WO2006064373A2 - Methods of purifying immunologlobulins - Google Patents

Abstract

The disclosure provides methods for purifying certain subclasses of immunoglobulins from biological fluids, such as, e.g., serum. The purification process involves the precipitation of non-immunoglobulin proteins with caprylic acid, followed by the precipitation of euglobulin immunoglobulins (e.g., IgM) by desalting. The methods are of use in a large-scale commercial production of various antibodies, e.g., anti-thymocyte antibodies as exemplified in the disclosure. Compositions produced by the methods disclosed as well as uses for such compositions are provided.

Description

METHODS OF PURIFYING IMMUNOLOGLOBULINS

[0001] This application claims priority to United States application No. 60/636,665, filed on December 16, 2004, which is hereby incorporated by reference.

Field of the Invention

[0002] This invention relates to protein production and purification methods and, more specifically, to immunoglobulin purification.

Background of the Invention

[0003] Purification of antibodies from sera and other biological fluids is commonly used in production of antibodies for many therapeutic, diagnostic, and research applications. Numerous methods for purifying immunoglobulins are available. The choice of the purification method depends on various parameters, such as the nature, quality, and quantity of the starting material; the amount and the type of the required antibody; the desired purity and yield; and finally, the cost. For a review of various immunoglobulin purification methods, see, e.g., Josic et al. (2001) Food Technol. Biotechnol., 39(3):215- 226.

[0004] Serum proteins can be roughly divided into globulins and albumin. The latter is the most abundant serum protein. Globulins, in turn, are divided into alpha, beta, and gamma globulins based on their electrophoretic properties. The gamma fraction includes various classes ("isotypes") of immunoglobulins (Ig), which in most animals include IgG, IgM, IgA, IgD, and IgE. IgG and IgA are further divided into subclasses which vary among species. For example, in human, IgG subclasses include IgG₁, IgG₂, IgG₃, and IgG₄; in mouse, IgG subclasses are IgGi, lgG_2a. lgG_2b, and IgG₃; while in rabbit, there is only one subclass of IgG, IgGi. For a detailed discussion on classification of immunoglobulins and their physico-chemical properties, see, e.g., R. Nezlin, The Immunoglobulins: Structure and Function, Academic Press, 1998. [0005] Caprylic acid has been used for immunoglobulin purification due to its ability to bind to the hydrophobic regions of certain serum proteins including albumin. Caprylic acid, CH₃(CH₂)SCOOH, is a fatty acid also known as 1-heptanecarboxylic, octanoic, octoic, and octic acid (see, also, CAS Reg. No. 124-07-2; and United States Patents No. 2,821 ,534 and No. 3,053,869).

[0006] The addition of caprylic acid to serum at an acidic pH causes albumin to precipitate, while leaving most immunoglobulins in solution irrespective of their class. For review of caprylic acid precipitation procedures, see, e.g., Temponi et al. (1989) Hybridoma, 8(1):85-94; Mahanty et al. (1989) Comp. Immun. Microbiol. Infect. Dis., 12(4):153-160; and McKinney et al. (1987) J. Immunol. Meth., 96:271-278; see, also, United States Patents No. 6,307,028 and 5,886,154.

[0007] Since caprylic acid precipitation does not allow purification of specific Ig (sub)classes, further purification steps are necessary to isolate desired Ig (sub)classes. Typically, undesirable Ig are removed by chromatography. However, chromatographic procedures are not optimal as they often lead to loss of desired Ig fraction (reduced yield).

[0008] Therefore, there exists a need for new methods of purifying specific immunoglobulins, particularly, the methods that can be used in a large-scale commercial production of antibodies.

SUMMARY OF THE INVENTION

[0009] The invention is based, in part, on the observation that certain subclasses of immunoglobulins, belonging to the group of blood proteins termed "euglobulins," require salts for solubility in water and, therefore, precipitate under low ionic strength conditions. Examples of euglobulin Ig include IgM, IgGβ, and IgG₂₀ (rat). The aforementioned property of euglobulins has been previously exploited for purification of monoclonal antibodies from ascites fluid by dialysis against demineralized water (see, e.g., Garcia-Gonzalez et al. (1988) J. Immunol. Meth., 111:17-23).

[0010] The present invention provides a method of purifying certain subclasses of immunoglobulins from a biological fluid, such as, e.g., serum or ascites fluid, or a partially purified fraction thereof. In the methods of the invention, the biological fluid or the fraction thereof contains the following components: (1 ) albumin, (2) at least one subclass of non-euglobulin Ig (e.g., IgGi_, gG₂, and IgG₄), (3) at least one subclass of euglobulin Ig (e.g., IgM, IgG₃, and lgG_2c). and optionally, (4) other proteins. [0011] The method of the invention includes:

(a) precipitating albumin from a biological fluid or partially purified fraction thereof by contacting said fluid or fraction with caprylic acid, thereby yielding a first precipitate and a first supernatant;

(b) precipitating euglobulin Ig from the first supernatant by desalting, thereby yielding a second precipitate and a second supernatant; and

(c) recovering non-euglobulin Ig from the second supernatant and/or recovering a euglobulin Ig from the second precipitate. [0012] The methods of the invention are useful, for example, for isolation of immunogen-specific IgG (referred to as "IgG antibodies") from hyperimmune animal sera. The method can also be employed, for example, for separation of monoclonal antibodies from the endogenous contaminating immunoglobulins in the ascites fluid. An application of the methods is illustrated in the Examples, which describe purification of anti-thymocyte IgG antibodies from rabbit hyperimmune serum.

[0013] The biological fluid or the fraction thereof used in the methods of the invention is further characterized in that it contains at least 1 mg/ml endogenous albumin; at least 0.1 mg/ml non-euglobulin Ig; and/or at least 0.01 mg/ml euglobulin Ig. In some embodiments, the partially purified fraction of the biological fluid is a decomplemented and/or heamadsorbed serum.

[0014] The invention further provides compositions produced by the methods of the invention (e.g., anti-thymocyte antibodies) as well as uses for these compositions.

[0015] The foregoing and following descriptions are illustrative and explanatory only and are not restrictive of the invention, as claimed. BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1 is a flow-chart illustrating an Ig purification process according to the methods of the invention.

[0017] Figure 2A shows a Coomassie™-stained gel with lanes as follows: (A) IEF standards; (B) albumin; (C) IgM; (D) commercially available rabbit anti-thymocyte antibody, Thyreoglobulin® (SangStat; batch 1); (E) control rabbit IgG (Sigma); (F) Thymoglobulin® (SangStat; batch 2); (G) rabbit anti-thymocyte IgG antibody purified using caprylic acid and euglobulin precipitations.

[0018] Figure 2B shows a Coomassie™-stained gel with lanes as follows: (A) IEF standards (Bio-Rad, No. 161-0310); (B) rabbit anti-thymocyte IgG purified using caprylic acid and euglobulin precipitations; (C) Tecelac™ (Biotest Pharma, Germany); (D) Fresenius™ (Fresenius AG, Germany)

[0019] Figure 3 shows results of capillary isoelectric focusing as follows: TH091 , commercially available rabbit anti-thymocyte antibody, Thymoglobulin® (SangStat); P6R3, rabbit anti-thymocyte IgG antibody purified using caprylic acid and euglobulin precipitations.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The invention provides a method of purifying certain subclasses of immunoglobulins from a biological fluid or partially purified fraction thereof. As used herein, the term "purified" and its cognates mean "free of others proteins endogenously present in the biological fluid from which the antibody is purified." The purity may be assessed by any suitable method, including, but not limited to, SDS-PAGE, capillary electrophoresis, and HPLC, agarose electrophoresis.

[0021] In the methods of the invention, the biological fluid or fraction thereof contains the following components: (1) albumin, (2) at least one subclass of non-euglobulin Ig (e.g., IgGi, gG₂, and IgG-i), (3) at least one subclass of euglobulin Ig (e.g., IgM, lgG₃, and IgG₂₀), and optionally, (4) other proteins. (If an immunoglobulin class includes only one subclass, the terms "class" and "subclasses" are used interchangeably with respect to that immunoglobulin class.) [0022] Fig. 1 provides a general outline of one embodiment of the Ig purification process according to the methods of the invention. The process of the invention may comprise other steps not shown in the figure; not all of the shown steps are required to practice the invention as claimed.

[0023] Step 1 in Fig. 1 includes obtaining a biological fluid or a partially purified fraction thereof. The biological fluid or the fraction thereof used in the methods of the invention is further characterized in that it contains at least 1 , 5, 10, 20, or 50 mg/ml endogenous albumin; at least 0.1 , 0.3, 0.5, 0.7, 1 , 1.5, 3, or 5 mg/ml non-euglobulin Ig; and/or at least 0.01 , 0.05, 0.1 , 0.3, 0.5, or 1 mg/ml euglobulin Ig. Examples of biological fluids that can be used in the methods of the invention include blood, plasma, serum (including nonimmune and hyperimmune sera), lymphatic fluid, ascites fluid, and milk. Such a biological fluid can be obtained from any mammal, including, e.g., human, monkey, chimpanzee, mouse, rat, rabbit, bovine, sheep, horse, swine, pig, and goat. The fluid may be obtained from an immunized animal or from a transgenic animal which expresses an engineered immunologlublin (e.g., as described in C. Borrebaeck (ed.) Antibody Engineering, 2nd ed., Oxford University Press, 1995.) The illustrative Examples below describe purification of IgG antibodies from hyperimmune anti-thymocyte rabbit serum from rabbit immunized with human thymocytes. Alternatively, hyperimmune serum may be obtained from animals immunized with isolated T cells (e.g., Jurkat cells), B cells, or with another suitable cell type, transfected or untransfected.

[0024] Isolation methods for various biological fluids are well known in the art.

[0025] Steps 2-4 shown in Fig. 1 represent an example of the initial partial purification and conditioning of a biological fluid. In general, partial purification may include, for example, removal of particulate matter and specific impurities, removal/addition of stabilizers, protease inhibitors, detergents, solvents, anti-microbial compounds, etc. For example, a biological fluid used in the methods of the invention may be chromatographically prepurified or subjected to Conn's fractionation. Examples of methods for partial purification are known and are described here and in, e.g., Antibody Purification: Handbook, Amersham Pharmacia Biotech, No. 18-1037-46, edition AA; and J. Curling, Methods of Plasma Protein Fractionation, Academic Press, London, 1980.

[0026] In case of the blood and blood-derived biological fluids, it may be desirable to remove complement. Decomplementatioπ may be accomplished, for example, by heating the fluid to 55-60 °C for 30-60 min. As illustrated in the Examples, the serum can be decomplemented by heating to 57±1⁰C for 40-45 min.

[0027] In case of hyperimmune biological fluids such as, e.g., serum from immunized animals, it may desirable to remove antibodies specific to the undesirable antigenic components. The removal of such components may be accomplished by affinity purification with the undesirable antigen. As illustrated in the Examples, heamadsorbtion may be performed to remove anti-red blood cell antibodies from the rabbit hyperimmune serum obtained from rabbits which were immunized with human thymocytes. If decomplementation is performed, heamadsorbtion may be after decomplementation.

[0028] Partial purification may further include other conditioning steps, such as, e.g., sample concentration and/or dilution in order to adjust salt and/or protein concentration. For example, in some embodiments, before precipitation with caprylic acid, the biological fluid is adjusted to total protein concentration of 1-100, 1-50, 1-20, 5-20, 5-15, or 10 mg/ml by an appropriate dilution/concentration. As illustrated in the Examples, the decomplemented heamadsorbed rabbit serum may be diluted 3-5-fold prior to the caprylic acid precipitation in order to bring the protein concentration to 5-20 mg/ml.

[0029] Conditioning may further include adjustment of the pH prior to caprylic acid purification. The caprylic acid precipitation requires acidic pH (generally, pH < 7). Accordingly, pH of biological fluid may be adjusted with an acidic buffer (e.g., an acetic buffer) prior to the addition of caprylic acid. For example, pH of biological fluid is adjusted to 3.8-4.8, 4.1-4.5, or such that after the addition of caprylic acid, pH of the solution is 3.6-4.6, 4-4.3. As illustrated in the Examples, the decomplemented heamadsorbed serum may be adjusted to pH 4.4 prior to the addition of caprylic acid, or such that after the addition of caprylic acid, pH of the solution is 4.2. [0030] In step 5 shown in Fig. 1, caprylic acid is added to the biological fluid or partially purified fraction thereof at the final concentration of 100-1000, 100-500, 100-300, 100-200, 150-175 mM. The term "caprylic acid," as used herein, also refers to salts thereof, where appropriate. As illustrated in the Examples, caprylic acid may be added to the partially purified, conditioned serum at a final concentration of 160 mM (0.25 % v/v). The addition of caprylic acid yields a precipitate ("first precipitate") containing albumin and other mostly non-immunoglobulin and some immunoglobulin proteins, whereas a substantial fraction of Ig remains soluble in the supernatant ("first supernatant"). Following this procedure, more than 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of endogenous albumin in the biological fluid may be precipitated by caprylic acid.

[0031] In step 6 shown Fig. 1, the soluble Ig fraction is collected. This may be accomplished by conventional techniques, such centrifugation, filtration, etc. As illustrated in the Examples, the precipitate is first pelletted by centrifugation. Then, the supernatant is collected and filtered through an appropriate filter (e.g., 3.0 and 0.22 μm filters as shown in the Examples) in order to remove any remaining insoluble aggregates. Any suitable filter can be used, e.g., filters manufactured by Pall, Inc. (East Hills, NY), Sartorius (Goettingen, Germany), and Millipore (Billerica, MA).

[0032] The soluble protein fraction remaining after caprylic acid precipitation, contains both euglobulin and non-euglobulin Ig. As illustrated in the Examples, after caprylic acid precipitation the soluble fraction contains

[0033] In step 7 shown in Fig. 1 , euglobulin Ig is precipitated by desalting the solution. Desalting may be accomplished by any suitable method, including, but not limited to, conventional dialysis, diafiltration, and size exclusion chromatography (SEC). Desalting methods are well known.

[0034] "Diafiltration" is a dilution of a sample with water or buffer followed by concentration by means of filtration with a size-discriminating filter/membrane. Since most immunoglobulins have molecular weight of at least 150 kDa, generally, any suitable filter with a size cut-off of 150 kDa or less, e.g., 30 kDa, can be used. If desired, the solution may be concentrated prior to diafiltering. [0035] In some embodiments, desalting is accomplished by diafiltration with demineralized water or dialysis against demineralized water. The term "demineralized" refers to "deionized," "pure," or "distilled" water, or otherwise to water of low ionic strength whose electrical conductivity is 10, 5, 1 , 0.5 mS/cm or less. Desalting is carried out at least to a point at which euglobulin precipitates, e.g., until electrical conductivity of the solution being desalted is 15, 12, 10, 7 mS/cm or less. (All conductivity values refer to measurements at room temperature and neutral pH.)

[0036] Following desalting, πon-euglobulin Ig and/or euglobulin Ig₁ are recovered in steps 8 and 9 shown in Fig. 1.

[0037] Non-euglobulin Ig, e.g., Igd, is recovered from the soluble Ig fraction, whereas euglobulin Ig (e.g., IgM and lgG3) may be recovered from the precipitate. The recovery of euglobulin Ig from a precipitate is exemplified in, e.g., Garcia-Gonzalez et al. (1988) J. Immunol. Meth., 111:17-23). The recovery of non-euglobulin Ig from the soluble fraction requires removal of the precipitate which can be accomplished by any conventional techniques, such centrifugation, filtration, etc. For example, the precipitate is first pelletted by centrifugation, and the supernatant is then collected and filtered. The solution may then be subjected to further processing. As illustrated in the Examples, the supernatant containing anti-thymocyte IgGi is subjected to sodium phosphate precipitation of immunoglobulin. Thereafter, anti-thymocyte antibodies may be reconstituted in a desired buffer. Optionally, the antibody product made according to the method of the invention can be sterilized.

[0038] The invention further provides compositions produced by the methods of the invention (e.g., anti-thymocyte antibodies) and uses for these compositions. As seen in Figs. 2A and 2B and described in the Examples, a larger portion of immunoglobulins purified from rabbit anti-thymocyte serum purified according to the methods of the invention, constitutes an acidic fraction (pi < 6.5) which is not observed in commercially available rabbit anti- thymocyte IgG antibody Thymoglobulin® (Fig. 2A, lanes D and F) as well as other anti-thymocyte products including Fresenius™ ( Fig. 2B₁ lane D), ATGam™, porcine ATG, and Tecelac™ (Fig. 2B, lane C). An example of the pi profile of an anti-thymocyte antibody product according to the invention is shown in Fig. 2A, lane G₁ and Fig. 2B, lane D. Accordingly, in some embodiments, the invention provides a novel thymoglobulin composition with the pi profile substantially as shown in lane G of Fig. 2A, lane G, and Fig. 2B, lane B.

[0039] Anti-thymocyte antibodies induce immunosuppression as a result of T-cell depletion and immune modulation (see, e.g., Bonnefoy- Bernard et al. (1991) Transplantation, 51:669-673). Accordingly, the invention further provides a pharmaceutical composition comprising antibodies produced according to the methods of the invention. Acceptable pharmaceutical formulations and excipients are known (see, e.g., 2004 Physicians' Desk Reference® (PDR) (2003) Thomson Healthcare, 58th ed; Gennado et al., (2000), 20th ed., Lippincott, Williams & Wϊlkins) Remington: The Science and Practice of Pharmacy). For example, a pharmaceutical composition may formulated to contain glycine, mannitol, polysorbate 80, and sodium chloride as excipients (e.g., as currently used in the IV-compatible formulation of Thymoglobulin®). The composition may be supplied in solution or as a lyophilized powder.

[0040] Use of the pharmaceutical composition in various immunological and lymphoproliferative disorders and conditions in which inhibition of lymphoblast or lymphocyte (e.g., T cells) growth is desirable. Examples of disorders include acute or chronic rejection of a transplant (e.g., in the treatment of renal transplant acute rejection in conjunction with concomitant with immunosuppression), aplastic anemia, and steroid resistant graft-versus-host disease (GVHD).

[0041] The following examples provide illustrative embodiments of the invention. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the present invention. Such modifications and variations are encompassed within the scope of the invention. The Examples do not in any way limit the invention. EXAMPLES

Example 1: Preparation of anti-thymocyte serum

[0042] Specific Pathogen Free (SPF) rabbits New Zealand/California rabbits were immunized with human thymocytes. Two injections are performed: the first at Day 0 (50 million cells subcutaneously) and the second at Day 14 (50 million cells intravenously). Bleedings were performed at Day 20±1 and Day 22±1 (ear of the animal), and Day 25±1 (intracardiac bleeding).

[0043] Serum from rabbits immunized with human thymocytes was homogenized for 10 minutes; pH was adjusted to 7.0±0.2 with 1 N NaOH and 2N HCI; and the serum was incubated for 40 min at 57±1⁰C and then cooled to 22±3⁰C.

Example 2: Removal of complement and anti-RBC antibodies from the anti-thymocyte serum

[0044] Decomplementation — Serum from rabbits immunized with human thymocytes was homogenized for 10 minutes. The pH was then adjusted to 7.0±0.2 with 1 N NaOH and 2N HCI and the serum was incubated for 40 min at 57±1 "C and then cooled to 22±3⁰C.

[0045] Removal of anti-red blood cell (RBC) antibodies — 115 mg red blood cells were added to 1 g of the decomplemented serum. The solution was incubated at room temperature (RT) for 60±15 min with constant stirring. Following the incubation, the solution was centrifuged at 1337±10g for 5 minutes at 10±2⁰C. The resulting supernatant was collected and subjected to a second haemadsorption. The final supernatant was filtered on a 3 μm membrane (Millipore, No. SSWPO4700).

Example 3: Caprylic acid precipitation

[0046] Three volumes of 60 mM acetate buffer, pH 4.0, were added to one volume of haemadsorbed serum prepared as described in Example 2. The pH of the solution was then adjusted to 4.4±0.1 with 1N NaOH and 2N HCI. Caprylic acid was then added to the diluted serum (25 μl of caprylic acid per one milliliter of the diluted serum). The solution was stirred for 30 min at RT and then centrifuged at 10,000g, also at RT. The supernatant was collected and filtered first through a 3 μm membrane (Millipore, No. SSWPO4700) and then through a 0.22 μm membrane (Millipore, No. CVWPO4700).

Example 4: Removal of IgM

[0047] Ultrafiltration — The solution, produced according to Example 3, was concentrated to half the volume by ultrafiltration with a Biomax® 30 kDa membrane ((Millipore®, No. PXBO30A50)

[0048] Diafiltration — The concentrated solution was then diafϊltered with 12 volumes of demineralized water such that the conductivity of the solution reached 10 mS/cm or lower. Following diafiltration, the solution was centrifuged at 1O₁OOOg for 15 min at RT to remove precipitated IgM, and the supernatant was collected.

Example 5: Recovery of IgG

[0049] Sodium sulfate precipitation — Four mg/ml of sodium phosphate was added to the supernatant prepared as described in Example 4. The solution was stirred for 15 minutes at RT. The pH of the solution was then adjusted to pH 7.4±0.2 with 1 N NaOH and 2N HCI. Thereafter, a 210 g/l solution of sodium sulfate in the amount of 15.66 ml per milliliter of the supernatant was added slowly, with gentle stirring. The solution was incubated for additional 30 min at RT and then centrifuged at 10,000g for 30 min, also at RT.

[0050] Following centhfugation, the supernatant was removed, and the precipitate was washed with 190 g/l sodium sulfate, then stirred for 30 min and centrifuged at 10,000g for 30 min at RT. The supernatant was removed and the pellet was suspended in the 22.5 g/l glycine buffer (pH 6>7±0.2) in the amount of 6.6 ml of the buffer per gram of pellet. The resuspended solution was then filtered through a CUNO™ filter and the sodium sulfate precipitation was repeated.

[0051] At the end of the second sodium sulfate precipitation, the pellet was resuspended in the glycine buffer (10 g/l glycine, 2 g/l NaCI) and filtered through the CUNO™ filter and a 0.22 μm filter. The filtrate was then concentrated using a 10 kDa membrane (Pellicon XL™ filter, 50 cm² Biomax™ membrane (Millipore, No. PXBO10A50). The concentrated solution was diafiltered with 4 volumes of the glycine buffer (10 g/l glycine, 2 g/l NaCI, resistivity 220-340 Ohπrcm, pH 6.0±0.4). The overall IgG yield (the amount of IgG in the final solution vs. in the initial serum) was about 68%.

Example 6A: Isoelectric Focusing

[0052] An aliquot the concentrated anti-thymocyte IgG solution produced according to Example 5, was subjected to isoelectic focusing using pre-cast IEF gels (pH 3-10, Invitrogen, No. EC66555) along with other samples.

[0053] Fig. 2A shows a Coomassie™-stained gel with lanes as follows: (A) IEF standards (Bio-Rad, No. 161-0310); (B) albumin; (C) IgM; (D) commercially available rabbit anti-human thymocyte antibody, Thymoglobulin® (SangStat; batch 1); (E) control rabbit IgG (Sigma); (F) Thymoglobulin® (SangStat; batch 2); (G) rabbit anti-thymocyte IgG purified using caprylic acid and euglobulin precipitations. Fig. 2B shows a Coomassie™-stained gel with lanes as follows: (A) IEF standards (Bio-Rad, No. 161-0310); (B) rabbit anti-thymocyte IgG purified using caprylic acid and euglobulin precipitations; (C) Tecelac™; (D) Fresenius™.

[0054] As seen in Figs. 2A and 2B, a larger portion of all recovered material constitutes an acidic fraction (pi < 6.5) as opposed to commercially available anti-thymocyte products.

Example 6B: Capillary Isoelectric Focusing

[0055] The capillary (Beckman; neutral capillary, 50 μm by 30.2 cm, effective length 20 cm) was washed with 10 mM H₃PO₄ for 1 min at 30 psi. The acid was removed by rinsing the capillary with water for 1 min at 30 psi. The capillary was then filled with a sample containing 2% ampholyte mixture (pH 3 - 10) in a polymer gel for 1.5 min at 30 psi. 91 mM phosphoric acid was used as anolyte, and 20 mM sodium hydroxide was used as catholyte. A 15 kV voltage was applied for 4 min. The voltage then was increased to 21 kV and the isoelectic focusing was performed for 50 min at 0.5 psi at room temperature. Detection was performed using UV detector at 280 nm. [0056] Figure 3 shows the results of capillary isoelectric focusing as follows: TH091 , commercially available rabbit anti-thymocyte antibody, Thymoglobυlin® (SangStat); P6R3, rabbit anti-thymocyte IgG antibody purified using caprylic acid and euglobulin precipitations. The clEF results confirm that a larger portion of all recovered material constitutes an acidic fraction (pi < 6.5) as opposed to commercially available anti-thymocyte products.

[0057] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention. All publications, patents, and biological sequences cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

[0058] Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, treatment conditions, and so forth used in the specification, including claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of purifying immunoglobulin from a biological fluid or a partially purified or conditioned fraction thereof; said fluid or fraction thereof comprising (1) albumin, (2) at least one subclass of non-euglobulin Ig, and (3) at least one class of euglobulin Ig; the method comprising:

(a) precipitating albumin from said fluid or fraction thereof by adding caprylic acid to the fluid or the fraction thereof, thereby yielding a first precipitate and a first supernatant,

(b) precipitating euglobulin Ig from the first supernatant by desalting, thereby yielding a second precipitate and a second supernatant; and

(c) recovering non-euglobulin Ig from the second supernatant and/or recovering a euglobulin Ig from the second precipitate.

2. The method of claim 1 , wherein the biological fluid or fraction thereof contains one or more of:

(a) at least 1 mg/ml endogenous albumin;

(b) at least 0.1 mg/ml non-euglobulin Ig; and

(c) at least 0.01 mg/ml euglobulin Ig.

3. The method of claim 1 , wherein the biological fluid is a from a mammal selected from a group consisting of human, monkey, chimpanzee, mouse, rat, rabbit, bovine, sheep, horse, swine, pig, and goat.

4. The method of claim 1 , wherein the biological fluid is serum or ascites fluid.

5. The method of claim 4, wherein the serum or ascites fluid is hyperimmune. 6. The method of claim 5, wherein the hyperimmune serum is an anti-thymocyte serum.

7. The method of claim 1 , wherein the fraction of the biological fluid is a decomplemented serum.

8. The method of claim 1 , wherein the fraction of the biological fluid is a heamadsorbed serum.

9. The method of claim 1 , wherein the euglobulin Ig is selected from the group consisting of IgM₁ lgG3, and IgG₂₀.

10. The method of claim 1, wherein the non-euglobulin Ig is IgG.

11. The method of claim 10, wherein the IgG is IgG₁.

12. The method of claim 1 , wherein the fraction of the biological fluid is a decomplemented, heamadsorbed rabbit anti-thymocyte serum which is, optionally, diluted.

13. The method of claim 12, wherein the fraction is diluted at least 2-fold.

14. The method of claim 12, wherein the fraction is diluted with an acidic buffer.

15. The method of claim 14, wherein the acidic buffer is acetate buffer.

16. The method of claim 1, wherein caprylic acid is added in step (a) at a final concentration of 50 mM or more.

17. The method of claim 1 , wherein the biological fluid or fraction thereof is brought to pH 3.8 to 4.8 before the addition of caprylic acid. 1'β. The method of claim 1, wherein desalting is achieved by dialysis or diafiltration.

1|9. The method of claim 1 , wherein desalting is achieved by size exclusion chromatography.

20. The method of claim 1 , wherein the conductivity of the solution desalted is 15 mS/cm or less.

21. The method of claim 1 , wherein the method does not comprise chromatographic separation of immunoglobulin classes.

22. The method of claim 1 , the non-euglobulin Ig is recovered from the second supernatant is achieved by precipitation with sodium sulfate.

23. A method of purifying an IgG antibody from serum, the method comprising:

(a) adding caprylic acid to a partially purified or conditioned fraction of the serum with thereby yielding a first precipitate and a first supernatant;

(b) dialyzing the first supernatant against demineralized water thereby yielding a second precipitate and a second supernatant; and

(c) recovering the IgG antibody from the second supernatant.

24. The method of claim 23, wherein the Ig antibody is an anti- thymocyte antibody.

25. A method of purifying an anti-thymocyte antibody from hyperimmune rabbit serum comprising:

(a) partially purifying and conditioning the serum;

(b) adding caprylic acid to the partially purified and conditioned serum of (a), thereby yielding a precipitate and a supernatant; (c) desalting the supernatant of (b) such that the euglobins precipitate, thereby yielding a second precipitate and a second supernatant; and

(d) recovering the anti-thymocyte antibody from the second supernatant.

26. The method of claim 25, wherein the step (a) comprises decomplementation.

27. The method of claim 25, wherein the decomplementation comprises heating the serum to 50 to 60⁰C.

28. The method of claim 25, wherein the step (a) comprises heamadsorbtion.

29. The method of claim 25, wherein the step (a) comprises dilution with an acetate buffer.

30. The method of claim 25, wherein caprylic acid is added at final concentration of 100-200 mM.

31. The method of claim 25, wherein recovering of step (d) comprises sodium sulfate precipitation.

32. The method of claim 25, further comprising preparing pharmaceutical composition comprising the recovered antibody.

33. A composition prepared by the method of any one of preceding claims.

34. The composition of claim 33, wherein the composition comprises an anti-thymocyte antibody. 35. An anti-thymocyte antibody composition having a pi profile substantially as shown in Fig. 2A, lane G, or Fig. 2B, lane B.

36. An anti-thymocyte antibody composition having a pi profile substantially as shown in Fig. 3, graph P6R3.

37. Use of the composition of claim 34, 35, or 36 for treatment of renal transplant acute or chronic rejection of a transplant, aplastic anemia, or steroid resistant graft-versus-host disease.