Methods for purifying immunoglobulin G and uses thereof

Abstract

The present disclosure relates to a method for purifying Immunoglobulin G (IgG) and other proteins, such as albumin, from plasma or fractions thereof using an affinity chromatography resin containing a ligand capable of specifically binding to the CH3 domain of human IgG. The disclosure also relates to formulations and uses of the plasma protein products produced from the method.

Description

[Technical field]

[0001] Related Application Data This application claims priority from Australian Patent Application No. 2021902332, filed on July 29, 2021, and entitled "Method of purifying immunoglobulin G and uses thereof," U.S. Patent Application No. 63 / 227,329, filed on July 29, 2021, and entitled "Method of purifying immunoglobulin G and uses thereof," and U.S. Patent Application No. 63 / 365,530, filed on May 31, 2022, and entitled "Method of purifying immunoglobulin G and uses thereof."

[0002] Sequence Listing This application is filed with a Sequence Listing in electronic format, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to methods for purifying immunoglobulin G (IgG) and other proteins, such as albumin, from plasma, and to formulations and uses of the plasma protein products. [Background technology]

[0004] Immunoglobulin G (IgG) is one of the most abundant proteins in plasma and is responsible for, for example, neutralization of toxins, activation of complement and opsonization. IgG purified from human plasma is used for prophylactic prevention of infectious diseases in immunocompromised patients, replacement therapy of antibody deficiencies in patients, and treatment of conditions associated with immunodeficiency, inflammatory and autoimmune diseases and acute infections in patients. Plasma-derived immunoglobulins have become the major plasma product and consumption is increasing worldwide. Human immunoglobulin products, both hyperimmune (or "specific") and normal (or "non-specific") types, are composed primarily of IgG. Hyperimmune immunoglobulin products include hepatitis B, tetanus, varicella-zoster, and rabies immunoglobulins; each containing a known concentration of a specific antibody. The antibody specificity of normal polyvalent human immunoglobulins (IG) reflects the antibody specificity of the donor population. A list of FDA-approved IGs is shown at https: / / www.fda.gov / vaccines-blood-biologics / approved-blood-products / immune-globulins. Currently, there are commercially available intravenous IG (IVIG) products (usually 5% or 10% (w / v) stabilized solutions), such as Privigen® (CSL Behring), Flebogamma® (Grifols), Gamunex®-C (Grifols), Gammagard® (Takeda), and Octagam® (Octapharma). More recently, subcutaneous IG (SCIG) administration has become available. Commercially available SCIG products (usually 10%, 16.5% or 20% (w / v) stabilized solutions) include Hizentra® (CSL Behring), Gamunex®-C (Grifols), Xembify® (Grifols), Cutaquig® (Octapharma), and Cuvitru® (Takeda). Other IG products are administered intramuscularly (IMIG).

[0005] IG products contain mainly IgG with a defined distribution of IgG subclasses: IgG1, IgG2, IgG3 and IgG4. However, IG products may differ in various respects: IgG monomer, dimer and aggregate concentrations; IgA and IgM content; stabilizers; additives; and impurity (proteases such as factor XI / XIa) levels. It is recognized that IgA may cause anaphylactic reactions in IgA-deficient patients. For this reason, it is desirable for IG products to contain low amounts of IgA. The attributes of IgG-containing IG products must also meet the requirements of local and / or regional pharmacopoeias in order to be registered in the respective jurisdictions (e.g., 1999).

[0006] Existing methods for purifying IgG from plasma and its fractions include chromatographic (e.g., affinity chromatography, anion exchange chromatography, hydrophobic interaction chromatography, SE-HPLC) and non-chromatographic (precipitation and liquid extraction) purification methods. The major obstacles of existing methods are the high cost and time involved in purifying IgG, the requirement to co-purify other proteins (e.g., albumin and clotting factors) from the same plasma or plasma fraction, and the need to ensure that the product is of suitable quality (e.g., purity and stability) for therapeutic use. For example, affinity resins used in affinity chromatography have relatively low binding capacities, and chromatographic purification from average size batches can reach volumes of hundreds of liters (in contrast, plasma fractions are typically thousands of liters), requiring huge capital investments in the amount of resin used, infrastructure for handling and packing the chromatography columns, and running costs. Currently, up to 70-75% of the IgG present in plasma can be recovered from plasma using existing technologies. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Human Normal Immunoglobulin for Subcutaneous Administration, Ph. Eur. monograph 2788 Summary of the Invention [Problem to be solved by the invention]

[0008] It will therefore be apparent to those skilled in the art that there is a need in the art for improved methods of purifying IgG from plasma or fractions thereof. [Means for solving the problem]

[0009] The present disclosure is based on the inventors' identification of a method for purifying IgG from plasma or a fraction thereof with high yield (e.g., 75% or more). The method also allows IgG to be recovered from plasma or a fraction thereof with high purity (e.g., 95% or more). In particular, the inventors have found that the use of sequential affinity chromatography (e.g., simulated moving bed (SMB) chromatography) with an affinity chromatography resin containing a ligand capable of specifically binding to the CH3 domain of human IgG allows for the purification of IgG from plasma with high yield and purity while minimizing the impact on the IgG subclass distribution (i.e., IgG1, IgG2, IgG3 and IgG4) compared to existing products. Furthermore, the inventors have found that the method is further improved by the use of specific washing and regeneration buffers. The method advantageously uses less chromatography buffer and allows the affinity resin to be reused multiple times (at least 50 cycles), further reducing the cost of purifying IgG from plasma or a fraction thereof.

[0010] Thus, the findings of the present inventors provide the basis for a method for producing an IgG-enriched preparation. This finding also provides the basis for a pharmaceutical composition comprising the IgG-enriched preparation, and for the use of the composition or the IgG for the treatment, prevention, and / or delay of progression of a condition in a subject, such as primary immunodeficiency disease, chronic inflammatory demyelinating polyneuropathy, and chronic immune thrombocytopenic purpura.

[0011] The present disclosure provides an affinity chromatography resin comprising a ligand capable of specifically binding to the CH3 domain of human IgG.

[0012] The disclosure also provides a method for purifying IgG from plasma or a fraction thereof using affinity chromatography, comprising binding the IgG to an affinity chromatography resin comprising a ligand capable of specifically binding to the CH3 domain of human IgG, and recovering the IgG.

[0013] The disclosure further provides a method of producing an IgG-enriched preparation from plasma or a fraction thereof using affinity chromatography, comprising binding the IgG to an affinity chromatography resin comprising a ligand capable of specifically binding to the CH3 domain of human IgG, and recovering the IgG.

[0014] The present disclosure provides a method for purifying IgG from plasma or a fraction thereof using sequential affinity chromatography, comprising binding the IgG to an affinity chromatography resin comprising a ligand capable of specifically binding to the CH3 domain of human IgG, and recovering the IgG.

[0015] The disclosure also provides a method for producing an IgG-enriched preparation from plasma or a fraction thereof using sequential affinity chromatography, comprising binding the IgG to an affinity chromatography resin comprising a ligand capable of specifically binding to the CH3 domain of human IgG, and recovering the IgG.

[0016] In one example, the resin comprises a ligand comprising a single domain [VHH] antibody fragment from camelid, e.g., the ligand is a VHH antibody fragment, in one example, the ligand does not comprise a CH1 domain.

[0017] In one example, the resin comprises a matrix selected from the group consisting of a cross-linked poly(styrene-divinylbenzene) matrix and an agarose-based matrix. For example, the matrix is ​​a cross-linked poly(styrene-divinylbenzene) matrix. In another example, the matrix is ​​an agarose-based matrix.

[0018] In one example, the resin comprises a ligand capable of specifically binding to the CH3 domain of human IgG, where the ligand is conjugated to a cross-linked poly(styrene-divinylbenzene) matrix. For example, the resin comprises a ligand comprising a VHH antibody fragment conjugated to a cross-linked poly(styrene-divinylbenzene) matrix.

[0019] In one example, the resin comprises a ligand capable of specifically binding to the CH3 domain of human IgG and an agarose-based matrix, for example, the resin comprises a ligand comprising a VHH antibody fragment conjugated to an agarose-based matrix.

[0020] In one example, the resin comprises a VHH antigen binding protein comprising the amino acid sequence set forth in SEQ ID NO: 1, or a sequence which comprises at least 50% amino acid identity to the sequence set forth in SEQ ID NO: 1. In one example, the VHH antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1. In one example, the VHH antigen binding protein comprises a sequence which comprises at least 50% amino acid identity to the sequence set forth in SEQ ID NO: 1.

[0021] In one example, the resin comprises a VHH antigen binding protein comprising a framework region comprising the amino acid sequence set forth in SEQ ID NO: 1 or a sequence comprising at least 50% amino acid identity to the sequence set forth in SEQ ID NO: 1. In one example, the framework region comprises the amino acid sequence set forth in SEQ ID NO: 1. In another example, the framework region comprises a sequence comprising at least 50% amino acid identity to the sequence set forth in SEQ ID NO: 1.

[0022] In one example, the resin comprises a VHH antigen binding protein comprising an amino acid sequence comprising four framework regions, FR1, FR2, FR3 and FR4, and three complementarity determining regions, CDR1, CDR2 and CDR3, operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein: a) CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO:2, or an amino acid sequence which differs from SEQ ID NO:2 at one or two amino acid residues; b) CDR2 has an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:3; and c) CDR3 has an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4; and wherein each of the framework regions has at least 50% amino acid identity to the framework amino acid sequence of any one of SEQ ID NO:1; wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of SEQ ID NO:1.

[0023] In one example, the resin comprises a VHH antigen binding protein comprising an amino acid sequence comprising four framework regions, FR1, FR2, FR3 and FR4, and three complementarity determining regions, CDR1, CDR2 and CDR3, operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein; a) CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NO:2, or an amino acid sequence which differs from SEQ ID NO:2 at one or two amino acid residues; b) CDR2 has an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:3; and c) CDR3 has an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 4; and wherein each of the framework regions has at least 50% amino acid identity with any one of the framework amino acid sequences of SEQ ID NO: 1; wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of SEQ ID NO:1, and wherein the antigen binding protein specifically binds to the Fc domain of a human IgG molecule and does not bind to an IgG molecule of murine or bovine origin.

[0024] In one example, the resin comprises a VHH antigen binding protein comprising a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, or an amino acid sequence which differs from SEQ ID NO:2 at one or two amino acid residues.

[0025] In one example, the resin comprises a VHH antigen binding protein comprising a CDR2 that comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3.

[0026] In one example, the resin comprises a VHH antigen binding protein comprising a CDR3 that comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4.

[0027] In one example, the method further comprises washing the resin with a wash buffer. For example, the method comprises washing the resin with a wash buffer prior to recovering the IgG. For example, the method comprises washing the resin with a wash buffer prior to recovering the bound IgG.

[0028] In one example, the method further comprises washing the resin with a wash buffer as part of recovering the IgG. For example, the method further comprises washing the resin with a wash buffer as part of recovering the bound IgG. In such a method, the washing may remove unbound or weakly bound IgG from the resin. Such unbound or weakly bound IgG may be discarded before recovering the bound IgG. Alternatively, unbound or weakly bound IgG is recovered. In one example, bound, unbound, and weakly bound IgG are recovered. In one example, bound and weakly bound IgG are recovered. In another example, unbound and weakly bound IgG are not recovered.

[0029] In one example, the method includes washing the resin with a wash buffer before recovering the IgG. For example, the method includes washing impurities from the resin with a wash buffer and recovering the IgG. In one example, the method includes washing the resin with a wash buffer before recovering the IgG and collecting the flow-through. In one example, the flow-through includes impurities. In one example, the method includes washing the resin with a wash buffer before recovering the IgG and collecting the impurities in the flow-through. For example, the method includes recovering the impurities from the resin with a wash buffer. In one example, the impurities and the IgG are recovered. For example, the impurities and the IgG are recovered together. In another example, the impurities and the IgG are recovered separately.

[0030] In one example, the method includes recovering a wash fraction. For example, the method includes recovering a wash fraction prior to recovering the IgG. In one example, the wash fraction includes impurities. In one example, the wash fraction includes IgG. For example, the wash fraction includes unbound IgG. In one example, the wash fraction includes weakly bound IgG. In another example, the wash fraction includes unbound and weakly bound IgG. In one example, the wash fraction includes impurities and IgG.

[0031] In one example, the impurities include albumin (α-globulins and / or β-globulins), plasma lipids, plasma proteins, proteases (e.g., serine proteases, kallikrein, plasmin and FXa), serine protease inhibitors (e.g., C1 inhibitor, α-1-antitrypsin and antithrombin), IgA and IgM, Factor VIII, fibrinogen, von Willebrand factor, activated clotting factors (e.g., FXa, FIXa, FVIIa and thrombin), Factor XIII, contact system factors (e.g., FXIa, FXIIa and plasma kallikrein), PKA, Factor IX, prothrombin complex, C1 esterase inhibitor, protein C, antithrombin III, RhD immunoglobulin and / or platelet membrane microparticles.

[0032] In one example, plasma protein products are produced using the methods described herein.

[0033] In one example, the plasma protein product is an IgG concentrated preparation. In another example, the plasma protein product comprises purified IgG.

[0034] In one example, plasma protein products are produced using bound, unbound and / or weakly binding IgG. For example, plasma protein products are produced using bound, unbound and / or weakly binding IgG. In one example, plasma protein products are produced using bound IgG. For example, plasma protein products are produced using bound IgG. In one example, plasma protein products are produced using unbound IgG. For example, plasma protein products are produced using unbound IgG. In one example, plasma protein products are produced using weakly binding IgG. For example, plasma protein products are produced using weakly binding IgG. In one example, plasma protein products are produced using bound and weakly binding IgG. For example, plasma protein products are produced using bound and weakly binding IgG. In one example, plasma protein products are produced using unbound and weakly binding IgG. For example, plasma protein products are produced using unbound and weakly binding IgG. In one example, plasma protein products are produced using bound and unbound IgG. For example, plasma protein products are produced using bound and unbound IgG. In one example, plasma protein products are produced using bound, unbound and weakly binding IgG. For example, conjugated, unconjugated and weakly conjugated IgG are used to produce plasma protein products.

[0035] In one example, a plasma protein product is produced using impurities, e.g., the impurities are recovered and used to produce the plasma protein product.

[0036] In one example, the plasma protein product is selected from the group consisting of albumin, serine proteases, plasmin, FXa, alpha-1-antitrypsin, IgA, IgM, factor VIII, fibrinogen, von Willebrand factor, activated clotting factors, factor XIII, contact system factors, PKA, factor IX, prothrombin complex, C1 esterase inhibitor, protein C, antithrombin III, RhD immunoglobulin protein products.

[0037] In one example, the activated coagulation factor is selected from the group consisting of FXa, FIXa, FVIIa and thrombin.For example, the activated coagulation factor is FXa.For example, the activated coagulation factor is FIXa.For example, the activated blood coagulation factor is FVIIa.For example, the activated coagulation factor is thrombin.

[0038] In one example, the contact factor protein is selected from the group consisting of FXIa, FXIIa and kallikrein.For example, the contact factor protein is FXIa.For example, the contact factor protein is FXII.For example, the contact factor protein is kallikrein.

[0039] In one example, the plasma protein product is an albumin protein product. In one example, the plasma protein product is a serine protease protein product. In one example, the plasma protein product is a plasmin protein product. In one example, the plasma protein product is a FXa protein product. In one example, the plasma protein product is an alpha-1-antitrypsin protein product. In one example, the plasma protein product is an IgA protein product. In one example, the plasma protein product is an IgM protein product. In one example, the plasma protein product is a factor VIII protein product. In one example, the plasma protein product is a fibrinogen protein product. In one example, the plasma protein product is a von Willebrand factor protein product. In one example, the plasma protein product is an activated coagulation factor protein product. For example, the plasma protein product is a FXa protein product. For example, the plasma protein product is a FIXa protein product. For example, the plasma protein product is a FVIIa protein product. For example, the plasma protein product is a thrombin protein product.

[0040] In one example, the plasma protein product is a factor XIII protein product. In one example, the plasma protein product is a contact system factor protein product. For example, the plasma protein product is a FXIa protein product. For example, the plasma protein product is a FXII protein product. For example, the plasma protein product is a kallikrein plasma product. In one example, the plasma protein product is a PKA protein product. In one example, the plasma protein product is a factor IX protein product. In one example, the plasma protein product is a prothrombin complex protein product. In one example, the plasma protein product is a C1 esterase inhibitor protein product. In one example, the plasma protein product is a protein C protein product. In one example, the plasma protein product is an antithrombin III protein product. In one example, the plasma protein product is a RhD immunoglobulin protein product.

[0041] In one example, the wash buffer has a dissociation constant (pKa) of 6.8-8.5 at a pH of 5-9 and 25° C. In one example, the wash buffer has a dissociation constant (pKa) of 6.8-8.5 at a pH of 5-10 and 25° C.

[0042] In one example, the wash buffer has a pH of 5 to 10. In one example, the wash buffer has a pH of 5 to 9. For example, the wash buffer may have a pH of 5, or 5.1, or 5.2, or 5.3, or 5.4, or 5.5, or 5.6, or 5.7, or 5.8, or 5.9, or 6.0, or 6.1, or 6.2, or 6.3, or 6.4, or 6.5, or 6.6, or 6.7, or 6.8, or 6.9, or 7, or 7.1, or 7.2, or 7.3, or 7.4, or is 7.5, or 7.6, or 7.7, or 7.8, or 7.9, or 8.0, or 8.1, or 8.2, or 8.3, or 8.4, or 8.5, or 8.6, or 8.7, or 8.8, or 8.9, or 9.0, or 9.1, or 9.2, or 9.3, or 9.4, or 9.5, or 9.6, or 9.7, or 9.8, or 9.9, or 10.0.

[0043] In one example, the wash buffer has a pH of 7-10 and a dissociation constant (pKa) of 6.8-8.5 at 25°C.

[0044] In one example, the wash buffer has a pH of 7-8 and a dissociation constant (pKa) of 6.8-8.5 at 25°C.

[0045] In one example, the wash buffer has a pH of 7 to 8. For example, the wash buffer has a pH of 7, or 7.1, or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7, or 7.8, or 7.9, or 8.0. In one example, the wash buffer has a pH of 7.4.

[0046] In one example, the wash buffer has a pH of 7.4 to 7.8. For example, the wash buffer has a pH of 7.4, or 7.5, or 7.6, or 7.7, or 7.8.

[0047] In one example, the wash buffer has a pKa of 6.8 to 8.5 at 25° C. For example, the wash buffer has a pKa of 6.8, or 6.9, or 7.0, or 7.1, or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7, or 7.8, or 7.9, or 8.0, or 8.1, or 8.2, or 8.3, or 8.4, or 8.5 at 25° C.

[0048] In one example, the wash buffer has a pKa of 7.21 at 25°C.

[0049] In one example, the wash buffer has a pH of 7.4 and a dissociation constant (pKa) of 7.21 at 25°C.

[0050] In one example, the wash buffer may contain sodium dihydrogen phosphate, sodium citrate, imidazole, Tris, glycylglycine, 3-morpholinopropane-1-sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), bis[(2-hydroxyethyl)amino]acetic acid (Bicine), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sulfite, , 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), 4-(N-morpholino)butanesulfonic acid (MOBS), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), Tricine, triethanolamine (TEA) and combinations thereof. For example, the wash buffer is a sodium dihydrogen phosphate buffer. For example, the wash buffer is an imidazole buffer. In another example, the wash buffer is a Tris buffer. In a further example, the wash buffer is a glycylglycine buffer. In one example, the wash buffer is a MOPS buffer. In another example, the wash buffer is a PIPES buffer. In a further example, the wash buffer is a TES buffer. In one example, the wash buffer is a Bicine buffer. In another example, the wash buffer is a sulfite buffer. In a further example, the wash buffer is an EPPS buffer. In one example, the wash buffer is a HEPPSO buffer. In another example, the wash buffer is a MOBS buffer. In a further example, the wash buffer is a POPSO buffer. In one example, the wash buffer is a TAPSO buffer. In another example, the wash buffer is a Tricine buffer. In a further example, the wash buffer is a TEA buffer. In one example, the wash buffer is a sodium citrate buffer.

[0051] In one example, the buffering agent of the wash buffer is at a concentration of 5 mM to 200 mM. For example, the buffering agent of the wash buffer is at a concentration of 5 mM to 10 mM, or 5 mM to 20 mM, or 5 mM to 50 mM, or 50 mM to 100 mM, or 100 mM to 150 mM, or 150 mM to 200 mM. In another example, the buffering agent of the wash buffer is at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM, or 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM, or 105 mM, or 150 ... 50 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM.

[0052] In one example, the buffering agent in the wash buffer is at a concentration of 5 mM.

[0053] In one example, the buffering agent in the wash buffer is at a concentration of 20 mM.

[0054] In one example, the buffering agent in the wash buffer is at a concentration of 50 mM.

[0055] In one example, the buffering agent in the wash buffer is at a concentration of 100 mM.

[0056] In one example, the buffering agent in the wash buffer is at a concentration of 150 mM.

[0057] In one example, the buffering agent in the wash buffer is at a concentration of 200 mM.

[0058] In one example, the wash buffer further comprises sodium chloride. For example, the wash buffer further comprises sodium chloride at a concentration of up to 1000 mM. In one example, the sodium chloride is at a concentration of 5 mM to 50 mM. For example, the sodium chloride is at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM. In another example, the sodium chloride is at a concentration of 50 mM to 100 mM. For example, the sodium chloride is at a concentration of 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM. In one example, the sodium chloride is at a concentration of 100 to 200 mM. For example, sodium chloride is at a concentration of 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM. In other examples, sodium chloride is at a concentration of 200 to 300 mM. For example, sodium chloride is at a concentration of 200 mM, or 225 mM, or 250 mM, or 275 mM, or 300 mM. In further examples, sodium chloride is at a concentration of 300 mM to 400 mM. For example, sodium chloride is at a concentration of 300 mM, or 325 mM, or 350 mM, or 375 mM, or 400 mM. In one example, sodium chloride is at a concentration of 400 mM to 500 mM. For example, sodium chloride is at a concentration of 400 mM, or 425 mM, or 450 mM, or 475 mM, or 400 mM. In another example, sodium chloride is at a concentration of 500 mM to 1000 mM. For example, sodium chloride is at a concentration of 500 mM, or 550 mM, or 600 mM, or 650 mM, or 700 mM, or 750 mM, or 800 mM, or 850 mM, or 900 mM, or 950 mM, or 1000 mM. In one example, sodium chloride is at a concentration of less than 1000 mM.For example, sodium chloride is at a concentration of 500 mM.

[0059] In one example, the wash buffer further comprises sodium chloride, where the sodium chloride is at a concentration of 145 mM.

[0060] In one example, the wash buffer further comprises sodium chloride, where the sodium chloride is at a concentration of 500 mM.

[0061] In one example, the wash buffer contains 20 mM sodium dihydrogen phosphate, 145 mM sodium chloride, and has a pH of 7.4.

[0062] In one example, the wash buffer contains 20 mM sodium dihydrogen phosphate, 500 mM sodium chloride, pH 7.4.

[0063] In one example, the wash buffer further comprises a divalent salt. For example, the wash buffer further comprises a divalent salt at a concentration of up to 1000 mM. In one example, the divalent salt is at a concentration of 5 mM to 50 mM. For example, the divalent salt is at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM. In another example, the divalent salt is at a concentration of 50 mM to 100 mM. For example, the divalent salt is at a concentration of 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM. In one example, the divalent salt is at a concentration of 100 to 200 mM. For example, the divalent salt is at a concentration of 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM. In other examples, the divalent salt is at a concentration of 200 mM to 300 mM. For example, the divalent salt is at a concentration of 200 mM, or 225 mM, or 250 mM, or 275 mM, or 300 mM. In further examples, the divalent salt is at a concentration of 300 mM to 400 mM. For example, the divalent salt is at a concentration of 300 mM, or 325 mM, or 350 mM, or 375 mM, or 400 mM. In one example, the divalent salt is at a concentration of 400 mM to 500 mM. For example, the divalent salt is at a concentration of 400 mM, or 425 mM, or 450 mM, or 475 mM, or 400 mM. In another example, the divalent salt is at a concentration of 500 mM to 1000 mM. For example, the divalent salt is at a concentration of 500 mM, or 550 mM, or 600 mM, or 650 mM, or 700 mM, or 750 mM, or 800 mM, or 850 mM, or 900 mM, or 950 mM, or 1000 mM. In one example, the divalent salt is at a concentration of 500 mM. In one example, the divalent salt is at a concentration of less than 1000 mM.

[0064] In one example, the wash buffer comprises sodium chloride and / or a divalent salt at a concentration of up to 1000 mM. For example, the wash buffer comprises sodium chloride and / or a divalent salt at a concentration of about 500 mM.

[0065] In one example, the divalent salt is selected from the group consisting of magnesium chloride, calcium chloride, barium chloride, copper(II) chloride, nickel chloride, manganese chloride, and combinations thereof. For example, the divalent salt is magnesium chloride. In one example, the divalent salt is calcium chloride. In another example, the divalent salt is barium chloride. In a further example, the divalent salt is copper chloride. In one example, the divalent salt is nickel chloride. In another example, the divalent salt is manganese chloride.

[0066] In one example, the method includes recovering the IgG by eluting the IgG from the resin with an elution buffer. For example, the method includes recovering the bound IgG by eluting the bound IgG from the resin with an elution buffer.

[0067] In one example, the elution buffer has a pH of 3 to 5. For example, the elution buffer has a pH of 3, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5, or 3.6, or 3.7, or 3.8, or 3.9, or 4, or 4.1, or 4.2, or 4.3, or 4.4, or 4.5, or 4.6, or 4.7, or 4.8, or 4.9, or 5.

[0068] In one example, the elution buffer has a pH of 4.

[0069] In one example, the elution buffer comprises a buffer selected from the group consisting of sodium acetate, acetic acid, and sodium citrate. In one example, the elution buffer comprises sodium acetate, acetic acid, sodium citrate, and sodium dihydrogen phosphate. In one example, the elution buffer is or comprises a sodium phosphate buffer and / or an acetate buffer. For example, the elution buffer comprises sodium acetate. For example, the elution buffer comprises acetic acid. For example, the elution buffer comprises sodium citrate. For example, the elution buffer comprises sodium dihydrogen phosphate.

[0070] In one example, the buffering agent of the elution buffer is at a concentration of 5 mM to 200 mM. For example, the buffering agent of the elution buffer is at a concentration of 5 mM to 10 mM, or 5 mM to 20 mM, or 5 mM to 50 mM, or 50 mM to 100 mM, or 100 mM to 150 mM, or 150 mM to 200 mM. For example, the buffering agent of the wash buffer is at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM, or 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM, or 105 mM. M, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM.

[0071] In one example, the buffering agent in the elution buffer is at a concentration of 5 mM.

[0072] In one example, the buffering agent in the elution buffer is at a concentration of 20 mM.

[0073] In one example, the buffering agent in the elution buffer is at a concentration of 50 mM.

[0074] In one example, the buffering agent in the elution buffer is at a concentration of 100 mM.

[0075] In one example, the buffer in the elution buffer is at a concentration of 150 mM.

[0076] In one example, the buffer in the elution buffer is at a concentration of 200 mM.

[0077] In one example, the elution buffer is or comprises an acetate buffer, for example a sodium acetate buffer.

[0078] In one example, the elution buffer is or comprises a phosphate buffer and / or an acetate buffer. For example, the elution buffer is or comprises a sodium dihydrogen phosphate and sodium acetate buffer. In one example, the elution buffer is or comprises a phosphate buffer.

[0079] In one example, the elution buffer is or comprises an acetate buffer having a pH of 3 to 5. For example, the elution buffer is or comprises an acetate buffer having a pH of 3, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5, or 3.6, or 3.7, or 3.8, or 3.9, or 4, or 4.1, or 4.2, or 4.3, or 4.4, or 4.5, or 4.6, or 4.7, or 4.8, or 4.9, or 5. For example, the elution buffer is or comprises an acetate buffer having a pH of 4. For example, the elution buffer is or comprises a sodium acetate buffer having a pH of 4.

[0080] In one example, the elution buffer is or comprises a phosphate and / or acetate buffer having a pH of 3 to 5. For example, the elution buffer is or comprises a phosphate and / or acetate buffer having a pH of 3, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5, or 3.6, or 3.7, or 3.8, or 3.9, or 4, or 4.1, or 4.2, or 4.3, or 4.4, or 4.5, or 4.6, or 4.7, or 4.8, or 4.9, or 5. In one example, the elution buffer is or comprises a phosphate and / or acetate buffer having a pH of 4.

[0081] In one example, the elution buffer is or comprises a phosphate buffer having a pH of 3 to 5. For example, the elution buffer is or comprises a phosphate buffer having a pH of 3, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5, or 3.6, or 3.7, or 3.8, or 3.9, or 4, or 4.1, or 4.2, or 4.3, or 4.4, or 4.5, or 4.6, or 4.7, or 4.8, or 4.9, or 5. In one example, the elution buffer is or comprises a phosphate buffer having a pH of 4.

[0082] In one example, the elution buffer comprises 10 mM, or 11 mM, or 12 mM, or 13 mM, or 14 mM, or 15 mM, or 16 mM, or 17 mM, or 18 mM, or 19 mM, or 20 mM phosphate and / or acetate buffer.

[0083] In one example, the elution buffer comprises 10 mM, or 11 mM, or 12 mM, or 13 mM, or 14 mM, or 15 mM, or 16 mM, or 17 mM, or 18 mM, or 19 mM, or 20 mM acetate buffer. For example, the elution buffer comprises 20 mM acetate buffer. In one example, the elution buffer comprises 20 mM sodium acetate buffer.

[0084] In one example, the elution buffer comprises 10 mM, or 11 mM, or 12 mM, or 13 mM, or 14 mM, or 15 mM, or 16 mM, or 17 mM, or 18 mM, or 19 mM, or 20 mM phosphate buffer. For example, the elution buffer comprises 20 mM phosphate buffer. In one example, the elution buffer comprises 20 mM sodium phosphate buffer.

[0085] In one example, the elution buffer contains 20 mM sodium acetate at pH 4.

[0086] In one example, the elution buffer further comprises sodium chloride. For example, the elution buffer further comprises sodium chloride at a concentration of up to 150 mM. In one example, the sodium chloride is at a concentration of 50-100 mM. For example, the sodium chloride is at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In another example, the sodium chloride is at a concentration of 100-150 mM. For example, sodium chloride is at a concentration of 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, or 150 mM.

[0087] In one example, the elution buffer further comprises a divalent salt. For example, the elution buffer further comprises a divalent salt at a concentration of up to 150 mM. In one example, the divalent salt is at a concentration of 50-100 mM. For example, the divalent salt is at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In another example, the concentration of the divalent salt is 100-150 mM. For example, the divalent salt is at a concentration of 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, or 150 mM.

[0088] In one example, the divalent salt is selected from the group consisting of magnesium chloride, calcium chloride, barium chloride, copper(II) chloride, nickel chloride, manganese chloride, and combinations thereof. For example, the divalent salt is magnesium chloride. For example, the divalent salt is calcium chloride. In one example, the divalent salt is barium chloride. In another example, the divalent salt is copper chloride. In a further example, the divalent salt is nickel chloride. In one example, the divalent salt is manganese chloride.

[0089] In one example, the method further comprises equilibrating the resin with an equilibration buffer, e.g., equilibrating the resin prior to loading the plasma or a fraction thereof containing IgG onto the resin.

[0090] In one example, the method further comprises equilibrating the resin with an equilibration buffer having a pH of 5 to 9. For example, the equilibration buffer has a pH of 5, or 5.1, or 5.2, or 5.3, or 5.4, or 5.5, or 5.6, or 5.7, or 5.8, or 5.9, or 6.0, or 6.1, or 6.2, or 6.3, or 6.4, or 6.5, or 6.6, or 6.7, or 6.8, or 6.9, or 7, or 7.1, or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7, or 7.8, or 7.9, or 8.0, or 8.1, or 8.2, or 8.3, or 8.4, or 8.5, or 8.6, or 8.7, or 8.8, or 8.9, or 9.0.

[0091] In one example, the method further comprises equilibrating the resin with an equilibration buffer having a pH of 7 to 8. For example, the equilibration buffer has a pH of 7, or 7.1, or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7, or 7.8, or 7.9, or 8. In one example, the equilibration buffer has a pH of 7.4.

[0092] In one example, the equilibration buffer comprises a buffer selected from the group consisting of sodium dihydrogen phosphate, sodium citrate, imidazole, Tris, glycylglycine, MOPS, PIPES, TES, Bicine, HEPES, EPPS, HEPPSO, MOBS, POPSO, TAPSO, Tricine, TEA, and combinations thereof. For example, the equilibration buffer is a sodium dihydrogen phosphate buffer. In another example, the equilibration buffer is a sodium citrate buffer. In a further example, the equilibration buffer is an imidazole buffer. In one example, the equilibration buffer is a Tris buffer. In another example, the equilibration buffer is a glycylglycine buffer. In a further example, the equilibration buffer is a MOPS buffer. In one example, the equilibration buffer is a PIPES buffer. In another example, the equilibration buffer is a TES buffer. In a further example, the equilibration buffer is a Bicine buffer. In one example, the equilibration buffer is a sulfite buffer. In another example, the equilibration buffer is an EPPS buffer. In a further example, the equilibration buffer is a HEPPSO buffer. In one example, the equilibration buffer is a MOBS buffer. In another example, the equilibration buffer is a POPSO buffer. In a further example, the equilibration buffer is a TAPSO buffer. In one example, the equilibration buffer is a Tricine buffer. In another example, the equilibration buffer is a TEA buffer.

[0093] In one example, the buffering agent of the equilibration buffer is at a concentration of 5 mM to 200 mM. In one example, the buffering agent of the equilibration buffer is at a concentration of 5 mM to 50 mM, for example, at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM. In another example, the buffering agent of the equilibration buffer is at a concentration of 50 mM to 100 mM, for example, at a concentration of 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM. In a further example, the equilibration buffer is at a concentration of 100 mM to 150 mM, such as 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM. In one example, the equilibration buffer is at a concentration of 150 mM to 200 mM, such as 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM.

[0094] In one example, the buffering agent in the equilibration buffer is at a concentration of 5 mM.

[0095] In one example, the buffering agent in the equilibration buffer is at a concentration of 20 mM.

[0096] In one example, the buffering agent in the equilibration buffer is at a concentration of 50 mM.

[0097] In one example, the buffering agent in the equilibration buffer is at a concentration of 100 mM.

[0098] In one example, the buffering agent in the equilibration buffer is at a concentration of 150 mM.

[0099] In one example, the buffering agent in the equilibration buffer is at a concentration of 200 mM.

[0100] In one example, the equilibration buffer further comprises sodium chloride. For example, the equilibration buffer further comprises sodium chloride at a concentration of up to 1000 mM. In one example, the sodium chloride is at a concentration of 5 mM to 50 mM. For example, the sodium chloride is at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM. In another example, the sodium chloride is at a concentration of 50 mM to 100 mM. For example, the sodium chloride is at a concentration of 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM. In one example, the sodium chloride is at a concentration of 100 to 200 mM. For example, sodium chloride is at a concentration of 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM. In other examples, sodium chloride is at a concentration of 200 to 300 mM. For example, sodium chloride is at a concentration of 200 mM, or 225 mM, or 250 mM, or 275 mM, or 300 mM. In further examples, sodium chloride is at a concentration of 300 mM to 400 mM. For example, sodium chloride is at a concentration of 300 mM, or 325 mM, or 350 mM, or 375 mM, or 400 mM. In one example, sodium chloride is at a concentration of 400 mM to 500 mM. For example, sodium chloride is at a concentration of 400 mM, or 425 mM, or 450 mM, or 475 mM, or 400 mM. In another example, sodium chloride is at a concentration of 500 mM to 1000 mM. For example, sodium chloride is at a concentration of 500 mM, or 550 mM, or 600 mM, or 650 mM, or 700 mM, or 750 mM, or 800 mM, or 850 mM, or 900 mM, or 950 mM, or 1000 mM. In one example, sodium chloride is at a concentration of less than 1000 mM.For example, sodium chloride is at a concentration of 500 mM.

[0101] In one example, the equilibration buffer further comprises sodium chloride, where the sodium chloride is at a concentration of 145 mM.

[0102] In one example, the equilibration buffer further comprises sodium chloride, where the sodium chloride is at a concentration of 500 mM.

[0103] In one example, the equilibration buffer further comprises a divalent salt. For example, the equilibration buffer further comprises a divalent salt at a concentration of up to 1000 mM. In one example, the divalent salt is at a concentration of 5 mM to 50 mM. For example, the divalent salt is at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM. In another example, the divalent salt is at a concentration of 50 mM to 100 mM. For example, the divalent salt is at a concentration of 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM. In one example, the divalent salt is at a concentration of 100 to 200 mM. For example, the divalent salt is at a concentration of 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM. In other examples, the divalent salt is at a concentration of 200 mM to 300 mM. For example, the divalent salt is at a concentration of 200 mM, or 225 mM, or 250 mM, or 275 mM, or 300 mM. In further examples, the divalent salt is at a concentration of 300 mM to 400 mM. For example, the divalent salt is at a concentration of 300 mM, or 325 mM, or 350 mM, or 375 mM, or 400 mM. In one example, the divalent salt is at a concentration of 400 mM to 500 mM. For example, the divalent salt is at a concentration of 400 mM, or 425 mM, or 450 mM, or 475 mM, or 400 mM. In another example, the divalent salt is at a concentration of 500 mM to 1000 mM. For example, the divalent salt is at a concentration of 500 mM, or 550 mM, or 600 mM, or 650 mM, or 700 mM, or 750 mM, or 800 mM, or 850 mM, or 900 mM, or 950 mM, or 1000 mM. In one example, the divalent salt is at a concentration of less than 1000 mM. For example, the divalent salt is at a concentration of 500 mM.

[0104] In one example, the divalent salt is selected from the group consisting of magnesium chloride, calcium chloride, barium chloride, copper(II) chloride, nickel chloride, manganese chloride, and combinations thereof. For example, the divalent salt is magnesium chloride. In one example, the divalent salt is calcium chloride. In another example, the divalent salt is barium chloride. In a further example, the divalent salt is copper chloride. In one example, the divalent salt is nickel chloride. In another example, the divalent salt is manganese chloride.

[0105] In one example, the composition of the equilibration buffer is the same as that of the wash buffer. For example, the equilibration buffer contains 20 mM sodium dihydrogen phosphate, 145 mM sodium chloride, and a pH of 7.4. In another example, the equilibration buffer contains 20 mM sodium dihydrogen phosphate, 500 mM sodium chloride, and a pH of 7.4.

[0106] In one example, the resin is equilibrated i) after stripping the resin, or ii) without stripping the resin. For example, the resin is equilibrated after stripping the resin. In another example, the resin is equilibrated without stripping.

[0107] In one example, the method further comprises, after stripping the resin, equilibrating the resin with an equilibration buffer having a pH of 7-8.

[0108] In one example, the method optionally includes stripping the resin with a stripping buffer after recovering the IgG from the resin. For example, the method further includes stripping the resin with a stripping buffer after recovering the IgG from the resin. In another example, the method does not include stripping the resin with a stripping buffer after recovering the IgG from the resin. For example, the resin is not stripped after recovering the IgG from the resin.

[0109] In one example, the stripping buffer has a pH of 2 to 3. For example, the stripping buffer has a pH of 2, or 2.1, or 2.2, or 2.3, 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3. In one example, the pH of the stripping buffer is 2.5.

[0110] In one example, the stripping buffer comprises a buffer selected from the group consisting of sodium dihydrogen phosphate, glycine and sodium citrate. For example, the stripping buffer comprises sodium dihydrogen phosphate. For example, the stripping buffer comprises glycine. For example, the stripping buffer comprises sodium citrate.

[0111] In one example, the buffering agent of the stripping buffer has a concentration of 10 mM to 500 mM. For example, the buffering agent of the stripping buffer has a concentration of 10 mM to 20 mM, or 10 mM to 50 mM, or 10 mM to 100 mM, or 10 mM to 200 mM, or 10 mM to 300 mM, or 10 mM to 400 mM. For example, the buffering agent of the stripping buffer has a concentration of 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM, or 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM, or 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM, or 210 mM , or 220 mM, or 230 mM, or 240 mM, or 250 mM, or 260 mM, or 270 mM, or 280 mM, or 290 mM, or 300 mM, or 210 mM, or 220 mM, or 230 mM, or 240 mM, or 250 mM, or 260 mM, or 270 mM, or 280 mM, or 290 mM, or 300 mM, or 310 mM M, or 320 mM, or 330 mM, or 340 mM, or 350 mM, or 360 mM, or 370 mM, or 380 mM, or 390 mM, or 400 mM, or 410 mM, or 420 mM, or 430 mM, or 440 mM, or 450 mM, or 460 mM, or 470 mM, or 480 mM, or 490 mM, or 500 mM.

[0112] In one example, the buffering agent in the stripping buffer is at a concentration of 5 mM.

[0113] In one example, the buffering agent of the stripping buffer is at a concentration of 20 mM.

[0114] In one example, the buffering agent of the stripping buffer is at a concentration of 50 mM.

[0115] In one example, the buffering agent of the stripping buffer is at a concentration of 100 mM.

[0116] In one example, the buffering agent of the stripping buffer is at a concentration of 150 mM.

[0117] In one example, the buffering agent of the stripping buffer is at a concentration of 200 mM.

[0118] In one example, the buffering agent of the stripping buffer is at a concentration of 250 mM.

[0119] In one example, the buffering agent of the stripping buffer is at a concentration of 300 mM.

[0120] In one example, the buffering agent of the stripping buffer is at a concentration of 350 mM.

[0121] In one example, the buffering agent of the stripping buffer is at a concentration of 400 mM.

[0122] In one example, the buffering agent of the stripping buffer is at a concentration of 450 mM.

[0123] In one example, the buffering agent of the stripping buffer is at a concentration of 500 mM.

[0124] In one example, the stripping buffer contains 20 mM sodium dihydrogen phosphate, pH 2.5.

[0125] In one example, the stripping buffer further comprises sodium chloride. For example, the stripping buffer further comprises sodium chloride at a concentration of up to 1000 mM. In one example, the sodium chloride is at a concentration of 5 mM to 50 mM. For example, the sodium chloride is at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM. In another example, the sodium chloride is at a concentration of 50 mM to 100 mM. For example, the sodium chloride is at a concentration of 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM. In one example, the sodium chloride is at a concentration of 100 to 200 mM. For example, sodium chloride is at a concentration of 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM. In other examples, sodium chloride is at a concentration of 200 to 300 mM. For example, sodium chloride is at a concentration of 200 mM, or 225 mM, or 250 mM, or 275 mM, or 300 mM. In further examples, sodium chloride is at a concentration of 300 mM to 400 mM. For example, sodium chloride is at a concentration of 300 mM, or 325 mM, or 350 mM, or 375 mM, or 400 mM. In one example, sodium chloride is at a concentration of 400 mM to 500 mM. For example, sodium chloride is at a concentration of 400 mM, or 425 mM, or 450 mM, or 475 mM, or 400 mM. In another example, sodium chloride is at a concentration of 500 mM to 1000 mM. For example, sodium chloride is at a concentration of 500 mM, or 550 mM, or 600 mM, or 650 mM, or 700 mM, or 750 mM, or 800 mM, or 850 mM, or 900 mM, or 950 mM, or 1000 mM. In one example, sodium chloride is at a concentration of less than 1000 mM.

[0126] In one example, the stripping buffer further comprises a divalent salt. For example, the stripping buffer further comprises a divalent salt at a concentration of up to 1000 mM. In one example, the divalent salt is at a concentration of 5 mM to 50 mM. For example, the divalent salt is at a concentration of 5 mM, or 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM. In another example, the divalent salt is at a concentration of 50 mM to 100 mM. For example, the divalent salt is at a concentration of 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, or 95 mM, or 100 mM. In one example, the divalent salt is at a concentration of 100 to 200 mM. For example, the divalent salt is at a concentration of 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM, or 155 mM, or 160 mM, or 165 mM, or 170 mM, or 175 mM, or 180 mM, or 185 mM, or 190 mM, or 195 mM, or 200 mM. In other examples, the divalent salt is at a concentration of 200 mM to 300 mM. For example, the divalent salt is at a concentration of 200 mM, or 225 mM, or 250 mM, or 275 mM, or 300 mM. In further examples, the divalent salt is at a concentration of 300 mM to 400 mM. For example, the divalent salt is at a concentration of 300 mM, or 325 mM, or 350 mM, or 375 mM, or 400 mM. In one example, the divalent salt is at a concentration of 400 mM to 500 mM. For example, the divalent salt is at a concentration of 400 mM, or 425 mM, or 450 mM, or 475 mM, or 400 mM. In another example, the divalent salt is at a concentration of 500 mM to 1000 mM. For example, the divalent salt is at a concentration of 500 mM, or 550 mM, or 600 mM, or 650 mM, or 700 mM, or 750 mM, or 800 mM, or 850 mM, or 900 mM, or 950 mM, or 1000 mM. In one example, the divalent salt is at a concentration of less than 1000 mM.

[0127] In one example, the divalent salt is selected from the group consisting of magnesium chloride, calcium chloride, barium chloride, copper(II) chloride, nickel chloride, manganese chloride, and combinations thereof. For example, the divalent salt is magnesium chloride. For example, the divalent salt is calcium chloride. For example, the divalent salt is barium chloride. For example, the divalent salt is copper chloride. For example, the divalent salt is nickel chloride. For example, the divalent salt is manganese chloride.

[0128] In one example, the resin is equilibrated. In one example, the resin is equilibrated after stripping the resin. In one example, the method further comprises equilibrating the resin with an equilibration buffer having a pH of 7-8 after stripping the resin.

[0129] In one example, the method includes: a) equilibrating the resin with an equilibration buffer having a pH of 7-8; b) after recovering the bound IgG from the resin, stripping the resin with a stripping buffer having a pH between 2 and 3; and / or c) Stripping the resin and then equilibrating the resin with equilibration buffer. Includes.

[0130] In one example, the resin is equilibrated without stripping. For example, the method includes equilibrating the resin with an equilibration buffer after recovering bound IgG from the resin, without stripping the resin with a stripping buffer. In one example, the method includes equilibrating the resin with an equilibration buffer having a pH of 7-8 after recovering bound IgG from the resin.

[0131] In one example, the method further comprises regenerating the resin.

[0132] In one example, the method further comprises sterilizing the resin.

[0133] In one example, the method includes loading plasma or a fraction thereof onto an affinity chromatography resin.

[0134] In one example, plasma or its fraction contacts with resin during loading of plasma or its fraction.For example, plasma or its fraction contacts with resin for at least 0.25 minutes, or 0.5 minutes, or 1 minute, or 1.5 minutes, or 2 minutes, or 2.5 minutes, or 3 minutes, or 3.5 minutes, or 4 minutes, or 4.5 minutes, or 5 minutes.For example, plasma or its fraction contacts with resin for 0.1 minutes, 0.25 minutes, 0.3 minutes, 0.35 minutes, 0.4 minutes, 0.45 minutes, 0.5 minutes, or 1 minute, or 1.5 minutes, or 2 minutes, or 2.5 minutes, or 3 minutes, or 3.5 minutes, or 4 minutes, or 4.5 minutes, or 5 minutes.

[0135] In one example, the plasma or fraction thereof is in contact with the resin for up to 5 minutes during loading of the plasma or fraction thereof.

[0136] In one example, the plasma or fraction thereof is contacted with the resin for 0.25 to 5 minutes during loading of the plasma or fraction thereof. For example, during loading, the plasma or fraction thereof is contacted with the resin for 0.25 minutes, 0.3 minutes, 0.35 minutes, 0.4 minutes, 0.45 minutes, 0.5 minutes, or 1 minute, or 1.5 minutes, or 2 minutes, or 2.5 minutes, or 3 minutes, or 3.5 minutes, or 4 minutes, or 4.5 minutes, or 5 minutes. In one example, during loading, the plasma or fraction thereof is contacted with the resin for at least 0.25 minutes.

[0137] In one example, the buffer is contacted with the resin for at least 0.1 minutes in one or more non-loading phase(s) of the method.

[0138] In one example, during one or more non-loading phase(s) of the continuous chromatography method, the buffer is in contact with the resin for up to 5 minutes.

[0139] In one example, during one or more non-loading phase(s) of the continuous chromatography method, the buffer contacts the resin for 0.1 to 5 minutes, e.g., the buffer contacts the resin for at least 0.1 minutes, or 0.25 minutes, or 0.5 minutes, or 1 minute, or 1.5 minutes, or 2 minutes, or 2.5 minutes, or 3 minutes, or 3.5 minutes, or 4 minutes, or 4.5 minutes, or 5 minutes.

[0140] In one example, the non-loading phase is selected from the group consisting of an equilibration phase, a wash phase, an elution phase, a strip phase, a re-equilibration phase and combinations thereof.

[0141] In one example, the unloading phase is a balancing phase.

[0142] In one example, the non-loading phase is a wash phase.

[0143] In one example, the non-loading phase is a dissolution phase.

[0144] In one example, the non-loading phase is a strip phase.

[0145] In one example, the unloading phase is a re-equilibration phase.

[0146] In one example, the buffer that contacts the resin during one or more non-loading phase(s) of the continuous chromatography method is selected from the group consisting of an equilibration buffer, a wash buffer, a stripping buffer, and a re-equilibration buffer. For example, the buffer is an equilibration buffer. In another example, the buffer is a wash buffer. In a further example, the buffer is a stripping buffer. In one example, the buffer is a re-equilibration buffer.

[0147] In one example, the equilibration buffer is contacted with the resin for 0.1 to 5 minutes.

[0148] In one example, the wash buffer is in contact with the resin for 0.1 to 5 minutes.

[0149] In one example, the elution buffer is contacted with the resin for 0.1 to 5 minutes.

[0150] In one example, the stripping buffer is in contact with the resin for 0.1 to 5 minutes.

[0151] In one example, the method includes contacting the resin with a volume of elution buffer less than a column volume (CV) before recovering the bound IgG from the resin. For example, the method includes a "pre-elution" phase in which the resin is contacted with a volume of elution buffer less than a column volume (CV) before recovering the bound IgG from the resin. In one example, the method includes washing the resin with a volume of elution buffer less than a CV before recovering the bound IgG from the resin. For example, the volume of elution buffer used to wash the resin before recovering the bound IgG from the resin is up to 0.5 CV. For example, the volume of elution buffer used to wash the resin before recovering the bound IgG from the resin is 0.5 to 1.0 CV. For example, the volume of elution buffer used to wash the resin before recovering the bound IgG from the resin is 0.1 CV, or 0.2 CV, or 0.3 CV, or 0.4 CV, or 0.5 CV, or 0.6 CV, or 0.7 CV, or 0.8 CV, or 0.9 CV.

[0152] In one example, the volume of the elution buffer is 0.1 CV.

[0153] In one example, the volume of the elution buffer is 0.2 CV.

[0154] In one example, the volume of the elution buffer is 0.3 CV.

[0155] In one example, the volume of the elution buffer is 0.4 CV.

[0156] In one example, the volume of the elution buffer is 0.5 CV.

[0157] In one example, the volume of the elution buffer is 0.6 CV.

[0158] In one example, the volume of the elution buffer is 0.7 CV.

[0159] In one example, the volume of the elution buffer is 0.8 CV.

[0160] In one example, the volume of the elution buffer is 0.9 CV.

[0161] In one example, the method comprises contacting the resin with a volume of elution buffer less than the CV, followed by elution of bound IgG from the resin.

[0162] It will be apparent to those skilled in the art that IgG is normally present in plasma at a concentration of 5-15 g / L.

[0163] In one example, the plasma fraction is selected from the group consisting of cryo-rich plasma, cryo-opa plasma, supernatant I (SNI), Cohn fraction II (Fr II), Cohn fraction II+III (Fr II+III), Cohn fraction I+II+III (FrI+II+III), Kisler / Nitschmann precipitate A (KN A), Kisler / Nitschmann precipitate B (KN B), Kisler / Nitschmann supernatant precipitate B (KN B+1), and combinations thereof. In one example, the plasma fraction is cryo-rich plasma. For example, the plasma fraction is cryo-opa plasma. For example, the plasma fraction is supernatant I (SN I). For example, the plasma fraction is Cohn fraction II (Fr II). For example, the plasma fraction is Cohn fraction II+III (Fr II+III). For example, the plasma fraction is Cohn fraction I+II+III (FrI+II+III). For example, the plasma fraction is Kissler / Nitschmann Precipitate A (KN A). For example, the plasma fraction is Kissler / Nitschmann Precipitate B (KN B). For example, the plasma fraction is Kissler / Nitschmann Supernatant Precipitate B (KN B+1).

[0164] In one example, the plasma fraction is a suspension paste. For example, the suspension paste is selected from the group consisting of Kohn fraction II (Fr II), Kohn fraction II+III (Fr II+III), Kohn fraction I+II+III (FrI+II+III), Kisler / Nitschmann precipitate A (KN A), Kisler / Nitschmann precipitate B (KN B), Kisler / Nitschmann supernatant precipitate B (KN B+1), and combinations thereof. For example, the suspension paste is Kohn fraction II (Fr II) paste. In one example, the suspension paste is Kohn fraction II+III (Fr II+III) paste. In another example, the suspension paste is Kohn fraction I+II+III (FrI+II+III) paste. In another example, the suspension paste is Kisler / Nitschmann precipitate A (KN A) paste. In another example, the suspension paste is Kisler / Nitschmann precipitate B (KN B) paste. In a further example, the suspension paste is Kisler / Nitschmann Supernatant Precipitate B (KN B+1) paste.

[0165] In one example, the plasma fraction is selected from the group consisting of a mammalian plasma fraction, a human plasma fraction, an equine plasma fraction, and a bovine plasma fraction.

[0166] In one example, the plasma fraction is a mammalian plasma fraction.

[0167] In one example, the plasma fraction is a human plasma fraction.

[0168] In one example, the plasma fraction is a horse plasma fraction.

[0169] In one example, the plasma fraction is a bovine plasma fraction.

[0170] In one example, the plasma fraction is a bovine plasma fraction containing human polyclonal antibodies.

[0171] In one example, the plasma or a fraction thereof is clarified. Methods for clarifying the plasma or a fraction thereof will be apparent to those skilled in the art and / or described herein. For example, the plasma or a fraction thereof is clarified by passing the plasma or a fraction thereof through a filter. For example, a depth filter or a membrane filter can be used. For example, the plasma or a fraction thereof is passed through a combination of filters. For example, the combination can be a combination of 1.2 μm and 0.45 / 0.22 μm membrane filters. For example, the plasma or a fraction thereof is clarified by passing the plasma or a fraction thereof through a depth filter (e.g., a BECO® depth filter). In one example, the plasma or a fraction thereof is clarified by passing the plasma or a fraction thereof through a filter press (e.g., a BECO® Integra plate or compact plate) that includes one or more depth filter(s). In one example, the filter press further includes one or more filter aid(s) (e.g., a cellulose-based filter aid such as Diacel® 150). In one example, the plasma or a fraction thereof is clarified by passing the plasma or a fraction thereof through a lipid-specific filter (e.g., a Zeta Plus™ DEL series filter). For example, the plasma fraction is a clarified supernatant I (SN I). For example, the plasma fraction is a clarified Cohn fraction II (Fr II). For example, the plasma fraction is a clarified Cohn fraction II+III (Fr II+III). For example, the plasma fraction is a clarified Cohn fraction I+II+III (FrI+II+III). For example, the plasma fraction is a clarified Kisler / Nitschmann precipitate A (KN A). For example, the plasma fraction is a clarified Kisler / Nitschmann precipitate B (KN B). For example, the plasma fraction is a clarified Kisler / Nitschmann supernatant precipitate B (KN B+1).

[0172] In one example, the plasma is clarified cryo-rich plasma.

[0173] In one example, the plasma fraction is clarified cryopreserved plasma.

[0174] In one example, the plasma or a fraction thereof is warmed to a first temperature of about 32° C. and then cooled to a second temperature of about 21° C. prior to the successive affinity chromatography steps. In one example, the plasma or a fraction thereof is at a first temperature of about 32° C. and then at a second temperature of about 21° C. prior to the successive affinity chromatography steps.

[0175] In one example, the plasma or a fraction thereof is at a temperature in the range of 2° C. to 35° C. prior to the successive affinity chromatography steps. In one example, the plasma or a fraction thereof is at a temperature in the range of 2° C. to 28° C. prior to the successive affinity chromatography steps. For example, the plasma or a fraction thereof is at a temperature in the range of 10° C. to 28° C., for example, 10° C., or 11° C., or 12° C., or 13° C., or 14° C., 15° C., or 16° C., or 17° C., or 18° C., or 19° C., or 20° C., or 21° C., or 22° C., or 23° C., or 24° C., or 25° C., or 26° C., or 27° C., or 28° C. , or 11°C, or 12°C, or 13°C, or 14°C, 15°C, or 16°C, or 17°C, or 18°C, or 19°C, or 20°C, or 21°C, or 22°C, or 23°C, or 24°C, or 25°C, or 26°C, or 27°C, or 28°C, or 29°C, or 30°C, or 31°C, or 32°C, or 33°C, or 34°C, or 35°C. In one example, the plasma or fraction thereof is at a temperature in the range of 2°C to 35°C prior to loading onto the sequential affinity chromatography resin. In one example, the plasma or fraction thereof is at a temperature in the range of 2°C to 28°C prior to loading onto the sequential affinity chromatography resin. For example, the temperature is in the range of 10°C to 35°C. For example, the plasma or fraction thereof is at a temperature in the range of 30°C to 35°C. For example, the plasma or fraction thereof is at a temperature of at least 32°C. For example, the plasma or fraction thereof is at a temperature in the range of 32°C to 35°C. In one example, the plasma or fraction thereof is at a temperature of 32°C. For example, the temperature is in the range of 10°C to 28°C. In one example, the plasma or fraction thereof is at a temperature in the range of 2°C to 25°C. For example, the temperature is in the range of 10°C to 25°C. In one example, the plasma or fraction thereof is at a temperature in the range of 20°C to 25°C. For example, the plasma or fraction thereof is at a temperature of 21°C. In one example, the plasma or fraction thereof is at a temperature in the range of 2°C to 20°C. For example, the temperature is in the range of 10°C to 20°C. In one example, the plasma or fraction thereof is at a temperature in the range of 2°C to 18°C. For example, the temperature is in the range of 10°C to 18°C. In one example, the plasma or fraction thereof is at a temperature in the range of 2°C to 15°C.For example, the temperature is in the range of 10° C. to 15° C. In one example, the plasma or fraction thereof is at a temperature in the range of 2° C. to 10° C. For example, the plasma or fraction thereof is at a temperature of 2° C., or 3° C., or 4° C., or 5° C., or 6° C., or 7° C., or 8° C., or 9° C., or 10° C.

[0176] In one example, the plasma or fraction thereof is at a temperature of 2°C.

[0177] In one example, the plasma or fraction thereof is at a temperature of 10°C.

[0178] In one example, the plasma or fraction thereof is at a temperature of 18°C.

[0179] In one example, the plasma or fraction thereof is at a temperature of 21°C.

[0180] In one example, the plasma or fraction thereof is at a temperature of 28°C.

[0181] In one example, the plasma or fraction thereof is at a temperature of 32°C.

[0182] In one example, the plasma or fraction thereof is at that temperature for up to 48 hours. For example, the plasma or fraction thereof is held at that temperature for up to 48 hours before loading the plasma or fraction thereof onto a continuous affinity chromatography resin. In one example, the plasma or fraction thereof is held at that temperature for up to 2 hours, or 4 hours, or 6 hours, or 8 hours, or 10 hours, or 12 hours, or 14 hours, or 16 hours, or 18 hours, or 20 hours, or 22 hours, or 24 hours, or 26 hours, or 28 hours, or 30 hours, or 32 hours, or 34 hours, or 36 hours, or 38 hours, or 40 hours, or 42 hours, or 44 hours, or 46 hours before loading. For example, the plasma or fraction thereof is held at that temperature for 0-2 hours, 2-24 hours, or 4-24 hours, or 8-24 hours, or 12-24 hours, or 18-24 hours, or 24-48 hours, or 36-48 hours prior to loading.

[0183] In one example, the plasma or a fraction thereof is at a first temperature in the range of 30°C to 38°C and then at a second temperature in the range of 2°C to 28°C prior to the successive affinity chromatography steps. For example, the plasma or a fraction thereof is warmed to a first temperature in the range of 30°C to 38°C and then cooled to a second temperature in the range of 2°C to 28°C prior to the successive affinity chromatography steps. In one example, the plasma or a fraction thereof is warmed to a first temperature in the range of 30°C to 35°C and then cooled to a second temperature in the range of 18°C ​​to 25°C prior to the successive affinity chromatography steps. For example, the plasma or a fraction thereof is warmed to a first temperature of about 30°C, or about 31°C, or about 32°C, or about 33°C, or about 34°C, or about 35°C. In one example, the plasma or fraction thereof is cooled to a second temperature of about 18°C, or about 19°C, or about 20°C, or about 21°C, or about 22°C, or about 23°C, or about 24°C, or about 25°C.

[0184] In one example, the plasma or fraction thereof is at the first and / or second temperature for up to 48 hours. For example, the plasma or fraction thereof is held at the first and / or second temperature for up to 48 hours before loading the plasma or fraction thereof onto the sequential affinity chromatography resin. In one example, the plasma or fraction thereof is held at the first and / or second temperature for up to 2 hours, or 4 hours, or 6 hours, or 8 hours, or 10 hours, or 12 hours, or 14 hours, or 16 hours, or 18 hours, or 20 hours, or 22 hours, or 24 hours, or 26 hours, or 28 hours, or 30 hours, or 32 hours, or 34 hours, or 36 hours, or 38 hours, or 40 hours, or 42 hours, or 44 hours, or 46 hours before loading. For example, the plasma or fraction thereof is held at the first and / or second temperature for 0-2 hours, 2-24 hours, or 4-24 hours, or 8-24 hours, or 12-24 hours, or 18-24 hours, or 24-48 hours, or 36-48 hours prior to loading.

[0185] In one example, the continuous affinity chromatography is selected from the group consisting of simulated moving bed (SMB) chromatography, cyclic countercurrent chromatography (PCC), continuous countercurrent tangential chromatography (CCTC), and continuous countercurrent spiral chromatography (CCSC).

[0186] In one example, the continuous affinity chromatography is simulated moving bed (SMB) chromatography. In another example, the continuous affinity chromatography is cyclic countercurrent chromatography (PCC). In a further example, the continuous affinity chromatography is continuous countercurrent tangential chromatography (CCTC). In one example, the continuous affinity chromatography is continuous countercurrent spiral chromatography (CCSC).

[0187] In one example, the resin is in the form of a slurry, e.g., the resin includes resin particles in the form of a slurry.

[0188] In one example, the slurry is passed through one or more columns, each of which contains a membrane, for example, the membrane is a hollow fiber membrane.

[0189] In one example, the slurry is passed through a series of two or more columns containing membranes, for example, the slurry is passed through 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12 columns.

[0190] In one example, the slurry is passed through a series of two columns.

[0191] In one example, the slurry is passed through a series of three columns.

[0192] In one example, the slurry is passed through a series of four columns.

[0193] In one example, the slurry is passed through a series of five columns.

[0194] In one example, the slurry is passed through a series of six columns.

[0195] In one example, the slurry is passed through a series of seven columns.

[0196] In one example, the slurry is passed through a series of eight columns.

[0197] In one example, the slurry is passed through a series of nine columns.

[0198] In one example, the slurry is passed through a series of 10 columns.

[0199] In one example, the slurry is passed through a series of 11 columns.

[0200] In one example, the slurry is passed through a series of 12 columns.

[0201] In one example, the resin is packed into one or more columns, each column containing one or more zones. For example, the resin is packed into a series of two or more columns. For example, the resin is packed into a series of two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve columns.

[0202] In one example, the resin is packed into two columns in series.

[0203] In one example, the resin is packed into a series of three columns.

[0204] In one example, the resin is packed into a series of four columns.

[0205] In one example, the resin is packed into a series of five columns.

[0206] In one example, the resin is packed into a series of six columns.

[0207] In one example, the resin is packed into a series of seven columns.

[0208] In one example, the resin is packed into a series of eight columns.

[0209] In one example, the resin is packed into a series of nine columns.

[0210] In one example, the resin is packed into a series of 10 columns.

[0211] In one example, the resin is packed into a series of 11 columns.

[0212] In one example, the resin is packed into a series of 12 columns.

[0213] For example, the zone is selected from the group consisting of an equilibration zone, a binding zone, a wash zone, an elution zone, a stripping zone, and combinations thereof. In other examples, the zone is selected from the group consisting of an equilibration zone, a binding zone, a wash zone, an elution zone, and combinations thereof. In one example, the zone is an equilibration zone. In another example, the zone is a binding zone. In a further example, the zone is a wash zone. In one example, the zone is an elution zone. In another example, the zone is a stripping zone. In one example, there is no stripping zone. In a further example, the zone is a wash / elution zone. In one example, the zone is an equilibration / binding zone. In another example, the zone is a binding / wash zone.

[0214] In one example, the resin is packed into one or more column(s), each column containing one zone.

[0215] In one example, the resin is packed into one or more column(s), each column containing two zones.

[0216] In one example, the resin is packed into one or more column(s), each column containing four zones.

[0217] In one example, two or more columns are fluidly connected and separated by a fluid conduit that includes inlet and outlet valves.

[0218] In one example, the resin is packed into a first column followed by one or more subsequent column(s).

[0219] In one example, the first column is loaded with IgG at a concentration that exceeds the dynamic binding capacity (DBC) of the resin.

[0220] Determining the DBC of resin is clear to those skilled in the art and / or described herein.For example, the DBC of resin can be determined by loading IgG into a column and monitoring the concentration of unbound IgG flowing through the column, for example by UV tracing in a chromatography system.For example, the DBC of resin is 5mg, or 10mg, or 20mg, or 30mg, or 40mg, or 50mg, or 60mg, or 70mg of IgG per mL of resin.

[0221] In one example, the DBC of the resin is at least 5 mg IgG per mL of resin.

[0222] In one example, the DBC of the resin is at least 10 mg IgG per mL of resin.

[0223] In one example, the DBC of the resin is at least 20 mg IgG per mL of resin.

[0224] In one example, the DBC of the resin is 40 mg IgG per mL of resin.

[0225] In one example, the first column is loaded with IgG at a concentration of greater than 5 mg, or 10 mg, or 20 mg, or 30 mg, or 40 mg, or 50 mg, or 60 mg, or 70 mg per mL of resin.

[0226] In one example, the first column is loaded with IgG at a concentration up to the DBC of the resin, for example, the first column is loaded with IgG at a concentration of up to 5 mg, or 10 mg, or 20 mg, or 30 mg, or 40 mg per mL of resin.

[0227] In one example, the first column is loaded with IgG at a concentration of greater than 5 mg IgG per mL of resin.

[0228] In one example, the first column is loaded with IgG at a concentration of greater than 10 mg IgG per mL of resin.

[0229] In one example, the first column is loaded with IgG at a concentration of greater than 20 mg IgG per mL of resin.

[0230] In one example, the first column is loaded with IgG at a concentration of up to 40 mg IgG per mL of resin.

[0231] In one example, one or more subsequent column(s) are loaded with IgG at a concentration up to the DBC of the resin.

[0232] In one example, one or more subsequent column(s) are loaded with IgG at a concentration of up to 5 mg, or 10 mg, or 20 mg, or 30 mg, or 40 mg of IgG per mL of resin.

[0233] In one example, one or more subsequent column(s) are loaded with IgG at a concentration of up to 20 mg IgG per mL of resin.

[0234] In one example, one or more subsequent column(s) are loaded with IgG at a concentration of up to 30 mg IgG per mL of resin.

[0235] In one example, one or more subsequent column(s) are loaded with IgG at a concentration of up to 40 mg IgG per mL of resin.

[0236] In one example, the method further comprises washing unbound IgG from the first column to one or more subsequent column(s) with a wash buffer and recovering bound IgG. For example, bound IgG is recovered from the first column and one or more subsequent column(s). For example, bound IgG is recovered from the first column without a wash step. For example, bound IgG is recovered from the first column by eluting bound IgG with an elution buffer as described herein. For example, bound IgG is recovered from one or more subsequent column(s) after washing with a wash buffer as described herein. For example, bound IgG is recovered from one or more subsequent column(s) after washing the resin with a wash buffer and eluting bound IgG with an elution buffer as described herein.

[0237] In one example, the method further comprises washing the one or more subsequent column(s) with a wash buffer described herein and recovering the bound IgG from the one or more subsequent column(s).

[0238] In one example, the method further comprises stripping and / or equilibrating the first column at which point the bound IgG is recovered from the one or more subsequent column(s). In one example, the method further comprises equilibrating the first column at which point the bound IgG is recovered from the one or more subsequent column(s). For example, the method does not include stripping the first column at which point the bound IgG is recovered from the one or more subsequent column(s).

[0239] In one example, the method further comprises stripping and / or equilibrating one or more subsequent column(s) at the time the bound IgG is recovered from the first column. In one example, the method further comprises equilibrating one or more subsequent column(s) at the time the bound IgG is recovered from the first column. For example, the method does not include stripping one or more subsequent column(s) at the time the bound IgG is recovered from the first column.

[0240] In one example, the method further comprises stripping and / or equilibrating the first column at a time when one or more subsequent column(s) are washed with a wash buffer as described herein. In one example, the method further comprises equilibrating the first column at a time when one or more subsequent column(s) are washed with a wash buffer as described herein. For example, the method does not include stripping the first column at a time when one or more subsequent column(s) are washed with a wash buffer as described herein.

[0241] In one example, the method further comprises stripping and / or equilibrating one or more subsequent column(s) at the time the first column is washed with a wash buffer described herein. In one example, the method further comprises equilibrating one or more subsequent column(s) at the time the first column is washed with a wash buffer described herein. For example, the method does not include stripping one or more subsequent column(s) at the time the first column is washed with a wash buffer described herein.

[0242] In one example, the resin has a total bed height of at least 2 cm. For example, the resin has a total bed height of 2 cm to 30 cm. For example, the resin has a total bed height between 10 cm to 30 cm. For example, the resin has a total bed height of 30 cm to 70 cm. For example, the resin has a total bed height of 2 cm, or 6 cm, or 10 cm, or 15 cm, or 20 cm, or 25 cm, or 30 cm, or 35 cm, or 40 cm, or 45 cm, or 50 cm, or 55 cm, or 60 cm, or 65 cm, or 70 cm.

[0243] In one example, the resin has a total bed height of at least 2 cm.

[0244] In one example, the resin has a total bed height of 6 cm.

[0245] In one example, the resin has a total bed height of 20 cm.

[0246] In one example, the resin has a total bed height of 30 cm.

[0247] In one example, the resin has a total bed height of 50 cm.

[0248] In one example, the resin has a total bed height of 70 cm.

[0249] In one example, the column has a diameter of 5 cm to 200 cm. For example, the column has a diameter of 5 cm, or 10 cm, or 20 cm, or 30 cm, or 40 cm, or 50 cm, or 60 cm, or 70 cm, or 80 cm, or 90 cm, or 100 cm, or 110 cm, or 120 cm, or 130 cm, or 140 cm, or 150 cm, or 160 cm, or 170 cm, or 180 cm, or 190 cm, or 200 cm.

[0250] In one example, the column has a diameter of 5 cm.

[0251] In one example, the column has a diameter of 20 cm.

[0252] In one example, the column has a diameter of 50 cm.

[0253] In one example, the column has a diameter of 100 cm.

[0254] In one example, the column has a diameter of 200 cm.

[0255] In one example, the method further comprises one or more steps selected from ethanol precipitation, octanoic acid fractionation, membrane or resin chromatography (e.g., ion exchange chromatography, hydrophobic interaction chromatography, isoagglutinin affinity chromatography), viral inactivation, viral filtration and ultrafiltration / diafiltration, where the step(s) are performed before or after the successive affinity chromatography step. For example, the method further comprises ethanol precipitation. For example, the method further comprises octanoic acid fractionation. For example, the method further comprises membrane or resin chromatography. For example, the method further comprises ion exchange chromatography. For example, the method further comprises anion exchange chromatography. For example, the method further comprises cation exchange chromatography. For example, the method comprises hydrophobic interaction chromatography. For example, the method comprises isoagglutinin affinity chromatography. For example, the method further comprises viral inactivation. For example, the method further comprises nanofiltration. For example, the method further comprises ultrafiltration / diafiltration.

[0256] In one example, the method further comprises anion exchange chromatography and viral filtration.

[0257] In one example, the method further comprises low pH incubation, depth filtration, anion exchange chromatography and viral filtration.

[0258] In one example, the low pH incubation is performed in the presence of a detergent. For example, the method further comprises a low pH incubation in the presence of a detergent.

[0259] In one example, the method further comprises ion exchange chromatography, the ion exchange chromatography step comprising an anion exchange chromatography step using an anion exchange resin operated in a flow-through mode.

[0260] In one example, the flow-through and / or post-wash eluate are collected. For example, the flow-through is collected. In another example, the post-wash eluate is collected. In one example, the flow-through and post-wash eluate are collected. It will be apparent to one skilled in the art that only the flow-through and post-wash are collected (i.e., pooled), and not the elution phase.

[0261] In one example, the anion exchange resin is selected from the group consisting of a weak anion exchanger, a strong anion exchanger and a mixed mode anion exchanger.

[0262] In one example, the anion exchange resin is a weak anion exchanger.

[0263] In one example, the anion exchange resin is a mixed mode anion exchanger.

[0264] In one example, the anion exchange resin is a strong anion exchanger. In one example, the ion exchange chromatography step includes an anion exchange chromatography step using a strong anion exchange resin operated in a flow-through mode. In one example, the strong anion exchange resin includes a matrix consisting of a poly(styrene-divinylbenzene) matrix. In one example, the strong anion exchange resin includes quaternized polyethyleneimine functional groups.

[0265] In one example, the anion exchange resin is washed with a pre-equilibration buffer prior to equilibration. It will be clear to one skilled in the art that the pre-equilibration step is only performed for the first run and / or after storage of the resin.

[0266] In one example, the pre-equilibration buffer is selected from the group consisting of monobasic sodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), phosphoric acid (H3PO4), and combinations thereof.

[0267] In one example, the pre-equilibration buffer comprises Na2HPO4.

[0268] In one example, the pre-equilibration buffer comprises H3PO4.

[0269] In one example, the pre-equilibration buffer comprises NaH2PO4.

[0270] In one example, the pre-equilibration buffer includes Na2HPO4 and NaH2PO4.

[0271] In one example, the pre-equilibration buffer contains a buffer having a concentration ranging from 50 mM to 150 mM. For example, the concentration is 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM. In one example, the pre-equilibration buffer contains a buffer having a concentration of 100 mM.

[0272] In one example, the pre-equilibration buffer contains NaH2PO4 at a concentration ranging from 50 mM to 150 mM. For example, the pre-equilibration buffer contains NaH2PO4 at a concentration of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM. In one example, the pre-equilibration buffer contains NaH2PO4 at a concentration of 100 mM.

[0273] In one example, the pre-equilibration buffer has a pH in the range of 5.8 to 6.6. For example, the pre-equilibration buffer has a pH of about 5.8, or about 5.9, or about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6. In one example, the pre-equilibration buffer has a pH of 6.2.

[0274] In one example, the pre-equilibration buffer further comprises a salt. For example, the pre-equilibration buffer further comprises sodium chloride. In one example, the sodium chloride has a concentration in the range of 100 mM to 1000 mM. For example, the pre-equilibration buffer comprises sodium chloride at a concentration of 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM. In one example, the pre-equilibration buffer comprises sodium chloride at a concentration of 1000 mM.

[0275] In one example, the anion exchange resin is pre-equilibrated with a pre-equilibration buffer containing 1000 mM NaH2PO4 and 1000 mM sodium chloride, pH 6.2. In one example, the anion exchange resin is pre-equilibrated with a pre-equilibration buffer containing 100 mM NaH2PO4 and 1000 mM sodium chloride, pH 6.2.

[0276] In one example, the volume of the pre-equilibration buffer is at least 2 CV. For example, the volume of the pre-equilibration buffer is 2 CV, or 3 CV, or 4 CV, or 5 CV, or 6 CV, or 7 CV, or 8 CV, or 9 CV, or 10 CV. In one example, the volume of the pre-equilibration buffer is 2 CV to 10 CV.

[0277] In one example, the volume of the pre-equilibration buffer is at least 10 CV. For example, the volume of the pre-equilibration buffer is 10 CV, or 11 CV, or 12 CV, or 13 CV, or 14 CV, or 15 CV, or 16 CV, or 17 CV, or 18 CV, or 19 CV, or 20 CV. In one example, the volume of the pre-equilibration buffer is 15 CV.

[0278] In one example, the anion exchange resin is equilibrated with an equilibration buffer selected from the group consisting of monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), phosphoric acid (H3PO4), sodium citrate, 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, L-histidine, and combinations thereof.

[0279] In one example, the anion exchange resin is equilibrated with an equilibration buffer containing Na2HPO4.

[0280] In one example, the anion exchange resin is equilibrated with an equilibration buffer containing H3PO4.

[0281] In one example, the anion exchange resin is equilibrated with an equilibration buffer containing NaH2PO4.

[0282] In one example, the anion exchange resin is equilibrated with an equilibration buffer containing Na2HPO4 and NaH2PO4.

[0283] In one example, the anion exchange resin is equilibrated with an equilibration buffer containing MES.

[0284] In one example, the anion exchange resin is equilibrated with an equilibration buffer that contains sodium citrate.

[0285] In one example, the anion exchange resin is equilibrated with an equilibration buffer containing Bis-Tris.

[0286] In one example, the anion exchange resin is equilibrated with an equilibration buffer containing L-histidine.

[0287] In one example, the equilibration buffer has a concentration ranging from 5 mM to 50 mM. For example, the equilibration buffer has a concentration of 5 mM, or 10 mM, or 20 mM, or 30 mM, or 40 mM, or 50 mM. In one example, the equilibration buffer has a concentration of 5 mM. In another example, the equilibration buffer has a concentration of 10 mM. In a further example, the equilibration buffer has a concentration of 20 mM. In one example, the equilibration buffer has a concentration of 30 mM. In another example, the equilibration buffer has a concentration of 40 mM. In a further example, the equilibration buffer has a concentration of 50 mM.

[0288] In one example, the equilibration buffer contains NaH2PO4 at a concentration ranging from 5 mM to 50 mM. In one example, the equilibration buffer contains NaH2PO4 at a concentration ranging from 10 mM to 50 mM. For example, the equilibration buffer contains NaH2PO4 at a concentration of 10 mM, 20 mM, 30 mM, 40 mM, 50 mM. In one example, the equilibration buffer contains NaH2PO4 at a concentration of 5 mM. In one example, the equilibration buffer contains NaH2PO4 at a concentration of 10 mM.

[0289] In one example, the equilibration buffer has a pH in the range of 5.8 to 6.6. For example, the equilibration buffer has a pH of about 5.8, or about 5.9, or about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6. In one example, the equilibration is at a pH of 6.2.

[0290] In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising a phosphate buffer at a pH in the range of 5.8-6.6. In one example, the equilibration buffer comprises a phosphate buffer at pH 6.0. In one example, the equilibration buffer comprises a phosphate buffer at pH 6.2. In one example, the equilibration buffer comprises a phosphate buffer at pH 6.6. In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising 5 mM NaH2PO4, pH 6.2. In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising 10 mM NaH2PO4, pH 6.2.

[0291] In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising an MES buffer having a pH in the range of 5.8 to 6.6. In one example, the equilibration buffer comprises an MES buffer pH 6.0. In one example, the equilibration buffer comprises an MES buffer pH 6.2. In one example, the equilibration buffer comprises an MES buffer pH 6.6.

[0292] In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising a Bis-Tris buffer at a pH range of 5.8 to 6.6. In one example, the equilibration buffer comprises a Bis-Tris buffer pH 6.0. In one example, the equilibration buffer comprises a Bis-Tris buffer pH 6.2. In one example, the equilibration buffer comprises a Bis-Tris buffer pH 6.6.

[0293] In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising an L-histidine buffer at a pH in the range of 5.8 to 6.6. In one example, the equilibration buffer comprises an L-histidine buffer at pH 6.0. In one example, the equilibration buffer comprises an L-histidine buffer at pH 6.2. In one example, the equilibration buffer comprises an L-histidine buffer at pH 6.6.

[0294] In one example, the volume of the equilibration buffer is at least 2 CV. For example, the volume of the equilibration buffer is 2 CV, or 3 CV, or 4 CV, or 5 CV, or 6 CV, or 7 CV, or 8 CV, or 9 CV, or 10 CV. In one example, the volume of the equilibration buffer is 2 CV to 10 CV.

[0295] In one example, the volume of the equilibration buffer is at least 10 CV. For example, the volume of the equilibration buffer is 10 CV, or 11 CV, or 12 CV, or 13 CV, or 14 CV, or 15 CV, or 16 CV, or 17 CV, or 18 CV, or 19 CV, or 20 CV. In one example, the volume of the equilibration buffer is 15 CV.

[0296] In one example, the anion exchange resin is loaded with IgG at a concentration ranging from 5 g IgG per L of resin to 15 g IgG per L of resin. For example, the resin is loaded with 5 g, or 6 g, or 7 g, or 8 g, or 9 g, or 10 g, or 11 g, or 12 g, or 13 g, or 14 g, or 15 g of IgG per L of resin. In one example, the resin is loaded with 15 g of IgG per L of resin.

[0297] In one example, the anion exchange resin is loaded with IgG at a concentration ranging from 5 g IgG per L load to 15 g IgG per L load. For example, the resin is loaded with 5 g / L, or 6 g / L, or 7 g / L, or 8 g / L, or 9 g / L, or 10 g / L, or 11 g / L, or 12 g / L, or 13 g / L, or 14 g / L, or 15 g / L of IgG. In one example, the resin is loaded with 15 g IgG per L load.

[0298] In one example, the anion exchange chromatography step includes a post-load wash buffer selected from the group consisting of phosphate buffer, sodium citrate buffer, 2-(N-morpholino)ethanesulfonic acid buffer, acetate buffer, Bis-tris buffer, and L-histidine buffer. In one example, the anion exchange chromatography step includes a post-load wash buffer selected from the group consisting of phosphate buffer, sodium citrate buffer, and acetate buffer.

[0299] In one example, the post-loading wash buffer has a concentration ranging from 5 mM to 50 mM. In one example, the post-loading wash buffer has a concentration ranging from 10 mM to 50 mM. For example, the post-loading wash buffer has a concentration of 10 mM, 20 mM, 30 mM, 40 mM, 50 mM. In one example, the post-loading wash buffer has a concentration of 5 mM. In one example, the post-loading wash buffer has a concentration of 10 mM.

[0300] In one example, the post-loading wash buffer comprises a phosphate buffer, for example, the phosphate buffer is selected from the group consisting of monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), phosphoric acid (H3PO4), and combinations thereof.

[0301] In one example, the post-load wash buffer contains Na2HPO4.

[0302] In one example, the post-load wash buffer comprises H3PO4.

[0303] In one example, the post-loading wash buffer comprises NaH2PO4. For example, the post-loading wash buffer comprises 5 mM NaH2PO4. In another example, the post-loading wash buffer comprises 10 mM NaH2PO4.

[0304] In one example, the post-load wash buffer comprises Na2HPO4 and NaH2PO4.

[0305] In one example, the post-load wash buffer comprises a sodium citrate buffer.

[0306] In one example, the post-loading wash buffer comprises an acetate buffer. For example, the post-loading wash buffer comprises sodium acetate. For example, the post-loading wash buffer comprises 5 mM acetate. In another example, the post-loading wash buffer comprises 10 mM acetate.

[0307] In one example, the post-loading wash buffer comprises a phosphate buffer and an acetate buffer. For example, the post-loading wash buffer comprises NaH2PO4 and sodium acetate. For example, the post-loading wash buffer comprises 5 mM NaH2PO4 and 10 mM sodium acetate.

[0308] In one example, the post-loading wash buffer comprises MES buffer.

[0309] In one example, the post-load wash buffer is a Bis-Tris buffer.

[0310] In one example, the post-loading wash buffer is an L-histidine buffer.

[0311] In one example, the post-loading wash buffer has a pH in the range of 5.0 to about 8.0. For example, the post-loading wash buffer has a pH in the range of 5.5 to 7.0. In another example, the post-loading wash buffer has a pH in the range of 5.8 to 6.6. For example, the post-loading wash buffer has a pH of about 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or 6.6. In one example, the post-loading wash buffer has a pH of 6.0. In one example, the post-loading wash buffer has a pH of 6.2. In another example, the post-loading wash buffer has a pH of 6.6.

[0312] In one example, the post-load wash buffer further comprises a salt, for example, the salt is sodium chloride.

[0313] In one example, the post-load wash buffer is salt-free.

[0314] In one example, sodium chloride has a concentration of 0 mM to 200 mM. In one example, sodium chloride has a concentration of 0 mM to 50 mM. In one example, sodium chloride has a concentration of 0 mM to 100 mM. For example, sodium chloride has a concentration of 20 mM to 150 mM. In one example, sodium chloride has a concentration of 20 mM to 80 mM. For example, sodium chloride has a concentration of about 20 mM, or 30 mM, or 40 mM, or 50 mM, or 60 mM, or 70 mM, or 80 mM. In one example, sodium chloride has a concentration of about 20 mM. In one example, sodium chloride has a concentration of about 25 mM. For example, sodium chloride has a concentration of 50 mM. In one example, sodium chloride has a concentration of about 70 mM. In one example, sodium chloride has a concentration of 120 mM to 200 mM. For example, sodium chloride has a concentration of 150 mM. In another example, the sodium chloride is at a concentration of 200 mM.

[0315] In one example, the post-load wash buffer comprises a phosphate buffer at a pH range of 5.8 to 6.6.

[0316] In one example, the post-load wash buffer comprises phosphate buffer pH 6.0.

[0317] In one example, the post-load wash buffer comprises phosphate buffer pH 6.2.

[0318] In one example, the post-load wash buffer comprises phosphate buffer pH 6.6.

[0319] In one example, the post-load wash buffer comprises phosphate buffer and 0 mM sodium chloride pH 6.0.

[0320] In one example, the post-load wash buffer comprises phosphate buffer and 20 mM sodium chloride pH 6.0.

[0321] In one example, the post-load wash buffer comprises phosphate buffer and 50 mM sodium chloride pH 6.0.

[0322] In one example, the post-load wash buffer comprises phosphate buffer and 0 mM sodium chloride pH 6.6.

[0323] In one example, the post-load wash buffer comprises phosphate buffer and 25 mM sodium chloride pH 6.6.

[0324] In one example, the post-load wash buffer comprises phosphate buffer and 50 mM sodium chloride pH 6.6.

[0325] In one example, the post-load wash buffer comprises phosphate buffer and 0 mM sodium chloride pH 6.2.

[0326] In one example, the post-load wash buffer comprises MES buffer at a pH range of 5.8 to 6.6.

[0327] In one example, the post-load wash buffer comprises MES buffer pH 6.0.

[0328] In one example, the post-load wash buffer comprises MES buffer pH 6.6.

[0329] In one example, the post-load wash buffer comprises MES buffer and 20 mM sodium chloride pH 6.0.

[0330] In one example, the post-load wash buffer comprises MES buffer and 50 mM sodium chloride pH 6.0.

[0331] In one example, the post-load wash buffer comprises MES buffer and 25 mM sodium chloride pH 6.6.

[0332] In one example, the post-load wash buffer comprises MES buffer and 50 mM sodium chloride pH 6.6.

[0333] In one example, the post-load wash buffer comprises a sodium citrate buffer at a pH range of 5.8 to 6.6.

[0334] In one example, the post-load wash buffer comprises sodium citrate buffer pH 6.0.

[0335] In one example, the post-load wash buffer comprises sodium citrate buffer pH 6.6.

[0336] In one example, the post-load wash buffer comprises sodium citrate buffer and 20 mM sodium chloride pH 6.0.

[0337] In one example, the post-load wash buffer comprises sodium citrate buffer and 50 mM sodium chloride pH 6.0.

[0338] In one example, the post-load wash buffer comprises sodium citrate buffer and 25 mM sodium chloride pH 6.6.

[0339] In one example, the post-load wash buffer comprises sodium citrate buffer and 50 mM sodium chloride pH 6.6.

[0340] In one example, the post-load wash buffer comprises a sodium acetate buffer at a pH range of 5.8 to 6.6.

[0341] In one example, the post-load wash buffer comprises sodium acetate buffer pH 6.0.

[0342] In one example, the post-load wash buffer comprises sodium acetate buffer pH 6.2.

[0343] In one example, the post-load wash buffer comprises sodium acetate buffer pH 6.6.

[0344] In one example, the post-load wash buffer comprises sodium acetate buffer and 0 mM sodium chloride pH 6.0.

[0345] In one example, the post-load wash buffer comprises sodium acetate buffer and 20 mM sodium chloride pH 6.0.

[0346] In one example, the post-load wash buffer comprises sodium acetate buffer and 50 mM sodium chloride pH 6.0.

[0347] In one example, the post-load wash buffer comprises sodium acetate buffer and 0 mM sodium chloride pH 6.6.

[0348] In one example, the post-load wash buffer comprises sodium acetate buffer and 25 mM sodium chloride pH 6.6.

[0349] In one example, the post-load wash buffer comprises sodium acetate buffer and 50 mM sodium chloride pH 6.6.

[0350] In one example, the post-load wash buffer comprises sodium acetate buffer and 0 mM sodium chloride pH 6.2.

[0351] In one example, the post-load wash buffer comprises a phosphate and sodium acetate buffer at a pH range of 5.8 to 6.6.

[0352] In one example, the post-load wash buffer comprises a phosphate and sodium acetate buffer pH 6.0.

[0353] In one example, the post-load wash buffer comprises a phosphate and sodium acetate buffer pH 6.2.

[0354] In one example, the post-load wash buffer comprises a phosphate and sodium acetate buffer pH 6.6.

[0355] In one example, the post-load wash buffer comprises phosphate and sodium acetate buffer and 0 mM sodium chloride pH 6.0.

[0356] In one example, the post-load wash buffer comprises phosphate and sodium acetate buffer and 20 mM sodium chloride pH 6.0.

[0357] In one example, the post-load wash buffer comprises phosphate and sodium acetate buffer and 50 mM sodium chloride pH 6.0.

[0358] In one example, the post-load wash buffer comprises phosphate and sodium acetate buffer and 0 mM sodium chloride pH 6.6.

[0359] In one example, the post-load wash buffer comprises a phosphate and sodium acetate buffer and 25 mM sodium chloride pH 6.6.

[0360] In one example, the post-load wash buffer comprises phosphate and sodium acetate buffer and 50 mM sodium chloride pH 6.6.

[0361] In one example, the post-load wash buffer comprises phosphate and sodium acetate buffer and 0 mM sodium chloride pH 6.2.

[0362] In one example, the post-load wash buffer comprises a Bis-Tris buffer at a pH range of 5.8 to 6.6.

[0363] In one example, the post-load wash buffer comprises Bis-Tris buffer pH 6.0.

[0364] In one example, the post-load wash buffer comprises Bis-Tris buffer pH 6.6.

[0365] In one example, the post-load wash buffer comprises Bis-Tris buffer and 20 mM sodium chloride pH 6.0.

[0366] In one example, the post-load wash buffer comprises Bis-Tris buffer and 50 mM sodium chloride pH 6.0.

[0367] In one example, the post-load wash buffer comprises Bis-Tris buffer and 25 mM sodium chloride pH 6.6.

[0368] In one example, the post-load wash buffer comprises Bis-Tris buffer and 50 mM sodium chloride pH 6.6.

[0369] In one example, the post-load wash buffer comprises an L-histidine buffer at a pH in the range of 5.8 to 6.6.

[0370] In one example, the post-load wash buffer comprises L-histidine buffer pH 6.0.

[0371] In one example, the post-load wash buffer comprises L-histidine buffer pH 6.6.

[0372] In one example, the post-load wash buffer comprises L-histidine buffer and 20 mM sodium chloride pH 6.0.

[0373] In one example, the post-load wash buffer comprises L-histidine buffer and 50 mM sodium chloride pH 6.0.

[0374] In one example, the post-load wash buffer comprises L-histidine buffer and 25 mM sodium chloride pH 6.6.

[0375] In one example, the post-load wash buffer comprises L-histidine buffer and 50 mM sodium chloride pH 6.6.

[0376] In one example, the volume of the post-loading wash buffer is 1-5 CV. For example, the volume of the post-loading wash buffer is 1 CV, or 2 CV, or 3 CV, or 4 CV, or 5 CV. In one example, the volume of the post-loading wash buffer is 3 CV.

[0377] In one example, the anion exchange resin is regenerated with a regeneration buffer selected from the group consisting of sodium chloride, sodium dihydrogen phosphate, sodium hydroxide, acetic acid and combinations thereof.

[0378] In one example, the anion exchange resin is regenerated with a regeneration buffer selected from the group consisting of sodium chloride, phosphate buffer, sodium hydroxide buffer, acetate buffer and combinations thereof.

[0379] In one example, the anion exchange resin is regenerated with a regeneration buffer comprising a phosphate buffer, for example, the phosphate buffer is selected from the group consisting of monosodium phosphate (NaH2PO4), disodium phosphate (Na2HPO4), phosphoric acid (H3PO4), and combinations thereof.

[0380] In one example, the regeneration buffer comprises Na2HPO4.

[0381] In one example, the renaturation buffer comprises H3PO4.

[0382] In one example, the regeneration buffer comprises NaH2PO4.

[0383] In one example, the regeneration buffer comprises Na2HPO4 and NaH2PO4.

[0384] In one example, the regeneration buffer comprises sodium chloride. In another example, the regeneration buffer comprises sodium hydroxide. In a further example, the regeneration buffer comprises acetic acid.

[0385] In one example, the regeneration buffer comprises sodium chloride and a phosphate buffer. In one example, the regeneration buffer consists of sodium chloride and sodium dihydrogen phosphate (NaH2PO4). In one example, the regeneration buffer comprises sodium chloride and Na2HPO4. In one example, the regeneration buffer comprises sodium chloride and H3PO4. In one example, the regeneration buffer comprises sodium chloride, Na2HPO4 and NaH2PO4.

[0386] In one example, the renaturation buffer contains 1 M sodium chloride and 10 mM sodium dihydrogen phosphate, pH 6.2.

[0387] In one example, the renaturation buffer contains 1 M sodium chloride and 10 mM Na2HPO4, pH 6.2.

[0388] In one example, the regeneration buffer contains 1 M sodium chloride and 10 mM H3PO4, pH 6.2.

[0389] In one example, the regeneration buffer contains 1 M sodium chloride and 10 mM Na2HPO4 and NaH2PO4, pH 6.2.

[0390] In one example, the renaturation buffer comprises 1 M sodium chloride and 100 mM sodium dihydrogen phosphate, pH 6.2.

[0391] In one example, the renaturation buffer contains 1 M sodium chloride and 100 mM Na2HPO4, pH 6.2.

[0392] In one example, the regeneration buffer contains 1 M sodium chloride and 100 mM H3PO4, pH 6.2.

[0393] In one example, the regeneration buffer contains 1 M sodium chloride and 100 mM Na2HPO4 and NaH2PO4, pH 6.2.

[0394] In one example, the regeneration buffer contains 0.5 M sodium hydroxide.

[0395] In one example, the renaturation buffer contains 1 M acetic acid.

[0396] In one example, the volume of the regeneration buffer is 1 to 10 CV. For example, the volume of the regeneration buffer is 1 CV, or 2 CV, or 3 CV, or 4 CV, or 5 CV, or 6 CV, or 7 CV, or 8 CV, or 9 CV, or 10 CV. In one example, the volume of the regeneration buffer is 5 CV.

[0397] Suitable regeneration methods will be apparent to those skilled in the art and / or are described herein.

[0398] In one example, at least 75% of the IgG is recovered from the plasma or a fraction thereof. In another example, at least 75% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method. For example, at least 75% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method without further purification steps. For example, at least 75% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method with further purification steps. For example, at least 75% of the IgG is recovered from the plasma or a fraction thereof after an ion exchange chromatography step. In one example, at least 75% of the IgG is recovered from the plasma or a fraction thereof after an anion exchange chromatography step. In one example, at least 75% of the IgG is recovered from the plasma or a fraction thereof, where the IgG is derived from at least 500 kg of plasma or a fraction thereof. For example, at least 75% of the IgG is recovered from large-scale purification of the plasma or a fraction thereof. For example, 75%, or 76%, or 77%, or 78%, or 79% of the IgG is recovered from the plasma or fraction thereof. In one example, 75% of the IgG is recovered from the plasma or fraction thereof. In one example, 76% of the IgG is recovered from the plasma or fraction thereof. In one example, 77% of the IgG is recovered from the plasma or fraction thereof. In one example, 78% of the IgG is recovered from the plasma or fraction thereof. In another example, 79% of the IgG is recovered from the plasma or fraction thereof.

[0399] In one example, at least 80% of the IgG is recovered from the plasma or a fraction thereof. In another example, at least 80% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method. For example, at least 80% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method without further purification steps. For example, at least 80% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method with further purification steps. For example, at least 80% of the IgG is recovered from the plasma or a fraction thereof after an ion exchange chromatography step. In one example, at least 80% of the IgG is recovered from the plasma or a fraction thereof after an anion exchange chromatography step. In one example, at least 80% of the IgG is recovered from the plasma or a fraction thereof, where the IgG is derived from at least 500 kg of plasma or a fraction thereof. For example, at least 80% of the IgG is recovered from large-scale purification of the plasma or a fraction thereof. For example, 80%, or 81%, or 82%, or 83%, or 84% of the IgG is recovered from the plasma or fraction thereof. In one example, 80% of the IgG is recovered from the plasma or fraction thereof. In one example, 81% of the IgG is recovered from the plasma or fraction thereof. In one example, 82% of the IgG is recovered from the plasma or fraction thereof. In one example, 83% of the IgG is recovered from the plasma or fraction thereof. In another example, 84% of the IgG is recovered from the plasma or fraction thereof.

[0400] In one example, at least 85% of the IgG is recovered from the plasma or a fraction thereof. In another example, at least 85% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method. For example, at least 85% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method without further purification steps. For example, at least 85% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method with further purification steps. For example, at least 85% of the IgG is recovered from the plasma or a fraction thereof after an ion exchange chromatography step. In one example, at least 85% of the IgG is recovered from the plasma or a fraction thereof after an anion exchange chromatography step. In one example, at least 85% of the IgG is recovered from the plasma or a fraction thereof, where the IgG is derived from at least 500 kg of plasma or a fraction thereof. For example, at least 85% of the IgG is recovered from large-scale purification of the plasma or a fraction thereof. For example, 85%, or 86%, or 87%, or 88%, or 89% of the IgG is recovered from the plasma or fraction thereof. In one example, 85% of the IgG is recovered from the plasma or fraction thereof. In one example, 86% of the IgG is recovered from the plasma or fraction thereof. In one example, 87% of the IgG is recovered from the plasma or fraction thereof. In one example, 88% of the IgG is recovered from the plasma or fraction thereof. In another example, 89% of the IgG is recovered from the plasma or fraction thereof.

[0401] In one example, at least 90% of the IgG is recovered from the plasma or a fraction thereof. In another example, at least 90% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method. For example, at least 90% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method without further purification steps. For example, at least 90% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method with further purification steps. For example, at least 90% of the IgG is recovered from the plasma or a fraction thereof after an ion exchange chromatography step. In one example, at least 90% of the IgG is recovered from the plasma or a fraction thereof after an anion exchange chromatography step. In one example, at least 90% of the IgG is recovered from the plasma or a fraction thereof, where the IgG is derived from at least 500 kg of plasma or a fraction thereof. For example, at least 90% of the IgG is recovered from large-scale purification of the plasma or a fraction thereof. For example, 90%, or 91%, or 92%, or 93%, or 94% of the IgG is recovered from the plasma or fraction thereof. In one example, 90% of the IgG is recovered from the plasma or fraction thereof. In one example, 91% of the IgG is recovered from the plasma or fraction thereof. In one example, 92% of the IgG is recovered from the plasma or fraction thereof. In one example, 93% of the IgG is recovered from the plasma or fraction thereof. In another example, 94% of the IgG is recovered from the plasma or fraction thereof.

[0402] In one example, at least 95% of the IgG is recovered from the plasma or a fraction thereof. In another example, at least 95% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method. For example, at least 95% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method without further purification steps. For example, at least 95% of the IgG is recovered from the plasma or a fraction thereof according to a continuous chromatography method with further purification steps. For example, at least 95% of the IgG is recovered from the plasma or a fraction thereof after an ion exchange chromatography step. In one example, at least 95% of the IgG is recovered from the plasma or a fraction thereof after an anion exchange chromatography step. In one example, at least 95% of the IgG is recovered from the plasma or a fraction thereof, where the IgG is derived from at least 500 kg of plasma or a fraction thereof. For example, at least 95% of the IgG is recovered from large-scale purification of the plasma or a fraction thereof. For example, 95%, or 96%, or 97%, or 98%, or 99% of the IgG is recovered from the plasma or fraction thereof. In one example, 95% of the IgG is recovered from the plasma or fraction thereof. In one example, 96% of the IgG is recovered from the plasma or fraction thereof. In one example, 97% of the IgG is recovered from the plasma or fraction thereof. In one example, 98% of the IgG is recovered from the plasma or fraction thereof. In another example, 99% of the IgG is recovered from the plasma or fraction thereof.

[0403] In one example, the eluted IgG has a purity of at least 95%. In another example, the eluted IgG has a purity of at least 95% according to a continuous chromatography method. In one example, the eluted IgG has a purity of at least 95% according to a continuous chromatography method without further purification steps. In one example, the eluted IgG has a purity of at least 95% according to a continuous chromatography method with further purification steps. In one example, the eluted IgG with a purity of at least 95% is derived from at least 500 kg of plasma or a fraction thereof. For example, the eluted IgG with a purity of at least 95% is recovered from a large-scale purification of plasma or a fraction thereof. For example, the eluted IgG has a purity of 95%, 96%, 97%, 98% or 99%. In one example, the eluted IgG has a purity of 95%. In one example, the eluted IgG has a purity of 96%. In one example, the eluted IgG has a purity of 97%.

[0404] In one example, the eluted IgG has a purity of at least 98%. In another example, the eluted IgG has a purity of at least 98% according to a continuous chromatography method. In one example, the eluted IgG has a purity of at least 98% according to a continuous chromatography method without further purification steps. In one example, the eluted IgG has a purity of at least 98% according to a continuous chromatography method with further purification steps. In one example, the eluted IgG with a purity of at least 98% is derived from at least 500 kg of plasma or a fraction thereof. For example, the eluted IgG with a purity of at least 98% is recovered from a large-scale purification of plasma or a fraction thereof. For example, the eluted IgG has a purity of 98% or 99%.

[0405] In one example, the method is carried out on a large scale.For example, the method is carried out on an industrial or commercial scale.The method carried out on an industrial or commercial scale is clear to those skilled in the art and / or described herein.For example, the method carried out on an industrial scale includes the large-scale purification of IgG from plasma or its fraction.

[0406] In one example, the large-scale purification of IgG is carried out using at least 500 kg of plasma or a fraction thereof. For example, the large-scale purification of IgG is carried out using 500 kg to 1000 kg, or 1000 kg to 2500 kg, or 2500 kg to 5000 kg, or 5000 kg to 7500 kg, or 7500 kg, or 10000 kg, or 10000 kg to 12500 kg, or 12500 kg to 15000 kg of plasma or a fraction thereof. In one example, the large-scale purification of IgG is carried out using at least 1000 kg, or 2500 kg, or 5000 kg, or 7500 kg, or 10000 kg, or 12500 kg, or 15000 kg of plasma or a fraction thereof. In one example, the large-scale purification of IgG is carried out using at least 1000 kg of plasma or a fraction thereof. In one example, the large-scale purification of IgG is carried out using at least 2500 kg of plasma of that fraction. In one example, the large-scale purification of IgG is carried out using at least 5000 kg of plasma of that fraction. In one example, the large-scale purification of IgG is carried out using at least 7500 kg of plasma of that fraction. In one example, the large-scale purification of IgG is carried out using at least 10000 kg of plasma of that fraction. In one example, the large-scale purification of IgG is carried out using at least 12500 kg of plasma of that fraction. In one example, the large-scale purification of IgG is carried out using at least 15000 kg of plasma of that fraction.

[0407] In one example, the method further comprises formulating the purified IgG into a pharmaceutical composition.

[0408] The present disclosure further provides a method for purifying IgG from plasma or a fraction thereof using SMB chromatography, comprising: a) equilibrating an affinity chromatography resin comprising a cross-linked poly(styrene-divinylbenzene) matrix and a ligand capable of specifically binding to the CH3 domain of human IgG with a 20 mM phosphate equilibration buffer having a pH of 7-8; b) binding IgG from the plasma or a fraction thereof to a resin; c) washing the resin with a 20 mM phosphate wash buffer having a pH of 7 to 8; and d) eluting the bound IgG with a 20 mM acetate elution buffer having a pH between 3 and 5. Includes; Here, steps a)-d) may be repeated on an affinity chromatography resin, where the affinity chromatography resin is packed in a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet and an outlet valve, and optionally, the method further comprises stripping the resin with a 20 mM phosphate wash buffer having a pH of 2-3.

[0409] The present disclosure further provides a method for purifying IgG from plasma or a fraction thereof using SMB chromatography, comprising: a) equilibrating an affinity chromatography resin comprising a cross-linked poly(styrene-divinylbenzene) matrix and a ligand capable of specifically binding to the CH3 domain of human IgG with an equilibration buffer comprising 20 mM sodium dihydrogen phosphate buffer, 145 mM sodium chloride, and having a pH of 7-8; b) binding IgG from the plasma or a fraction thereof to a resin; c) washing the resin with a wash buffer comprising 20 mM sodium dihydrogen phosphate buffer, 145 mM sodium chloride and having a pH of 7 to 8; and d) eluting the bound IgG with a 20 mM acetate elution buffer having a pH between 3 and 5. Includes; Here, steps a)-d) may be repeated on an affinity chromatography resin, where the affinity chromatography resin is packed in a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet and an outlet valve, and optionally, the method further comprises stripping the resin with a 20 mM phosphate wash buffer having a pH of 2-3.

[0410] The present disclosure further provides a method for purifying IgG from plasma or a fraction thereof using SMB chromatography, comprising: a) equilibrating an affinity chromatography resin comprising a cross-linked poly(styrene-divinylbenzene) matrix and a ligand capable of specifically binding to the CH3 domain of human IgG with an equilibration buffer comprising 20 mM sodium dihydrogen phosphate buffer, 500 mM sodium chloride, and having a pH of 7-8; b) binding IgG from the plasma or a fraction thereof to a resin; c) washing the resin with a wash buffer containing 20 mM sodium dihydrogen phosphate buffer, 500 mM sodium chloride and having a pH of 7 to 8; and d) eluting the bound IgG with a 20 mM acetate elution buffer having a pH between 3 and 5. Includes; Here, steps a)-d) may be repeated on an affinity chromatography resin, where the affinity chromatography resin is packed in a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet and an outlet valve, and optionally, the method further comprises stripping the resin with a 20 mM phosphate wash buffer having a pH of 2-3.

[0411] In one example, the method does not include stripping the resin.

[0412] The present disclosure further provides a method for purifying IgG from plasma or a fraction thereof using SMB chromatography, comprising: a) equilibrating an affinity chromatography resin comprising a cross-linked poly(styrene-divinylbenzene) matrix and a ligand capable of specifically binding to the CH3 domain of human IgG with a 20 mM phosphate equilibration buffer having a pH of 7-8; b) binding IgG from the plasma or a fraction thereof to a resin; c) washing the resin with a 20 mM phosphate wash buffer having a pH of 7 to 8; and d) eluting the bound IgG with a 20 mM acetate elution buffer having a pH between 3 and 5. Includes; Here, steps a) to d) may be repeated on an affinity chromatography resin, where the affinity chromatography resin is packed in a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet and an outlet valve, and the method does not include stripping the resin.

[0413] The present disclosure further provides a method for purifying IgG from plasma or a fraction thereof using SMB chromatography, comprising: a) equilibrating an affinity chromatography resin containing a cross-linked poly(styrene-divinylbenzene) matrix and a ligand capable of specifically binding to the CH3 domain of human IgG with a 20 mM phosphate equilibration buffer having a pH of 7-8; b) binding IgG from the plasma or a fraction thereof to a resin; c) washing the resin with a 20 mM phosphate wash buffer having a pH of 7 to 8; and d) eluting the bound IgG with a 20 mM acetate or phosphate elution buffer having a pH between 3 and 5. Includes; Here, steps a) to d) may be repeated on an affinity chromatography resin, where the affinity chromatography resin is packed in a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet and an outlet valve, and the method optionally does not include stripping the resin.

[0414] The present disclosure further provides a method for purifying IgG from plasma or a fraction thereof using SMB chromatography, comprising: a) equilibrating an affinity chromatography resin comprising a cross-linked poly(styrene-divinylbenzene) matrix and a ligand capable of specifically binding to the CH3 domain of human IgG with an equilibration buffer comprising 20 mM sodium dihydrogen phosphate buffer, 145 mM sodium chloride, and having a pH of 7-8; b) binding IgG from the plasma or a fraction thereof to a resin; c) washing the resin with a wash buffer containing 20 mM sodium dihydrogen phosphate buffer, 145 mM sodium chloride, and having a pH of 7 to 8; and d) eluting the bound IgG with a 20 mM acetate elution buffer having a pH between 3 and 5. Includes; Here, steps a) to d) may be repeated on an affinity chromatography resin, where the affinity chromatography resin is packed in a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet and an outlet valve, and the method does not include stripping the resin.

[0415] The present disclosure further provides a method for purifying IgG from plasma or a fraction thereof using SMB chromatography, comprising: a) equilibrating an affinity chromatography resin comprising a cross-linked poly(styrene-divinylbenzene) matrix and a ligand capable of specifically binding to the CH3 domain of human IgG with an equilibration buffer comprising 20 mM sodium dihydrogen phosphate buffer, 500 mM sodium chloride, and having a pH of 7-8; b) binding IgG from the plasma or a fraction thereof to a resin; c) washing the resin with Who Wash Buffer containing 20 mM sodium dihydrogen phosphate buffer, 500 mM sodium chloride, pH 7-8; and d) eluting the bound IgG with a 20 mM acetate elution buffer having a pH between 3 and 5. Includes; Here, steps a) to d) may be repeated on an affinity chromatography resin, where the affinity chromatography resin is packed in a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet and an outlet valve, and the method does not include stripping the resin.

[0416] In one example, the method is repeated on the resin for at least 50 cycles. For example, the method is repeated on the resin for at least 50 cycles per batch of plasma or a fraction thereof. In one example, the method is repeated on the resin for 50-80 cycles, 60-80 cycles, 70-80 cycles per batch of plasma or a fraction thereof. For example, the method is repeated on the resin for at least 60 cycles, or 65 cycles, or 70 cycles, or 75 cycles, or 80 cycles per batch of plasma or a fraction thereof.

[0417] In one example, the method is repeated on the resin for 50 cycles per batch of plasma or a fraction thereof.

[0418] In one example, the method is repeated on the resin for 60 cycles per batch of plasma or a fraction thereof.

[0419] In one example, the method is repeated on the resin for 70 cycles per batch of plasma or a fraction thereof.

[0420] In one example, the method is repeated on the resin for 80 cycles per batch of plasma or a fraction thereof.

[0421] In one example, the method is repeated on the resin with multiple batches of plasma or a fraction thereof. For example, the method is repeated on the resin with at least two batches of plasma or a fraction thereof. In one example, the method is repeated on the resin with 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 batches of plasma or a fraction thereof. In one example, the method is repeated on the resin with 4-10 batches of plasma or a fraction thereof.

[0422] In one example, the method is repeated on the resin for up to 800 total cycles. For example, the resin is reused for up to 800 total cycles. In one example, the method is repeated on the resin for up to 100, or 200, or 300, or 400, or 500, or 600, or 700 total cycles. For example, the method is repeated on the resin for up to 100 total cycles. For example, the method is repeated on the resin for up to 200 total cycles. For example, the method is repeated on the resin for up to 300 total cycles. For example, the method is repeated on the resin for up to 400 total cycles. For example, the method is repeated on the resin for up to 500 total cycles. For example, the method is repeated on the resin for up to 600 total cycles. For example, the method is repeated on the resin for up to 700 total cycles.

[0423] In one example, the method is repeated on the resin for 100-200 cycles, or 200-300 cycles, or 200-500 cycles, or 500-800 cycles.

[0424] In one example, the process is repeated for 200 cycles on the resin.

[0425] In one example, the process is repeated for 300 cycles on the resin.

[0426] In one example, the process is repeated for 400 cycles on the resin.

[0427] In one example, the process is repeated for 500 cycles on the resin.

[0428] In one example, the process is repeated for 600 cycles on the resin.

[0429] In one example, the process is repeated on the resin for 700 cycles.

[0430] In one example, the process is repeated 800 cycles on the resin.

[0431] In one example, the method is repeated on the resin for 200-500 cycles. For example, the resin is reused for up to 200-500 cycles in total. In one example, the resin is reused for up to 500 cycles in total with up to 10 batches of plasma or fractions thereof.

[0432] In one example, the disinfection step is performed on the resin after each cycle. In another example, the disinfection step is performed on the resin after multiple cycles. For example, the disinfection step is performed on the resin after at least 50 cycles. In one example, the disinfection step is performed on the resin after at least 100 cycles. In another example, the disinfection step is performed on the resin after at least 150 cycles. In a further example, the disinfection step is performed on the resin after at least 200 cycles. In one example, the disinfection step is performed on the resin after each batch of plasma or a fraction thereof. For example, the disinfection step is performed on the resin between each batch of plasma or a fraction thereof, i.e., before each batch of plasma or a fraction thereof is loaded onto the resin.

[0433] Suitable disinfection methods will be known to those of skill in the art and / or described herein.

[0434] In one example, the method reduces the DBC of the resin. For example, reusing the resin reduces the DBC of the resin. In one example, the DBC of the resin is reduced by up to 80%. For example, the DBC of the resin is reduced by up to 75%, or 70%, or 65%, or 60%, or 55%, or 40%, or 45%, or 40%, or 35%, or 30%, or 25%, or 20%, or 15%, or 10%, or 5%.

[0435] In one example, the resin is reused until the DBC of the resin has decreased by up to 80%.

[0436] In one example, the method reduces the DBC of the resin by 80%, e.g., the resin is reused until the DBC of the resin is reduced by 80%.

[0437] In one example, the method reduces the DBC of the resin by 70%, e.g., the resin is reused until the DBC of the resin is reduced by 70%.

[0438] In one example, the method reduces the DBC of the resin by 60%, e.g., the resin is reused until the DBC of the resin is reduced by 60%.

[0439] In one example, the method reduces the DBC of the resin by 50%, e.g., the resin is reused until the DBC of the resin is reduced by 50%.

[0440] In one example, the method reduces the DBC of the resin by 40%, e.g., the resin is reused until the DBC of the resin is reduced by 40%.

[0441] In one example, the method reduces the DBC of the resin by 30%, e.g., the resin is reused until the DBC of the resin is reduced by 30%.

[0442] In one example, the method reduces the DBC of the resin by 20%, e.g., the resin is reused until the DBC of the resin is reduced by 20%.

[0443] In one example, the method reduces the DBC of the resin by 10%, e.g., the resin is reused until the DBC of the resin is reduced by 10%.

[0444] The present disclosure also provides pharmaceutical compositions comprising IgG purified or produced by the methods described herein. For example, the pharmaceutical composition comprises IgG purified or produced by the methods described herein and a pharma- ceutically acceptable carrier.

[0445] In one example, the pharmaceutical composition comprises at least 1% (w / v) purified IgG. For example, the pharmaceutical composition comprises 1% (w / v) purified IgG. In another example, the pharmaceutical composition comprises 5% (w / v) purified IgG. In one example, the pharmaceutical composition comprises 10-30% (w / v) purified IgG. For example, the pharmaceutical composition comprises 10% (w / v) purified IgG. In one example, the pharmaceutical composition comprises 16.5% (w / v) purified IgG. In another example, the pharmaceutical composition comprises 20% (w / v) purified IgG. In one example, the pharmaceutical composition comprises 25% (w / v) purified IgG. In another example, the pharmaceutical composition comprises 30% (w / v) purified IgG.

[0446] In one example, the IgG content in the pharmaceutical composition is at least 95% (w / w) of the total amount of protein in the composition. For example, the IgG content in the pharmaceutical composition is 95% (w / w) of the total amount of protein in the composition. In another example, the IgG content in the pharmaceutical composition is 96% (w / w) of the total amount of protein in the composition. In a further example, the IgG content in the pharmaceutical composition is 97% (w / w) of the total amount of protein in the composition. In one example, the IgG content in the pharmaceutical composition is 98% (w / w) of the total amount of protein in the composition. In a further example, the IgG content in the pharmaceutical composition is 99% (w / w) of the total amount of protein in the composition.

[0447] In one example, the pharmaceutical composition comprises 100 mg / mL of total human plasma protein. In one example, the pharmaceutical composition comprises 20 g / 100 mL of total human plasma protein.

[0448] In one example, the pharmaceutical composition comprises immunoglobulin G (IgG) of at least 95% purity. For example, the pharmaceutical composition comprises immunoglobulin G (IgG) of at least 96% purity. In another example, the pharmaceutical composition comprises immunoglobulin G (IgG) of at least 97% purity. In another example, the pharmaceutical composition comprises immunoglobulin G (IgG) of at least 98% purity. In another example, the pharmaceutical composition comprises immunoglobulin G (IgG) of at least 99% purity.

[0449] In one example, the pharmaceutical composition comprises an IgG1 subclass distribution of at least 60%. For example, the pharmaceutical composition comprises an IgG1 subclass distribution of at least 65%.

[0450] In one example, the pharmaceutical composition comprises an IgG2 subclass distribution of less than 30%. For example, the pharmaceutical composition comprises an IgG2 subclass distribution of less than 28%.

[0451] In one example, the pharmaceutical composition comprises an IgG3 subclass distribution of less than 5%. For example, the pharmaceutical composition comprises an IgG3 subclass distribution of less than 4%.

[0452] In one example, the pharmaceutical composition comprises an IgG4 subclass distribution of less than 5%. For example, the pharmaceutical composition comprises an IgG4 subclass distribution of less than 3%.

[0453] In one example, the pharmaceutical composition comprises an IgG subclass distribution similar to normal human plasma, for example, 69% IgG1, 26% IgG2, 3% IgG3 and 2% IgG4.

[0454] In one example, the pharmaceutical composition comprises a nominal osmolality of about 300 mOsm / kg to 400 mOsm / kg. In one example, the pharmaceutical composition comprises a nominal osmolality of 380 mOsm / kg. For example, the pharmaceutical composition comprises a nominal osmolality of about 300 mOsm / kg to 350 mOsm / kg. In one example, the pharmaceutical composition comprises a nominal osmolality of 320 mOsm / kg.

[0455] In one example, the pharmaceutical composition comprises a pH of 4 to 5.5. For example, the pharmaceutical composition comprises a pH of 4.5 to 5.0. In one example, the pharmaceutical composition comprises a pH of 4.6 to 5.0. For example, the pharmaceutical composition comprises a pH of 4.6. In one example, the pharmaceutical composition comprises a pH of 4.7. In another example, the pharmaceutical composition comprises a pH of 4.8. In a further example, the pharmaceutical composition comprises a pH of 4.9. In one example, the pharmaceutical composition comprises a pH of 5.0.

[0456] In one example, the pharmaceutical composition further comprises 200 mmol / L to 300 mmol / L of L-proline. For example, the pharmaceutical composition further comprises 225 mmol / L to 275 mmol / L of L-proline. In one example, the pharmaceutical composition further comprises 240 mmol / L to 260 mmol / L of L-proline. For example, the pharmaceutical composition further comprises 250 mmol / L of L-proline.

[0457] In one example, the pharmaceutical composition comprises a sodium content of ≦1 mmol / L.

[0458] In one example, the pharmaceutical composition comprises an IgA content of ≦0.05 mg / mL. For example, the pharmaceutical composition comprises an IgA content of ≦0.04 mg / mL, or ≦0.03 mg / mL. In one example, the pharmaceutical composition comprises an IgA content of ≦0.025 mg / mL. In one example, the pharmaceutical composition comprises an IgA content of ≦0.01 mg / mL. For example, the pharmaceutical composition comprises an IgA content of ≦0.009 mg / mL.

[0459] In one example, the pharmaceutical composition comprises an IgA content of ≦0.1 mg / g IgG. In one example, the pharmaceutical composition comprises an IgA content of ≦0.09 mg / g IgG.

[0460] In one example, the pharmaceutical composition comprises an IgM content of ≦10 mg / L. For example, an IgM content of ≦10 mg / L, ≦9 mg / L, ≦8 mg / L, ≦7 mg / L, ≦6 mg / L, ≦5 mg / L, ≦4 mg / L, ≦3 mg / L, ≦2 mg / L. In one example, the pharmaceutical composition comprises an IgM content of ≦2 mg / L. In one example, the pharmaceutical composition comprises an IgM content of ≦1 mg / L. In one example, the pharmaceutical composition comprises an IgM content of ≦0.5 mg / L. For example, the pharmaceutical composition comprises an IgM content of <0.17 mg / L.

[0461] In one example, the pharmaceutical composition comprises an IgM content of < 2 μg / g IgG. In one example, the pharmaceutical composition comprises an IgM content of < 1.9 μg / g IgG.

[0462] In one example, the pharmaceutical composition comprises an albumin content of ≦0.50 mg / mL. For example, the pharmaceutical composition comprises an albumin content of ≦0.40 mg / mL. In one example, the pharmaceutical composition comprises an albumin content of ≦0.30 mg / mL. In one example, the pharmaceutical composition comprises an albumin content of ≦0.20 mg / mL. In one example, the pharmaceutical composition comprises an albumin content of ≦0.10 mg / mL. For example, the pharmaceutical composition comprises an albumin content of ≦0.09 mg / mL. In one example, the pharmaceutical composition comprises an albumin content of ≦0.08 mg / mL. In one example, the pharmaceutical composition comprises an albumin content of ≦0.07 mg / mL.

[0463] In one example, the pharmaceutical composition comprises an albumin content of ≦1 mg / g IgG. In one example, the pharmaceutical composition comprises an albumin content of ≦0.80 mg / g IgG.

[0464] In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of ≦35 IU / mL. In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of ≦30 IU / mL. In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of ≦50 IU / mL. In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of ≦20 IU / mL. For example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of ≦15 IU / mL. In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of ≦10 IU / mL.

[0465] The present disclosure also provides a pharmaceutical composition as described herein for use in treating, preventing and / or delaying the progression of a condition in a subject.For example, the present disclosure provides a pharmaceutical composition as described herein for use in treating a condition in a subject.In another example, the present disclosure provides a pharmaceutical composition as described herein for use in preventing a condition in a subject.In a further example, the present disclosure provides a pharmaceutical composition as described herein for use in delaying the progression of a condition in a subject.

[0466] In some examples, the pharmaceutical composition is present in a vial, a pre-filled syringe, or an autoinjector device.

[0467] The present disclosure also provides a pre-filled syringe comprising the pharmaceutical composition described herein.

[0468] The present disclosure also provides an autoinjector device comprising a pharmaceutical composition described herein.

[0469] In one example, the composition of the present disclosure is administered subcutaneously to a subject in need thereof. In another example, the composition of the present disclosure is administered intravenously to a subject in need thereof.

[0470] In one example, the compositions of the present disclosure are self-administered.

[0471] In one example, the compositions of the present disclosure are self-administered subcutaneously.

[0472] In one example, the composition of the present disclosure is provided in a pre-filled syringe.

[0473] In one example, the compositions of the present disclosure are self-administered subcutaneously with a pre-filled syringe.

[0474] The present disclosure further provides for the use of IgG purified or produced by the methods described herein in the manufacture of a medicament for treating, preventing and / or delaying the progression of a condition in a subject. For example, the present disclosure provides for the use of IgG purified or produced by the methods described herein in the manufacture of a medicament for treating a condition in a subject. In another example, the present disclosure provides for the use of IgG purified or produced by the methods described herein in the manufacture of a medicament for preventing a condition in a subject. In a further example, the present disclosure provides for the use of IgG purified or produced by the methods described herein in the manufacture of a medicament for delaying the progression of a condition in a subject.

[0475] The present disclosure also provides a method for treating, preventing, and / or delaying the progression of a condition in a subject, comprising administering to the subject a pharmaceutical composition of the present disclosure.For example, the present disclosure provides a method for treating a condition in a subject.In another example, the present disclosure provides a method for preventing a condition in a subject.In a further example, the present disclosure provides a method for delaying the progression of a condition in a subject.

[0476] The present disclosure also provides a kit for use in treating or preventing or delaying the progression of a condition in a subject, comprising: (a) at least one pharmaceutical composition described herein; (b) instructions for using the kit in treating or preventing or delaying a condition in a subject; and (c) optionally, at least one further therapeutically active compound or agent; A kit comprising:

[0477] In one example, the condition is an immune deficiency, an autoimmune disease, or an acute infectious disease. For example, the condition may be allogeneic bone marrow transplantation, chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP), childhood HIV, primary immune deficiency, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP), kidney transplantation with high antibody recipients or ABO incompatible donors, chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves' ophthalmopathy, Guillain-Barre syndrome, muscular dystrophy, inclusion body myositis, Lambert-Eaton syndrome, lupus erythematosus, multifocal motor neuropathy, or the like. Qi, multiple sclerosis (MS), myasthenia gravis, neonatal alloimmune thrombocytopenia, parvovirus B19 infection, pemphigus, post-transfusion purpura, renal transplant rejection, spontaneous abortion, miscarriage, stiff neck syndrome, opsoclonus-myoclonus, severe sepsis and septic shock in critically ill adults, toxic epidermal necrolysis, chronic lymphocytic leukemia, multiple myeloma, X-linked agammaglobulinemia, hypogammaglobulinemia, primary immunodeficiency, RRMS, Alzheimer's disease, and Parkinson's disease.

[0478] In one example, the condition is selected from the group consisting of primary immunodeficiency (PI), chronic inflammatory demyelinating polyneuropathy (CIDP), and chronic immune thrombocytopenic purpura (ITP).

[0479] In one example, the condition is a primary immunodeficiency (PI).

[0480] In one example, the condition is chronic inflammatory demyelinating polyneuropathy (CIDP).

[0481] In one example, the condition is chronic immune thrombocytopenic purpura (ITP).

[0482] In one example of any of the methods described herein, the subject is a mammal, for example a primate, such as a human. [Brief description of the drawings]

[0483] [Figure 1] (A) SDS-PAGE gel image of clarified cryopur plasma FcXP POROS® eluate (FcXP) under reducing (left) and non-reducing (right) conditions, and (B) a table of protein impurities identified in the eluate from the SDS-PAGE gel run. [Diagram 2] 2D-DIGE gel image of proteins in the eluate. [Diagram 3] FIG. 1 is a graphical representation showing the IgG subclass distribution of cryo-rich plasma (CRP) and cryo-poor plasma (CPP), CRP and CPP eluates (i.e., eluates from FcXP resin) prior to use in the methods described herein. [Figure 4] 1 is a graphical representation showing the static binding capacity of FcXP POROS® resin in consecutive runs at bed heights of 6 cm (LTS1) and 20 cm (LTS2). [Figure 5A] Graphs showing the procoagulant activity of (A) plasma and (B) cryopreserved plasma (CPP) as determined by NaPTT assay as a function of temperature over time or as a result of filtration. Clotting time was set to >150 seconds. [Figure 5B]Graphs showing the procoagulant activity of (A) plasma and (B) cryopreserved plasma (CPP) as determined by NaPTT assay as a function of temperature over time or as a result of filtration. Clotting time was set to >150 seconds. [Figure 6A] FIG. 1 is a graphical representation showing the proteolytic activity of thrombin (S-2238), general serine protease (S-2288), kallikrein (S-2302), plasmin (S-2251), and FXa (S-2765) as a function of temperature over time in (A) plasma and (B) cryopreserved plasma (CPP). [Figure 6B] FIG. 1 is a graphical representation showing the proteolytic activity of thrombin (S-2238), general serine protease (S-2288), kallikrein (S-2302), plasmin (S-2251), and FXa (S-2765) as a function of temperature over time in (A) plasma and (B) cryopreserved plasma (CPP). [Figure 7] 1 is a graphical representation showing viral inactivation of CRP with N-octyl-β-D-glucopyranoside. [Figure 8] (A) Temperature-dependent volume-normalized ratio of cryoprecipitate in samples thawed at different temperatures; and (B) hold time test schematic to evaluate optimal melting and hold time temperatures, respectively. [Figure 9] 1 is a series of graphical representations showing backpressure during an SMB process with (A) a strip phase and (B) without a strip phase. [Figure 10] 1 is a series of graphical representations showing (A) the decrease in proteolytic activity of eluates (i.e., eluates from FcXP resin) with increasing conductivity of the wash buffer, and (B) the decrease in proteolytic activity of eluates (i.e., eluates from FcXP resin) from normal and cryopreserved plasma (CPP) as a result of increasing the conductivity of the wash buffer from 145 mM sodium chloride to 500 mM sodium chloride. [Figure 11]1 is a series of graphical representations showing (A) IgG yield, (B) product purity using the Lapchip assay, and (C) albumin, IgA and IgM levels in normal plasma and cryopur plasma (CPP) for elutions (i.e., elutions from FcXP resin) using wash buffers containing 145 mM or 500 mM sodium chloride.

[0484] Array table key SEQ ID NO:1 is the amino acid sequence of the VHH fragment. SEQ ID NO:2 is the amino acid sequence of CDR1 of the VHH fragment. SEQ ID NO:3 is the amino acid sequence of CDR2 of the VHH fragment. SEQ ID NO: 4 is the amino acid sequence of the CDR3 of the VHH fragment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0485] overview Throughout this specification, unless specifically stated otherwise or the context requires otherwise, a reference to a single step, composition of matter, group of steps or composition of matter shall be deemed to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps or composition of matter.

[0486] Those skilled in the art will understand that the present disclosure can be affected by variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions and compounds referred to or shown herein, individually or collectively, and any and all combinations, or any two or more of said steps or features.

[0487] The present disclosure is not to be limited in scope by the specific examples described herein, which are for the purposes of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0488] Any embodiment of the present disclosure herein shall be deemed to apply mutatis mutandis to other embodiments of the present disclosure, unless specifically stated otherwise.

[0489] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0490] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be deemed to provide explicit support for both meanings or either meaning.

[0491] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of other elements, integers or steps, or group of elements, integers or steps.

[0492] As used herein, the term "derived from" shall be deemed to indicate that the specified integer may be derived from a particular origin, although not necessarily directly from that origin.

[0493] Additionally, as used herein, the singular forms "a," "and," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0494] Selected Definitions The terms "purify" or "purifying" or "purification" shall be taken to mean the complete or partial removal of at least one impurity present in plasma or a fraction thereof, thereby improving the purity level of IgG in solution.

[0495] The term "impurity" or "impurites" shall be considered to include one or more components in plasma or fractions thereof other than IgG. For example, impurities include albumin (α-globulins and / or β-globulins), plasma lipids, plasma proteins, proteases (e.g., serine proteases, kallikrein, plasmin and FXa), serine protease inhibitors (e.g., C1 inhibitor, α-1-antitrypsin and antithrombin), IgA and IgM, factor VIII, fibrinogen, von Willebrand factor, activated clotting factors (e.g., FXa, FIXa, FVIIa and thrombin), factor XIII, contact system factors (e.g., FXIa, FXIIa and plasma kallikrein), PKA, factor IX, prothrombin complex, C1 esterase inhibitor, protein C, antithrombin III, RhD immunoglobulin and platelet membrane microparticles.

[0496] The term "immunoglobulin G (IgG)", also known as "gamma globulin" or "immunoglobulin", shall be taken to mean an antibody of isotype G. There are several subclasses of IgG, e.g., IgG1, IgG2, IgG3 and IgG4.

[0497] The term "plasma" is intended to mean the straw-colored / light yellow component of blood obtained from one or more blood donors. Methods for obtaining plasma from a donor will be apparent to one of skill in the art and / or are described herein. For example, plasma may be obtained by removing red blood cells from donated blood. For example, plasma may be obtained by plasmapheresis.

[0498] The term "plasma fraction" or "fraction thereof" refers to plasma that has been fractionated to isolate one or more desired protein components from the plasma. For example, plasma can be fractionated to isolate a cryoprecipitate (proteins that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold) and a cryosupernatant (also known as cryoprecipitate plasma). For example, plasma can be fractionated by ethanol precipitation to produce an IgG-containing Oncre fraction, a Cohn fraction, an ammonium sulfate precipitate, or precipitate A (KN A), B (KN B), and a supernatant precipitate B (KN B+1) from the plasma, as described in U.S. Pat. No. 3,301,842. Plasma fractions include the II+III precipitate produced according to the Cohn method, such as method 6, Cohn et al., J. Am; Chem. Soc., 68(3), 459-475(1946), method 9, Oncley et al., J. Am; Chem. Soc., 71, 541-550 (1946), or the I+II+III precipitate of method 10, Cohn et al., J. Am; Chem. Soc., 72, 465-474(1950); and Deutsch et al., J. Biol. Chem. 164, 109-118(1946), or precipitates A-B and supernatant precipitate B of Nitschmann and Kistler Vox Sang. 7, 414-424(1962); Helv. Chim. Acta 37, 866-873(1954). For example, plasma can be fractionated by octanoic acid fractionation as described in European Application No. 893450. Typically, the Cohn fraction, and Kissler / Nitschmann precipitates A (KN A), B (KN B), and supernatant precipitate B (KN B+1) are present as suspended pastes. Other purification techniques, including chromatography, may also be used.

[0499] The term "cryo-precipitate" or "cryo-precipitates" refers to proteins in plasma that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold. Cryoprecipitates include factor VIII, fibrinogen, von Willebrand factor, factor XIII, and platelet membrane microparticles.

[0500] The term "cryoprecipitate plasma" shall be taken to mean plasma from which the cryoprecipitate has been removed.

[0501] The term "cryo-rich plasma" shall be taken to mean plasma that contains components typically found in cryoprecipitates.

[0502] The term "clarified" or "clarifying" is intended to mean the process of passing plasma or a fraction thereof through appropriate filters (e.g., depth filters and / or 1.2 and 0.45 / 0.22 μm membrane filters) to remove one or more impurities prior to use in the methods described herein.

[0503] The term "dissociation constant" refers to the pKa of a buffer. pKa=-log 10 (Ka), where Ka is the acid dissociation constant of the buffering agent in the buffer. For example, a wash buffer of 20 mM sodium dihydrogen phosphate, 40 mM sodium chloride at pH 7.4 contains sodium dihydrogen phosphate as the buffering agent. Phosphate has three dissociation constants (pKa1: 2.16, pKa2: 7.21, pKa3: 12.32).

[0504] The term "affinity chromatography resin" shall be taken to mean a resin comprising an affinity chromatography ligand (e.g., a camelid-derived single domain [VHH] antibody fragment) bound to a matrix, e.g., as described herein. Exemplary affinity chromatography resins for use in the methods described herein include POROS® CaptureSelect® FcXP affinity resin (Thermo Fisher) and CaptureSelect® FcXP agarose affinity resin (Thermo Fisher). Further exemplary affinity chromatography resins include resins having an amino acid sequence encoded by SEQ ID NO: 1 or a variant thereof that specifically binds to the CH3 domain of human IgG. Exemplary affinity chromatography resins are also described in US Pat. No. 10,259,886.

[0505] The terms "specifically binds," "specifically binding," or "binds specifically" shall be taken to mean that a protein of the disclosure reacts or associates with a particular antigen or cell expressing it more frequently, more rapidly, more persistently, and / or with higher affinity than it reacts with alternative antigens or cells. For example, a ligand capable of specifically binding to the CH3 domain of human IgG will bind with substantially greater affinity (e.g., 1.5-fold, or 2-fold, or 5-fold, or 10-fold, or 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 150-fold, or 200-fold) than it binds to other antigens. Generally, but not necessarily, reference to binding shall mean specific binding, and each term shall be understood to clearly support the other term.

[0506] The term "ligand" shall be taken to mean a molecule immobilized on the matrix of an affinity chromatography resin that specifically binds to the CH3 domain of human IgG. For example, the ligand is a single domain [VHH] antibody fragment of camelid origin.

[0507] The term "enriched preparation" shall be considered to include an eluate, solution or pharmaceutical composition as described herein. The concentrated preparations of the present disclosure contain IgG of higher purity compared to IgG in plasma or fractions thereof.

[0508] The term "Camelidae-derived single domain [VHH] antibody fragment" shall be taken to mean the VHH domain of a Camelidae antibody. Camelidae antibodies are antibodies produced from camels and llamas, which lack the CH1 domain normally present in human immunoglobulins and have only one VHH domain. Exemplary affinity chromatography resins containing Camelidae-derived [VHH] antibody fragments include CaptureSelect® antibody affinity chromatography resins (Thermo Fisher). For example, CaptureSelect® FcXL affinity resin, POROS® CaptureSelect® FcXP affinity resin, CaptureSelect IgG-CH1 affinity resin, and CaptureSelect FcXP agarose affinity resin. Further exemplary affinity chromatography resins include IgSelect® affinity resin (Cytiva), HiTrap® IgSelect® affinity resin (Cytiva), Pierce® Protein G Agarose affinity resin (Thermo Fisher), and Protein G Sepharose 4 Fast Flow affinity resin (Cytiva).

[0509] The term "matrix" shall be taken to mean a support to which a ligand is immobilized. Exemplary matrices are cross-linked poly(styrene-divinylbenzene) matrices and agarose-based matrices.

[0510] The term "dynamic binding capacity" or "DBC" of a chromatography resin shall be considered to refer to the maximum amount of IgG that the resin will bind under operating conditions before significant breakthrough of unbound IgG occurs.

[0511] The term "per mL of resin" shall be deemed to refer to per mL of wet packed volume of resin.

[0512] The term "bed height" shall be taken to mean the height to which the affinity chromatography resin is packed in the column. It will be clear to one skilled in the art that reference to "total bed height" refers to the bed height of all columns in a continuous chromatography setup.

[0513] The term "non-loading phase" shall be taken to mean a phase other than the loading phase of a continuous chromatographic method. For example, a non-loading phase may mean an equilibration phase, a wash phase, an elution phase, a strip phase and / or a re-equilibration phase.

[0514] The term "cycle" shall be taken to mean one round of equilibration, IgG loading, binding, elution, stripping, sanitization, and / or regeneration performed on the resin.

[0515] The term "purity" shall be taken to mean the proportion of IgG relative to the total protein content of purified IgG, IgG enriched preparations and pharmaceutical compositions, expressed as a percentage.

[0516] The terms "industrial or commercial scale" or "large scale" or "manufacturing scale" shall be deemed to refer to the amount of product produced in batches intended for clinical trials, formulation, sale and / or distribution to the public. For example, industrial scale refers to the large-scale purification of IgG from plasma or a fraction thereof to produce a plasma protein product.

[0517] The term "plasma protein product" shall be taken to refer to preparations, compositions and / or protein products that contain plasma proteins (e.g., impurities such as IgG or albumin) derived from the purification of plasma or a fraction thereof. Typically, the plasma proteins are the predominant proteins in the plasma protein product.

[0518] The term "pharmaceutical composition" shall be taken to mean a formulation of IgG with compounds generally accepted in the art for the delivery of IgG to a mammal. Exemplary compounds include all pharma- ceutically acceptable carriers, diluents or excipients thereof.

[0519] The terms "treat" or "treatment" or "treating" shall be taken to mean administering a therapeutically effective amount of IgG such that one or more symptoms or characteristics of a condition in a subject are ameliorated, or such that the subject is no longer clinically diagnosed as having the condition.

[0520] The terms "preventing," "prevent," or "prevention" include providing prophylaxis with respect to the occurrence or recurrence of a particular condition in a subject. A subject may be predisposed to or at risk of developing a condition, but may not yet be diagnosed with said condition.

[0521] As used herein, the phrase "slowing the progression of" includes reducing or slowing the progression of the condition and / or at least one symptom of the condition in a subject.

[0522] The term "condition" shall be taken to mean a state or condition of a subject requiring treatment with IgG. Exemplary conditions include, but are not limited to, primary immunodeficiency (PI), chronic inflammatory demyelinating polyneuropathy (CIDP), and chronic immune thrombocytopenic purpura (ITP).

[0523] The term "subject" shall be taken to mean any animal, e.g., a mammal, including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0524] Sequential Affinity Chromatography The present disclosure provides a method for purifying IgG from plasma or a fraction thereof using sequential affinity chromatography.

[0525] The term "continuous affinity chromatography" shall be taken to mean a chromatographic method comprising one or more columns (columns) packed with the same affinity resin, where each column comprises one or more zones. A zone is a column, or a region of a column, that contains a resin in which one or more chromatographic steps can be performed. For example, a zone is selected from the group consisting of an equilibration zone, a binding zone, a washing zone, an elution zone, a stripping zone, or a combination thereof. In one example, a zone is selected from the group consisting of an equilibration zone, a binding zone, a washing zone, an elution zone, or a combination thereof.

[0526] Sequential affinity chromatography involving two or more columns involves columns connected in an arrangement that allows the columns to be operated in series and / or in parallel. In principle, the first column and / or subsequent columns can be loaded with IgG while other columns (or other zones of columns) are simultaneously equilibrating, washing, eluting, and / or regenerating. Examples of sequential affinity chromatography will be apparent to those skilled in the art and / or are described herein.

[0527] Examples of columns that can be used to perform the continuous chromatography method are clear to those skilled in the art and / or are described herein.For example, the continuous chromatography method can be performed using Tricorn 5 / 100 (Cytiva).In another example, the continuous chromatography method can be performed using BioSMB PD System (Sartorius).

[0528] Simulated Moving Bed (SMB) Chromatography In one example, the sequential affinity chromatography is simulated moving bed (SMB) chromatography. The term "simulated moving bed chromatography" or "SMB chromatography" refers to a chromatography method first described in U.S. Pat. No. 2,985,589. Examples of SMB chromatography setups and / or equipment will be apparent to those skilled in the art and / or are described herein. The simulated moving bed concept involves using multiple small columns (rather than one large column) containing solid sorbents (e.g., affinity resins) and simultaneously performing one or more sequential chromatography steps (i.e., equilibration, binding, washing, elution or stripping) on ​​different columns in a continuous loop.

[0529] In an example of an SMB chromatography setup, the columns are arranged in four sections, with one or more columns per section. Two inlet streams (feed and eluate) and two outlet streams (extract and raffinate) are directed alternately through the column ring. The inlet and outlet positions are switched in liquid flow direction at regular time intervals to simulate countercurrent movement of the columns. One or more columns of the SMB chromatography setup are loaded with the feed (containing the adsorbent components (extract)) and the extract binds to the resin in the column. Meanwhile, the less adsorbent components in the feed (raffinate) pass through the column. The raffinate is loaded onto one or more subsequent column(s) or removed from the SMB chromatography system as waste. The eluate is loaded onto the column and the extract is collected. For example, more feed can be loaded onto one or more subsequent column(s) while the eluate is collected from the first column.

[0530] Suitable washing and elution buffers having the features of the present disclosure will be apparent to those skilled in the art and / or are described herein.In one example, the washing buffer comprises 20 mM sodium dihydrogen phosphate, 145 mM sodium chloride, and has a pH of 7.4.In one example, the washing buffer comprises 20 mM sodium dihydrogen phosphate, 500 mM sodium chloride, and has a pH of 7.4.

[0531] The resin in SMB chromatography may undergo multiple (e.g., 50) cycles of resin equilibration, IgG loading, binding, elution, stripping, disinfection, and / or regeneration for each batch of plasma or fraction thereof used. Multiple batch runs (e.g., 4-10 batches) may be performed using SMB chromatography. The total life time of the resin in SMB chromatography may range from 200-500 cycles (if not more) before the resin becomes unusable. Generally, resin regeneration is performed to allow the resin to be used multiple times.

[0532] Periodic Countercurrent Chromatography (PCC) In one example, the continuous affinity chromatography is cyclic countercurrent chromatography (PCC). Examples of PCC setups and / or devices will be apparent to those skilled in the art and / or are described herein. The concept of PCC involves the use of multiple columns containing solid sorbents (e.g., affinity resins) to perform chromatographic steps in parallel in a quasi-continuous manner. Buffers used in the binding, washing, and / or elution steps flow countercurrently to the affinity resin.

[0533] An example of a PCC setup involves the use of two columns. In a first step, the sample is loaded onto the first column above the DBC of the resin, such that unbound products (e.g., IgG) pass through the first column and are captured by the second column. In a second step, the first column is washed, eluted, washed and / or re-equilibrated independently of the second column being loaded with additional sample. In a third step, additional sample is loaded onto the second column above the DBC of the resin, such that unbound products pass through the second column and are captured by the first column. In a fourth step, the second column is washed, eluted, washed and / or re-equilibrated independently of the first column being loaded with additional sample. The process steps are cycled continuously between the two columns.

[0534] Other examples of PCC setups involve the use of multiple columns, for example, as a variation of the above PCC setup, multiple columns can be used to capture unbound product, simulating the use of a large column.

[0535] Continuous Countercurrent Tangential Chromatography (CCTC) In one example, the continuous affinity chromatography is continuous countercurrent tangential chromatography (CCTC). Examples of CCTC setups and / or equipment will be apparent to those skilled in the art and / or are described herein. The CCTC concept involves using an affinity resin in the form of a slurry, which is continuously directed through a number of static mixers and hollow fiber membranes that separate the fluid phase from the resin. CCTC is usually performed at low pressure.

[0536] One example of a CCTC process involves binding, first wash, second wash, elution, stripping and / or equilibration steps. Other examples of CCTC processes include binding, first wash, second wash, elution and / or equilibration steps. For example, a CCTC process does not include a stripping step. The sample (e.g., plasma or a fraction thereof) and affinity resin are passed through a static mixer and hollow fiber membrane in the binding step. Impurities are removed in the flow-through of the hollow fiber membrane in the washing step, and the resin-bound product (i.e., IgG) is retained on the membrane. The hollow fibers retain the resin and allow the product to pass through in the elution step. The resin is stripped and / or equilibrated, and the process is repeated.

[0537] Continuous Countercurrent Spiral Chromatography (CCSC) In one example, the continuous affinity chromatography is continuous countercurrent spiral chromatography (CCSC). Examples of CCSC setups and / or equipment will be apparent to those skilled in the art and / or are described herein. The CCSC concept involves the use of a compact rotating coil separation column mounted on the rotating frame of a centrifuge. There are two currently available separation column designs: the spiral disk assembly and the spiral tube support assembly.

[0538] An exemplary CCSC process involves a coiled separation column rotating synchronously around its own axis (e.g., 1,000-1,200 rpm) while rotating around the central axis of a centrifuge. The mobile phase can be passed through the rotor of the centrifuge without a rotating seal, resulting in mass retention of the stationary phase while mixing the two phases along the length of the column, resulting in highly efficient solute separation.

[0539] Affinity Chromatography Resins The present disclosure provides a method for purifying immunoglobulin G (IgG) from plasma or a fraction thereof using an affinity chromatography resin. The affinity resin of the present disclosure comprises a ligand capable of specifically binding to the CH3 domain of human IgG.

[0540] Suitable affinity chromatography resins will be apparent to those skilled in the art and / or described herein. In one example, the resin comprises a ligand of a single domain [VHH] antibody fragment from camelid. Those skilled in the art will recognize that a ligand based on a single domain [VHH] antibody fragment from camelid can specifically bind to all subclasses of IgG (IgG1, IgG2, IgG3, IgG4). Exemplary resins are CaptureSelect® FcXP affinity chromatography resin (Thermo Fisher), CaptureSelect® FcXL affinity resin (Thermo Fisher), CaptureSelect® IgG-CH1 affinity resin (Thermo Fisher), and CaptureSelect® FcXP agarose affinity resin (Thermo Fisher). Further exemplary affinity chromatography resins include IgSelect® affinity resin (Cytiva), HiTrap® IgSelect® affinity resin (Cytiva), Pierce® Protein G Agarose affinity resin (Thermo Fisher), and Protein G Sepharose 4 Fast Flow affinity resin (Cytiva).

[0541] In one example, the affinity chromatography resin comprises a camelid-derived single domain [VHH] antibody fragment and a cross-linked poly(styrene-divinylbenzene) matrix. For example, the affinity chromatography resin is POROS® CaptureSelect® FcXP affinity resin (Thermo Fisher). The cross-linked poly(styrene-divinylbenzene) matrix allows the resin to withstand pressures up to 100 bar.

[0542] In one example, the affinity chromatography resin comprises a camelid-derived single domain [VHH] antibody fragment and an agarose-based matrix, for example, the affinity chromatography resin is CaptureSelect FcXP agarose affinity resin (Thermo Fisher).

[0543] In one example, the sequential affinity chromatography process is carried out at a pressure ranging from about 2 to about 5 bar. For example, the sequential affinity chromatography process is carried out at a pressure ranging from about 3 to about 4 bar. In one example, the sequential affinity chromatography process is carried out at a pressure ranging from about 3.25 to about 3.5 bar.

[0544] buffer solution The present disclosure provides a continuous affinity chromatography method using a buffer that allows efficient binding of IgG to and recovery from the resin. Generally, the plasma or a fraction thereof is at neutral pH (pH about 7.4). The resin is equilibrated with an equilibration buffer and / or washed with a wash buffer having a buffer range covering neutral pH. Suitable wash buffers include buffers with a dissociation constant (pKa) of 6.8-8.5 at 25°C.

[0545] An exemplary buffer for the equilibration and / or wash buffer is sodium dihydrogen phosphate, the phosphate component of which has three dissociation constants (pKa: 2.16, 7.21 and 12.32). Phosphate has a dissociation constant at about the pH of the elution and / or stripping buffer used in the sequential affinity chromatography method. However, phosphate does not have a dissociation constant between the pH of the equilibration and / or wash buffer (high pH) and the pH of the elution and / or stripping buffer (low pH) used in the sequential affinity chromatography method. This allows for quicker switching between the wash and elution steps, and the stripping and equilibration steps, resulting in sharper peaks and shorter chromatographic phases. The advantage of using such equilibration and / or wash buffers is that smaller amounts of buffer can be used, thereby improving the efficiency of the sequential affinity chromatography method.

[0546] Other suitable buffers for the equilibration and / or washing buffers include imidazole (pKa:7.0), Tris (pKa:8.30), glycylglycine (pKa:8.40), MOPS (pKa:7.2), PIPES (pKa:6.8), TES (pKa:7.40), bicine (pKa:8.35), HEPES (pKa:7.55), EPPS (pKa:8.00), HEPPSO (pKa:7.85), MOBS (pKa:7.60), POPSO (pKa:7.78), TAPSO (pKa:7.61), Tricine (pKa:8.05), TEA (pKa:7.76).

[0547] Analysis of IgG composition Methods for determining yield, purity and IgG subclass distribution will be apparent to one of skill in the art and / or are described herein.

[0548] In one example, purity is determined by SDS-PAGE and MALDI-TOF-MS peptide fingerprint analysis. Briefly, purified IgG, IgG concentrated preparations or IgG-containing pharmaceutical compositions described herein are loaded onto an appropriate SDS-PAGE gel (e.g., 8-16% TRIS-glycine) under reducing and non-reducing conditions along with protein size markers and a positive control for IgG (e.g., Privigen). Proteins are separated based on size and protein bands of interest are isolated, processed, and analyzed by MALDI-TOF-MS.

[0549] In other examples, impurities in the IgG enriched preparations or IgG-containing pharmaceutical compositions described herein are measured by enzyme-linked immunosorbent assay (ELISA) using an impurity (e.g., IgA) specific antibody. For example, ELISA is performed using commercially available methods. In one example, the purity, yield and / or subclass distribution of IgG is determined by nephelometry. In one example, the purity of IgG is determined by nephelometry. In one example, the yield of IgG is determined by nephelometry. In one example, the subclass distribution of IgG is determined by nephelometry. For example, the light scattering pattern of purified IgG, the IgG enriched preparations or IgG-containing pharmaceutical compositions described herein is measured by nephelometry and compared to the light scattering profile of a composition with a known IgG subclass distribution.

[0550] Stability of plasma and its fractions The suitability of plasma or a fraction thereof for loading onto an affinity resin as described herein can be determined by assessing the procoagulant activity, proteolytic activity and particle size of the plasma or a fraction thereof. Methods for assessing procoagulant activity, proteolytic activity and particle size will be apparent to one of skill in the art and / or are described herein. Briefly, the plasma or a fraction thereof is freeze / thawed in one or more cycles, stored at 2°C to 32°C (e.g., 2°C, 10°C, 18°C, 21°C, 28°C or 32°C) for 24 hours or up to 48 hours, and analyzed using one or more methods described below. In one example, the plasma or a fraction thereof is thawed in one or more cycles at a temperature of 32°C, stored for 24 hours or up to 48 hours, and analyzed using one or more methods described below. In another example, the plasma or a fraction thereof is thawed in one or more cycles at a temperature of 32°C, stored for 24 hours or up to 48 hours, and analyzed using one or more methods described below, and then cooled and stored at a temperature of 21°C. In one example, the plasma or a fraction thereof is thawed at a temperature of 32° C. and at a temperature of 21° C. prior to sequential affinity chromatography.

[0551] In one example, the procoagulant activity in plasma or a fraction thereof can be determined using an in vitro clotting assay, such as the activated partial thromboplastin time (NaPTT) assay. The NaPTT assay measures the rate at which one or more clotting factors (e.g., fibrinogen, prothrombin, proaccelerin, antihemophilic factor, Stuart-Prower factor, plasma thromboplastin precursor factor, and Hegeman factor) are activated or formed in plasma or a fraction thereof when a clotting activator (e.g., silica, kaolin, ellagic acid) is added to the assay.

[0552] In one example, proteolytic activity in plasma or a fraction thereof can be assessed by measuring the activities of thrombin, general serine protease, kallikrein, plasmin and FXa using commercially available kits such as, for example, Thrombin Activity Assay Kit (S-2238), General Serine Protease Assay Kit (S-2288), Kallikrein Activity Assay Kit (S-2302), Plasmin Activity Assay Kit (S-2251) and FXa Activity Kit (S-2765).

[0553] In one example, the size of any particles in plasma or a fraction thereof is assessed by microflow imaging (MFI) and the polydispersity index is calculated, the calculation of which will be apparent to one skilled in the art.

[0554] Additional purification steps The additional purification step can be performed before or after the consecutive chromatography steps. In one example, the additional purification step can be performed before the consecutive chromatography steps. In one example, the additional purification step can be performed after the consecutive chromatography steps.

[0555] In one example, the method further comprises one or more steps selected from the group consisting of ethanol precipitation, octanoic acid fractionation, ion exchange chromatography, viral inactivation, viral filtration and ultrafiltration / diafiltration. Additional purification steps will be apparent to one of skill in the art and / or are described herein.

[0556] In one example, the method further comprises ethanol precipitation. For example, cold ethanol can be used to isolate and concentrate IgG by removing albumin, α- and β-globulin from plasma or its fractions. For example, as described in WO2011 / 149472.

[0557] In one example, the method further comprises immunoaffinity chromatography. For example, the method further comprises isoagglutinin affinity chromatography using Eshmuno anti-A and anti-B resin. For example, isoagglutinin affinity chromatography can be used to remove isoagglutinins A and B.

[0558] In one example, the method further comprises octanoic acid fractionation. Octanoic acid can be used to remove plasma lipids and plasma proteins (other than IgG). For example, as described in WO2011 / 131787.

[0559] In one example, the method further comprises ion exchange chromatography. In one example, the ion exchange chromatography is anion exchange chromatography. For example, anion exchange chromatography can be used to remove IgA, remaining IgM and other plasma components (other than IgG).

[0560] The anion exchanger can be a resin-based anion exchanger, an anion exchange membrane adsorbent, or any other type of anion exchanger having a positively charged substrate for capturing negatively charged particles. In one example, the anion exchanger is an anion exchange membrane adsorbent. In another example, the anion exchanger is a resin-based anion exchanger. In a further example, the anion exchanger is a monolith anion exchanger.

[0561] In one example, the method further comprises anion exchange chromatography using a resin-based anion exchanger. For example, the anion exchange chromatography resin is a strong anion exchanger. In one example, the strong anion exchange resin comprises a matrix consisting of a poly(styrene-divinylbenzene) matrix. In one example, the strong anion exchanger comprises a quaternized polyethyleneimine functional group. Suitable resin-based anion exchanges will be apparent to those skilled in the art, and include, for example, POROS™ HQ 50.

[0562] In one example, the anion exchange chromatography step is performed in flow-through mode. In another example, the anion exchange chromatography step is performed in bind-and-elute mode.

[0563] In one example, the anion exchange chromatography step includes a buffer selected from the group consisting of sodium citrate, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium dihydrogen phosphate, Bis-Tris, phosphate, L-histidine, and combinations thereof. In one example, the anion exchange chromatography step includes a buffer that includes an MES buffer. In another example, the anion exchange chromatography step includes a phosphate buffer.

[0564] In one example, the method further comprises viral inactivation. For example, viral inactivation can be performed by adjusting the solution to a low pH. The low pH can be a pH between 2 and 4. In one example, low pH viral inactivation is performed in the presence of caprylate. In another example, viral inactivation can be performed by contacting the plasma or a fraction thereof, or the IgG-enriched preparation or IgG-containing pharmaceutical composition with n-octyl-β-D-glucopyranoside (OG), thereby forming an OG-IgG mixture. In a further example, low pH viral inactivation is performed in the presence of N,N-dimethylmyristylamine N-oxide (TDAO).

[0565] In a further example, viral inactivation can be achieved by subjecting plasma or a fraction thereof, or an IgG-enriched preparation or an IgG-containing pharmaceutical composition to a solvent-detergent inactivation process. Suitable solvent-detergent treatments will be apparent to those skilled in the art and include, for example, environmentally friendly detergents. Exemplary environmentally friendly detergents suitable for use in the present disclosure, particularly for use in inactivating lipid-enveloped viruses, include N,N-dimethylmyristylamine N-oxide (TDAO), polysorbate 80 (PS80), polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100-reduced), and non-ionic surfactants prepared from glucose and alcohol (e.g., Simulsol™ formulations). In one example, the detergent is N,N-dimethylmyristylamine N-oxide (TDAO). In one example, the detergent is polysorbate 80. In another example, the detergent is polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100-reduced). In a further example, the surfactant is a non-ionic surfactant prepared from glucose and alcohol.

[0566] In one example, the OG concentration in the OG-IgG mixture is in the range of 25 mM to 80 mM. For example, the OG concentration in the OG-IgG mixture is in the range of 25 mM to 50 mM, or 50 mM to 80 mM, or 30 mM to 60 mM. For example, the OG concentration in the OG-IgG mixture is 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM, or 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM.

[0567] In one example, the OG concentration in the OG-IgG mixture is 30 mM.

[0568] In one example, the plasma or a fraction thereof, or the IgG-enriched preparation or IgG-containing pharmaceutical composition is contacted with OG for up to 15 minutes. For example, the plasma or a fraction thereof, or the IgG-enriched preparation or IgG-containing pharmaceutical composition is contacted with OG for up to 0.5 minutes, or 1 minute, or 1.5 minutes, or 2 minutes, or 2.5 minutes, or 3 minutes, or 3.5 minutes, or 4 minutes, or 4.5 minutes, or 5 minutes, or 5.5 minutes, or 6 minutes, or 6.5 minutes, or 7 minutes, or 7.5 minutes, or 8 minutes, or 8.5 minutes, or 9 minutes, or 9.5 minutes, or 10 minutes, or 10.5 minutes, or 11 minutes, or 11.5 minutes, or 12 minutes, or 12.5 minutes, or 13 minutes, or 13.5 minutes, or 14 minutes, or 14.5 minutes, or 15 minutes.

[0569] In one example, the plasma or a fraction thereof, or the IgG-enriched preparation or the IgG-containing pharmaceutical composition is contacted with OG at a temperature ranging from 2° C. to 28° C. For example, the plasma or a fraction thereof, or the IgG-enriched preparation or the IgG-containing pharmaceutical composition is contacted with OG at a temperature ranging from 2° C. to 8° C., or from 2° C. to 28° C., or from 2° C. to 25° C., or from 2° C. to 20° C., or from 2° C. to 18° C., or from 2° C. to 15° C., or from 2° C. to 10° C. For example, the plasma or a fraction thereof, or the IgG enriched preparation or the IgG-containing pharmaceutical composition is contacted with OG at a temperature of 2°C, or 3°C, or 4°C, or 5°C, or 6°C, or 7°C, or 8°C, or 9°C, or 10°C, or 11°C, or 12°C, or 13°C, or 14°C, 15°C, or 16°C, or 17°C, or 18°C, or 19°C, or 20°C, or 21°C, or 22°C, or 23°C, or 24°C, or 25°C, or 26°C, or 27°C, or 28°C.

[0570] In one example, the plasma or a fraction thereof, or the IgG enriched preparation or IgG-containing pharmaceutical composition is contacted with OG at a temperature of 2°C.

[0571] In one example, the plasma or a fraction thereof, or the IgG enriched preparation or IgG-containing pharmaceutical composition is contacted with OG at a temperature of 8°C.

[0572] In one example, the plasma or a fraction thereof, or the IgG-enriched preparation or IgG-containing pharmaceutical composition is contacted with OG at a temperature of 10°C.

[0573] In one example, the plasma or a fraction thereof, or the IgG enriched preparation or IgG-containing pharmaceutical composition is contacted with OG at a temperature of 18°C.

[0574] In one example, the plasma or a fraction thereof, or the IgG enriched preparation or IgG-containing pharmaceutical composition is contacted with OG at a temperature of 28°C.

[0575] In one example, the method further includes virus filtration. For example, a virus filtration membrane with a pore size of 15-20 nm can be used to remove microorganisms and viruses from the solution or eluate or pharmaceutical composition. Exemplary nanofilters include Planova S20N (Asahi), Virosart HC (Sartorius) and Planova 20N (Asahi).

[0576] In one example, the method further comprises UF / DF. Exemplary UF / DF membranes are Pellicon 2 Cassettes (Millipore) or Polyethersulfone or Hydrosart cassettes (Sartorius).

[0577] Pharmaceutical Compositions The purified IgG (co-active ingredient) of the present disclosure is useful for formulation into pharmaceutical compositions for parenteral administration, such as intravenous or subcutaneous administration, for therapeutic and prophylactic treatments.

[0578] The composition for administration generally comprises a solution of the purified IgG of the present disclosure dissolved in a pharma- ceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers, such as buffered saline, may be used. The composition may optionally contain a pharma- ceutically acceptable carrier to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0579] The concentration of the purified IgG of the present disclosure in these formulations can vary widely and is selected primarily based on fluid volumes, viscosities, and body weight, depending on the particular mode of administration selected and the needs of the patient. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives. For example, the pharmaceutical composition includes proline as a stabilizer.

[0580] Suitable pharmaceutical compositions according to the present disclosure will generally comprise an amount of purified IgG of the present disclosure mixed with an acceptable pharmaceutical carrier, such as a sterile aqueous solution, to provide a range of final concentrations depending on the intended use. Preparation techniques are generally known in the art, as exemplified in Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980.

[0581] For example, the IgG concentration of the pharmaceutical composition is 1-5% w / v, 5-15% w / v, or 8-12% w / v. For example, the IgG concentration of the pharmaceutical composition is 1%, 2%, 3%, 4%, 5%, or 6%, or 7%, or 8%, or 9%, or 10%, or 11%, or 12%, or 13%, or 14%, or 15% w / v. For intravenous use, 1% w / v (i.e., 10 g IgG / L) may be used. For intravenous use, 10% w / v (i.e., 100 g IgG / L) may be used.

[0582] For subcutaneous administration, higher concentrations can be used, for example, 15-35% w / v, or 20-30% w / v. In one example, the IgG concentration of the pharmaceutical composition is 16%, or 17%, or 18%, or 19%, or 20%, or 21%, or 22%, or 23%, or 24%, or 25%, or 26% w / v.

[0583] How to use As discussed herein, the disclosure provides methods of treating, preventing and / or delaying progression of a condition in a subject comprising administering to the subject an IgG or pharmaceutical formulation.

[0584] In one example, the condition is selected from the group consisting of primary immunodeficiency (PI), chronic inflammatory demyelinating polyneuropathy (CIDP), and chronic immune thrombocytopenic purpura (ITP). EXAMPLES

[0585] Example 1: Affinity resin The affinity chromatography resins POROS® CaptureSelect® FcXP affinity resin (Thermo Fisher) and CaptureSelect® FcXP agarose affinity resin (Thermo Fisher) were used to evaluate the suitability of the resin for capturing IgG from plasma or its fractions. Both affinity chromatography resins contain a ligand capable of binding to the CH3 domain of human IgG, specifically single domain [VHH] antibody fragments of camelid origin, and a matrix of either cross-linked poly(styrene-divinylbenzene) or agarose. The affinity chromatography resins were packed into Cytiva Tricorn columns (5 mm diameter) and chromatography was performed on an Akta avant 25 system (Cytiva).

[0586] Cryo-rich plasma (CRP) and cryo-purified plasma (CPP) (CSL Behring) were warmed to 37°C and filtered through a 0.22 μm bottle-top filter. Affinity chromatography resins were evaluated using the chromatography run conditions and buffers provided by Thermo Fischer, as shown in Table 1 below.

[0587] [Table 1]

[0588] Purification of IgG from CRP and CPP using POROS® CaptureSelect® FcXP affinity resin resulted in higher IgG yields and slightly purer eluates compared to using CaptureSelect® FcXP agarose affinity resin. Based on these initial results, the suitability of POROS® CaptureSelect® FcXP affinity resin for use in sequential affinity chromatography was further evaluated.

[0589] Example 2: Buffer Composition A disadvantage of the wash buffers in Table 1 is that the buffer citrate has three dissociation constants (pKa1: 3.13, pKa2: 4.76, pKa3: 6.4) and is not adequately buffered at the pH of 7.4 required for equilibration. Furthermore, pH shifts during wash and elution, and during stripping and equilibration with citrate-containing buffers (listed in Table 1), result in inefficient pH changes and extension phases during chromatography.

[0590] To determine a suitable buffer composition that allows for rapid adjustment of pH during continuous affinity chromatography, the experiment performed in Example 1 was repeated using POROS® CaptureSelect® FcXP affinity resin using the chromatography buffers in Table 2 instead of Table 1.

[0591] [Table 2]

[0592] The sodium dihydrogen phosphate present in the wash buffer has three dissociation constants (pKa1: 2.16, pKa2: 7.21, and pKa3: 12.32) covering the pH range of 7.4 required for resin equilibration. It was found that increasing the buffer concentration of sodium dihydrogen phosphate to 20 mM eliminated the effects of insufficient buffering during the experiment. However, lower concentrations of sodium dihydrogen phosphate may be used in the wash buffer.

[0593] The results showed that the use of sodium dihydrogen phosphate allowed for rapid switching between low pH during elution and stripping and high pH during equilibration and washing, resulting in sharper peaks and shorter phases in sequential affinity chromatography runs.

[0594] No difference in the binding behavior of IgG was observed between pH 7.4 and 7.8, which allows CRP and CPP with a pH in this range to be applied to the resin without pH adjustment.

[0595] Example 3: Bed Height and Flow Rate The bed height and flow rate used in the chromatographic process were evaluated to determine conditions that allow for the reduction of the loading and non-loading phases. The experiment described in Example 2 was repeated with different bed heights of the resin and different flow rates of plasma or fractions thereof and elution buffer applied to the resin.

[0596] A bed height of 20 cm showed a significantly higher purification efficiency of IgG from plasma or its fractions compared to lower bed heights, which allowed for a short non-loading phase. Lowering the flow rate during elution to 150 cm / hr (contact time 5 min) did not show a significant effect compared to elution at 2400 cm / h. Surprisingly, it was found that there was no significant decrease in IgG binding when the loading flow rate at which plasma or its fractions were applied to the resin was increased to 2400 cm / hr (contact time 0.5 min).

[0597] The optimized conditions for purifying IgG from plasma or its fractions, determined by extensive experimentation, are summarized below in Table 3. The selected combination of buffer composition, resin, bed height, and flow rate reduced the amount of waste, flow-through, and product in each of the non-loading phases (i.e., equilibration, wash, elution, strip, and re-equilibration).

[0598] [Table 3]

[0599] When a column with a bed height of 20 cm was used, the total volume of the unloaded phase was 8.6 CV, whereas the loaded capacity of the clarified CPP was 4.3 CV. The unloaded / loaded CV ratio was 2, allowing the SMB setup with only three columns. Similar results were observed using a column bed height of approximately 6 cm. In experiments performed with a column with a bed height of approximately 6 cm, the total volume of the unloaded phase was 7 CV, whereas the loaded capacity of the clarified CPP was 3.8. The unloaded / loaded CV ratio was 1.8, allowing the SMB setup with only three columns instead of four. This result shows that reducing the volume of the unloaded phase allows the use of fewer columns in the purification method, regardless of the bed height.

[0600] Furthermore, IgG was eluted in 1.8 CV, resulting in a concentration factor of 2.4. The cycle time (excluding pump ramp-up and pump wash) was approximately 6.5 min.

[0601] Further concentration can be achieved by overlapping the elution and stripping phases, as can the wash and elution phases. The buffer requirement can be further reduced by increasing the buffer concentration or pH during re-equilibration, resulting in a durably unloaded / loaded CV ratio below 2, even as the loading amount of plasma or its fractions decreases with resin aging.

[0602] Example 4: Elution Profile purity SDS-PAGE gel electrophoresis was performed to qualitatively determine the purity of the eluates of Examples 2 and 3. Samples (8 μg and 16 μg) of IgG purified from clarified CPPs using FcXP resin (FcXP) according to Examples 2 and 3 were run on an 8-16% TRIS-glycine SDS-PAGE gel under reducing and non-reducing conditions (Figure 1). A protein marker (M) See Blue Plus 2 Marker (Invitrogen) was also included. SDS-PAGE gels were Coomassie stained. The purity of IgG from clarified CPPs purified using POROS® CaptureSelect® FcXP affinity resin was determined to be 98.7% by SDS-PAGE.

[0603] MALDI-TOF-MS peptide mass fingerprinting was performed to determine the impurity profile of the eluates of Examples 2 and 3. Visible bands (indicated by arrows in FIG. 1) were isolated and used in MALDI-TOF-MS peptide mass fingerprinting to determine the identity of the protein in each band. Impurities were identified in the MALDI-TOF-MS peptide mass fingerprinting (indicated by arrows A-F in FIG. 1) and are summarized in Table 4. The remaining bands (arrows without letter designation in FIG. 1) were identified as IgG. The most abundant impurities in the FcXP sample were IgM, albumin, and apolipoprotein A-1 (arrows B, C, and F in FIG. 1). The other three components of the complement system were minor impurities (arrows A, D, and E in FIG. 1 and bands A, D, and E in Table 4).

[0604] [Table 4]

[0605] SE-HPLC showed that the average monomer and dimer content of IgG in the eluate was 98.1%, the polymer content was 0.9%, and the fragment content was 1.0%.

[0606] 2D differential gel electrophoresis (2D DIGE) was performed to determine the isoelectric point (pI) of impurities in the eluate relative to IgG. Samples of purified IgG from clarified CPPs (FcXP POROS® eluate) were loaded with Sci5 and Sci3 dyes, respectively, and loaded onto 2D-SDS-PAGE. Separation of the samples by isoelectric point was performed at pH 3-10, followed by size separation under reducing conditions in the presence of SDS (Figure 2). IgG was identified to have an isoelectric point at pH 7-9 for the FcXP POROS® eluate samples (Figure 2). IgM and albumin were identified to have a pH of approximately 6.5 in the eluate (circles in Figure 2), lower than the isoelectric point of IgG.

[0607] The results indicate that it is possible to remove IgM and albumin from the eluate without significant loss of IgG using an ion exchange polishing step.

[0608] IgG subclass distribution Immunonephelometry was performed to determine whether the affinity resin used for IgG purification affected the subclass distribution of IgG in the eluates of Examples 2 and 3. IgG subclass distribution was measured for purified IgG from CRP, CPP, CRP or CPP using POROS® CaptureSelect® FcXP (Figure 3). Subclass distribution was calculated as the relative portion of IgG classes to the sum of all classes (IgGx / (IgG1+IgG2+IgG3+IgG4)).

[0609] yield The yield was measured using immunonephelometry. In the eluates from plasma in Examples 2 and 3, at least 95% of the IgG was recovered, and was as high as 96%.

[0610] Example 5: Purification Cycle To investigate the effect of purification cycles on POROS® CaptureSelect® FcXP affinity resin, multiple purification cycles were performed in succession and the binding capacity of the resin was measured. At various time points during multiple purification cycles, the breakthrough behavior of pure IgG was measured and used to calculate the remaining binding capacity of the resin. The loss of binding capacity can be attributed to aging of the resin.

[0611] CRP or CPP was applied to the resin (6 cm or 20 cm bed height), plasma was allowed to contact the resin for 0.5 min for each phase, and the binding capacity of the resin was measured over time. Figure 4 shows that there is no difference in resin aging between SMB chromatography runs performed with 6 cm and 20 cm bed height resin. The decrease in binding capacity follows a linear trend with an average slope of less than 5% per 100 runs, indicating minimal resin aging even after 100 runs. This result indicates that the POROS® CaptureSelect FcXP® affinity resin is suitable for use in the SMB chromatography setup under the conditions described in Examples 2 and 3.

[0612] Example 6: Stability of Plasma and Its Fractions The stability of CRP and CPP for use in the methods described herein was evaluated. CRP and CPP were freeze-thawed up to two times and / or filtered using a 0.22 μm filter. Processed CRP and CPP were stored at 10° C., 18° C., or 28° C. for 24 or 48 hours. The IgG content and IgG subclass distribution of the samples were measured by immunonephelometry. The results in Tables 5 and 6 indicate that the IgG content and IgG subclass distribution of CRP and CPP are not affected by filtration, temperature, time, and freeze / thaw.

[0613] [Table 5]

[0614] [Table 6]

[0615] To measure the clotting of proteins in plasma and CPPs, procoagulant activity was measured using the non-activated partial thromboplastin time (NaPTT) assay. Clotting time was set to >150 seconds in the NaPTT assay. Procoagulant activity was observed in plasma and CPPs at 28°C for 24 or 48 hours (Figure 5). No procoagulant activity was observed in plasma and CPPs after filtration, freeze / thawing, or at temperatures of 10°C or 18°C ​​for 24 or 48 hours.

[0616] To measure proteolytic activity in plasma and CPPs, the activities of thrombin, general serine protease, kallikrein, plasmin, and FXa were measured using chromogenic substrate assays (thrombin: S-2238; general serine protease: S-2288; kallikrein: S-2302; plasmin: S-2251; and FXa: S-2765). Proteolytic activity was observed in plasma and CPPs at 24 or 48 hours at 28°C (Figure 6). No proteolytic activity was observed in plasma and CPPs after filtration, freeze / thawing, or at temperatures of 10°C or 18°C ​​for 24 or 48 hours.

[0617] The stability of CRP and CPP was also evaluated by measuring the change in particle size within the samples as measured by microflow imaging (MFI) and dynamic light scattering (DLS), and the polydispersity index of the samples was also calculated. Tables 8 and 9 show that the samples were extensively polydispersed (>0.4). Tables 7 and 8 show that the polydispersity index of the samples at 10°C and 18°C ​​did not differ at 4 and 24 hours, but increased obviously at 48 hours. The trend index of plasma did not differ significantly with temperature and time, indicating that the plasma samples were more stable with time and temperature compared to CPP.

[0618] [Table 7]

[0619] [Table 8]

[0620] These results indicate that CRP and CPP undergo particle formation upon storage at higher temperatures for longer periods. Suitable temperatures for storage of plasma or fractions thereof may be between 10°C and 18°C ​​for up to 48 hours.

[0621] Example 7: Evaluation of viral inactivation using n-octyl-β-D-glucopyranoside N-octyl-β-D-glucopyranoside (OG) was evaluated to determine the virus reduction potential of CRP prior to IgG purification. Vesicular stomatitis virus (VSV) was added at a 1:20 dilution to thawed and homogenized CRP (50 mg / ml) and EMEM medium (control). Aliquots of VSV-spiked CRP and VSV-spiked EMEM medium were withdrawn before the addition of OG. OG was added to VSV-spiked CRP and VSV-spiked EMEM medium such that the final OG concentration in the mixture was 30 mM and mixed by pipetting for approximately 10 seconds. The mixture was incubated at 5°C with VSV-spiked EDEM medium and VSV-spiked plasma not spiked with OG. Incubation samples were withdrawn at 15, 30, and 60 min. To neutralize the activity of OG, samples were diluted 10-fold with medium.

[0622] Aliquots of 100 μL of OG-treated VSV-spiked CRP, OG-treated VSV-spiked EDEM medium, VSV-spiked CRP, VSV-spiked EDEM medium, and controls were titrated (10-fold serial dilutions ~ 10) into 150 μL of African green monkey kidney cell (Vero-PH) preculture suspension in standard 96-well microplates (Nunc, flat-bottom wells). -6Negative controls included CRP and EDEM media. Positive controls included the VSV stock used for spiking and a control virus stock with acceptable VSV titers achieved based on previous results of characterization of the VSV virus stock.

[0623] Plates were incubated at 37 °C, 3-5% CO2 and examined for virus-specific cytopathogenic effects (CPE) using a microscope over a period of 7 days. Cell cultures titrated in the negative control were required to be free of CPE.

[0624] Infectious titers were calculated according to the Spearman-Karber method and expressed as log10 CCID50 / mL (50% cell culture infectious dose per mL). If no infectious virus was detected in a microtitration starting from, for example, a 1:10 dilution, the viral titer was recorded as <1.5 log10 CCID50 / mL. To lower the limit of detection, 1 mL of post-treated Vero-PH cells at a 1:10 dilution was inoculated into four T25 flasks. If all four T25 cultures were negative for infectious virus, the resulting infectious titer was recorded as <0.5 log10 CCID50 / mL.

[0625] A robust reduction of VSV was observed when a final OG concentration of 30 mM was present in OG-treated, VSV-spiked CRP incubated at 5° C. The log10 reduction factor (LRF) under this condition (i.e., 30 mM final OG at 5° C.) was greater than 5.3 (Figure 7). The LRF is the ratio of the viral load in the starting material (e.g., CRP) before processing and the viral load in the final material (e.g., OG-treated CRP) after processing, taking into account both the sample volume and viral titer before and after processing.

[0626] Example 8: Optimization of plasma thawing To evaluate the optimal temperature for plasma thawing, the temperature at which cryoprecipitates were observed was determined. Briefly, cryo-rich plasma (CRP) was gradually heated from 15°C to 38°C after thawing, and fractions of CRP were centrifuged at 20°C, 25°C, 28°C, 30°C, 32°C, and 37°C, and the pellets were weighed depending on the temperature. At approximately 30°C, only nonspecific aggregates were present, and no cryoprecipitates were present.

[0627] To determine the optimal temperature, the activity of von Willebrand factor (vWF), one of the major components of cryoprecipitates, was measured in each pellet. As shown in Figure 8A and Table 8, thawing at 32°C resulted in robust pellet formation and activity of vWF in the supernatant.

[0628] [Table 9]

[0629] To further confirm plasma thaw at 32° C., retention time studies were performed as shown in FIG. 8B and the resulting eluates were assayed for proteolytic activity using the NaPTT assay.

[0630] As shown in Table 9, when the eluate was filtered at 32° C., no proteolytic activity was observed for up to 48 hours.

[0631] [Table 10]

[0632] Samples were also analyzed for particulate levels using dynamic light scattering. No differences in polydispersity index (PDI) were observed between samples, with all samples having a PDI < 0.6 (data not shown).

[0633] Turbidity was also assessed over time, and visible degradation was observed at all temperatures (i.e., 14°C, 21°C, and 32°C) after 48 hours. At 24 hours, no visible degradation was observed at 14°C, but almost none at 32°C. A distinct cycle per cycle pressure increase during chromatography was also observed at 48 hours in plasma held at 32°C compared to 14°C (data not shown).

[0634] Despite the visible degradation, no effect on IgG content was observed, particularly when samples were thawed and stored at higher temperatures, with no observed decrease in IgG content over time.

[0635] Example 9: Optimization of a sequential affinity chromatography method Strip phase removal In the SMB process, an increase in single-column backpressure was observed due to denaturation of proteins accumulated on the column due to harsh conditions (i.e., pH 2.5) during the strip phase. Therefore, to examine whether removing the strip phase could reduce the increase in backpressure, the SMB process was performed without removing the strip phase.

[0636] As shown in Figure 9, the removal of the strip phase resulted in a reduction in the backpressure rise during the SMB process and a stable pressure.

[0637] Increased conductivity of the cleaning phase The conductivity of the wash phase was screened to see if it could reduce protease activity in the eluate.

[0638] As shown in Figure 10, increasing the sodium chloride concentration in the wash buffer from 145 mM to 500 mM reduced protease activity in the eluate below the detection limit. Increasing the conductivity of the wash buffer increased the elution of additional proteins (e.g., components of the complement system), but no impact on IgG yield and / or overall product purity was observed with wash buffers of higher conductivity (Figure 11). Purity levels were comparable to the commercially available Privigen by Labchip assay and nephelometry (Figure 11B and Figure 11C).

[0639] Example 10: Anion Exchange Polishing Step The use of POROS™ HQ 50 anion exchange resin as a polishing step was evaluated. The resin was operated in flow-through mode.

[0640] First, the resins were screened using MES and phosphate buffers for their effect on impurity removal.

[0641] The FcXP eluate was subjected to UF / DF in 10 mM MES or phosphate buffer pH 6.0, 0 mM NaCl before being loaded onto an anion exchange chromatography column. The flow-through and post-wash were collected and analyzed separately.

[0642] As shown in Table 10, MES and phosphate buffers provided acceptable IgG yields and acceptable impurity removal.

[0643] Phosphate buffer at pH 6.0 (low conductivity) provided the best results for the development goal of impurity removal.

[0644] [Table 11]

[0645] Example 11: Optimization and Design of Experiments (DoE) studies of equilibration and loading buffers for the anion exchange polishing step Buffer solution evaluation test

[0646] [Table 12]

[0647] Samples were loaded according to Example 10 above. Briefly, the FcXP eluate was rebuffered and loaded onto a Poros™ HQ50. The flow-through and post-wash were collected and analyzed separately.

[0648] Citrate buffers removed IgA well over a wide range of both NaCl and pH. Acceptable IgA removal was achieved with low salt phosphate.

[0649] Phosphate buffers removed IgM well over a wide range of both NaCl and pH.

[0650] Bis-Tris and histidine provided good albumin removal over a wide range of both NaCl and pH. Phosphate provided good albumin removal at low salt.

[0651] Experimental Design Phosphate Buffer

[0652] [Table 13]

[0653] Preliminary recommendations for equilibration and loading included 0 mM NaCl and pH 6.2, with the load material in pilot-scale runs having a purity of approximately 93.5% and an impurity concentration of 40.4-71.6 mg / g IgG. In BioSMB runs, the load material had a purity of approximately 97% and an impurity / IgG concentration of 12.0-20.4 mg / g IgG, with the optimal concentration for impurity removal being 5-15 g / L IgG. Resin loads were 25-42 mg impurities per mL of resin.

[0654] Preliminary recommendations for post-wash conditions include 0 mM NaCl and pH 6.0.

[0655] Example 12: Anion Exchange Polishing Step MES and phosphate buffers were further screened for their effect on impurity removal in the POROS™ HQ 50 anion exchange polish step. The process was carried out as described above in Example 10 using MES buffer pH 6.0 with 20 mM or 50 mM NaCl, MES buffer pH 6.6 with 25 mM or 50 mM NaCl, and phosphate buffer pH 6.2 with 0 mM NaCl.

[0656] In MES and phosphate buffers, IgG yields were acceptable, and impurity removal was acceptable in the flow-through (no post-wash).

[0657] [Table 14]

[0658] Example 13: Scale-up of SMB FcXP Chromatography Step A four-column setup in SMB mode using 1 cm internal diameter columns was performed without resin stripping and alternating wash phases using the following conditions:

[0659] [Table 15]

[0660] After each batch, all columns were regenerated with 20% EtOH and 20 mM NaOH before being used for the next batch.

[0661] The IgG yield of the FcXP eluate ranged from 98.3 to 99.1%, and the total IgG recovery of all fractions ranged from 99.4 to 100.3%. The purity of the FcXP eluate was also assessed by Labchip, and the purity of the product was 96.4 to 96.9%.

[0662] The purification process yielded 9.55-10.4 g / L of IgG.

[0663] Impurities in the FcXP eluate that were measured included IgM (47.85-51.90 mg / L; 4.8-5.3 mg / gm IgG), IgA (68.8-76.55 mg / L; 7-8 mg / gm IgG) and albumin (55.3-78.05 mg / L; 5.8-7.6 mg / gm IgG).

[0664] Example 14: Scale-up process run with cryo-rich plasma Pooled cryo-rich plasma from 30 donors was thawed and clarified through 1.2 μm and 0.45 μm+0.2 μm filters before being used for downstream processing.

[0665] The thawed and filtered plasma was purified and processed according to the following process: 1. FcXP SMB Chromatography 2. Concentration and buffer exchange by UF / DF 3. pH shift and filtration 4. Anion Exchange Chromatography (using POROS™ HQ50) 5. Isoagglutinin affinity chromatography 6. Viral inactivation 7. UF / DF 8. Formulation to 100g / L IgG

[0666] Three separate runs were performed, with IgG recoveries from steps 1–6 ranging from 83–96% and total process recoveries ranging from 81–94%.

[0667] As a result of the purification process, the IgG subclass distribution of the final formulated bulk product was 67-69% IgG1, 24-27% IgG2, 3-4% IgG3, and 2-3% IgG4.

[0668] The purity of the products after isoagglutinin chromatography was also assessed by Labchip, and the purity of the products was 97–98.5%.

[0669] The flow-through and post-wash of POROS 50HQ were evaluated for impurities including IgM (<1.86-<2.55 μg / gm IgG), IgA (0.109-0.111 mg / gm IgG), and albumin (0.59-0.74 mg / gm IgG). Yields were also determined to be 93-97%.

[0670] The formulated bulk product was also evaluated for impurities including IgM (<0.17 mg / L; <1.74 to <1.86 μg / gm IgG), IgA (8.46 to 8.76 mg / L; 0.089 to 0.093 mg / gm IgG) and albumin (62.75 to 67.60 mg / L; 0.64 to 0.74 mg / gm IgG). FcXP ligand was <10 ppm (below the limit of detection).

[0671] Removal of isoagglutinin was also evaluated after FcXP SMB chromatography (Iso-A titer 1:16; Iso-B titer 1:32), POROS HQ50 chromatography (Iso-A titer 1:4; Iso-B titer 1:8), and isoagglutinin chromatography (Iso-A titer 1:0; Iso-B titer 1:0).

[0672] array SEQ ID NO: 1 is the amino acid sequence of the VHH fragment QVQLQESGGGLVQAGDSLRISCRASGLTVNDLYMGWFRQAPGKEREFVGRVTPGDNTDYTYYVDSVKGRFTISRDSAKNTVYLQMNSLKLEDTAVYLCAGRRFGSSEWDYWGQGTQVTVSS SEQ ID NO: 2 is the amino acid sequence of CDR1 of the VHH fragment GLTVNDLYMG SEQ ID NO: 3 is the amino acid sequence of CDR2 of the VHH fragment RVTPGDNTDYTYYVDSVK SEQ ID NO: 4 is the amino acid sequence of the CDR3 of the VHH fragment RRFGSSEWDY

Claims

1. A method for purifying immunoglobulin G (IgG) from plasma or a fraction thereof using continuous affinity chromatography, the method comprising binding IgG to an affinity chromatography resin comprising a ligand that specifically binds to the CH3 domain of human IgG, and recovering the IgG.

2. A method for producing an immunoglobulin G (IgG) enriched preparation from plasma or a fraction thereof using continuous affinity chromatography, the method comprising binding IgG to an affinity chromatography resin comprising a ligand that specifically binds to the CH3 domain of human IgG, and recovering the IgG.

3. The method according to claim 1, wherein the ligand comprises a single domain [VHH] antibody fragment derived from a camel.

4. The method according to claim 1, wherein the resin comprises a matrix selected from the group consisting of a crosslinked poly(styrene-divinylbenzene) matrix and an agarose-based matrix.

5. The method according to claim 1, wherein the ligand comprises a VHH antibody fragment conjugated to a crosslinked poly(styrene-divinylbenzene) matrix.

6. The method according to claim 1, further comprising washing the resin with a washing buffer having a pH of 5 to 10 and a dissociation constant (pKa) of 6.8 to 8.5 at 25°C.

7. The method according to claim 1, further comprising eluting the bound IgG from the resin with an elution buffer having a pH of 3 to 5.

8. The method according to claim 1, wherein the plasma or a fraction thereof is contacted with the resin for 0.1 to 5 minutes.

9. The method according to claim 1, wherein the plasma fraction is selected from the group consisting of cryo-rich plasma, cryo-poor plasma, supernatant I (SN I), cone fraction II (FrII), cone fraction II+III (Fr II+III), cone fraction I+II+III (FrI+II+III), Kistler / Nitschmann precipitate A (KN A), Kistler / Nitschmann precipitate B (KN B), Kistler / Nitschmann supernatant precipitate B (KN B+1), and combinations thereof.

10. The method according to claim 1, wherein the plasma or a fraction thereof is thawed at a temperature of at least 32°C.

11. The method according to claim 1, wherein the plasma or a fraction thereof prior to continuous affinity chromatography is at a temperature in the range of 2°C to 28°C.

12. The method according to claim 11, wherein the plasma or a fraction thereof before continuous affinity chromatography is at a temperature of 21 °C.

13. The method according to claim 11, wherein the plasma or a fraction thereof is at said temperature for a maximum of 48 hours.

14. The method according to claim 1, wherein the washing buffer contains a buffer selected from the group consisting of sodium dihydrogen phosphate, imidazole, Tris, glycylglycine, 3-morpholinopropane-1-sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), bis[(2-hydroxyethyl)amino]acetic acid (bicine), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sulfurous acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-(2-hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), 4-(N-morpholino)butanesulfonic acid (MOBS), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), tricine, triethanolamine (TEA) and combinations thereof.

15. The method according to claim 14, wherein the buffer is at a concentration of 5 mM to 200 mM.

16. The method according to claim 1, wherein the washing buffer further contains sodium chloride and / or divalent salts at a concentration of up to 1000 mM.

17. The method according to claim 1, wherein the washing buffer contains 20 mM sodium dihydrogen phosphate, 500 mM sodium chloride and has a pH of 7.

4.

18. The method according to claim 1, wherein the elution buffer is a phosphate buffer and / or an acetate buffer having a pH of 3 to 5, or contains these.

19. The method according to claim 1, wherein the elution buffer is in contact with the resin for a maximum of 5 minutes.

20. The method according to claim 1, further comprising equilibrating the resin with an equilibration buffer having a pH of 7 to 8.

21. The method according to claim 20, wherein the equilibration buffer contains 20 mM sodium dihydrogen phosphate, 500 mM sodium chloride and has a pH of 7.

4.

22. The method according to claim 20, wherein the resin is equilibrated i) after stripping the resin or ii) without stripping the resin.

23. The method according to claim 1, further comprising equilibrating the resin with an equilibration buffer having a pH of 7 to 8 after stripping the resin.

24. The method according to claim 1, wherein the continuous affinity chromatography is selected from the group consisting of simulated moving bed (SMB) chromatography, periodic countercurrent chromatography (PCC), continuous countercurrent tangential chromatography (CCTC), and continuous countercurrent spiral chromatography (CCSC).

25. The method according to claim 1, wherein the resin is in the form of a slurry or the resin is packed in one or more columns, each column containing one or more zones.

26. The method according to claim 25, wherein the zone is selected from the group consisting of an equilibration zone, a binding zone, a washing zone, an elution zone, or a combination thereof.

27. The method according to claim 25, wherein the columns are fluidly connected and separated by a fluid conduit including inlet and outlet valves.

28. The method according to claim 25, wherein the resin is packed in a series of three columns, each column being a separate zone.

29. The method according to claim 1, wherein the resin is packed in a first column and one or more subsequent columns.

30. The method according to claim 29, wherein IgG is loaded onto the first column at a concentration exceeding the dynamic binding capacity (DBC) of the resin.

31. The method according to claim 30, wherein the DBC of the resin is at least 5 mg of IgG per mL of resin.

32. The method according to claim 29, wherein one or more subsequent columns are loaded with IgG at a concentration up to the DBC of the resin.

33. The method according to claim 29, wherein one or more subsequent columns are loaded with IgG at a maximum IgG concentration of 40 mg per mL of resin.

34. The method according to claim 1, wherein the resin has a total bed height of 2 cm to 30 cm.

35. The method according to claim 1, further comprising regenerating the resin.

36. The method according to claim 1, further comprising disinfecting the resin.

37. The method according to claim 1, further comprising one or more steps selected from the group consisting of ethanol precipitation, octanoic acid fractionation, ion exchange chromatography, virus inactivation, virus filtration, and ultrafiltration / diafiltration.

38. The method according to claim 37, wherein the ion exchange chromatography step comprises an anion exchange chromatography step using a strong anion exchange resin operating in a flow-through mode.

39. The method according to claim 38, wherein the strong anion exchange resin comprises a matrix consisting of a poly(styrene-divinylbenzene) matrix.

40. The method according to claim 38, wherein the strong anion exchange resin comprises a quaternized polyethyleneimine functional group.

41. The method according to claim 38, wherein the anion exchange chromatography step comprises a post-loading wash buffer selected from the group consisting of phosphate buffer, sodium citrate buffer, 2-(N-morpholino)ethanesulfonic acid buffer, acetate buffer, Bis-tris buffer, and L-histidine buffer.

42. The method according to claim 41, wherein the post-loading wash buffer comprises a phosphate buffer having a pH in the range of 5.8 to 6.

6.

43. The method according to claim 41, wherein the post-loading wash buffer further comprises sodium chloride at a concentration of 0 mM to 50 mM.

44. The method according to claim 1, wherein at least 75% of IgG is recovered from plasma or a fraction thereof.

45. The method according to claim 1, wherein the eluted IgG has a purity of at least 95%.

46. The method according to claim 1, further comprising formulating IgG into a pharmaceutical composition.

47. A method for purifying immunoglobulin G (IgG) from plasma or a fraction thereof using simulated moving bed (SMB) chromatography, comprising: a) equilibrating an affinity chromatography resin comprising a crosslinked poly(styrene-divinylbenzene) matrix and a ligand capable of specifically binding to the CH3 domain of human IgG with a 20 mM phosphate equilibration buffer having a pH of 7 to 8; b) binding IgG from plasma or a fraction thereof to the resin; c) washing the resin with a 20 mM phosphate wash buffer having a pH of 7 to 8; and d) eluting the bound IgG with a 20 mM acetate or phosphate elution buffer having a pH of 3 to 5 comprising; Steps a) to d) may be repeated on an affinity chromatography resin, the affinity chromatography resin being packed in a series of two or more fluid-connected columns separated by a fluid conduit including inlet and outlet valves, optionally the method not including stripping the resin, a method.

48. Use of IgG purified or produced by the method according to any one of claims 1 to 47 in the manufacture of a medicament for the treatment, prevention and / or delay of progression of a condition in a subject.

49. Use according to claim 48, wherein the condition is selected from the group consisting of primary immunodeficiency (PI), chronic inflammatory demyelinating polyneuropathy (CIDP), and chronic immune thrombocytopenic purpura (ITP).

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