CN114014906A - Method for purifying hydrophobic protein by using cation exchange chromatography - Google Patents

Abstract

The invention discloses a method for purifying hydrophobic protein by cation exchange chromatography. The chromatography steps of the method comprise balancing, loading, washing and eluting, wherein cation eluent used in the chromatography process does not contain strong electrolyte components, and the conductivity of the cation flushing liquid is higher than that of a loaded sample; the salts used in the cation balancing solution, the washing solution and the eluent comprise weak acid salts and/or phosphate salts. The method can effectively remove HCP and polymers, avoid peak splitting or multi-peak phenomenon of cation chromatography elution peak, and the yield and purity of the collected bispecific antibody are higher and can both reach more than 90% (even can reach more than 99%).

Description

Method for purifying hydrophobic protein by using cation exchange chromatography

Technical Field

The invention relates to the technical field of antibody purification, in particular to a method for purifying hydrophobic protein by using a cation exchange chromatography.

Background

Hydrophobicity is an important physicochemical property of an antibody molecule, and the strength of the hydrophobicity of the antibody molecule has an important influence on the selection and process development of a filler for cation exchange chromatography. Generally, antibody molecules with stronger hydrophobicity are more prone to have the problems of elution multiple peaks, poorer polymer removal effect, poor stability, low yield and the like in the cation exchange chromatography purification process. The hydrophobic antibody is specifically described below as an example.

Through research and development for more than 30 years, monoclonal antibody (mAb) drugs have made great progress in the field of treatment of tumors and autoimmune diseases, and at the same time, have also become the development direction with the fastest growth rate and the most promising in the field of medicine, and bring new hopes for patients who are ineffective in conventional treatment. At present, the affinity, stability, biological activity and treatment effect of the medicine are greatly improved through genetic engineering modification and effective quality control.

Bispecific antibodies (BsAb) enable a single molecule to bind to two distinct targets simultaneously, and better therapeutic efficacy of Bispecific antibodies than monoclonal antibody combinations is observed for various indications including tumors and infectious diseases. The combination of two targets at the same time can realize the unique action mechanism that the monoclonal antibody can not realize, and the interest of the bispecific antibody as a therapeutic drug is increasing. Chinese patents (application publication nos. CN110305210A and CN101896504B) disclose methods for designing and preparing bispecific antibodies. Chinese patent (application publication No. CN107922476A) discloses a method for isolating these bispecific antibodies from a mixture comprising monospecific antibodies with two kappa light chains or parts thereof and monospecific antibodies with two lambda light chains or parts thereof.

Downstream purification is considered to be one of the most challenging stages in the industrial production of monoclonal antibody drugs, and currently, a three-step purification strategy is widely adopted: the method comprises the steps of sample capture, moderate purification and fine purification, and the strategy is complex in process and strict in operation requirements, so that the purification cost generally accounts for 50-80% of the total production cost. The first step of sample capture is usually protein A affinity chromatography, and the medium and fine purifications can be ion exchange chromatography and hydrophobic chromatography. Downstream processing of bivalent bispecific antibodies likely utilizes the same processing strategy as monoclonal antibodies. Although different platform processes for purification of monoclonal antibodies employ these several chromatographic methods, different parameters and different filler selections can produce completely different results.

Cation exchange Chromatography (CEX) is widely used in the purification process of platform monoclonal antibodies due to its high loading, selectivity to impurities, expandability and robustness, and is mainly used to remove high polymers, as well as host proteins and DNA. The isoelectric point of mabs is generally neutral or weakly basic, so cation exchange packing is generally suitable for operating in the bind-elute mode in antibody purification processes, mabs bind to the resin under low conductivity conditions, and the pH is below the isoelectric point of the target molecule. Elution of the mAb by increasing conductivity or pH can be achieved by linear gradient or step elution to predetermined conditions. Impurities, particularly high polymers, are generally more tightly bound on the CEX packing than the mAb product and can be separated from the desired components by adjusting the elution conditions and the collection range.

CEX is generally considered a gentle operation, unlikely to cause conformational changes in proteins, as it is based primarily on electrostatic interactions. In most cases, the binding protein elutes from the CEX column in a unimodal form, with the salt concentration eluted depending on the pH and gradient slope. However, the use of cation exchange chromatography is sometimes influenced by the chemical properties of the protein, leading to unexpected results. Gillespy et al found that loading a highly purified glycosylated antibody onto a Cation exchange column and eluting it with a salt gradient resulted in two elution peaks (Gillespie R, Nguyen T, Macneil S et al. catalysis surface-mediated attenuation of an aggregated immunoglobulin (IgG1). J Chromatogr A,2012,1251: 101-one 110), one containing almost completely the monomeric form of the antibody and the other containing an increased percentage of aggregated species formed within the column. The molecular mechanism of the phenomenon that multiple elution peaks occur during elution by cation and anion exchange chromatography is generally thought to be that adsorption on the surface of the filler leads to local changes in the dynamic conformation of the surface of the protein molecule (Kimer L K, Pabst T M, Hunter A K, etc.. Chromatographic biochemical antibodies on the surface of exchange columns. II. biomolecular catalysts. J. chromatograph A,2019,1601: 133. 144.). The solvent exposure area of monoclonal antibodies bound to CEX packing increases over time, gradually increasing the aggregation of unstable intermediates upon elution, leading to a bimodal elution phenomenon (Guo J, Carta G. underfolding and aggregation of a glycosylated monomeric antibody on a cation exchange column. part II. protein structures by hydrogen exchange mass spectrometry. J chromatography A,2014,1356: 129-. Reversible protonation of histidine residues in monoclonal antibody molecules also produces different bimodal elutions on different cationic fillers (Luo H, Cao M, Newell K et al. Double-peak elution profile of a monoclonal antibody in exchange chromatography, J chromatography A,2015,1424: 92-101). Certain monoclonal antibody molecules aggregate on reversible changes in conformation and irreversible columns on CEX columns, and also cause elution of three peaks (Guo J, Creasy A D, Barker G et al. Surface induced three-peak interaction detector of a monoclonal antibody degradation interaction chromatography. J chromatography A,2016,1474: 85-94). Luo et al reported that NaCl in an IgG2 monoclonal antibody induced a reversible auto-association strongly bound to a CEX column during linear salt gradient elution and co-eluted with aggregates, resulting in lower purity of the eluted product and significant peak splitting (Luo H, Macapagal N, Newell K, et al. Effects of salt-induced reversible selection on the elution phase of monoclonal antibody in exchange chromatography. J Chromatoger A,2014,1362: 186-193).

The appearance of multiple peaks on a CEX column is common in linear salt gradient elution mode, and similar phenomena occur with some mAb molecules eluting at salt level (Guo J, Carta G. underfolding and aggregation of a glycosylated monomeric antibody on a cation exchange column. part II. protein structures by hydrogen chromatography. J chromatography A,2014,1356: 129-. The appearance of multiple elution peaks on the CEX column, in addition to the nature of the mAb molecule itself, also affects the chemistry and structure of the cation exchange packing, as is more evident in bimodal elution profiles of polymer grafted resins such as Capto S Impact and Eshmuno CPX (Farys M, Gibson D, Lewis AP et al, Isotype dependent on-column non-reversible aggregation of anionic antigens Biotechnol Bioeng,2018,115(5): 1279-.

Although the capture behavior of bispecific antibody molecules on protein a fillers is similar to that of conventional mabs, due to the multi-domain structure and flexibility of single chain variable fragment (scFv) attachment of bispecific antibody molecules, as well as complex impurity composition, cation exchange chromatography may differ significantly, and different conformations may bind to the Chromatographic surface in different ways, leading to more complex Chromatographic phenomena, including the possibility of multimodal elution (Kimerer L K, Pabst T M, Hunter a K et al. At present, the CEX chromatography purification of the bispecific antibody mostly adopts an mAb platform purification method, which is easy to cause the abnormal phenomenon of cation elution peak and has the defects of complex process, low carrying capacity, low product yield, low purity and the like.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for purifying hydrophobic protein by using cation exchange chromatography, which aims to overcome the defects of aggregation, low yield, low purity and the like in the process of purifying hydrophobic antibody molecules in the prior art, the method can effectively remove HCP and polymers, avoid peak splitting or multimodal phenomena of cation chromatography elution peaks, and ensure that the yield and the purity of the collected hydrophobic protein (such as bispecific antibody) are higher and can reach more than 90 percent (even more than 99 percent).

The invention mainly solves the technical problems through the following technical scheme.

One of the technical schemes of the invention is as follows: a method for purifying hydrophobic protein by cation exchange chromatography comprises the steps of balancing, loading, washing and eluting, wherein cation eluent used in the chromatography process does not contain strong electrolyte components, and the conductivity of the cation flushing liquid is higher than that of the loaded sample;

the salts used in the cation balancing solution, the washing solution and the eluent comprise weak acid salts and/or phosphate salts.

Preferably, the filler used for cation exchange chromatography contains a compound selected from sulfonic acid group (SO)₃^2-) A strong cation exchanger containing a group selected from the group consisting of a sulfomethyl group (S), a sulfopropyl group (SP) and a phosphoric group (P), or a weak cation exchanger containing an ion exchange group selected from the group consisting of a carboxymethyl group (CM) and a carboxyl group (COO-), preferably containing a sulfonic acid group^-Or sulfopropyl, etc. The cation exchange chromatographic packing used in the present invention preferably includes, but is not limited to, Capto S Impact and Capto MMC from GE, POROS 50XS and POROS 50HS from Thermofisor, Fractogel EMD SO3 from Merck Millipore^-(M) and Eshmuno CPX, Nanogel-50SP, Suzhou Nami, and the like; preferably Capto S Impact or Poros 50HS fillers.

Cation exchange chromatography as described in the present invention may be conventional in the art, preferably using a bind-elute mode.

The cation balancing solution, the flushing solution and the eluent can be non-strong acid salt, preferably acetic acid-acetate buffer solution, citric acid-citrate buffer solution, phosphate buffer solution or histidine buffer solution, and more preferably acetic acid-acetate buffer solution; preferably, none of the cation exchange solution, the rinse solution and the eluent contains strong electrolyte components.

In the present invention, the elution may be conventional in the art, and preferably is a salt gradient elution or a salt isocratic elution. Wherein, the salt gradient elution is preferably a salt linear gradient elution.

Preferably, the conductivity of the eluent eluted by the salt isocratic elution is 12.55-17.6 mS/cm, and the pH value is 4.8-5.5.

Preferably, the conductivity of the eluent eluted by the salt linear gradient is 19.0-25.0 mS/cm, and the pH value is 5.0-5.4.

Wherein: when the filler is Capto S Impact, the range of the conductivity of the eluent for salt isocratic elution is preferably 13.5-15.5 mS/cm; when the filler is Poros 50HS, the conductivity range of the eluent for isocratic elution of the salt is preferably 12.55-14.75 mS/cm.

In the present invention, the pH of the cation balance liquid is preferably 4.9 to 5.5, and more preferably 5.0; the conductivity is preferably 3.0 to 10.5mS/cm, more preferably 5.7 to 10.5mS/cm, and still more preferably 8.0 mS/cm.

In the present invention, the pH of the rinsing liquid is preferably 4.9 to 5.5, more preferably 5.0; the conductivity is preferably 3.0 to 10.5mS/cm, more preferably 5.7 to 10.5mS/cm, and still more preferably 8.0 mS/cm.

The conductivity of the sample is preferably 3.74-10.5 mS/cm, more preferably 3.74-7.55 mS/cm.

The hydrophobic protein in the invention is an antibody, such as a bispecific antibody or a monoclonal antibody; the bispecific antibody is preferably OX40/PD-L1 bispecific antibody; more preferably a single domain antigen binding site that specifically binds PD-L1 and a Fab fragment that specifically bindsOX 40.

Preferably, the bispecific antibody comprises polypeptide chain 1: VH-CH1-CH2-CH 3-linker-VHH, and polypeptide chain 2: VL-CL; preferably, CDRs 1-3 of the VHH are respectively shown as SEQ ID NO 1-3 in the sequence table, amino acid sequences of CDRs 1-CDR 3 of the VH are respectively shown as SEQ ID NO 4-6, and CDRs 1-CDR 3 of the VL are respectively shown as SEQ ID NO 7-9.

Wherein the linker preferably comprises the amino acid sequence (Gly)₄Ser) n, wherein n is a positive integer equal to or greater than 1, for example, n is a positive integer from 1 to 7, for example, n is 1, 2, 3, 4, 5 or 6.

The amino acid sequence of the VH is preferably shown as SEQ ID NO 10 in the sequence table.

The amino acid sequence of VL is preferably shown as SEQ ID NO. 11 in the sequence list.

The amino acid sequence of the VHH is preferably shown as SEQ ID NO. 12 in the sequence table.

Preferably, thepolypeptide chain 1 comprises the sequence shown as SEQ ID NO. 13 or a variant thereof which retains the function of the sequence; more preferably, the variant has more than 90% identity, preferably more than 95%, more preferably more than 99% identity to the sequence shown in SEQ ID NO 13.

Preferably, thepolypeptide chain 2 comprises the sequence shown as SEQ ID NO. 14 or a variant thereof which retains the function of the sequence; more preferably, the variant has more than 90% identity, preferably more than 95%, more preferably more than 99% identity to the sequence shown in SEQ ID NO. 14.

In a preferred embodiment of the invention, the OX40/PD-L1 bispecific antibody is a fully human bispecific antibody obtained from CHO cell culture; preferably, the OX40/PD-L1 bispecific antibody is more hydrophobic than ipilimumab.

The numbers in this disclosure are approximate, regardless of whether the word "about" or "approximately" is used. The numerical values of the salt concentration and the protein concentration may differ by ± 10%, the numerical value of the pH may differ by ± 0.1, and the numerical value of the conductivity may differ by ± 0.5. Whenever any one of the salt concentration and the protein concentration is disclosed as having N₁Numerical values of any value having N₁The number of +/10% values will be explicitly disclosed, where +/means plus or minus, and N is₁10% to N₁A range between + 10% is also disclosed. For example, a cleaning solution for cationic fillers of 0.5mol/L NaOH has a value of 0.5moI/L +/-10% is also disclosed, while concentrations between 0.5 mol/L-10% and 0.5mol/L + 10% are also within the disclosed ranges, i.e., 0.45-0.55 mol/L and values therebetween, are all within the inclusion range of cationic CIP solutions. Also whenever the numerical values of the numbers shown for pH and conductance are disclosed, respectively, N₂And N₃When simultaneously disclosed is N₂A range of + -0.1 and N₃A range of ± 0.5.

Defining:

the "hydrophobic protein" referred to in the present invention is not particularly limited, and includes any hydrophobic protein that can be purified from cells by using the ion exchange chromatography method of the present invention. In addition, the "hydrophobic protein" does not refer to a specific value or range of hydrophobicity, but refers to any hydrophobicity that renders a target protein insoluble in aqueous solution by binding to a cell structure, or self-association, and allows the protein to be purified by the ion exchange chromatography method of the present invention.

The term "strongly hydrophobic protein" as used herein refers to a protein whose peak time is not earlier than that of Ipiimumab under the chromatographic conditions of HIC-HPLC described in section 1.3 of the test materials and test methods, preferably not earlier than that of OX40/PD-L1 bispecific antibody under the same conditions.

The definition of "antibody" in the present invention refers to any immunoglobulin, complex or fragment form thereof. The term includes, but is not limited to, monoclonal or polyclonal antibodies to IgA, IgD, IgE, IgG, and IgM, including naturally or genetically modified by human origin, chimerism, synthesis, recombination, hybridization, mutation, and the like. In an embodiment of the invention, the antibody may be a monoclonal antibody IgG of fully human origin.

The definition of "monoclonal antibody" or "monoclonal antibody" in the present invention refers to an antibody synthesized by a single effector B cell directed against a particular epitope. It can be murine, chimeric, humanized and fully human monoclonal antibodies, depending on the stage of development.

The definition of "bispecific antibody" or "double antibody" in the present invention refers to an artificial engineered antibody that can simultaneously bind two specific epitopes or target proteins, has the ability to simultaneously bind two different epitopes, and can perform some special biological functions. In embodiments of the invention, the OX40/PD-L1 bispecific antibody can be a fully human monoclonal antibody in which both the variable and constant regions of the antibody are human. OX40 (also known as CD134, TNFRSF4, and ACT35) is a member of the cell surface glycoprotein and Tumor Necrosis Factor (TNF) receptor superfamily, expressed on T lymphocytes and provides costimulatory signals for the proliferation and survival of activated T cells. PD-L1 (also known as differentiation antigen cluster 274(CD274) or B7 homolog 1 (B7-H1)) is a 40kDa type I transmembrane protein. PD-L1 binds to its receptor PD-1 present on activated T cells, down-regulating T cell activation. In embodiments of the invention, the OX40/PD-L1 bispecific antibody comprises a single domain antigen binding site that specifically binds PD-L1 and a Fab fragment that specifically bindsOX 40.

The definition of "CHO cell" in the present invention refers to mammalian Chinese hamster ovary cells, which are the most and most successful cell types for expressing foreign proteins, and are commonly used mammalian host cells. When a recombinant expression vector encoding an antibody gene is introduced into a CHO cell, the host cell can be cultured under appropriate conditions so that it expresses or secretes the antibody into the medium, resulting in a mixture containing the antibody of interest. In embodiments of the invention, a "CHO cell" may be a Chinese hamster ovary cell used to express an OX40/PD-L1 bispecific antibody.

The term "impurity" refers to a substance that is different from the desired antibody product. Contaminants include, but are not limited to: host cell material such as Host Cell Proteins (HCPs) and DNA; a variant, fragment, aggregate or derivative of the desired antibody; cell culture media components.

The term "wash buffer" is used herein to refer to the buffer that flows through the chromatography material after loading the composition and before eluting the bispecific antibody of interest. The wash buffer can be used to remove one or more impurities from the chromatography packing without substantially eluting the desired antibody product.

The term "elution buffer" is used to elute the antibody of interest from the solid phase. Herein, the elution buffer has a higher conductivity or a higher pH relative to the wash buffer such that the desired antibody product is eluted from the chromatography medium.

The term "ion exchange" or "ion exchange chromatography" refers to a chromatographic method for separating solutes using an ion exchanger as a stationary phase based on the difference in electrostatic interaction forces between the dotted solutes and the ion exchanger. Depending on the nature of the ion exchanger, it can be classified as anionicA sub-exchanger and a cation exchanger. The former has exchange capacity to anions, and active groups have positive charges; the latter has an exchange effect on cations, and the active groups are negatively charged. According to the pH range of the ion exchange capacity, the ion exchanger is divided into strong and weak: the strong ion exchanger has the ion exchange function and large pH value range, and the ionization rate is less influenced by pH; the weak ion exchanger has a small pH value range of ion exchange action, and the ionization rate is greatly influenced by pH. In the present embodiment, "cation chromatography" or "cation exchange chromatography" both refer to chromatography performed using a cation exchanger. In the above step, since the antibody has a high isoelectric point and is positively charged in a buffer at a pH lower than the isoelectric point, the cation exchanger can separate the target product by binding different cations in the solution with different strengths. The cation exchanger according to the present invention typically has a sulfonic acid group (SO)₃^2-) Strong cation exchangers of sulfomethyl (S), Sulfopropyl (SP), phosphate (P) and the like and polymers containing Carboxymethyl (CM), Carboxyl (COO)^-) Weak cation exchangers of plasma exchange groups; preferably containing SO₃^2-Or a strong cation exchanger of the group SP or the like, more preferably Capto S Impact orPoros 50 HS.

The term "polymer" is understood to mean a non-covalent association of the same antibody molecule, a molecule formed by the association of two or more antibodies. The antibody may be composed of homogeneous or heterogeneous multiple polypeptides to which a single chain antibody is covalently bound (e.g., disulfide bonds). The dimer is a non-specific binding of two IgG molecules. The formation of multimers is closely related to the distorting influence of antibody structure. For example, high salt, extreme pH induction, and interaction of antibody molecules with filler surface groups can all result in denaturation of the antibody to form multimers. The fraction of the polymer that flows off the SEC analytical column is usually observed as one or more peaks preceding the main peak in the SEC-HPLC chromatogram.

The definition of "loading" in the present invention refers to the operation of feeding the sample to be separated into the chromatography column by means of a sample pump or manually so as to make it contact with the chromatography packing. In embodiments of the invention, loading may be an operation in which an appropriately treated sample of OX40/PD-L1 bispecific antibody is added to a CEX chromatography column.

The definition of 'gradient elution' used in the invention is to program the composition of a mobile phase, such as the polarity, ionic strength, pH value and the like of a solvent in one analysis period, and is used for analyzing complex samples with large component number and large property difference. Gradient elution can reduce analysis time, increase resolution, improve peak shape, increase detection sensitivity, but often causes baseline drift and decreases reproducibility. The gradient elution of the two solvent compositions can be mixed to any degree, i.e. there are a number of elution profiles: linear gradients, concave gradients, convex gradients, and stepped gradients, with linear gradients being most commonly used. In embodiments of the invention, the elution step of cation exchange chromatography may employ a linear gradient based on salt concentration and a linear gradient of pH.

The definition of "isocratic elution" used in the present invention refers to an elution mode in which the composition ratio and flow rate of an elution buffer are constant during the elution process of cation exchange chromatography.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the method can effectively remove HCP and polymers, avoid peak splitting or multi-peak phenomenon of cation chromatography elution peak, and the yield and purity of the collected bispecific antibody are higher and can both reach more than 90% (even can reach more than 99%).

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic structural diagram of an anti-OX 40/PD-L1 bispecific antibody.

FIG. 2A is the linear gradient elution peak of salt for the purification of bispecific antibody using cation exchangechromatography packing POROS 50 HS.

FIG. 2B is a graph of purity of the components of the eluate from cation exchange chromatography and HCP as a function of harvest volume; the cation exchange chromatography packing was POROS 50 HS.

FIG. 3 is a chromatogram of a bispecific antibody purified using cation exchangechromatography packing POROS 50 XS.

FIG. 4 is a graph of the distribution of monomer and polymer content of bispecific antibody in the components of the cation pool.

Figure 5 is a salt linear gradient elution purification bispecific antibody chromatogram of POROS 50HS packing.

Figure 6 is a salt linear gradient elution purification bispecific antibody chromatogram of Capto MMC packing.

FIG. 7 is a graph showing the trend of elution volumes of monomer content and polymer content of fractions collected in stages during elution of bispecific antibody on four different cation exchange chromatography packing materials.

FIG. 8 is a cation chromatography profile in pH linear gradient elution mode.

FIG. 9 is a cation chromatography pattern in pH isocratic gradient elution mode; the elution buffer was 50mM tris-HCl +50mM NaCl, pH7.2, conductivity 8.52 mS/cm.

FIG. 10 is a cation chromatography profile in pH isocratic gradient elution mode; the elution buffer was 25mM tris-HCl +25mM NaCl, pH7.2, conductivity 4.74 mS/cm.

FIG. 11 is a cation chromatogram. Filling: POROS 50HS, linear elution, elution buffer 500mmol/L acetic acid-sodium acetate, pH5.0, 22.3 mS/cm.

FIG. 12 is a cation chromatogram. Filling: capto S Impact, linear elution, 500mmol/L acetic acid-sodium acetate, pH5.0, 22.3 mS/cm.

FIG. 13 is a cation chromatogram. Filling: capto S Impact, isocratic elution, 50mmol/L acetic acid-sodium acetate (pH5.0) in both equilibration buffer and wash buffer, and 325mmol/L acetic acid-sodium acetate (pH5.0) in elution buffer.

FIG. 14 is a cation chromatogram. Filling: capto S Impact, isocratic elution, equilibration buffer and wash buffer were 150mmol/L acetic acid-sodium acetate (pH5.0), and elution buffer was 325mmol/L acetic acid-sodium acetate (pH 5.0).

FIG. 15 is a cation chromatogram. Filling: capto S Impact, linear gradient elution, 50mmol/L acetate-sodium acetate (pH5.0) wash buffer, 500mmol/L acetate-sodium acetate (pH5.0) elution buffer.

Detailed Description

The specific implementation mode of the invention comprises the following steps:

1) the cation sample is subjected to clarification filtration or centrifugal separation on CHO cell culture fluid expressing the bispecific antibody to remove cells and cell debris to obtain clarified liquid;

2) purifying the clarified liquid by Protein A affinity chromatography filler, and collecting affinity eluent;

3) adjusting the pH of the affinity eluent to 4.9-5.5, or adjusting the pH of the affinity eluent to 3.5-3.6 for low-pH inactivation, standing at room temperature for 60-180 min, adjusting the pH to 4.9-5.5, adjusting the conductivity of the samples obtained by the two modes, and filtering the samples by using a 0.22 mu m filter to obtain a cation sample;

in addition, the samples of the affinity eluent with the adjusted pH value and the samples of the low-pH inactivated collecting liquid which are processed by the purification steps of deep adsorption filtration, anion exchange chromatography, hydrophobic chromatography and the like can also be used as cation sample loading samples after pH adjustment and conductivity adjustment.

4) And (3) performing cation exchange chromatography, wherein a cation balancing solution balances 3-5 column volumes of a chromatographic column and then starts to sample, elution is directly started after the sample loading is finished or the elution is started after 2-5 column volumes of a washing buffer solution are washed, and elution components are collected.

In some embodiments, the bispecific antibody of step 1) is characterized by: comprising a single domain antigen binding site that specifically binds PD-L1 and a Fab fragment that specifically bindsOX 40.

In some embodiments, the clarification filtration of step 1) may be performed using commercially available membranes such as 3M, Sartorius, Merck Millipore, PALL, and the like.

In some embodiments, the centrifugation step 1) is performed by centrifuging the CHO cell culture fluid at 4000-9000 g. In the embodiment of the invention, the centrifugation is carried out on a Beckman Avanti J-E centrifuge and is carried out for 10-15 min at 4 ℃.

In some embodiments, the affinity chromatography packing material of step 2) can be MabSelect SureLX (GE), MabSelect prism A (GE), UniMab Protein A (Nami, Suzhou),

AF-rProtein A HC-650F (Tosoh), Amsphere A3(JSR), KanCapA 3G (Kaneka), POROS MabCapture A Select (Thermofisher), Eshmuno A (EMD Merck), Praesto AP (Purolite) filler.

In some embodiments, the reagent used for adjusting pH from low to high in step 3) is 1-2 mol/L Tris, 0.1-1.0 mol/L NaOH, 0.5mol/L dipotassium hydrogen phosphate solution (pH 9.5), 0.2-0.5 mol/L glycine-sodium hydroxide solution (pH 10.5), 1-2 mol/L arginine solution (pH 9.0), or the like; the pH is adjusted from high to low by using 1-3 mol/L acetic acid solution, 1-3M citric acid solution, 0.1-1.0 mol/L HCl, 0.2-0.5 mol/L glycine-hydrochloric acid solution (pH 2.3) and the like.

In some embodiments, the cation sample in step 3) is treated with ultrapure water, cation equilibrium liquid or a mixture of both to adjust the conductivity, and the conductivity is in the range of 4.0-10.0 mS/cm, preferably 4.0-7.5 mS/cm.

In some embodiments, the packing material for cation exchange chromatography in step 4) includes, but is not limited to, Capto S Impact and Capto MMC from GE, POROS 50XS and POROS 50HS from thermodissher, and octogel EMD SO3 from Merck Millipore^-(M) and Eshmuno CPX, Nanogel-50SP, Nami, Suzhou, etc.

Preferably, the cation exchange chromatography medium is equilibrated with an equilibration buffer prior to loading. More preferably, the cation exchange chromatography column is subjected to equilibrium treatment by using 3-5 CV of equilibrium buffer solution.

Preferably, the pH of the equilibration buffer is 4.9-5.5, preferably 5.0. The equilibration buffer includes but is not limited to acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, phosphate buffer, preferably acetic acid-sodium acetate buffer.

In some embodiments, the pH of the wash buffer for the cation exchange chromatography in step 4) is 4.9 to 5.5, preferably 5.0, and the conductivity is 3 to 10mS/cm, preferably 6 to 10 mS/cm. The washing buffer includes but is not limited to acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, phosphate buffer, preferably acetic acid-sodium acetate buffer.

In some embodiments, the pH of the elution buffer for cation exchange chromatography in step 4) is 4.8 to 7.2, preferably 5.0 to 5.5. The elution buffer includes but is not limited to acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, phosphate buffer, preferably acetic acid-sodium acetate buffer.

In some embodiments, the elution of bispecific antibody in step 4) may employ salt linear gradient elution, salt isocratic elution, pH linear gradient elution, pH isocratic elution, and pH and salt dual gradient elution, preferably salt linear gradient elution and salt isocratic elution, more preferably salt linear gradient elution.

In some embodiments, the collection conditions for the eluted sample in step 4) are: and during elution, starting collection when the ultraviolet absorption value at 280nm is increased to 100mAU/mm, and stopping collection when the ultraviolet absorption value at 280nm is reduced to 200-650 mAU/mm.

The water used in the examples of the present invention was ultrapure water having a resistivity of 18.0M Ω · cm (25 ℃).

The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

1.1 bispecific antibody (hereinafter referred to as double antibody)

The diabody is a bispecific antibody which simultaneously binds to PD-L1 and OX40 as disclosed in PCT/CN 2020/073959. The entire contents of the PCT application are hereby incorporated by reference for the purposes of this application.

The antibody subtype of the double antibody is IgG2, the isoelectric point of the antibody is 8.0-8.6, and the molecular weight of the antibody is 174.3 kDa.

The double antibody is an anti-OX 40/PD-L1 bispecific antibody with the structure shown in figure 1, wherein antigen A is OX40, and antigen B is PD-L1.

Wherein, the specific sequence in the double antibody is as follows:

the amino acid sequence of VH-CH1-CH2-CH 3-linker-VHH (i.e., IGN-LP peptide chain #1) in FIG. 1 is shown in SEQ ID NO: 13; the amino acid sequence of the VH ofanti-OX 40 antibody ADI-20057 in FIG. 1 is shown in SEQ ID NO: 10; the amino acid sequence of CH1 in FIG. 1 is shown as SEQ ID NO: 16; the amino acid sequence of Fc (i.e. CH2-CH3) in FIG. 1 is shown as SEQ ID NO: 17; the amino acid sequence of the linker in FIG. 1 is shown as SEQ ID NO 15; the amino acid sequence of VHH of the anti-PD-L1 single domain antibody in FIG. 1 is shown as SEQ ID NO 12; the amino acid sequence of VL-CL (i.e., IGN-LP peptide chain #2) in FIG. 1 is shown in SEQ ID NO: 14; the amino acid sequence of the VL ofanti-OX 40 antibody ADI-20057 in FIG. 1 is shown in SEQ ID NO: 11; the amino acid sequence of CL in FIG. 1 is shown in SEQ ID NO 18.

Further, the amino acid sequences of CDR 1-CDR 3 of the anti-PD-L1 single domain antibody included in the double antibody are respectively shown in SEQ ID NO. 1-3; the amino acid sequences of HCDR 1-HCDR 3 of the anti-OX 40 antibody ADI-20057 contained in the double antibody are respectively shown as SEQ ID NO. 4-6; the amino acid sequences of LCDR 1-LCDR 3 of the anti-OX 40 antibody ADI-20057 contained in the double antibody are respectively shown in SEQ ID NO. 7-9.

In one embodiment, the dual antibody is an anti-OX 40/PD-L1 bispecific antibody recombinantly expressed in 293 cells or CHO cells.

1.2 hydrophobicity validation of double antibodies

The following chromatographic conditions were set:

the hydrophobicity of the OX40/PD-L1 bispecific antibody was tested by HIC-HPLC method. Analysis was performed using a Waters e 2695 or agilent 1260 hplc. Selecting an Iplilimumab antibody to compare the retention time with the hydrophobicity of the sample to be detected, wherein the longer the retention time is, the stronger the protein hydrophobicity is.

Chromatographic analysis conditions: using a MabPacTM HIC-10LC column (manufacturer: Thermo, cat # 088480); mobile phase A: 1.8mol/L ammonium sulfate, 100mmol/L NaH2PO4.2H2O, pH 6.5, mobile phase B: a mixed solution of 90% volume fraction 100mmol/L NaH2PO4.2H2O (pH 6.5) solution and 10% isopropanol; flow rate: 1.0 ml/min; elution procedure: 100% A → 0% A within 0 to 20 min; 100% B within 20-25 min; 100% A within 25-30 min; collecting time: 30 min; sample introduction amount: 10-20 mul; column temperature: 25 ℃; detection wavelength: 280 nm.

As a result, under the chromatographic conditions described above, the peak time of Iplilimumab was about 28 minutes, and the peak time of OX40/PD-L1 bispecific antibody was about 31 minutes.

Example 2

(1) Preparation of cation Loading sample

CHO cell culture fluid expressing OX40/PD-L1 bispecific antibody was centrifuged at 9000g of centrifugal force for 15min using a Beckman Avanti J-E centrifuge, and the supernatant was filtered through a 0.22 μm filter to obtain a clarified solution, which was then subjected to affinity chromatography purification using a MabSelect SuRe LX (GE) packing, the pH of the affinity pool was adjusted to 4.9, the conductivity was adjusted to 4.0mS/cm using ultrapure water, and the supernatant was filtered through a 0.22 μm filter to obtain a cation supernatant. The above cation-loaded sample had a protein content of 10.9g/L, a purity of 96.0% (SEC-HPLC method, all purity data below are determined by this method), and an HCP content of 3232.9 ppm.

(2) Cation exchange chromatography

Merck Millipore filling with high resolution POROS 50HS filler from Thermofisiher

L LaboratoryColumn VL 11X 250 Column, Column height 21.3cm, Column volume 22.6 mL. After the chromatographic column is balanced by 5 Column Volumes (CV) through an equilibrium buffer solution (50mmol/L acetic acid-sodium acetate, pH 4.9), an AKTA pure M purification system (the optical path of an ultraviolet detector is 2mm) is adopted for loading, the loading capacity of a filler is 50g/L, the retention time of the sample during loading is 6min, namely 3.77ml/min, after the sample loading is finished, 3CV (50mmol/L acetic acid-sodium acetate, pH5.5) is used, salt linear gradient elution (0-100% solution B, 20CV) is carried out by using a washing buffer solution and an elution buffer solution (solution B: 50mmol/L acetic acid-sodium acetate, 0.5mol/L NaCl, pH5.5), the collection is started when the ultraviolet absorption value of 280nm wavelength is increased to 100mAU/mm, the collection is carried out by adopting tube separation, 5.0ml is collected by each tube, and the collection is stopped when the ultraviolet absorption value of 280nm wavelength is reduced to 100 mAU/mm. And regenerating 3CV by using a buffer solution containing 1mol/L sodium chloride after the elution is finished, washing for 15-30 min by using 0.5mol/L NaOH solution, and then storing by using 10mmol/L NaOH or 20% ethanol, wherein the regeneration, washing and storage methods in subsequent implementation are the same as those in the embodiment and are not repeated.

(3) Detection and analysis

And detecting the protein content, the purity and the HCP content of the collected liquid in each tube, wherein the protein content is detected by adopting NanoDrop2000, and the HCP content is detected by adopting an ELISA kit purchased from a commercial channel.

As shown in FIG. 2A, the chromatogram of cation elution shows that the elution peak has a double peak, and when the collection is stopped when the elution peak falls to about 400mAU, that is, when the collection is stopped when about 45.2ml (2CV) is collected, the yield is 82.4% and the purity is 95.2%. If the collection stopping point is set at about 1.33CV (corresponding to about 1300mAU of ultraviolet absorption at 280 nm), the yield is 77.1%, the purity is 98.5%, and the yield and the purity are low. The total polymer content of the collected liquid exceeds that of the sample by calculating and stopping the collection from the beginning of collection to the vicinity of 400mAU, which indicates that new polymers are generated in the cation elution process. Purity and HCP changes with elution column volume as shown in figure 2B, the purity of the bispecific antibody undergoes a process of decreasing followed by increasing and then decreasing with increasing salt concentration, resulting in a lower purity of the pool. The HCP content of the fractions of the pool in each fraction of the eluate increased gradually with increasing salt concentration, and most of the HCP was removed when the collection was stopped at around 45.2ml (2 CV).

Example 3

(1) Preparation of cation Loading sample

CHO cell culture fluid expressing OX40/PD-L1 bispecific antibody was filtered through a deep adsorption membrane of Millistak D0HC and X0HC from Merck mileore, purified by affinity chromatography using Protein A packing, and the pH of the affinity collection fluid was adjusted to 5.0 with 2mol/L Tris solution, adjusted to 3.0mS/cm with ultra pure water, and filtered through a 0.22 μm filter to obtain a cation sample. The protein content of the cation sample is 17.0g/L, the purity is 96.9 percent, and the HCP content is 60.7 ppm.

(2) Cation exchange chromatography

Merck Millipore fill with Thermofisiher high-load POROS 50XS filler

L LaboratoryColumn VL 11X 250 Column, Column height 9.5cm, Column volume 10.1 mL. After the chromatographic column is balanced for 5CV through an equilibrium buffer solution (50mmol/L acetic acid-sodium acetate, pH5.0), an AKTA pure 150L purification system (the optical path of an ultraviolet detector is 2mm) is adopted for loading, the loading capacity of the loading is 37g/L filler, the loading flow rate is 1.68ml/min, 3CV of a washing buffer solution (50mmol/L acetic acid-sodium acetate, pH5.0) is adopted after the loading is finished, salt linear gradient elution (0-100% B solution, 10CV) is carried out by using the washing buffer solution and an elution buffer solution (B solution: 50mmol/L acetic acid-sodium acetate, 0.5mol/L NaCl, pH5.0) is carried out, the collection is started when the ultraviolet absorption value at the wavelength of 280nm is increased to 100mAU/mm, the collection is carried out by using a branch pipe, 3.4ml (1/3CV) is collected by each pipe, and the collection is stopped when the ultraviolet absorption value at the wavelength of 280nm is reduced to 100 mAU/mm.

(3) Detection and analysis

The chromatogram of cation elution is shown in fig. 3, the elution curve of the bispecific antibody is similar to that of example 1, elution and collection are started when the ratio of the components of the eluent is 20-30%, and the peak height of the elution peak is double (the peak height of the 2 nd elution peak is lower), and the maximum elution protein concentration is reached at 0.6-0.7 CV. The distribution of the monomer and polymer contents of the bispecific antibody in each component of the cation collecting solution is shown in FIG. 4, and the peaks of the fluctuation of the monomer content and the peaks of the fluctuation of the polymer content overlap, so that the polymer in the cation collecting solution is difficult to remove, the yield and purity of the elution peak at 1CV are respectively 78.2% and 98.1%, and the yield and purity of the elution peak at 1.33CV are respectively 88.4% and 96.9%. The total polymer content of all the pool fractions exceeded that of the sample load, indicating that a new polymer was produced during the cation elution as in example 1.

Example 4

(1) Preparation of cation Loading sample

CHO cell culture fluid expressing OX40/PD-L1 bispecific antibody was filtered through deep adsorption membranes of SUPRAcap HP PDE8 and PDE2 from PALL corporation, then affinity chromatography was performed using MabSelect prism packing, the pH of the affinity collection fluid was adjusted to 6.0 with 2mol/L Tris solution, filtered through a 0.22 μm filter and then stored at 4 ℃ in a refrigerator, adjusted to 5.0 with 2mol/L acetic acid, adjusted to 3.76mS/cm with ultrapure water, and filtered through a 0.22 μm filter to obtain a cation sample. The protein content of the cation sample is 4.4g/L, the purity is 96.8 percent, and the HCP content is 896.8 ppm.

(2) Cation exchange chromatography

Selecting 5 different CEX fillers, POROS 50HS, Capto S Impact, Capto MMC and Factogel EMD SO^3-(M) and Nanogel-50SP were subjected to cation exchange chromatography test, the buffer, operating procedure and collection conditions of cation chromatography were the same as those of example 2, and tube collection was adopted, and the retention time of elution of loaded sample was 6min for 1 tube of 1/3CV collection;

the chromatographic columns all adopt Omnifit 006EZ-06-25-FF columns (the inner diameter is 6.6mm, the length of a column tube is 250mm) of the company Diba, wherein POROS 50HS packing is filled in thechromatographic column 1, the height of the column is 19.2cm, and CV is 6.57 mL; wherein the middle layerThechromatographic column 2 is filled with a Capto S Impact filler, the height of the chromatographic column is 18cm, and the CV is 6.16 mL; wherein thechromatographic column 3 is filled with Factogel EMD SO^3-(M) a packing, column height 19cm, CV 6.5 mL; wherein thechromatographic column 4 is filled with Nanogel-50SP filler, the column height is 20.5cm, and CV is 6.97 mL; the chromatographic column 5 is filled with Capto MMC filler, the height of the column is 19.5cm, and CV is 6.67 mL.

(3) Detection and analysis

The cation chromatogram of POROS 50HS packing is shown in fig. 5, the double peak phenomenon in example 1 and example 2 does not appear in the elution peak, the purity and the yield are high, and when the peak is cut off at the collection volume of 2CV (the ultraviolet absorption value is about 600mAU), the yield is 88.9 percent, and the purity is 98.7 percent. The chromatogram of Capto MMC filler is shown in FIG. 6, the bispecific antibody molecule has strong hydrophobicity, and is firmly combined with the filler, no sample is collected in the elution process, and the bispecific antibody molecule is difficult to elute in the regeneration liquid containing 1mol/L NaCl (pH7.2). Capto S Impact and Factogel EMD SO^3-The chromatography pattern of the (M) packing was similar to that of POROS 50HS packing, while that of Nanogel-50SP packing was similar to that of example 1, and the results of the tests for several packings are shown in Table 1.

TABLE 1 results of testing equal-volume mixtures of the first 2 column volumes of the different cationic packings

For salt linear gradient purification of bispecific antibodies using different packing, the polymer content and monomer content of the fractionated pools as a function of the collection volume are shown in FIG. 7, and the polymer content begins to increase rapidly at 1CV collection volume using POROS 50XS packing, resulting in a lower purity of the first 2CV collection mixtures than the sample load. The purity of the first 2CV pool mixtures was higher with POROS 50HS packing, which is better suited for cationic salt linear gradient elution of bispecific antibodies.

Example 5

(1) Preparation of cation Loading sample

The cell culture fluid was clarified and filtered using Zeta Plus 90ZB05A and Emphaze AEX membranes of 3M, purified by affinity chromatography using MabSelect prism A packing, the pH of the affinity collection fluid was adjusted to 7.2 with 2mol/L Tris solution and then subjected to anion exchange chromatography, the pH of the anion collection fluid was adjusted to 5.17 with 1mol/L dilute hydrochloric acid, the conductivity was adjusted to 3.56mS/cm with ultrapure water, and the mixture was filtered through a 0.22 μ M filter and used as a cation sample. The protein content of the cation sample is 4.4g/L, the purity is 97.1 percent, the HCP content is 9.4ppm, and the conductivity is 3.56 mS/cm.

(2) Cation exchange chromatography

The effect of cation exchange chromatography on bispecific antibody purification was examined using a pH linear gradient elution. The pH gradient elution adopts a filler Factogel EMD SO^3-(M) Omnifit column was packed with 19cm high CV 6.5mL and washed with 0.5mol/L NaOH before use, and specific chromatographic conditions are shown in Table 2 below, using stepwise collection with one tube per 1/3 CV.

TABLE 2 chromatographic conditions for cations using pH Linear gradient elution mode

N/A indicates not applicable

(3) Detection assay

As shown in fig. 8, when the fraction of the eluent reaches 100%, the elution peak has not dropped to 200mAU, and the elution condition of 100% fraction of the eluent is continuously maintained until the preset collection stop condition is reached, the total collection volume is 8.3CV, and the elution force is weak. The conductivity of the equilibrium solution and the eluent is respectively 3.0mS/cm and 6.0mS/cm, the conductivity at the initial collection of the salt gradient elution in theembodiments 1, 2 and 3 is generally between 10 and 15mS/cm, and the increase of the conductivity of the eluent to the interval can improve the elution force. The detection result shows that the polymers are distributed on the front side and the rear side of an elution peak, part of the polymers are eluted from the column at low pH and low conductivity, collected liquids of the 3 rd to 20 th tubes are mixed in equal proportion, and the yield and the purity are respectively 79.6% and 98.6% through detection, and the yield is relatively low.

Example 6

(1) Cationic loading sample preparation

CHO cell culture fluid was filtered through deep adsorption membrane cartridges (Millistak C0HC and X0HC, Merck Millipore Inc.) and then purified by affinity chromatography using MabSelect prism A packing, pH was adjusted back to about 5.0 with 1M Tris, conductivity was adjusted to about 3.9mS/cm by dilution with ultrapure water, and the cell culture fluid was filtered through a 0.22 μ M filter to obtain a cation-loaded sample. The concentration of thesample 1 is 12.8g/L, the pH value is 5.03, and the conductivity is 3.94 mS/cm;sample 2 had a concentration of 14.4g/L, pH 5.02, and conductivity 3.85 mS/cm. Both samples had a purity of 97.4% and an HCP content of 1323.9 ppm.

(2) Cation exchange chromatography

The effect of cation exchange chromatography on bispecific antibody purification was examined using a pH isocratic elution format. POROS adopting cation prepacked column^TM GoPure^TMHS (POROS 50HS packing, inner diameter 12mm, height 5cm, column volume 5.7mL),sample 1 andsample 2 were loaded with 28mL and 25mL, respectively. The equilibration solutions ofsample 1 andsample 2 were 50mmol/L acetate-sodium acetate buffer solution (pH5.0), the washing solutions were 20mmol/L acetate-sodium acetate buffer solution (pH5.0), and the eluents were 50mmol/L Tris-HCl, 50mmol/L NaCl (pH7.2, conductivity 8.52mS/cm) and 25mmol/L NaCl (pH7.2, conductivity 4.74mS/cm), respectively, 10CV was eluted, and the collection conditions and other chromatographic conditions were the same as in example 4.

(3) Detection assay

As shown in fig. 9 and 10, the cation chromatogram has large pH fluctuation after the start of elution, and the bispecific antibody molecules bound to the cation column are not eluted by 10CV of eluent with low conductivity (fig. 10), and although the bispecific antibody molecules are eluted by 10CV of eluent with high conductivity, the elution volume is less than 3CV, and the yield is high, the purity of the antibody molecules is relatively low, the removal effect of HCP is poor, the total volume of the 1 st to 6 th tubes is 2CV, the yield is 93.8%, the purity is 97.1%, and the content of HCP is 371 ppm. Therefore, the effect of purifying the bispecific antibody by adopting a pH isocratic elution mode for POROS 50HS filler purification is poor, and the conductivity of the eluent is between 4.74 and 8.52 mS/cm.

Example 7

(1) Preparation of cation Loading sample

The cell culture fluid was clarified and filtered under the same conditions as in example 2, purified by affinity chromatography as in example 3, and the affinity collection fluid was subjected to pH inactivation treatment (adjustment of the pH of the sample to 3.5, treatment at room temperature for 65min, adjustment of the pH back to 6.0), further subjected to deep adsorption filtration (ADF) treatment, adjustment of the pH of the ADF collection fluid to 5.0, adjustment of the conductivity with ultrapure water, and filtration through a 0.22 μm filter to give a cation loading sample.

(2) Cation exchange chromatography

The purification effect of the cationic filler POROS 50HS under several different conditions was tested using an isocratic elution mode. Test condition 1: the concentration of the sample to be loaded is 7.6g/L, the purity is 97.8 percent, the HCP content is 32.3ppm, the conductivity is 5.92mS/cm, and the loading capacity is 50 g/L. The chromatographic column is aGE XK 26X 40 column (CV is 132.5ml), the linear flow rate during loading and elution is 300cm/h, the equilibrium eluent and the washing eluent are both 50mmol/L acetic acid-sodium acetate buffer solution (pH5.0, conductivity is about 3.0mS/cm), the eluent is 50mmol/L acetic acid-sodium acetate +150mmol/L NaCl (pH5.0, conductivity is about 17.39mS/cm), the elution is 5CV, the ultraviolet absorption value rises to 200mAU, the collection starts, and the ultraviolet absorption value falls to 400mAU and the collection stops.

Test condition 2: the sample was loaded at a concentration of 7.44g/L, pH 5.06, conductivity 3.91mS/cm, purity 97.2%,GE XK 16X 40 column (CV ═ 40ml), and the eluent was 50mmol/L acetic acid-sodium acetate +120mmol/L NaCl (pH5.0, conductivity about 14.75mS/cm), and the other parameters were the same as those intest condition 1.

Test condition 3: the sample concentration of the sample was 13.8g/L, pH 5.04, conductivity 3.17mS/cm, purity 97.2%, HCP content 8.5 ppm. The column was Merck Millipore VL11 column, the linear flow rate was 200cm/h for loading and elution, the eluent was 50mmol/L acetic acid-sodium acetate +110mmol/L NaCl (pH5.0, conductivity about 12.55mS/cm), and 6CV was eluted, otherwise the conditions were the same as those undertest condition 1.

(3) Detection assay

The detection results of the three isocratic elution test conditions are shown in table 3, when the conductivity of the eluent is 17.39mS/cm, the yield is high, but the effect of removing the polymer is poor, the purity of the collected liquid is poorer than that of the sample, and the situation that the bispecific antibody generates a new polymer in the elution process due to relatively strong elution conditions is possible; the yield is reduced when the conductivity is 14.75mS/cm, but the purity is better; the yield is higher at the conductivity of 12.55mS/cm, but the yield is lower, the collection volume is larger, the sample is not eluted from the column after 6 CVs are finished, and if the elution volume is increased, the collection volume is larger, which is not beneficial to the subsequent technological process operation. By combining the analysis, when the filler POROS 50HS adopts isocratic elution to purify the bispecific antibody, the conductivity needs to be controlled between 12.55-14.75 mS/cm.

TABLE 3 comparison of the test results of three isocratic elution test conditions

Example 8

(1) Preparation of cation Loading sample

The cation load was prepared as in example 1, with a load concentration of 8.4g/L, pH 5.07, 4.16mS/cm, 97.2% purity, and an HCP content of 1846.5 ppm.

(2) Cation exchange chromatography

NaCl is generally used in the elution buffer for downstream cation purification, and the previous examples show that in most cases, the elution solution containing strong electrolyte NaCl has not ideal effect no matter linear gradient elution or isocratic elution, and the present example adopts weak acid salt acetic acid-sodium acetate buffer as the only component of the elution buffer solution to examine the purification effect on bispecific antibody.

Test condition 1: capto S Impact packing, VL11 column (CV ═ 23.0ml), loading 70 g/L; test condition 2: POROS 50HS packing, VL11 column (CV 17.4ml), loading 50 g/L. The equilibration and elution solutions for the two test conditions were as in example 6, the elution buffer was 500mmol/L acetic acid-sodium acetate (pH5.0), 10CV was eluted with a linear gradient (0-100%), and the collection conditions and other chromatographic procedures were as in example 6.

(3) Detection assay

The cation chromatograms for both test conditions are shown in fig. 11 and 12, and neither cation elutes a peak after the main peak, with no prominent peaks as in examples 1 and 2, and with a larger elution volume than with the elution buffer containing NaCl. The POROS 50HS packing was used with a broad peak and a large collection volume of about 6 CVs, while the Capto S Impact packing was used under the same collection conditions with a total collection volume of about 3.7 CVs. The detection results of the two salt linear gradient elution test conditions are shown in table 4, the yield and purity of the two fillers are both higher than those of the fillers obtained by linear elution with a salt containing NaCl (as in examples 1, 2 and 3), and when the collection stopping point is set between 700 and 800mAU, the yield of the POROS 50HS filler reaches 92.4 percent, and the purity reaches 99 percent; when the collection stopping point is set between 500 and 600mAU, the yield of the Capto S Impact filler reaches 89.2 percent, and the purity reaches 99.5 percent.

TABLE 4 comparison of test results for two salt linear gradient elution test conditions

Example 9

(1) Preparation of cation Loading sample

Preparation of cation loaded samples as in example 6, sample information obtained from a number of different batch processes is shown in table 5.

TABLE 5 cationic sample information Table

(2) Cation exchange chromatography

The Capto S Impact packing is adopted to test the effect of purifying OX40/PD-L1 bispecific antibody under various chromatographic conditions, the conductivity variation range of eluent is 12.7-17.6 mS/cm, the pH variation range is 4.8-5.5, isocratic elution mode is adopted, and part of cation chromatographic buffer solution and part of test conditions are shown in tables 6 and 7. Except 13# in Table 7, a GE XK26 column (CV is 114.1mL) is used, and VL11 chromatography columns are adopted for the rest, the CV is 17-23.0 mL, and the elution volume number is 5-6 CV.

TABLE 6 cationic chromatography buffer information

Table 7 partial cation chromatography test condition information

(3) Detection and analysis

The detection results of part of test conditions are shown in Table 8, and comparison of the test conditions from #1 to # 3 shows that the conductivity of the eluent is within the range of 13.5-15.5 mS/cm, the yield and purity of the eluted collected liquid are high, and the purity of the eluted collected liquid can reach more than 99.0% when the collection stopping condition is set between 400 and 800 mAU. The conductivity of the eluents oftest condition #1 andtest condition # 3 are relatively close, and the pH oftest condition #1 is lower than that oftest condition # 3, resulting in a lower yield oftest condition # 3. Through comparison oftest conditions 4# to 7#, the conductivity of the eluent is in the range of 13.8-17.3 mS/cm, the purity of the eluted collected liquid is reduced along with the increase of the conductivity, the conductivity is higher in the range of 13.8-14.4, and the effect of removing polymers is better; comparing test conditions # 5 and # 8, it was found that the conductivity of the loaded samples affected elution, and at a conductivity of 10.0mS/cm, the peak did not elute completely at the set 5CV elution, whereas at the same batch of samples, which would have a conductivity of 7.55mS/cm, the peak was normal.

Comparing test conditions 9# and 10# it was found that the low conductivity sample (3.74mS/cm) was applied with different equilibrium and elution conditions, the yield, purity and HCP content of the collected solution were not different, but the peak shape of the elution peak was greatly different, and the chromatograms of test conditions 9# and 10# are shown in FIG. 13 and FIG. 14. At lower conductivity of the equilibration and wash solutions, e.g., 3.0mS/cm, the elution peak "occurred during elution (FIG. 13), and when fractions collected in fractions were run through the same procedure again, the same elution peak" occurred, possibly due to histidine protonation or other reversible changes that caused the molecular conformation of the bispecific antibody during elution. The elution peak shape was similar to that of the other test conditions when the conductivity of the equilibration and wash buffers was increased.

Comparing test conditions 11# and 12#, it is found that the lower pH of the eluent can cause that the antibody molecules can not be combined on the filler and can not be eluted, so that the sample can not be collected, the pH of the eluent is more than 5.2, the conductivity is in the range of 16.6-17.6 mS/cm, the yield is higher, but the effect of removing polymers is hardly generated.

Compared with the test conditions 13# to 15#, the conductivity of the equilibration buffer and the washing buffer within 5.7-10.5 mS/cm has no influence on the purity of an eluted sample, but the yield is slightly higher when the conductivity is about 8.0.

In combination with the above analysis, the conductivity of the sample, the conductivity of the equilibration buffer and the wash buffer, and the conductivity and pH of the eluent have a large influence on the yield and purity of the sample, and should be controlled within a reasonable interval.

TABLE 8 comparison of test results of part of test conditions

Test conditions	Collection Volume (CV)	Yield (%)	Purity (%)	HCP content (ppm)
					1#	2.3	99.9	98.5	2.3
2#	3.3	99.3	98.9	2.4
					3#	2.7	91.2	99.1	3.6
4#	3.0	80.9	98.8	0.9
					5#	3.0	80.1	99.5	1.0
6#	2.7	82.2	98.5	1.5
					7#	2.0	81.4	98.2	1.1
8#	3.6	60.0	99.6	1.0
					9#	2.7	93.3	99.2	Less than 0.2
10#	2.7	93.3	99.2	Less than 0.2
					11#	0	N/A	N/A	N/A
12#	2.2	99.5	92.4	3.1
					13#	4.1	90.6	98.6	9.7
14#	4.1	93.8	98.7	8.9
					15#	4.2	92.5	98.7	7.4

Note: N/A indicates not applicable, no sample was collected during the elution phase under 11# test conditions.

Example 10

(1) Preparation of cation Loading sample

Cation loading sample preparation as in example 6, cation loading sample information: the concentration is 14g/L, the pH is 5.01, the concentration is 6.88mS/cm, and the purity is 97.6%.

(2) Cation exchange chromatography

Using Capto S Impact packing, VL11 column, CV 21.52 mL; the pH value of the elution buffer solution is 5.0-5.4, the conductivity of the eluent is 22.0-27.0 mS/cm, the washing solution is 150mmol/L acetic acid-sodium acetate (pH5.0), and the four eluents respectively correspond to the test conditions of 1# to 4 #: (1)500mmol/L acetic acid-sodium acetate (pH 5.0); (2)475mmol/L acetic acid-sodium acetate (pH5.0); (3)500mmol/L acetic acid-sodium acetate (pH 5.2); (4)525mmol/L acetic acid-sodium acetate (pH 5.4). The loading capacity of the sample is 80g/L, after the sample loading is finished, the washing liquid and the eluent are eluted by adopting a linear gradient of 20-80%, and the elution volume is 10 CV.Test condition 1 the collection conditions were the same as in example 6, andtest conditions 2 to 4 were carried out with the initial collection point being an elution peak ultraviolet absorption value of 200mAU and the final collection point being an elution peak ultraviolet absorption value of 1200 mAU.

(3) Detection assay

The cation chromatogram undertest condition 1 is shown in fig. 15, and the morphology of the falling portion of the elution peak is different from that of the cation chromatogram 12 in example 7, and the collected volume is larger. The yields and purities of the cation collections under the four test conditions are shown in Table 9, with the yield greater than 91.0% and the purity greater than 99.1% when the UV280 value for terminating collection is set between 888-1340 mAU. When the terminated UV280 collection was set at 1200mAU, the comparison of the yield and purity of the four test conditions found thattest condition #2 was the best. The yield of four tests was greater than 90.0% and the purity was greater than 99.0% when the collection of UV280 terminated at 1300mAU stopped. By adopting the method of the embodiment, the parameters of the eluent are in a wider operation range (the conductivity range is 19.0-27.0 mS/cm, the pH value is 5.0-5.4), the yield and the purity of the collecting solution are higher, and the conductivity of the collecting solution is in a range of 13.5-15.5 mS/cm.

TABLE 9 yield and purity of the cation harvest under four test conditions

Example 11

(1) Preparation of cation Loading sample

Cation loading sample preparation as in example 6, cation loading sample information: the concentration was 10.1g/L, pH 5.05, 3.58mS/cm, SEC-HPLC purity 95.5%.

(2) Cation exchange chromatography

Using Capto S Impact packing, VL11 column, CV 21.52 mL; equilibration buffer, wash buffer and eluent a: 50mM acetic acid-sodium acetate, pH 4.93, 2.57 mS/cm; eluent B: (B1)50mM acetic acid-sodium acetate, 500mM KCl, pH5.0, 60.0 mS/cm; (B2)50mM acetic acid-sodium acetate, 250mM Na₂SO₄pH5.0, 63.0 mS/cm; cation exchange chromatography procedure: balancing 5CV, loading 80g/L, washing 3CV, eluting 20CV in linear gradient of 0-100% B, and collectingCollecting 200mAU-1200mAU components, and regenerating 3CV andCIP 3 CV.

(3) Detection assay

The yields of cation pools using eluents B1 and B2 were 95.7% and 99.6%, respectively, and the SEC-HPLC purities were 96.8% and 96.0%, respectively. The above results show that the cation eluent component contains strong electrolyte such as KCl and Na₂SO₄The yield of cation pool was higher when salt linear gradient elution was used, but the polymer removal was not good, similar to the result obtained when the cation eluent composition contained NaCl in the previous example.

Example 12

(1) Preparation of cation Loading sample

Cation loading sample preparation as in example 6, cation loading sample information: the concentration was 8.2g/L, pH 4.95, 6.61mS/cm, SEC-HPLC purity 98.0%.

(2) Cation exchange chromatography

Using Capto S Impact packing, VL11 column, CV 21.52 mL; experiment 1: equilibration buffer, wash buffer and eluent a 1: 60mM histidine hydrochloride, pH 4.96, 4.77 mS/cm; eluent B1: 400mM histidine hydrochloride, pH 5.41, 20.5 mS/cm; experiment 2: equilibration buffer, wash buffer and eluent a 2: 50mM citric acid-sodium citrate, pH5.0, 6.83 mS/cm; eluent B2: 400mM citric acid-sodium citrate, pH5.0, 6.83 mS/cm; cation exchange chromatography procedure: balancing 5CV, loading 80g/L, washing 3CV, eluting 20CV by 0-100% B linear gradient, collecting 200mAU-1200mAU component, regenerating 3CV,CIP 3 CV.

(3) Detection assay

The yields of cation pools using eluents B1 and B2 were 93.5% and 91.8%, respectively, the SEC-HPLC purities were 98.1% and 98.4%, respectively, and the collection volumes were 2.2CV and 2.1CV, respectively. The results show that when the cation eluent contains weak acid salts, such as 400mM histidine hydrochloride and sodium citrate, and the salt linear gradient elution is adopted, the yield of the cation collecting solution is higher, but the removal effect on polymers is not obvious, and the method can be related to the higher purity of the sample. The elution volume is larger than that of an acetic acid-sodium acetate system (about 3CV), which shows that the elution force of the buffer solution system adopting two linear gradient elutions in the case is stronger, the concentration of histidine hydrochloride and sodium citrate in the eluent is reduced or the collection condition is changed, so that the removal capability of the polymer can be improved.

Claims (10)

1. A method for purifying hydrophobic protein by cation exchange chromatography, wherein the chromatography steps comprise equilibration, loading, washing and elution, and the method is characterized in that cation eluent used in the chromatography process does not contain strong electrolyte components, and the conductivity of the cation flushing liquid is higher than that of the loading sample;

the salts used in the cation balancing solution, the washing solution and the eluent comprise weak acid salts and/or phosphate salts.

2. The method of claim 1, wherein the filler used in the cation exchange chromatography comprises a filler selected from the group consisting of sulfonic acid groups (SO)₃^2-) A strong cation exchanger containing a group selected from the group consisting of a sulfomethyl group (S), a sulfopropyl group (SP) and a phosphoric group (P), or a weak cation exchanger containing an ion exchange group selected from the group consisting of a carboxymethyl group (CM) and a carboxyl group (COO-), preferably a strong cation exchanger containing a sulfopropyl group or a sulfomethyl group;

and/or, said cation exchange chromatography employs a bind-elute mode;

and/or the cation balancing solution, the flushing solution and the eluent are acetic acid-acetate buffer solution, citric acid-citrate buffer solution, phosphate buffer solution or histidine buffer solution, preferably acetic acid-acetate buffer solution; preferably, the cation balance liquid, the washing liquid and the eluent do not contain strong electrolyte components;

and/or the elution is salt gradient elution or salt isocratic elution, and the salt gradient elution is preferably salt linear gradient elution.

3. The method according to claim 2, wherein the conductivity of the eluent for salt isocratic elution is 12.55-17.6 mS/cm, and the pH value is 4.8-5.5;

the conductivity of the eluent eluted by the salt linear gradient is 19.0-25.0 mS/cm, and the pH value is 5.0-5.4.

4. The method according to claim 3, wherein when the filler is Capto S Impact, the conductivity of the salt isocratic elution eluate is in the range of 13.5 to 15.5 mS/cm; when the filler is Poros 50HS, the conductivity range of the salt isocratic elution liquid is 12.55-14.75 mS/cm.

5. The method according to claim 1, wherein the pH of the rinsing liquid is 4.9 to 5.5, preferably 5.0; the conductivity is 3.0-10.5 mS/cm, preferably 5.7-10.5 mS/cm, and more preferably 8.0 mS/cm;

and/or the pH value of the equilibrium buffer solution is 4.9-5.5, preferably 5.0; the conductivity is 3.0-10.5 mS/cm, preferably 5.7-10.5 mS/cm, and more preferably 8.0 mS/cm;

and/or the conductivity of the sample is 3.74-10.5 mS/cm, preferably 3.74-7.55 mS/cm.

6. The method of any one of claims 1 to 5, wherein the hydrophobic protein is an antibody, such as a bispecific antibody or a monoclonal antibody; the bispecific antibody is preferably OX40/PD-L1 bispecific antibody; more preferably a single domain antigen binding site that specifically binds PD-L1 and a Fab fragment that specifically binds OX 40.

7. The method of claim 6, wherein the bispecific antibody comprises polypeptide chain 1: VH-CH1-CH2-CH 3-linker-VHH, and polypeptide chain 2: VL-CL; preferably, CDRs 1-3 of the VHH are respectively shown as SEQ ID NO 1-3 in the sequence table, amino acid sequences of CDRs 1-CDR 3 of the VH are respectively shown as SEQ ID NO 4-6, and CDRs 1-CDR 3 of the VL are respectively shown as SEQ ID NO 7-9.

8. The method of claim 7, wherein said linker comprises an amino acid sequence (Gly)₄Ser)n，Wherein n is a positive integer equal to or greater than 1, for example, n is a positive integer of 1-7, for example, n is 1, 2, 3, 4, 5 or 6, and the amino acid sequence of the linker is preferably shown as SEQ ID NO. 15 in the sequence table;

and/or the amino acid sequence of the VH is shown as SEQ ID NO 10 in the sequence table;

and/or the amino acid sequence of the VL is shown as SEQ ID NO. 11 in a sequence table;

and/or the amino acid sequence of the VHH is shown as SEQ ID NO 12 in the sequence table;

and/or the amino acid sequence of CH1 is shown as SEQ ID NO 16 in the sequence table;

and/or the amino acid sequence of CH2-CH3 is shown as SEQ ID NO 17 in the sequence table;

and/or the amino acid sequence of the CL is shown as SEQ ID NO 18 in the sequence table.

9. The method of claim 8, wherein the polypeptide chain 1 comprises the sequence set forth in SEQ ID No. 13 or a variant thereof that retains the function of said sequence; preferably, the variant has more than 90% identity, preferably more than 95%, more preferably more than 99% identity with the sequence shown as SEQ ID NO. 13;

and/or, the polypeptide chain 2 comprises a sequence as shown in SEQ ID NO. 14 or a variant thereof which retains the function of the sequence; preferably, the variant has more than 90% identity, preferably more than 95%, more preferably more than 99% identity to the sequence shown in SEQ ID NO. 14.

10. The method of claim 9, wherein the OX40/PD-L1 bispecific antibody is a fully human bispecific antibody obtained from CHO cell culture;

preferably, the OX40/PD-L1 bispecific antibody is more hydrophobic than ipilimumab.

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