Production of clinical human IgG products by affinity chromatography

Abstract

An affinity chromatography method is provided for preparing an IgG preparation comprising one or more of IgG1, IgG2, IgG3, and IgG4 from an IgG-containing starting material. The method comprises contacting the IgG-containing starting material with a first chromatography medium comprising a first solid support to which protein A is bound to form a protein A-bound fraction of an IgG subclass having protein A-binding affinity and forming a first flow-through comprising an IgG subclass that does not bind to protein A, and contacting the first flow-through with a first affinity ligand chromatography medium comprising a second solid support to which a first affinity ligand having IgG-binding affinity is bound to form a second flow-through comprising the first affinity ligand-bound IgG subclass and an IgG subclass that does not bind to the first affinity ligand-bound IgG subclass.

Description

[Background technology]

[0001] 2. Background of the Invention Human plasma-derived immunoglobulin products were first used in 1952 to treat immune deficiencies. Initially, the method of choice was to administer IgG intramuscularly or subcutaneously. However, to inject the larger volumes of IgG needed to effectively treat a variety of diseases, intravenously administered products containing lower concentrations of IgG (50 mg / mL) were developed. Typically, intravenous immunoglobulin (IVIG) contains pooled immunoglobulin G (IgG) derived from the plasma of over a thousand blood donors. IVIG, which generally contains >95% unmodified IgG with intact Fc-dependent effector functions and only trace amounts of immunoglobulin A (IgA) or immunoglobulin M (IgM), is a sterile, purified IgG product primarily used to treat three major categories of disease states: 1. immunodeficiencies characterized by low antibody levels, such as X-linked agammaglobulinemia, hypogammaglobulinemia (primary immunodeficiencies), and acquired immunocompromised diseases (secondary immunodeficiencies), 2. inflammatory and autoimmune diseases, and 3. acute infections.

[0002] Many IVIG manufacturers offer a variety of IVIG products. Compared to older freeze-dried IVIG products, which contain only 50 mg / mL of protein in solution after reconstitution, the latest developments offer ready-to-use, sterile, liquid preparations of highly purified and concentrated human IgG antibodies in various volumes and concentrations. Because IgG products such as IVIG are produced from pooled human plasma, pathogen contamination from donor blood (especially viruses known to cause various diseases in humans) is a serious problem in the manufacturing process. Another important consideration for IgG products is the stability during storage, especially as a ready-to-use preparation. Compared to IVIG, subcutaneously administrable immunoglobulin preparations have the advantage of homecare treatment and fewer side effects.

[0003] In the fourth of a series of seminal papers published on the preparation and characterization of serum and plasma proteins, Cohn et al. (J. Am. Chem. Soc., 1946, 68(3):459-475) (Non-Patent Document 1) first described a method for alcohol fractionation of plasma proteins (Method 6) that allowed the isolation of an IgG-rich fraction from human plasma. A few years later, Oncley et al. (J. Am. Chem. Soc., 1949, 71(2):541-550) (Non-Patent Document 2) extended the Cohn method by publishing a method (Method 9) that led to the isolation of a purer IgG preparation.

[0004] Although these methods laid the industry-wide foundation for plasma-derived blood factors, they failed to provide IgG preparations of sufficiently high concentration for the treatment of several immune-related diseases, including Kawasaki syndrome, immune thrombocytopenic purpura, and primary immune deficiencies. Therefore, additional methodologies were developed using various techniques, such as ion exchange chromatography, to provide IgG preparations of higher purity and concentration. Ion exchange chromatography was first employed for this purpose by Hoppe et al. (Munch Med Wochenschr 1967(34):1749-1752) (Non-Patent Document 3) and Falksveden (Swedish Patent No.348942) (Non-Patent Document 4), as well as Falksveden and Lundblad (Methods of Plasma Protein Fractionation 1980) (Non-Patent Document 5).

[0005] Various modern methods use column chromatography plus precipitation steps such as caprylic acid precipitation (Lebing et al., Vox Sang 2003(84):193-201) and Cohn fraction (I+) II+III ethanol precipitation (Tanaka et al., Braz J Med Biol Res 2000(33)37-30). More recently, Teschner et al. (Vox Sang, 2007(92):42-55) described a method for producing a 10% IVIG product that first removes cryoprecipitate from pooled plasma, then performs a modified Cohn-Oncley cold ethanol fractionation, followed by intermediate S / D treatment, ion exchange chromatography, nanofiltration, and optionally ultrafiltration / diafiltration.

[0006] Despite the purity, safety, and yield achieved by these IgG isolation methods, the yield of IgG recovered from plasma can still be improved. For example, Teschner et al. reported that their method increased IgG yield to 65% (Teschner et al., supra). Although an improvement over previously used methods, recovery of this amount of IgG still means that at least about one-third of the IgG present in the pooled plasma fraction is lost during the isolation process.

[0007] Due to the limited supply of plasma available for the production of plasma-derived products, the isolation of several blood proteins from a common starting plasma pool can be achieved by integrating the purification into a single framework. For example, IgG is commonly enriched by the formation of Cohn fraction II+III precipitates or Kistler-Nitschmann precipitate A, and the corresponding supernatants are then used for the production of alpha-1-antitrypsin and albumin. Similarly, several methods have been described for the production of other plasma proteins, such as factor H, from by-products formed during the production of IgG immunoglobulins, including WO2008 / 113589 and WO2011 / 011753.

[0008] Therefore, there is a need for improved and more efficient methods for producing therapeutic IgG products.

[0009] Furthermore, a need remains for methods to obtain highly concentrated IgG preparations suitable for subcutaneous and / or intramuscular administration.

[0010] Most protein purification methods involve some form of chromatography, whereby molecules in a solution (mobile phase) are separated based on differences in their chemical or physical interactions with a solid material (stationary phase). Gel filtration (also called size-exclusion chromatography or SEC) uses a porous resin material to separate (i.e., physically exclude) molecules based on size. In ion-exchange chromatography, molecules are separated according to the strength of their overall ionic interactions (i.e., non-specific interactions) with the solid-phase material. Although affinity chromatography methods are widely used for protein purification, the method has not been deployed for large-scale, industrial-scale purification of plasma proteins such as IgG.

[0011] Affinity chromatography (also called affinity purification) relies on specific binding interactions between molecules. A specific ligand is chemically immobilized or "bound" to a solid support, so that as a complex mixture passes through the column, molecules with specific binding affinity to the ligand will bind. After other sample components are washed away, the bound molecule is stripped from the support, resulting in the molecule being purified from the original sample.

[0012] A variety of affinity ligands useful for protein production and purification are known in the art. Protein A and Protein G are the most commonly used capture proteins in the purification of human antibodies. However, Protein A and Protein G have several drawbacks, such as high cost, low stability, and the possibility of contaminating the product by hydrolysis and release of peptide fragments. In general, affinity resins derived from Protein A and / or Protein G do not effectively bind all IgG subclasses of human IgG fractions. Therefore, there is a need for an efficient and preferably compact method that allows binding of a wider range of human IgG subclasses by combining Protein A or Protein G derived resins with one or more IgG binding resins based on the complementary binding principle.

[0013] The present invention addresses these and other needs by providing an efficient and effective chromatographic method for producing IgG, which in one embodiment avoids the rigorous and time-consuming ethanol fractionation process that is currently standard in the plasma product field. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] WO2008 / 113589 [Patent Document 2] WO2011 / 011753 [Non-patent literature]

[0015] [Non-Patent Document 1] Cohn et al. (J.Am.Chem.Soc.,1946,68(3):459-475) [Non-Patent Document 2] Oncley et al. (J.Am.Chem.Soc.,1949,71(2):541-550) [Non-Patent Document 3] Hoppe et al. (Munch Med Wochenschr 1967(34):1749-1752) [Non-Patent Document 4] Falksveden (Swedish Patent No.348942) [Non-Patent Document 5] Falksveden and Lundblad (Methods of Plasma Protein Fractionation 1980) [Non-Patent Document 6] Lebing et al., Vox Sang 2003(84):193-201 [Non-Patent Document 7] Tanaka et al.,Braz J Med Biol Res 2000(33)37-30 [Non-Patent Document 8] Teschner et al.(Vox Sang,2007(92):42-55) Summary of the Invention

[0016] There remains a need for a more efficient, rapid, high-yield process for purifying IgG from feedstocks. Since individual IgG isoforms are characterized by different affinities for their conjugated epitopes, a method that allows for easy capture and / or purification of one or more isotypes would allow for increased affinity of a particular isotype and its utilization in therapeutic IgG preparations. The present invention provides these and other advantages by providing a new method of performing IgG-specific affinity chromatography on feedstocks containing one or more IgG isoforms.

[0017] Quite surprisingly, the inventors have discovered that by performing two or more affinity capture chromatography steps on a plasma product starting material, e.g., cryo-supernatant, it is possible to rapidly and efficiently produce high-purity (e.g., >80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%) IgG products in which the IgG subtype profile is substantially identical to the plasma IgG subtype distribution, in high yield. Thus, the present invention provides a method for producing IgG preparations with IgG subtype profiles, impurity profiles, and combinations thereof that are essentially identical to currently approved and commercially available IgG preparations, without using classical plasma fractionation techniques (e.g., Cohn fractionation). This unexpected discovery represents a significant advance in the field of isolating IgG fractions from bulk plasma.

[0018] In an exemplary embodiment, an aqueous IgG solution is provided that comprises at least about 99.5% IgG and less than about 0.5% impurities, the impurities comprising factor XI with an activity of less than about 0.0025E / mg total IgG, preferably less than about 0.0020E / mg total IgG, more preferably less than about 0.0015E / mg total IgG, and most preferably less than about 0.0012E / mg total IgG. In a further exemplary embodiment, an aqueous IgG solution is provided that comprises at least about 99.5% IgG and less than about 0.5% impurities, the impurities comprising complement C3 at a level of less than about 3.5 μg / mg total IgG, preferably less than about 3.0 μg / mg total IgG, more preferably less than about 2.5 μg / mg total IgG, and most preferably less than about 2.2 μg / mg total IgG. In an exemplary embodiment, an aqueous IgG solution is provided comprising at least about 99.5% IgG and less than about 0.5% impurities, including complement C3 and factor XI as impurities, wherein complement C3 is present at a level of less than about 3.5 μg / mg total IgG, preferably less than about 3.0 μg / mg total IgG, more preferably less than about 2.5 μg / mg total IgG, and most preferably less than about 2.2 μg / mg total IgG, and factor XI is present with an activity of less than about 0.0025E / mg total IgG, preferably less than about 0.0020E / mg total IgG, more preferably less than about 0.0015E / mg total IgG, and most preferably less than about 0.0012E / mg total IgG. In an exemplary embodiment, an aqueous IgG solution is provided comprising at least about 99.5% IgG and less than about 0.5% impurities, including complement C3 and factor XI as impurities, where complement C3 is present at a level of less than about 2.5 μg / mg total IgG and factor XI is present at an activity of less than about 0.0015 E / mg total IgG.

[0019] One embodiment of the present invention is directed to an affinity chromatography method for preparing an IgG preparation comprising one or more of IgG1, IgG2, IgG3 and IgG4 in any combination from an IgG-containing starting material. In an exemplary embodiment, the method provides an IgG preparation having a predetermined pattern / ratio of IgG subtypes, a predetermined impurity pattern, and combinations thereof. In an exemplary embodiment, the pattern / ratio is essentially identical to the pattern / ratio of plasma IgG subtype distribution.

[0020] What was surprising and unexpected was that the second column, i.e., the column following the Protein A column, bound a significant fraction of IgG subtypes that did not bind to the Protein A column. In an exemplary embodiment, the medium of the second column is an antibody. Exemplary antibodies include those that can bind to the common region of one or more IgG subclasses. The antibody can be a full-length monoclonal antibody, or an antibody fragment (e.g., Fab, Fab2, single chain, camelid antibody fragment, or peptide). In an exemplary embodiment, the column is CaptureSelect™ CH1-XL affinity matrix (ThermoFisher), or a column with similar IgG binding selectivity.

[0021] Another unexpected result of the present invention was the discovery by the inventors that an on-column solvent / detergent viral inactivation (SD_VI) step can be performed on the column-bound protein material without adversely affecting the fidelity of the separation aspect of the process. This discovery further improves the efficiency and compactness of the process of the present invention, especially compared to standard fractionation procedures in the art, by incorporating the SD_VI step directly into an established unit operation, rather than performing it as a separate step on material separated from the chromatographic medium. Indeed, the improved purity of IgG preparations in which an on-column SD_VI step was incorporated into the purification was even more unexpected, since this step was not thought to have any effect beyond viral inactivation. The SD_VI step depletes or removes impurities other than those derived from viruses.

[0022] In an exemplary embodiment, the SD_VI step depletes or removes at least one or more of albumin, apolipoprotein AI, complement C3, haptoglobin, fibrinogen, complement C4-B, complement C1s accessory component, complement C1r accessory component, apolipoprotein B-100, alpha-2-macroglobulin, which in an exemplary embodiment are found in the wash solution from the SD_VI step.

[0023] In various embodiments, the SD_VI step is performed following the IQEQ step, in which the target therapeutic protein (e.g., IgG) is captured on the chromatographic medium and a flow-through is generated, and prior to elution of the target therapeutic protein from the chromatographic medium.

[0024] The exemplary method of the invention can be carried out continuously or as a single unit operation using an array of two or more different IgG-binding affinity resins (tandem arrays) or on a skid allowing for fully automated continuous chromatography. The principles are not limited to IgG but can be applied to purify other proteins, e.g., plasma proteins such as albumin, A1PI, FVIII, etc.

[0025] Exemplary embodiments of the invention described herein provide the advantage that purification of immunoglobulins, such as human IgG fractions, from feedstocks, such as plasma and / or plasma fractions (e.g., cryoprecipitate supernatants), having a wide range of IgG subclass composition is superior to methods using single Protein A or Protein G derived or alternative IgG binding affinity purification, with the advantage that the unit operations required for finalization of the purified IgG product as an administrable drug are significantly reduced due to the high purity of the resulting IgG subclass composition.

[0026] An exemplary method includes (a) contacting the IgG-containing starting material with a first chromatography medium comprising a first solid support to which protein A is bound, thereby forming a protein A bound fraction of an IgG subclass having protein A binding affinity, and forming a first flow-through comprising an IgG subclass not bound to protein A; and (b) contacting the first flow-through with a first affinity ligand chromatography medium comprising a second solid support to which a first affinity ligand having IgG binding affinity is bound, thereby forming a protein A bound fraction of an IgG subclass not bound to protein A. (c) eluting the Protein A bound fraction from the first chromatography medium to form a first eluate and contacting the first eluate with a first affinity ligand chromatography medium to form a second flow-through comprising an IgG subclass that does not bind to the Protein A class; and (d) eluting a member selected from the first affinity ligand binding IgG subclass, the second affinity ligand binding IgG subclass, and combinations thereof from the first affinity ligand column, thereby forming a second eluate.

[0027] An exemplary affinity ligand chromatography medium comprises a VHH antibody fragment.

[0028] In an exemplary embodiment, the method further comprises (e) mixing the first flow-through and the second eluate on the first affinity ligand chromatography medium.

[0029] In an exemplary embodiment, the method includes (f) contacting the second eluate of (d) with a second affinity ligand chromatography medium comprising a third solid support having a second affinity ligand bound thereto, thereby forming a second flow-through comprising the third affinity ligand binding IgG subclass and a fourth IgG subclass that does not bind to the second affinity ligand chromatography medium; and (g) eluting a member selected from the second affinity ligand binding IgG subclass, the fourth IgG subclass, and combinations thereof from the second affinity ligand chromatography medium, thereby forming a third eluate.

[0030] In an exemplary embodiment, the method includes (h) prior to (g), mixing the first flow-through and the second eluate on a second affinity ligand chromatography medium.

[0031] In various embodiments, the method includes, prior to (i)(b), contacting the first chromatographic medium with a buffer / detergent mixture that does not elute the protein A-binding fraction of the IgG subclass having protein A-binding affinity from the first chromatographic medium.

[0032] In some embodiments, the method includes, prior to (j)(g), contacting the second affinity ligand chromatography medium with a buffer / detergent mixture that does not elute the second affinity ligand-bound IgG subclass from the second affinity ligand chromatography medium.

[0033] An exemplary embodiment of the present invention is directed to an affinity chromatography method for preparing an IgG preparation enriched in a first IgG subclass having low protein A binding affinity from a plasma-derived IgG solution depleted of protein A-binding IgG subclasses. The method comprises: (a) contacting the IgG solution depleted of protein A-binding IgG subclasses with a first affinity ligand chromatography medium comprising a first solid support to which a first affinity ligand having IgG-binding affinity is bound, thereby forming a first bound fraction of the first IgG subclass having low protein A binding affinity and a first flow-through; and (b) eluting the first bound fraction of the first IgG subclass having low protein A binding affinity, thereby forming a first eluate comprising the IgG preparation enriched in the first IgG subclass having low protein A binding affinity.

[0034] In an exemplary embodiment, the method further comprises (c) prior to (b), contacting the first bound fraction of the first IgG subclass having low Protein A binding affinity with a buffer / detergent solution and thereafter removing the detergent solution from the first affinity ligand chromatography medium.

[0035] In an exemplary embodiment, the method further comprises: (d) subsequent to (b), contacting the first eluate with a second chromatographic medium comprising a solid support having an affinity ligand attached thereto having affinity for a second IgG subclass with lower Protein A binding affinity, thereby forming a first bound fraction of the second IgG subclass with lower Protein A binding affinity, and (e) eluting the first bound fraction of the second IgG subclass with lower Protein A binding affinity from the second chromatographic medium, thereby forming a second eluate.

[0036] In some embodiments, the method further comprises (f) prior to (e), contacting the first bound fraction of the second IgG subclass having lower Protein A binding affinity with a buffer / detergent solution and then removing the detergent solution from the first chromatographic medium.

[0037] In various embodiments, the method further comprises (g) prior to(a), contacting the plasma-derived IgG solution with a protein A chromatography medium comprising protein A bound to a solid support, thereby forming a protein A binding fraction of an IgG subclass having a high affinity for protein A and an IgG preparation enriched in a first IgG subclass having a low protein A binding affinity.

[0038] In an exemplary embodiment, the method further comprises (h) eluting the Protein A binding fraction of IgG subclasses having high affinity for Protein A from the Protein A chromatography medium, thereby forming a third eluate.

[0039] An exemplary method includes combining two or more of the first eluate, the second eluate, and the third eluate.

[0040] Other aspects and advantages of the present invention will become apparent from the following detailed description, the figures, and the appended claims. [Brief description of the drawings]

[0041] [Figure 1] UV profile of Protein A-bound IgG subcluster tandem column I (Protein A and CH1-XL). [Diagram 2] UV profile overlaid with flow-through (FT) of non-Protein A binding subclasses on IgG Capture Select resin. [Diagram 3]Overlaid UV profile of elution peaks of non-Protein A binding subclasses on IgG Capture Select resin. [Figure 4] This is a chromatogram of IGC02. [Diagram 5] Chromatogram IGC01S. [Figure 6] Chromatogram IGC04. [Figure 7] SDS-PAGE silver stain profile of IGC04 silver stain. 1. Load, cryo supernatant, diluted 1:560. 2. Flow-through, diluted 1:500. 3. WSD, on-column SD_VI, diluted 1:10. 4. Wash 2, diluted 1:5. 5. Wash 3, diluted 1:5. 6. Eluate, diluted 1:100. 7. Eluate, diluted 1:50. 8. Strip, diluted 1:10. NuPAGE 4-12% Bis-Tris Midi Gel 1.0mm. 20 well Catalog No. WG1402BX10, 50 μl of sample was added to non-reducing SDS running buffer LDS-SB:NP0007, 20 μl of iodoacetamide and incubated for 30 minutes at +18-26° C., +50 μl of buffer LDS-SB:NP0007 incubated for 10 minutes at 70° C. Lanes 8 and 9 show the surprisingly high level of purity of the eluate. [Figure 8] Chromatogram of IGC06. Conductivity (red): UV280 (blue) UV254 (purple) pH: green. [Figure 9]SDS-PAGE silver stain profile of IGC06 silver stain. 1. Mol weight marker. 2. Blank. 3. Load, cryo supernatant, diluted 1:560. 4. Flow through, diluted 1:500. 5. WSD, on-column SD_VI, diluted 1:10. 6. Wash 2, diluted 1:5. 7. Wash 3, diluted 1:5. 8. Eluate, IgG fraction diluted 1:100. 9. Eluate, IgG fraction diluted 1:50. 10. Strip, diluted 1:10. NuPAGE 4-12% Bis-Tris Midi Gel 1.0mm. 20 well Cat. No. WG1402BX10. Non-reducing SDS running buffer LDS-SB:NP0007. 50μl sample added to 20μl iodoacetamide and incubated at +18-26°C for 30 minutes. + 50 μl of buffer LDS-SB:NP0007 incubated at 70° C. for 10 min. [Figure 10] FIG. 1 is a model flow diagram for loading test material (10% polyclonal immunoglobulin) onto a Protein A column. [Figure 11] FIG. 11 is a model flow diagram showing the eluate from FIG. 10 being dispensed into separate columns, each containing a separate affinity chromatography medium. The eluate from each of these columns is collected separately. [Figure 12] A model flow diagram showing test material (cryosupernatant) loaded onto a Protein A column. The eluate from this column is loaded onto a column of affinity chromatography media (e.g., CaptureSelect CH1-XL). [Figure 13] FIG. 1 is a model flow diagram showing eluate from Protein A chromatography of cryo-supernatant loaded onto an exemplary affinity ligand chromatography media (CH1-XL). [Figure 14] A model flow diagram showing cryo-supernatants loaded onto a tandem array of Protein A and affinity chromatography media. A wash step is performed on the array after loading the cryo-supernatant. The eluate contains a broad range of IgG subclasses. [Figure 15]Model flow diagram of a three-column array (TRIDEM) of Protein A, CaptureSelect CH1-XL, and CaptureSelect IgG3. Cryoprecipitate was loaded onto the array and a wash step was performed. The eluate contains a broad range of IgG subclasses. [Figure 16] 1 is a schematic of exemplary tandem and tridem chromatography arrays. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description of the Invention A. Introduction Provided herein are exemplary embodiments for purifying one or more IgG isotypes from a feedstock using a process incorporating at least one affinity ligand-based affinity chromatography method. Exemplary IgG preparations of the invention are prepared from an IgG-containing starting material, e.g., cryoprecipitate supernatant, to provide IgG preparations that contain purified immunoglobulins.

[0043] An exemplary method includes: (a) contacting the IgG-containing starting material with a first chromatography medium comprising a first solid support to which protein A is bound, thereby forming a protein A bound fraction of an IgG subclass having protein A binding affinity, and forming a first flow-through comprising an IgG subclass not bound to protein A; and (b) contacting the first flow-through with a first affinity ligand chromatography medium comprising a second solid support to which a first affinity ligand having binding affinity for a selected immunoglobulin is bound, thereby forming a protein A bound fraction of an IgG subclass having protein A binding affinity, and a first flow-through comprising an IgG subclass not bound to protein A. (c) eluting the Protein A bound fraction from the first chromatography medium to form a first eluate and contacting the first eluate with the first affinity ligand chromatography medium to form a second affinity ligand binding IgG subclass and a second flow-through comprising the IgG subclass that does not bind to the ligand; and (d) eluting a member selected from the first affinity ligand binding IgG subclass, the second affinity ligand binding IgG subclass, and combinations thereof from the first affinity ligand column, thereby forming a second eluate.

[0044] Protein A-based chromatographic media do not bind all subclasses of IgG with equal efficiency. In an exemplary embodiment, the present invention provides a method and system for isolating and / or purifying IgG fractions and IgG subtypes from plasma starting material using affinity ligand-based affinity resins. Exemplary resins for use in the present invention include those relatively recently developed for the purification of antibody fusion proteins. These resins bind IgG at different sites (modes) and are therefore used to complement and enhance the use of Protein A resins or to overcome shortcomings of Protein A-based resins.

[0045] In addition, the affinity chromatography-based approach of the present invention can be formatted in continuous or semi-continuous modes to allow for high throughput.

[0046] Exemplary embodiments include dedicated wash steps, and on-column solvent / detergent, viral inactivation steps ("on-column" SD_VI processing), thereby reducing the total number of unit operations compared to traditional IgG fractionation and purification. The on-column SD_VI steps can be performed on any column or multiple columns in the array.

[0047] An exemplary process of the invention includes at least two viral inactivation / depletion steps (eg, a dedicated SD_VI, an acidic hold step, nanofiltration 35 nm).

[0048] In various embodiments, the invention also provides IgG preparations produced using the methods and / or systems of the invention.

[0049] B. Definition Although the terms used herein are believed to be well understood by those of ordinary skill in the art, definitions are set forth herein to facilitate description of the subject matter disclosed herein.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are described herein.

[0051] The terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. The use of the terms "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0052] All references in this disclosure to singular features or limitations are intended to include the corresponding plural feature(s) or limitation(s), and vice versa, unless otherwise stated or clearly indicated to the contrary by the context in which the reference is made.

[0053] As used herein, all combinations of method or process steps can be performed in any order unless otherwise stated or clearly suggested to the contrary by the context in which the referenced combination is made.

[0054] The methods and devices of the present disclosure may include, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, including their components, as well as any additional or optional components or limitations described herein or that are otherwise useful.

[0055] Unless otherwise indicated, all numbers expressing properties such as physical dimensions, amounts of ingredients, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0056] As used herein, a range can be expressed as from "about" one particular value and / or to "about" another particular value. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0057] For clarity, not all of the routine features of the implementations described herein are shown and described. It will be understood that in the development of any such actual implementation, numerous implementation-specific decisions will be made to achieve the developer's particular goals, e.g., compliance with application- and business-related constraints, and that these particular goals will vary from implementation to implementation and from developer to developer. Moreover, it will be understood that such a development effort may be complex and time-consuming, but would nevertheless be a routine undertaking of engineering for those of ordinary skill in the art having the benefit of this disclosure.

[0058] Many modifications and variations of the exemplary embodiments described in this disclosure can be made without departing from the spirit and scope of the exemplary embodiments, as will be apparent to those skilled in the art. The specific exemplary embodiments described herein are provided by way of example only, and the disclosure is intended to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (generally, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc., incorporated herein by reference) as well as chemical methods. In addition, Harlow & Lane, A Laboratory Manual Cold Spring Harbor, NY, refers to standard immunological techniques.

[0060] The term "IgG-containing starting material" as used herein includes plasma, cryo-poor plasma, fractions for plasma fractionation processes, such as Cohn fractions, and recombinant IgG preparations, as well as other feedstock sources for the disclosed processes that contain one or more IgG. In some embodiments, the term refers to a human IgG-containing starting material, although the invention is not limited to the use of human-derived IgG-containing starting materials.

[0061] The term "low protein A binding affinity," as used herein, refers to an Ig species that does not bind to or is easily washed off of a chromatographic medium containing immobilized protein A. Exemplary species with low protein A binding affinity are those species in which the bound fraction is essentially completely eluted from a protein A column at a pH greater than 5, an eluent conductivity greater than about 5 mS / cm, or a combination thereof.

[0062] As used herein, the term "antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or genes, or fragments thereof, that specifically binds and recognizes an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0063] An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The variable light chain (V L ) and variable heavy chain (V H ) refer to each of those light and heavy chains.

[0064] The terms "antibody" and "immunoglobulin" are used interchangeably in some embodiments to refer to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies, e.g., naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains, CHL, CH2 and CH3. In some antibodies, e.g., naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The heavy chain may or may not have a C-terminal lysine. The term "antibody" may include bispecific or multispecific antibodies. An exemplary affinity ligand is an antibody or a fragment thereof.

[0065] Exemplary antibodies (immunoglobulins) include human IgA, IgD, IgG, IgE or IgM. The antibody may be an IgG1, IgG2, IgG3 and IgG4 antibody. As used herein, "IgG" in some embodiments has the structure of a naturally occurring IgG antibody, i.e., has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an IgG antibody may consist of two heavy chains (HC) and two light chains (LC), each of which is linked by the same number and position of disulfide bridges that occur in a naturally occurring Ig antibody (unless the antibody has been mutated to modify the disulfide bridges).

[0066] Immunoglobulins may be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into subclasses in certain species, namely IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. There are several allotypes of immunoglobulins, such as IgG1, that differ from each other by up to a few amino acids. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, namely monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and fully synthetic antibodies.

[0067] Camelid antibodies are sometimes used in affinity purification procedures. A significant proportion of camelid antibodies are single domain antibodies, which interact with their antigens through a single heavy chain binding domain that is devoid of light chains. This domain is also called "VHH" or "VHH antibody" or "VHH domain". Recombinant VHH antibodies present an intact antigen binding domain of minimal size. The absence of a VL domain allows VHH antibodies to achieve greater structural flexibility than VH domains associated with VL domains. Furthermore, the complementarity determining regions (CDRs) of VHHs, especially CDR3, are statistically longer than those of conventional VH-VL antibodies (Muyldermans S., J. Biotechnol., 2001, 74, 277-302).

[0068] An exemplary affinity ligand is a VHH ligand. By "VHH ligand" is meant a single domain heavy chain antibody or antibody fragment derived from Camelidae. In general, VHH ligands have heavy chains derived from immunoglobulins that naturally lack light chains, which combine together to form a multivalent single polypeptide. This polypeptide retains the antigen binding affinity of the whole parent immunoglobulin, but is much smaller in size, making it less immunogenic. VHH ligands are described in detail, for example, in Frenken et al., J. Biotechnol. 78:11-21 (2000), van der Linden et al., Biochem Biophys. Acta. 1431:37-46 (1999), Spinelli et al., Biochemistry 39:1217-1222 (2000), U.S. Patent Application Publication Nos. 20030078402, 2004 / 0142432, and U.S. Patent Nos. 6,399,763 and 6,670,453.

[0069] Exemplary VHH antibodies (and fragments) for use as chromatography affinity ligands in the methods of the invention include those having affinity for at least one isotype of IgG. Exemplary VHH antibodies for use in the methods of the invention are selective for at least a first isotype of IgG over a second isotype.

[0070] The term "antigen-binding portion" of an antibody, fragment thereof (or affinity ligand as used herein) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding function" of an antibody include: (i) a Fab fragment (fragment from papain cleavage) or similar monovalent fragment consisting of the VL, VH, LC, and CH1 domains; (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of the VH domain; (vi) an isolated complementarity determining region (CDR); and (vii) a combination of two or more isolated CDRs, optionally joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0071] As used herein, the term "ultrafiltration (UF)" encompasses a variety of membrane filtration methods in which hydrostatic pressure forces a liquid through a semi-permeable membrane. Water and low molecular mass solutes pass through the membrane while suspended solids and solutes of high molecular mass are retained. This separation process is characterized by the separation of macromolecules (10 3 ~10 6 Ultrafiltration membranes are often used to purify and concentrate solutions (1000 kDa), especially protein solutions. Several ultrafiltration membranes are available, depending on the size of the molecules they retain. Ultrafiltration is typically characterized by membrane pore sizes of 1-1000 kDa and operating pressures of 0.01-10 bar, and is particularly useful for the separation of colloids, such as separating proteins from small molecules like sugars and salts.

[0072] As used herein, the term "diafiltration" is performed with the same membranes as ultrafiltration and is tangential flow filtration. During diafiltration, buffer is introduced into a recycle tank and filtrate is removed from the unit operation. In processes where product is present in excess (e.g., IgG), diafiltration washes components from the product pool into the filtrate, thereby exchanging the buffer and reducing the concentration of undesirable species.

[0073] The term "chromatography" refers to a technique for separating a protein of interest (e.g., an antibody) from other molecules (e.g., contaminants) present in a mixture. Typically, the protein of interest is separated from other molecules (e.g., contaminants) as a result of differences in the speed at which individual molecules of the mixture move in a stationary medium under the influence of a mobile phase, or differences in binding and elution processes. The terms "matrix" or "chromatographic matrix" are used interchangeably herein and refer to any type of adsorbent, resin, or solid phase that separates the protein of interest (e.g., an Fc region-containing protein such as an immunoglobulin) from other molecules present in the mixture in a separation process. Non-limiting examples include particulate, monolithic, or fibrous resins, and membranes that can be placed in columns or cartridges. Examples of materials for forming matrices include polysaccharides (such as agarose and cellulose), as well as other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles, and derivatives of any of the above. Examples of typical matrix types suitable for the disclosed method are cation exchange resins, affinity resins, anion exchange resins, or mixed-mode resins. A "ligand" is a functional group that binds to a chromatography matrix and determines the binding properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity ligands, bioaffinity groups, and mixed-mode groups (combinations of the above). Some preferred ligands that can be used herein include, but are not limited to, strong cation exchange groups such as sulfopropyl, sulfonic acid, strong anion exchange groups such as trimethylammonium chloride, weak cation exchange groups such as carboxylic acid, weak anion exchange groups such as N5N diethylamino or DEAE, hydrophobic interaction groups such as phenyl, butyl, propyl, hexyl, and affinity ligands such as protein A, protein G, protein L, etc.

[0074] The term "affinity chromatography" refers to a protein separation technique in which a protein of interest (e.g., a protein of interest or an Fc region comprising an antibody) specifically binds to a ligand specific for the protein of interest. Such ligands are commonly referred to as affinity or biospecific ligands. In some embodiments, the affinity ligand (e.g., protein A or a functional variant thereof) is covalently attached to a chromatography matrix material and is accessible to the protein of interest in solution when the solution contacts the chromatography matrix. The protein of interest generally retains its specific binding affinity to the affinity ligand during the chromatography step, while other solutes and / or proteins in the mixture do not significantly or specifically bind to the ligand. Binding of the protein of interest to the immobilized ligand allows contaminating proteins or protein impurities to pass through the chromatography matrix while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound protein of interest is then removed in active form from the immobilized ligand under appropriate conditions (e.g., low pH, high pH, ​​high salt, competing ligands, etc.) and passed through the chromatography column with an elution buffer, removing the contaminating proteins or protein impurities that were previously passed through the column. Either component can be used as a ligand for purifying the respective specific binding protein, e.g., an antibody. However, in various methods according to the present disclosure, Protein A, G, A / G and / or L are used as ligands for the Fc region containing target protein. Elution conditions of the target protein (e.g., an Fc region containing protein) from the affinity ligand (e.g., Protein A) can be readily determined by one of skill in the art. In some embodiments, Protein G or Protein L, or functional variants thereof, can be used as the biospecific ligand. In some embodiments, a biospecific ligand such as Protein A is used at a pH range of 5-9 to bind to the protein containing the Fc region, and the biospecific ligand / target protein complex is washed or re-equilibrated, followed by elution with a buffer having a pH of about 4 or less that contains at least one salt.

[0075] The term "affinity ligand" refers to a ligand capable of capturing one or more IgG species. An exemplary affinity ligand is selected from one IgG species in a mixture of species, and a method is provided in which one or more IgG species can be purified from a mixture containing one or more IgG species by use of a column, filter, or other medium containing the affinity ligand. An exemplary affinity ligand is a VHH antibody or a fragment thereof.

[0076] The terms "purifying," "separating," or "isolating," as used interchangeably herein, refer to increasing the purity of a protein of interest from a composition or sample that contains the protein of interest and one or more impurities. Typically, the degree of purity of a protein of interest is increased by removing (completely or partially) at least one impurity from the composition. "Purifying" and its equivalents refer to one or more steps performed to separate a therapeutic protein from one or more other impurities (e.g., bulk impurities) or components present in a fluid (e.g., plasma, Cohn fraction, liquid culture medium proteins, or one or more other components present in or secreted from mammalian cells (e.g., DNA, RNA, other proteins, endotoxins, viruses, etc.)) that contains the therapeutic protein. For example, purification can be performed during or after an initial capture step. Purification can be performed using resins, membranes, or any other solid support that bind either the therapeutic protein or the contaminants (e.g., using affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography, or molecular sieve chromatography). Therapeutic proteins can be purified from fluids containing the therapeutic proteins using at least one chromatography column and / or chromatographic membrane (e.g., any of the chromatography columns or chromatographic membranes described herein).

[0077] For example, immunoglobulins can be purified by removing contaminating non-immunoglobulin proteins. They are also purified by removing immunoglobulins other than IgG. Removal of non-immunoglobulin proteins and / or removal of immunoglobulins other than IgG will increase the percentage of desired IgG in the feedstock. Purity can be measured by standard assays known in the art or described herein, including SDS-PAGE followed by Coomassie Blue staining, and chromatographic methods (e.g., size-exclusion chromatography (SEC) on an HPLC system). The purity of an IgG sample can be calculated, for example, from an SDS PAGE gel after scanning using a Kodak Image Station 1000 or equivalent system, or by analysis of the SEC chromatogram by software on a Shimadzu HPLC system. A sample is considered pure when it is at least 90%, 95%, or 99% free of components other than the desired product (e.g., immunoglobulin).

[0078] Preferably, the method results in an Ig preparation that is at least 80%, 85%, 90%, 95%, or 99% or more pure. "Pure" refers to a preparation of one or more Ig isotypes that is reduced in contaminating non-Ig proteins, or to a preparation of a first Ig isotype that is substantially free of contaminating Igs of a second isotype. In an exemplary embodiment, the invention provides a preparation of an IgG subtype that is enriched for that subtype compared to a preparation of that subtype purified using only Protein A.

[0079] The term "capture" refers to a step performed to partially purify or isolate, optionally concentrate, and optionally stabilize a therapeutic protein (e.g., at least or about 5%, e.g., at least or about 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, at least about 95%, or at least about 99% pure by weight) from one or more other components present in a mixture containing the protein, e.g., IgG-containing starting material. Typically, capture is performed using a resin that binds the therapeutic protein (e.g., by using affinity chromatography). Non-limiting methods for capturing a therapeutic protein from a liquid are described herein and other methods are known in the art. The therapeutic protein may be captured from a liquid medium using at least one chromatography column and / or chromatographic membrane (e.g., any of the chromatography columns and / or chromatographic membranes described herein). In an exemplary embodiment, the invention provides a method for enriching a preparation in a selected Ig isotype or IgG subtype by capturing one or more isotypes and / or subtypes other than those desired to be enriched or enriched on an affinity column (other than Protein A).

[0080] As used herein, the term "buffer" refers to a substance whose presence in a solution increases the amount of acid or alkali that must be added to produce a unit change in pH. A buffer resists changes in pH through the action of its acid-base conjugate components. Buffers for use with biological reagents are generally capable of maintaining a constant hydrogen ion concentration so that the pH of the solution is within the physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases.

[0081] As used herein, the term "chromatography column" or "column" in reference to chromatography refers to a container, often in the form of a cylinder or hollow pillar, filled with a chromatography matrix or resin, which is a material that provides the physical and / or chemical properties used in purification.

[0082] As used herein, the term "flow-through" or "flow-through mode" refers to a general purification approach in which contaminants are removed from a mixture during chromatography because the contaminants are retained by the chromatographic process and are bound to the chromatographic medium, usually in a column. The protein of interest is purified because it does not bind (or binds less strongly than the contaminants) to the chromatographic medium, usually the chromatographic medium in a column, but instead flows out and is collected. "Flow-through" also refers to the liquid collected from this chromatographic mode. After the liquid medium containing the protein of interest passes through the column, impurities bound to the column are optionally "stripped" or removed from the column, so that the column can then be regenerated for another chromatography run. This approach differs from "bind and elute" or "bind and elute mode," in which the target protein of interest is retained on the chromatographic medium in the column and the impurities flow through the column. This process then involves the specific elution of the protein of interest using different column conditions that prevent the binding of the protein of interest to the chromatographic medium, usually the resin in the column.

[0083] The term "polishing" is a term of art and refers to a step carried out to remove residual trace or minor contaminants or impurities from a fluid containing a therapeutic protein close to the final desired purity. For example, polishing can be performed by passing a fluid containing IgG through a chromatography column(s) or membrane absorber(s) that selectively binds either the IgG (the target therapeutic protein) or minor contaminants or impurities present in the fluid containing the target therapeutic protein. In such an example, the eluate / filtrate of the chromatography column(s) or membrane absorber(s) contains the therapeutic protein.

[0084] As used herein, the term "contaminant" or "impurity" is used in the broadest sense to cover any undesirable component or compound in a mixture. Contaminant proteins include, but are not limited to, those naturally produced by the donor or host cells, as well as proteins (e.g., proteolytic fragments) and other process-related contaminants that are related to or derived from the protein of interest. In certain embodiments, the contaminant precipitate is separated from the cell culture using another means, such as centrifugation, sterile filtration, depth filtration, and tangential flow filtration. In various embodiments, the contaminant is an Ig isotype or IgG subtype other than the isotype or IgG subtype desired to be enriched in the formulation by performing the method of the present invention to produce such formulation. Exemplary contaminants that are depleted or removed during the process of the present disclosure include one or more of albumin, apolipoprotein AI, complement C3, haptoglobin, fibrinogen, complement C4-B, complement C1s accessory component, complement C1r accessory component, apolipoprotein B-100, and alpha-2-macroglobulin. In an exemplary embodiment, these contaminants are removed during the on-column solvent / surfactant step.

[0085] The term "loading buffer" refers to a buffer used to prepare and load a mixture or other sample onto a chromatography unit.

[0086] As used herein, "eluate" refers to the fluid exiting a chromatography column or membrane that contains a detectable amount of protein.

[0087] As used herein, a "therapeutic drug substance" refers to a substance comprising a protein (e.g., IgG) that has been sufficiently enriched, purified or separated by the methods of the invention from contaminating proteins, lipids, nucleic acids (e.g., contaminating proteins, lipids, and nucleic acids present in a liquid culture medium or derived from a host cell (e.g., a mammalian, yeast, or bacterial host cell), as well as biological contaminants (e.g., viral and bacterial contaminants). An exemplary therapeutic drug substance can be formulated into a pharmaceutical product without further substantial purification and / or decontamination steps.

[0088] The term "unit operation" refers to a functional step that can be performed in a process of the invention for producing a therapeutic protein drug substance from a feedstock. For example, a unit operation can be filtration (e.g., removal of contaminating bacteria, yeast viruses, or mycobacteria, and / or particulate matter from a fluid containing the therapeutic protein), capture, chromatography, purification, retention or storage, polishing, viral inactivation, adjustment of the ionic concentration and / or pH of a fluid containing the therapeutic protein, and / or removal of unwanted salts.

[0089] In some embodiments, the chromatography system disclosed herein is on a skid.As used herein, "skid" refers to a three-dimensional solid structure that can serve as a platform or carrier for the system described herein.A skid can provide mobility to the system or a part thereof if it includes one or more structures (e.g., wheels, rollers, etc.) that allow it to move.

[0090] The term "multi-column chromatography system" or "MCCS" refers to a system of a total of two or more interconnected or switchable chromatography columns and / or chromatographic membranes. A non-limiting example of a multi-column chromatography system is a cyclic countercurrent chromatography system (PCC) that includes a total of two or more interconnected or switchable chromatography columns and / or chromatographic membranes. Further examples of multi-column chromatography systems are described herein and known in the art. An exemplary MCCS of the present invention includes a Protein A column fluidly coupled to an affinity ligand column, which may itself be fluidly coupled, directly or indirectly, to one or more additional affinity ligand columns.

[0091] In some embodiments, the method and system recovers at least about 40%, 50%, 55%, or 60%, and up to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least about 99% of the amount of protein contained in the feedstock loaded onto the first column, of the desired protein, e.g., IgG or another selected Ig isoform, or IgG subtype, from the feedstock. In some embodiments, the purification recovery is at least about 60% to about 70%. In exemplary embodiments, the purification recovery is at least about 98% or at least about 99% of the amount of protein contained in the feedstock loaded onto the first column. In various embodiments, the IgG purification recovery is at least about 98% or at least about 99% of the amount of IgG contained in the feedstock loaded onto the first column. In exemplary embodiments, the purification recovery of one or more of IgG1, IgG2, IgG3, and / or IgG4 is at least about 98% or at least about 99% of the amount of relevant IgG contained in the feedstock loaded onto the first column.

[0092] As used herein, the term "mixing" refers to the act of dispersing two or more separate compounds or substances in a solution or suspension by any form of agitation. The term "mixing" as used in this application does not require that all components in the solution or suspension be completely evenly distributed as a result.

[0093] As used herein, the term "solvent" encompasses any liquid substance capable of dissolving or dispersing one or more other substances. Solvents can be naturally occurring inorganic substances such as water, or organic liquids such as ethanol, acetone, methyl acetate, ethyl acetate, hexane, petroleum ether, etc. As used in the term "solvent detergent treatment," solvent refers to an organic solvent (e.g., tri-N-butyl phosphate) that is part of the solvent detergent mixture used to inactivate lipid-enveloped viruses in solution. An exemplary solvent / detergent process is performed on a column to remove plasma protein contaminants as described above, in addition to virus-derived contaminants.

[0094] As used herein, the term "surfactant" is used interchangeably with the term "surfactant" or "surface acting agent" in this application. Surfactants are typically organic compounds that are amphiphilic, i.e., contain both hydrophobic groups ("tails") and hydrophilic groups ("heads"), which make them soluble in both organic solvents and water. Surfactants can be classified by the presence of a group in their head that carries a formal charge. Nonionic surfactants have no charged groups in their head, while ionic surfactants carry a net charge in their head. Zwitterionic surfactants contain a head that has two oppositely charged groups.Some examples of common surfactants are anionic (based on sulfate, sulfonate or carboxylate anions): perfluorooctanoic acid (PFOA or PFO), perfluorooctane sulfonate (PFOS), sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfates, sodium laureth sulfate (also known as sodium lauryl ether sulfate or SLES), alkylbenzene sulfonates; cationic (based on quaternary ammonium cations), cetyltrimethylammonium bromide (CTAB) aka hexadecyltrimethylammonium bromide, and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT); long chain fatty acids and their bases: caprylate, caprylic acid, heptanoat, hexanoic acid, heptanoic acid acid), nanoacid, and decanoic acid; zwitterionic (amphoteric): dodecyl betaine, cocamidopropyl betaine, cocoamphoglycinate; nonionic: alkyl poly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) and poly(propylene oxide) (commercially known as Poloxamers or Poloxamines), and alkyl polyglucosides including octyl glucoside, decyl maltoside, fatty alcohols (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates (Tween 20, Tween 80, etc.), Triton detergents, and dodecyl dimethylamine oxide. In an exemplary embodiment, the detergent is combined with a solvent and the combination is utilized to perform a viral inactivation step on a protein preparation in the methods of the invention. In various embodiments, this viral inactivation step is carried out while the preparation of the invention is on the chromatography column utilized in the method of the invention.

[0095] As used herein, "Protein A" refers to an affinity ligand that is a 49 kDa surface protein found naturally in the cell wall of the bacterium Staphylococcus aureus. The protein binds immunoglobulins and contains five homologous Ig-binding domains. Affinity chromatography media that include immobilized Protein A are known in the art. As will be appreciated by those of skill in the art, in variations of the methods disclosed herein, other affinity ligands, such as Protein G, Protein A / G, and Protein L, can be utilized in place of or in addition to Protein A and are considered to be within the scope of the present invention. Portions of the present disclosure that explicitly disclose Protein A also relate to embodiments in which one or more of Protein G, Protein A / G, and / or Protein L are utilized in place of or in addition to Protein A.

[0096] As used herein, the term "not significantly eluting" refers to the properties of an aqueous solution when a protein immobilized on a chromatography medium is contacted with an aqueous solution, such as a buffer, solvent / detergent, or other mixture, i.e., the solution does not elute any protein or only a very small amount of protein is eluted from the chromatography medium. In exemplary embodiments, less than about 5%, less than about 3%, less than about 1%, or less than about 0.1% of the protein is eluted from the chromatography medium by the solution. An exemplary solution that does not significantly elute the protein is a solvent / detergent solution. An exemplary protein that is not eluted from the chromatography medium by a solvent / detergent solution is IgG. In exemplary embodiments, "not significantly eluting" means that less than about 5% of the IgG immobilized on the chromatography medium is eluted from the chromatography medium by contact with the solvent / detergent solution.

[0097] As used herein, a table entry marked with an "x" indicates that the associated component has not been analyzed.

[0098] C. Abbreviation BDS, bulk drug substance; CRS, cryo-supernatant (supernatant after first precipitation in the COHN fraction); CV, column volume; DFM, data file management (protocol system); E, eluate fraction; EDTA, ethylenediaminetetraacetic acid; EtOH, ethanol; Fc, antibody FC domain; FT, flow-through; HPLC, high performance liquid chromatography; IgG, immunogamma globulin; IgG1, IgG2, IgG3, IgG4, immunogamma globulin subclasses 1 to 4; IQEQ, equilibration buffer (an exemplary IQEQ allows for further purification of plasma proteins from the flow-through of one or more affinity columns after absorption of IgG) ; e.g., 10 mM Na acetate, 10 mM Tris HCl, 120 mM NaCl, pH 7.2); MAB, monoclonal antibody; MWCO, molecular weight cut-off; SD_VI, solvent detergent-viral inactivation, e.g., SD_VI_MIX Triton X 10.55 g / Polysorbate 80 3.21 g / TnBP 2.91 g / kg buffer containing 10 mM Na acetate, 10 mM Tris HCl, 120 mM NaCl); SEC, size exclusion chromatography; TnBP, tri-N-butyl phosphate; vWF, von Willebrand factor; W, wash fraction; WB, Western blot; WSD, wash solvent / detergent.

[0099] C. Embodiment In an exemplary embodiment, the invention is directed to an affinity chromatography method for preparing an IgG preparation comprising one or more of IgG1, IgG2, IgG3 and IgG4 from a feedstock, e.g., an IgG-containing starting material, the method comprising: (a) contacting the feedstock with a first chromatography medium comprising a first solid support to which protein A is bound, thereby forming a protein A-bound fraction of an IgG subclass having protein A-binding affinity, and forming a first flow-through comprising an IgG subclass not bound to protein A; and (b) contacting the first flow-through with a first affinity ligand chromatography medium comprising a second solid support to which a first affinity ligand having IgG-binding affinity is bound, thereby forming a protein A-bound fraction of an IgG subclass not bound to protein A. (c) eluting the Protein A bound fraction from the first chromatography medium to form a first eluate and contacting the first eluate with a first affinity ligand chromatography medium to form a second flow-through comprising an IgG subclass that does not bind to Protein A; and (d) eluting a member selected from the first affinity ligand binding IgG subclass, the second affinity ligand binding IgG subclass, and combinations thereof from the first affinity ligand column, thereby forming a second eluate.

[0100] In an exemplary embodiment, the method further comprises (e) mixing the first flow-through and the second eluate on the first affinity ligand chromatography medium.

[0101] In an exemplary embodiment, the method includes (f) contacting the second eluate of (d) with a second affinity ligand chromatography medium comprising a third solid support having a second affinity ligand bound thereto, thereby forming a second flow-through comprising the third affinity ligand binding IgG subclass and a fourth IgG subclass that does not bind to the second affinity ligand chromatography medium; and (g) eluting a member selected from the second affinity ligand binding IgG subclass, the fourth IgG subclass, and combinations thereof from the second affinity ligand chromatography medium, thereby forming a third eluate.

[0102] In an exemplary embodiment, the method includes (h) prior to (g), mixing the first flow-through and the second eluate on a second affinity ligand chromatography medium.

[0103] In various embodiments, the method includes, prior to (i)(b), contacting the first chromatographic medium with a buffer / detergent mixture that does not elute the protein A-binding fraction of the IgG subclass having protein A-binding affinity from the first chromatographic medium.

[0104] In some embodiments, the method includes, prior to (j)(g), contacting the second affinity ligand chromatography medium with a buffer / detergent mixture that does not elute the second affinity ligand-bound IgG subclass from the second affinity ligand chromatography medium.

[0105] Exemplary embodiments include dedicated wash steps and on-column solvent / detergent, viral inactivation steps ("on-column" SD_VI processing), thereby reducing the total number of unit operations compared to traditional IgG fractionation and purification. The on-column SD_VI step can be performed on any column or multiple columns in the array. In various embodiments, on-column SD_VI further improves the efficiency and compactness of the inventive process, especially compared to standard fractionation procedures in the art, by incorporating the SD_VI step directly into established unit operations rather than performing it as a separate step on material separated from the chromatographic media. Indeed, the improved purity of IgG preparations in which the on-column SD_VI step was incorporated into the purification was even more unexpected, since this step was not expected to have any effect beyond viral inactivation. The SD_VI step depletes or removes impurities other than those derived from viruses.

[0106] In various embodiments, the SD_VI step is performed following the IQEQ step, in which the target therapeutic protein (e.g., IgG) is captured on the chromatographic medium and a flow-through is generated, and prior to elution of the target therapeutic protein from the chromatographic medium.

[0107] In an exemplary embodiment, the SD_VI step depletes or removes at least one of albumin, apolipoprotein AI, complement C3, haptoglobin, fibrinogen, complement C4-B, complement C1s accessory component, complement C1r accessory component, apolipoprotein B-100, alpha-2-macroglobulin, which in an exemplary embodiment are found in the wash solution from the SD_VI step.

[0108] In an exemplary embodiment, the buffer / detergent mixture includes acetate, Tris, Triton X, tri(n-butyl) phosphate (TnBP).

[0109] In an exemplary embodiment, the buffer / detergent mixture comprises a buffer component having about 10 mM sodium acetate, about 10 mM TrisHCl, and about 120 mM NaCl, and a surfactant component having about 10.55 g Triton X, 3.21 g Polysorbate 80, and about 2.91 g TnBP per kg of buffer solution.

[0110] The methods and systems of the present invention provide preparations with significantly increased IgG content compared to the feedstock.

[0111] In an exemplary embodiment, the IgG in the formulation is at least about 90% wt / wt of the protein content of the IgG formulations of the invention.

[0112] In an exemplary embodiment, the IgG1 in the formulation is about 40% to about 90% wt / wt of the protein content of the formulation.

[0113] In an exemplary embodiment, the IgG2 in the formulation is about 15% to about 55% wt / wt of the protein content of the formulation.

[0114] In an exemplary embodiment, the IgG3 in the formulation is from about 0.5% to about 50% wt / wt of the protein content of the formulation.

[0115] In an exemplary embodiment, the IgG4 in the formulation is from about 0.1% to about 20% wt / wt of the protein content of the formulation.

[0116] Exemplary methods of the invention provide highly pure IgG preparations. In various embodiments, the invention provides highly pure preparations of one isoform of IgG.

[0117] In exemplary embodiments, the albumin in the formulation is in an amount of less than about 5 g per L of the formulation, e.g., less than about 2 g, less than about 1 g, less than about 0.4 g, less than about 0.2 g, less than about 0.1 g, or less than about 0.05 g.

[0118] In an exemplary embodiment, the activity of Factor XI in the formulation is less than about 0.01 E / mLt, such as less than about 0.005 E / mLt, such as less than about 0.001 E / mLt.

[0119] In an exemplary embodiment, alpha-2-macroglobulin is present in the formulation in an amount less than about 0.2 g, e.g., less than about 0.1 g, e.g., less than about 0.05 g per L of the formulation.

[0120] In exemplary embodiments, transferrin is present in the formulation in an amount less than about 0.4 g, less than about 0.2 g, e.g., less than about 0.09 g per liter of the formulation.

[0121] In an exemplary embodiment, the formulation has a PL1 activity of less than about 10 nm / mL min.

[0122] In exemplary embodiments, fibrinogen is present in the formulation in an amount of less than about 60 μg, less than about 35 μg, e.g., less than about 13 μg per mL of the formulation.

[0123] In an exemplary embodiment, the IgG-containing starting material is a cryosupernatant.

[0124] In an exemplary embodiment, each column contains a member selected from a first chromatography medium comprising a first solid support having protein A, a first affinity ligand chromatography medium comprising a second solid support having a first affinity ligand bound thereto having IgG binding affinity, and combinations thereof.

[0125] In an exemplary embodiment, the invention provides a system and a method of using the system, in which two or more of the chromatography columns containing protein A, one or more of the affinity ligand chromatography media, and combinations thereof are in fluid communication in a column array, with two or more of the columns containing a second chromatography medium and a second affinity ligand chromatography medium. The columns are optionally fluidly coupled by fluid inlets, fluid outlets, and tubing connecting one or more outlets from one or more columns to one or more inlets of one or more columns. The columns in the array can be in fluid communication in series, parallel, or a combination of these configurations.

[0126] In an exemplary embodiment, the outlet of the first column is in fluid communication with the inlet of the second column, such that the eluate from the first column can be loaded directly into the inlet of the second column and thus onto the chromatographic medium contained therein.

[0127] In an exemplary embodiment, the outlet of the column bearing protein A is in fluid communication with the inlet of the first affinity ligand chromatography medium.

[0128] In an exemplary embodiment, the outlet of the column containing the second chromatographic medium to which the first affinity ligand is bound is in fluid communication with the inlet of the column containing the second affinity ligand chromatographic medium.

[0129] In various embodiments, the outlet of the first column is in switchable communication with the inlet of one or more second columns. In an exemplary embodiment, the outlet(s) of the one or more first columns is in switchable communication with the inlet(s) of one or more second columns.

[0130] In an exemplary embodiment, the Protein A-binding IgG subclass depleted IgG solution comprises a member selected from IgG1, IgG2, IgG3, IgG4, and combinations thereof.

[0131] In another exemplary embodiment, the first chromatographic medium has selective affinity for the CH1 domain of human IgG antibodies.

[0132] In an exemplary embodiment, the first chromatographic medium has a selective affinity for the CH1 domain of a member selected from IgG1 and IgG3 that is greater than its selective affinity for the CH1 domain of a member selected from IgG2 and IgG4.

[0133] In an exemplary embodiment, eluting from the first chromatography medium, the first affinity ligand chromatography medium, and / or the second affinity ligand chromatography medium utilizes an eluent that includes glycine.

[0134] In an exemplary embodiment, the glycine concentration is about 50 mM to about 150 mM. In an exemplary embodiment, the glycine concentration is about 130 mM.

[0135] Other eluents that may be used in the present invention include, but are not limited to, citrate, acetate, MES, HCl at a pH below 4.0, and combinations thereof.

[0136] An exemplary embodiment of the invention is directed to an affinity chromatography method for preparing an IgG preparation enriched in at least a first IgG subclass with low protein A binding affinity from a plasma-derived IgG solution depleted of protein A-binding IgG subclasses. The method comprises: (a) contacting the IgG solution depleted of protein A-binding IgG subclasses with a first affinity ligand chromatography medium comprising a first solid support to which a first affinity ligand having IgG-binding affinity is bound, thereby forming a first bound fraction of the first IgG subclass with low protein A binding affinity and a first flow-through; and (b) eluting the first bound fraction of the first IgG subclass with low protein A binding affinity, thereby forming a first eluate comprising the IgG preparation enriched in the first IgG subclass with low protein A binding affinity.

[0137] An exemplary derived IgG solution depleted of protein A-binding IgG subclasses is prepared by passing the IgG-containing starting material over a chromatography medium containing protein A, allowing the IgG subclass(es) with protein A affinity to bind, and collecting the IgG subclasses that do not have sufficient protein A affinity to remain bound to protein A during this process, typically in the flow-through from the chromatography medium.

[0138] In an exemplary embodiment, the method further comprises (c) prior to (b), contacting the first bound fraction of the first IgG subclass having low Protein A binding affinity with a buffer / detergent solution and thereafter removing the detergent solution from the first affinity ligand chromatography medium.

[0139] In an exemplary embodiment, the method further comprises: (d) subsequent to (b), contacting the first eluate with a second chromatographic medium comprising a solid support having an affinity ligand attached thereto having affinity for a second IgG subclass with lower Protein A binding affinity, thereby forming a first bound fraction of the second IgG subclass with lower Protein A binding affinity, and (e) eluting the first bound fraction of the second IgG subclass with lower Protein A binding affinity from the second chromatographic medium, thereby forming a second eluate.

[0140] In some embodiments, the method further comprises (f) prior to (e), contacting the first bound fraction of the second IgG subclass having lower Protein A binding affinity with a buffer / detergent solution and then removing the detergent solution from the first chromatographic medium.

[0141] In various embodiments, the method further comprises (g) prior to(a), contacting the plasma-derived IgG solution with a protein A chromatography medium comprising protein A bound to a solid support, thereby forming a protein A binding fraction of an IgG subclass having a high affinity for protein A and an IgG preparation enriched in a first IgG subclass having a low protein A binding affinity.

[0142] In an exemplary embodiment, the method further comprises (h) eluting the Protein A binding fraction of IgG subclasses having high affinity for Protein A from the Protein A chromatography medium, thereby forming a third eluate.

[0143] An exemplary method includes combining two or more of the first eluate, the second eluate, and the third eluate.

[0144] Exemplary methods of the invention use two, three, or more columns in series. The starting protein mixture is applied to the inlet of the first column (e.g., protein A), then a preselected amount of wash solution is passed through the column to remove unbound IgG and impurities, and in some embodiments, the protein population in the mixture is passed through the first column and advanced to one or more of the subsequent columns. The flow out of the terminal column is optionally collected. Following the wash cycle, an eluent (in some embodiments, a slightly acidic eluent) is passed through the inlet of the first column, and the protein population bound to the chromatographic medium is eluted into the second column, and the protein population bound to the chromatographic medium in the second column is eluted through the end of the device, and in some embodiments, into a third column packed with a third chromatographic medium, and so on. In the elution step, the protein population bound to the chromatographic medium (e.g., the second affinity ligand medium) of the first column is desorbed from the medium, sent to the second column, and bound to the second chromatographic medium of the second column. The same applies to proteins adsorbed to the second medium, which are desorbed and sent to the third column in embodiments where a third column is present, or optionally collected at the end of the second column in embodiments where a third column is not present, or collected at the end of the third column, and the above process is optionally repeated for the third and subsequent columns.

[0145] In exemplary embodiments, the flow-through and / or eluate from each column or subset of columns may be collected separately or pooled before being loaded onto a subsequent column. Also within the scope of the invention are embodiments in which a combination of columns in series and one or more individual column(s) are utilized.

[0146] Also provided herein is an IgG formulation prepared by the method of the invention. In an exemplary embodiment, the formulation has an IgG subtype profile essentially identical to that of human plasma. In an exemplary embodiment, the formulation has an impurity profile essentially identical to that of human plasma. In an exemplary embodiment, the formulation has both an IgG subtype profile and an impurity profile essentially identical to that of a commercially available polyclonal IgG formulation, e.g., Gammgard liquid, Kiovig, Cuvitru.

[0147] The aqueous IgG solutions according to the invention may be further processed by one or more ion exchange steps and by formulation into a final pharma- ceutically acceptable formulation.

[0148] In an exemplary embodiment, an aqueous IgG solution is provided comprising at least about 99.5% IgG and less than about 0.5% impurities, including Factor XI with an activity of less than about 0.0025E / mg total IgG, preferably less than about 0.0020E / mg total IgG, more preferably less than about 0.0015E / mg total IgG, and most preferably less than about 0.0012E / mg total IgG.

[0149] In an exemplary embodiment, an aqueous IgG solution is provided that contains at least about 99.5% IgG and less than about 0.5% impurities, including complement C3 at a level of less than about 3.5 μg / mg total IgG, preferably less than about 3.0 μg / mg total IgG, more preferably less than about 2.5 μg / mg total IgG, and most preferably less than about 2.2 μg / mg total IgG.

[0150] In an exemplary embodiment, an aqueous IgG solution is provided comprising at least about 99.5% IgG and less than about 0.5% impurities, including complement C3 and factor XI as impurities, wherein complement C3 is present at a level of less than about 3.5 μg / mg total IgG, preferably less than about 3.0 μg / mg total IgG, more preferably less than about 2.5 μg / mg total IgG, and most preferably less than about 2.2 μg / mg total IgG, and factor XI is present with an activity of less than about 0.0025E / mg total IgG, preferably less than about 0.0020E / mg total IgG, more preferably less than about 0.0015E / mg total IgG, and most preferably less than about 0.0012E / mg total IgG. In an exemplary embodiment, an aqueous IgG solution is provided comprising at least about 99.5% IgG and less than about 0.5% impurities, including complement C3 and factor XI as impurities, where complement C3 is present at a level of less than about 2.5 μg / mg total IgG and factor XI is present at an activity of less than about 0.0015 E / mg total IgG.

[0151] The following examples are provided to illustrate certain exemplary embodiments of the invention and should not be construed as limiting the scope of the invention in any way. Affinity purification methods were evaluated for their ability to develop next-generation human IgG products that are comparable to or better than currently available IgG products. To date, affinity purification has not been used on a large scale to replace fractionation to purify IgG or other proteins from IgG-containing starting materials. EXAMPLES

[0152] Example 1 1.1 Experimental Overview Initial experiments were performed using purified plasma-derived polyclonal IgG as starting material, with the aim to rapidly gather information on benchmarking defined IgG products already in clinical use.

[0153] Within the first screen, several potential candidates for a second affinity purification step were evaluated in parallel as independent chromatographic runs. Additionally, a tandem approach was carried out as described below. 1. Capture of the Major Part of IgG by Protein A-Derived Resin 2. Capture residual IgG present in the Protein A capture flow-through (FT) with an alternative IgG-binding affinity resin

[0154] In a next step, the resin identified as providing the best binding properties (capacity) was selected for further experimental setup, in which the capture of IgG from cryo-supernatants was evaluated.

[0155] The first series of experiments was designed to screen combinations of Protein A and resin in a "tandem" array to capture the full spectrum of IgG subclasses. As starting material, a liquid purified plasma-derived polyclonal IgG preparation was used.

[0156] A second series of experiments was designed to directly optimize the IgG subclass spectrum using modified Cohn cryosupernatant as the starting material.

[0157] 1.2 Biological materials 1. Purified plasma-derived polyclonal IgG 2. Cryosupernatant

[0158] 1.3 Buffers All buffers used in the experiments were prepared in the business department. Buffers were stored at +2 to +8°C.

[0159] 1.4 Resin Selection For direct capture, MabSelect Prism A (GE Healthcare) was chosen due to its high resistance to sodium hydroxide.

[0160] Cryosupernatant contains all blood coagulation factors except FVIII and vWF, which are reduced after cryoprecipitation, a fact that can cause partial coagulation and precipitation on the resin and thus necessitates efficient regeneration and washing procedures.

[0161] The resin chosen for capture of the remaining IgG subpopulations is a commercially available chromatography resin provided by Thermo Fisher.

[0162] [Table 1]

[0163] 1.5 Evaluation of single unit operations / clusters

[0164] [Table 2]

[0165] 1.6 Chromatography scheme of Phase 1 (PrismA affinity) A chromatography column of the following dimensions was used for the Protein A capture step: Column: 3.2cm h=7.5cm A=8.04cm 2 V=60.3ml

[0166] [Table 3]

[0167] 1.7 Screening Phase / Experimental Results Affinity of purified IgG-containing starting material to MAbSelectPrismA Chromatography runs on the Protein A capture column were performed as previously described in the Phase 1 chromatography scheme, with a calculated column loading of 30 mg IgG per ml of resin.

[0168] The flow-through UV profile is shown in Figure 1. The low absorbance indicates high binding efficiency of human IgG in this experimental setup using IgG-containing starting material.

[0169] Flow-through fraction (UV280 signal) run of PrismA using IgG-containing starting material.

[0170] This flow-through fraction is collected and used as the load for further screening of affinity resins with different specificities for immunoglobulin subpopulations, as described in resin selection.

[0171] 1.8 IgG Content and Subclass Distribution of Load and Flow-Through (FT) Fractions of MabSelect Prism A Resin

[0172] [Table 4]

[0173] Example 2 2.1 Chromatography scheme for Phase 2 (resin screening) The following chromatography columns were used for the resin screening runs with Protein A capture flow-through: Column: 10cm h=3~4cm A=0.79cm 2 V=3.5~4.5ml

[0174] The chromatographic procedures for these runs are listed in Chromatographic Procedures for the Resin Screening Runs.

[0175] 2.2 Chromatographic procedures for resin screening runs

[0176] [Table 5]

[0177] 2.3 CaptureSelect Flow-Through Evaluation UV 280 nm Signal Figure 2 shows the flow-through profiles of the screened resins. Different UV280 signal heights were observed, for example with anti-Lamba or anti-CH1-XL. The lower the flow-through UV level, the higher the UV elution peak.

[0178] 2.4 CaptureSelect Dissolution Evaluation UV 280 nm Signal Figure 3 shows the elution profiles of the screened resins. Different UV280 signal heights and areas were observed. CaptureSelect CH1-XL shows the highest peak and the largest peak area.

[0179] Based on these data, the CaptureSelect resins were ranked: the most efficient resin was CaptureSelect CH1-XL.

[0180] 2.5 Data Evaluation a. Summary of peak areas of CaptureSelect affinity elution of PrismA flow-through of purified IgG-containing starting material. Table 6 shows the peak areas (UV280nm) of the different affinity columns tested. The results of the analysis of Protein A resin are listed below the IgG content and subclass distribution of the load and flow-through (FT) fractions of the MabSelect Prism A resin. IgG2 and IgG4 are captured almost quantitatively, while IgG1 and IgG3 are captured less efficiently. These subtypes are captured by the second resin. CaptureSelect CH1-XL was confirmed to bind the majority of the remaining IgG subspecies from the MabSelect PrismA flow-through. This resin was introduced as the second column and the following experiments were performed.

[0181] [Table 6]

[0182] Example 3 3.1 Evaluation of CaptureSelect resin for binding of remaining IgG subclasses in MabSelect PrismA flow-through The following table summarizes the recovery of IgG1 and IgG3 from the flow-through fractions of MabSelect Prism A using different CaptureSelect columns. Only the flow-through and elution fractions are listed. Some may be in the flow-through fractions (below detection limit) and in post-wash and elution fractions (not analyzed).

[0183] [Table 7]

[0184] [Table 8]

[0185] [Table 9]

[0186] [Table 10]

[0187] [Table 11]

[0188] [Table 12]

[0189] Example 4 4.1 Variant IGC02 a. Buffer composition used in IGC02

[0190] [Table 13]

[0191] 4.2 IGC02 execution IGC02 was performed in two steps: first, affinity was run on MABSelect PrismA and, as a second step, the MABSelect PrismA flow-through was applied to CaptureSelect CH1_XL.

[0192] Cryosupernatant from the Cohn fractionation process was introduced into the purification process without any further treatment prior to the experiment.

[0193] a. First step MAB-SelectPrismA (GE Healthcare, Catalog number: 17-5498-02, ID: 3.2cm h=7.5cm A=8.04cm 2 V = 60.3 ml) was equilibrated with IQEQ buffer and then the cryo-supernatant was applied to the column with a contact time of approximately 6 min.

[0194] After loading, a first wash with 2 CV of buffer IQEQ was performed to remove unbound proteins from the resin (added to the flow-through). SD_VI on-column treatment was performed by applying IQEQ_VI buffer containing a surfactant composition of 10.55 g Triton X-100, 3.21 g Polysorbate 80, and 2.91 g TnBP in 1 kg IQEQ buffer (corresponding to 1% / 0.3% / 0.3% Triton X100 / Polysorbate 80 / TnBP). SD_VI on-column was performed by contacting the bound proteins with IQEQ_VI buffer at a flow rate of 4 ml / min within 4 CV for at least about 60 min. The wash is optionally collected for analysis.

[0195] Wash 2 was performed with IQEQ buffer to further deplete impurities and wash out the SD_VI reagent at 10 CV and a flow rate of 10 ml / min. The wash is optionally collected for analysis.

[0196] Elution was performed by applying 5 CV of E1 buffer (100 mM glycine, pH 3.2). The first step (strip) of the washing procedure was carried out with E2 buffer (100 mM glycine, pH 2.7).

[0197] After re-equilibration with 4 CV of IQEQ buffer, the column was regenerated and stored.

[0198] For playback and saving procedures, please refer to the playback procedures.

[0199] Example 5 5.1 Columns MABSelectPrismA GE Healthcare, Catalog Number: 17-5498-02 Lot number: 10264190 ID:3.2cm h=7.5cm A=8.04cm 2 V=60.3ml (target)

[0200] [Table 14]

[0201] a. Chromatogram IGC02 Please refer to Figure 4.

[0202] 5.1 Second step IGC01S CaptureSelect CH1-XL (Thermo Fisher, Catalog Number: 2943452050, ID: 1.0cm h=3.8cm A=0.79cm 2 V = 3.0 ml) was equilibrated with IQEQ buffer, after which the MABSelect Prism A flow-through (IGC02) was applied to the column with a contact time of approximately 10 min.

[0203] After loading, a first wash with 5 CV of buffer IQEQ was performed to remove unbound protein from the resin (added to the flow-through). SD_VI on column was performed by applying IQEQ_VI buffer containing a surfactant composition of 10.55 g Triton X-100, 3.21 g Polysorbate 80, and 2.91 g TnBP in 1 kg IQEQ buffer. SD_VI on column was performed by contacting the bound protein with IQEQ_VI buffer within 10 CV (100 min) at a flow rate of 0.3 ml / min for a minimum of 60 min.

[0204] Wash 2 was performed using IQEQ buffer to further remove impurities and wash out the SD_VI reagent at 20 CV and a flow rate of 0.8 ml / min.

[0205] Elution was performed by applying 10 CV of E2 buffer (100 mM glycine, pH 2.7).

[0206] After re-equilibration with 10 CV of IQEQ buffer, the column was regenerated and stored.

[0207] For playback and saving procedures, please refer to the playback procedures.

[0208] 5.2 Columns CaptureSelect CH1XL Thermo Fisher, catalog number: 2943452050 Lot number: 170426-01 ID:10cm h=3.8cm A=0.79cm 2 V=3.0ml

[0209] [Table 15]

[0210] a. Chromatogram IGC01S Please refer to Figure 5.

[0211] 5.3 IGC02 execution results

[0212] [Table 16]

[0213] [Table 17]

[0214] Example 6 6.1 Variant IGC04 a. Buffer composition used in Example 6

[0215] [Table 18]

[0216] 6.2 IGC04 Cryosupernatant from Cohn was used as the starting material for the purification process without any further treatment prior to the experiment. The experiment was carried out at +2 to +8°C.

[0217] MAB-SelectPrismA (GE Healthcare, Catalog number: 17-5498-02, ID: 3.2cm h=7.5cm A=8.04cm 2 V=60.3ml) and CaptureSelect CH1-XL Thermo Fisher, Catalog Number: 2943452050) ID: 2.2cm h=3.6cm A=3.81cm 2 After equilibration of the V = 13.7 ml array in series (flow-through of the first column loaded directly onto the second column), the cryo-supernatant was applied to the CH1-XL column with a contact time of approximately 6 min [2.3 ml / min].

[0218] After loading, a first wash with 2 CV of buffer IQEQ was performed to remove unbound proteins from the resin. SD_VI of the column process was performed by applying IQEQ_VI buffer containing a detergent composition containing 10.55 g TritonX-100, 3.21 g polysorbate 80, and 2.91 g TnBP to 1 kg of IQEQ buffer (the components of the detergent composition are mixed for about 15 minutes and then added to IQEQ buffer while mixing). SD_VI on the column is performed by contacting the bound proteins with up to 4 CV of IQEQ_VI buffer at a flow rate of 4 ml / min for a minimum of 60 minutes. For the SD_VI step: 16.6 g / Kg of SD_VI_MIX is added to 10 mM Na Acetate, 10 mM Tris HCl, 120 mM NaCl, pH 7.2. SD_VI_MIX was prepared by mixing the detergent / solvent in the ratio 10.55 g TritonX100 / 3.21 g Polysorbate 80 / 2.91 g TnPB (tri-n-butyl phosphate) for a minimum of 15 min and added to the buffer while mixing.

[0219] Wash 2 was performed using W2 buffer to further remove impurities and wash away the SD_VI reagent at 5 CV and a flow rate of 10 ml / min. The third and final wash step was performed by applying 6 CV of IQEQ buffer at a flow rate of 10 ml / min.

[0220] Elution was performed by applying 5 CV of E1 buffer (100 mM glycine, pH 3.2).

[0221] The first step of the washing procedure (strip) was performed with E2 buffer (100 mM glycine, pH 2.7).

[0222] After re-equilibration with 4 CV of IQEQ buffer, the columns were regenerated and stored separately.

[0223] CV is the total volume of both columns (CH1-XL and CaptureSelect CH1-XL). For regeneration and saving procedures, see Regeneration Procedures.

[0224] Example 7 The eluate from Example 6 (IGC04) was treated as described below. This example is referred to as Example 6-Variant IGC04.

[0225] 7.1 Tandem column array Column 1 MABSelectPrismA GE Healthcare, Catalog Number: 17-5498-02 Lot number: 10264190 ID:3.2cm h=7.5cm A=8.04cm 2 V=60.3ml (target) b. Column 2 CaptureSelect CH1XL Thermo Fisher, catalog number: 2943452050 Lot number: 170426-01 ID:2.2cm h=3.6cm A=3.81cm 2 V=13.7ml Column volume 1+2: 74.0 ml Program: 74.506 (defined as column 1+2 + connection - 1 CV)

[0226] [Table 19]

[0227] c. Chromatogram IGC04 Please refer to Figure 6.

[0228] 7.2 Results and Data of Example 7

[0229] [Table 20]

[0230] [Table 21]

[0231] Product / Impurity Profile LC-MS data were generated for the protein profile of IMAX_FC_CRS_04WSD (wash buffer with SD_VI reagent) and of the eluate IMAX_FC_CRS_04 (two columns in series elution: MABSelectPrismA and CapturSelect CH1_XL) representing the intermediate product fraction after affinity purification. The impurity profile of SD_VI of the column virus inactivation wash fraction and the product / impurity profile of the eluate were determined by RP-HPLC-ESI-MS / MS. Samples were precipitated with TCA, reduced, alkylated, digested with trypsin and then analyzed on a Thermo UltiMateCap-HPLC coupled to an Eclipse Orbitrap (RP-HPLC-ESI-MSMS analysis). Data were analyzed with the "Proteome Discoverer" software. Results consist of the protein composition and the relative protein abundance of each sample (label-free quantification).

[0232] result The results are shown in Tables 22 and 23. All different detected variants of immunoglobulins were combined into one protein entry "immunoglobulin" and all respective area% values ​​were compiled. Accession numbers are from UniProtKB. An area% value of 0.00% means that the protein could be detected and identified, but the relative amount is below the threshold of 0.01%.

[0233] conclusion The IMAX_FC_CRS_04 WSD fraction ("wash fraction") contains impurities that are depleted during SD_VI on the column. The "wash fraction" sample has a protein composition of 66.96% immunoglobulins and 33.04% other proteins. It was possible to detect and identify 150 different proteins. (Table 22). In the WSD, there was essentially 0% serum amyloid A-2 protein, prenylcysteine ​​oxidase 1, leucine-rich alpha 2 glycoprotein, coagulation factor XI, proteoglycan 4, carboxypeptidase B2, sex hormone binding globulin, adiponectin, hyaluronic acid binding protein 2, neutrophil defensin 1, Ras-related protein Rap-1b, transforming growth factor beta-induced protein ig-h3, integrin beta 3, properdin, or carboxypeptidase N catalytic chain.

[0234] The "eluate" sample has a protein composition of 99.57% immunoglobulins and 0.43% other proteins. 26 different proteins could be detected and identified. (Table 23). This eluate represents a highly pure human IgG pool (>99%), superior to current commercially available IgG products. The affinity eluate contained essentially 0% alpha-1-acid glycoprotein 2, transthyretin, apolipoprotein B-100, C4b binding protein alpha chain, fibronectin, histidine-rich glycoprotein, complement C5, or pregnancy zone proteins.

[0235] [Table 22] TIFF2025509267000024.tif235165TIFF2025509267000025.tif235165TIFF2025509267000026.tif235165TIFF2025509267 000027.tif235165TIFF2025509267000028.tif235165TIFF2025509267000029.tif235165TIFF2025509267000030.tif71165

[0236] [Table 23] TIFF2025509267000032.tif54165

[0237] [Table 24]

[0238] a. SDS PAGE silver staining profile Please refer to Figure 7.

[0239] Example 8 8.1 a. Buffer composition used in Example 8

[0240] [Table 25]

[0241] 8.1.Execution Cryosupernatant from the Cohn fractionation process was used as the starting material for the purification process without any further treatment before the experiment. MAB-Select Prism A (GE Healthcare, Catalog Number: 17-5498-02, ID: 3.2 cm h=7.5 cm A=8.04 cm 2 V=60.3ml) and CaptureSelect CH1-XL Thermo Fisher, Catalog Number: 2943452050, ID: 2.2cm h=3.6cm A=3.81cm 2 V=13.7ml) and CaptureSelect IgG3 (Thermo Fisher, Catalog No.: 191304010, ID: 2.2cm h=3.1cm A=3.81cm 2 After equilibration of an array of three columns in series (V = 11.8 ml), the cryosupernatant was applied to the CH1-XL column for approximately 6 min [2.3 ml / min].

[0242] After loading, a first wash with 2 CV of buffer IQEQ was performed to remove unbound protein from the resin. SD_VI on column was performed by applying IQEQ_VI buffer containing a surfactant composition of 10.55 g Triton X-100, 3.21 g Polysorbate 80, and 2.91 g TnBP in 1 kg of IQEQ buffer. SD_VI on column was performed by contacting the bound protein with up to 4 CV of IQEQ_VI buffer at a flow rate of 4 ml / min for a minimum of 60 min.

[0243] Wash 2 was performed using W2 buffer to further remove impurities and wash away the SD_VI reagent at 5 CV, flow rate 10 ml / min. The third and final wash step was performed by applying 6 CV of IQEQ buffer at a flow rate of 10 ml / min.

[0244] Elution was performed by applying 5 CV of E1 buffer (100 mM glycine, pH 3.2). The first step (strip) of the washing procedure was carried out with E2 buffer (100 mM glycine, pH 2.7). After re-equilibration with 4 CV of IQEQ buffer, the columns were regenerated and stored separately.

[0245] 8.2 Tridem column array: Column 1 MABSelectPrismA GE Healthcare, Catalog Number: 17-5498-02 Lot number: 10264190 ID:3.2cm h=7.5cm A=8.04cm 2 V=60.3ml

[0246] b. Column 2 CaptureSelect CH1XL Thermo Fisher, catalog number: 2943452050 Lot number: 170426-01 ID:2.2cm h=3.6cm A=3.81cm 2 V=13.7ml

[0247] c. Column 3 Thermo Fisher, Catalog Number: 191304010 Lot number: 151012-01 ID:2.2cm h=3.1cm A=3.81cm 2 V=11.8ml Column volume 1+2+3: 85.8 ml Program: 86.4 (column 1+2+3+ connection)

[0248] 8.3 Chromatograms Please refer to Figure 8.

[0249] 8.4 Results and Data of the Execution a. Comparison of load and eluate yield IgG subclass distribution The Tridem array provides a highly efficient method for purifying the Cohn fraction into an IgG pattern that is comparable to the IgG subclass distribution in plasma. The ratio of IgG subclasses is nearly identical to the pattern in commercial IVIG products. Only the amount of IgG4 is higher. SDS-PAGE silver staining confirms the high purity of the affinity eluate.

[0250] [Table 26]

[0251] b. Impurity profile of affinity eluate

[0252] [Table 27]

[0253] b. SDS PAGE silver staining profile Please refer to Figure 9.

[0254] By using a combination of two and / or three columns containing complementary immunoglobulin-binding resins in series, a broad pattern of IgG subclasses was obtained from IgG-containing starting material (cryosupernatant, gammaguard liquid). The purity of the resulting intermediate (eluate) was surprisingly high and comparable to affinity purification steps used in monoclonal antibody purification.

[0255] Example 9 9.1 Equipment An AKTA was used in these experiments. The system was equipped with probes that allowed electronic recording and online monitoring of UV absorption (2 mm flow cell), conductivity, pressure, temperature, and pH. The chromatography unit was operated using the system software Unicorn 7.3.

[0256] [Table 28]

[0257] 9.2 Regeneration Procedure a.CaptoSelectPRISM A was typically regenerated using 1 M NaOH, applying 2 CV for a total of 30 min.

[0258] [Table 29]

[0259] b. CaptureSelect CH1-XL was regenerated using 50% (w / w) ethylene glycol, 10% (w / w) ethanolamine, 0.1% polysorbate 80, pH 8.5 (PDAM buffer) by applying 4 CV for a total of 60 min.

[0260] [Table 30]

[0261] Techniques and processes that can be applied in conjunction with or in addition to the affinity chromatography manufacturing process disclosed herein include US10,125,189 (Method for producing highly concentrated immunoglobulin preparations for subcutaneous use), US9,468,675 (Removal of serine proteases by treatment with fine silicon dioxide), US8,993,734 (Method for producing immunoglobulin preparations with improved yields), US9,782,477 (Fraction I-IV-1 precipitation of immunoglobulins from plasma), US20150133636 (Purification of biological molecules), US20170218012 (Integrated continuous manufacturing of therapeutic protein drug substances). All of the above references are incorporated herein by reference in their entirety.

[0262] While the present invention has been described with respect to specific exemplary embodiments and examples, it will be understood that the embodiments disclosed herein are for illustrative purposes only, and that various modifications and changes can be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims.

[0263] References All references cited herein, including the following documents, are hereby incorporated by reference in their entirety, including all patents, patent applications, and non-patent documents referenced below or elsewhere in this specification. TIFF2025509267000040.tif38160

Claims

1. 1. An affinity chromatography method for preparing an IgG preparation comprising one or more of IgG1, IgG2, IgG3, and IgG4 from an IgG-containing starting material, comprising: (a) contacting the IgG-containing starting material with a first chromatographic medium comprising a first solid support having protein A bound thereto, thereby forming a protein A bound fraction of an IgG subclass having protein A binding affinity, and forming a first flow-through comprising an IgG subclass that does not bind to protein A; (b) contacting the first flow-through with a first affinity ligand chromatography medium comprising a second solid support having a first affinity ligand bound thereto having IgG-binding affinity, thereby forming a second flow-through comprising the first affinity ligand-bound IgG subclass and an IgG subclass that does not bind to the first affinity ligand-bound IgG subclass; (c) eluting the Protein A bound fraction from the first chromatography medium to form a first eluate and contacting the first eluate with the first affinity ligand chromatography medium to form a second affinity ligand-bound IgG subclass and a second flow-through; (d) eluting members selected from the first affinity ligand-binding IgG subclass, the second affinity ligand-binding IgG subclass, and combinations thereof from the first affinity ligand column, thereby forming a second eluate; The method comprising:

2. The method of claim 1 , wherein the first flow-through and the second eluate are mixed on the first affinity ligand chromatography medium.

3. (e) contacting the second eluate of (d) with a second affinity ligand chromatography medium comprising a third solid support having a second affinity ligand bound thereto having IgG-binding affinity, thereby forming a second flow-through comprising the third affinity ligand-bound IgG subclass and a fourth IgG subclass that does not bind to the second affinity chromatography medium; (f) eluting a member selected from the second affinity ligand-bound IgG subclass, the fourth IgG subclass, and combinations thereof from the second affinity ligand chromatography medium, thereby forming a third eluate. The method of claim 2 , further comprising:

4. The method of claim 3 , wherein the first flow-through and the second eluate are mixed on the second affinity ligand chromatography medium.

5. (g) prior to (b), contacting said first chromatographic medium with a buffer / detergent mixture that does not allow said Protein A-binding fraction of an IgG subclass having Protein A-binding affinity to be eluted from said first chromatographic medium. The method of claim 1 further comprising:

6. (h) prior to (f), contacting the second affinity ligand chromatography medium with a buffer / detergent mixture that does not significantly elute the second affinity ligand-bound IgG subclass from the second affinity ligand chromatography medium. The method of claim 3 further comprising:

7. The method of claim 5 or claim 6, wherein the buffer / detergent mixture comprises acetate, Tris, Triton X, tri(n-butyl)phosphate (TnBP).

8. The buffer / surfactant mixture is a buffer composition having about 10 mM sodium acetate, about 10 mM Tris-HCl, and about 120 mM NaCl; A surfactant component having about 10.55 g Triton X, 3.21 g Polysorbate 80, and about 2.91 g TnBP per kg of said buffer solution (about 16.6 g SD_VI reagent per kg of buffer); The method of claim 7, comprising:

9. 2. The method of any one of the preceding claims, wherein the IgG in the formulation is at least about 90% (wt / wt) of the protein content of the formulation.

10. 13. The method of any one of the preceding claims, wherein the IgG1 in the formulation is from about 40% to about 90% (wt / wt) of the protein content of the formulation.

11. 13. The method of any one of the preceding claims, wherein the IgG1 in the formulation is from about 50% to about 70% (wt / wt) of the protein content of the formulation.

12. 13. The method of any one of the preceding claims, wherein the IgG2 in the formulation is from about 10% to about 50% (wt / wt) of the protein content of the formulation.

13. 13. The method of any one of the preceding claims, wherein the IgG2 in the formulation is from about 27% to about 37% (wt / wt) of the protein content of the formulation.

14. 13. The method of any one of the preceding claims, wherein the IgG3 in the formulation is from about 0.1% to about 50% (wt / wt) of the protein content of the formulation.

15. 13. The method of any one of the preceding claims, wherein the IgG3 in the formulation is from about 0.5% to about 8% (wt / wt) of the protein content of the formulation.

16. 13. The method of any one of the preceding claims, wherein the IgG4 in the formulation is from about 3% to about 70% (wt / wt) of the protein content of the formulation.

17. 13. The method of any one of the preceding claims, wherein the IgG4 in the formulation is from about 0.5% to about 8% (wt / wt) of the protein content of the formulation.

18. (i) the IgG1 in the formulation is about 50% to about 70% (wt / wt) of the protein content of the formulation; (ii) the IgG2 in the formulation is about 27% to about 37% (wt / wt) of the protein content of the formulation; (iii) the IgG3 in the formulation is from about 2.5% to about 8% (wt / wt) of the protein content of the formulation; (iv) the IgG4 in the formulation is from about 3% to about 70% (wt / wt) of the protein content of the formulation; 10. A method according to any one of the preceding claims.

19. 10. The method of any one of the preceding claims, wherein the albumin in the formulation is in an amount of less than about 5 g per liter of the formulation.

20. 2. The method of any one of the preceding claims, wherein the factor XI activity in the formulation is less than about 0.01 E / mL.

21. 13. The method of any one of the preceding claims, wherein alpha-2-macroglobulin is present in the formulation in an amount of less than about 0.2 g per liter of formulation.

22. 10. The method of any one of the preceding claims, wherein the transferrin present in the formulation is in an amount of less than about 0.09 g / L of the formulation.

23. 2. The method of any one of the preceding claims, wherein the PL1 activity of the preparation is less than about 10 nm / mL min.

24. 10. The method of any one of the preceding claims, wherein the fibrinogen present in the formulation is in an amount of less than about 35 μg per mL of the formulation.

25. 2. The method of any one of the preceding claims, wherein the IgG-containing starting material is a cryosupernatant.

26. 2. The method of any one of the preceding claims, wherein an individual column comprises a member selected from the first chromatography medium comprising a first solid support having protein A, the first affinity ligand chromatography medium comprising a second solid support having a first affinity ligand bound thereto having IgG binding affinity, and combinations thereof.

27. 27. The method of claim 26, wherein two or more of the columns are in fluid communication in an array, and two or more of the columns comprise a second chromatographic medium and a second affinity ligand chromatographic medium.

28. 28. The method of claim 27, wherein the outlet of the first column is in fluid communication with the inlet of the second column.

29. 30. The method of claim 28, wherein the outlet of the column having protein A is in fluid communication with the inlet of the first affinity ligand chromatography medium.

30. 28. The method of claim 27, wherein the outlet of the column containing the second chromatography medium is in fluid communication with the inlet of the column containing the second affinity ligand chromatography medium.

31. 10. The method of any one of the preceding claims, wherein the IgG solution depleted of Protein A binding IgG subclasses comprises members selected from IgG1, IgG2, IgG3, IgG4, and combinations thereof.

32. 2. The method of any one of the preceding claims, wherein the first chromatographic medium has selective affinity for the CH1 domain of a human IgG antibody.

33. 10. The method of any one of the preceding claims, wherein the first chromatography medium has a selective affinity for the CH1 domain of a member selected from IgG1 and IgG3, which selective affinity is greater than the selective affinity for the CH1 domain of a member selected from IgG2 and IgG4.

34. 2. The method of any one of the preceding claims, wherein eluting from the first chromatography medium, the first affinity ligand chromatography medium, and the second affinity ligand chromatography medium utilizes an eluent comprising glycine.

35. 35. The method of claim 34, wherein the glycine concentration is from about 50 mM to about 150 mM.

36. 1. An affinity chromatography method for preparing an IgG preparation enriched in a first IgG subclass having low protein A binding affinity from a plasma-derived IgG solution depleted of protein A-binding IgG subclasses, comprising the steps of: (a) contacting the IgG solution depleted of Protein A-binding IgG subclass with a first affinity ligand chromatography medium comprising a first solid support having a first affinity ligand having IgG-binding affinity bound thereto, thereby forming a first bound fraction of the first IgG subclass having low Protein A-binding affinity and a first flow-through; (b) eluting the first bound fraction of the first IgG subclass having low Protein A binding affinity, thereby forming a first eluate comprising the IgG preparation enriched in the first IgG subclass having low Protein A binding; The method comprising:

37. (c) prior to (b), contacting the first bound fraction of the first IgG subclass having low Protein A binding affinity with a buffer / detergent solution, and thereafter removing the detergent solution from the first affinity ligand chromatography medium.

37. The method of claim 36, further comprising:

38. (d) subsequent to (b), contacting the first eluate with a second chromatographic medium comprising a solid support having an affinity ligand bound thereto having affinity for a second IgG subclass having a lower Protein A binding affinity, thereby forming a first bound fraction of the second IgG subclass having a lower Protein A binding affinity; (e) eluting the first bound fraction of the second IgG subclass having a lower Protein A binding affinity from the second chromatography medium, thereby forming a second eluate; 37. The method of claim 36, further comprising:

39. (f) prior to (e), contacting the first bound fraction of the second IgG subclass having a lower Protein A binding affinity with a buffer / detergent solution, and thereafter removing the detergent solution from the first chromatographic medium.

40. The method of claim 38, further comprising:

40. (g) prior to (a), contacting the plasma-derived IgG solution with a protein A chromatography medium comprising protein A bound to a solid support, thereby forming a protein A-binding fraction of an IgG subclass having a high affinity for protein A and an IgG preparation enriched in a first IgG subclass having a low protein A-binding affinity.

37. The method of claim 36, further comprising:

41. (h) eluting the Protein A bound fraction of IgG subclasses having high affinity for Protein A from the Protein A chromatography medium, thereby forming a third eluate.

37. The method of claim 36, further comprising:

42. 42. The method of any one of claims 36-41, further comprising combining two or more of the first eluate, the second eluate, and the third eluate.

43. 1. An aqueous IgG solution comprising at least about 99.5% IgG and less than about 0.5% impurities, the impurities including factor XI with an activity of less than about 0.0025E / mg total IgG, preferably less than about 0.0020E / mg total IgG, more preferably less than about 0.0015E / mg total IgG, and most preferably less than about 0.0012E / mg total IgG.

44. 1. An aqueous IgG solution comprising at least about 99.5% IgG and less than about 0.5% impurities, the impurities including complement C3 at a level of less than about 3.5 μg / mg of total IgG, preferably less than about 3.0 μg / mg of total IgG, more preferably less than about 2.5 μg / mg of total IgG, and most preferably less than about 2.2 μg / mg of total IgG.

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