CN117769570A

CN117769570A - Method for purifying immunoglobulin G and use thereof

Info

Publication number: CN117769570A
Application number: CN202280052984.0A
Authority: CN
Inventors: R·阿南迪; P·多林格; L·霍勒; M·纽恩费尔特; M·波拉廷斯卡; H·N·维尔卡; K·安德斯; N·舒尔茨; D·博莱玛
Original assignee: CSL Behring AG
Current assignee: CSL Behring AG
Priority date: 2021-07-29
Filing date: 2022-07-29
Publication date: 2024-03-26

Abstract

The present disclosure relates to methods of purifying immunoglobulin G (IgG) and other proteins (e.g., albumin) from plasma or fractions thereof using affinity chromatography resins comprising ligands capable of specifically binding to the CH3 domain of human IgG. The disclosure also relates to formulations and uses of plasma protein products produced by the method.

Description

Method for purifying immunoglobulin G and use thereof

Data of related applications

The present application claims priority from australian patent application No. 2021902332 entitled "Method of purifying immunoglobulin G and uses thereof" filed on 29 of 7 th 2021, from U.S. patent application No. 63/227,329 entitled "Method of purifying immunoglobulin G and uses thereof" filed on 29 of 7 th 2021, and from U.S. patent application No. 63/365,530 entitled "Method of purifying immunoglobulin G and uses thereof" filed on 31 of 5 th 2022.

Sequence listing

The present application is filed with a sequence listing in electronic form. The entire contents of the sequence listing are incorporated herein by reference.

FIELD

The present disclosure relates to methods of purifying immunoglobulin G (IgG) and other proteins, such as albumin, from plasma, and formulations and uses of plasma protein products thereof.

Background

Immunoglobulin G (IgG) is the most abundant protein in plasmaOne, and is responsible for, for example, toxin neutralization, complement activation, and opsonization. IgG purified from human plasma is used for prophylactic prevention of infection in immunodeficient patients, replacement therapy of antibody deficiency 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 global consumption is increasing. Human immunoglobulin products (including hyperimmune (or "specific") and normal (or "non-specific")) consist 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 in normal multivalent human Immunoglobulin (IG) is similar to that in the donor population. https:// www.fda.gov/vaccines-blood-biologics/applied-blood-products/immune-globulins provide an FDA approved IG list. There are several commercial intravenous IG (IVIG) products (typically 5% or 10% (w/v) stable solutions) including (CSL Behring)、(Grifols)、-C(Grifols)、(Takeda) and->(Octaphaharma). Recently, subcutaneous IG (SCIG) administration has become possible. The commercial SCIG product (usually a 10%, 16.5% or 20% (w/v) stable solution) comprises +.>(CSL Behring)、-C(Grifols)、(Grifols)、(Octaphaharma) and +.>(Takeda). Other IG products are administered intramuscularly (IMIG).

The IG product contains mainly IgG and has a well-defined IgG subclass distribution: igG1, igG2, igG3 and IgG4. However, IG products may differ in different respects: igG monomer, dimer, and aggregate concentrations; igA and IgM content; a stabilizer; an additive; and impurity levels (e.g., proteases such as factor XI/XIa). Regarding IgA, it is recognized that it may lead to allergic reactions in IgA deficient patients. Thus, it is desirable for IG products to contain lower amounts of IgA. The properties of IgG-containing IG products must also meet local and/or regional pharmacopoeia requirements to be registered in the respective jurisdiction (e.g. subcutaneous injection of human normal immunoglobulins, european pharmacopoeia monograph 2788).

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 main hurdles of the existing methods are the high cost and time involved in IgG purification, the need 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 has the proper quality (e.g. purity and stability) for therapeutic use. For example, affinity resins used in affinity chromatography may have relatively low binding capacity, and chromatographic purification of batches of average size can reach volumes of hundreds of liters (in contrast, plasma fractions are typically thousands of liters), which is a significant capital investment in the amount of resin used, the infrastructure of the process and package chromatographic columns, and the running cost. Currently, up to 70-75% of IgG present in plasma can be recovered from plasma using prior art techniques.

Thus, it will be apparent to the skilled artisan that there is a need in the art for improved methods of purifying IgG from plasma or fractions thereof.

Disclosure of Invention

The present disclosure is based on the inventors' identification of methods for purifying IgG from plasma or fractions thereof in high yields (e.g.,. Gtoreq.75%). The method also allows recovery of IgG from plasma or fractions thereof in high purity (e.g., > 95%). In particular, the inventors found that the use of continuous affinity chromatography (e.g. Simulated Moving Bed (SMB) chromatography) using an affinity chromatography resin comprising a ligand capable of specifically binding the CH3 domain of human IgG results in purification of IgG in high yields and purity from plasma with minimal impact on IgG subclass distribution (i.e. IgG1, igG2, igG3 and IgG 4) compared to existing products. In addition, the inventors have found that the method is further improved by using certain wash and regeneration buffers. This method advantageously enables the use of smaller volumes of chromatographic buffer and the repeated use of affinity resins (at least 50 cycles) multiple times, thereby further reducing the cost of purifying IgG from plasma or fractions thereof.

Thus, the inventors' findings provide a basis for a method of producing IgG-enriched formulations. These findings also provide a basis for pharmaceutical compositions comprising IgG-enriched formulations and the use of the compositions or IgG for treating, preventing, and/or delaying progression of conditions (e.g., primary immunodeficiency, chronic inflammatory demyelinating polyneuropathy, and chronic immune thrombocytopenic purpura) in a subject.

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

The present disclosure also provides methods of purifying IgG from plasma or fractions thereof using affinity chromatography, the methods comprising binding IgG to an affinity chromatography resin comprising a ligand capable of specifically binding the CH3 domain of human IgG, and collecting IgG.

The present disclosure also provides methods of producing an IgG-enriched preparation from plasma or a fraction thereof using affinity chromatography, the method comprising binding IgG to an affinity chromatography resin comprising a ligand capable of specifically binding the CH3 domain of human IgG, and collecting IgG.

The present disclosure provides methods of purifying IgG from plasma or fractions thereof using continuous affinity chromatography, comprising binding IgG to an affinity chromatography resin comprising a ligand capable of specifically binding to the CH3 domain of human IgG, and collecting IgG.

The present disclosure also provides methods of producing an 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 capable of specifically binding the CH3 domain of human IgG, and collecting IgG.

In one example, the resin comprises a ligand comprising a single domain [ VHH ] antibody fragment of camelid origin. For example, the ligand is a VHH antibody fragment. In one example, the ligand does not comprise a CH1 domain.

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 crosslinked poly (styrene-divinylbenzene) matrix. In another example, the matrix is an agarose-based matrix.

In one example, the resin comprises a ligand capable of specifically binding to the CH3 domain of human IgG, wherein 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.

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.

In one example, the resin comprises a VHH antigen binding protein comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence as set forth in SEQ ID NO:1, and a sequence having at least 50% amino acid identity. In one example, the VHH antigen binding protein comprises SEQ ID NO:1, and a polypeptide comprising the amino acid sequence shown in (1). In one example, the VHH antigen binding protein comprises an amino acid sequence that hybridizes to SEQ ID NO:1, and a sequence having at least 50% amino acid identity.

In one example, the resin comprises a VHH antigen binding protein comprising a framework region comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence as set forth in SEQ ID NO:1, and a sequence having at least 50% amino acid identity. In one example, the framework region comprises SEQ ID NO:1, and a polypeptide comprising the amino acid sequence shown in (1). In another example, the framework region comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, and a sequence having at least 50% amino acid identity.

In one example, the resin comprises a VHH antigen binding protein comprising an amino acid sequence comprising 4 framework regions FR1, FR2, FR3, and FR4, and 3 complementarity determining regions CDR1, CDR2, and CDR3 operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein:

a) CDR1 has a sequence selected from SEQ ID NOs: 2 or at one or two amino acid residues with SEQ ID NO:2, the amino acid sequences of the different amino acid sequences;

b) CDR2 has the amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 80% sequence identity to the amino acid sequence of seq id no; and, a step of, in the first embodiment,

c) CDR3 has the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 80% sequence identity to the amino acid sequence of seq id no; and wherein each framework region hybridizes to SEQ ID NO:1, the framework amino acid sequence of any one of 1 has at least 50% amino acid identity; and

Wherein each framework region hybridizes to SEQ ID NO:1 has at least 50% amino acid identity to the framework amino acid sequence.

c) CDR3 has the amino acid sequence of SEQ ID NO:4, an amino acid sequence having at least 80% sequence identity to the amino acid sequence of seq id no; and wherein each framework region hybridizes to SEQ ID NO:1, and the framework amino acid sequence of any one of 1 has at least 50% amino acid identity, and

wherein each framework region hybridizes to SEQ ID NO:1, and wherein the antigen binding protein specifically binds to an Fc domain of a human IgG molecule and does not bind to an IgG molecule of murine or bovine origin.

In one example, the resin comprises a VHH antigen binding protein comprising CDR1, the CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 2 or at one or two amino acid residues with SEQ ID NO:2, and a different amino acid sequence.

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

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

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 collection of IgG. For example, the method comprises washing the resin with a wash buffer prior to collection of bound IgG.

In one example, the method further comprises washing the resin with a wash buffer as part of collecting IgG. For example, the method further comprises washing the resin with a wash buffer as part of collecting the bound IgG. In such a method, washing may remove unbound or weakly bound IgG from the resin. Such unbound or weakly bound IgG may be discarded prior to collection of bound IgG. Alternatively, unbound or weakly bound IgG is collected. In one example, bound, unbound, and weakly bound IgG are collected. In one example, bound and weakly bound IgG are collected. In another example, unbound and weakly bound IgG is not collected.

In one example, the method includes washing the resin with a wash buffer prior to collecting IgG. For example, the method includes washing impurities from the resin with a wash buffer and collecting IgG. In one example, the method includes washing the resin with a wash buffer prior to collecting IgG and collecting the flow-through. In one example, the flow-through comprises impurities. In one example, the method includes washing the resin with a wash buffer prior to collecting IgG and collecting impurities in the flow-through. For example, the method includes collecting impurities from the resin with a wash buffer. In one example, impurities and IgG are collected. For example, impurities and IgG are collected together. In another example, the impurity and IgG are collected separately.

In one example, the method includes collecting a wash fraction. For example, the method comprises collecting the wash fraction prior to collecting IgG. In one example, the wash fraction comprises impurities. In one example, the wash fraction comprises IgG. For example, the wash fraction comprises unbound IgG. In one example, the wash fraction comprises weakly bound IgG. In another example, the wash fraction comprises unbound and weakly bound IgG. In one example, the wash fraction comprises impurities and IgG.

In one example, the impurities include albumin (α -globulin and/or β -globulin), plasma lipids, plasma proteins, proteases (e.g., serine proteases, kallikrein, plasmin, and FXa), serine protease inhibitors (e.g., C1 inhibitors, 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 inhibitors, protein C, antithrombin III, rhD immunoglobulins, and/or platelet membrane particulates.

In one example, a plasma protein product is produced using the methods described herein.

In one example, the plasma protein product is an IgG-rich formulation. In another example, the plasma protein product comprises purified IgG.

In one example, the plasma protein product is produced using bound, unbound, and/or weakly bound IgG. For example, bound, unbound and/or weakly bound IgG is used to produce plasma protein products. In one example, the plasma protein product is produced using conjugated IgG. For example, conjugated IgG is used to produce plasma protein products. In one example, the plasma protein product is produced using unbound IgG. For example, unbound IgG is used to produce plasma protein products. In one example, the plasma protein product is produced using weakly bound IgG. For example, weakly bound IgG is used to produce plasma protein products. In one example, the plasma protein product is produced using bound and weakly bound IgG. For example, bound and weakly bound IgG are used to produce plasma protein products. In one example, the plasma protein product is produced using unbound and weakly bound IgG. For example, unbound and weakly bound IgG is used to produce a plasma protein product. In one example, bound and unbound IgG are used to produce a plasma protein product. For example, bound and unbound IgG are used to produce plasma protein products. In one example, the plasma protein product is produced using bound, unbound, and weakly bound IgG. For example, bound, unbound and weakly bound IgG are used to produce plasma protein products.

In one example, the plasma protein product is produced using impurities. For example, impurities are collected and used to produce plasma protein products.

In one example, the plasma protein product is selected from albumin, serine protease, plasmin, FXa, alpha-1-antitrypsin, igA, igM, factor VIII, fibrinogen, von willebrand factor, activated clotting factor, factor XIII, contact system factor, PKA, factor IX, prothrombin complex, C1 esterase inhibitor, protein C, antithrombin III, rhD immunoglobulin product.

In one example, the activated clotting factor is selected from FXa, FIXa, FVIIa and thrombin. For example, the activated clotting factor is FXa. For example, the activated coagulation factor is FIXa. For example, the activated clotting factor is FVIIa. For example, the activated clotting factor is thrombin.

In one example, the contact system factor protein is selected from FXIa, FXIIa and kallikrein. For example, the contact system factor protein is FXIa. For example, the contact system factor protein is FXII. For example, the contact system factor protein is kallikrein.

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 clotting 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.

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 the 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 product.

In one example, the wash buffer has a pH of 5 to 9 and a dissociation constant (pKa) of 6.8 to 8.5 at 25 ℃. In one example, the wash buffer has a pH of 5 to 10 and a dissociation constant (pKa) of 6.8 to 8.5 at 25 ℃.

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 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 9.0, or 9.1, or 9.2, or 9.3, or 9.4, or 9.5, or 9.7, or 9.0, or 9.10 or 9.0.

In one example, the wash buffer has a pH of 7 to 10 and a dissociation constant (pKa) of 6.8 to 8.5 at 25 ℃.

In one example, the wash buffer has a pH of 7 to 8 and a dissociation constant (pKa) of 6.8 to 8.5 at 25 ℃.

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 pH of the wash buffer is 7.4.

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.

In one example, the wash buffer has a pKa of 6.8 to 8.5 at 25 ℃. 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 ℃.

In one example, the wash buffer has a pKa of 7.21 at 25 ℃.

In one example, the wash buffer has a pH of 7.4 at 25℃and a dissociation constant (pKa) of 7.21.

In one example, the wash buffer comprises a buffer selected from the group consisting of: 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-piperazine ethanesulfonic acid (HEPES), sulfurous acid, 4- (2-hydroxyethyl) -1-piperazine propanesulfonic 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 (taco), tricine, triethanolamine (TEA), and combinations thereof. For example, the wash buffer is sodium dihydrogen phosphate buffer. For example, the wash buffer is an imidazole buffer. In another example, the wash buffer is 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 PIPES buffer. In a further example, the wash buffer is a TES buffer. In one example, the wash buffer is Bicine buffer. In another example, the wash buffer is a sulfite buffer. In a further example, the wash buffer is EPPS buffer. In one example, the wash buffer is 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 Tricine-buffer. In a further example, the wash buffer is TEA buffer. In one example, the wash buffer is sodium citrate buffer.

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

In one example, the concentration of the buffer of the wash buffer is 5mM.

In one example, the concentration of the buffer of the wash buffer is 20mM.

In one example, the concentration of the buffer of the wash buffer is 50mM.

In one example, the concentration of the buffer of the wash buffer is 100mM.

In one example, the concentration of the buffer of the wash buffer is 150mM.

In one example, the concentration of the buffer of the wash buffer is 200mM.

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

In one example, the wash buffer further comprises sodium chloride, wherein the concentration of sodium chloride is 145mM.

In one example, the wash buffer further comprises sodium chloride, wherein the concentration of sodium chloride is 500mM.

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

In one example, the wash buffer comprises 20mM sodium dihydrogen phosphate, 500mM sodium chloride, and has a pH of 7.4.

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

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

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 cupric chloride. In one example, the divalent salt is nickel chloride. In another example, the divalent salt is manganese chloride.

In one example, the method includes collecting IgG by eluting IgG from the resin with an elution buffer. For example, the method includes collecting the bound IgG by eluting the bound IgG from the resin with an elution buffer.

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.

In one example, the pH of the elution buffer is 4.

In one example, the elution buffer comprises a buffer selected from 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.

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

In one example, the concentration of the buffer of the elution buffer is 5mM.

In one example, the concentration of the buffer of the elution buffer is 20mM.

In one example, the concentration of the buffer of the elution buffer is 50mM.

In one example, the concentration of the buffer of the elution buffer is 100mM.

In one example, the concentration of the buffer of the elution buffer is 150mM.

In one example, the concentration of the buffer of the elution buffer is 200mM.

In one example, the elution buffer is or comprises an acetate buffer. For example, sodium acetate buffer.

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 sodium dihydrogen phosphate and sodium acetate buffers. In one example, the elution buffer is or comprises a phosphate buffer.

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. In one example, the elution buffer is or comprises an acetate buffer at pH 4. For example, the elution buffer is or comprises a sodium acetate buffer at pH 4.

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 at pH 4.

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 at pH 4.

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

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

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

In one example, the elution buffer comprises 20mM sodium acetate at pH 4.

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

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

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 cupric chloride. In a further example, the divalent salt is nickel chloride. In one example, the divalent salt is manganese chloride.

In one example, the method further comprises equilibrating the resin with an equilibration buffer. For example, the resin is equilibrated prior to loading the plasma or IgG-containing fraction thereof onto the resin.

In one example, the method further comprises equilibrating the resin with an equilibration buffer having a pH of 5 to 9. For example, the pH of the equilibration buffer is 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.

In one example, the method further comprises equilibrating the resin with an equilibration buffer having a pH of 7 to 8. For example, the pH of the equilibration buffer is 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 pH of the equilibration buffer is 7.4.

In one example, the equilibration buffer comprises a buffering agent 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 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 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 PIPES buffer. In another example, the equilibration buffer is a TES buffer. In a further example, the equilibration buffer is 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 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 Tricine-buffer. In another example, the equilibration buffer is TEA buffer.

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

In one example, the concentration of the buffer of the equilibration buffer is 5mM.

In one example, the concentration of the buffer of the equilibration buffer is 20mM.

In one example, the concentration of the buffer of the equilibration buffer is 50mM.

In one example, the concentration of the buffer of the equilibration buffer is 100mM.

In one example, the concentration of the buffer of the equilibration buffer is 150mM.

In one example, the concentration of the buffer of the equilibration buffer is 200mM.

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

In one example, the equilibration buffer further comprises sodium chloride, wherein the concentration of sodium chloride is 145mM.

In one example, the equilibration buffer further comprises sodium chloride, wherein the concentration of sodium chloride is 500mM.

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

In one example, the composition of the equilibration buffer is the same as the wash buffer. For example, the equilibration buffer contained 20mM sodium dihydrogen phosphate, 145mM sodium chloride and pH 7.4. In another example, the equilibration buffer comprises 20mM sodium dihydrogen phosphate, 500mM sodium chloride and pH 7.4.

In one example, the resin is equilibrated i) after desorption of the resin or ii) without desorption of the resin. For example, the resin is equilibrated after desorption of the resin. In another example, the resin is equilibrated without desorbing the resin.

In one example, the method further comprises equilibrating the resin with an equilibration buffer having a pH of 7 to 8 after desorbing the resin.

In one example, the method optionally includes desorbing the resin with a desorption buffer after collecting IgG from the resin. For example, the method further comprises desorbing the resin with a desorption buffer after collecting IgG from the resin. In another example, the method does not include desorbing the resin with a desorption buffer after IgG is collected from the resin. For example, after IgG is collected from the resin, the resin is not desorbed.

In one example, the desorption buffer has a pH of 2 to 3. For example, the desorption 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 desorption buffer is 2.5.

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

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

In one example, the concentration of buffer of the desorption buffer is 5mM.

In one example, the concentration of buffer of the desorption buffer is 20mM.

In one example, the concentration of buffer of the desorption buffer is 50mM.

In one example, the concentration of buffer of the desorption buffer is 100mM.

In one example, the concentration of buffer of the desorption buffer is 150mM.

In one example, the concentration of buffer of the desorption buffer is 200mM.

In one example, the concentration of buffer of the desorption buffer is 250mM.

In one example, the concentration of buffer of the desorption buffer is 300mM.

In one example, the concentration of buffer of the desorption buffer is 350mM.

In one example, the concentration of buffer of the desorption buffer is 400mM.

In one example, the concentration of buffer of the desorption buffer is 450mM.

In one example, the concentration of buffer of the desorption buffer is 500mM.

In one example, the desorption buffer comprises 20mM sodium dihydrogen phosphate and has a pH of 2.5.

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

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

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 cupric chloride. For example, the divalent salt is nickel chloride. For example, the divalent salt is manganese chloride.

In one example, the resin is equilibrated. In one example, the resin is equilibrated after desorption of the resin. In one example, the method further comprises equilibrating the resin with an equilibration buffer having a pH of 7 to 8 after desorbing the resin.

In one example, the method includes:

a) Equilibrating the resin with an equilibration buffer having a pH of 7 to 8;

b) After the bound IgG was collected from the resin, the resin was desorbed with a desorption buffer at pH 2 to 3; and/or

c) After desorption of the resin, the resin is equilibrated with an equilibration buffer.

In one example, the resin is equilibrated without desorbing the resin. For example, the method includes equilibrating the resin with an equilibration buffer and desorbing the resin without a desorption buffer after the bound IgG is collected from the resin. In one example, the method includes equilibrating the resin with an equilibration buffer having a pH of 7 to 8 after the bound IgG is collected from the resin.

In one example, the method further comprises regenerating the resin.

In one example, the method further comprises sterilizing the resin.

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

In one example, the plasma or fraction thereof is contacted with the resin for at least 0.1 minutes during loading of the plasma or fraction thereof. For example, the plasma or fraction thereof is contacted with the 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, the plasma or fraction thereof is contacted with the resin for 0.1, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, or 1, or 1.5, or 2, or 2.5, or 3, or 3.5, or 4, or 4.5, or 5 minutes.

In one example, the plasma or fraction thereof is contacted with the resin for up to 5 minutes during loading of the plasma or fraction thereof.

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 contacts 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 contacts the resin for at least 0.25 minutes.

In one example, the buffer is contacted with the resin for at least 0.1 minutes during one or more non-loaded phases of the method.

In one example, the buffer is contacted with the resin for up to 5 minutes during one or more non-loaded phases of the continuous chromatographic method.

In one example, the buffer is contacted with the resin during one or more non-loaded phases of the continuous chromatographic method for 0.1 to 5 minutes. For example, the buffer is contacted with the resin for at least 0.1 minute, or 0.25 minute, or 0.5 minute, or 1 minute, or 1.5 minute, 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, the non-loaded phase is selected from the group consisting of an equilibration phase, a wash phase, an elution phase, a desorption phase, a re-equilibration phase, and combinations thereof.

In one example, the no-load phase is a balancing phase.

In one example, the non-loaded phase is a wash phase.

In one example, the non-loaded phase is an elution phase.

In one example, the non-loaded phase is a desorption phase.

In one example, the no-load phase is a rebalancing phase.

In one example, the buffer that contacts the resin during one or more non-loaded phases of the continuous chromatographic method is selected from the group consisting of equilibration buffer, wash buffer, desorption buffer, and 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 desorption buffer. In one example, the buffer is a rebalancing buffer.

In one example, the equilibration buffer contacts the resin for 0.1 to 5 minutes.

In one example, the wash buffer contacts the resin for 0.1 to 5 minutes.

In one example, the elution buffer contacts the resin for 0.1 to 5 minutes.

In one example, the desorption buffer is contacted with the resin for 0.1 to 5 minutes.

In one example, the method includes contacting the resin with a volume of elution buffer less than the Column Volume (CV) prior to collecting bound IgG from the resin. For example, the method includes a "pre-elution" stage of contacting the resin with a volume of elution buffer less than the Column Volume (CV) prior to collecting bound IgG from the resin. In one example, the method includes washing the resin with a volume of elution buffer less than CV prior to collecting bound IgG from the resin. For example, the volume of elution buffer used to wash the resin prior to collection of bound IgG from the resin is at most 0.5 CV. For example, the volume of elution buffer used to wash the resin prior to collection of bound IgG from the resin is 0.5 to 1.0 CV. For example, the volume of elution buffer used to wash the resin prior to collection of 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.

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

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

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

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

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

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

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

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

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

In one example, the method includes eluting bound IgG from the resin after performing the step of contacting the resin with an elution buffer of less than the CV volume.

It will be apparent to the skilled person that IgG is typically present in plasma at a concentration of 5-15g/L plasma.

In one example, the plasma fraction is selected from the group consisting of cryoprecipitated-rich plasma, cryoprecipitated-poor plasma, supernatant I (SN I), cohn fraction II (Fr II), cohn fraction ii+iii (Fr ii+iii), cohn fraction i+ii+iii (Fr i+ii+iii), kistler/Nitschmann precipitate a (KN a), kistler/Nitschmann precipitate B (KN B), kistler/Nitschmann precipitate of supernatant B (KN b+1), and combinations thereof. In one example, the plasma fraction is cryoprecipitated-rich plasma. For example, the plasma fraction is cryoprecipitated lean 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 (Fr I+II+III). For example, the plasma fraction is Kistler/Nitschmann precipitate A (KN A). For example, the plasma fraction is Kistler/Nitschmann precipitate B (KN B). For example, the plasma fraction is Kistler/Nitschmann precipitate of supernatant B (KN B+1).

In one example, the plasma fraction is a suspension paste. For example, the suspension paste is selected from the group consisting of Cohn fraction II (Fr II), cohn fraction II+III (Fr II+III), cohn fraction I+II+III (Fr I+II+III), kistler/Nitschmann precipitate A (KN A), kistler/Nitschmann precipitate B (KN B), kistler/Nitschmann precipitate of supernatant B (KN B+1), and combinations thereof. For example, the suspension paste is a Cohn fraction II (Fr II) paste. In one example, the suspension paste is a Cohn fraction ii+iii (Fr ii+iii) paste. In another example, the suspension paste is a Cohn fraction i+ii+iii (Fr i+ii+iii) paste. In another example, the suspension paste is a Kistler/Nitschmann precipitate A (KN A) paste. In another example, the suspension paste is a Kistler/Nitschmann precipitate B (KN B) paste. In a further example, the suspension paste is a Kistler/Nitschmann precipitate (KN B+1) paste of supernatant B.

In one example, the plasma fraction is selected from the group consisting of a mammalian plasma fraction, a human plasma fraction, a horse plasma fraction, and a bovine plasma fraction.

In one example, the plasma fraction is a mammalian plasma fraction.

In one example, the plasma fraction is a human plasma fraction.

In one example, the plasma fraction is a horse plasma fraction.

In one example, the plasma fraction is a bovine plasma fraction.

In one example, the plasma fraction is a bovine plasma fraction comprising human polyclonal antibodies.

In one example, the plasma or fraction thereof is clarified. Clarifying formulation for plasma or fractions thereofThe methods are obvious to the skilled person and/or described herein. For example, the plasma or fraction thereof is clarified by passing the plasma or fraction thereof through a filter. For example, a depth filter or a membrane filter may be used. For example, passing the plasma or fractions thereof through a combination of filters. For example, the combination may be a 1.2 and 0.45/0.22 μm membrane filter combination. For example, by passing the plasma or fractions thereof through a depth filter (e.gDepth filter) to clarify the plasma or fractions thereof. In one example, the plasma or fractions thereof is produced by passing the plasma through a filter press comprising one or more depth filters (e.g.)>Integrated or compact plate) to clarify the plasma or fractions thereof. In one example, the filter press further comprises one or more filter aids (e.g. cellulose-based filter aids, e.g.)>150). In one example, the plasma or fraction thereof is purified by passing it through a lipid-specific filter (e.g. Zeta Plus ^TM DEL series filter) to clarify the plasma or fractions thereof. For example, the plasma fraction is clarified supernatant I (SN I). For example, the plasma fraction is clarified Cohn fraction II (Fr II). For example, the plasma fraction is clarified Cohn fraction II+III (Fr II+III). For example, the plasma fraction is clarified Cohn fraction I+II+III (Fr I+II+III). For example, the plasma fraction is clarified Kistler/Nitschmann precipitate A (KN A). For example, the plasma fraction is clarified Kistler/Nitschmann precipitate B (KN B). For example, the plasma fraction is the Kistler/Nitschmann precipitate of clarified supernatant B (KN B+1).

In one example, the plasma is clarified cryoprecipitated-rich plasma.

In one example, the plasma fraction is clarified cryoprecipitated lean plasma.

In one example, the plasma or fraction thereof is heated to a first temperature of about 32 ℃ and then cooled to a second temperature of about 21 ℃ prior to the continuous affinity chromatography step. In one example, the plasma or fraction thereof is at a first temperature of about 32 ℃ and then at a second temperature of about 21 ℃ prior to the continuous affinity chromatography step.

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

In one example, the plasma or fraction thereof is at a temperature of 2 ℃.

In one example, the plasma or fraction thereof is at a temperature of 10 ℃.

In one example, the plasma or fraction thereof is at a temperature of 18 ℃.

In one example, the plasma or fraction thereof is at a temperature of 21 ℃.

In one example, the plasma or fraction thereof is at a temperature of 28 ℃.

In one example, the plasma or fraction thereof is at a temperature of 32 ℃.

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 maintained at this temperature for up to 48 hours before loading the plasma or fraction thereof onto the continuous affinity chromatography resin. In one example, the plasma or fraction thereof is maintained 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 prior to loading. For example, the plasma or fraction thereof is maintained at this temperature for 0 to 2 hours, 2 to 24 hours, or 4 to 24 hours, or 8 to 24 hours, or 12 to 24 hours, or 18 to 24 hours, or 24 hours to 48 hours, or 36 to 48 hours prior to loading.

In one example, the plasma or fraction thereof is at a first temperature in the range of 30 ℃ to 38 ℃ and then at a second temperature in the range of 2 ℃ to 28 ℃ prior to the continuous affinity chromatography step. For example, prior to the continuous affinity chromatography step, the plasma or fraction thereof is heated to a first temperature in the range of 30 ℃ to 38 ℃ and then cooled to a second temperature in the range of 2 ℃ to 28 ℃. In one example, the plasma or fraction thereof is heated to a first temperature in the range of 30 ℃ to 35 ℃ and then cooled to a second temperature in the range of 18 ℃ to 25 ℃ prior to the continuous affinity chromatography step. For example, the plasma or fraction thereof is heated to a first temperature of about 30 ℃, or about 31 ℃, or about 32 ℃, or about 33 ℃, or about 34 ℃, or about 35 ℃. In one example, the plasma or fraction thereof is cooled to a second temperature of about 18 ℃, or about 19 ℃, or about 20 ℃, or about 21 ℃, or about 22 ℃, or about 23 ℃, or about 24 ℃, or about 25 ℃.

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 maintained at the first and/or second temperature for up to 48 hours before loading the plasma or fraction thereof onto the continuous affinity chromatography resin. In one example, the plasma or fraction thereof is maintained 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 prior to loading. For example, the plasma or fraction thereof is maintained at the first and/or second temperature for 0 to 2 hours, 2 to 24 hours, or 4 to 24 hours, or 8 to 24 hours, or 12 to 24 hours, or 18 to 24 hours, or 24 to 48 hours, or 36 to 48 hours prior to loading.

In one example, the continuous affinity chromatography is selected from Simulated Moving Bed (SMB) chromatography, periodic Countercurrent Chromatography (PCC), continuous Countercurrent Tangential Chromatography (CCTC), and continuous countercurrent helical chromatography (CCSC).

In one example, the continuous affinity chromatography is Simulated Moving Bed (SMB) chromatography. In another example, the continuous affinity chromatography is Periodic 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 helical chromatography (ccs).

In one example, the resin is in the form of a slurry. For example, the resin contains resin particles in the form of a slurry.

In one example, the slurry is passed through one or more columns, wherein each column comprises a membrane. For example, the membrane is a hollow fiber membrane.

In one example, the slurry is passed through a series of two or more columns containing membranes. For example, the slurry is passed through two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve columns.

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

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

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

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

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

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

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

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

In one example, the slurry is passed through a series of ten columns.

In one example, the slurry is passed through a series of eleven columns.

In one example, the slurry is passed through a series of twelve columns.

In one example, the resin is packed into one or more columns, where each column includes one or more regions. 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.

In one example, the resin is packed into a series of two columns.

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

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

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

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

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

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

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

In one example, the resin is packed into a series of ten columns.

In one example, the resin is packed into a series of eleven columns.

In one example, the resin is packed into a series of twelve columns.

For example, the zone is selected from the group consisting of equilibration zone, binding zone, wash zone, elution zone, desorption zone, and combinations thereof. In another example, the region is selected from: an equilibration zone, a binding zone, a wash zone, an elution zone, and combinations thereof. In one example, the region is a balancing area. In another example, the region is a bonding region. In a further example, the zone is a wash zone. In one example, the region is an elution zone. In another example, the zone is a desorption zone. In one example, there is no desorption zone. In another example, the zone is a wash/elution zone. In one example, the region is a balance/binding region. In another example, the zone is a binding/washing zone.

In one example, the resin is packed into one or more columns, where each column includes a region.

In one example, the resin is packed into one or more columns, where each column includes two regions.

In one example, the resin is packed into one or more columns, where each column includes four regions.

In one example, two or more columns are fluidly connected and separated by a fluid conduit that includes an inlet valve and an outlet valve.

In one example, the resin is packed into a first column and one or more subsequent columns.

In one example, the first column supports IgG at a concentration higher than the Dynamic Binding Capacity (DBC) of the resin.

Determining the DBC of a resin is obvious to a skilled artisan and/or described herein. For example, the DBC of the resin can be determined by loading IgG onto the column and monitoring the concentration of unbound IgG as it flows through the column, such as by UV tracking of the chromatographic system. For example, the DBC of the resin is 5mg, or 10mg, or 20mg, or 30mg, or 40mg, or 50mg, or 60mg, or 70mg IgG per mL of resin.

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

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

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

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

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

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

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

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

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

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

In one example, one or more subsequent columns are loaded with IgG at a concentration of DBC that is up to the resin.

In one example, one or more subsequent columns load IgG at a concentration of up to 5mg, or 10mg, or 20mg, or 30mg, or 40mg IgG per mL of resin.

In one example, one or more subsequent columns are loaded with IgG at a concentration of up to 20mg IgG per mL of resin.

In one example, one or more subsequent columns are loaded with IgG at a concentration of up to 30mg IgG per mL of resin.

In one example, one or more subsequent columns are loaded with IgG at a concentration of up to 40mg IgG per mL of resin.

In one example, the method further comprises washing unbound IgG from the first column to one or more subsequent columns with a wash buffer and collecting bound IgG. For example, bound IgG is collected from the first and one or more subsequent columns. For example, bound IgG is collected from the first column without a washing step. Bound IgG is collected from the first column, for example, by eluting the bound IgG with an elution buffer as described herein. For example, bound IgG is collected from one or more subsequent columns after washing with the wash buffers described herein. For example, after washing the resin with a wash buffer and eluting the bound IgG with an elution buffer as described herein, the bound IgG is collected from one or more subsequent columns.

In one example, the method further comprises washing one or more subsequent columns with a wash buffer described herein and collecting bound IgG from the one or more subsequent columns.

In one example, the method further comprises desorbing and/or equilibrating the first column when collecting bound IgG from one or more subsequent columns. In one example, the method further comprises equilibrating the first column when the bound IgG is collected from one or more subsequent columns. For example, the method does not include desorbing the first column when bound IgG is collected from one or more subsequent columns.

In one example, the method further comprises desorbing and/or equilibrating one or more subsequent columns while collecting bound IgG from the first column. In one example, the method further comprises equilibrating one or more subsequent columns when the bound IgG is collected from the first column. For example, the method does not include desorbing one or more subsequent columns when the bound IgG is collected from the first column.

In one example, the method further comprises desorbing and/or equilibrate the first column while washing one or more subsequent columns with a washing buffer as described herein. In one example, the method further comprises equilibrating the first column while washing one or more subsequent columns with a wash buffer as described herein. For example, the method does not include desorbing the first column while washing one or more subsequent columns with a wash buffer as described herein.

In one example, the method further comprises desorbing and/or equilibrate one or more subsequent columns while washing the first column with a washing buffer as described herein. In one example, the method further comprises equilibrating one or more subsequent columns while washing the first column with a wash buffer as described herein. For example, the method does not include desorbing one or more subsequent columns while washing the first column with a wash buffer as described herein.

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

In one example, the total bed height of the resin is at least 2cm.

In one example, the total bed height of the resin is 6cm.

In one example, the total bed height of the resin is 20cm.

In one example, the total bed height of the resin is 30cm.

In one example, the total bed height of the resin is 50cm.

In one example, the total bed height of the resin is 70cm.

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

In one example, the diameter of the column is 5cm.

In one example, the diameter of the column is 20cm.

In one example, the diameter of the column is 50cm.

In one example, the diameter of the column is 100cm.

In one example, the diameter of the column is 200cm.

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, lectin affinity chromatography), virus inactivation, virus filtration, and ultrafiltration/diafiltration, wherein the steps are performed before or after the successive affinity chromatography steps. For example, the method further comprises ethanol precipitation. For example, the method further comprises octanoic acid fractionation. For example, the method further comprises membrane chromatography 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 includes hydrophobic interaction chromatography. For example, the method includes cognate lectin 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.

In one example, the method further comprises anion exchange chromatography and virus filtration.

In one example, the method further comprises low pH incubation, depth filtration, anion exchange chromatography, and virus filtration.

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

In one example, the method further comprises ion exchange chromatography, wherein the ion exchange chromatography step comprises an anion exchange chromatography step using an anion exchange resin operating in a flow-through mode.

In one example, the flow-through and/or post-wash eluate is 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 eluents are collected. It will be apparent to those skilled in the art that only the flow-through phase and the post-wash phase are collected (i.e., combined), and the wash phase is not collected.

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.

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

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

In one example, the anion exchange resin is a strong anion exchanger. In one example, the ion exchange chromatography step comprises an anion exchange chromatography step using a strong anion exchange resin operating in a flow-through mode. In one example, the strong anion exchange resin comprises a matrix composed of a poly (styrene-divinylbenzene) matrix. In one example, the strong anion exchange resin comprises quaternized polyethyleneimine functional groups.

In one example, the anion exchange resin is washed with a pre-equilibration buffer prior to equilibration. It is obvious to the skilled person that the pre-equilibration step is only performed after the first run and/or after the resin storage.

In one example, the pre-equilibration buffer is selected from the group consisting of monosodium phosphate (NaH) ₂ PO ₄ ) Disodium phosphate (Na) ₂ HPO ₄ ) Phosphoric acid (H) ₃ PO ₄ ) And combinations thereof.

In one example, the pre-equilibration buffer comprises Na ₂ HPO ₄ 。

In one example, the pre-equilibration buffer comprises H ₃ PO ₄ 。

In one example, the pre-equilibration buffer comprises NaH ₂ PO ₄ 。

In one example, the pre-equilibration buffer comprises Na ₂ HPO ₄ And NaH ₂ PO ₄ 。

In one example, the pre-equilibration buffer comprises a concentration of buffer in the range of 50mM to 150mM. For example, the concentration is 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM. In one example, the pre-equilibration buffer comprises a concentration of 100mM buffer.

In one example, the pre-equilibration buffer comprises NaH at a concentration in the range of 50mM to 150mM ₂ PO ₄ . For example, the pre-equilibration buffer comprises NaH at a concentration of 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM ₂ PO ₄ . In one example, the pre-equilibration buffer comprises NaH at a concentration of 100mM ₂ PO ₄ 。

In one example, the pH of the pre-equilibration buffer is in the range of 5.8 to 6.6. For example, the pH of the pre-equilibration buffer is 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-equilibrium pH is 6.2.

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

In one example, the anion exchange resin is pre-equilibrated with a pre-equilibration buffer comprising 1000mM NaH ₂ PO ₄ And 1000mM sodium chloride, pH 6.2. In one example, the sample is pre-equilibrated with a pre-equilibration bufferThe anion exchange resin is balanced, and the pre-balancing buffer solution comprises 100mM NaH ₂ PO ₄ And 1000mM sodium chloride, pH 6.2.

In one example, the volume of pre-equilibration buffer is at least 2 CV. For example, the pre-equilibration buffer has a volume of 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 pre-equilibration buffer has a volume of 2 to 10 CVs.

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

In one example, the catalyst is selected from monosodium phosphate (NaH ₂ PO ₄ ) Disodium phosphate (Na) ₂ HPO ₄ ) Phosphoric acid (H) ₃ PO ₄ ) Sodium citrate, 2- (N-morpholino) ethanesulfonic acid (MES), bis-Tris, L-histidine, and combinations thereof.

In one example, the anion exchange resin comprises Na ₂ HPO ₄ Is balanced by the equilibration buffer.

In one example, the anion exchange resin comprises H ₃ PO ₄ Is balanced by the equilibration buffer.

In one example, the anion exchange resin comprises NaH ₂ PO ₄ Is balanced by the equilibration buffer.

In one example, the anion exchange resin comprises Na ₂ HPO ₄ And NaH ₂ PO ₄ Is balanced by the equilibration buffer.

In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising MES.

In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising sodium citrate.

In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising Bis-Tris.

In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising L-histidine.

In one example, the concentration of the equilibration buffer is in the range of 5mM to 50mM. For example, the concentration of the equilibration buffer is 5mM, or 10mM, or 20mM, or 30mM, or 40mM, or 50mM. In one example, the concentration of equilibration buffer is 5mM. In another example, the concentration of equilibration buffer is 10mM. In a further example, the concentration of the equilibration buffer is 20mM. In one example, the concentration of equilibration buffer is 30mM. In another example, the concentration of the equilibration buffer is 40mM. In a further example, the concentration of the equilibration buffer is 50mM.

In one example, the equilibration buffer comprises NaH at a concentration in the range of 5mM to 50mM ₂ PO ₄ . In one example, the equilibration buffer comprises NaH at a concentration in the range of 10mM to 50mM ₂ PO ₄ . For example, the equilibration buffer comprises NaH at a concentration of 10mM, 20mM, 30mM, 40mM, 50mM ₂ PO ₄ . In one example, the equilibration buffer comprises NaH at a concentration of 5mM ₂ PO ₄ . In one example, the equilibration buffer comprises NaH at a concentration of 10mM ₂ PO ₄ 。

In one example, the pH of the equilibration buffer is in the range of 5.8 to 6.6. For example, the pH of the equilibration buffer is 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 performed at a pH of 6.2.

In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising a phosphate buffer having a pH in the range of 5.8 to 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, a solution containing 5mM NaH ₂ PO ₄ The anion exchange resin was equilibrated with an equilibration buffer at pH 6.2. In one example, the method comprises 10m M NaH ₂ PO ₄ The anion exchange resin was equilibrated with an equilibration buffer at pH 6.2.

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 a MES buffer at ph 6.0. In one example, the equilibration buffer comprises a MES buffer at ph 6.2. In one example, the equilibration buffer comprises a MES buffer at ph 6.6.

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

In one example, the anion exchange resin is equilibrated with an equilibration buffer comprising an L-histidine buffer having 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.

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

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

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

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

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

In one example, the concentration of the post-load wash buffer is in the range of 5mM to 50mM. In one example, the concentration of the post-load wash buffer is in the range of 10mM to 50mM. For example, the concentration of the post-load wash buffer is 10mM, 20mM, 30mM, 40mM, 50mM. In one example, the concentration of the post-load wash buffer is 5mM. In one example, the concentration of the post-load wash buffer is 10mM.

In one example, the post-load wash buffer comprises a phosphate buffer. For example, the phosphate buffer is selected from monosodium phosphate (NaH) ₂ PO ₄ ) Disodium phosphate (Na) ₂ HPO ₄ ) Phosphoric acid (H) ₃ PO ₄ ) And combinations thereof.

In one example, the post-load wash buffer comprises Na ₂ HPO ₄ 。

In one example, the post-load wash buffer comprises H ₃ PO ₄ 。

In one example, the post-load wash buffer comprises NaH ₂ PO ₄ . For example, the post-load wash buffer comprises 5mM NaH ₂ PO ₄ . In another example, the post-load wash buffer comprises 10mM NaH ₂ PO ₄ 。

In one example, the post-load wash buffer comprises Na ₂ HPO ₄ And NaH ₂ PO ₄ 。

In one example, the post-load wash buffer comprises sodium citrate buffer.

In one example, the post-load wash buffer comprises an acetate buffer. For example, the post-load wash buffer comprises sodium acetate. For example, the post-load wash buffer comprises 5mM acetic acid. In another example, the post-load wash buffer comprises 10mM acetic acid.

In one example, the post-load wash buffer comprises a phosphate buffer and an acetate buffer. For example, the post-load wash buffer comprises NaH ₂ PO ₄ And sodium acetate. For example, the post-load wash buffer comprises 5mM NaH ₂ PO ₄ And 10mM sodium acetate.

In one example, the post-load wash buffer comprises MES buffer.

In one example, the post-load wash buffer is Bis-Tris buffer.

In one example, the post-load wash buffer is an L-histidine buffer.

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

In one example, the post-load wash buffer further comprises a salt. For example, the salt is sodium chloride.

In one example, the post-load wash buffer does not comprise salt.

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

In one example, the post-load wash buffer comprises a phosphate buffer having a pH in the range of 5.8 to 6.6.

In one example, the post-load wash buffer comprises phosphate buffer at ph 6.0.

In one example, the post-load wash buffer comprises phosphate buffer at ph 6.2.

In one example, the post-load wash buffer comprises phosphate buffer at ph 6.6.

In one example, the post-load wash buffer comprises phosphate buffer at pH6.0 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate buffer at pH6.0 and 20mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate buffer at pH6.0 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate buffer at pH6.6 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate buffer at pH6.6 and 25mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate buffer at pH6.6 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate buffer at pH6.2 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises an MES buffer having a pH in the range of 5.8 to 6.6.

In one example, the post-load wash buffer comprises MES buffer at ph 6.0.

In one example, the post-load wash buffer comprises MES buffer at ph 6.6.

In one example, the post-load wash buffer comprises MES buffer at pH6.0 and 20mM sodium chloride.

In one example, the post-load wash buffer comprises MES buffer at pH6.0 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises MES buffer at pH6.6 and 25mM sodium chloride.

In one example, the post-load wash buffer comprises MES buffer at pH6.6 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises sodium citrate buffer having a pH in the range of 5.8 to 6.6.

In one example, the post-load wash buffer comprises sodium citrate buffer at ph 6.0.

In one example, the post-load wash buffer comprises sodium citrate buffer at ph 6.6.

In one example, the post-load wash buffer comprises sodium citrate buffer at pH6.0 and 20mM sodium chloride.

In one example, the post-load wash buffer comprises sodium citrate buffer at pH6.0 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises sodium citrate buffer at pH6.6 and 25mM sodium chloride.

In one example, the post-load wash buffer comprises sodium citrate buffer at pH6.6 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises a sodium acetate buffer having a pH in the range of 5.8 to 6.6.

In one example, the post-load wash buffer comprises sodium acetate buffer at ph 6.0.

In one example, the post-load wash buffer comprises sodium acetate buffer at ph 6.2.

In one example, the post-load wash buffer comprises sodium acetate buffer at ph 6.6.

In one example, the post-load wash buffer comprises sodium acetate buffer at pH6.0 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises sodium acetate buffer at pH6.0 and 20mM sodium chloride.

In one example, the post-load wash buffer comprises sodium acetate buffer at pH6.0 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises sodium acetate buffer at pH6.6 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises sodium acetate buffer at pH6.6 and 25mM sodium chloride.

In one example, the post-load wash buffer comprises sodium acetate buffer at pH6.6 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises sodium acetate buffer at pH6.2 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers having a pH in the range of 5.8 to 6.6.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at ph 6.0.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at ph 6.2.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at ph 6.6.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at pH6.0 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at pH6.0 and 20mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at pH6.0 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at pH6.6 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at pH6.6 and 25mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at pH6.6 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises phosphate and sodium acetate buffers at pH6.2 and 0mM sodium chloride.

In one example, the post-load wash buffer comprises Bis-Tris buffer having a pH in the range of 5.8 to 6.6.

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

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

In one example, the post-load wash buffer comprises Bis-Tris buffer at pH6.0 and 20mM sodium chloride.

In one example, the post-load wash buffer comprises Bis-Tris buffer at pH6.0 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises Bis-Tris buffer at pH6.6 and 25mM sodium chloride.

In one example, the post-load wash buffer comprises Bis-Tris buffer at pH6.6 and 50mM sodium chloride.

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

In one example, the post-load wash buffer comprises an L-histidine buffer at pH 6.0.

In one example, the post-load wash buffer comprises an L-histidine buffer at pH 6.6.

In one example, the post-load wash buffer comprises L-histidine buffer at pH6.0 and 20mM sodium chloride.

In one example, the post-load wash buffer comprises L-histidine buffer at pH6.0 and 50mM sodium chloride.

In one example, the post-load wash buffer comprises L-histidine buffer at pH6.6 and 25mM sodium chloride.

In one example, the post-load wash buffer comprises L-histidine buffer at pH6.6 and 50mM sodium chloride.

In one example, the volume of wash buffer after loading is 1 to 5 CVs. For example, the volume of wash buffer after loading is 1 CV, or 2 CV, or 3 CV, or 4 CV, or 5 CV. In one example, the volume of wash buffer after loading is 3 CVs.

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

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.

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 monosodium phosphate (NaH) ₂ PO ₄ ) Disodium phosphate (Na) ₂ HPO ₄ ) Phosphoric acid (H) ₃ PO ₄ ) And combinations thereof.

In one example, the regeneration buffer comprises Na ₂ HPO ₄ 。

In one example, the regeneration buffer comprises H ₃ PO ₄ 。

In one example, the regeneration buffer comprises NaH ₂ PO ₄ 。

In one example, the regeneration buffer comprises Na ₂ HPO ₄ And NaH ₂ PO ₄ 。

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.

In one exampleThe regeneration buffer comprises sodium chloride and phosphate buffer. In one example, the regeneration buffer comprises sodium chloride and sodium dihydrogen phosphate (NaH ₂ PO ₄ ). In one example, the regeneration buffer comprises sodium chloride and Na ₂ HPO ₄ . In one example, the regeneration buffer comprises sodium chloride and H ₃ PO ₄ . In one example, the regeneration buffer comprises sodium chloride and Na ₂ HPO ₄ And NaH ₂ PO ₄ 。

In one example, the regeneration buffer comprises 1M sodium chloride and 10mM sodium dihydrogen phosphate, pH6.2.

In one example, the regeneration buffer comprises 1M sodium chloride and 10mM Na ₂ HPO ₄ ，pH6.2。

In one example, the regeneration buffer comprises 1M sodium chloride and 10mM H ₃ PO ₄ ，pH6.2。

In one example, the regeneration buffer comprises 1M sodium chloride and 10mM Na ₂ HPO ₄ And NaH ₂ PO ₄ ，pH6.2。

In one example, the regeneration buffer comprises 1M sodium chloride and 100mM sodium dihydrogen phosphate, pH6.2.

In one example, the regeneration buffer comprises 1M sodium chloride and 100mM Na ₂ HPO ₄ ，pH6.2。

In one example, the regeneration buffer comprises 1M sodium chloride and 100mM H ₃ PO ₄ ，pH6.2。

In one example, the regeneration buffer comprises 1M sodium chloride and 100mM Na ₂ HPO ₄ And NaH ₂ PO ₄ ，pH6.2。

In one example, the regeneration buffer comprises 0.5M sodium hydroxide.

In one example, the regeneration buffer comprises 1M acetic acid.

In one example, the volume of regeneration buffer is 1 to 10 CVs. For example, the volume of 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 regeneration buffer is 5 CVs.

Suitable regeneration methods will be apparent to the skilled artisan and/or described herein.

In one example, at least 75% IgG is recovered from the plasma or fraction thereof. In another example, at least 75% IgG is recovered from the plasma or fraction thereof after continuous chromatography. For example, at least 75% of IgG is recovered from the plasma or fraction thereof after continuous chromatography without further purification steps. For example, at least 75% of IgG is recovered from the plasma or fraction thereof after continuous chromatography with further purification steps. For example, at least 75% of IgG is recovered from the plasma or fraction thereof after the ion exchange chromatography step. In one example, at least 75% of the IgG is recovered from the plasma or fraction thereof after the anion exchange chromatography step. In one example, at least 75% of the IgG is recovered from the plasma or fraction thereof, wherein the IgG is derived from at least 500kg of plasma or fraction thereof. For example, at least 75% of IgG is recovered from large-scale purification of plasma or fractions thereof. For example, 75%, or 76%, or 77%, or 78%, or 79% IgG is recovered from the plasma or fraction thereof. In one example, 75% IgG is recovered from plasma or fractions thereof. In one example, 76% IgG is recovered from plasma or fractions thereof. In one example, 77% IgG is recovered from plasma or fractions thereof. In one example, 78% IgG is recovered from plasma or fractions thereof. In another example, 79% IgG is recovered from plasma or fractions thereof.

In one example, at least 80% IgG is recovered from the plasma or fraction thereof. In another example, at least 80% IgG is recovered from the plasma or fraction thereof after continuous chromatography. For example, at least 80% of IgG is recovered from the plasma or fraction thereof after continuous chromatography without further purification steps. For example, at least 80% of the IgG is recovered from the plasma or fraction thereof after continuous chromatography with further purification steps. For example, at least 80% of the IgG is recovered from the plasma or fraction thereof after the ion exchange chromatography step. In one example, at least 80% of the IgG is recovered from the plasma or fraction thereof after the anion exchange chromatography step. In one example, at least 80% of the IgG is recovered from the plasma or fraction thereof, wherein the IgG is derived from at least 500kg of plasma or fraction thereof. For example, at least 80% of IgG is recovered from large-scale purification of plasma or fractions thereof. For example, 80%, or 81%, or 82%, or 83%, or 84% IgG is recovered from the plasma or fraction thereof. In one example, 80% IgG is recovered from plasma or fractions thereof. In one example, 81% IgG is recovered from plasma or fractions thereof. In one example, 82% of the IgG is recovered from the plasma or fraction thereof. In one example, 83% IgG is recovered from the plasma or fraction thereof. In another example, 84% IgG is recovered from plasma or fractions thereof.

In one example, at least 85% IgG is recovered from the plasma or fraction thereof. In another example, at least 85% IgG is recovered from the plasma or fraction thereof after continuous chromatography. For example, at least 85% of IgG is recovered from the plasma or fraction thereof after continuous chromatography without further purification steps. For example, at least 85% of the IgG is recovered from the plasma or fraction thereof after continuous chromatography with further purification steps. For example, at least 85% of the IgG is recovered from the plasma or fraction thereof after the ion exchange chromatography step. In one example, at least 85% of the IgG is recovered from the plasma or fraction thereof after the anion exchange chromatography step. In one example, at least 85% of the IgG is recovered from the plasma or fraction thereof, wherein the IgG is derived from at least 500kg of plasma or fraction thereof. For example, at least 85% of IgG is recovered from large-scale purification of plasma or fractions thereof. For example, 85%, or 86%, or 87%, or 88%, or 89% of IgG is recovered from the plasma or fraction thereof. In one example, 85% IgG is recovered from plasma or fractions thereof. In one example, 86% IgG is recovered from plasma or fractions thereof. In one example, 87% IgG is recovered from plasma or fractions thereof. In one example, 88% IgG is recovered from plasma or fractions thereof. In another example, 89% IgG is recovered from plasma or fractions thereof.

In one example, at least 90% of the IgG is recovered from the plasma or fraction thereof. In another example, at least 90% of the IgG is recovered from the plasma or fraction thereof after continuous chromatography. For example, at least 90% of the IgG is recovered from the plasma or fraction thereof after continuous chromatography without further purification steps. For example, at least 90% of IgG is recovered from the plasma or fraction thereof after continuous chromatography with further purification steps. For example, at least 90% of the IgG is recovered from the plasma or fraction thereof after the ion exchange chromatography step. In one example, at least 90% of the IgG is recovered from the plasma or fraction thereof after the anion exchange chromatography step. In one example, at least 90% of the IgG is recovered from the plasma or fraction thereof, wherein the IgG is derived from at least 500kg of plasma or fraction thereof. For example, at least 90% of IgG is recovered from large-scale purification of plasma or fractions thereof. For example, 90%, or 91%, or 92%, or 93%, or 94% IgG is recovered from the plasma or fraction thereof. In one example, 90% IgG is recovered from plasma or fractions thereof. In one example, 91% IgG is recovered from plasma or fractions thereof. In one example, 92% IgG is recovered from plasma or fractions thereof. In one example, 93% of the IgG is recovered from the plasma or fraction thereof. In another example, 94% IgG is recovered from plasma or fractions thereof.

In one example, at least 95% IgG is recovered from the plasma or fraction thereof. In another example, at least 95% IgG is recovered from the plasma or fraction thereof after continuous chromatography. For example, at least 95% of the IgG is recovered from the plasma or fraction thereof after continuous chromatography without further purification steps. For example, at least 95% of IgG is recovered from the plasma or fraction thereof after continuous chromatography with further purification steps. For example, at least 95% of the IgG is recovered from the plasma or fraction thereof after the ion exchange chromatography step. In one example, at least 95% of the IgG is recovered from the plasma or fraction thereof after the anion exchange chromatography step. In one example, at least 95% of the IgG is recovered from the plasma or fraction thereof, wherein the IgG is derived from at least 500kg of plasma or fraction thereof. For example, at least 95% of IgG is recovered from large-scale purification of plasma or fractions thereof. For example, 95%, or 96%, or 97%, or 98%, or 99% IgG is recovered from the plasma or fractions thereof. In one example, 95% IgG is recovered from plasma or fractions thereof. In one example, 96% IgG is recovered from plasma or fractions thereof. In one example, 97% IgG is recovered from plasma or fractions thereof. In one example, 98% IgG is recovered from plasma or fractions thereof. In another example, 99% IgG is recovered from plasma or fractions thereof.

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% after continuous chromatography. In one example, the eluted IgG has a purity of at least 95% after continuous chromatography without further purification steps. In one example, the eluted IgG has a purity of at least 95% after a continuous chromatographic process with further purification steps. In one example, the eluted IgG with a purity of at least 95% is derived from at least 500kg plasma or fraction thereof. For example, eluted IgG having a purity of at least 95% is recovered from large scale purification of plasma or fractions thereof. For example, the purity of eluted IgG is 95%, 96%, 97%, 98% or 99%. In one example, the purity of the eluted IgG is 95%. In one example, the purity of the eluted IgG is 96%. In one example, the purity of the eluted IgG was 97%.

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% after continuous chromatography. In one example, the eluted IgG has a purity of at least 98% after continuous chromatography without further purification steps. In one example, the eluted IgG has a purity of at least 98% after continuous chromatography with further purification steps. In one example, the eluted IgG with a purity of at least 98% is derived from at least 500kg plasma or fraction thereof. For example, eluted IgG having a purity of at least 98% is recovered from large scale purification of plasma or fractions thereof. For example, the purity of eluted IgG is 98% or 99%.

In one example, the method is performed on a large scale. For example, the process is carried out on an industrial or commercial scale. Methods performed on an industrial or commercial scale are apparent to the skilled artisan and/or are described herein. For example, methods performed on an industrial scale include large scale purification of IgG from plasma or fractions thereof.

In one example, large scale purification of IgG is performed using at least 500kg plasma or fractions thereof. For example, large scale purification of IgG is performed using 500kg to 1000kg, or 1000kg to 2500kg, or 2500kg to 5000kg, or 5000kg to 7500kg, or 10000kg to 12500kg, or 12500kg to 15000kg of plasma or fractions thereof. In one example, large scale purification of IgG is performed using at least 1000kg, or 2500kg, or 5000kg, or 7500kg, or 10000kg, or 12500kg, or 15000kg of plasma or fractions thereof. In one example, large scale purification of IgG is performed using at least 1000kg of plasma or fractions thereof. In one example, large scale purification of IgG is performed using at least 2500kg of plasma or fractions thereof. In one example, large scale purification of IgG is performed using at least 5000kg of plasma or fractions thereof. In one example, large scale purification of IgG is performed using at least 7500kg of plasma or fractions thereof. In one example, large scale purification of IgG is performed using at least 10000kg of plasma or fractions thereof. In one example, large scale purification of IgG is performed using at least 12500kg of plasma or fractions thereof. In one example, large scale purification of IgG is performed using at least 15000kg of plasma or fractions thereof.

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

The present disclosure also provides a method of purifying IgG from plasma or fractions thereof using SMB chromatography, the method 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 20mM phosphate equilibration buffer at a pH of 7 to 8;

b) Binding IgG from plasma or fractions thereof to the resin;

c) Washing the resin with 20mM phosphate wash buffer pH 7 to 8; and

d) Eluting the bound IgG with 20mM acetate elution buffer at pH 3 to 5;

wherein steps a) to d) may be repeated on an affinity chromatography resin, and wherein the affinity chromatography resin is packed into a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet valve and an outlet valve, and optionally the method further comprises desorbing the resin with 20mM phosphate wash buffer at a pH of 2 to 3.

a) 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 is equilibrated with an equilibration buffer comprising 20mM sodium dihydrogen phosphate buffer, 145mM sodium chloride and pH 7 to 8;

b) Binding IgG from plasma or fractions thereof to the resin;

c) Washing the resin with a wash buffer comprising 20mM sodium dihydrogen phosphate buffer, 145mM sodium chloride and having a pH of 7 to 8; and

d) Eluting the bound IgG with 20mM acetate elution buffer at pH 3 to 5;

a) 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 is equilibrated with an equilibration buffer comprising 20mM sodium dihydrogen phosphate buffer, 500mM sodium chloride and pH 7 to 8;

b) Binding IgG from plasma or fractions thereof to the resin;

c) Washing the resin with a wash buffer comprising 20mM sodium dihydrogen phosphate buffer, 500mM sodium chloride and having a pH of 7 to 8; and

d) Eluting the bound IgG with 20mM acetate elution buffer at pH 3 to 5;

In one example, the method does not include desorbing the resin.

b) Binding IgG from plasma or fractions thereof to the resin;

c) Washing the resin with 20mM phosphate wash buffer pH 7 to 8; and

d) Eluting the bound IgG with 20mM acetate elution buffer at pH 3 to 5;

wherein steps a) to d) may be repeated on an affinity chromatography resin, and wherein the affinity chromatography resin is packed into a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet valve and an outlet valve, and wherein the method does not comprise desorbing the resin.

b) Binding IgG from plasma or fractions thereof to the resin;

c) Washing the resin with 20mM phosphate wash buffer pH 7 to 8; and

d) Eluting the bound IgG with 20mM acetate or phosphate elution buffer at pH 3 to 5;

wherein steps a) to d) may be repeated on an affinity chromatography resin, and wherein the affinity chromatography resin is packed into a series of two or more fluidly connected columns separated by a fluid conduit comprising an inlet valve and an outlet valve, and optionally wherein the method does not comprise desorbing the resin.

b) Binding IgG from plasma or fractions thereof to the resin;

d) Eluting the bound IgG with 20mM acetate elution buffer at pH 3 to 5;

b) Binding IgG from plasma or fractions thereof to the resin;

d) Eluting the bound IgG with 20mM acetate elution buffer at pH 3 to 5;

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

In one example, the method is repeated for 50 cycles on the resin for each batch of plasma or fraction thereof.

In one example, the method is repeated for 60 cycles on the resin for each batch of plasma or fraction thereof.

In one example, the method is repeated for 70 cycles on the resin for each batch of plasma or fraction thereof.

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

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

In one example, the process is repeated on the resin for a total of up to 800 cycles. For example, the resin is reused for a total number of up to 800 cycles. In one example, the method is repeated on the resin for up to a total of 100, or 200, or 300, or 400, or 500, or 600, or 700 cycles. For example, the process is repeated on the resin for up to a total of 100 cycles. For example, the process is repeated on the resin for up to a total of 200 cycles. For example, the process is repeated on the resin for up to a total of 300 cycles. For example, the process is repeated on the resin for up to a total of 400 cycles. For example, the process is repeated on the resin for up to a total of 500 cycles. For example, the process is repeated on the resin for up to a total of 600 cycles. For example, the process is repeated on the resin for up to a total of 700 cycles.

In one example, the process is repeated on the resin for 100 to 200 cycles, or 200 to 300 cycles, or 200 to 500 cycles, or 500 to 800 cycles.

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

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

In one example, the process is repeated 400 cycles on the resin.

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

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

In one example, the process is repeated 700 cycles on the resin.

In one example, the process is repeated for 800 cycles on the resin.

In one example, the process is repeated for 200 to 500 cycles on the resin. For example, the resin is reused for a total number of up to 200 to 500 cycles. In one example, up to 10 batches of plasma or fractions thereof are used to reuse the resin for up to a total of 500 cycles.

In one example, the resin is subjected to a sterilization step after each individual cycle. In another example, the resin is subjected to a sterilization step after a plurality of cycles. For example, the resin is subjected to a sterilization step after at least 50 cycles. In one example, the resin is subjected to a sterilization step after at least 100 cycles. In another example, the resin is subjected to a sterilization step after at least 150 cycles. In a further example, the resin is subjected to a sterilization step after at least 200 cycles. In one example, the resin is subjected to a sterilization step after each batch of plasma or fraction thereof. For example, the resin is subjected to a sterilization step between each batch of plasma or fractions thereof, i.e., prior to loading each batch of plasma or fractions thereof onto the resin.

Suitable sterilization methods are known to the skilled person and/or are described herein.

In one example, the method reduces the DBC of the resin. For example, repeated use of the resin lowers 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%.

In one example, the resin is reused until the DBC of the resin is reduced by up to 80%.

In one example, the method reduces the DBC of the resin by 80%. For example, the resin is reused until the DBC of the resin is reduced by 80%.

In one example, the method reduces the DBC of the resin by 70%. For example, the resin is reused until the DBC of the resin is reduced by 70%.

In one example, the method reduces the DBC of the resin by 60%. For example, the resin is reused until the DBC of the resin is reduced by 60%.

In one example, the method reduces the DBC of the resin by 50%. For example, the resin is reused until the DBC of the resin is reduced by 50%.

In one example, the method reduces the DBC of the resin by 40%. For example, the resin is reused until the DBC of the resin is reduced by 40%.

In one example, the method reduces the DBC of the resin by 30%. For example, the resin is reused until the DBC of the resin is reduced by 30%.

In one example, the method reduces the DBC of the resin by 20%. For example, the resin is reused until the DBC of the resin is reduced by 20%.

In one example, the method reduces the DBC of the resin by 10%. For example, the resin is reused until the DBC of the resin is reduced by 10%.

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 pharmaceutically acceptable carrier.

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% to 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.

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

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

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

In one example, the pharmaceutical composition comprises at least 60% IgG1 subclass distribution. For example, the pharmaceutical composition comprises at least 65% IgG1 subclass distribution.

In one example, the pharmaceutical composition comprises less than 30% IgG2 subclass distribution. For example, the pharmaceutical composition comprises less than 28% IgG2 subclass distribution.

In one example, the pharmaceutical composition comprises less than 5% IgG3 subclass distribution. For example, the pharmaceutical composition comprises less than 4% IgG3 subclass distribution.

In one example, the pharmaceutical composition comprises less than 5% IgG4 subclass distribution. For example, the pharmaceutical composition comprises less than 3% IgG4 subclass distribution.

In one example, the pharmaceutical composition comprises an IgG subclass distribution similar to that of normal human plasma, e.g., 69% IgG ₁ 、26％ IgG ₂ 、3％ IgG ₃ And 2% IgG ₄ 。

In one example, the pharmaceutical composition comprises a nominal osmotic pressure of about 300mOsm/kg to 400 mOsm/kg. In one example, the pharmaceutical composition comprises a nominal osmotic pressure of 380 mOsm/kg. For example, the pharmaceutical composition comprises a nominal osmotic pressure of about 300mOsm/kg to 350 mOsm/kg. In one example, the pharmaceutical composition comprises a nominal osmotic pressure of 320 mOsm/kg.

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 pH of the pharmaceutical composition is 4.6. In one example, the pH of the pharmaceutical composition is 4.7. In another example, the pH of the pharmaceutical composition is 4.8. In a further example, the pH of the pharmaceutical composition is 4.9. In one example, the pH of the pharmaceutical composition is 5.0.

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

In one example, the pharmaceutical composition comprises a sodium content of 1mmol/L or less.

In one example, the pharmaceutical composition comprises an IgA content of less than or equal to 0.05 mg/mL. For example, the pharmaceutical composition comprises an IgA content of 0.04mg/mL or 0.03 mg/mL. In one example, the pharmaceutical composition comprises an IgA content of less than or equal to 0.025 mg/mL. In one example, the pharmaceutical composition comprises an IgA content of less than or equal to 0.01 mg/mL. For example, the pharmaceutical composition comprises an IgA content of less than or equal to 0.009 mg/mL.

In one example, the pharmaceutical composition comprises an IgA content of less than or equal to 0.1mg/g IgG. In one example, the pharmaceutical composition comprises an IgA content of less than or equal to 0.09mg/g IgG.

In one example, the pharmaceutical composition comprises an IgM content of 10mg/L or less. For example, igM content is 10mg/L or less, 9mg/L or less, 8mg/L or less, 7mg/L or less, 6mg/L or less, 5mg/L or less, 4mg/L or less, 3mg/L or less, and 2mg/L or less. In one example, the pharmaceutical composition comprises an IgM content of 2mg/L or less. In one example, the pharmaceutical composition comprises an IgM content of 1mg/L or less. In one example, the pharmaceutical composition comprises an IgM content of less than or equal to 0.5 mg/L. For example, the pharmaceutical composition comprises an IgM content of <0.17 mg/L.

In one example, the pharmaceutical composition comprises an IgM content of less than or equal to 2 μg/g IgG. In one example, the pharmaceutical composition comprises an IgM content of less than or equal to 1.9 μg/g IgG.

In one example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.50 mg/mL. For example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.40 mg/mL. In one example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.30 mg/mL. In one example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.20 mg/mL. In one example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.10 mg/mL. For example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.09 mg/mL. In one example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.08 mg/mL. In one example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.07 mg/mL.

In one example, the pharmaceutical composition comprises an albumin content of 1mg/g IgG or less. In one example, the pharmaceutical composition comprises an albumin content of less than or equal to 0.80mg/g IgG.

In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of less than or equal to 35 IU/mL. In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of less than or equal to 30 IU/mL. In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of less than or equal to 50 IU/mL. In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of less than or equal to 20 IU/mL. For example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of 15IU/mL or less. In one example, the pharmaceutical composition comprises a prekallikrein activator (PKA) level of less than or equal to 10 IU/mL.

The present disclosure also provides pharmaceutical compositions described herein for use in treating, preventing, and/or delaying 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.

In some examples, the pharmaceutical composition is present in a vial, a prefilled syringe, or an automatic injection device.

The present disclosure also provides a prefilled syringe comprising the pharmaceutical composition described herein.

The present disclosure also provides an automatic injection device comprising the pharmaceutical composition described herein.

In one example, the compositions of the present disclosure are administered subcutaneously to a subject in need thereof. In another example, the compositions of the present disclosure are administered intravenously to a subject in need thereof.

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

In one example, the compositions of the present disclosure are administered subcutaneously by themselves.

In one example, the compositions of the present disclosure are provided in a prefilled syringe.

In one example, the compositions of the present disclosure are administered subcutaneously by itself with a prefilled syringe.

The present disclosure also provides 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 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 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 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.

The present disclosure also provides methods of treating, preventing, and/or delaying 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 methods of treating a condition in a subject. In another example, the present disclosure provides a method of preventing a condition in a subject. In further examples, the present disclosure provides methods of delaying the progression of a condition in a subject.

The present disclosure also provides a kit for treating or preventing a condition in a subject or delaying the progression of a condition in a subject, the kit comprising:

(a) At least one pharmaceutical composition described herein;

(b) Instructions for using the kit to treat or prevent or delay a condition in a subject; and

(c) Optionally, at least one additional therapeutically active compound or drug.

In one example, the condition is an immunodeficiency, an autoimmune disease, or an acute infection. For example, the condition is allogeneic bone marrow transplantation, chronic lymphocytic leukemia, idiopathic Thrombocytopenic Purpura (ITP), childhood HIV, primary immunodeficiency, kawasaki disease, chronic Inflammatory Demyelinating Polyneuropathy (CIDP), renal transplantation of high antibody recipients or ABO incompatible donors, chronic fatigue syndrome, clostridium difficile colitis, dermatomyositis and polymyositis, graves' eye disease, guillain-barre syndrome, muscular dystrophy, inclusion body myositis, lambert-Eaton syndrome, lupus erythematosus, multifocal motor neuropathy, multiple Sclerosis (MS), myasthenia gravis, neonatal alloimmune thrombocytopenia, parvovirus B19 infection, pemphigus, post transfusion purpura, renal transplant rejection, natural abortion, stiff person syndrome, eye contracture, severe sepsis and hemorrhagic shock of severe cases, toxic epidermolysis, chronic myelogenous leukemia, multiple myeloma, X-globulinemia, multiple sclerosis, rrzematosis, rrm, and immunodeficiency, and multiple sclerosis.

In one example, the condition is selected from the group consisting of primary immunodeficiency disease (PI), chronic Inflammatory Demyelinating Polyneuropathy (CIDP), and chronic Immune Thrombocytopenic Purpura (ITP).

In one example, the condition is primary immunodeficiency disease (PI).

In one example, the condition is Chronic Inflammatory Demyelinating Polyneuropathy (CIDP).

In one example, the condition is chronic Immune Thrombocytopenic Purpura (ITP).

In one example of any of the methods described herein, the subject is a mammal, e.g., a primate, e.g., a human.

Brief description of the drawings

FIG. 1 is a graph of (A) clarified cryoprecipitated depleted plasma FcXP under reducing (left) and non-reducing (right) conditionsSDS-PAGE gel images of the eluate (FcXP), and (B) from the protein impurity tables identified in the SDS-PAGE gel run eluate.

FIG. 2 is a 2D-DIGE gel image of proteins in an eluate.

Fig. 3 is a graph showing IgG subclass distribution of Cryoprecipitated Rich Plasma (CRP) and cryoprecipitated depleted plasma (CPP), CRP, and CPP eluate (i.e., eluate from FcXP resin) prior to use in the methods described herein.

FIG. 4 is a graph showing FcXP at bed heights of 6cm (LTS 1) and 20cm (LTS 2) Graphical representation of the static binding capacity of a resin in continuous operation.

Fig. 5 is a graph showing procoagulant activity of (a) plasma and (B) cryoprecipitated-poor plasma (CPP) as a result of temperature change over time or filtration as determined by the natt assay. The setting time was set to >150s.

FIG. 6 is a graph showing proteolytic activity of thrombin (S-2238), general serine protease (S-2288), kallikrein (S-2302), plasmin (S-2251) and FXa (S-2765) in (A) plasma and (B) cryoprecipitated depleted plasma (CPP) as a result of temperature change over time.

FIG. 7 is a graph showing viral inactivation of CRP using N-octyl-beta-D-glucopyranoside.

FIG. 8 is a graph showing the temperature-dependent volume normalization ratio of cold precipitate in (A) samples thawed at different temperatures; and (B) illustrations of a holding time study schematic for evaluating optimal thaw and holding time temperatures, respectively.

Fig. 9 is a series of diagrams showing backpressure during an SMB process with (a) a desorption stage and (B) no desorption stage.

Fig. 10 is a series of graphs showing (a) a decrease in proteolytic activity in the eluate (i.e., the eluate from the FcXP resin) with increasing wash buffer conductivity, and (B) a decrease in proteolytic activity in the eluate (i.e., the eluate from the FcXP resin) from normal plasma and cryoprecipitated depleted plasma (CPP) due to an increase in wash buffer conductivity from 145mM sodium chloride to 500mM sodium chloride.

FIG. 11 is a series of graphs showing (A) IgG yield, (B) product purity as determined using Lapchip, and (C) albumin, igA, and IgM levels in normal plasma and cryoprecipitated depleted plasma (CPP) in an eluate using a wash buffer containing 145mM or 500mM sodium chloride (i.e., an eluate from a FcXP resin).

Sequence listing keywords

SEQ ID NO:1 is the amino acid sequence of a 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 CDR3 of the VHH fragment

Detailed Description

General description

Throughout this specification, unless the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter should be taken to encompass one or more (i.e. one or more) of such steps, compositions of matter, group of steps or group of compositions of matter.

It will be appreciated by those skilled in the art that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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

Any examples of the disclosure herein should be considered as applicable to any other examples of the disclosure mutatis mutandis unless specifically stated otherwise.

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

The term "and/or", e.g. "X and/or Y", is understood to mean "X and Y" or "X or Y", and is to be taken as providing explicit support for both meanings or for either meaning.

Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein, the term "derived from" should be understood to indicate that a specified whole may be obtained from a particular source, although not necessarily directly from that source.

Furthermore, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Selected definition

The term "purified" or "pure" is understood to mean that at least one impurity present in the plasma or a fraction thereof is completely or partially removed, thereby increasing the purity level of IgG in the solution.

The term "impurities" should be understood to include one or more components other than IgG in the plasma or fraction thereof. For example, impurities may include albumin (α -globulin and/or β -globulin), plasma lipids, plasma proteins, proteases (e.g., serine proteases, kallikrein, plasmin, and FXa), serine protease inhibitors (e.g., C1 inhibitors, 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 complexes, C1 esterase inhibitors, protein C, antithrombin III, rhD immunoglobulins, and platelet membrane microparticles.

The term "immunoglobulin G (IgG)", also known as "gamma globulin" or "immunoglobulin", is understood to mean antibodies of isotype G. IgG has several subclasses, such as IgG1, igG2, igG3, and IgG4.

The term "plasma" refers to the straw-colored/yellowish component of blood obtained from one or more blood donors. Methods of obtaining plasma from a donor are obvious to the skilled artisan and/or are described herein. For example, plasma is obtained by removing red blood cells from donated blood. For example, plasma is obtained by plasmapheresis.

The term "plasma fraction" or "fraction thereof" refers to plasma that has been fractionated to separate one or more desired protein components from the plasma. For example, plasma can be fractionated to separate cryoprecipitate (proteins that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed under cold conditions) and cryoprecipitate supernatant (also known as cryoprecipitate-depleted plasma). For example, plasma may be fractionated by ethanol precipitation to produce an on cley fraction, cohn fraction, ammonium sulfate precipitate or precipitate a (KN a), B (KN B) and supernatant B precipitate (knb+1) containing IgG from the plasma, as described in us patent 3,301,842. The plasma fraction includes a ii+iii precipitate, or i+ii+iii precipitate, produced according to the Cohn method (e.g., 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)), 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 Nitschmann and Kistler Vox Sang.7,414-424 (1962); helv.Chim. Acta 37,866-873 (1954) precipitate A, B and supernatant B precipitate. For example, plasma may be fractionated by octanoic acid fractionation as described in european application 893450. Typically, the Cohn fraction and the Kistler/Nitschmann precipitate A (KN A), B (KN B) and the precipitate of supernatant B (KN B+1) are present as a suspension paste. Other purification techniques, including chromatography, may be used.

The term "cryoprecipitate" or "cryoprecipitate" refers to proteins in plasma that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed under cold conditions. Cryoprecipitates include factor VIII, fibrinogen, von willebrand factor, factor XIII and platelet membrane microparticles.

The term "cryoprecipitated lean plasma" is understood to mean plasma from which cryoprecipitate has been removed.

The term "cryoprecipitated-rich plasma" is understood to mean a plasma comprising the components typically found in cryoprecipitates.

The term "clarified" or "clarified" is understood to mean a process of passing the plasma or a fraction thereof through a suitable filter (e.g. a depth filter and/or a 1.2 and 0.45/0.22 μm membrane filter) to remove one or more impurities prior to use in the methods described herein.

The term "dissociation constant" refers to the pKa of the buffer. pka= -log ₁₀ (Ka), wherein Ka is the acid dissociation constant of the buffer. For example, a wash buffer of 20mM sodium dihydrogen phosphate, 40mM sodium chloride, pH 7.4, contains sodium dihydrogen phosphate as a buffer. Phosphoric acid has three dissociation constants (pKa 1:2.16, pKa2:7.21, pKa3: 12.32).

The term "affinity chromatography resin" is understood to mean a resin comprising affinity chromatography ligands (e.g. single domain of camelidae origin [ VHH ]Antibody fragments). Exemplary affinity chromatography resins for use in the methods described herein includeFcXP affinity resin (Thermo Fisher) and +.>FcXP agarose affinity resin (Thermo Fisher). Other exemplary affinity chromatography resins include those having a CH3 domain that specifically binds human IgG consisting of SEQ ID NO:1 or a variant thereof. An exemplary affinity chromatography resin is also described in US 10259886.

The terms "specific binding," "specific binding," or "specifically binding" are understood to mean that the proteins of the present disclosure react or associate with a particular antigen or cell expressing it more frequently, more rapidly, for a longer duration, and/or with greater affinity than the replacement antigen or cell. For example, a ligand is capable of specifically binding to a CH3 domain of human IgG 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 to other antigens. In general, but not necessarily, references to binding means specific binding, and each term should be understood to provide explicit support for the other terms.

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

The term "enriched formulation" should be understood to include an eluent, solution or pharmaceutical composition as described herein. The enriched formulations of the present disclosure comprise IgG of higher purity than IgG in plasma or fractions thereof.

The term "single domain of camelidae origin [ VHH ]]Antibody fragments "are understood to mean the VHH domain of a camelidae antibody. Camelid antibodies are antibodies produced by camels and llamas, which do not have the CH1 domain normally present in human immunoglobulins and only have one VHH domain. Comprising a camelid-derived [ VHH ]]Exemplary affinity chromatography resins for antibody fragments includeAntibody affinity chromatography resin (Thermo Fisher). For example, a->FcXL affinity resin,/->FcXP affinity resin, captureSelect IgG-CH1 affinity resin, and CaptureSelect FcXP agarose affinity resin. Other exemplary affinity chromatography resins include +.>Affinity resin (Cytiva), ->Affinity resin (Cytiva), ->Protein G agarose affinity resin (Thermo Fisher) and Protein G agarose 4 fast flow affinity resin (Cytiva).

The term "matrix" is understood to mean a support to which ligands are immobilized. Exemplary matrices are crosslinked poly (styrene-divinylbenzene) matrices and agarose-based matrices.

The term "dynamic binding capacity" or "DBC" of a chromatography resin should be taken to mean the maximum amount of IgG that the resin will bind under operating conditions before significant breakthrough of unbound IgG occurs.

The term "per mL resin" is understood to mean a wet fill volume of resin per mL.

The term "bed height" is understood to mean the height at which the column is filled with affinity chromatography resin. It will be apparent to the skilled person that reference to "total bed height" refers to the bed height of all columns in a continuous chromatographic setup.

The term "non-loaded phase" is understood to mean a phase other than the loaded phase of continuous chromatography. For example, the non-loaded phase may refer to an equilibration phase, a wash phase, an elution phase, a desorption phase, and/or a re-equilibration phase.

The term "circulation" is understood to mean a round of equilibration, igG loading, binding, elution, desorption, sterilization and/or regeneration performed on a resin.

The term "purity" refers to the ratio of IgG to the total protein content of purified IgG, igG-enriched formulations and pharmaceutical compositions, expressed as a percentage.

The term "industrial or commercial scale" or "large scale" or "manufacturing scale" refers to the quantity of product produced in a batch designed for clinical testing, formulation, sale, and/or distribution to the public. For example, industrial scale refers to large scale purification of IgG from plasma or fractions thereof to produce a plasma protein product.

The term "plasma protein product" refers to a preparation, composition and/or protein product comprising purified plasma protein (e.g., igG or impurities such as albumin) derived from plasma or fractions thereof. Typically, plasma proteins are the major proteins in plasma protein products.

The term "pharmaceutical composition" is understood to mean a formulation of IgG with a compound commonly accepted in the art for delivering IgG to a mammal. Exemplary compounds include all pharmaceutically acceptable carriers, diluents or excipients thereof.

The terms "treat," "treating" or "treatment" are understood to mean administration of a therapeutically effective amount of IgG such that one or more symptoms or features of a condition in a subject are reduced or the subject is no longer clinically diagnosed as having the disorder.

The terms "preventing," "preventing" or "preventing" include providing prophylaxis against the occurrence or recurrence of a particular condition in a subject. The subject may be susceptible to or at risk of developing a condition but has not yet been diagnosed with the condition.

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

The term "condition" is understood to refer to the presence or health of a subject in need of treatment with IgG. Exemplary conditions include, but are not limited to, primary immunodeficiency disease (PI), chronic Inflammatory Demyelinating Polyneuropathy (CIDP), and chronic Immune Thrombocytopenic Purpura (ITP).

The term "subject" is understood to mean any animal, including humans, such as mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

Continuous affinity chromatography

The present disclosure provides methods for purifying IgG from plasma or fractions thereof using continuous affinity chromatography.

The term "continuous affinity chromatography" is understood to mean a chromatography method comprising one or more columns filled with the same affinity resin, wherein each column comprises one or more zones. The zone is a column or a zone of a column containing resin in which one or more chromatographic steps may be performed. For example, the zone is selected from the group consisting of equilibration zone, binding zone, wash zone, elution zone, desorption zone, or a combination thereof. In one example, the region is selected from the group consisting of an equilibration zone, a binding zone, a wash zone, an elution zone, or a combination thereof.

Continuous affinity chromatography involving more than one column involves columns connected in an arrangement that allows the columns to operate in series and/or parallel. In principle, igG may be loaded onto the first and/or subsequent columns, while other columns (or other regions of the columns) undergo equilibration, washing, elution and/or regeneration simultaneously. Examples of continuous affinity chromatography are apparent to the skilled artisan and/or described herein.

Examples of columns useful for performing continuous chromatography are apparent to the skilled artisan and/or described herein. For example, continuous chromatography can be performed using Tricorn5/100 (Cytiva). In another example, continuous chromatography may be performed using the biosba PD system (Sartorius).

Simulated Moving Bed (SMB) chromatography

In one example, the continuous affinity chromatography is Simulated Moving Bed (SMB) chromatography. The term "simulated moving bed chromatography" or "SMB chromatography" refers to chromatography described first in U.S. patent 2,985,589. Examples of SMB chromatographic settings and/or devices will be apparent to those skilled in the art and/or described herein. The concept of a simulated moving bed involves using a plurality of smaller columns (instead of one large column) containing solid absorbent (e.g. affinity resin) and performing one or more consecutive chromatographic steps (i.e. equilibration, binding, washing, elution or desorption) simultaneously on different columns in a continuous loop.

One example of an SMB chromatographic setup is to arrange the columns into four sections, each section having one or more columns. Two inlet streams (feed and eluent) and two outlet streams (extract and raffinate) are introduced into and withdrawn from the collar in alternating order. The inlet and outlet positions are switched at regular time intervals along the direction of liquid flow, thereby simulating countercurrent movement of the column. The feed (containing the adsorbable component (extract)) is loaded onto one or more columns of an SMB chromatographic apparatus, the extract being bound to the resin within the column. At the same time, less of the adsorbed components (raffinate) in the feed passed through the column. The raffinate may be loaded onto one or more subsequent columns or removed from the SMB chromatography system as a waste stream. The eluate is loaded onto a column to collect the extract. For example, the eluent may be collected from a first column while more feed is loaded onto one or more subsequent columns.

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

The resin in the SMB chromatography may undergo multiple cycles (e.g., 50 cycles) of resin equilibration, igG loading, binding, elution, desorption, sterilization, and/or regeneration for each batch of plasma or fraction thereof used. Multiple batch runs (e.g., 4 to 10 batches) can be performed using SMB chromatography. The total lifetime of the resin in the SMB chromatography may be in the range of 200 to 500 cycles (if not more) before the resin is unusable. Resin regeneration is typically performed to allow for multiple uses of the resin.

Periodic Countercurrent Chromatography (PCC)

In one example, the continuous affinity chromatography is Periodic Countercurrent Chromatography (PCC). Examples of PCC settings and/or devices will be apparent to those skilled in the art and/or described herein. The concept of PCC involves the use of multiple columns containing solid absorbents (e.g., affinity resins) and performing the chromatographic steps in parallel in a quasi-continuous manner. The buffer used for the binding, washing and/or elution step flows counter-currently to the affinity resin.

One example of a PCC setup involves the use of two columns. In the first step, the sample is loaded onto the first column above the resin DBC so that unbound product (e.g., igG) breaks through the first column and is captured by the second column. In the second step, the first column is washed, eluted, cleaned and/or rebalanced independently of the second column loaded with further sample. In the third step, additional sample is loaded onto the second column above the resin DBC so that unbound product breaks through the second column and is captured by the first column. In a fourth step, the second column is washed, eluted, cleaned and/or rebalanced independently of the first column loaded with further sample. The process steps are continuously cycled between the two columns.

Another example of a PCC arrangement involves the use of multiple columns. For example, a variation of the PCC arrangement described above may involve the use of multiple columns to capture unbound product, which simulates the use of large columns.

Continuous Countercurrent Tangential Chromatography (CCTC)

In one example, the continuous affinity chromatography is Continuous Countercurrent Tangential Chromatography (CCTC). Examples of CCTC settings and/or devices will be apparent to those skilled in the art and/or described herein. The concept of CCTC involves the use of an affinity resin in the form of a slurry, wherein the slurry is continuously directed through a plurality of static mixers and hollow fiber membranes, which separate the fluid phase from the resin. CCTC is typically performed at low pressure.

Examples of CCTC processes involve binding, first washing, second washing, elution, desorption and/or equilibration steps. Another example of a CCTC process involves binding, first washing, second washing, elution, and/or equilibration steps. For example, CCTC processes do not involve a desorption step. In the binding step, the sample (e.g., plasma or a fraction thereof) and the affinity resin are passed through a static mixer and a hollow fiber membrane. During the washing step, impurities are removed in the flow-through of the hollow fiber membranes, while the resin bound product (i.e., igG) is retained by the membranes. The hollow fibers retain the resin and allow the product to flow through during the elution step. The resin is desorbed and/or equilibrated and the process is repeated.

Continuous Countercurrent Screw Chromatography (CCSC)

In one example, the continuous affinity chromatography is continuous countercurrent helical chromatography (ccs). Examples of CCSC settings and/or devices will be apparent to those skilled in the art and/or described herein. The concept of CCSC involves the use of compact rotating coil separation columns mounted on a centrifuge rotating frame. There are two types of separation column designs currently: a spiral tray assembly and a spiral tube support assembly.

An exemplary CCSC process involves a coiled separation column that rotates about the central axis of the centrifuge while it rotates synchronously about its own axis (e.g., 1,000 to 1,200 rpm). The mobile phase can pass through the centrifuge rotor without rotary sealing and a large amount of stationary phase is retained while the two phases mix along the length of the column to produce efficient solute separation.

Affinity chromatography resin

The present disclosure provides methods of purifying immunoglobulin G (IgG) from plasma or fractions thereof using affinity chromatography resins. The affinity resins of the present disclosure comprise ligands capable of specifically binding to the CH3 domain of human IgG.

Suitable affinity chromatography resins will be apparent to the skilled artisan and/or are described herein. In one example, the resin comprises a single domain [ VHH ] of camelid origin]Ligand of antibody fragment. The skilled person will appreciate that the single domain [ VHH ] based on camelid origin]The ligands of the antibody fragments are capable of specifically binding to all subclasses of IgG (IgG 1, igG2, igG3, igG 4). Exemplary resins areFcXP affinity chromatography resin (Thermo Fisher),. About.>FcXL affinity resin (Thermo Fisher), and->IgG-CH1 affinity resin (Thermo Fisher) and +.>FcXP agarose affinity resin (Thermo Fisher). Other exemplary affinity chromatography resins include +.>Affinity resin (Cytiva), ->Affinity resin (Cytiva),Protein G agarose affinity resin (Thermo Fisher) and Protein G agarose 4 fast flow affinity resin (Cytiva).

In one example, the affinity chromatography resin comprises a single domain [ VHH ] of camelid origin ]Antibody fragments and cross-linked poly (styrene-divinylbenzene) matrices. For example, the affinity chromatography resin isFcXP affinity resin (Thermo Fisher). The crosslinked poly (styrene-divinylbenzene) matrix allows the resin to withstand pressures up to 100 bar.

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

In one example, the continuous affinity chromatography process is performed at a pressure in the range of about 2 bar to about 5 bar. For example, the continuous affinity chromatography process is performed at a pressure in the range of about 3 bar to about 4 bar. In one example, the continuous affinity chromatography process is performed at a pressure in the range of about 3.25 bar to about 3.5 bar.

Buffer solution

The present disclosure provides a continuous affinity chromatography method using a buffer that enables IgG to bind efficiently to the resin and collect IgG from the resin. Typically, the plasma or fraction thereof is at neutral pH (pH of 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 having dissociation constants (pKa) of 6.8 to 8.5 at 25 ℃.

An exemplary buffer for the equilibration and/or wash buffer is sodium dihydrogen phosphate, wherein the phosphate component of sodium dihydrogen phosphate has three dissociation constants (pKa: 2.16, 7.21, and 12.32). Phosphoric acid has a dissociation constant near the pH of the elution and/or desorption buffer used in the continuous affinity chromatography method. However, phosphoric acid does not have a dissociation constant between the pH of the equilibration and/or wash buffer (higher pH) and the elution and/or desorption buffer (lower pH) used in continuous affinity chromatography methods. This enables a fast switching between the washing and elution steps and the desorption and equilibration steps, resulting in a more defined peak and a shorter chromatographic phase. The advantage of using such equilibration and/or wash buffers is that smaller buffer volumes can be used, thereby increasing the efficiency of the continuous affinity chromatography process.

Other suitable buffers for the equilibration and/or wash 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), HEPSO (pKa: 7.85), MOBS (pKa: 7.60), POPSO (pKa: 7.78), TAPSO (pKa: 7.61), tricine (pKa: 8.05), TEA (pKa: 7.76).

IgG fraction analysis

Methods of determining yield, purity, and IgG subclass distribution are obvious to the skilled artisan and/or are described herein.

In one example, purity is determined by SDS-PAGE and MALDI-TOF-MS peptide fingerprinting. Briefly, purified IgG, igG-enriched formulations or IgG-containing pharmaceutical compositions described herein are loaded onto a suitable SDS-PAGE gel (e.g., 8-16% tris-glycine) under reducing and non-reducing conditions, along with a protein size marker and a positive control for IgG (e.g., privigen). Proteins were separated based on size and protein bands of interest were separated, processed and analyzed by MALDI-TOF-MS.

In another example, impurities in an IgG-enriched formulation or an IgG-containing pharmaceutical composition described herein are measured in an enzyme-linked immunosorbent assay (ELISA) using an impurity (e.g., igA) specific antibody. For example, ELISA is performed using a commercially available method. 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, igG-enriched formulations, or IgG-containing pharmaceutical compositions described herein is measured by nephelometry and compared to the light scattering pattern of compositions having a known IgG subclass distribution.

Stability of plasma and fractions thereof

The stability of the plasma or fraction thereof for loading onto the affinity resins described herein can be determined by assessing procoagulant activity, proteolytic activity and particle size of the plasma or fraction thereof. Methods for assessing procoagulant activity, proteolytic activity, and particle size are apparent to the skilled artisan and/or are described herein. Briefly, the plasma or fraction thereof is frozen/thawed in one or more cycles, stored at 2 ℃ to 32 ℃ (e.g., 2 ℃, 10 ℃, 18 ℃, 21 ℃, 28 ℃, or 32 ℃) for 24 or up to 48 hours, and analyzed using one or more methods described below. In one example, the plasma or fraction thereof is thawed in one or more cycles at a temperature of 32 ℃, stored for 24 or up to 48 hours and analyzed using one or more of the methods described below. In another example, the plasma or fraction thereof is thawed in one or more cycles at a temperature of 32 ℃, stored for 24 or up to 48 hours and analyzed using one or more methods described below, and then cooled and stored at a temperature of 21 ℃. In one example, the plasma or fraction thereof is thawed at a temperature of 32 ℃ and a temperature of 21 ℃ prior to continuous affinity chromatography.

In one example, procoagulant activity in plasma or fractions thereof can be determined using an in vitro coagulation assay, such as an activated partial thromboplastin time (natt) assay. The natptt assay measures the rate at which one or more coagulation factors (e.g., fibrinogen, prothrombin, procoagulant, antihemophilic factor, stuart-pro factor, plasma thromboplastin precursor, and Hegeman factor) are activated or formed in plasma or fractions thereof when a coagulation activator (e.g., silica, kaolin, ellagic acid) is added to the assay.

In one example, proteolytic activity in plasma or fractions thereof can be assessed by measuring the activity of thrombin, general serine proteases, kallikrein, plasmin and FXa, for example using commercially available kits such as 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).

In one example, the size of any particle in the plasma or fraction thereof is assessed by microfluidic imaging (MFI) and the polydispersity index is calculated. The calculation of the polydispersity index is obvious to the skilled person.

Additional purification step

The additional purification step may be performed before or after the successive chromatographic steps. In one example, an additional purification step may be performed prior to the continuous chromatography step. In one example, an additional purification step may be performed after the continuous chromatography step.

In one example, the method further comprises one or more steps selected from ethanol precipitation, octanoic acid fractionation, ion exchange chromatography, virus inactivation, virus filtration, and ultrafiltration/diafiltration. Additional purification steps will be apparent to the skilled artisan and/or are described herein.

In one example, the method further comprises ethanol precipitation. For example, cold ethanol can be used to isolate and enrich IgG by removing albumin and α -and β -globulins from plasma or fractions thereof. For example as described in WO 2011/149772.

In one example, the method further comprises immunoaffinity chromatography. For example, the method further comprises cognate lectin affinity chromatography using Eshmuno anti-a and anti-B resins. For example, lectin affinity chromatography can be used to remove lectins a and B.

In one example, the method further comprises octanoic acid fractionation. Octanoic acid can be used to remove plasma lipids and plasma proteins (except IgG). For example as described in WO 2011/131787.

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 (in addition to IgG).

The anion exchanger may be a resin-based anion exchanger, an anion exchange membrane adsorbent, or any other form of anion exchanger having a positively charged matrix 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 monolithic anion exchanger.

In one example, the method further comprises anion exchange chromatography using a resin-based anion exchanger. For example, anion exchange chromatography resins are strong anion exchangers. In one example, the strong anion exchange resin comprises a matrix composed of a poly (styrene-divinylbenzene) matrix. In one example, the strong anion exchanger comprises quaternized polyethyleneimine functional groups. Suitable resin-based anion exchange are apparent to the skilled artisan and include, for example, POROS ^TM HQ 50。

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 a binding and elution mode.

In one example, the anion exchange chromatography step comprises 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 comprises a buffer comprising MES buffer. In another example, the anion exchange chromatography step comprises phosphate buffer.

In one example, the method further comprises viral inactivation. For example, viral inactivation may be achieved by adjusting the solution to a low pH. The low pH may be a pH of 2 to 4. In one example, low pH viral inactivation is performed in the presence of caprylate. In another example, viral inactivation may be achieved by contacting the plasma or fraction thereof, or an IgG-rich formulation or an 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-dimethyl myristamine N-oxide (TDAO).

In a further example, viral inactivation may be achieved by exposing the plasma or fraction thereof, or an IgG-enriched formulation or IgG-containing pharmaceutical composition to a solvent-detergent inactivation step. Suitable solvent-detergent treatments are obvious to the skilled person and include, for example, environmentally friendly detergents. Exemplary environmentally friendly detergents suitable for use in the present disclosure and particularly for inactivating lipid-enveloped viruses include N, N-dimethylmyristamine N-oxide (TDAO), polysorbate 80 (PS 80), polyoxyethylene (10) isooctyl cyclohexyl etherX-100-reduced form), and nonionic surfactants prepared from glucose and alcohols (e.g., simulsol ^TM A preparation). In one example, the detergent is N, N-dimethylmyristamine N-oxide (TDAO). In one example, the detergent is polysorbate 80. In another example, the detergent is polyoxyethylene (10) isooctyl cyclohexyl ether (++>X-100-reduced form). In a further example, the detergent is a nonionic surfactant prepared from glucose and an alcohol.

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

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

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

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

In one example, plasma or a fraction thereof, or an IgG-rich formulation or an IgG-containing pharmaceutical composition is contacted with OG at a temperature of 2 ℃.

In one example, plasma or a fraction thereof, or an IgG-rich formulation or an IgG-containing pharmaceutical composition is contacted with OG at a temperature of 8 ℃.

In one example, plasma or a fraction thereof, or an IgG-rich formulation or an IgG-containing pharmaceutical composition is contacted with OG at a temperature of 10 ℃.

In one example, plasma or a fraction thereof, or an IgG-rich formulation or an IgG-containing pharmaceutical composition is contacted with OG at a temperature of 18 ℃.

In one example, plasma or a fraction thereof, or an IgG-rich formulation or an IgG-containing pharmaceutical composition is contacted with OG at a temperature of 28 ℃.

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

In one example, the method further comprises ultrafiltration/diafiltration. Exemplary ultrafiltration/diafiltration membranes are Pellicon 2Cassettes (Millipore) or polyethersulfone or Hydrosart cassettes (Sartorius).

Pharmaceutical composition

Purified IgG of the present disclosure (synonymous with active ingredient) can be used to formulate pharmaceutical compositions for parenteral (e.g., intravenous administration or subcutaneous administration) for therapeutic and prophylactic treatment.

Compositions for administration typically comprise a solution of purified IgG of the present disclosure dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline and the like. The composition may contain pharmaceutically acceptable carriers, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., as needed, approaching physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

The concentration of purified IgG of the present disclosure in these formulations can vary widely and will be selected based primarily on fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the patient. The carrier may contain small amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives. For example, the pharmaceutical composition comprises proline as a stabilizer.

Suitable pharmaceutical compositions according to the present disclosure generally comprise an amount of purified IgG of the present disclosure admixed with an acceptable pharmaceutical carrier (e.g., sterile aqueous solution) to give a range of final concentrations depending on the intended use. Preparation techniques are well known in the art, as exemplified by Remington's Pharmaceutical Sciences,16th Ed.Mack Publishing Company,1980.

For example, the IgG concentration of the pharmaceutical composition is 1 to 5% w/v, 5 to 15% w/v, or 8 to 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., 10g IgG/L) may be used. For intravenous use, 10% w/v (i.e., 100g IgG/L) may be used.

For subcutaneous administration, higher concentrations may be used. For example 15 to 35% w/v, or 20 to 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.

Application method

As discussed herein, the present disclosure provides methods of treating, preventing, and/or delaying progression of a condition in a subject comprising administering IgG or a pharmaceutical formulation to the subject.

Examples

Example 1: affinity resin

Affinity chromatography resinFcXP affinity resin (Thermo Fisher) andFcXP agarose affinity resin (Thermo Fisher) was used for evaluation purposesSuitable resins that capture IgG from plasma or fractions thereof. Both affinity chromatography resins contain ligands capable of binding to the CH3 domain of human IgG (in particular single domain of camelid origin [ VHH]Antibody fragments), and cross-linked poly (styrene-divinylbenzene) or agarose matrices. Filling affinity chromatography resin in Cytiva's Tricorn column (diameter 5 mm) and at +.>The chromatograms were performed on the avant 25 system (cytova).

Cryoprecipitated Rich Plasma (CRP) and cryoprecipitated depleted plasma (CPP) (prepared by CSL Behring) were heated to 37 ℃ and filtered through a 0.22 μm vial top filter. The affinity chromatography resins were evaluated using the chromatographic run conditions and buffers provided by Thermo Fischer as described in table 1 below.

Table 1 is used to evaluate affinity chromatography buffers of FcXP resins for purification of IgG from human CCP and CPP.

Chromatographic phase	Buffer solution
		Balancing	10mM citrate buffer, 150mM sodium chloride pH7.4
Column washing	10mM citrate buffer, 150mM sodium chloride pH7.4
		Elution	20mM sodium acetate buffer, pH4.0
Column desorption	100mM citric acid bufferFlushing liquid, pH2.0
		Neutralization of the eluent	1M Tris base stock, diluted to 20mM in final samples

And use ofFcXP agarose affinity resin was used as compared to +.>FcXP affinity resin purification of IgG from CRP and CPP gave higher yields of recovered IgG and the purity of the eluate was slightly higher. Based on these preliminary results, further evaluation of +.>Applicability of FcXP affinity resin in continuous affinity chromatography.

Example 2: buffer composition

The disadvantage of the wash buffers of Table 1 is that the buffer citric acid has three dissociation constants (pKa 1:3.13, pKa2:4.76, pKa 3:6.4) and is not suitable for buffering at pH7.4 required for equilibration. Furthermore, washing and elution with a buffer comprising citric acid (as provided in table 1) and pH shift between desorption and equilibration lead to ineffective pH change and prolonged phases during chromatography.

To determine the proper buffer composition to enable rapid pH adjustment during continuous affinity chromatography, use is made ofFcXP affinity resin the experiment performed in example 1 was repeated and the chromatographic buffer of table 2 was used instead of the chromatographic buffer of table 1.

Table 2: for evaluation of IgG purification from human CRP and CPP Affinity chromatography buffers for FcXP affinity resins.

Chromatographic phase	Buffer solution
		Balancing	20mM sodium dihydrogen phosphate buffer, 145mM sodium chloride, pH7.4
Column washing	20mM sodium dihydrogen phosphate buffer, 145mM sodium chloride, pH7.4
		Elution	20mM sodium acetate buffer, pH4.0
Column desorption	20mM sodium dihydrogen phosphate buffer, pH2.5

The sodium dihydrogen phosphate present in the wash buffer had three dissociation constants (pKa 1:2.16, pKa2:7.21 and pKa 3:12.32) which covered a pH of 7.4 required for resin equilibration. Increasing the buffer concentration of sodium dihydrogen phosphate to 20mM was found to exclude the effect of under-buffering during the experiment. However, lower concentrations of sodium dihydrogen phosphate may be used in the wash buffer.

The results show that the use of sodium dihydrogen phosphate can be switched rapidly between a low pH during elution and desorption and a high pH during equilibration and washing, resulting in a more definite peak and shorter phase in continuous affinity chromatography runs.

No difference in binding behavior of IgG was observed between pH7.4 and 7.8, thus allowing CRP and CPP with pH in this range to be applied to the resin without adjusting pH.

Example 3: bed height and flow rate

To determine conditions that can reduce the loaded and unloaded phases, the bed height and flow rate used in the chromatographic process were evaluated. The experiment described in example 2 was repeated at different resin bed heights and using different flow rates of plasma or fractions thereof and elution buffer applied to the resin.

A bed height of 20cm shows significantly higher efficiency in purifying IgG from plasma or fractions thereof compared to a lower bed height, thus achieving a shorter non-loaded phase. The flow rate reduced to 150cm/hr (5 min contact time) during elution did not show a significant effect compared to 2400cm/h elution. Surprisingly, it was found that the load flow rate at which plasma or fractions thereof was applied to the resin was increased to 2400cm/hr (0.5 minute contact time) without significant reduction in IgG binding.

The optimized conditions for purification of IgG from plasma or fractions thereof, as determined by a number of experiments, are summarized in table 3 below. The combination of buffer composition, resin, bed height and flow rate selected results in a reduction in volume of waste liquid, flow through and product during each non-loaded stage (i.e., equilibration, wash, elution, desorption and rebalancing).

Table 3: usingOptimization of FcXP affinity resin purification of IgG from human CRP and CPP. The height of the bed in a column of diameter 5mm was about 20cm. The contact time was 0.5 minutes resulting in a flow rate of up to 2400 cm/hr.

Using a 20cm bed height column, the combined volume for the unloaded stage was 8.6 CV, while the load volume for the clarified CPP was 4.3 CV. The non-load/load CV ratio is 2, and the SMB setting can be realized by only three columns. Similar results were observed with a bed height of about 6 cm. In experiments performed in columns of approximately 6cm bed height, the combined volume of the unloaded stage was 7 CV, while the loaded volume of the clarified CPP was 3.8. The no load/load CV ratio was 1.8, enabling an SMB setup containing only 3 columns instead of 4. The results show that reducing the volume of the non-loaded stage allows the use of fewer columns in the purification process, regardless of bed height.

Furthermore, igG eluted at 1.8 CVs, giving a concentration factor of 2.4. The cycle time (excluding pump acceleration and pump wash) was about 6.5 minutes.

As with the wash and elution stages, further densification can be achieved by overlapping elution and desorption stages. An increase in buffer concentration or pH during rebalancing will further reduce the buffer needed, making the non-load/load CV ratio permanently below 2, even if the loading of plasma or fractions thereof is reduced due to resin aging.

Example 4: eluent profile

Purity of

To qualitatively determine the purity of the eluates of examples 2 and 3, SDS-PAGE gel electrophoresis was performed. Samples (8. Mu.g and 16. Mu.g) of IgG purified from clarified CPP using FcXP resin (FcXP) according to examples 2 and 3 were electrophoresed on 8-16% TRIS-glycine SDS-PAGE gels under reducing and non-reducing conditions (FIG. 1). Protein marker (M) See Blue Plus2Marker (Invitrogen) is also included. SDS-PAGE gels were subjected to Coomassie staining. As determined by SDS-PAGE, use is made ofThe IgG purity in FcXP affinity resin purified clarified CPP was 98.7%.

To determine the impurity profile of the eluents of examples 2 and 3, MALDI-TOF-MS peptide mass fingerprinting was performed. Visible bands (marked with arrows in fig. 1) were separated and used for MALDI-TOF-MS peptide mass fingerprinting to determine the identity of the protein at each band. Impurities were identified by MALDI-TOF-MS peptide mass fingerprinting (labeled with arrows A-F in FIG. 1) and summarized in Table 4. The remaining bands (arrows with no letters designated in fig. 1) were identified as IgG. The most abundant impurities in FcXP samples were IgM, albumin and apolipoprotein a-1 (arrows B, C and F in fig. 1). The other three components of the complement system are small amounts of impurities (arrows A, D and E in fig. 1 and bands A, D and E in table 4).

Table 4: authentication from MALDI-TOF-MSCaptureSelect FcXP impurity +.>

SE-HPLC showed 98.1% average monomer and dimer content of IgG in the eluate, with 0.9% polymer and 1.0% fragment content.

To determine the isoelectric point (pI) of the impurities in the eluate compared to IgG, two-dimensional differential gel electrophoresis (2 DDIGE) was performed. From clarified CPP (FcXPEluent) purified IgG samples were loaded with Sci5 and Sci3 dyes, respectively, and onto 2D-SDS-PAGE. The samples were separated at isoelectric points between pH 3 and 10 and then size separated in the presence of SDS under reducing conditions (fig. 2). FcXP->IgG in the eluate samples were identified as having isoelectric points between pH 7 and 9 (fig. 2). IgM and albumin in the eluate were identified as having a pH of about 6.5 (circles in fig. 2), which is below the isoelectric point of IgG.

The results show that IgM and albumin can be removed from the eluate without loss of significant amounts of IgG using an ion exchange purification step.

IgG subclass distribution

To determine if the affinity resin used to purify IgG affects the subclass distribution of IgG in the eluate of examples 2 and 3, an immunoturbidimetry was performed. Using FcXP determines IgG subclass distribution of CRP, CPP, igG purified from CRP, or CPP (fig. 3). Subclass distribution is calculated by the relative fraction of IgG class to the sum of all classes (IgGx/(igg1+igg2+igg3+igg4)).

Yield rate

The yields were determined using immunoturbidimetry. At least 95% of the IgG recovered in the plasma eluate of example 2 and example 3 was at most 96%.

Example 5: purification cycle

To determine the purification cycle pairThe effect of FcXP affinity resin was followed by multiple purification cycles and the binding capacity of the resin was measured. At various points during the multiple purification cycles, breakthrough behavior of pure IgG was determined and used to calculate the remaining binding capacity of the resin. The loss of binding capacity may be due to aging of the resin.

CRP or CPP was applied to the resin (bed height 6cm or 20 cm) to allow plasma to contact the resin for 0.5 min at each stage and the binding capacity of the resin was measured over time. FIG. 4 shows that there is no difference in resin aging when SMB chromatography is performed using resins of 6cm and 20cm bed height. The decrease in binding capacity follows a linear trend with every 100 runs<An average slope of 5% indicates that the resin has little aging even after 100 runs. Results demonstrate that CaptureSelectAffinity resins are suitable for use in the SMB chromatographic settings under the conditions described in examples 2 and 3.

Example 6: stability of plasma and fractions thereof

The stability of CRP and CPP used 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. The treated CRP and CPP were stored at 10 ℃, 18 ℃ or 28 ℃ for 24 or 48 hours. The IgG content and IgG subclass distribution of the samples were determined by immunonephelometry. The results in tables 5 and 6 show that the IgG content and IgG subclass distribution of CRP and CPP are not affected by filtration, temperature, time and freeze/thaw.

Table 5: igG content of CRP with and without filtration, freezing/thawing, and storage at set time and temperature.

Table 6: igG content of CPP with and without filtration, freeze/thaw and storage at set time and temperature.

To determine the clotting of proteins in plasma and CPP, the procoagulant activity was determined using a non-activated partial thromboplastin time (natt) assay. The clotting time in the natptt assay was set to >150 seconds. Procoagulant activity in plasma and CPP was observed at 28 ℃ for 24 or 48 hours (fig. 5). Procoagulant activity was not observed in plasma and CPP that were filtered, frozen/thawed or at temperatures of 10 ℃ or 18 ℃ for 24 or 48 hours.

To determine proteolytic activity in plasma and CPP, the activities of thrombin, general serine protease, kallikrein, plasmin and FXa (thrombin: S-2238; general serine protease: S-2288; kallikrein: S-2302; plasmin: S-2251; and FXa: S-2765) were determined using chromogenic substrate assays. Proteolytic activity in plasma and CPP was observed at 28 ℃ for 24 or 48 hours (fig. 6). No proteolytic activity was observed in plasma and CPP filtered, frozen/thawed or at temperatures of 10 ℃ or 18 ℃ for 24 or 48 hours.

The stability of CRP and CPP was also assessed by measuring the change in particle size within the sample by micro-flow imaging (MFI) and Dynamic Light Scattering (DLS), and the polydispersity index of the sample was calculated. Tables 8 and 9 show that the samples have a broad polydispersity (> 0.4). Tables 7 and 8 show that the samples have no difference in polydispersity index at 10 ℃ and 18 ℃ for 4 hours or 24 hours, but increase significantly at 48 hours. There was no significant difference in the trend index of plasma over temperature and time, indicating that plasma samples were more stable over time and temperature compared to CPP.

Table 7: particle analysis of CPP stored, with and without filtration at set times and temperatures.

Sample of	Time (hr)	Temperature (. Degree. C.)	Polydispersity index
				CPP before filtration	0		0.8022
CPP after filtration	0		0.5168
				CPP	4	10	0.506
CPP	4	18	0.5216
				CPP	4	28	0.5673
CPP	24	10	0.5268
				CPP	24	18	0.5391
CPP	24	28	0.7055
				CPP	48	10	0.8131
CPP	48	18	0.8005
				CPP	48	28	0.667

Table 7: particle analysis of CRP stored, with and without filtration at set times and temperatures.

The results show that particles are formed in CRP and CPP at higher temperatures and longer storage times. Suitable temperatures for storing the plasma or fractions thereof may be from 10 ℃ to 18 ℃ for up to 48 hours.

Example 7: evaluation of viral inactivation Using n-octyl-beta-D-glucopyranoside

N-octyl- β -D-glucopyranoside (OG) was evaluated to determine its viral reduction ability in CRP prior to IgG purification. Thawed and homogenized CRP (50 mg/ml) and EMEM medium (control) were spiked with 1:20 dilution of Vesicular Stomatitis Virus (VSV). Aliquots of VSV-labeled CRP and VSV-labeled EMEM medium prior to addition of OG were collected. OG was added to VSV-labeled CRP and VSV-labeled EMEM medium such that the final OG concentration in the mixture was 30mM and mixed by pipetting for about 10 seconds. The mixture was incubated with OG-free VSV plus standard EDEM medium and VSV contaminated plasma at 5 ℃. Incubated samples were collected at 15, 30 and 60 min time points. The samples were diluted 10-fold in culture medium to neutralize the activity of OG.

mu.L of OG treated VSV-labeled CRP, OG treated VSV-labeled EDEM medium, VSV-labeled CRP, VSV-labeled EDEM medium and control aliquots were titrated (ten-fold serial dilutions to 10) ^-6 Diluted) onto 150 μl of preculture suspension of african green monkey kidney cells (Vero-PH) in a standard 96 well microplate (Nunc, flat bottom well). The negative controls used were CRP and EDEM medium. The positive control used was a VSV stock for addition of the label, as well as a control virus stock with acceptable VSV titres obtained based on previous results of VSV virus stock characterization.

Plates were incubated at 37 ℃, 3-5% co2, and examined for virus-specific cytopathic effect (CPE) using microscopy for 7 days. Cell cultures titrated with negative controls were required to be CPE-free.

According to Spearman-The method calculates the infection titer and is expressed as log10CCID50/mL (50% cell culture infection dose/mL). If no infectious virus is detected by microtiter, e.g. starting from a 1:10 dilution, the virus titer is given as<1.5log10CC ID50/mL. To lower the limit of detection, 1mL of 1:10 diluted post-treated Vero-PH cells were inoculated into 4T 25 flasks. When all 4T 25 cultures were negative for infectious virus, the resulting infectious titer was recorded as <0.5log10 CCID50/mL。

A significant reduction in VSV was observed when a final OG concentration of 30mM was present in the OG-treated VSV-plus-standard CRP incubated at 5 ℃. Under these conditions (i.e., 30mM final concentration of OG at 5 ℃), the log10 reduction factor (LRF). Gtoreq.5.3 (FIG. 7). LRF is the ratio of viral load in pre-treated starting material (e.g., CRP) to viral load in post-treated final material (e.g., OG-treated CRP) and takes into account sample volume and viral titer before and after treatment.

Example 8: optimization of plasma thawing

To evaluate the optimal temperature for plasma thawing, the temperature at which cryoprecipitation was observed was determined. Briefly, cryoprecipitated plasma (CRP) was gradually heated from 15 ℃ to 38 ℃ after thawing, and a portion of CRP was centrifuged at 20 ℃, 25 ℃, 28 ℃, 30 ℃,32 ℃ and 37 ℃ and the temperature dependent pellet was weighed. At-30 ℃, only non-specific aggregates were present and no cryoprecipitation was present.

To determine the optimal temperature, the activity of von willebrand factor (vWF) (one of the major components of cryoprecipitation) in each pellet was determined. As shown in fig. 8A and table 8, thawing at 32 ℃ resulted in robust precipitate formation and vWF activity in the supernatant.

Table 8: determination of vWF Activity in CRP precipitate at different temperatures

To further confirm plasma thawing at 32 ℃, a hold time study was performed as shown in fig. 8B, and the resulting eluate was assayed for proteolytic activity using the natt assay.

As shown in table 9, no proteolytic activity was observed for up to 48 hours when the eluate was filtered at 32 ℃.

Table 9: proteolytic activity after thawing CRP at 32 ℃ versus 14 °

The particulate level in the sample was also analyzed using dynamic light scattering. No difference in polydispersity index (PDI) was observed between samples with PDI <0.6 in all samples (data not shown).

Turbidity was also assessed over time and significant degradation was observed at all temperatures (i.e. 14 ℃, 21 ℃ and 32 ℃) after 48 hours. At 24 hours, no significant degradation was observed at 14 ℃, whereas most degradation was observed at 32 ℃. For plasma maintained at 32 ℃, a significant cycle-by-cycle pressure increase during chromatography was also observed at 48 hours compared to 14 ℃ (data not shown).

No effect on IgG content was observed despite significant degradation. In particular, the IgG content does not decrease over time when the sample is thawed and stored at higher temperatures.

Example 9: optimization of continuous affinity chromatography methods

Removal of desorption stage

During the SMB process, an increase in backpressure of the individual column was observed due to denaturation of the protein accumulated on the column by severe conditions during the desorption phase (i.e. ph 2.5). Therefore, to investigate whether removal of the desorption stage reduced the increase in back pressure, the SMB process was run without the desorption stage.

As shown in fig. 9, removal of the desorption stage resulted in a decrease in back pressure increase and pressure stabilization during SMB.

Increasing the conductivity of the wash stage

The conductivity of the wash stage was screened to investigate whether protease activity in the eluate could be reduced.

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

Example 10: anion exchange refining step

For use of POROS ^TM- HQ50 anion exchange resin was evaluated as a refining step. The resin is operated in a flow-through mode.

Initially, the resin was screened for effect on impurity consumption using MES and phosphate buffer.

The FcXP eluate was UF/DF in 10mM MES or phosphate buffer ph6.0, 0mM NaCl prior to loading onto the anion exchange chromatography column. The flow-through and post-wash solutions were collected and analyzed, respectively.

As shown in table 10, MES buffer and phosphate buffer resulted in acceptable IgG yields and acceptable impurity consumption.

phosphate buffer at pH6.0 (low conductivity) produced the best results in terms of development goals for impurity consumption.

Table 10: buffer screening

Example 11: anion exchange refining step Balancing and load buffer optimization studies and design of experiments (DoE) studies

Buffer evaluation study

Buffer and conditions evaluated:

the sample was loaded according to example 10 above. Briefly, the FcXP eluate was re-buffered and loaded onto Poros ^TM HQ 50. The flow-through and post-wash solutions were collected and analyzed, respectively.

Citrate buffer consumed IgA well over a broad NaCl and pH range. Acceptable IgA consumption in phosphate is achieved at low salt.

Phosphate buffer consumed IgM well over a broad range of NaCl and pH.

Bis-Tris and histidine consume albumin well over a broad NaCl and pH range. Good albumin consumption in phosphate at low salt.

Design of experiments with phosphate buffer

The following conditions were evaluated:

x：0-40mM

y：pH 5.8-6.6

preliminary suggestions for equilibration and loading included 0mM NaCl and pH6.2, the loaded material in pilot scale runs had a purity of about 93.5% and an impurity concentration of 40.4-71.6mg/g IgG. In a BioSMB run, the support material had a purity of about 97%, an impurity/IgG concentration of 12.0-20.4mg/g IgG and a concentration of 5-15g/L IgG for optimal impurity consumption. The loading of the resin is 25-42mg of impurities per mL of resin.

Preliminary suggestions of post-wash conditions included 0mM NaCl and pH6.0.

Example 12: anion exchange refining step

Further screening of MES and phosphate buffer against POROS ^TM Effect of impurity consumption in HQ 50 anion exchange refining step. As described in example 10 above, this procedure was performed using MES buffer pH6.0 with 20mM or 50mM NaCl, MES buffer pH6.6 with 25mM or 50mM NaCl, and phosphate buffer pH6.2 with 0mM NaCl.

MES buffer and phosphate buffer produced acceptable IgG yields, as well as acceptable impurity consumption in the flow-through (no post-wash).

Example 13: magnification of SMB FcXP chromatographic step

The 4 column set-up in SMB mode using a 1cm inner diameter column was performed without resin desorption stage and alternating wash stage using the following conditions:

buffer 1:20mM NaH ₂ PO ₄ 、500mM NaCl，pH7.4；

Buffer 2:20mM acetic acid, pH4.0

After each batch, all columns were regenerated using 20% etoh and 20mM NaOH and used to process the next batch.

The IgG yield in FcXP eluate was 98.3-99.1% and the total IgG recovery in all fractions was 99.4-100.3%. The purity of the FcXP eluate was also evaluated by Labchip and the product purity was 96.4-96.9%.

The purification process produced 9.55-10.4g/L IgG.

Impurities in the FcXP eluate were determined, including IgM (47.85-51.90 mg/L;4.8-5.3mg/gm IgG), igA (68.8-76.55 mg/L;7-8mg/gm IgG) and albumin (55.3-78.05 mg/L;5.8-7.6mg/gm IgG).

Example 14: amplification process run using cryoprecipitated rich plasma

Pooled cryoprecipitated-rich plasma from 30 donors was thawed and clarified through 1.2 μm and 0.45 μm+0.2 μm filters in series prior to use in downstream processing.

The thawed and filtered plasma was purified and processed as follows:

FcXP SMB chromatography

2. Concentration and buffer exchange by UF/DF

pH Change and filtration

4. Anion exchange chromatography (using POROS ^TM- HQ50)

5. Affinity chromatography of lectin of the same family

6. Virus inactivation

7.UF/DF

8. Formulated to 100g/L IgG

3 separate runs were performed with IgG recovery in steps 1-6 of 83-96% and total process recovery of 81-94%.

The purification process results in a distribution of IgG subclasses of 67-69% IgG1, 24-27% IgG2, 3-4% IgG3, and 2-3% IgG4 in the final formulated bulk product.

The purity of the product after lectin chromatography was also assessed by Labchip, where the purity of the product was 97-98.5%.

The POROS 50HQ flow-through and post-wash solutions 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). The yield was also determined to be 93-97%.

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

The lectin consumption was also assessed after FcXP SMB chromatography (Iso-A titres 1:16; iso-B titres 1:32), POROS HQ50 chromatography (Iso-A titres 1:4; iso-B titres 1:8) and after cognate lectin chromatography (Iso-A titres 1:0; iso-B titres 1:0).

Sequence(s)

SEQ ID NO:1 is the amino acid sequence of a 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 CDR3 of the VHH fragment

RRFGSSEWDY

Claims

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

2. A method of producing an immunoglobulin G (IgG) -rich preparation from plasma or a fraction thereof using continuous affinity chromatography, the method comprising binding the IgG to an affinity chromatography resin comprising a ligand capable of specifically binding the CH3 domain of human IgG, and collecting the IgG.

3. The method of claim 1 or 2, wherein the ligand comprises a single domain [ VHH ] antibody fragment of camelid origin.

4. A method according to any one of claims 1 to 3, wherein the resin comprises a matrix selected from the group consisting of a cross-linked poly (styrene-divinylbenzene) matrix and an agarose-based matrix.

5. The method of any one of claims 1 to 4, wherein the ligand comprises a VHH antibody fragment conjugated to a cross-linked poly (styrene-divinylbenzene) matrix.

6. The method of any one of claims 1 to 5, wherein the method further comprises washing the resin with a wash buffer having a pH of 5 to 10 and a dissociation constant (pKa) of 6.8 to 8.5 at 25 ℃.

7. The method of any one of claims 1 to 6, wherein the method further comprises eluting bound IgG from the resin with an elution buffer having a pH of 3 to 5.

8. The method of any one of claims 1 to 7, wherein the plasma or fraction thereof is contacted with the resin for 0.1 to 5 minutes.

9. The method of any one of claims 1 to 8, wherein the plasma fraction is selected from the group consisting of cryoprecipitated-rich plasma, cryoprecipitated-poor plasma, supernatant I (SNI), cohn fraction II (Fr II), cohn fraction ii+iii (Fr ii+iii), cohn fraction i+ii+iii (Fr i+ii+iii), kistler/Nitschmann precipitate a (KN a), kistler/Nitschmann precipitate B (KN B), kistler/Nitschmann precipitate of supernatant B (KN b+1), and combinations thereof.

10. The method of any one of claims 1 to 9, wherein the plasma or fraction thereof is thawed at a temperature of at least 32 ℃.

11. The method of any one of claims 1 to 10, wherein the plasma or fraction thereof is at a temperature in the range of 2 ℃ to 28 ℃ prior to the continuous affinity chromatography.

12. The method of claim 11, wherein the plasma or fraction thereof is at a temperature of 21 ℃ prior to the continuous affinity chromatography.

13. The method of claim 11 or 12, wherein the plasma or fraction thereof is at the temperature for up to 48 hours.

14. The method of any one of claims 1 to 13, wherein the wash buffer comprises 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-piperazine ethanesulfonic acid (HEPES), sulfurous acid, 4- (2-hydroxyethyl) -1-piperazine propanesulfonic 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 (taco), tricine, triethanolamine (TEA), and combinations thereof.

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

16. The method of any one of claims 1 to 15, wherein the wash buffer further comprises sodium chloride and/or divalent salt at a concentration of up to 1000 mM.

17. The method of any one of claims 1 to 16, wherein the wash buffer comprises 20mM sodium dihydrogen phosphate, 500mM sodium chloride and has a pH of 7.4.

18. The method of any one of claims 1 to 17, wherein the elution buffer is or comprises a phosphate buffer and/or an acetate buffer having a pH of 3 to 5.

19. The method of any one of claims 1 to 18, wherein the elution buffer contacts the resin for up to 5 minutes.

20. The method of any one of claims 1 to 19, wherein the method further comprises equilibrating the resin with an equilibration buffer having a pH of 7 to 8.

21. The method of claim 20, wherein the equilibration buffer comprises 20mM sodium dihydrogen phosphate, 500mM sodium chloride and pH 7.4.

22. The method of claim 20 or 21, wherein i) after desorbing the resin or ii) balancing the resin without desorbing the resin.

23. The method of any one of claims 1 to 22, wherein the method further comprises equilibrating the resin with an equilibration buffer having a pH of 7 to 8 after desorbing the resin.

24. The method of any one of claims 1 to 23, 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 helical chromatography (CCSC).

25. The method of any one of claims 1 to 24, wherein the resin is in the form of a slurry or the resin is packed into one or more columns, wherein each column comprises one or more regions.

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

27. The method of claim 25 or 26, wherein the columns are fluidly connected and separated by a fluid conduit comprising an inlet valve and an outlet valve.

28. The method of any one of claims 25 to 27, wherein the resin is packed into a series of three columns, wherein each column is a separate zone.

29. The method of any one of claims 1 to 28, wherein the resin is packed into a first column and one or more subsequent columns.

30. The method of claim 29, wherein the first column is loaded with IgG at a concentration higher than the Dynamic Binding Capacity (DBC) of the resin.

31. The method of claim 30, wherein the resin has a DBC of at least 5mg igg per mL of resin.

32. The method of any one of claims 29 to 31, wherein the one or more subsequent columns are loaded with IgG at a concentration of DBC of at most the resin.

33. The method of any one of claims 29 to 32, wherein the one or more subsequent columns are loaded with IgG at a concentration of up to 40mg IgG per mL of resin.

34. The method of any one of claims 1 to 33, wherein the resin has a total bed height of 2cm to 30 cm.

35. The method of any one of claims 1 to 34, wherein the method further comprises regenerating the resin.

36. The method of any one of claims 1 to 35, wherein the method further comprises sterilizing the resin.

37. The method of any one of claims 1 to 36, wherein the method further comprises 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 of 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 of claim 38, wherein the strong anion exchange resin comprises a matrix consisting of a poly (styrene-divinylbenzene) matrix.

40. The method of claim 38 or 39, wherein the strong anion exchange resin comprises quaternized polyethyleneimine functional groups.

41. The method of claim 38 to 40, wherein the anion exchange chromatography step comprises a post-load wash buffer selected from the group consisting of phosphate buffer, sodium citrate buffer, 2- (N-morpholino) ethanesulfonic acid buffer, acetic acid buffer, bis-tris buffer, and L-histidine buffer.

42. The method of claim 41, wherein the post-load wash buffer comprises a phosphate buffer having a pH in the range of 5.8 to 6.6.

43. The method of claim 41 or 42, wherein the post-load wash buffer further comprises sodium chloride at a concentration of 0mM to 50 mM.

44. The method of any one of claims 1 to 43, wherein at least 75% of said IgG is recovered from said plasma or fraction thereof.

45. The method of any one of claims 1 to 44, wherein the eluted IgG has a purity of at least 95%.

46. The method of any one of claims 1 to 45, wherein the method further comprises formulating the IgG into a pharmaceutical composition.

47. A method of purifying immunoglobulin G (IgG) from plasma or a fraction thereof using Simulated Moving Bed (SMB) chromatography, the method comprising:

b) Binding IgG from plasma or fractions thereof to the resin;

c) Washing the resin with 20mM phosphate wash buffer pH 7 to 8; and

48. A pharmaceutical composition comprising IgG purified or produced by the method of any one of claims 1 to 47.

49. The pharmaceutical composition of claim 48, wherein the pharmaceutical composition comprises 100mg/mL total human plasma protein.

50. The pharmaceutical composition of claim 48, wherein the pharmaceutical composition comprises 20g/100mL total human plasma protein.

51. The pharmaceutical composition of any one of claims 48 to 50, wherein the pharmaceutical composition comprises at least 98% pure immunoglobulin G (IgG).

52. The pharmaceutical composition of any one of claims 48 to 51, wherein said pharmaceutical composition comprises a nominal osmotic pressure of 320 mOsm/kg.

53. The pharmaceutical composition of any one of claims 48 to 52, wherein said pharmaceutical composition comprises a pH of 4.6 to 5.0.

54. The pharmaceutical composition of any one of claims 48 to 53, wherein the pharmaceutical composition comprises a pH of 4.8.

55. The pharmaceutical composition of any one of claims 48 to 54, wherein the pharmaceutical composition further comprises 250 mmol/L-proline.

56. The pharmaceutical composition of any one of claims 48 to 55, wherein the pharmaceutical composition comprises a sodium content of 1mmol/L or less.

57. The pharmaceutical composition of any one of claims 48 to 56, wherein the pharmaceutical composition comprises an IgA content of less than or equal to 0.05 mg/mL.

58. The pharmaceutical composition of any one of claims 48 to 57, wherein the pharmaceutical composition comprises a prekallikrein activator (PKA) level of less than or equal to 35 IU/mL.

59. Use of IgG purified or produced by the method of any one of claims 1 to 47 in the manufacture of a medicament for treating, preventing and/or delaying the progression of a condition in a subject.

60. The pharmaceutical composition of any one of claims 48 to 58 for use in treating, preventing and/or delaying the progression of a condition in a subject.

61. A method of treating, preventing and/or delaying progression of a condition in a subject, the method comprising administering to the subject a pharmaceutical composition of any one of claims 48 to 58.

62. The use of claim 59 or the pharmaceutical composition of claim 60 or the method of claim 61, wherein said condition is selected from the group consisting of primary immunodeficiency disease (PI), chronic Inflammatory Demyelinating Polyneuropathy (CIDP) and chronic Immune Thrombocytopenic Purpura (ITP).