exponentially growing state. Serial subculture is characterized in that the volume of culture is always increasing stepwise, there can be multiple harvests, the cells continue to grow and the process can continue for as long as desired. [000145] In the chemostat and turbidostat methods, the extracted medium contains cells. Thus, the cells remaining in the cell culture vessel must grow to maintain a steady state. In the chemostat method, the growth rate is typically controlled by controlling the dilution rate i.e. the rate at which fresh medium is added. The cells are cultured at a sub-maximal growth rate, which is achieved by restricting the dilution rate. The growth rate is typically high. In contrast, in the turbidostat method, the dilution rate is set to permit the maximum growth rate that the cells can achieve at the given operating conditions, such as pH and temperature. [000146] In an intensified fed-batch culture, culture vessels, reactors or chambers, of any of various capacities are used to grow suspensions of mammalian host cells, e.g., CHO cells. Each culture vessel is connected via inlets to an array of porous tangential flow filters which in turn are connected via outlets back to the culture vessel. After cell growth, the JUST1781 PCT International Patent Application suspensions of host cells and growth medium are pumped through the array of porous tangential flow filters to concentrate the cell suspension. The cell suspension is recycled through the filters and culture vessel allowing a portion of the old growth medium (and its serum components, if any) to be removed. A supply of fresh sterile serum-free expression medium is added to the concentrated cell suspension to maintain a nominal volume in the culture vessel. The recombinant protein of interest, e.g., an antibody, is produced subsequently by the host cells suspended in the expression medium and is secreted by the cells into the expression medium from which it can be harvested by standard techniques. (See, e.g., Zijlstra et al., Process for the culturing of cells, US8119368, US8222001, US8440458). [000147] In a perfusion or continuous culture, the extracted medium is depleted of cells, because most of the cells are retained in the culture vessel, for example, by being retained on a membrane through which the extracted medium flows. However, typically such a membrane retains 100% of cells, and so a proportion are removed when the medium is extracted. Alternatively, sonic cell separation technology achieves separation of cells from the media matrix with high-frequency, resonant ultrasonic waves rather than using a physical barrier, unlike tangential-flow filtration (TFF) or alternating tangential flow filtration (ATF); the cells are held back using an acoustic field as the bioprocess fluid flows through an open channel. The use of acoustic waves allows differentiation of particles of equal size, and thus the technology can be used for the separation of particles from the nano- to macro- scales. (See, e.g., Challenger, C.A., An acoustic wave-based technology for cell harvesting applications may help enable continuous manufacturing, BioPharm International 30(9):30 (2017)). Regardless of the technology employed to separate the cells from the extracted medium, it may not be crucial to operate perfusion cultures at very high growth rates, as the majority of the cells are retained in the culture vessel. [000148] Continuous cultures, particularly repeated batch, chemostat and turbidostat cultures, are typically operated at high growth rates. According to common practice, it is typical to seek to maintain growth rates at maximum, or close to maximum, in an effort to obtain maximum volumetric productivity. Volumetric productivity is measured in units of protein quantity or activity per volume of culture per time interval. Higher cell growth equates to a higher volume of culture being produced per day and so is conventionally considered to reflect a higher volumetric productivity. A suitable fully continuous process can have a perfusion bioreactor coupled to recombinant protein harvesting and protein JUST1781 PCT International Patent Application purification steps, for example, a multi-column chromatography capture step, followed by flow-through virus inactivation, multi-column intermediate purification, a flow-through membrane adsorber polishing step, continuous virus filtration and a final ultrafiltration step operated in continuous mode. (See, e.g., Crowley et al., Process for cell culturing by continuous perfusion and alternating tangential flow, US8206981). [000149] The cell density is commonly monitored in cell cultures. In principle, a high cell density would be considered to be desirable since, provided that the productivity per cell is maintained, this should lead to a higher productivity per bioreactor volume. However, increasing the cell density can actually be harmful to the cells, and the productivity per cell is reduced. There is therefore a need to monitor cell density. To date, in mammalian cell culture processes, this has been done by extracting samples of the culture and analyzing them under a microscope or using a cell counting device such as the CASY TT device sold by Scharfe System GmbH, Reutlingen, Germany. It can be advantageous to analyze the cell density by means of a suitable probe introduced into the bioreactor itself (or into a loop through which the medium and suspended cells are passed and then returned to the bioreactor). Such probes are available commercially from Aber Instruments, for example the Biomass Monitor 220, 210220 or 230. The cells in the culture act as tiny capacitors under the influence of an electric field, since the non-conducting cell membrane allows a build-up of charge. The resulting capacitance can be measured; it is dependent upon the cell type and is directly proportional to the concentration of viable cells. A probe of 10 to 25 mm diameter uses two electrodes to apply a radio frequency field to the biomass and a second pair of electrodes to measure the resulting capacitance of the polarized cells. Electronic processing of the resulting signal produces an output which is an accurate measurement of the concentration of viable cells. The system is insensitive to cells with leaky membranes, the medium, gas bubbles and debris. Alternatively, cell viability can be measured by use of a vital dye (or vital stain) to stain small-aliquot samples of culture sampled periodically, and microscopically enumerated to determine viable cell count. For example Trypan blue is a vital dye commonly used for this purpose. Automated cell counters supplied by Beckman (e.g., Vi-Cell™ XR) and other companies are available. Examples include cell counting instruments made by other manufacturers, e.g., Nova Biomedical, Olympus, Thermo Fisher Scientific and Eppendorf. Cells can also be counted using flow cytometry or manually by using a hemocytometer. [000150] Typically, a viable cell density can be used from 1.0 x 10⁶ to 2.0 x 10⁷, or up to about 5 x 10⁷ cells/mL. It is known that increasing the concentration of cells toward the JUST1781 PCT International Patent Application higher end of the preferred ranges can improve volumetric productivity. Nevertheless, ranges of cell density including any of the above point values as lower or higher ends of a range are envisaged. [000151] The culture is typically carried out in a bioreactor, which is usually a stainless steel, glass or plastic vessel of 0.01 (i.e., 10-mL) to 10000 (ten thousand) litres capacity, for example, 0.01, 0.015, 0.10, 0.25, 0.30, 0.35, 1, 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, 500, 1000, 2500, 5000 or 8000 liters. The vessel is usually rigid but flexible plastic bags or bioreactor liners can be used. These flexible plastic bioreactor bags and liners are generally of the “single use” type. [000152] Upon culturing the host cells, the recombinant polypeptide or protein, can be produced intracellularly, in the periplasmic space, or, preferably, directly secreted into the medium. Harvesting the recombinant protein involves separating it from particulate matter that can include host cells, cell aggregates, and/or lysed cell fragments, into a cell-free supernatant fraction that is free of host cells and cellular debris. Such cellular debris is removed, for example, by centrifugation or microfiltration. After the recombinant protein, e.g., recombinant antibodies, is separated from the host cells and/or other particulate debris, harvesting the recombinant protein into a cell-free supernatant fraction can optionally involve capture of the recombinant protein by one or more chromatographic capture steps that can partially purify and/or concentrate the protein, such as Protein A or Protein G or Protein L affinity chromatography. (See, e.g., Frank, M. B., “Antibody Binding to Protein A and Protein G beads” 5. In: Frank, M. B., ed., Molecular Biology Protocols. Oklahoma City (1997)). [000153] After harvesting the cell culture fluid comprising a recombinant protein of interest (e.g., an antibody or antibody fragment or fusion protein), can be further purified from the cell-free supernatant fraction. Typically, the purification of recombinant proteins is usually accomplished by an optional series of chromatographic steps such as anion exchange chromatography, cation exchange chromatography, affinity chromatography (using Protein A or Protein G or Protein L as an affinity ligand), hydrophobic interaction chromatography, hydroxy apatite chromatography and size exclusion chromatography. Further, the purification process may comprise one or more ultra-, nano- or diafiltration steps, and/or, optionally, an acidic viral inactivation step. Other optional known techniques for protein purification such JUST1781 PCT International Patent Application as ethanol precipitation, Reverse Phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also possible depending on the antibody to be recovered. [000154] The present method involves harvesting the one or more proteins of interest (such as, but not limited to recombinant antibodies or fusion proteins) that are present in the culture supernatant, or in a cell-free extract, and then purifying the cell-free supernatant fraction by affinity chromatography to purify IgG present in the cell-free supernatant fraction. In this step, affinity chromatography involves loading the cell-free supernatant fraction onto an affinity chromatography matrix having conjugated moieties with particular affinity for immunoglobulin molecules that may be of interest. In embodiments including an immunoglobulin Fc domain, such as some fusion proteins (e.g., petibodies), such conjugated moieties can include, e.g., Protein A, and/or Protein G, and/or Protein L, or anti-kappa antibodies with an affinity for Fab antibody fragments, or anti-his antibodies, or glutathione, or another suitable matrix-conjugated antibody that specifically binds an immunoglobulin epitope of interest. For example, a Protein A matrix can be used to purify proteins that include polypeptides based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Also useful in purifying embodiments (e.g., fusion proteins) comprising an immunoglobulin Fc domain are engineered versions of Protein A that are multimers (typically tetramers, pentamers or hexamers) of a single domain which has been modified to improve its characteristics for industrial applications. “Protein A” is an approximately 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus; Protein A is encoded by the spa gene of S. aureus, and its expression in S. aureus is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. (See, Fournier, B., and Klier, A, Protein A gene expression is regulated by DNA supercoiling which is modified by the ArlS–ArlR two-component system of Staphylococcus aureus, Microbiology 150:3807-19 (2004)). Protein A (Spa gene product) is useful in biochemical research and industry because of its ability to bind immunoglobulins. Protein A is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It has been shown via crystallographic refinement that the primary binding site for Protein A is on the Fc region, between the CH2 and CH3 domains. (Deisenhofer, J., Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of Protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution, Biochemistry 20 (9): 2361–70 (1981)). In addition, Protein A binds human IgG molecules containing IgG F(ab')₂ fragments from the JUST1781 PCT International Patent Application human VH3 gene family. (See, Sasso EH, Silverman GJ, Mannik M, Human IgA and IgG F(ab')2 that bind to staphylococcal Protein A belong to the VHIII subgroup, Journal of Immunology.147 (6): 1877–83 (1991)). Protein A is typically produced and purified in industrial fermentation for use in immunology, biological research and industrial applications. Natural (or native) Protein A can be cultured in Staphylococcus aureus and contains the five homologous antibody binding regions described above and a C-terminal region for cell wall attachment. Recombinant versions of Protein A, typically produced in Escherichia coli, are also useful for purposes of the invention. For use in the present invention, Protein A matrix can be obtained commercially in various embodiments (e.g., Protein A-Sepharose^® from Staphylococcus aureus, from Sigma-Aldrich; MabSelect^TM Protein A, MabSelect SuRe^® Protein A, MabSelect SuRe^® LX, and Protein A Sepharose^® FF from GE Healthcare Life Sciences; Eshmuno^® A Protein A from EMD Millipore; Toyopearl^® AF-rProtein A from Tosoh Bioscience; POROS^® Protein A from Thermo Fisher Scientific; CaptivA^® Protein A affinity resin from Repligen). Recombinant versions of Protein A commonly contain the five homologous antibody binding domains, but for purposes of the present invention can vary in other parts of the structure in order to facilitate covalent coupling to substrates, e.g., resins (such as, but not limited to, agarose). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al, EMBO J.5: 15671575 (1986)). Also available commercially (e.g., from Molecular Cloning Laboratories (MCLAB) or Protein Specialists (Prospec)), is recombinant Protein G, an immunoglobulin-binding protein derived from the cell wall of certain strains of beta-hemolytic streptococci. It binds with high affinity to the Fc portion of various classes and subclasses of immunoglobulins from a variety of species. The albumin and cell surface binding domains have been eliminated from Recombinant Protein G to reduce nonspecific binding, although the Fc binding domain is still present and, therefore, can be used to separate IgG from crude samples. The recombinant Protein G is produced in Escherichia coli using sequence from Streptococcus C1-C2-C3. The Protein G contains 200 amino acids (190-384 and five additional residues not including methionine) having a molecular mass of 21.8kDa. The Protein-G migrates on SDS-PAGE around 32kDa. [000155] Encompassed within the term “matrix” are resins, beads, nanoparticles, nanofibers, hydrogels, membranes, and monoliths, or any other physical matrix, bearing a relevant covalently bound chromatographic ligand (e.g., Protein A, Protein G, or other affinity chromatographic ligand, such as a target ligand, an antibody targeting an epitope tag, a charged moiety, or a hydrophobic moiety, etc.) for purposes of the inventive method. The JUST1781 PCT International Patent Application matrix to which the affinity target ligand is attached is most often agarose, but other matrices are available. For example, mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the protein comprises a C_H3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. An affinity chromatography matrix may be placed or packed into a column useful for the purification of proteins. Loading of the cell-free supernatant fraction onto the affinity chromatography matrix preferably occurs at about neutral pH. [000156] The specific target ligand of interest can be covalently conjugated to an affinity chromatography matrix, e.g., for protein purification purposes. The affinity matrix with the target ligand covalently attached should have sufficient binding capacity to account for the required mass sufficient to be detected by a mass spectrometer, if desired. This can be achieved with appropriately dense conjugation reactive moieties on the matrix (e.g., resin and/or resin bed size in the column). In producing and storing the conjugated affinity chromatography matrix reagent for future use in this step, the stability of a particular conjugated affinity chromatography matrix needs to be considered with regards to the conjugated target ligand itself or the mode by which the ligand is attached to the matrix. Ligand affinity conjugation instability and degradation of the conjugated affinity chromatography matrix reagent during storage can result in decreased product yields and/or binding artifacts leading to difficult data analysis or misinterpretation. The practitioner should exercise caution with respect to the appropriate storage conditions and quality control employed to ensure the effective quality of the affinity chromatography matrix before use in the inventive method of producing or expressing the protein(s) of interest. [000157] The term “to bind” or “binding” a molecule to an affinity chromatography matrix comprising a covalently-conjugated target moiety, e.g., Protein A or a Protein A matrix, or Protein G or a Protein G matrix, or a particular conjugated target ligand of interest, means exposing the molecule to the affinity chromatography target moiety, under appropriate conditions (e.g., pH and selected salt/buffer composition), such that the molecule is reversibly immobilized in, or on, the affinity chromatography matrix (e.g., a Protein A- or Protein G- conjugated or target ligand-conjugated) by virtue of its binding affinity to the target moiety under those conditions, regardless of the physical mechanism of affinity that may be involved. (See, e.g., Jendeberg, L. et al., The Mechanism of Binding Staphylococcal Protein A to Immunoglobin G Does Not Involve Helix Unwinding, Biochemistry 35(1): 22–31 (1996); JUST1781 PCT International Patent Application Nelson, J.T. et al., Mechanism of Immobilized Protein A Binding to Immunoglobulin G on Nanosensor Array Surfaces, Anal. Chem., 87(16):8186–8193 (2015)). [000158] The term "buffer" or "buffered solution" refers to solutions which resist changes in pH by the action of its conjugate acid-base range. Examples of useful buffers that control pH at ranges of about pH 4 to about pH 8 include phosphate, bicarbonate, acetate, MES, citrate, Tris, bis-tris, histidine, arginine, succinate, citrate, glutamate, and lactate, or a combination of two or more of these, or other mineral acid or organic acid buffers. Salts containing sodium, ammonium, and potassium cations are often used in making a buffered solution. [000159] “Under physiological conditions,” with respect to the expression of a protein of interest by a host cell, refer to cell culture conditions that allow the cell to produce gene products, including the presence of permissive physicochemical conditions of temperature, pH, osmolality, oxygenation, nutrients, micronutrients, growth factor(s), and/or hormones. [000160] "Under physiological conditions," with respect to incubating buffers and immunoglobulins, or other binding assay reagents, means incubation under conditions of temperature, pH, and ionic strength, that permit a biochemical reaction, such as a non- covalent binding reaction, to occur. Typically, the temperature is at room or ambient temperature up to about 37°C and at pH 6.5-7.5. [000161] "Physiologically acceptable salt" of a composition of matter, for example a salt of a protein of interest, e.g., a fusion protein, or another immunoglobulin, such as an antibody, or any other protein of interest, or a salt of an amino acid, such as, but not limited to, a lysine, histidine, or proline salt, means any salt, or salts, that are known or later discovered to be pharmaceutically acceptable. Some non-limiting examples of pharmaceutically acceptable salts are: acetate salts; trifluoroacetate salts; hydrohalides, such as hydrochloride (e.g., monohydrochloride or dihydrochloride salts) and hydrobromide salts; sulfate salts; citrate salts; maleate salts; tartrate salts; glycolate salts; gluconate salts; succinate salts; mesylate salts; besylate salts; salts of gallic acid esters (gallic acid is also known as 3,4, 5 trihydroxybenzoic acid) such as pentagalloylglucose (PGG) and epigallocatechin gallate (EGCG), salts of cholesteryl sulfate, pamoate salts, tannate salts, and oxalate salts. JUST1781 PCT International Patent Application [000162] A "reaction mixture" is an aqueous mixture containing all the reagents and factors necessary, which under physiological conditions of incubation, permit an in vitro biochemical reaction of interest to occur, such as a covalent or non-covalent binding reaction. [000163] A "stable" formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon processing (e.g., ultrafiltration, diafiltration, other filtering steps, vial filling), transportation, and/or storage of the antibody drug substance and/or drug product. Together, the physical, chemical and biological stability of the protein in a formulation embody the “stability” of the protein formulation, which is specific to the conditions under which the formulated protein product is stored. For instance, a drug product stored at subzero temperatures would be expected to have no significant change in either chemical, physical or biological activity while a drug product stored at 40°C would be expected to have changes in its physical, chemical and biological activity with the degree of change dependent on the time of storage for the drug substance or drug product. The configuration of the protein formulation can also influence the rate of change. For instance, aggregate formation is highly influenced by protein concentration with higher rates of aggregation observed with higher protein concentration. Excipients are also known to affect stability of the drug product with, for example, addition of salt increasing the rate of aggregation for some proteins while other excipients such as sucrose are known to decrease the rate of aggregation during storage. Instability is also greatly influenced by pH giving rise to both higher and lower rates of degradation depending on the type of modification and pH dependence. [000164] Various analytical techniques for measuring protein stability are available in the art and are reviewed, e.g., in Wang, W. (1999), Instability, stabilization and formulation of liquid protein pharmaceuticals, Int J Pharm 185:129-188. Stability can be measured at a selected temperature for a selected time period. For rapid screening, for example, the formulation can be kept at 40°C for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8°C, generally the formulation should be stable at 30°C for at least 1 month, or 40°C for at least a week, and/or stable at 2-8°C for at least two years. [000165] A protein "retains its physical stability" in a formulation if it shows minimal signs of changes to the secondary and/or tertiary structure (i.e., intrinsic structure), or aggregation, and/or precipitation and/or denaturation upon visual examination of color and/or JUST1781 PCT International Patent Application clarity, or as measured by UV light scattering or by size exclusion chromatography, or other suitable methods. Physical instability of a protein, i.e., loss of physical stability, can be caused by oligomerization resulting in dimer and higher order aggregates, subvisible, and visible particle formation, and precipitation. The degree of physical degradation can be ascertained using varying techniques depending on the type of degradant of interest. Dimers and higher order soluble aggregates can be quantified using size exclusion chromatography, while subvisible particles may be quantified using light scattering, light obscuration or other suitable techniques. [000166] A protein "retains its chemical stability" in a formulation, if the chemical stability at a given time is such that covalent bonds are not made or broken, resulting in changes to the primary structure of the protein component, e.g., antibody. Changes to the primary structure may result in modifications of the secondary and/or tertiary and/or quaternary structure of the protein and may result in formation of aggregates or reversal of aggregates already formed. Typical chemical modifications can include isomerization, deamidation, N-terminal cyclization, backbone hydrolysis, methionine oxidation, tryptophan oxidation, histidine oxidation, beta-elimination, disulfide formation, disulfide scrambling, disulfide cleavage, and other changes resulting in changes to the primary structure including D-amino acid formation. Chemical instability, i.e., loss of chemical stability, may be interrogated by a variety of techniques including ion-exchange chromatography, capillary isoelectric focusing, analysis of peptide digests and multiple types of mass spectrometric techniques. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical alteration may involve size modification (e.g. clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and/or matrix- assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for example. Other types of chemical alteration include charge alteration (e.g. occurring as a result of deamidation) which can be evaluated by charge-based methods, such as, but not limited to, ion-exchange chromatography, capillary isoelectric focusing, or peptide mapping. [000167] Loss of physical and/or chemical stability may result in changes to biological activity as either an increase or decrease of a biological activity of interest, depending on the modification and the protein being modified. A protein "retains its biological activity" in a buffered solution or formulation, if the biological activity of the protein at a given time is within about 30% of the biological activity exhibited at the time the formulation was prepared. Activity is considered decreased if the activity is less than 70% of its starting value. JUST1781 PCT International Patent Application Biological assays may include both in vivo and in vitro based assays such as ligand binding, potency, cell proliferation or other surrogate measure of its biopharmaceutical activity. [000168] Quantification of the protein product of interest is often useful or necessary to track in a production or manufacturing process. An antibody that specifically binds a domain of the protein of interest can therefore be useful for these purposes. [000169] In some embodiments, the protein of interest is an antigen-binding protein, such as an antibody. The term "antibody", or interchangeably “Ab,” is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies (including human, humanized or chimeric antibodies), monomeric, homodimeric, and heterodimeric antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), single domain antibodies (sdAbs), and antibody fragments that can bind antigen (e.g., Fab, Fab', F(ab')2, Fv, single chain antibodies, diabodies), comprising complementarity determining regions (CDRs) of the foregoing as long as they exhibit the desired biological activity. Multimers or aggregates of intact molecules and/or fragments, including chemically derivatized antibodies, are contemplated. The term “antibody” encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.Antibodies of any isotype class or subclass, including IgG, IgM, IgD, IgA, and IgE, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, or any allotype, are contemplated. Different isotypes have different effector functions; for example, IgGl and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC) activity. [000170] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies that are antigen binding proteins are highly specific binders, being directed against an individual antigenic site or epitope, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different epitopes. Nonlimiting examples of monoclonal antibodies include JUST1781 PCT International Patent Application murine, rabbit, rat, chicken, chimeric, humanized, or human antibodies, fully assembled antibodies, multispecific antibodies (including bispecific antibodies), antibody fragments that can bind an antigen (including, Fab, Fab', F(ab)2, Fv, single chain antibodies, diabodies), maxibodies, nanobodies, and recombinant peptides comprising CDRs of the foregoing as long as they exhibit the desired biological activity, or variants or derivatives thereof. [000171] The modifier "monoclonal" indicates the character of the sdAb or antibody as being obtained from a substantially homogeneous population of sdAb or antibodies, and is not to be construed as requiring production of the sdAb or antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624- 628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. [000172] In a conventional "antibody" (i.e., homodimeric antibody), each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain of about 220 amino acids (about 25 kDa) and one "heavy" chain of about 440 amino acids (about 50-70 kDa). The amino-terminal portion of each chain includes a "variable" ("V") region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. The variable region differs among different antibodies. The constant region is the same among different antibodies. Within the variable region of each heavy or light chain, there are three hypervariable subregions that help determine the antibody's specificity for antigen in the case of an antibody that is an antigen binding protein. The variable domain residues between the hypervariable regions are called the framework residues and generally are somewhat homologous among different antibodies. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. Human light chains are classified as kappa (κ) and lambda (λ) light chains. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). An "antibody" also encompasses a recombinantly made antibody, and antibodies that are glycosylated or lacking glycosylation. JUST1781 PCT International Patent Application [000173] The term "light chain" or "immunoglobulin light chain" includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, C_L. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda chains. [000174] The term "heavy chain" or "immunoglobulin heavy chain" includes a full- length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, C_H1, C_H2, and C_H3. The V_H domain is at the amino-terminus of the polypeptide, and the CH domains are at the carboxyl-terminus, with the CH3 being closest to the carboxy-terminus of the polypeptide. Heavy chains are classified as mu (µ), delta (δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Heavy chains may be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE. Several of these may be further divided into subclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Different IgG isotypes may have different effector functions (mediated by the Fc region), such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (Fc.gamma.Rs) on the surface of immune effector cells such as natural killers and macrophages, leading to the phagocytosis or lysis of the targeted cells. In CDC, the antibodies kill the targeted cells by triggering the complement cascade at the cell surface. [000175] An "Fc region", or used interchangeably herein, "Fc domain" or "immunoglobulin Fc domain", contains two heavy chain fragments, which in a full antibody comprise the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C_H3 domains. [000176] The term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule. [000177] For a detailed description of the structure and generation of antibodies, see Roth, D. B., and Craig, N. L., Cell, 94:411-414 (1998), herein incorporated by reference in its entirety. Briefly, the process for generating DNA encoding the heavy and light chain immunoglobulin sequences occurs primarily in developing B-cells. Prior to the rearranging JUST1781 PCT International Patent Application and joining of various immunoglobulin gene segments, the V, D, J and constant (C) gene segments are found generally in relatively close proximity on a single chromosome. During B-cell-differentiation, one of each of the appropriate family members of the V, D, J (or only V and J in the case of light chain genes) gene segments are recombined to form functionally rearranged variable regions of the heavy and light immunoglobulin genes. This gene segment rearrangement process appears to be sequential. First, heavy chain D-to-J joints are made, followed by heavy chain V-to-DJ joints and light chain V-to-J joints. In addition to the rearrangement of V, D and J segments, further diversity is generated in the primary repertoire of immunoglobulin heavy and light chains by way of variable recombination at the locations where the V and J segments in the light chain are joined and where the D and J segments of the heavy chain are joined. Such variation in the light chain typically occurs within the last codon of the V gene segment and the first codon of the J segment. Similar imprecision in joining occurs on the heavy chain chromosome between the D and JH segments and may extend over as many as 10 nucleotides. Furthermore, several nucleotides may be inserted between the D and JH and between the VH and D gene segments which are not encoded by genomic DNA. The addition of these nucleotides is known as N-region diversity. The net effect of such rearrangements in the variable region gene segments and the variable recombination which may occur during such joining is the production of a primary antibody repertoire. [000178] The term "hypervariable" region refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a complementarity determining region or CDR [i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as described by Kabat et al., Sequences of Proteins of Immunological Interest, th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]. Even a single CDR may recognize and bind antigen, although with a lower affinity than the entire antigen binding site containing all of the CDRs. [000179] An alternative definition of residues from a hypervariable "loop" is described by Chothia et al., J. Mol. Biol.196: 901-917 (1987) as residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain. JUST1781 PCT International Patent Application [000180] "Framework" or "FR" residues are those variable region residues other than the hypervariable region residues. [000181] "Antibody fragments" comprise a portion of an intact full length antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.,8(10):1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. [000182] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment which contains the constant region. The Fab fragment contains all of the variable domain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fc fragment displays carbohydrates and is responsible for many antibody effector functions (such as binding complement and cell receptors), that distinguish one class of antibody from another. [000183] Pepsin treatment yields an F(ab')2 fragment that has two "Single-chain Fv" or "scFv" antibody fragments comprising the V_H and V_L domains of antibody, wherein these domains are present in a single polypeptide chain. Fab fragments differ from Fab' fragments by the inclusion of a few additional residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994). [000184] A "Fab fragment" is comprised of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. [000185] A "Fab' fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule. JUST1781 PCT International Patent Application [000186] A "F(ab')₂ fragment" contains two light chains and two heavy chains containing a portion of the constant region between the C_H1 and C_H2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. [000187] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light- chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH VL dimer. A single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. [000188] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Pat. No.4,946,778 and No.5,260,203, the disclosures of which are incorporated by reference in their entireties. [000189] "Single-chain Fv" or "scFv" antibody fragments comprise the V_H and V_L domains of antibody, wherein these domains are present in a single polypeptide chain, and optionally comprising a polypeptide linker between the V_H and V_L domains that enables the Fv to form the desired structure for antigen binding (Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Nati. Acad. Sci. USA 85:5879-5883, 1988). An "Fd" fragment consists of the VH and CH1 domains. [000190] The term "diabodies" refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). [000191] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light JUST1781 PCT International Patent Application chain. In some instances, two or more V_H regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two V_H regions of a bivalent domain antibody may target the same or different antigens. [000192] The term "antigen binding protein" (ABP) includes antibodies or antibody fragments, as defined herein, that specifically bind a target ligand or antigen of interest. [000193] In general, an antigen binding protein, e.g., an immunoglobulin protein, or an antibody or antibody fragment, "specifically binds" to a target ligand or antigen of interest when it has a significantly higher binding affinity for, and consequently is capable of distinguishing, that target ligand or antigen, compared to its affinity for other unrelated proteins, under similar binding assay conditions. There are two expressions called “equilibrium dissociation constant” (abbreviated herein “KD” or “Kd”), which are commonly used to define the affinity of a protein for a ligand. The value of K_D is a kinetic term; it is the ratio of the off-rate (kback) and on-rate (kforward) constants. The formula for the value of KD is the following: [000194] (I) [000195] KD = kback/kforward = koff/kon= (kd/ka), where [000196] (II) [000197] kback = koff = kd, i.e., the “dissociation rate constant,” and [000198] (III) [000199] kforward = kon = ka, i.e., the “association rate constant.” [000200] On the other hand, the value of Kd is calculated by the following formula: [000201] (IV) [000202] Kd = ([total binding sites] x [total ligand])/[PL]. [000203] For an antigen-binding protein with a single binding site, Kd can be calculated from the concentrations of the antigen-binding protein (P), the ligand (L) and the P-L complex (PL), at equilibrium, with Kd = [P][L]/[PL]. [000204] The values of KD and Kd are typically equivalent for a binding protein having a single binding site. Typically, an antigen binding protein is said to "specifically bind" its target antigen when the equilibrium dissociation constant (Kd or KD) is 10^-8 M or lower. The JUST1781 PCT International Patent Application antigen binding protein specifically binds antigen with "high affinity" when the equilibrium dissociation constant is 10^-9 M or lower, and with "very high affinity" when the K_d or K_D is 10^-10 M or lower. A number of nanobodies are disclosed herein having different affinities to fentanyl, as well as to carfentanil. Differing sensitivities or affinities to a plurality or multiplicity of different opioids amongst nanobody species can be useful for distinguishing between each opioid species. For example, in a multiplexed assay, differing affinities to a single target (e.g., fentanyl and/or carfentanil) can also be useful. Of particular importance are uses in which a plurality of species of nanobodies are simultaneously deployed, in which differing affinities to the target can be helpful to ascertain analyte information (such as concentration) from a single preparation of sample, whereas an inconvenient dilution series may need to be prepared if only one species of nanobody were to be used. Other uses of relatively low-affinity nanobodies can include those in which relatively weak binding is desirable, for examples, when it is desirable to easily dissociate the nanobody-antigen pair in order to recycle the nanobodies. In addition, antibodies with relatively lower fentanyl affinity can have other desirable properties, such as but not limited to, a relatively higher affinity to other opioid species, or an increased stability to environmental conditions (e.g., temperature or pH). [000205] The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity" means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) must be addressed by a particular mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides. JUST1781 PCT International Patent Application Using a computer program such as BLAST or FASTA, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol.5, supp.3 (1978)] can be used in conjunction with the computer program. For example, the percent identity can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences. In calculating percent identity, the sequences being compared are aligned in a way that gives the largest match between the sequences. [000206] The GCG program package is a computer program that can be used to determine percent identity; the GCG program package includes GAP (Devereux et al., 1984, Nucl. Acid Res.12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align the two polypeptides or two polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the "matched span", as determined by the algorithm). A gap opening penalty (which is calculated as 3.times. the average diagonal, wherein the "average diagonal" is the average of the diagonal of the comparison matrix being used; the "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm. [000207] Recommended parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program include the following: Algorithm: Needleman et al., 1970, J. Mol. Biol.48:443-453; [000208] Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra; JUST1781 PCT International Patent Application [000209] Gap Penalty: 12 (but with no penalty for end gaps); [000210] Gap Length Penalty: 4; [000211] Threshold of Similarity: 0. [000212] Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide. [000213] The term "modification" when used in connection with proteins of interest, include, but are not limited to, one or more amino acid changes (including substitutions, insertions or deletions); chemical modifications; covalent modification by conjugation to therapeutic or diagnostic agents; labeling (e.g., with radionuclides or various enzymes); covalent polymer attachment such as PEGylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of non-natural amino acids. By methods known to the skilled artisan, proteins, can be “engineered” or modified for improved target affinity, selectivity, stability, and/or manufacturability before the coding sequence of the “engineered” protein is included in the expression cassette. [000214] The term "derivative," when used in connection with proteins of interest, refers to proteins that are covalently modified by conjugation to therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as PEGylation (derivatization with polyethylene glycol) and insertion or substitution of natural or non-natural amino acids. [000215] Conservatively modified forms of the antigen-binding proteins disclosed herein are also contemplated as being embodiments of the present invention. A "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non-native or non-canonical residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis" (see, for example, MacLennan et al, Acta Physiol. Scand. JUST1781 PCT International Patent Application SuppL, 643:55-67 (1998); Sasaki et al, 1998, Adv. Biophys.35: 1-24 (1998), which discuss alanine scanning mutagenesis). [000216] Desired amino acid substitutions (whether conservative or non- conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptide sequence, or to increase or decrease the affinity of the peptide or vehicle-conjugated peptide molecules described herein. [000217] Naturally occurring residues may be divided into classes based on common side chain properties: [000218] 1) hydrophobic: norleucine (Nle), Met, Ala, Val, Leu, Ile; [000219] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; [000220] 3) acidic: Asp, Glu; [000221] 4) basic: His, Lys, Arg; [000222] 5) residues that influence chain orientation: Gly, Pro; and [000223] 6) aromatic: Trp, Tyr, Phe. [000224] Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues (e.g., norleucine (Nle)), which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. [000225] In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). JUST1781 PCT International Patent Application [000226] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al, 1982, J. Mol. Biol.157: 105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making conservative amino acid substitutions based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included. [000227] It is also understood in the art that the conservative amino acid substitutions of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein. [000228] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as "epitopic core regions." [000229] Examples of conservative amino acid substitutions include the substitution of one non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, norleucine (Nle), alanine, or methionine for another, the substitution of one polar (hydrophilic) amino acid residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic amino acid residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase "conservative amino JUST1781 PCT International Patent Application acid substitution" also includes the use of a chemically derivatized residue in place of a non- derivatized residue, provided that such polypeptide displays the requisite bioactivity. Other exemplary amino acid substitutions that can be useful in accordance with the present invention are set forth in Table A below. [000230] Table A. Some Useful Conservative Amino Acid Substitutions. Conventional three-letter abbreviations are shown. “Nle” means norleucine. Original Exemplary Residue Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe, Nle Leu Ile, Val, Met, Ala, Phe, Nle Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala, Nle [000231] Cloning DNA [000232] Cloning of DNA is carried out using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, which is incorporated herein by reference). For example, a cDNA library may be constructed by reverse transcription of polyA+ mRNA, preferably membrane-associated mRNA, and the library screened using probes specific for human immunoglobulin polypeptide gene JUST1781 PCT International Patent Application sequences. In one embodiment, however, the polymerase chain reaction (PCR) is used to amplify cDNAs (or portions of full-length cDNAs) encoding an immunoglobulin gene segment of interest (e.g., a light or heavy chain variable segment). The amplified sequences can be readily cloned into any suitable vector, e.g., expression vectors, minigene vectors, or phage display vectors. It will be appreciated that the particular method of cloning used is not critical, so long as it is possible to determine the sequence of some portion of the polypeptide of interest, e.g., antibody sequences. [000233] One source for antibody nucleic acids is a hybridoma produced by obtaining a B cell from an animal immunized with the antigen of interest and fusing it to an immortal cell. Alternatively, nucleic acid can be isolated from B cells (or whole spleen) of the immunized animal. Yet another source of nucleic acids encoding antibodies is a library of such nucleic acids generated, for example, through phage display technology. Polynucleotides encoding peptides of interest, e.g., variable region peptides with desired binding characteristics, can be identified by standard techniques such as panning. [000234] Sequencing of DNA is carried out using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, which is incorporated herein by reference). By comparing the sequence of the cloned nucleic acid with published sequences of genes and cDNAs, one of skill will readily be able to determine sequence identity, depending on the region sequenced. One source of gene sequence information is the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD. [000235] Embodiments of the present invention can involve so-called “Next- generation” sequencing, as a preferred method for confirming the presence of all engineered DNA constructs prior to the transfection step(s). (See, e.g., Buermans, H. P. J., & den Dunnen, J. T., Next generation sequencing technology: Advances and applications, Biochimica et Biophysica Acta - Molecular Basis of Disease 1842(10): 1932–1941 (2014)). Sanger will provide an indication of the species present but not as individual designs. Sequencing will validate that the absence of any species was not due to their absence as a DNA construct. There is a possibility that some engineered designs will not be expressed and secreted at high enough levels to survive all processing steps and be detected by mass spectrometry. This may result because certain engineered antibody variant designs are JUST1781 PCT International Patent Application unstable, but such variants will not likely be viable as therapeutics anyway. This can be viewed as part of the screening process, however, since typically it is desirable to find antibody variant candidates that do express well for manufacturing purposes. [000236] Chemical synthesis of parts or the whole of a coding region containing codons reflecting desires protein changes can be cloned into an expression vector by either restriction digest and ligation of 5' and 3' ends of fragments or the entire open reading frame (ORF), containing nucleotide overhangs that are generated by restriction enzyme digestion and which are compatible to the destination vector. The fragments or inserts are typically ligated into the destination vector using a T4 ligase or other common enzyme. Other useful methods are similar to the above except that the cut site for the restriction enzyme is at location different from the recognition sequence. Alternatively, isothermal assembly (i.e., “Gibson Assembly”) can be employed, in which nucleotide overhangs are generated during synthesis of fragments or ORFs; digestion by exonucleases is employed. Alternatively, nucleotide overhangs can be ligated ex vivo by a ligase or polymerase or in vivo by intracellular processes. [000237] Alternatively, homologous recombination can be employed, similar to isothermal assembly, except exonuclease activity of T4 DNA ligase can used on both insert and vector and ligation can be performed in vivo. [000238] Another useful cloning method is the so-called “TOPO” method, in which a complete insert containing a 3' adenosine overhang (generated by Taq polymerase) is present, and Topoisomerase I ligates the insert into a TOPO vector. [000239] Another useful cloning method is degenerate or error-prone PCR exploiting degenerate primers and/or a thermally stable low-fidelity polymerase caused by the polymerase within certain reaction conditions. Fragments or inserts are then cloned into an expression vector. [000240] The above are merely examples of known cloning techniques, and the skilled practitioner knows how to employ any other suitable cloning techniques. [000241] Isolated DNA can be operably linked to control sequences or placed into expression vectors, which are then transfected into host cells that do not otherwise produce the protein(s) of interest, to direct the synthesis of the protein(s) in the recombinant host cells. [000242] Nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader JUST1781 PCT International Patent Application is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. [000243] Many vectors are known in the art. Vector components can include one or more of the following: a signal sequence (that may, for example, direct secretion of the expressed protein by the recombinant host cells); an origin of replication, one or more selection marker or reporter genes (that may, for example, encode a fluorescent protein, such as a green fluorescent protein (GFP), an enhanced green fluorescent protein (EGFP), a red- shifted green fluorescent protein (rs-GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), a cyan fluorescent protein (e.g., CyOFP1), mini Singlet Oxygen Generator (miniSOG), a luminescent protein (e.g., luciferase), or the like, or may confer antibiotic or other drug resistance, or complement auxotrophic deficiencies of the host cells or supply critical nutrients not available in the medium, e.g., dihydrofolate reductase or glutamine synthetase selection markers), an enhancer element, a promoter, and a transcription termination sequence, all of which are well known in the art. [000244] Protein expression and Cell Culture [000245] Embodiments of the inventive method for recombinantly expressing one or more proteins of interest in a cultured eukaryotic host cell line involves culturing the host cells. Such cultured cells are typically made by recombinant DNA technology involving transient or stable transfection, e.g., the pooled plasmid constructs (expression vectors) from the cloning step can be transfected into a plurality of host cells (e.g., mammalian, e.g., HEK 293 or CHO cell, or insect cells, or microbial host cells, e.g., yeast cells, or algal or microalgal cells) for expression using a cationic lipid, polyethylenimine, Lipofectamine^TM, or ExpiFectamine^TM, or electroporation. The skilled practitioner is aware of numerous suitable means for transfecting to achieve expression of recombinant antibodies. Alternatively, methods for stable genomic integration of expressions cassettes encoding the protein of interest can be JUST1781 PCT International Patent Application employed to make a production cell line of protein-secreting mammalian cells. (See, e.g., Zhang, Crispr-Cas Systems and Methods for Altering Expression Of Gene Products, WO2014093661 A2; Frendewey et al., Methods and Compositions for the Targeted Modification of a Genome, US9228208 B2; Church et al., Multiplex Automated Genome Engineering, WO2008052101A2, US8153432 B2; Bradley et al., Methods Cells and Organisms, US2015/0079680 A1; Begemann et al., Compositions and Methods for Modifying Genomes, WO2017141173A2; Gill et al., Nucleic acid-guided nucleases, US9982279 B1; Minshull et al., Enhanced nucleic acid constructs for eukaryotic gene expression, US9428767B2, US9580697B2, US9574209B2; Minshull et al., DNA Vectors, Transposons And Transposases For Eukaryotic Genome Modification, US10041077B2; McGrew et al., Hybrid Promoter and Uses Thereof, US 11028410B2; McGrew et al., Expression from Transposon-Based Vectors and Uses, US 11098310B2; McGrew et al., Inducible Expression From Transposon-Based Vectors and Uses, US 2019/0185881A1). [000246] The transfectant or transformant host cells can be provided with an additional recombinant expression cassette for expressing a selectable marker, for example, but not limited to, one or more of the following: glutamine synthase, dihydrofolate reductase, puromycin-N acetyl transferase, blasticidin-S deaminase, hygromycin phosphotransferase, aminoglycoside phosphotransferase, nourseothircin N-acetyl transferase, or a protein that binds to zeocin. [000247] The protein of interest is typically obtained by culturing the transfected or transformed host cells under physiological conditions allowing the cells to express recombinant proteins. Most conveniently, the expressed recombinant proteins are directly secreted into the extracellular culture medium (by employing appropriate secretory-directing signal peptides) and are harvested therefrom; otherwise additional steps will be needed to isolate the expressed antibodies from a cell extract. [000248] Useful secretory signal peptide (SP) sequences are known in the art, and these can be added, adjacent or distal, to a coding sequence for a protein of interest, herein, for the purpose of facilitating secretion of the inventive antigen-binding protein. An example of a useful SP sequence is the IGKV1-39*01 SP signal peptide: [000249] MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:6). Other examples of useful SP sequences include: [000250] MEAPAQLLFLLLLWLPDTTG (SEQ ID NO:7), JUST1781 PCT International Patent Application [000251] MEWTWRVLFLVAAATGAHS (SEQ ID NO:8), [000252] METPAQLLFLLLLWLPDTTG (SEQ ID NO:9), [000253] MKHLWFFLLLVAAPRWVLS (SEQ ID NO:10), [000254] MEWSWVFLFFLSVTTGVHS (SEQ ID NO:11), but any other suitable signal peptide sequence may be employed within the scope of the invention. [000255] The desired scale of the recombinant expression will be dependent on the type of expression system and the desired quantity of protein production. Some expression systems such as ExpiCHO^TM usually produce higher yields as compared to some earlier HEK293 technologies. A smaller scale ExpiCHO^TM might then suffice as compared to an HEK293 system. Efficiency of transfection can also be a consideration in choosing an appropriate expression system. Electroporation can be a suitable method given its effectiveness, relative low cost and the fact that high-throughput during this step is not critical. Additionally, the ratio of immunoglobulin light chain to heavy chain can be varied during the co-transfection to improve expression of certain variants. The product yield for a given variant has to be sufficient to survive numerous handling steps and produce a signal high enough to be detected by the chosen fluorescence detector. [000256] In general, the transfected or transformed host cells are typically cultured by any conventional type of culture, such as batch, fed-batch, intensified fed-batch, or continuous. Suitable continuous cultures included repeated batch, chemostat, turbidostat or perfusion culture with product and cell retention or solely cell retention. Bioreactors for protein production, which can be reusable or single-use bioreactors, typically can contain a volume of liquid culture medium of about 50 L to about 4000 L (e.g., 50 L, 60 L, 75 L, 100 L, 250 L, 500 L, 650 L, 750 L, 1000 L, 1250 L, 1500 L, 1750 L, 2000 L, 2250 L, 2500 L, 2750 L, 3000 L, 3250 L, 3500 L, 3750 L, or 4000 L), as desired. [000257] The host cells used to produce the protein of interest or “POI” (e.g., non- glycosylated or glycosylated proteins) in the invention can be cultured in a variety of media suitable for the type of host cell chosen. Commercially available media such as Ham's F10 (Sigma-Aldrich), Minimal Essential Medium (MEM) (Sigma-Aldrich), RPMI-1640 (Sigma- Aldrich), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich) can be suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz.58: 44 (1979), Barnes et al., Anal. Biochem.102: 255 (1980), U.S. Patent Nos. JUST1781 PCT International Patent Application 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Patent Re. No.30,985 can be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as Gentamycin™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source, such that the physiological conditions of the cell in, or on, the medium promote expression of the protein of interest by the host cell; any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. [000258] The ordinarily skilled artisan is familiar with useful culture conditions, such as temperature (for mammalian cells, typically, but not necessarily, about 37° ± 1°C), pH (typically, but not necessarily, the cell culture medium is maintained within the range of about pH 6.5-7.5), oxygenation, and the like. By "culturing at" or "maintaining at" a predetermined culture condition, is meant that the process control systems are set at a particular value for that condition, in other words the intended volume, target temperature, pH, oxygenation level, or the like, maintained at predetermined set points for each parameter, within a narrow range (i.e., “narrow deadband”) most optimal for the cell line and protein product of interest. Clearly, there will be small variations of the temperature, pH, or other culture condition over time, and from location to location through the culture vessel (i.e., the bioreactor). (See, also, e.g., Oguchi et al., pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture, Cytotechnology.52(3):199–207 (2006); Al- Fageeh et al., The cold-shock response in cultured mammalian cells: Harnessing the response for the improvement of recombinant protein production, Biotechnol. Bioeng.93:829–835 (2006); Marchant, R.J. et al., Metabolic rates, growth phase, and mRNA levels influence cell- specific antibody production levels from in vitro cultured mammalian cells at sub- physiological temperatures, Mol. Biotechnol.39:69–77 (2008)). [000259] Digital control units and sensory monitors are available commercially or can be constructed by the skilled artisan for use with cell culture bioreactors. Alternative digital control units (DCU) control and monitor the cell culture process are available commercially, made by companies such as B. Braun, New Brunswick, Sartorius, or Thermo Fisher Scientific. Other on-line or off-line analyses can include off-gas measurements by mass JUST1781 PCT International Patent Application spectrometry, in-depth determination of media composition (amino acids, vitamins, trace minerals) and expanded examination of cellular metabolites in addition to CO₂ and lactic acid. [000260] Examples of epitope tags include the flu HA tag polypeptide and its antibody 12CA5 [Field et al, Mol. Cell. Biol.8: 2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al, Mol. Cell. Biol.5(12): 3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al, Protein Engineering 3(6): 547-553 (1990)]. Other exemplary tags are a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation. Other labels and tags, such as the FLAG^® tag (Eastman Kodak, Rochester, NY) are well known and routinely used in the art. [000261] Thus an epitope tag peptide, can be used to facilitate purification and/or detection of the antigen-binding protein of the invention. In some cases, the tagging peptide is detectable by itself (e.g. fluorescent tags such as GFP) while in other cases the tagging peptide is detectable because it specifically binds a detectable molecule (in turn, the detectable molecule can be directly detectable, e.g. fluorescent, or it may be detected by specific binding to it of a detectable molecule, i.e. a scaffold of molecules may be required for detection). If used for purification and/or indirect detection, such a peptide is usually designed (or found) to have a high affinity to a readily, or even commercially, available antibody molecule. Such peptides are often derived from a species unrelated to the species where the polypeptides is intended to be used to avoid any cross reaction, especially during detection. The molecule binding the tagging peptide may be selected for its detectability and/or for ease of immobilization and/or recovery in purification processes. Common tagging peptide include HA-tag (a short peptide from human influenza hemagglutinin), Flag-tag, His- tag or hexa-histidine (SEQ ID NO:20) (comprising at least 6 histidine residues) and the Strep- tag (comprising eight amino acids and which is readily bound by commercially available Strep-tactin (an engineered streptavidin) and antibodies). In particular embodiments, the VHH- comprising polypeptide of the invention comprises a Strep-tag fused C-terminally to the V_HH, particularly intercalated between the VHH and effector peptide. The Strep-tag system for one- step purification and high-affinity detection or capturing of proteins can be useful in immunoaffinity purification of the inventive proteins. (See, e.g., Schmidt et al., “The Strep- tag system for one-step purification and high-affinity detection or capturing of proteins,” Nature Protocols volume 2, pages 1528-1535 (2007)). JUST1781 PCT International Patent Application [000262] Some particular, non-limiting, embodiments of amino acid substitution variants protein(s) of interest, such as immunoglobulins, including antibodies and antibody fragments are exemplified below. [000263] Any cysteine residue not involved in maintaining the proper conformation of the protein of interest, e.g., an immunoglobulin, also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the immunoglobulin to improve its stability (particularly where the immunoglobulin is an antibody fragment such as an Fv fragment). [000264] In certain instances, immunoglobulin variants are prepared with the intent to modify those amino acid residues which are directly involved in epitope binding in a starting sequence. In other embodiments, modification of residues which are not directly involved in epitope binding or residues not involved in epitope binding in any way, is desirable, for purposes discussed herein. Mutagenesis within any of the CDR regions and/or framework regions of a protein of interest is contemplated. [000265] In order to determine which antigen binding protein amino acid residues are important for epitope recognition and binding, alanine scanning mutagenesis can be performed to produce substitution variants. See, for example, Cunningham et al, Science, 244: 1081-1085 (1989), the disclosure of which is incorporated herein by reference in its entirety. In this method, individual amino acid residues are replaced one-at-a-time with an alanine residue and the resulting antibody is screened for its ability to bind its specific epitope relative to the unmodified polypeptide. Modified antigen binding proteins with reduced binding capacity are sequenced to determine which residue was changed, indicating its significance in binding or biological properties. [000266] Substitution variants of antigen binding proteins can be prepared by affinity maturation wherein random amino acid changes are introduced into the parent polypeptide sequence. See, for example, Ouwehand et al, Vox Sang 74 (Suppl 2):223-232, 1998; Rader et al, Proc. Natl. Acad. Sci. USA 95 :8910-8915, 1998; DaU'Acqua et al, Curr. Opin. Struct. Biol.8:443-450, 1998, the disclosures of which are incorporated herein by reference in their entireties. Affinity maturation involves preparing and screening the antigen binding proteins, or variants thereof and selecting from the resulting variants those that have modified biological properties, such as increased binding affinity relative to the parent antigen binding protein. A convenient way for generating substitutional variants is affinity maturation using JUST1781 PCT International Patent Application phage display. Briefly, several hypervariable region sites are mutated to generate all possible amino substitutions at each site. The variants thus generated are expressed in a monovalent fashion on the surface of filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). See e.g., WO 92/01047, WO 93/112366, WO 95/15388 and WO 93/19172. [000267] Current affinity maturation methods belong to two mutagenesis categories: stochastic and nonstochastic. Error prone PCR, mutator bacterial strains (Low et al, J. Mol. Biol.260, 359-68, 1996), and saturation mutagenesis (Nishimiya et al, J. Biol. Chem.275: 12813-20, 2000; Chowdhury, P. S. Methods Mol. Biol.178, 269-85, 2002) are typical examples of stochastic mutagenesis methods (Rajpal et al, Proc Natl Acad Sci U S A. 102:8466-71, 2005). Nonstochastic techniques often use alanine-scanning or site-directed mutagenesis to generate limited collections of specific muteins. Some methods are described in further detail below. [00354] Affinity maturation via panning methods— Affinity maturation of recombinant antibodies is commonly performed through several rounds of panning of candidate antibodies in the presence of decreasing amounts of antigen. Decreasing the amount of antigen per round selects the antibodies with the highest affinity to the antigen thereby yielding antibodies of high affinity from a large pool of starting material. Affinity maturation via panning is well known in the art and is described, for example, in Huls et al. (Cancer Immunol Immunother.50: 163-71, 2001). Methods of affinity maturation using phage display technologies are described elsewhere herein and known in the art (see e.g., Daugherty et al, Proc Natl Acad Sci USA.97:2029-34, 2000). [000268] So-called “look-through mutagenesis” (LTM) provides a method for rapidly mapping an antibody-binding site. (Rajpal et al., “A general method for greatly improving the affinity of antibodies by using combinatorial libraries,” Proc Natl Acad Sci U S A 102(24):8466-71 (2005)). For LTM, nine amino acids, representative of the major side-chain chemistries provided by the 20 natural amino acids, are selected to dissect the functional side- chain contributions to binding at every position in all six CDRs of an antibody. LTM generates a positional series of single mutations within a CDR where each "wild type" residue is systematically substituted by one of nine selected amino acids. Mutated CDRs are combined to generate combinatorial single- chain variable fragment (scFv) libraries of increasing complexity and size without becoming prohibitive to the quantitative display of all JUST1781 PCT International Patent Application muteins. After positive selection, clones with improved binding are sequenced, and beneficial mutations are mapped. [00356] Error-prone PCR— Error-prone PCR involves the randomization of nucleic acids between different selection rounds. The randomization occurs at a low rate by the intrinsic error rate of the polymerase used but can be enhanced by error- prone PCR (Zaccolo et al, J. Mol. Biol.285:775-783, 1999) using a polymerase having a high intrinsic error rate during transcription (Hawkins et al., J Mol Biol.226:889-96, 1992). After the mutation cycles, clones with improved affinity for the antigen are selected using routine methods in the art. [000269] Techniques utilizing gene shuffling and directed evolution may also be used to prepare and screen antigen binding proteins, or variants thereof, for desired activity. For example, Jermutus et al, Proc Natl Acad Sci U S A., 98(l):75-80 (2001) showed that tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of scFvs; Fermer et al., Tumour Biol.2004 Jan- Apr;25(l-2):7-13 reported that use of phage display in combination with DNA shuffling raised affinity by almost three orders of magnitude. Dougherty et al., Proc Natl Acad Sci U S A.2000 Feb.29; 97(5):2029-2034 reported that (i) functional clones occur at an unexpectedly high frequency in hypermutated libraries, (ii) gain- of-function mutants are well represented in such libraries, and (iii) the majority of the scFv mutations leading to higher affinity correspond to residues distant from the binding site. [000270] Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen, or to use computer software to model such contact points. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, they are subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. [000271] Glycoproteins with modified carbohydrate [000272] Glycoprotein variants can also be produced that have a modified glycosylation pattern relative to the parent polypeptide, for example, adding or deleting one or more of the carbohydrate moieties bound to the immunoglobulin, and/or adding or deleting one or more glycosylation sites in the immunoglobulin. JUST1781 PCT International Patent Application [000273] Glycosylation of polypeptides, including antibodies, is typically either N- linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X- serine and asparagine -X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Thus, N-linked glycosylation sites may be added to an immunoglobulin by altering the amino acid sequence such that it contains one or more of these tripeptide sequences. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5 -hydroxy lysine may also be used. O-linked glycosylation sites may be added to an immunoglobulin by inserting or substituting one or more serine or threonine residues to the sequence of the original immunoglobulin or antibody. [000274] Altered Effector Function [000275] Embodiments of antigen-binding proteins of interest can be fused to Fc- containing domains. In such embodiments, cysteine residue(s) may be removed or introduced in the Fc region of an antibody or Fc-containing polypeptide, thereby eliminating or increasing interchain disulfide bond formation in this region. A homodimeric immunoglobulin thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med.176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric immunoglobulins or homodimeric antibodies (i.e., antibodies having two copies of a single LC monomer species and a single HC monomer species) may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, an immunoglobulin can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-CancerDrug Design 3: 219-230 (1989). [000276] It is also contemplated that one or more of the N-terminal 20 amino acid residues (e.g., a signal sequence) of the heavy and/or light chain are removed, or amino acid residues are deleted from the C-terminal, for example, amino acid sequences from which one, two, three, four or five amino acid residues are lacking from the N-terminal or C-terminal, or from both. JUST1781 PCT International Patent Application [00365] Modifications to increase serum half- life also may desirable, for example, by incorporation of or addition of a salvage receptor binding epitope (e.g., by mutation of the appropriate region or by incorporating the epitope into a peptide tag that is then fused to the immunoglobulin at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., W096/32478) or adding molecules such as PEG or other water soluble polymers, including polysaccharide polymers. [000277] The salvage receptor binding epitope preferably constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the immunoglobulin or fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the C_H2 domain of the Fc region (e.g., of an IgG) and transferred to the CH1, CH3, or VH region, or more than one such region, of the immunoglobulin or antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL region or VL region, or both, of the immunoglobulin fragment. See also International applications WO 97/34631 and WO 96/32478 which describe Fc variants and their interaction with the salvage receptor. [000278] Other sites and amino acid residue(s) of the constant region have been identified that are responsible for complement dependent cytotoxicity (CDC), such as the Clq binding site, and/or the antibody-dependent cellular cytotoxicity (ADCC) [see, e.g., Molec. Immunol.29 (5): 633-9 (1992); Shields et al, J. Biol. Chem., 276(9):6591-6604 (2001); Lazar et al, Proc. Nat'l. Acad. Sci.103(11): 4005 (2006) which describe the effect of mutations at specific positions, each of which is incorporated by reference herein in its entirety]. Mutation of residues within Fc receptor binding sites can result in altered (i.e. increased or decreased) effector function, such as altered affinity for Fc receptors, altered ADCC or CDC activity, or altered half-life. As described above, potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different subclass (e.g. replacing an IgGl residue with a corresponding IgG2 residue at that position). [000279] The invention also encompasses recombinant expression of proteins of interest, e.g., immunoglobulin molecules, including antibodies and antibody fragments, with altered carbohydrate structure resulting in altered effector activity, including antibody JUST1781 PCT International Patent Application molecules with absent or reduced fucosylation that exhibit improved ADCC activity. A variety of ways are known in the art to accomplish this. For example, ADCC effector activity is mediated by binding of the antibody molecule to the FcyRIII receptor, which has been shown to be dependent on the carbohydrate structure of the N-linked glycosylation at the Asn-297 of the CH2 domain. Non-fucosylated antibodies bind this receptor with increased affinity and trigger FcyRIII -mediated effector functions more efficiently than native, fucosylated antibodies. For example, recombinant production of non-fucosylated antibody in CHO cells in which the alpha- 1,6-fucosyl transferase enzyme has been knocked out results in antibody with 100-fold increased ADCC activity (Yamane-Ohnuki et al, Biotechnol Bioeng.2004 Sep 5;87(5):614- 22). Similar effects can be accomplished through decreasing the activity of this or other enzymes in the fucosylation pathway, e.g., through siRNA or antisense RNA treatment, engineering cell lines to knockout the enzyme(s), or culturing with selective glycosylation inhibitors (Rothman et al., Mol Immunol.1989 Dec;26(12): 1113-23). Some host cell strains, e.g. Lecl3 or rat hybridoma YB2/0 cell line naturally produce antibodies with lower fucosylation levels. Shields et al, J Biol Chem.2002 Jul 26;277(30):26733-40; Shinkawa et al, J Biol Chem.2003 Jan 31;278(5):3466-73. An increase in the level of bisected carbohydrate, e.g. through recombinantly producing antibody in cells that overexpress GnTIII enzyme, has also been determined to increase ADCC activity. Umana et al., Nat Biotechnol.1999 Feb; 17(2): 176-80. It has been predicted that the absence of only one of the two fucose residues may be sufficient to increase ADCC activity. (Ferrara et al., J Biol Chem.2005 Dec 5). [000280] By way of further illustration, the following numbered embodiments are encompassed by the present invention: [000281] Embodiment 1: A multiple-vector expression system encoding a pre- mRNA molecule and a pre-mRNA trans-splicing molecule (PTM), wherein: [000282] (A) a first vector encodes the pre-mRNA molecule, which comprises 5' to 3': [000283] (i) a promoter; [000284] (ii) a first coding sequence comprising a 5'-exonic coding fragment of a gene encoding a predetermined selectable protein marker; [000285] (iii) an intron, comprising a first splicing domain comprising: [000286] (a) a 5' donor acceptor splice site sequence; JUST1781 PCT International Patent Application [000287] (b) a target binding sequence; [000288] (c) a branch point (BP) sequence; [000289] (d) a polypyrimidine-rich sequence (PPT); and [000290] (e) a 3' acceptor splice site sequence; and [000291] (B) a second vector encoding the PTM, wherein the PTM comprises 5' to 3': [000292] (i) a binding sequence complementary to the target binding sequence in the pre-mRNA molecule; [000293] (ii) a second splicing domain comprising: [000294] (a) a binding domain sequence complementary to a portion of the target binding sequence in (A)(iii)(b), above; [000295] (b) a branch point (BP) sequence; [000296] (c)) a polypyrimidine-rich sequence (PPT); and [000297] (d) a 3' acceptor splice site sequence; and [000298] (iii) a second coding sequence comprising a 3'-exonic coding fragment of the gene encoding the predetermined selectable protein marker, sequential to the 5'-exonic coding fragment of the gene in (A)(ii), above, whereby in combination therewith, after a trans- splicing event, an operable coding sequence of the protein marker is obtained that is selectable in a eukaryotic cell. [000299] Embodiment 2: The multiple-vector expression system according to Embodiment 1, further comprising, in the first vector in subpart (A): [000300] (iv) at least one operable expression cassette each comprising: [000301] (a) a promoter; [000302] (b) optionally, a motif that is recognizable by a m⁶A methyltransferase writer complex; [000303] (c) optionally, an intron; [000304] (d) an open reading frame encoding a first exogenous protein of interest (POI-1) operably linked to the promoter; and JUST1781 PCT International Patent Application [000305] (e) a polyadenylation site operably linked 3’ to the open reading frame. [000306] Embodiment 3: The multiple-vector expression system according to any of Embodiments 1-2, further comprising, in the second coding sequence in the second vector encoding the PTM in subpart (B)(iii), above: [000307] (iv) at least one operable expression cassette comprising: [000308] (a) a promoter; [000309] (b) optionally, a motif that is recognizable by a m⁶A methyltransferase writer complex; [000310] (c) optionally, an intron; [000311] (d) an open reading frame encoding a second exogenous protein of interest (POI-2) operably linked to the promoter; and [000312] (e) a polyadenylation site operably linked 3’ to the open reading frame. [000313] Embodiment 4: The multiple-vector expression system according to any of Embodiments 1-3, wherein the promoter in subpart (A)(i), above, is a constitutive promoter. [000314] Embodiment 5: The multiple-vector expression system according to any of Embodiments 1-4, comprising a motif that is recognizable by a m6A methyltransferase writer complex, wherein the motif comprises at least one m6A nucleotide located 5’ to the open reading frame. [000315] Embodiment 6: The multiple-vector expression system according to any of Embodiments 2-5, wherein. one or more of the promoters in Embodiment 2(iv)(a), above, and/or Embodiment 3(iv)(a), above, are each an inducible promoter. [000316] Embodiment 7: The multiple-vector expression system according to Embodiment 6, wherein the inducible promoter(s) comprises one or more TetO sequences. [000317] Embodiment 8: The multiple-vector expression system according to any of Embodiments 1-7, further comprising, at the 5’ ends of each of the first vector and the second vector, a 5’ ITR; and comprising, at the 3’ ends of each of the first vector and the second vector, a 3’ ITR. JUST1781 PCT International Patent Application [000318] Embodiment 9: The multiple-vector expression system according to Embodiment 8, wherein the 5’ ITRs are 5’ PiggyBac ITRs and the 3’ ITRs are 3’ PiggyBac ITRs. [000319] Embodiment 10: The multiple-vector expression system according to Embodiment 8, wherein the 5’ ITRs are 5’ Sleeping Beauty ITRs and the 3’ ITRs are 3’ Sleeping Beauty ITRs. [000320] Embodiment 11: The multiple-vector system according to Embodiment 9, wherein the 5’ PiggyBac ITRs each comprise the nucleotide sequence of SEQ ID NO:17; and the 3’ PiggyBac ITRs each comprise the nucleotide sequence of SEQ ID NO:18. [000321] Embodiment 12: The multiple-vector system according to Embodiment 10, wherein the 5’ Sleeping Beauty ITRs each comprise the nucleotide sequence of SEQ ID NO:15; and the 3’ Sleeping Beauty ITRs each comprise the nucleotide sequence of SEQ ID NO:16. [000322] Embodiment 13: The multiple-vector expression system according to any of Embodiments 8-12, further comprising a vector comprising an expression cassette from which a transposase or recombinase recognizing the ITRs, is expressible, and capable of integrating the first vector and/or the second vector into a eukaryotic genome. [000323] Embodiment 14: The multiple-vector expression system according to any of Embodiments 3-13, wherein the POI-1 and POI-2 are different subunits of a polymeric protein. [000324] Embodiment 15: The multiple-vector expression system according to Embodiment 14, wherein the polymeric protein is an antigen-binding protein. [000325] Embodiment 16: The multiple-vector expression system according to any of Embodiments 13-15, wherein the polymeric protein is a bispecific antigen-binding protein. [000326] Embodiment 17: The multiple-vector expression system according to any of Embodiments 13-16, wherein the polymeric protein is an antibody. [000327] Embodiment 18: The multiple-vector expression system according to any of Embodiments 1-17, wherein the selectable protein marker is a glutamine synthetase, selectable in a eukaryotic host cell that lacks functional endogenous glutamine synthetase. JUST1781 PCT International Patent Application [000328] Embodiment 19: The multiple-vector expression system according to any of Embodiments 1-17, wherein the selectable protein marker is dihydrofolate reductase, (DHFR) or an antibiotic resistance marker, selectable in a eukaryotic host cell that lacks functional endogenous expression of the selectable protein marker. [000329] Embodiment 20: A cultured eukaryotic recombinant host cell that does not endogenously express a predetermined functional selectable protein marker, wherein the eukaryotic host cell comprises the multiple-vector expression system according to any of Embodiments 1-19, which does encode the selectable protein marker. [000330] Embodiment 21: The cultured eukaryotic recombinant host cell according to Embodiment 20, wherein the cell is a mammalian cell. [000331] Embodiment 22: The cultured eukaryotic recombinant host according to any of Embodiments 20-21, wherein the cell is a CHO cell. [000332] Embodiment 23: A method of recombinantly expressing one or more proteins of interest in a cultured eukaryotic host cell line, comprising: [000333] (a) transfecting a plurality of cultured eukaryotic cells of a parent cell line, with the multiple-vector expression system according to any of Embodiments 1-19, wherein the parent cell line lacks a functional gene encoding the predetermined selectable protein marker; [000334] (b) culturing, in vitro, under physiological conditions that allows expression of proteins, in a culture medium not permissive to growth of the parent cell line; [000335] (c) selecting viable transfected cells from (b); and [000336] (d) harvesting the one or more proteins of interest. [000337] Embodiment 24: A method of selecting eukaryotic host cells that recombinantly express a protein of interest, in vitro, comprising: [000338] (a) transfecting a plurality of cultured eukaryotic host cells with the multiple- vector expression system according to any of Embodiment s 1-19, wherein the cells lack a functional gene encoding the predetermined selectable protein marker; JUST1781 PCT International Patent Application [000339] (b) culturing, in vitro, under physiological conditions that allows expression of proteins, in a culture medium not permissive to growth of eukaryotic host cells that do not recombinantly express the selectable protein marker; and [000340] (c) selecting viable transfected cells from (b). [000341] Embodiment 25: The method of recombinantly expressing one or more proteins of interest in a eukaryotic host cell line according to Embodiment 23, wherein the eukaryotic host cell line is a CHO cell line. [000342] Embodiment 26: The method of selecting eukaryotic host cells that recombinantly express a protein of interest according to Embodiment 24, wherein the eukaryotic host cells are CHO cells. [000343] Embodiment 27: The method of recombinantly expressing one or more proteins of interest in a eukaryotic host cell line according to any of Embodiments 23 and 25, wherein the selectable protein marker is a glutamine synthetase selectable in the eukaryotic cell that lacks functional endogenous glutamine synthetase. [000344] Embodiment 28: The method of recombinantly expressing one or more proteins of interest in a eukaryotic host cell line according to any of Embodiments 23 and 25, wherein the selectable protein marker is dihydrofolate reductase (DHFR) in the eukaryotic cell that lacks functional endogenous DHFR. [000345] Embodiment 29: The method of recombinantly expressing one or more proteins of interest in a eukaryotic host cell line according to any of Embodiments 23 and 25, wherein the selectable protein marker is an antibiotic resistance marker. [000346] Embodiment 30: The method of selecting eukaryotic host cells that recombinantly express a protein of interest according to any of Embodiments 24 and 26, wherein the selectable protein marker is a glutamine synthetase selectable in the eukaryotic cell that lacks functional endogenous glutamine synthetase. [000347] Embodiment 31: The method of selecting eukaryotic host cells that recombinantly express a protein of interest according to any of Embodiments 24 and 26, wherein the selectable protein marker is a DHFR selectable in the eukaryotic cell that lacks functional endogenous DHFR. JUST1781 PCT International Patent Application [000348] Embodiment 32: The method of selecting eukaryotic host cells that recombinantly express a protein of interest according to any of Embodiments 24 and 26, wherein the selectable protein marker is an antibiotic resistance marker. [000349] The following working examples are illustrative and not to be construed in any way as limiting the scope of the invention. [000350] EXAMPLES [000351] Example 1. Design of split glutamine synthetase using mRNA trans-splicing [000352] The glutamine synthetase (GS) gene (see, full coding sequence SEQ ID NO:14) was split, between exons 3 and 4, onto two separate plasmids; the target plasmid had GS exons 1 through 3, while the pre-trans-splicing molecule (PTM) plasmid contained GS exons 4 through 7. The target plasmid contained an intron sequence containing all the elements required for recruitment of the spliceosome: a 5’ splice site, a branch point, polypyrimidine tract and a 3' splice site. This intron sequence was obtained from RHO intron 1. (Berger, A. et al., “Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa,” Molecular Therapy 23(5), 918-930 (2015)). The PTM plasmid contained a binding domain complementary to 150-bp of the rhodopsin pre-mRNA first intron on the target plasmid, an intron sequence with a branch point, polypyrimidine tract and a 3’ splice site, followed by GS exons 4-7. An artificial intron was designed and placed between exons 4 and 5. This 97-bp artificial intron similarly had all the elements required for splicing: a 5’ splice site, a branch point, polypyrimidine tract and 3’ splice site. (See, Figure 1A). [000353] The target and PTM plasmids were transfected in a 1:1 ratio by the long duration electroporation (LDE) method into a glutamine synthetase knock-out (“GS KO”) cell line. (See, Bodwell J, Swiff F and Richardson J (1999), “Long duration electroporation for achieving high level expression of glucocorticoid receptors in mammalian cell lines,” J. Steroid Biochem. Mol. Biol.68(1- 2): 77-82). Other ratios (1:2 and 1:3) were also tested and yielded similar results. Forty-eight (48) hours after transfection, cells were selected in liquid culture medium without glutamine. Only upon mRNA expression of both plasmids, and successful trans-splicing facilitated by binding of the PTM to the intron of the target plasmid, would a full-length GS mRNA be formed, and be translated into functional glutamine allowing the cells to survive. Up to two expression cassettes, each expression cassette containing a promoter driving expression of an antibody coding sequence, followed by a polyadenylation signal at the 3’ end, can be on each of the target and PTM plasmid (Figure 1B). With this configuration, there was the potential to express up to four different chains or polypeptides using the glutamine synthetase selection marker. JUST1781 PCT International Patent Application [000354] Materials and Methods. [000355] Expression vectors. All gene synthesis, codon optimization, subcloning, assemblies and plasmid DNA maxipreps were performed by GenScript (New Jersey), at our direction. Regulatory elements and design and construction of the pJV145 control plasmid expressing mAb1 have been described (Ong E, Smidt P, McGrew TJ, “Limiting the metabolic burden of recombinant protein expression during selection yields pools with higher expression levels,” Biotechnol Prog. Sep (2019); 35(5): e2839). [000356] Design of pJV350 (target) plasmid. The pJV350 plasmid was designed with a human CMV promoter driving expression of GS exons 1-3, followed by an intron containing the 5’ splice site, branch point, polypyrimidine tract, 3’ splice site, and rhodopsin exon, in a 5’ to 3’ manner. The intron and rhodopsin exon sequences in the target plasmid were obtained from Berger et al., 2015. (Berger, A. et al., “Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa,”. Molecular Therapy 23(5):918-930 (2015)). This cassette of the GS fragment and intron was subcloned into a transposon-based expression vector, to replace expression of the full-length GS gene (pJV145; See, Ong E, Smidt P, McGrew TJ, “Limiting the metabolic burden of recombinant protein expression during selection yields pools with higher expression levels,” Biotechnol Prog. Sep (2019); 35(5): e2839). The pJV350 plasmid also had another expression cassette consisting of a mouse CMV promoter driving expression of the heavy chain of mAb1, followed by a polyadenylation signal. All these described elements were flanked by 3’ and 5’ inverted terminal repeat (ITR) sequences (e.g., “Sleeping Beauty” ITRs: SEQ ID NO:16 and SEQ ID NO:15, respectively), which allow them to be integrated into TTAA chromosomal sites by the piggybac transposase or other suitable transposase. [000357] The 5' “Sleeping Beauty” ITR sequence that can be used is the following: TATACAGTTGAAGTCGGAAGTTTACATACACTTAAGttggagtcattaaaactcgtttttcaactactcca caaatttcttgttaacaaacaatagttttggcaagtcagttaggacatctactttgtgcatgacacaagtcatttttccaacaattgtttacaga cagattatttcacttataattcactgtatcacaatTCCAGTGGGTCAGAAGTTTACATACACTAAGTtgactgt gcctttaaacagcttggaaaattccagaaaatgatgtcatggctttag//SEQ ID NO:15. [000358] The 3' “Sleeping Beauty” ITR sequence that can be used is the following: agcttgtggaaggctactcgaaatgtttgacccaagttaaacaatttaaaggcaatgctaccaaatactaATTGAGTGTATGT AAACTTCTGACCCACTGGGaatgtgatgaaagaaataaaagctgaaatgaatcattctctctactattattctgatatttc acattcttaaaataaagtggtgatcctaactgacctaagacagggaatttttactaggattaaatgtcaggaattgtgaaaaagtgagttta aatgtatttggCTAAGGTGTATGTAAACTTCCGACTTCAACTGTAT//SEQ ID NO:16. JUST1781 PCT International Patent Application [000359] Alternatively, some other embodiments of the present invention can employ the “5’ PiggyBac ITR,” (which we used in the working Examples hereunder) comprising the nucleotide sequence of SEQ ID NO:17: CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATGCATTCTTGAAATATTGCTCTCT CTTTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCCGCTT GGAGCTCCCGTGAGGCGTGCTTGTCAATGCGGTAAGTGTCACTGATTTTGAACTAT AACGACCGCGTGAGTCAAAATGACGCATGATTATCTTTTACGTGACTTTTAAGATT TAACTCATACGATAATTATATTGTTATTTCATGTTCTACTTACGTGATAACTTATTATAT ATATATTTTCTTGTTATAGATATC//SEQ ID NO:17. For purposes of the invention “3’ PiggyBac ITR” comprises the nucleotide sequence of SEQ ID NO:18 (which we also used in the working Examples hereunder): GATAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTAATAAAT AAATAAACATAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATA ATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAA ACACATGCGTCAATTTTACACATGATTATCTTTAACGTACGTCACAATATGATTATCT TTCTAGGG//SEQ ID NO:18. [000360] N⁶-Methyladenosine (m6A) sequence. The m6A sequence was synthesized and subcloned into the 5’UTR of the heavy chain in the target plasmid, upstream of the kozak sequence. (Costello A, Lao NT, Barron N, Clynes, M, “Improved yield of rhEPO in CHO cells with synthetic 5’UTR,” Biotechnol Lett 41:231-239 (2019)). [000361] Design of pJV351 (PTM) plasmid. The pJV351 plasmid was designed with the human CMV promoter driving the expression of an intron containing the binding domain, branch point, polypyrimidine tract, and GS exons 4-7, including the artificial intron (SEQ ID NO:2) between exons 4 and 5. The binding domain sequence (SEQ ID NO:1) in the PTM plasmid was obtained from the Binding Domain 1 sequence used in Berger et al. (2015). (Berger, A. et al., “Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa,”. Molecular Therapy 23(5):918-930 (2015)): [000362] CACCATTCATGGTGATAGCCGGGCTGCTGTTTGTGCAGGGCTGGCAC TGAACACTGCCTTGATCTTATTTGGAGCAATATGCGCTTGTCTAATTTCACAGCAAG AAAACTGAGCTGAGGCTCAAAGAAGTCAAGCGCCCTGCTGGGGCG//SEQ ID NO:1. [000363] The intron containing the branch point and polypyrimidine tract sequences on the PTM plasmid were obtained from Lorain et al. (2010). (Lorain, S., Peccate, C., Le Hir, M., Garcia, L., JUST1781 PCT International Patent Application “Exon exchange approach to repair Duchenne dystrophin transcripts,” PLOS One, 5(5) e10894 (2010)). The artificial intron (97 bp; SEQ ID NO:2), located between GS exons 4 and 5, was designed to contain the 5’ splice site of the first intron of human beta globin and the last 82 bp of cystic fibrosis transmembrane conductance regulator (CFTR) intron 9, which includes a branch point sequence, polypyrimidine tract and 3’ splice site (Mansfield et al., “5’ exon replacement and repair by spliceosome-mediated RNA trans-splicing,” RNA 9:1290-1297 (2003)): GTTGGTATCAAGGTTAACAAGCATCTATTGAAAATATCTGACAAACTCATCTTTTATTTTTG ATGTGTGTGTGTGTGTGTGTGTGTTTTTTTAACAG//SEQ ID NO:2. [000364] This cassette of the GS fragment and intron was subcloned into our in-house expression vector , to replace expression of the full-length GS gene. The pJV350 plasmid also had another expression cassette consisting of a mouse CMV promoter driving expression of the light chain of mAb1, followed by a polyadenylation signal. All these described elements were flanked by 3’ and 5’ inverted terminal repeat (ITR) sequences (“sleeping beauty” ITRs: SEQ ID NO:16 and SEQ ID NO:15, respectively), which allow them to be integrated into TTAA chromosomal sites by the piggybac transposase. [000365] Design of pJV336 plasmid containing M-3406 antibody with hole mutations. This plasmid had all the trans-splicing elements similar to pJV350, but with two expression cassettes each consisting of a mouse CMV promoter driving expression of the IgG1 heavy chain of M-3406, followed by a polyadenylation signal and the second expression cassette also containing a mouse CMV promoter driving the expression of the kappa light chain of M- 3406. Hole mutations (Y349C, T366S, L368A, Y407V) were engineered into the CH3 domain of heavy chain of M-3406. All these described elements were flanked by 3’ and 5’ inverted terminal repeat (ITR) sequences (“sleeping beauty” ITRs: SEQ ID NO:16 and SEQ ID NO:15, respectively), which allow them to be integrated into TTAA chromosomal sites by the piggybac transposase. [000366] Design of pJV337 plasmid containing M-3376 antibody with knob mutations. The pJV337 plasmid had all the trans-splicing elements similar to pJV351, but with two expression cassettes each consisting of a mouse CMV promoter driving expression of the IgG1 heavy chain of M-3376, followed by a polyadenylation signal and the second expression cassette also containing of a mouse CMV promoter driving the expression of the kappa light chain of M-3376. Knob mutations (S354C, T366W) were engineered into the CH3 domain of heavy chain of M-3376. All these described elements were flanked by 3’ and 5’ inverted terminal repeat (ITR) sequences (“sleeping beauty” ITRs: SEQ ID NO:16 and JUST1781 PCT International Patent Application SEQ ID NO:15, respectively), which allowed them to be integrated into TTAA chromosomal sites by the piggybac transposase. [000367] Cell culture. An in-house generated CHO-K1 glutamine synthetase (GS) knockout (KO) host cell line (CL-72) was used. CL-72 host cells were cultured at 37^°C in a humidified 5% CO₂ incubator in CD OptiCHO™ medium (ThermoFisher Scientific, Catalog number: 12681011), supplemented with 4 mM glutamine. The inducible CHO-K1 GS KO host cell line (CL-130) was cultured in CD OptiCHO™ medium supplemented with 4 mM glutamine and 400 µg/mL G418. (See, Ong, E., Smidt, P., McGrew, T.J., “Limiting the metabolic burden of recombinant protein expression during selection yields pools with higher expression levels,” Biotechnol Prog. Sep (2019); 35(5): e2839 (2019)). [000368] Long duration electroporation (LDE) transfections and selection. Cells were transfected by electroporation using cuvettes using the following electroporation settings: Capacitance = 3175 uF, voltage = 200V, resistance = 725 Ohms, with a BTX ECM 680 electroporation system (BTX). A total of 18.3 µg of plasmid DNA was transfected together with 6.25 µg of piggybac transposase mRNA in EX-CELL^® 302 serum-free medium for CHO Cells (Sigma-Aldrich, Cat. No. 24326C) into 20 million cells per transfection. Trans-splicing vectors were transfected at a molar ratio of 1:1 along with 25 µg of salmon sperm DNA as carrier DNA. After electroporation, cells were transferred from the cuvette into CD OptiCHO™ medium (ThermoFisher Scientific, Catalog number: 12681011) supplemented with 4 mM glutamine in stationary T75 flasks and kept at 37^°C in a humidified CO2 incubator. Forty-eight (48) hours post-transfection, cells were counted by a trypan blue exclusion assay using a Vi-Cell XR cell viability analyzer (Beckman Coulter Life Sciences), and 15 million cells were put in fresh CD OptiCHO™ medium without glutamine at a density of 1 x 10⁶ cells/mL for minus glutamine selection. Transfections performed in the inducible GS KO cell line (CL-130) were selected in CD OptiCHO™ medium without glutamine supplemented with 400 µg/mL G418. Approximately every three to four days, cells counts were obtained to monitor cell viability and viable density, and medium was refreshed. Cells were transferred to 50-mL spin tubes once they had cell viabilities of greater than 60% and viable cell densities of greater than 1 x 10⁶ cells/mL, and then the cells were passaged every three or four days at cell densities of 0.4 x 10⁶ cells/mL or 0.2 x 10⁶ cells/mL, respectively. [000369] Fed-batch production assay. Cells were passaged at a density of 1 x 10⁶ cells/mL the day prior to the production assay. To seed cells for the production assay, cells were centrifuged at 1000 rpm for 5 mins, and resuspended at a cell density of 0.6 x 10⁶ cells/mL into 3mL of BAK004- 101 proprietary chemically-defined fed-batch production medium in 24-deep-well plates. On days 3, 6 and 8, cells were counted on a Guava^® easyCyte^TM flow cytometer (EMD Millipore), and glucose was JUST1781 PCT International Patent Application measured with a colorimetric assay using a SpectraMax^® M5 microplate reader (Molecular Devices). As needed, cells were supplemented with 50% glucose up to approximately 7 g/L on these days. Additionally, cells were also fed with 4.5% of the starting culture volume of Hyclone^TM CellBoost^TM 7a Supplement (Cytiva) and 0.5% of the starting culture volume with Hyclone^TM CellBoost^TM 7a Supplement (Cytiva) on days 3, 6 and 8 of production. On day 10 of production, supernatant from cell cultures were harvested by centrifugation at 2000 rpm for 10 mins, followed by filtration. Titer measurements were then obtained by affinity ultra high performance liquid chromatography (UHPLC) column using POROS^TM 20 A Protein A affinity resin (ThermoFisher Scientific). For the inducible GS KO cell line, 0.0125 µg/mL of doxycycline was added to the production medium at days 0, 3, 6, and 8 to induce expression of the mAb. [000370] DNA and RNA extraction. Two million cells were pelleted by centrifugation at 1000 rpm for 5 minutes, and supernatants were aspirated. Cell pellets were frozen at -70^°C. DNA and RNA from each cell pellet were extracted using the NucleoSpin TriPrep Mini kit (Macherey-Nagel, Cat. No.740966.50) according to the manufacturer’s instructions. Extracted DNA and RNA were quantified using the Nanodrop microvolume spectrophotometer (ThermoFisher Scientific), 1 µg of RNA was converted to cDNA using the QuantiTect Reverse Transcription kit (Qiagen) according to manufacturer’s instructions. [000371] PCR. Phusion Flash High-Fidelity PCR master mix (ThermoFisher Scientific) was used along with the following primer sequences to amplify the GS gene, using the manufacturer’s protocol. The forward primer sits on GS exon 3 on the target plasmid, whereas the reverse primer sits on GS exon 4 on the PTM plasmid. Only upon successful trans-splicing where a full-length GS mRNA is formed, will a PCR product of the right size be obtained using cDNA as a PCR template. The following primer sequences were used for PCR detection of successful trans-splicing: [000372] GS forward primer sequence: TGGCCTTCCAATGGCTTT//SEQ ID NO:3; and [000373] GS reverse primer sequence: CCCAAAGTCTTCACATACTCGAT//SEQ ID NO:4. [000374] Results [000375] Use of split glutamine synthetase in creating stable CHO-K1 GS-KO cell lines. As a proof of concept, a monoclonal antibody (mAb1) was expressed using the split GS method, where the target plasmid expressed the immunoglobulin heavy chain, and the PTM plasmid expressed the immunoglobulin light chain. Cell counts from minus glutamine selection and titers from a production assay were compared to a control where both chains and full-length GS were expressed on the same plasmid (denoted as pJV145; see, Figure 2A). Successful trans-splicing of GS was evaluated in two ways: detection of a PCR product from cDNA but not genomic DNA of cells transfected with the target and PTM plasmids (Figure 2B), and full recovery of GS KO cells from selection in media JUST1781 PCT International Patent Application without glutamine (Figure 2C and Figure 2D). While the cells containing pJV145 control fully recovered from selection by day 14, cells bearing the split GS took much longer to recover at day 21. [000376] Improved recovery in selection and titer. To improve recovery from minus glutamine selection, the experiment was repeated in an inducible GS-KO CHO-K1 cell line (CL-130). In this cell line, the antibody was not expressed during selection, in order to reduce the metabolic burden on cells during selection, thereby allowing for more efficient trans-splicing and expression of GS. Indeed, split GS samples fully recovered by day 14 of selection and had titers with values 70% of the control (Figure 3A, Figure 3B, and Figure 3C). The effect of the artificial intron in improving these results was also apparent. [000377] Use of an m6A motif sequence. The N⁶-methyadenosine (m6A) modification is the most abundant internal modification of mRNA in higher eukaryotes. It is a post-transcriptional modification in which a methyltransferase methylates the sixth nitrogen atom of adenine. The m6A affects a variety of processes including mRNA stability, export, translation, and splicing. Two m6A motif sequences were inserted into the 5’UTR of the heavy chain, upstream of the kozak sequence, on the target plasmid. (See, Costello A, Lao NT, Barron N, Clynes, M, “Improved yield of rhEPO in CHO cells with synthetic 5’UTR,” Biotechnol. Lett.41:231-239 (2019)). [000378] Surprisingly, samples with the m6A motif sequence 5’ of the HC coding region had improved recovery from selection, but similar titers in the inducible cell line (Figure 4A, Figure 4B, and Figure 4C). This surprising result indicates that the m6A motif in a sequence linked to the target sequence can influence trans-splicing of a distinct transcript. In the non-inducible CL-72 cell line, samples with the m6A motif sequence showed improved recovery during selection, and slightly higher titers (see, Figure 5A, Figure 5B, and Figure 5C). [000379] We next sought to determine whether alternate locations of the m6A motif can also influence trans-splicing of GS. Interestingly, when the m6A sequence was placed upstream (i.e., 5’) to the intron containing the binding domain on the PTM plasmid, it did not improve recovery from minus glutamine selection in the CL-130 cell line (see, Figure 6A, Figure 6B, and Figure 6C). We also tested the effect of m6A in other locations such as the 3’UTR of the heavy chain, 3’ of the intron on the target plasmid, 3’ of the intron on the PTM plasmid, and 5’ of the GS fragment on the target plasmid. All these modifications showed no improvements to recovery from minus glutamine selection, compared to samples without m6A. This implies a unique requirement for the m6A to be at a specific location, 5’ of the heavy chain on the target plasmid, for it to improve recovery from selection. JUST1781 PCT International Patent Application [000380] We also tested the ability of this trans-splicing method to express a second monoclonal antibody. M-2865 is an artificial humanoid IgG1 antibody generated in silico using a Generative Adversarial Network (GAN) (Amimeur et al., “Designing feature- controlled humanoid antibody discovery libraries using Generative Adversarial Networks,” BioRxiv 2020.04.12.024844 (2020)). The heavy chain of M-2865 was on the pJV350 plasmid, whereas the light chain was on the pJV351 PTM plasmid (see, Figure 6A). All other elements of the vectors were unmodified. Titers of M-2865 generated by the trans- splicing vectors are about 67% of the control (see, Figure 6B, Figure 6C and Figure 6D), where both heavy and light chains of M-2865 are on the same vector with intact GS, implying the successful use of this method for a second molecule in the CL-130 inducible (GS null) cell line (Figure 6D). This antibody was also expressed in the CL-72 cell line; similar to MS-202, titers were much lower in CL-72. [000381] Expression of a novel SARS-CoV2 bispecific antibody using the inventive methods using split GS selection. Bispecific antibodies are a promising approach to improving target specificity by binding two different antigens, and such bispecific antibodies have tremendous potential to improve therapy for a variety of diseases. We used the inventive method, with a split GS selection, to express a novel bispecific antibody comprising of two SARS-CoV2 monoclonal IgG1 antibodies M-3406 and M-3376, generated in silico from a Generative Adversarial Network developed in-house. The “knob-into-hole” format was used to drive heterodimerization of the heavy chains of the two different antibodies (see, Figure 7A and Figure 7B). To do this, amino acid mutations within the CH3 domain of the heavy chains were engineered to create a protuberance in the form of a “knob” at the interface of one of the antibodies and a corresponding cavity in the form of a “hole” in the second antibody. The different antibodies were encoded on separate vectors containing different fragments of GS that were co-transfected into the CL-130 and CL-165 inducible CHO (GS null) cell lines. Upon successful trans-splicing of GS during minus-glutamine selection, the recovered cells were put into a 10-day fed-batch production run. A titer of about 1 mg/mL was observed on day 10 of production (Figure 7C), which is within the range of titers in stable CHO lines reported in the literature. (See, e.g., Rajendra et al., “Transient and stable CHO expression, purification and characterization of novel hetero-dimeric bispecific IgG antibodies.” Biotechol. Progress 33(2) (2016); Gong and Wu, Gong and Wu (2023) “Efficient production of bispecific antibody – optimization of transfection strategy leads to high-level stable cell line generation of a Fabs-in-tandem immunoglobin,” Antibody Therapeutics, JUST1781 PCT International Patent Application 6(3):170-179 (2023)). Size exclusion chromatography was carried on the purified supernatants from the cell cultures of each of the CL-130 and CL-165 cell lines, and showed a >85% main peak of the bispecific antibody (Figure 7D). Reduced capillary electrophoresis (rCE) was also performed and showed the presence of light and heavy chains (Figure 7E). [000382] Discussion. The results disclosed herein demonstrate that using split GS expression by trans-splicing was effective to create stable CHO cell lines expressing a monoclonal antibody in a system in which the coding sequences of the immunoglobulin heavy and immunoglobulin light chains of the mAb were on separate plasmids. This was replicated with the expression of at least two different monoclonal antibodies. The inducible cell line had a lower selection pressure, since the antibody was not expressed during selection, thereby allowing the trans-spliced GS to be expressed more effectively. Antibody titers of up to about 70% of the control were achieved with the expression of full-length GS protein, and other selection markers can be split and expressed in the same manner with similar results; We repeated the experiment using CL-165, another inducible GS-KO CHO- K1 cell line. Both improved selection recovery and antibody titers from samples with the artificial intron were observed in this cell line as well, but they were not as great as seen with the CL-130 cell line; this implies the likelihood of a cell line dependent effect. [000383] We also successfully expressed a bispecific antibody using the inventive compositions and methods. The bispecific antibody had four different chains and in a knob- in-hole format. The advantage of using the invention, e.g., with a split GS selection, for expressing bispecific or multi-chain antibodies is that only one selection marker is used for stable expression, instead of using multiple selection markers. Rajendra et al. reported that stable expression of bispecific antibodies in CHO cells had titers between 0.4 g/L to 2.3 g/L, an expression range within which the present invention performs., and even better titers compared to other studies expressing knob-in-hole format bispecific antibodies in CHO cells (Rajendra et al., “Transient and stable CHO expression, purification and characterization of novel hetero-dimeric bispecific IgG antibodies.” Biotechol. Progress 33(2) (2016); Ong et al, “Vector design for enhancing expression level and assembly of knob-into-hole based FabscFv-Fc bispecific antibodies in CHO cells.” Antibody Therapeutics, 5(4):288-300 (2022)). [000384] We note that the location of the m6A motif upstream of the heavy chain on the target plasmid improved the recovery from selection in both CL-72 and CL-130 cell lines, JUST1781 PCT International Patent Application implying that the m6A motif can improve trans-splicing efficiency, when located upstream of the heavy chain on the target plasmid. When the motif was placed in alternate locations on either the target or PTM plasmid it did not improve recovery from selection. Although the present invention does not depend upon any particular mechanism of action, we speculate that the m6A motif sequence recruits splicing factors that facilitate trans-splicing.

Claims

JUST1780 U.S. Provisional Patent Application We claim: 1. A multiple-vector expression system encoding a pre-mRNA molecule and a pre- mRNA trans-splicing molecule (PTM), wherein: (A) a first vector encodes the pre-mRNA molecule, which comprises 5' to 3': (i) a promoter; (ii) a first coding sequence comprising a 5'-exonic coding fragment of a gene encoding a predetermined selectable protein marker; (iii) an intron, comprising a first splicing domain comprising: (a) a 5' donor acceptor splice site sequence; (b) a target binding sequence; (c) a branch point (BP) sequence; (d) a polypyrimidine-rich sequence (PPT); and (e) a 3' acceptor splice site sequence; and (B) a second vector encoding the PTM, wherein the PTM comprises 5' to 3': (i) a binding sequence complementary to the target binding sequence in the pre-mRNA molecule; (ii) a second splicing domain comprising: (a) a binding domain sequence complementary to a portion of the target binding sequence in (A)(iii)(b); (b) a branch point (BP) sequence; (c)) a polypyrimidine-rich sequence (PPT); and (d) a 3' acceptor splice site sequence; and (iii) a second coding sequence comprising a 3'-exonic coding fragment of the gene encoding the predetermined selectable protein marker, sequential to the 5'-exonic coding fragment of the gene in (A)(ii), whereby in combination therewith, after a trans-splicing event, an operable coding sequence of the protein marker is obtained that is selectable in a eukaryotic cell. JUST1780 U.S. Provisional Patent Application 2. The multiple-vector expression system according to Claim 1, further comprising, in the first vector in subpart (A): (iv) at least one operable expression cassette each comprising: (a) a promoter; (b) optionally, a motif that is recognizable by a m⁶A methyltransferase writer complex; (c) optionally, an intron; (d) an open reading frame encoding a first exogenous protein of interest (POI-1) operably linked to the promoter; and (e) a polyadenylation site operably linked 3’ to the open reading frame. 3. The multiple-vector expression system according to any of Claims 1-2, further comprising, in the second coding sequence in the second vector encoding the PTM in subpart (B)(iii): (iv) at least one operable expression cassette comprising: (a) a promoter; (b) optionally, a motif that is recognizable by a m⁶A methyltransferase writer complex; (c) optionally, an intron; (d) an open reading frame encoding a second exogenous protein of interest (POI-2) operably linked to the promoter; and (e) a polyadenylation site operably linked 3’ to the open reading frame. 4. The multiple-vector expression system according to any of Claims 1-3, wherein the promoter in subpart (A)(i) is a constitutive promoter. 5. The multiple-vector expression system according to any of Claims 1-4, comprising a motif that is recognizable by a m⁶A methyltransferase writer complex, wherein the motif comprises at least one m6A nucleotide located 5’ to the open reading frame. JUST1780 U.S. Provisional Patent Application 6. The multiple-vector expression system according to any of Claims 2-5, wherein. one or more of the promoters in Claim 2(iv)(a) and/or Claim 3(iv)(a) are each an inducible promoter. 7. The multiple-vector expression system according to Claim 6, wherein the inducible promoter(s) comprises one or more TetO sequences. 8. The multiple-vector expression system according to any of Claims 1-7, further comprising, at the 5’ ends of each of the first vector and the second vector, a 5’ ITR; and comprising, at the 3’ ends of each of the first vector and the second vector, a 3’ ITR. 9. The multiple-vector expression system according to Claim 8, wherein the 5’ ITRs are 5’ PiggyBac ITRs and the 3’ ITRs are 3’ PiggyBac ITRs. 10. The multiple-vector expression system according to Claim 8, wherein the 5’ ITRs are 5’ Sleeping Beauty ITRs and the 3’ ITRs are 3’ Sleeping Beauty ITRs. 11. The multiple-vector system according to Claim 9, wherein the 5’ PiggyBac ITRs each comprise the nucleotide sequence of SEQ ID NO:17; and the 3’ PiggyBac ITRs each comprise the nucleotide sequence of SEQ ID NO:18. 12. The multiple-vector system according to Claim 10, wherein the 5’ Sleeping Beauty ITRs each comprise the nucleotide sequence of SEQ ID NO:15; and the 3’ Sleeping Beauty ITRs each comprise the nucleotide sequence of SEQ ID NO:16. 13. The multiple-vector expression system according to any of Claims 8-12, further comprising a vector comprising an expression cassette from which a transposase or recombinase recognizing the ITRs, is expressible, and capable of integrating the first vector and/or the second vector into a eukaryotic genome. 14. The multiple-vector expression system according to any of Claims 3-13, wherein the POI-1 and POI-2 are different subunits of a polymeric protein. 15. The multiple-vector expression system according to Claim 14, wherein the polymeric protein is an antigen-binding protein. 16. The multiple-vector expression system according to any of Claims 13-15, wherein the polymeric protein is a bispecific antigen-binding protein. JUST1780 U.S. Provisional Patent Application 17. The multiple-vector expression system according to any of Claims 13-16, wherein the polymeric protein is an antibody. 18. The multiple-vector expression system according to any of Claims 1-17, wherein the selectable protein marker is a glutamine synthetase selectable in a eukaryotic host cell that lacks functional endogenous glutamine synthetase. 19. The multiple-vector expression system according to any of Claims 1-17, wherein the selectable protein marker is dihydrofolate reductase (DHFR) or an antibiotic resistance marker, selectable in a eukaryotic host cell that lacks endogenous expression of the selectable protein marker. 20. A cultured eukaryotic recombinant host cell that does not endogenously express a predetermined functional selectable protein marker, wherein the eukaryotic host cell comprises the multiple-vector expression system according to any of Claims 1-19. 21. The cultured eukaryotic recombinant host cell according to Claim 20, wherein the cell is a mammalian cell. 22. The cultured eukaryotic recombinant host according to any of Claims 20-21, wherein the cell is a CHO cell. 23. A method of recombinantly expressing one or more proteins of interest in a cultured eukaryotic host cell line, comprising: (a) transfecting a plurality of cultured eukaryotic cells of a parent cell line, with the multiple-vector expression system according to any of Claims 1-19, wherein the parent cell line lacks a functional gene encoding the predetermined selectable protein marker; (b) culturing, in vitro, under physiological conditions that allows expression of proteins, in a culture medium not permissive to growth of the parent cell line; (c) selecting viable transfected cells from (b); and (d) harvesting the one or more proteins of interest. 24. A method of selecting eukaryotic host cells that recombinantly express a protein of interest, in vitro, comprising: JUST1780 U.S. Provisional Patent Application (a) transfecting a plurality of cultured eukaryotic host cells with the multiple-vector expression system according to any of Claims 1-19, wherein the cells lack a functional gene encoding the predetermined selectable protein marker; (b) culturing, in vitro, under physiological conditions that allows expression of proteins, in a culture medium not permissive to growth of eukaryotic host cells that do not express the selectable protein marker; and (c) selecting viable transfected cells from (b). 25. The method of recombinantly expressing one or more proteins of interest in a eukaryotic host cell line according to Claim 23, wherein the eukaryotic host cell line is a CHO cell line. 26. The method of selecting eukaryotic host cells that recombinantly express a protein of interest according to Claim 24, wherein the eukaryotic host cells are CHO cells. 27. The method of recombinantly expressing one or more proteins of interest in a eukaryotic host cell line according to any of Claims 23 and 25, wherein the selectable protein marker is a glutamine synthetase selectable in the eukaryotic cell that lacks functional endogenous glutamine synthetase. 28. The method of recombinantly expressing one or more proteins of interest in a eukaryotic host cell line according to any of Claims 23 and 25, wherein the selectable protein marker is dihydrofolate reductase (DHFR) in the eukaryotic cell that lacks functional endogenous DHFR. 29. The method of recombinantly expressing one or more proteins of interest in a eukaryotic host cell line according to any of Claims 23 and 25, wherein the selectable protein marker is an antibiotic resistance marker. 30. The method of selecting eukaryotic host cells that recombinantly express a protein of interest according to any of Claims 24 and 26, wherein the selectable protein marker is a glutamine synthetase selectable in the eukaryotic cell that lacks functional endogenous glutamine synthetase. 31. The method of selecting eukaryotic host cells that recombinantly express a protein of interest according to any of Claims 24 and 26, wherein the selectable protein marker is a DHFR selectable in the eukaryotic cell that lacks functional endogenous DHFR. JUST1780 U.S. Provisional Patent Application 32. The method of selecting eukaryotic host cells that recombinantly express a protein of interest according to any of Claims 24 and 26, wherein the selectable protein marker is an antibiotic resistance marker.