Method for producing an immunoglobulin preparation from C-1 inhibitor-deficient plasma

Abstract

A method for preparing an immunoglobulin G (IgG)-enriched fraction from C1-INH-deficient plasma supernatant has been described. Isolation of the immunoglobulin G (IgG)-enriched fraction from C1-INH-deficient plasma supernatant provided an alternative starting material for the manufacturing process. In the present invention, the C1-INH-deficient plasma supernatant is treated with heparin before further processing.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 002,791, filed Mar. 31, 2020, which is incorporated herein by reference in its entirety.

Background Art

[0002] Plasma - derived blood products are used to treat not only various blood diseases but also diseases of other causes. For example, immunoglobulin (IgG) preparations derived from human plasma were first used in 1952 to treat immunodeficiency. Since then, IgG preparations have found widespread use in at least three major categories of medical conditions: (1) immunodeficiencies such as X - linked agammaglobulinemia, hypogammaglobulinemia (primary immunodeficiency), and acquired immunocompromised states (secondary immunodeficiency) characterized by low antibody levels, (2) inflammatory and autoimmune diseases, and (3) acute infectious diseases.

[0003] Although IVIG treatment can be very effective in dealing with primary immunodeficiency diseases, this therapy is not a cure for the disease but only a temporary replacement for antibodies not produced in the body. Thus, patients dependent on IVIG therapy typically need to repeat dosing about once a month for life. This necessity creates a large demand for the continuous production of IVIG compositions. However, unlike other biologic agents produced via in vitro expression of recombinant DNA vectors, IVIG is fractionated from human - provided blood and plasma. Therefore, the production volume of IVIG cannot be increased simply by increasing the production amount. Rather, the levels of commercially available IVIG are limited by the available supply of the blood and plasma provided.

[0004] Several methods of preparing IVIG are used by commercial suppliers of IVIG preparations. One common problem with current IVIG production methods is the substantial loss of IgG during the purification process, which is predicted to be at least 30% - 35% of the total IgG content of the starting material. One challenge is to increase the IgG yield while maintaining the quality of virus inactivation and the absence of impurities that can cause adverse reactions.

[0005] At current production levels of IVIG, even a seemingly small increase in yield is actually very significant. For example, at the 2007 production level, a 2% increase in efficiency (equivalent to an increase of 56 milligrams per liter) results in a 1.5 metric ton increase in IVIG.

[0006] When manufacturing and formulating plasma-derived biotherapeutics, various safety precautions need to be considered. These include methods for removing and / or inactivating blood-borne infectious pathogens (e.g., viral and bacterial pathogens), anti-complement activity, and other undesirable contaminants arising from the use of the provided plasma. Studies have suggested that administration of high levels of amidolytic activity can result in undesirable thromboembolic events (Wolberg AS et al., Coagulation factor XI is a contaminant in intravenous immunoglobulin preparations. Am J Hematol 2000;65:30 - 34 (Non-Patent Document 1); and Alving BM et al., Contact-activated factors: contaminants of immunoglobulins preparations with coagulant and vasoactive properties. J Lab Clin Med 1980;96:334 - 346 (Non-Patent Document 2); the disclosures of which are hereby incorporated by reference in their entirety for all purposes).

[0007] Throwing light on this concern was the recent voluntary withdrawal of Octagam® (Octapharma) in the United States, as well as the suspension of the marketing authorizations for Octagam® and Octagam 10% by the European Commission due to an increased reporting of thromboembolic events. The high level of amidolytic activity in the biologic due to serine protease and serine protease zymogen impurities, such as factors XI, XIa, XII, and XIIa, likely led to the increased thromboembolic events (FDA Notice: Voluntary Market Withdrawal - September 23, 2010 Octagam [Immune Globulin Intravenous (Human)] 5% Liquid Preparation; Octagam 50 mg / ml, solution - Octapharma France - Quarantine of all lots, published online by AFSSAPS on September 9, 2010 (Non-Patent Document 3); and Questions and answers on the suspension of the marketing authorizations for Octagam (human normal immunoglobulin 5% and 10%), published online by the European Medicines Agency on September 23, 2010 (Non-Patent Document 4)).

[0008] WO2014113659A1 (Patent Document 1) discloses a method for isolating one or more blood products from an inter-alpha inhibitor protein (IαIp)-deficient blood product raw material. The blood product is chromatographically separated from the IαIp-deficient cryo-depleted plasma by contacting the aforementioned IαIp-deficient cryo-depleted plasma with a DEAE carrier. This reference does not disclose the use of C1-INH-deficient plasma supernatant for the production of IgG. Nor does it disclose treating the plasma supernatant with heparin to thereby reduce the amidolytic activity and procoagulant activity in IgG.

[0009] Due to concerns about the limited supply of starting materials for IgG preparations and the significant loss of IgG in the purification process, there is an immediate need in the art to provide a method for increasing the availability of a significant amount of alternative starting materials for manufacturing IgG.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0012] The present invention solves those problems and other problems. In one embodiment, the present invention is based on the discovery that C1-INH-deficient plasma supernatant can be used as a starting material for the preparation of immunoglobulin G (IgG) enriched fractions, thus making available another starting material for the preparation of IgG. Recent concerns regarding the amidolytic content of those plasma-derived protein compositions that are paired with the occurrence of thromboembolic events in patients to whom the plasma-derived protein compositions are administered have highlighted the need in the art for ways to reduce serine proteases (e.g., FXIa and FXIIa) and serine protease zymogens (e.g., FXI and FXII) during the manufacture of those biopharmaceuticals. Advantageously, the present invention is based at least in part on the unexpected discovery that heparin can be used to reduce procoagulant activity and amidolytic activity to acceptable levels during the fractionation process. Also provided are plasma-derived protein pharmaceutical compositions having reduced serine protease activity, serine protease content, and / or serine protease zymogen content. Also provided is a method of treating or preventing a disease by administration of a composition of the present invention.

[0013] In one embodiment, the present invention provides a method for preparing an IgG enriched fraction from a C1-INH-deficient supernatant fraction containing immunoglobulin G (IgG). The method comprises (a) contacting the C1-INH-deficient supernatant fraction with heparin, thereby forming a heparin-added C1-INH-deficient fraction; (b) Isolate IgG from the heparin-added C1-INH-deficient fraction, thereby forming an IgG-enriched fraction and includes.

[0014] In one embodiment of the method described herein, the supernatant fraction is the supernatant produced by C1 inhibitor adsorption.

[0015] In an exemplary embodiment, the supernatant fraction is plasma supernatant.

[0016] In one embodiment, the plasma supernatant is C1-INH-deficient cryoprecipitated plasma.

[0017] In various embodiments, the plasma supernatant is derived from double-deficient cryoprecipitated plasma (DDCPP).

[0018] In an exemplary embodiment, the supernatant fraction is deficient in one or more other blood coagulation factors selected from Factor II, Factor VII, Factor IX, Factor X, and mixtures thereof.

[0019] In one embodiment, the supernatant fraction is concentrated to the protein value of normal plasma before further processing.

[0020] In an exemplary embodiment, heparin is added in an amount of about 1 to about 20 units per mL of the supernatant fraction.

[0021] In an exemplary embodiment, heparin is added in an amount of about 5 to about 10 units per mL of the supernatant fraction.

[0022] In one embodiment, heparin is added in an amount of about 5 units per mL of the supernatant fraction.

[0023] In various embodiments, heparin is added in an amount of about 10 units per mL of the supernatant fraction.

[0024] In some embodiments, the method (c)Removing C1-inhibitor (C1-INH) from the cryoprecipitate-free plasma fraction containing C1-INH, thereby forming a C1-INH-deficient supernatant fraction further comprises.

[0025] In one embodiment, the IgG-enriched fraction contains from about 60% to about 80% of the IgG content found in the supernatant fraction.

[0026] In one embodiment, the IgG-enriched fraction contains at least about 50% of the IgG content in the supernatant fraction.

[0027] In one embodiment of the method described above, the purity of γ-globulin in the IgG-enriched fraction is at least about 95%.

[0028] In one embodiment of the method described above, the purity of γ-globulin in the IgG-enriched fraction is from about 95% to about 99.9%.

[0029] In an exemplary embodiment, the present invention provides the following steps in any order or combination: (i) precipitating the heparin-added fraction with from about 6% to about 10% ethanol, such as aqueous ethanol, at a pH of from about 7.0 to about 7.5 to obtain a fraction I precipitate and a fraction I supernatant; (ii) precipitating IgG from the fraction I supernatant with from about 18% to about 27% ethanol, such as aqueous ethanol, at a pH of from about 6.7 to about 7.3 to form a fraction II+III precipitate and provides a method for isolating IgG from a heparin-added fraction, comprising one or more of the above.

[0030] In one embodiment of the method described above, the method further comprises precipitating IgG from the heparin-added fraction with from about 18% to about 27% ethanol, such as aqueous ethanol, at a pH of from about 6.7 to about 7.3 to form a fraction I+II+III precipitate.

[0031] In one embodiment, the method comprises the following steps in any order or combination: (iii) suspending the precipitate of fraction II+III or fraction I+II+III in a suspension buffer, thereby forming an IgG suspension; (iv) mixing finely divided silicon dioxide (SiO2) with the IgG suspension, for example, for at least about 30 minutes; (v) filtering the IgG suspension, thereby forming a filtrate and a filter cake; and further comprising one or more of:

[0032] In one embodiment, the method comprises the following steps in any order or combination: (vi) washing the filter cake with a washing buffer having a pH of about 4.9 to about 5.3 and at least 1 filter press dead volume, thereby forming a wash solution; (vii) combining the filtrate with the wash solution, thereby forming a mixed solution, and treating the mixed solution with a detergent; (viii) adjusting the pH of the mixed solution of step (vii) to about 7.0 and adding ethanol thereto to a final concentration of about 20% to about 30%, thereby forming a precipitate G precipitate; (ix) dissolving the precipitate G precipitate in an aqueous solution containing an agent selected from a solvent, a detergent, or a combination thereof, for example, incubating the solution for at least about 60 minutes to form an incubation solution; (x) passing the elution solution through a cation exchange chromatography column to elute the protein adsorbed on the column into the eluate; (xi) passing the incubation solution through an anion exchange chromatography column to generate a flow-through fraction; (x) passing the flow-through fraction through a nanofiltration membrane to generate a nanofiltrate; (xi) concentrating the nanofiltrate by ultrafiltration to generate a first ultrafiltrate; (xii) To produce a diafiltrate, diafiltering the first ultrafiltrate against a diafiltration buffer; (xiii) Concentrating the diafiltrate by ultrafiltration to produce a second ultrafiltrate having a protein concentration of from about 8% (w / v) to about 22% (w / v), thereby forming an IgG concentrate fraction; further comprising one or more of the following.

[0033] In one embodiment, the method includes adding SiO2 until a final concentration of SiO2 of about 0.02 to about 0.10 grams per gram of the precipitate of fraction II+III or fraction I+II+III is obtained.

[0034] In one embodiment, the method includes washing the filter cake with at least about 3 filter press dead volumes of wash buffer.

[0035] In one embodiment, the method includes washing the filter cake with at least about 2 filter press dead volumes of wash buffer.

[0036] In one embodiment, the method includes eluting at least one protein with at least about 35 mM sodium dihydrogen phosphate dihydrate.

[0037] In one embodiment, the diafiltration buffer contains from about 200 mM to about 300 mM glycine.

[0038] In one embodiment, the method further includes treating the IgG solution with a solvent and / or a detergent in at least one virus inactivation or removal step.

[0039] In one embodiment of the methods described above, the method further includes an incubation step at a low pH of from about 4.0 to about 5.2.

[0040] In one embodiment of the method described above, the method further includes an incubation step at a low pH of about 4.4 to about 4.9.

[0041] In an exemplary embodiment, the present invention provides a supernatant fraction after C1 inhibitor adsorption, which contains IgG, and the fraction is a defrosted plasma fraction lacking at least about 70% of the total C1-INH present in the defrosted plasma fraction.

[0042] In a fourth aspect, the present invention provides a pharmaceutical composition comprising an IgG-enriched fraction prepared according to the present invention.

[0043] In one embodiment, the composition contains at least about 80 to 220 grams of IgG per liter of the composition.

[0044] In one embodiment, the pH of the pharmaceutical composition is from about 4.4 to about 4.9. [The present invention 1001] A method for preparing an IgG-enriched fraction from a C1-INH-deficient supernatant fraction containing immunoglobulin G (IgG), comprising: (a) contacting the C1-INH-deficient supernatant fraction with heparin to thereby form a heparin-added fraction; (b) isolating IgG from the heparin-added fraction to thereby form an IgG-enriched fraction The method as described above. [The present invention 1002] The method of the present invention 1001, wherein the supernatant fraction is a supernatant after C1 inhibitor adsorption. [The present invention 1003] The method of the present invention 1001 or 1002, wherein the supernatant fraction is a plasma supernatant. [The present invention 1004] The method of the present invention 1003, wherein the plasma supernatant is C1-INH-deficient cryoprecipitate-free plasma. [The present invention 1005] The method according to any one of the present inventions 1001 to 1004, wherein the supernatant fraction lacks one or more of other blood coagulation factors selected from factors II, VII, IX, and X, or a mixture thereof. [The present invention 1006] The method according to any one of the present inventions 1001 to 1005, wherein the supernatant fraction is concentrated to the protein value of normal plasma before further treatment. [The present invention 1007] The method according to any one of the present inventions 1001 to 1006, wherein the heparin is added in an amount of about 1 to about 20 units per 1 ml of the supernatant fraction. [The present invention 1008] The method of the present invention 1007, wherein the heparin is added in an amount of about 5 units per 1 ml of the supernatant fraction. [The present invention 1009] The method of the present invention 1007, wherein the heparin is added in an amount of about 10 units per 1 ml of the supernatant fraction. [The present invention 1010] Before step (a), removing C1-INH esterase inhibitor (C1-INH) from the cryoprecipitate-free plasma fraction containing C1-INH to thereby form the C1-INH-deficient supernatant fraction The method according to any one of the present inventions 1001 to 1009, further comprising the above step. [The present invention 1011] The method according to any one of the present inventions 1001 to 1010, wherein the IgG-enriched fraction contains at least about 50% of the IgG content found in the supernatant fraction. [The present invention 1012] The method according to any one of the present inventions 1001 to 1011, wherein the purity of IgG in the IgG-enriched fraction is at least 95%. [The present invention 1013] Isolating IgG from the heparin-added fraction in (b) is (i) To obtain Fraction I precipitate and Fraction I supernatant, precipitate the heparin-added fraction at a pH of about 7.0 to 7.5 using about 6% to about 10% ethanol, (ii) To form Fraction II+III precipitate, precipitate IgG from the Fraction I supernatant at a pH of about 6.7 to about 7.3 using about 18% to about 27% alcohol A method according to any one of 1001 to 1012 of the present invention, comprising: [The present invention 1014] Isolating IgG from the heparin-added fraction in b) (i) To form Fraction I+II+III precipitate, precipitate IgG from the heparin-added fraction at a pH of about 6.7 to about 7.3 using about 18% to about 27% alcohol A method according to any one of 1001 to 1012 of the present invention, comprising: [The present invention 1015] (iii) Suspending the precipitate of Fraction II+III or Fraction I+II+III in a suspension buffer, thereby forming an IgG suspension, (iv) Mixing finely divided silicon dioxide (SiO 2 ) with the IgG suspension for at least about 30 minutes, (v) Filtering the IgG suspension, thereby forming a filtrate and a filter cake A method according to 1013 or 1014 of the present invention, further comprising: [The present invention 1016] (vi) Washing the filter cake with a washing buffer having a pH of about 4.9 to about 5.3 and at least 1 filter press dead volume, thereby forming a washing solution, (vii) Combining the filtrate with the washing solution, thereby forming a solution, and treating the solution with a cleaning agent, (viii) Adjusting the pH of the solution in step (vii) to about 7.0 and adding ethanol to a final concentration of about 20% to about 30%, thereby forming a precipitate G precipitate, (ix) Dissolving the precipitate G precipitate in an aqueous solution containing one solvent and / or one or more cleaning agents and holding the solution for at least 60 minutes, (x) Passing the solution through a cation exchange chromatography column and eluting the protein adsorbed on the column with an eluent, (xi) Passing the eluent through an anion exchange chromatography column to produce a flow-through effluent, (x) Passing the effluent through a nanofilter to produce a nanofiltrate, (xi) To produce a first ultrafiltrate, concentrating the nanofiltrate by ultrafiltration; (xii) To produce a diafiltrate, diafiltering the first ultrafiltrate against a diafiltration buffer; (xiii) To produce a second ultrafiltrate having a protein concentration of about 8% (w / v) to about 22% (w / v), concentrating the diafiltrate by ultrafiltration, thereby forming an IgG concentrated fraction; The method of the present invention 1015, further comprising. [The present invention 1017] (iv) Adding SiO to a final concentration of about 0.02 to about 0.10 grams per gram of precipitate of fraction II+III or fraction I+II+III; 2 The method of the present invention 1015 or 1016, comprising. [The present invention 1018] (vi) The method of the present invention 1016 or 1017, comprising washing the filter cake with at least two filter press dead volumes of washing buffer. [The present invention 1019] (x) The method according to any one of the present inventions 1016 to 1018, comprising eluting the protein with at least 35 mM of sodium dihydrogen phosphate dihydrate. [The present invention 1020] (xii) The diafiltration buffer in comprises about 200 mM to about 300 mM of glycine, the method according to any one of the present inventions 1016 to [Error! Reference source not found]. [The present invention 1021] (vii) Treating the solution with one solvent and / or one or more detergents comprises at least one step of virus inactivation or removal, the method according to any one of the present inventions 1016 to 1020. [The present invention 1022] The method of the present invention 1021, wherein the virus inactivation is a solvent / detergent (S / D) virus inactivation step. [The present invention 1023] The method according to any one of the present inventions 1016 to 1022, further comprising an incubation step at a low pH of about 4.0 to about 5.2. [The present invention 1024] The method according to any one of the present inventions 1016 to 1022, further comprising an incubation step at a low pH of about 4.4 to about 4.9. [The present invention 1025] The fraction is a supernatant fraction after C1 inhibitor adsorption containing IgG, and is a defrosted plasma fraction lacking at least about 70% of the total C1-INH present in the defrosted plasma fraction. [The present invention 1026] A pharmaceutical composition comprising an IgG concentrated fraction prepared according to the method of any one of the present inventions 1001 to 1024. [The present invention 1027] The composition is a pharmaceutical composition of the present invention 1026, containing at least about 80 to 220 grams of IgG per liter of the composition. [The present invention 1028] The pH of the pharmaceutical composition is about 4.4 to about 4.9, which is a pharmaceutical composition of the present invention 1026 or 1027.

BEST MODE FOR CARRYING OUT THE INVENTION

[0045] Detailed Description of the Invention A. Introduction Unlike other biopharmaceuticals produced through recombinant expression of DNA vectors within host cell lines, plasma-derived proteins are fractionated from human-provided blood and plasma. Thus, the supply of those formulations cannot be increased simply by increasing production volume. Rather, the levels of commercially available blood products are limited by the available supply of the blood and plasma provided. This dynamic results in a lack of availability of raw human plasma for the manufacture of new plasma-derived blood factors for which there is not yet a well-established commercial market, including complement factor H (CFH) and inter-alpha-trypsin inhibitor protein (IαIp).

[0046] Concerns about the amidolytic content of plasma-derived compositions have highlighted in the art the need for methods to reduce serine proteases (e.g., FXIa and FXIIa) and serine protease zymogens (e.g., FXI and FXII) during the manufacture of IgG and other biopharmaceuticals.

[0047] C1 inhibitor (C1-INH, C1 esterase inhibitor) is the most important physiological inhibitor of plasma kallikrein, factor XIa, and factor XIIa. Deficiency of C1 inhibitor results in the accumulation of those factors in starting materials for the manufacture of commercial IgG therapeutics such as GAMMAGARD® LIQUID (GGL), making it difficult to produce IgG preparations for intravenous administration without increasing the risk of thromboembolic events. Due to the complexity of immunoglobulin production from plasma supernatant after adsorption of C1 inhibitor, called double-deficient cryoprecipitate-poor plasma (DDCPP), native plasma supernatant is not used as a starting material for the manufacture of IgG. Thus, to ensure proper removal of plasma kallikrein, factor XIa, and factor XIIa at low concentrations of C1 inhibitor, a calculated amount of 10,000 IU / L of heparin is added to DDCPP before the alcohol fractionation process is initiated.

[0048] The present disclosure is based in part on the discovery that a C1-INH-deficient plasma supernatant, as well as a supernatant fraction deficient in one or more of the other blood coagulation factors selected from factors II, VII, IX, X, and mixtures thereof, can be used as a starting material for the preparation of an immunoglobulin G (IgG) enriched fraction, thus making available another starting material for the preparation of IgG. Advantageously, the present invention is based at least in part on the unexpected discovery that heparin can be used to increase the reduction of procoagulant activity during the fractionation process.

[0049] To overcome these issues, the inventors developed a process incorporating a purification step, e.g., an initial purification step, of co-precipitating C1-INH-deficient plasma supernatant with heparin, thereby forming a heparin-added fraction, and then isolating IgG from the heparin-added fraction. Thus, heparin-treated C1-INH-deficient plasma supernatant can be used as a starting material for the preparation of immunoglobulin G (IgG) enriched fractions, providing a new starting material for the preparation of IgG.

[0050] In certain embodiments, the present invention provides a method for the manufacture of IVIG with reduced procoagulant activity and amidolytic activity.

[0051] In some embodiments, the present invention provides IgG compositions prepared according to the improved manufacturing methods provided herein. Advantageously, those compositions can be prepared at a lower cost than currently available commercial formulations due to the improved yields afforded by the methods provided herein. Further, those compositions are as pure as, if not purer than, compositions manufactured using commercial methods. Importantly, those compositions are suitable for use in IVIG therapy for immunodeficiency, inflammatory and autoimmune diseases, and acute infections. In one embodiment, the IgG composition is 10% or about 10% IgG for intravenous administration. In another embodiment, the IgG composition is 20% or about 20% for subcutaneous or intramuscular administration.

[0052] In various embodiments, the present invention provides pharmaceutical compositions and formulations of IgG compositions prepared from C1-INH-deficient plasma supernatant as provided herein. In certain embodiments, those compositions and formulations provide improved properties compared to other IVIG compositions currently on the market. For example, in certain embodiments, the compositions and formulations provided herein are stable over a long period of time.

[0053] In an exemplary embodiment, the present invention provides a method of treating immunodeficiency, inflammatory and autoimmune diseases, and acute infections, comprising administration of an IgG composition prepared from C1-INH-deficient plasma supernatant. In various embodiments, the IgG composition is prepared by the methods of the present invention.

[0054] In WO2001046219A2, which describes the use of an anion exchanger at acidic pH (i.e., pH less than 7) to isolate C1-INH, exemplary methods for the production of a C1-INH esterase inhibitor (C1-INH)-containing composition can be found.

[0055] B. Definitions As used herein, the terms "intravenous IgG" or "IVIG therapy" generally refer to a therapeutic method of administering a pharmaceutical composition of IgG immunoglobulin to a patient intravenously, subcutaneously, or intramuscularly to treat conditions such as, for example, immunodeficiency, inflammatory diseases, and autoimmune diseases. The IgG immunoglobulin is typically pooled and prepared from plasma. Whole antibodies or fragments may be used. The IgG immunoglobulin may be formulated at a higher concentration (e.g., higher than 10%) for subcutaneous administration or may be formulated for intramuscular administration. This is particularly common for special IgG preparations that are prepared at a higher titer than the average titer for specific antigens (e.g., Rho D factor, pertussis toxin, tetanus toxin, botulinum toxin, rabies, etc.). For ease of discussion, such IgG compositions formulated for subcutaneous or intramuscular use are also included in the term "IVIG" in this application.

[0056] As used herein, the term "amide-degrading activity" refers to the ability of a polypeptide to catalyze the hydrolysis of at least one peptide bond within another polypeptide. The amide-degrading activity profile for an IgG immunoglobulin composition may be determined by examining it with various chromogenic substrates having different specificities for proteases found in human plasma, including but not limited to PL-1 (broad spectrum), S-2288 (broad spectrum), S-2266 (FXIa, glandular kallikrein), S-2222 (FXa, trypsin), S-2251 (plasmin), and S-2302 (kallikrein, FXIa, and FXIIa). Methods for determining the amide-degrading activity of a composition are known in the art, for example, as described by M. Etscheid et al. (Identification of kallikrein and FXIa as impurities in therapeutic immunoglobulins: implications for the safety and control of intravenous blood products, Vox Sang 2011; the disclosures of which are hereby expressly incorporated by reference in their entirety for all purposes).

[0057] As used herein, the term "antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene or multiple immunoglobulin genes that specifically binds to and recognizes a specimen (antigen), or a fragment thereof. The recognized immunoglobulin genes include numerous immunoglobulin variable region genes along with the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes. The light chains are classified as either kappa or lambda. The heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50 - 70 kD). The N-terminus of each chain defines a variable region consisting of about 100 - 110 or more amino acids that are primarily responsible for antigen recognition. The terms variable light chain (V L ) and variable heavy chain (V H ) refer to the respective light and heavy chains thereof.

[0058] As used herein, the term "ultrafiltration (UF)" encompasses various membrane filtration methods in which a hydrostatic pressure forces a liquid against a semipermeable membrane. High molecular weight suspended solids and solutes are retained while water and low molecular weight solutes pass through the membrane. This separation step is often used to purify and concentrate macromolecular (10 3 ~10 6 Da) solutions, particularly protein solutions. Depending on the size of the molecules they retain, several ultrafiltration membranes are available. Ultrafiltration is typically characterized by a membrane pore size of 1 - 1000 kDa and an operating pressure of 0.01 - 10 bar and is useful for the separation of colloids such as separating proteins from small molecules such as sugars and salts.

[0059] As used herein, the term "diafiltration" is carried out by the same membrane as ultrafiltration and is tangential flow filtration. During diafiltration, buffer is introduced into the recycle tank and filtrate is removed from the unit operation. In processes where the product is present in the retentate (e.g., IgG), diafiltration flushes components from the product pool into the filtrate, thereby exchanging the buffer and reducing the concentration of unwanted species.

[0060] As used herein, the term "about" represents an approximate range from the specified value. In some embodiments, the range is plus or minus 1% to 10% from the specified value. Thus, "about" encompasses plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% from the stated value. For example, the phrase "about 20%" encompasses the range of 18 - 22%.

[0061] As used herein, the term "solvent" encompasses any liquid substance capable of dissolving or dispersing one or more other substances. The solvent may be essentially inorganic, such as water, or it may be an organic liquid such as ethanol, acetone, methyl acetate, ethyl acetate, hexane, petroleum ether, etc. When used in the term "solvent detergent treatment", the solvent represents an organic solvent (e.g., tri - N - butyl phosphate) that is part of a solvent detergent mixture used to inactivate lipid - enveloped viruses in solution.

[0062] As used herein, the term "detergent" is used interchangeably with the term "surfactant" or "surface acting agent". A surfactant is typically an amphiphilic, i.e., an organic compound containing both a hydrophobic group ("tail") and a hydrophilic group ("head"), which render the surfactant soluble in both organic solvents and water. Surfactants can be classified by the presence of groups having a formal charge at their head. Nonionic surfactants have no charged groups at their head, while ionic surfactants have a net charge at their head. Zwitterionic surfactants contain a head with two oppositely charged groups.Some examples of common surfactants include anionic (based on anions of sulfates, sulfonates or carboxylates): perfluorooctanoic acid (PFOA or PFO), perfluorooctane sulfonic acid (PFOS), sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alkyl sulfates, sodium laureth sulfate (also known as sodium lauryl ether sulfate or SLES), alkylbenzene sulfonates; cationic (based on quaternary ammonium cations), cetyltrimethylammonium bromide (CTAB) also known as hexadecyltrimethylammonium bromide, and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT); long-chain fatty acids and their bases: including caprylates, caprylic acid, heptanoic acid, hexanoic acid, heptanoic acid, nonanoic acid, and decanoic acid, etc.; zwitterionic (amphoteric): dodecyl betaine, cocamidopropyl betaine, coco ampho glycinate; nonionic: alkyl poly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) and poly(propylene oxide) (commercially known as Poloxamers or Poloxamines), and alkyl polyglucosides including octyl glucoside, decyl maltoside, fatty alcohols (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates (Tween 20, Tween 80, etc.), Triton detergents, and dodecyldimethylamine oxide.

[0063] As used in this application, the term "spraying" refers to a means of delivering a liquid substance to a system in the form of fine droplets or a mist of the liquid substance, for example, during an alcohol precipitation step such as a precipitation step of modified Cohn Fractionation I or Cohn Fractionation II+III. Spraying may be accomplished by any pressurized device such as a container (e.g., a spray bottle) having a spray head or nozzle, and operates manually or automatically to produce a fine mist from the liquid. Typically, spraying is performed while the system receiving the liquid substance is continuously agitated or otherwise mixed so that the liquid is rapidly and uniformly dispersed within the system.

[0064] As used herein, "cryo-depleted plasma" refers to the supernatant formed after the cold precipitation (cryoprecipitation) of plasma or pooled plasma at a temperature near freezing, e.g., a temperature below about 10°C. In the context of the present invention, plasma may interchangeably refer to whole blood plasma (i.e., plasma separated from whole blood ex vivo) or component plasma (i.e., plasma collected via plasma exchange). Cryoprecipitation is generally performed by thawing a pre-frozen pooled plasma that has already been tested for safety and quality considerations, for example, but fresh plasma may also be used. Thawing is typically performed at a temperature of 6°C or less. After thawing of the plasma frozen at a low temperature is complete, centrifugation is performed at a low temperature (e.g., ≤6°C) to separate the solid cryoprecipitate from the liquid supernatant. Alternatively, the separation step may be performed by filtration instead of centrifugation.

[0065] As used herein, "Cohn pool" refers to the starting material used for fractionation of plasma samples or pooling of plasma samples. Cohn pools include whole plasma, cryo-depleted plasma samples, and pools of cryo-depleted plasma samples, which may or may not have undergone a pretreatment step. In certain embodiments, the Cohn pool is a cryo-depleted plasma sample from which one or more blood factors have been removed in a pretreatment step, such as adsorption to a solid phase (e.g., aluminum hydroxide, finely divided silicon dioxide, etc.) or a chromatography step (e.g., ion exchange or heparin affinity chromatography). To form a Cohn pool, various blood factors including, but not limited to, Factor Eight Inhibitor Bypass Activity (FEIBA), Factor IX complex, Factor VII concentrate, or antithrombin III complex may be isolated from the cryo-depleted plasma sample.

[0066] As used herein, the term "plasma sample" refers to any suitable starting material, such as whole blood plasma or component plasma, or a plasma fraction or plasma supernatant, or a plasma-derived protein preparation. Exemplary "plasma samples" include IgG from plasma or plasma fractions, IgG from cryo-depleted plasma, IgG from adsorption of C-1 esterase inhibitor from cryo-depleted plasma, and IgG from doubly depleted cryo-depleted plasma (DDCPP).

[0067] As used herein, "double depleted cryo-poor plasma (also known as DDCPP / C-1 esterase inhibitor depleted cryo-poor plasma)" refers to the adsorbed supernatant formed after adsorption of C1 inhibitor of cryo-poor plasma at a temperature close to freezing, for example, a temperature below about 8°C. The manufacturing process of GAMMAGARD® LIQUID (Baxter Healthcare Corporation, Westlake Village, CA) employs a modified Cohn-Oncley cold ethanol fractionation procedure to isolate an intermediate immunoglobulin G (IgG) fraction designated as precipitate G (PptG) from frozen human plasma pools. PptG is further purified through subsequent use of weak cation and weak anion exchange chromatography. Downstream purification of PptG includes three dedicated virus removal steps: solvent / detergent treatment, nanofiltration, and incubation at low pH and elevated temperature in the final formulation. The starting material for the ethanol fractionation step may undergo different adsorption steps to obtain intermediates for the purification of coagulation factors and plasma protein inhibitors. The adsorbed supernatant obtained after adsorption of C1 inhibitor in the manufacturing process of CINRYZE® is designated as double depleted cryo-poor plasma (DDCPP).

[0068] As used herein, the term "native or variant native" refers to the use of DDCPP without any adjustment / modification as the starting material, and "variant heparin" refers to the addition of 5000 IU heparin / L DDCPP or 10000 IU heparin / L DDCPP to the starting material. "Variant NaCl" refers to the addition of sodium chloride to increase the conductivity of DDCPP.

[0069] As used herein, the term "C1 inhibitor (C1-inh, C1 esterase inhibitor)" is a protease inhibitor belonging to the serpin superfamily. Its main function is the inhibition of the complement system to prevent spontaneous activation. C1 inhibitor is an acute-phase protein that circulates in the blood at a level of about 0.25 g / L. The level increases approximately two-fold during inflammation. C1 inhibitor irreversibly binds to and inactivates the C1r and C1s proteases within the C1 complex of the classical pathway of complement. The MASP-1 and MASP-2 proteases within the mannose-binding lectin (MBL) complex of the lectin pathway are also inactivated. Thus, C1 inhibitor prevents the proteolytic cleavage of the later complement components C4 and C2 by C1 and MBL. C1 inhibitor is named for its complement-inhibitory activity, but also inhibits proteases of the fibrinolytic, coagulation, and kinin pathways. Note that C1 inhibitor is the most important physiological inhibitor of plasma kallikrein, FXIa, and FXIIa.

[0070] 1. Preparation of C1-INH-deficient supernatant fraction The starting materials used to prepare the IgG enriched fraction are generally composed entirely of the supernatant after C1 inhibitor adsorption or frozen plasma after C1 inhibitor adsorption or non-frozen plasma after C1 inhibitor adsorption. Exemplary samples, e.g., plasma supernatants, are composed of the adsorbed supernatant obtained after adsorption of C1 inhibitor in the manufacturing process of CINRYZE®. The purification process typically begins by thawing pre-frozen pooled plasma, which is preferably already tested for safety and quality considerations. Thawing is typically performed at a temperature of 6°C or lower. After thawing of the frozen plasma at low temperature, centrifugation is performed while cooling (e.g., ≦6°C) to separate the solid frozen precipitate from the liquid supernatant. Alternatively, the separation step is performed by filtration instead of centrifugation. The liquid supernatant (the supernatant after cooling-insoluble proteins have been removed from fresh thawed plasma by centrifugation, also referred to as "decryo plasma") then undergoes one or more adsorption steps to obtain an intermediate for the purification of coagulation factors and plasma protein inhibitors. The adsorbed supernatant obtained after adsorption of C1 inhibitor from decryo plasma is also referred to as double deficient decryo plasma (DDCPP).

[0071] 2. Preparation of the heparin added fraction C1-INH deficient supernatant fractions are generally not considered ideal starting materials for the production of IgG because the lack of C1-INH results in the accumulation of plasma kallikrein, factor XIa, and factor XIIa. To ensure proper removal of these factors with a significantly reduced concentration of C1-INH, a calculated amount of heparin (5000 U / kg DDCPP or 10,000 U / kg DDCPP) is added to the C1-INH deficient supernatant fraction before the alcohol fractionation process is initiated. The final IgG product obtained is shown to contain a residual heparin concentration of less than 1 IU / mL.

[0072] 3. First precipitation event - Modified fraction I The starting material for fraction I was DDCPP (the supernatant after C1 inhibitor adsorption). DDCPP was typically cooled to about 0 ± 2 °C and the pH was adjusted to about 7.0 to about 7.5, preferably about 7.1 to about 7.3, most preferably about 7.2 by the addition of an acid, such as acetic acid. In one embodiment, the pH of the defrosted plasma is adjusted to a pH of about 7.2. Then, while stirring the plasma, pre-cooled ethanol is added to a target concentration of 8% v / v or about 8% v / v. At the same time, the temperature is further lowered to about -2 °C to about +2 °C. In a preferred embodiment, the temperature is lowered to -1.5 °C or about -1.5 °C to precipitate contaminating substances such as α2-macroglobulin, β 1A - and β 1C -globulin, fibrinogen, and factor VIII. Typically, the precipitation event includes a holding time of at least about 1 hour, although shorter or longer holding times may also be employed. The supernatant (supernatant I), which ideally contains most of the IgG content present in the DDCPP, is then collected by centrifugation, filtration, or another suitable method.

[0073] Compared to conventional methods employed as the first fractionation step for defrosted plasma (Cohn et al., supra; Oncley et al., supra), the present invention provides, in some embodiments, a method that results in an improved IgG yield from the supernatant I fraction. In one embodiment, the improved IgG yield is achieved by adding alcohol by spraying. In another embodiment, the improved IgG yield is achieved by adding a pH adjuster by spraying. In yet another embodiment, the improved IgG yield is achieved by adjusting the pH of the solution after the addition of alcohol. In related embodiments, the improved IgG yield is achieved by adjusting the pH of the solution during the addition of alcohol.

[0074] In one particular aspect, the improvement relates to a method of reducing the amount of IgG lost in the precipitate fraction of the first precipitation step. For example, in certain embodiments, the amount of IgG lost in the precipitate fraction of the first precipitation step is reduced as compared to the amount of IgG lost in the first precipitation step of the Cohn fractionation 6 protocol.

[0075] In certain embodiments, process improvement is achieved by adjusting the pH of the solution to between about 7.0 and about 7.5 after the addition of the precipitating alcohol. In other embodiments, the pH of the solution is adjusted to between about 7.1 and about 7.3 after the addition of the precipitating alcohol. In yet other embodiments, the pH of the solution is adjusted to about 7.0 or about 7.1, 7.2, 7.3, 7.4, or 7.5 after the addition of the precipitating alcohol. In a particular embodiment, the pH of the solution is adjusted to about 7.2 after the addition of the precipitating alcohol. Thus, in certain embodiments, the amount of IgG lost in the precipitate fraction of the first precipitation step is reduced as compared to a similar precipitation step where the pH of the solution is adjusted before rather than after the addition of the precipitating alcohol. In one embodiment, the pH is maintained at the desired pH during the precipitation hold or incubation time by continuously adjusting the pH of the solution. In one embodiment, the alcohol is ethanol.

[0076] In other certain embodiments, process improvement is achieved by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying rather than by a flowing addition. Thus, in certain embodiments, the amount of IgG lost in the precipitate fraction of the first precipitation step is reduced as compared to a similar precipitation step where the alcohol and / or the solution used to adjust the pH is introduced by a flowing addition. In one embodiment, the alcohol is ethanol.

[0077] In yet other certain embodiments, improvements are realized by adjusting the pH of the solution to about 7.0 to about 7.5. In preferred embodiments, the pH of the solution is adjusted to about 7.1 to about 7.3. In other embodiments, the pH of the solution is adjusted to about 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 after the addition of the precipitating alcohol, by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying rather than by a flowing addition. In certain embodiments, the pH of the solution is adjusted to about 7.2 after the addition of the precipitating alcohol, by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying rather than by a flowing addition. In one embodiment, the alcohol is ethanol.

[0078] 4. Second precipitation event - Modified Fraction II+III To further increase the IgG content and purity of the fraction, Supernatant I is subjected to a second precipitation step which is the Modified Cohn-Oncley Fraction II+III fraction. Generally, the pH of the solution is adjusted to a pH of about 6.6 to about 6.8. In preferred embodiments, the pH of the solution is adjusted to about 6.7. Then, while stirring the solution, alcohol, preferably ethanol, is added to the solution to a final concentration of about 20% to about 25% (v / v) to precipitate IgG within the fraction. In preferred embodiments, alcohol is added to a final concentration of about 25% (v / v) to precipitate IgG within the fraction. Generally, contaminating substances such as α1-lipoprotein, α1-antitrypsin, Gc-globulin, α 1X -glycoprotein, haptoglobin, ceruloplasmin, transferrin, hemopexin, fraction of Christmas factor, thyroxine-binding globulin, cholinesterase, hypertensinogen, and albumin do not precipitate depending on their state.

[0079] Before or simultaneously with the addition of alcohol, the solution is further cooled to about -7°C to about -9°C. In a preferred embodiment, the solution is cooled to a temperature of about -7°C. After completion of the alcohol addition, the pH of the solution is immediately adjusted to about 6.8 to about 7.0. In a preferred embodiment, the pH of the solution is adjusted to about 6.9. Typically, the precipitation event includes a hold time of at least about 10 hours, although shorter or longer hold times may also be employed. Thereafter, a precipitate (modified fraction II+III) containing ideally at least about 85%, preferably at least about 90%, more preferably at least about 95% of the IgG content present in the defrosted plasma is separated from the supernatant by centrifugation, filtration, or another suitable method and collected. Compared to conventional methods employed as a second fractionation step for defrosted plasma (Cohn et al., supra; Oncley et al., supra), the present invention provides, in some embodiments, a method that results in improved IgG yield within the modified fraction II+III precipitate. In related embodiments, the present invention provides a method that results in reduced loss of IgG within the modified fraction II+III supernatant.

[0080] As compared to conventional methods employed as a second fractionation step for removing cryoprecipitate (Cohn et al., supra; Oncley et al., supra), the present invention provides, in some embodiments, methods that result in improved IgG yields in the modified fraction II+III precipitate. In one embodiment, improvement is achieved by addition of alcohol by spraying. In another embodiment, improvement is achieved by addition of a pH adjuster by spraying. In another embodiment, improvement is achieved by adjusting the pH of the solution after addition of alcohol. In related embodiments, improvement is achieved by adjusting the pH of the solution during addition of alcohol. In another embodiment, improvement is achieved by increasing the concentration of alcohol (e.g., ethanol) up to about 25% (v / v). In another embodiment, improvement is achieved by lowering the temperature of the precipitation step to about -7°C to about -9°C. In a preferred embodiment, improvement is achieved by increasing the concentration of alcohol (e.g., ethanol) up to about 25% (v / v) and lowering the temperature to about -7°C to about -9°C. In comparison, both Cohn et al. and Oncley et al. perform precipitation at -5°C and Oncley et al. use 20% alcohol to reduce the level of contaminants in the precipitate. Advantageously, the methods provided herein allow for maximum IgG yields without high levels of contamination in the final product.

[0081] When the pH of the solution is adjusted to a pH of about 6.9 prior to addition of precipitation alcohol, it has been found that the pH of the solution changes from 6.9 to about 7.4 to about 7.7, due in part to protein precipitation. As the pH of the solution changes away from 6.9, precipitation of IgG becomes less favorable and precipitation of certain contaminants becomes more favorable. Advantageously, the inventors have found that by adjusting the pH of the solution after addition of precipitation alcohol, a higher percentage of IgG is recovered in the fraction II+III precipitate.

[0082] In various embodiments, the improvements realized by the present invention relate to methods in which the amount of IgG lost in the supernatant fraction of the modified fraction II+III precipitation step is reduced when compared to the same method in which the improvements of the present invention are not incorporated. In other words, the proportion of starting IgG present in the fraction II+III precipitate is increased. In certain embodiments, process improvements are realized by adjusting the pH of the solution to between about 6.7 and about 7.1 immediately after or during the addition of the precipitating alcohol. In some embodiments, process improvements are realized by continuously maintaining the pH of the solution between about 6.7 and about 7.1 during the precipitation and / or incubation period. In some embodiments, the pH of the solution is adjusted to between about 6.8 and about 7.0 immediately after or during the addition of the precipitating alcohol, or to a pH of about 6.7, 6.8, 6.9, 7.0, or 7.1 immediately after or during the addition of the precipitating alcohol. In a particular embodiment, the pH of the solution is adjusted to about 6.9 immediately after or during the addition of the precipitating alcohol. In certain embodiments, the pH of the solution is continuously maintained between about 6.8 and about 7.0 during the precipitation incubation period, or continuously maintained at a pH of about 6.9 during the precipitation incubation period. By applying the process parameters of the present invention, in certain embodiments, the amount of IgG lost in the supernatant fraction of a second precipitation step is reduced compared to a similar precipitation step in which the pH of the solution is adjusted before rather than after the addition of the precipitating alcohol, or a similar precipitation step in which the pH of the solution is not maintained throughout the precipitation incubation period. In one embodiment, the pH is maintained at the desired pH during the precipitation holding or incubation time by continuously adjusting the pH of the solution. In one embodiment, the alcohol is ethanol.

[0083] In some embodiments, process improvements are achieved by adding the solution used to adjust the precipitating alcohol and / or pH by spraying rather than by flowing addition. Thus, in certain embodiments, the amount of IgG lost in the supernatant fraction of the second precipitation step is reduced compared to a similar precipitation step in which the solution used to adjust the alcohol and / or pH is introduced in large amounts by flowing addition. In one embodiment, the alcohol is ethanol.

[0084] In another embodiment, process improvements are achieved by performing the precipitation step at a temperature of about -7°C to about -9°C. In one embodiment, the precipitation step is performed at a temperature of about -7°C. In an exemplary embodiment, the precipitation step is performed at a temperature of about -8°C. In various embodiments, the precipitation step is performed at a temperature of about -9°C. In certain embodiments, the alcohol concentration of the precipitation step is about 23% to about 27%. In a preferred embodiment, the alcohol concentration is about 24% to about 26%. In an exemplary embodiment, the alcohol concentration is about 25%. In some embodiments, the alcohol concentration may be 23%, 24%, 25%, 26%, or 27%, or about 23%, 24%, 25%, 26%, or 27%. In an exemplary embodiment, the second precipitation step is performed at a temperature of -7°C or about -7°C with an alcohol concentration of about 25%. In one embodiment, the alcohol is ethanol.

[0085] By increasing the alcohol concentration in the second precipitation from 20% to 25% as used in the above Oncley et al. and decreasing the incubation temperature from -5°C to about -7°C as used in the Cohn and Oncley methods, the surprising effect of a 5% to 6% increase in the IgG content in the modified fraction II+III precipitate is obtained.

[0086] In another embodiment, the pH of the solution is adjusted to about 6.7 to about 7.1, preferably 6.9 or about 6.9, immediately after or during the addition of the precipitating alcohol, the pH of the solution is maintained at about 6.7 to about 7.1, preferably 6.9 or about 6.9, and the pH is continuously adjusted during the precipitation incubation period, and the precipitating alcohol and / or the solution used to adjust the pH are added by spraying rather than by a flowing addition, whereby process improvements are achieved.

[0087] In an exemplary embodiment, the precipitation step is carried out at a temperature of about -7 °C to about -9 °C, for example -7 °C, and IgG is precipitated with an alcohol concentration of about 23% to about 27%, for example 25%, whereby process improvements are achieved. In various embodiments, process improvements are achieved by incorporating all of the improvements of the modified fraction II+III provided above into the process. In an exemplary embodiment, IgG is precipitated at a temperature of -7 °C with 25% ethanol added by spraying, and then the pH of the solution is adjusted to 6.9 after the addition of the precipitating alcohol, whereby process improvements are achieved. In yet another preferred embodiment, the pH of the solution is maintained at 6.9 throughout the precipitation incubation or holding time.

[0088] 5. Extraction of the Modified Fraction II+III Precipitate To solubilize the IgG content of the modified fraction II+III precipitate, a chilled extraction buffer is used to resuspend the fraction II+III precipitate at a ratio of about 1 part precipitate to about 15 parts extraction buffer. For example, other suitable resuspension ratios of about 1:8 to about 1:30, for example about 1:10 to about 1:20, about 1:12 to about 1:18, about 1:13 to about 1:17, about 1:14 to about 1:16 may be used. In certain embodiments, the resuspension ratio may be about 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, or higher.

[0089] Suitable solutions for the extraction of the Modification II+III precipitate generally have a pH of from about 4.0 to about 5.5. In certain embodiments, the solution has a pH of from about 4.5 to about 5.0. In some embodiments, the extraction solution has a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In an exemplary embodiment, the pH of the extraction buffer is about 4.5. In an exemplary embodiment, the pH of the extraction buffer is about 4.7. In an exemplary embodiment, the pH of the extraction buffer is about 4.9. Generally, buffers selected from, for example, acetate, citrate, monobasic phosphate, dibasic phosphate, and mixtures thereof can be used to meet their pH requirements. Suitable buffer concentrations typically range from about 5 to about 100 mM, or from about 10 to about 50 mM, or from about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM of buffer.

[0090] An exemplary extraction buffer has a conductivity of from about 0.5 mS·cm -1 to about 2.0 mS·cm -1 . For example, in certain embodiments, the conductivity of the extraction buffer is about 0.5 mS·cm -1 , or about 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 mS·cm -1 . Those skilled in the art know how to produce an extraction buffer having a suitable conductivity.

[0091] In one particular embodiment, an exemplary extraction buffer may be about 5 mM sodium monophosphate and about 5 mM acetate, having a pH of about 4.5±0.2 and a conductivity of about 0.7 - 0.9 mS / cm.

[0092] Generally, extraction is performed at about 0°C to about 10°C, or about 2°C to about 8°C. In certain embodiments, extraction may be performed at about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. In an exemplary embodiment, extraction is performed at about 2°C to about 10°C. Typically, the extraction step is carried out for about 60 to about 300 minutes, or about 120 to about 240 minutes, or about 150 to about 210 minutes while continuously stirring the suspension. In certain embodiments, the extraction step is carried out for about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or about 300 minutes. In a preferred embodiment, the extraction step is carried out for at least about 160 minutes while continuing the stirring.

[0093] In a method employing an extraction buffer containing 5 mM monobasic sodium phosphate, 5 mM acetate, and 0.051% to 0.06% glacial acetic acid (v / v), a significant increase in the yield in the final IgG composition can be obtained without compromising the purity of the final product. In a preferred embodiment, the fraction II+III precipitate is extracted at a pH of 4.5±0.2 or about 4.5±0.2 with a paste-to-buffer ratio of 1:15 or about 1:15.

[0094] Advantageously, compared to the current manufacturing process for GAMMAGARD® LIQUID (Baxter Healthcare) employing an extraction buffer containing 5 mM monobasic sodium phosphate, 5 mM acetate, and 0.051% glacial acetic acid (v / v), it has been found that by increasing the glacial acetic acid content up to 0.06% (v / v) or about 0.06% (v / v), a significant increase in the yield in the final IgG composition is obtained. Compared to the methods previously employed for the extraction of the precipitate formed by the second precipitation step (GAMMAGARD® LIQUID), the present invention provides, in some embodiments, a method that results in an improved IgG yield within the modified fraction II+III suspension.

[0095] In one embodiment, the improvement relates to a method of reducing the amount of IgG lost in the insoluble fraction of the modified fraction II+III precipitate. In one embodiment, process improvement is achieved by extracting the modified fraction II+III precipitate at a ratio of 1:15 (precipitate to buffer) with a solution containing 5 mM monobasic sodium phosphate, 5 mM acetate, and 0.06% glacial acetic acid (v / v). In another embodiment, improvement is achieved by maintaining the pH of the solution relatively constant during the extraction process. In one embodiment, the pH of the solution is maintained at about 4.1 to about 4.9 during the extraction process. In an exemplary embodiment, the pH of the solution is maintained at about 4.2 to about 4.8 during the extraction process. In some embodiments, the pH of the solution is maintained at about 4.3 to about 4.7 during the extraction process. In various embodiments, the pH of the solution is maintained at about 4.4 to about 4.6 during the extraction process. In some embodiments, the pH of the solution is maintained at 4.5 during the extraction process.

[0096] In an exemplary embodiment, the improvement relates to a method of increasing the amount of IgG solubilized from the fraction II+III precipitate in the fraction II+III dissolution step. In one embodiment, process improvement is achieved by solubilizing the fraction II+III precipitate in a dissolution buffer containing about 600 mL of glacial acetic acid per about 1000 L. In another embodiment, the improvement relates to a method of reducing impurities after the IgG in the fraction II+III precipitate has been solubilized. In one embodiment, process improvement is achieved by mixing finely divided silicon dioxide (SiO2) with the fraction II+III suspension for at least about 30 minutes.

[0097] 6. Pretreatment and Filtration of the Modified Fraction II+III Suspension To remove the undissolved fraction of the modified fraction II+III precipitate (i.e., the modified fraction II+III filter cake), the suspension is typically filtered using depth filtration. Depth filters that can be employed in the methods provided herein include metallic, glass, ceramic, organic (such as diatomaceous earth) depth filters, etc. Examples of suitable filters include, but are not limited to, Cuno 50SA, Cuno 90SA, and Cuno VR06 filters (Cuno). Alternatively, the separation step may be carried out by centrifugation instead of filtration.

[0098] The manufacturing process improvements described above minimize IgG loss in the initial steps of the purification process, but important impurities including PKA activity, amide-degrading activity, and fibrinogen content are much higher when the II+III paste is extracted at pH 4.5 or 4.6 compared to when extraction is carried out at a pH of approximately 4.9 - 5.0.

[0099] It has now been discovered that the purity of the IgG composition can be greatly enhanced by adding a pretreatment step prior to filtration / centrifugation to reduce the impurities extracted in the methods provided herein. In one embodiment, this pretreatment step includes the addition of finely divided silicon dioxide particles (e.g., fumed silica, Aerosil®). In an exemplary embodiment, this treatment is followed by an incubation period of 40 - 80 minutes, during which the suspension is constantly mixed. In certain embodiments, the incubation period is from about 50 to about 70 minutes. In various embodiments, the incubation period is about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 minutes or more. Generally, the treatment is carried out at about 0°C to about 10°C, or about 2°C to about 8°C. In certain embodiments, the treatment may be carried out at about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. In a particular embodiment, the treatment is carried out at about 2°C to about 10°C.

[0100] Fumed silica treatment is exemplified in WO2011150284A2. In this patent application, the fraction II+III precipitate is suspended and divided into two samples, one of which is clarified only with filter aid before filtration, and one of which is treated with fumed silica before the addition of filter aid and filtration. As can be seen in chromatograph and quantification data, the filtrate sample pretreated with fumed silica has a much higher IgG purity than the sample treated with filter aid only.

[0101] In certain embodiments, fumed silica is added at a concentration of about 20 g / kg of II+III paste to about 100 g / kg of II+III paste (e.g., for the modified fraction II+III precipitate extracted at a ratio of 1:15, fumed silica should be added at a concentration of about 20 g / 16 kg of II+III suspension to about 100 g / 16 kg of II+III suspension, or at a final concentration of about 0.125% (w / w) to about 0.625% (w / w)). In certain embodiments, fumed silica may be added at a concentration of about 20 g / kg of II+III paste, or about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / kg of II+III paste. In one particular embodiment, fumed silica (e.g., Aerosil 380 or its equivalent) is added to the modified fraction II+III suspension up to a final concentration of about 40 g / 16 kg of II+III. Mixing is carried out at about 2°C to about 8°C for at least about 50 to about 70 minutes.

[0102] In certain embodiments, SiO2 is added to the IgG composition at a concentration of from about 0.01 g / g protein to about 10 g / g protein. In another embodiment, SiO2 is added to the IgG composition at a concentration of from about 0.01 g / g protein to about 5 g / g protein. In another embodiment, SiO2 is added to the IgG composition at a concentration of from about 0.02 g / g protein to about 4 g / g protein. In one embodiment, SiO2 is added at a final concentration of at least 0.1 g per gram of total protein. In another specific embodiment, fumed silica is added at a concentration of at least 0.2 g per gram of total protein. In another specific embodiment, fumed silica is added at a concentration of at least 0.25 g per gram of total protein. In other specific embodiments, fumed silica is added at a concentration of at least 1 g per gram of total protein. In another specific embodiment, fumed silica is added at a concentration of at least 2 g per gram of total protein. In another specific embodiment, fumed silica is added at a concentration of at least 2.5 g per gram of total protein. In yet other specific embodiments, finely divided silicon dioxide is added at a concentration of at least 0.01 g / g total protein, or at least 0.02 g, 0.03 g, 0.04 g, 0.05 g, 0.06 g, 0.07 g, 0.08 g, 0.09 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1.0 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g, 5.0 g, 5.5 g, 6.0 g, 6.5 g, 7.0 g, 7.5 g, 8.0 g, 8.5 g, 9.0 g, 9.5 g, 10.0 g, or higher per gram of total protein.

[0103] In certain embodiments, a filter aid, such as Celpure C300 (Celpure) or Hyflo-Super-Cel (World Minerals), is added after silicon dioxide treatment to facilitate depth filtration. The filter aid may be added at a final concentration of from about 0.01 kg / kg of II+III paste to about 1.0 kg / kg of II+III paste, or from about 0.02 kg / kg of II+III paste to about 0.8 kg / kg of II+III paste, or from about 0.03 kg / kg of II+III paste to about 0.7 kg / kg of II+III paste. In other embodiments, the filter aid may be added at a final concentration of from about 0.01 kg / kg of II+III paste to about 0.07 kg / kg of II+III paste, or from about 0.02 kg / kg of II+III paste to about 0.06 kg / kg of II+III paste, or from about 0.03 kg / kg of II+III paste to about 0.05 kg / kg of II+III paste. In certain embodiments, the filter aid is added at a final concentration of about 0.01 kg / kg of II+III paste, or about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 kg / kg of II+III paste.

[0104] In prior methods of purifying IgG, a significant proportion of IgG has been lost during the filtration steps within the process. It has been found that the standard method of post-filtration washing, which used 1.8 dead volumes of suspension buffer to clean the filter press frames and lines, was insufficient for maximum recovery of IgG at this step. Surprisingly, it has been found that at least 3.0 dead volumes, such as 3.6 dead volumes, of suspension buffer was useful for efficient recovery of IgG in the modified fraction II+III clarified suspension. In certain embodiments, the filter press is washed with any suitable suspension buffer. In an exemplary embodiment, the wash buffer comprises, for example, 5 mM sodium monophosphate, 5 mM acetate, and 0.015% glacial acetic acid (v / v).

[0105] In one embodiment, the improvement relates to a method of reducing the amount of IgG lost during the fraction II+III suspension filtration step. In one embodiment, process improvement is achieved by backwashing the filter with at least about 3.6 bed volumes of a lysis buffer containing 150 mL of glacial acetic acid per 1000 L. In one embodiment, the pH of the backwash extraction buffer is from about 4.6 to about 5.3. In a preferred embodiment, the pH of the backwash buffer is from about 4.7 to about 5.2. In another preferred embodiment, the pH of the backwash buffer is from about 4.8 to about 5.1. In yet another preferred embodiment, the pH of the backwash buffer is from about 4.9 to about 5.0.

[0106] Compared to the methods previously employed for clarification of the suspension formed from the second precipitation step, the present invention provides, in some embodiments, a method that results in improved IgG yield and purity in the clarified fraction II+III suspension. In one aspect, the improvement relates to a method of reducing the amount of IgG lost within the modified fraction II+III filter cake. In another aspect, the improvement relates to a method of reducing the amount of impurities found within the clarified fraction II+III suspension.

[0107] In one embodiment, process improvements are achieved by including fumed silica treatment prior to filtration or centrifugal clarification of the Fraction II+III suspension. In certain embodiments, the fumed silica treatment includes the addition of from about 0.01 kg / kg of II+III paste to about 0.07 kg / kg of II+III paste, or from about 0.02 kg / kg of II+III paste to about 0.06 kg / kg of II+III paste, or from about 0.03 kg / kg of II+III paste to about 0.05 kg / kg of II+III paste, or about 0.02 kg / kg of II+III paste, 0.03 kg / kg of II+III paste, 0.04 kg / kg of II+III paste, 0.05 kg / kg of II+III paste, 0.06 kg / kg of II+III paste, 0.07 kg / kg of II+III paste, 0.08 kg / kg of II+III paste, 0.09 kg / kg of II+III paste, or 0.1 kg / kg of II+III paste, and the mixture is incubated at a temperature of from about 2°C to about 8°C for from about 50 minutes to about 70 minutes, or for 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 minutes or more. In another embodiment, process improvements are achieved by including fumed silica treatment that reduces the levels of residual fibrinogen, amidolytic activity, and / or prekallikrein activator activity. In certain embodiments, process improvements are achieved by including fumed silica treatment that reduces the levels of FXI, FXIa, FXII, and FXIIa in the immunoglobulin preparation.

[0108] In another embodiment, process improvements are realized by washing the depth filter with a filter dead volume of about 3 to about 5 volumes after completion of the modified fraction II+III suspension filtration step. In certain embodiments, the filter is washed with a filter dead volume of about 3.5 to about 4.5 volumes, or at least about 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 volumes. In a particular embodiment, the filter press is washed with at least about 3.6 dead volumes of suspension buffer.

[0109] 7. Detergent Treatment To remove further contaminants from the modified fraction II+III filtrate, the sample is then subjected to a detergent treatment. Methods for the detergent treatment of plasma-derived fractions are well known in the art. In general, any standard non-ionic detergent treatment may be used in conjunction with the methods provided herein. For example, an exemplary protocol for the detergent treatment is provided below.

[0110] Briefly summarized, in an exemplary embodiment, a detergent, such as polysorbate 80, is added to the modified fraction II+III filtrate with stirring at a final concentration of about 0.2% (w / v), and the sample is incubated at a temperature of about 2°C to about 8°C for at least about 30 minutes. Sodium citrate anhydrous is then mixed with the solution at a final concentration of about 8 g / L, and the sample is incubated for an additional 30 minutes with continuous stirring at a temperature of about 2 - 8°C.

[0111] In certain embodiments, any suitable non-ionic detergent is used. Examples of suitable non-ionic detergents include, but are not limited to, octyl glucoside, digitonin, C12E8, Lubrol, Triton X-100, Nonidet P-40, Tween-20 (i.e., polysorbate 20), Tween-80 (i.e., polysorbate 80), alkyl poly(ethylene oxide), Brij detergents, alkylphenol poly(ethylene oxide), poloxamer, octyl glucoside, and decyl maltoside, among others.

[0112] In one embodiment, process improvements are achieved by adding detergent reagents (e.g., polysorbate 80 and sodium citrate anhydrous) by spraying rather than by flowing addition. In other embodiments, the detergent reagent may be added as a solid to the modified fraction II+III filtrate and the sample may be mixed to ensure rapid dispersion of the additive. In certain embodiments, it is preferred to add the solid reagent by dispersing the solid over the non-localized surface area of the filtrate so that local over-concentration, such as in flowing addition, does not occur.

[0113] 8. The Third Precipitation Event - Precipitate G In an exemplary embodiment, a third precipitation is performed at a concentration of 25% alcohol to remove some residual small proteins, such as albumin and transferrin. Briefly summarized, the pH of the detergent-treated II+III filtrate is adjusted to about 6.8 to about 7.2, such as about 6.9 to about 7.1, such as about 7.0, with a suitable pH adjustment solution (e.g., 1M sodium hydroxide or 1M acetic acid). Then, chilled alcohol is added to the solution to a final concentration of about 25% (v / v), and the mixture is incubated at about -6°C to about -10°C with stirring for at least 1 hour to form a third precipitate (i.e., precipitate G). In one embodiment, the mixture is incubated for at least 2 hours, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more. In a preferred embodiment, the mixture is incubated for at least 2 hours. In an exemplary embodiment, the mixture is incubated for at least 4 hours. In some embodiments, the mixture is incubated for at least 8 hours.

[0114] In one embodiment, the process improvement of the present invention relates to a method of reducing the amount of IgG lost in the supernatant fraction of the third precipitation step. In certain embodiments, the process improvement is achieved by adjusting the pH of the solution to about 6.8 to about 7.2 immediately after or during the addition of the precipitating alcohol. In another embodiment, the process improvement is achieved by continuously maintaining the pH of the solution at about 6.8 to about 7.2 during the precipitation incubation period. In some embodiments, the pH of the solution is adjusted to about 6.9 to about 7.1 immediately after or during the addition of the precipitating alcohol, or to a pH of about 6.8, 6.9, 7.0, 7.1, or 7.2 immediately after or during the addition of the precipitating alcohol. In a particular embodiment, the pH of the solution is adjusted to about 7.0 immediately after or during the addition of the precipitating alcohol. In certain embodiments, the pH of the solution is continuously maintained at about 6.9 to about 7.1 during the precipitation incubation period, or continuously maintained at a pH of about 7.0 during the precipitation incubation period. According to the improved method, in certain embodiments, the amount of IgG lost in the supernatant fraction of the third precipitation step is reduced as compared to a similar precipitation step in which the pH of the solution is adjusted before rather than after the addition of the precipitating alcohol, or a similar precipitation step in which the pH of the solution is not adjusted and maintained throughout the precipitation incubation period. In one embodiment, the pH is maintained at the desired pH during the precipitation hold or incubation time by continuously adjusting the pH of the solution. In one embodiment, the alcohol is ethanol.

[0115] In some embodiments, the process improvement is achieved by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying rather than by a large volume of flowing addition. Thus, in certain embodiments, the amount of IgG lost in the supernatant fraction of the third precipitation step is reduced as compared to a similar precipitation step in which the alcohol and / or the solution used to adjust the pH is introduced by flowing addition. In one embodiment, the alcohol is ethanol.

[0116] 9. Suspension and Filtration of Precipitate G (PptG) To solubilize the IgG contained in Precipitate G, a cold extraction buffer is used to resuspend the PptG. Briefly summarized, to achieve an AU value of about 40 - 95 280-320 the Precipitate G is dissolved 1:3.5 in Water for Injection (WFI) at about 0°C to about 8°C. The final pH of the solution, which is then stirred for at least 2 hours, is adjusted to about 5.2 ± 0.2. In one embodiment, this pH adjustment is carried out with 1M acetic acid. To increase the solubility of the IgG, the conductivity of the suspension is increased to about 2.5 - about 6.0 mS / cm. In one embodiment, the conductivity is increased by the addition of sodium chloride. The suspended PptG solution is then filtered through a suitable depth filter having a nominal pore size of about 0.1μm to about 0.4μm to remove any undissolved particles. In one embodiment, the nominal pore size of the depth filter is about 0.2μm (e.g., Cuno VR06 filter or its equivalent) to obtain a clarified filtrate. In another embodiment, the suspended PptG solution is centrifuged to recover a clarified filtrate. A post-wash of the filter is performed using a sodium chloride solution having a conductivity of about 2.5 - about 6.0 mS / cm. Typically, a suitable solution for the extraction of Precipitate G contains WFI and a low conductivity buffer. In one embodiment, the low conductivity buffer has a conductivity of less than about 10 mS / cm. In a preferred embodiment, the low conductivity buffer has a conductivity of less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mS / cm. In a preferred embodiment, the low conductivity buffer has a conductivity of less than about 6 mS / cm. In another preferred embodiment, the low conductivity buffer has a conductivity of less than about 4 mS / cm. In another preferred embodiment, the low conductivity buffer has a conductivity of less than about 2 mS / cm.

[0117] 10. Solvent Detergent Treatment To inactivate the various viral contaminants that may be present in the plasma-derived product, the clarified PptG filtrate is then subjected to solvent detergent (S / D) treatment. Methods for detergent treatment of plasma-derived fractions are known in the art (see, for example, Pelletier JP et al., Best Pract Res Clin Haematol. 2006;19(1):205-42 for a review). In general, any standard S / D treatment may be used in conjunction with the methods provided herein. An exemplary protocol for S / D treatment is provided below.

[0118] Briefly, Triton X-100, Tween-20, and tri(n-butyl) phosphate (TNBP) are added to the clarified PptG filtrate at final concentrations of about 1.0%, 0.3%, and 0.3%, respectively. The mixture is then stirred at a temperature of about 18 °C to about 25 °C for at least about 1 hour.

[0119] In one embodiment, process improvements are achieved by adding the S / D reagents (e.g., Triton X-100, Tween-20, and TNBP) by spraying rather than by a large volume of flowing addition. In other embodiments, the detergent reagent may be added as a solid to the clarified PptG filtrate that is mixed to ensure rapid dispersion of the S / D components. In certain embodiments, it is preferred to add the solid reagent by dispersing the solid over the non-localized surface area of the filtrate so that local over-concentration does not occur, such as in flowing addition.

[0120] 11. Ion Exchange Chromatography For further purifying and concentrating IgG from the S / D-treated PptG filtrate, cation exchange and / or anion exchange chromatography may be employed. Methods for purifying and concentrating IgG using ion exchange chromatography are known in the art. For example, U.S. Patent No. 5,886,154 describes a method in which the fraction II+III precipitate is extracted at a low pH (about 3.8 to 4.5), followed by precipitation of IgG using caprylic acid and finally performing two anion exchange chromatography steps. U.S. Patent No. 6,069,236 describes a chromatography-based IgG purification scheme that does not rely at all on alcohol precipitation. International Publication No. WO2005 / 073252 describes an IgG purification method involving extraction of the fraction II+III precipitate, caprylic acid treatment, PEG treatment, and a single anion exchange chromatography step. U.S. Patent No. 7,186,410 describes an IgG purification method involving extraction of either the fraction I+II+III or fraction II precipitate, followed by a single anion exchange step performed at an alkaline pH. U.S. Patent No. 7,553,938 describes a method involving extraction of either the fraction I+II+III or fraction II+III precipitate, caprylic acid treatment, and either one or two anion exchange chromatography steps. U.S. Patent No. 6,093,324 describes a purification method involving the use of a macroporous anion exchange resin performed at a pH of about 6.0 to about 6.6. U.S. Patent No. 6,835,379 describes a purification method that relies on cation exchange chromatography without an alcohol fractionation. The disclosures of the above publications are hereby incorporated by reference in their entirety for all purposes.

[0121] In one embodiment of the method of the present invention, the S / D-treated PptG filtrate may be subjected to both cation exchange chromatography and anion exchange chromatography. For example, in one embodiment, the S / D-treated PptG filtrate passes through a cation exchange column that binds IgG in solution. The S / D reagent may then be washed away from the absorbed IgG, and the absorbed IgG is then eluted from the column with a high pH elution buffer having a pH of about 8.0 to 9.0. In this way, the cation exchange chromatography step may be used to remove the S / D reagent from the preparation, concentrate the solution containing IgG, or both. In certain embodiments, the pH elution buffer may have a pH of about 8.2 to about 8.8, or about 8.4 to about 8.6, or a pH of about 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In a preferred embodiment, the pH of the elution buffer is about 8.5 ± 0.1.

[0122] In certain embodiments, the eluate from the cation exchange column may be adjusted to a lower pH, for example, about 5.5 to about 6.5, and diluted with a suitable buffer, resulting in a reduction in the conductivity of the solution. In certain embodiments, the pH of the cation exchange eluate may be adjusted to a pH of about 5.7 to about 6.3, or about 5.9 to about 6.1, or a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In a preferred embodiment, the pH of the eluate is adjusted to a pH of about 6.0 ± 0.1. The eluate is then loaded onto an anion exchange column, which binds some of the contaminating substances contained in the preparation. The column flow-through fraction containing the IgG fraction is collected during column loading and washing. In certain embodiments, the ion exchange chromatography step of the present invention may be performed in column mode, batch mode, or a combination of the two.

[0123] In certain embodiments, process improvements are achieved by adding a solution used to adjust the pH by spraying rather than by a large amount of flowing addition.

[0124] 12. Nanofiltration and Ultrafiltration / Diafiltration To further reduce the viral load of the IgG compositions provided herein, the anion exchange column effluent is nanofiltrated in some embodiments using a suitable nanofiltration device. In certain embodiments, the nanofiltration device has an average pore size of from about 15 nm to about 200 nm. Examples of nanofilters suitable for this use include, but are not limited to, DVD, DV 50, DV 20 (Pall), Viresolve NFP, Viresolve NFR (Millipore), Planova 15N, 20N, 35N, and 75N (Planova). In certain embodiments, the nanofilter may have an average pore size of from about 15 nm to about 72 nm, or from about 19 nm to about 35 nm, or 15 nm, 19 nm, 35 nm, or 72 nm. In a preferred embodiment, the nanofilter has an average pore size of about 35 nm, such as the Asahi PLANOVA 35N filter or its equivalent.

[0125] Optionally, ultrafiltration / diafiltration may be performed to further concentrate the nanofiltrate. In one embodiment, an open channel membrane is used, along with specially designed post-washing and formulation near the end of the production process, to increase the protein concentration (200 mg / mL) of the resulting IgG composition by about two-fold compared to current IVIGs (e.g., GAMMAGARD® LIQUID) without affecting yield and storage stability. Most commercially available ultrafiltration membranes cannot reach a concentration of 200 mg / mL of IgG without significant protein loss. Those membranes are blocked prematurely and thus it is difficult to achieve proper post-washing. Therefore, an open channel membrane configuration needs to be used. Even with an open channel membrane, a specially designed post-washing procedure needs to be used to obtain the required concentration without significant protein loss (less than 2% loss). Even more surprisingly, the fact that the high protein concentration of 200 mg / mL does not reduce the virus inactivation ability of the low pH storage step.

[0126] Following nanofiltration, the filtrate may be further concentrated by ultrafiltration / diafiltration. In one embodiment, the nanofiltered solution is concentrated by ultrafiltration to a protein concentration (w / v) of from about 2% to about 10%. In certain embodiments, the ultrafiltration is performed in a cassette with an open-channel screen and the ultrafiltration membrane has a nominal molecular weight cut-off (NMWCO) of less than about 100 kDa, or less than about 90, 80, 70, 60, 50, 40, or 30 kDa. In a preferred embodiment, the ultrafiltration membrane has an NMWCO of 50 kDa or less.

[0127] Once the ultrafiltration step is complete, the concentrate may be further concentrated via diafiltration against a solution suitable for intravenous or intramuscular administration. In certain embodiments, the diafiltration solution may contain a stabilizer and / or a buffer. In a preferred embodiment, the stabilizer and buffer are present at a suitable concentration, for example, from about 0.20 M to about 0.30 M, or from about 0.22 M to about 0.28 M, or from 0.24 M to about 0.26 mM, or a concentration of glycine of about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In a preferred embodiment, the diafiltration buffer contains 0.25 M or about 0.25 M glycine.

[0128] Typically, the minimum exchange volume is at least 3 times the original concentrate volume, or at least about 4, 5, 6, 7, 8, or 9 times or more the original concentrate volume. The IgG solution is concentrated to a final protein concentration of about 5% to about 25% (w / v), or about 6% to about 18% (w / v), or about 7% to about 16% (w / v), or about 8% to about 14% (w / v), or about 9% to about 12%, or to a final concentration of about 5%, or 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% or more. In one embodiment, a final protein concentration of at least about 23% is achieved without adding a post-wash fraction to the concentrated solution. In another embodiment, a final protein concentration of at least about 24% is achieved without adding a post-wash fraction to the concentrated solution. A final protein concentration of at least about 25% is achieved without adding a post-wash fraction to the concentrated solution. Typically, at the end of the concentration step, the pH of the solution is about 4.6 to 5.1.

[0129] In an exemplary embodiment, the pH of the IgG composition is adjusted to about 4.5 prior to ultrafiltration. The solution is concentrated to a protein concentration of 5 ± 2% w / v through ultrafiltration. The UF membrane has a nominal molecular weight cut-off (NMWCO) of 50,000 Daltons or less (Millipore Pellicon Polyethersulfone membrane). The concentrate is diafiltered against 10 volumes of a 0.25 M glycine solution, pH 4.5 ± 0.2. Through the ultra-diafiltration operation, the solution is maintained at a temperature of about 2°C to about 8°C. After diafiltration, the solution is concentrated to a protein concentration of at least 11% (w / v).

[0130] 13. Formulation When the diafiltration step is completed, the protein concentration of the solution is adjusted with the diafiltration buffer to a final concentration of about 5% to about 20% (w / v), or about 6% to about 18% (w / v), or about 7% to about 16% (w / v), or about 8% to about 14% (w / v), or about 9% to about 12%, or a final concentration of about 5%, or 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In an exemplary embodiment, the final protein concentration of the solution is about 9% to about 11%, for example, 10%.

[0131] In various embodiments, the formulated bulk solution is further sterilized by filtration through a membrane filter having an absolute pore size of about 0.22 microns or less, for example, about 0.2 microns. The solution is optionally aseptically dispensed into the final container for proper sealing, along with the samples to be tested.

[0132] In one embodiment, the IgG composition is further adjusted to a concentration of about 10.2 ± 0.2% (w / v) with the diafiltration buffer. The pH is adjusted to about 4.4 to about 4.9 if necessary. Finally, the solution is sterile filtered and incubated at about 30 °C for 3 weeks.

[0133] 14. Method of treatment As routinely practiced in modern medicine, sterile preparations of concentrated immunoglobulins (particularly, IgG) are used to treat three main classes of medical conditions: immunodeficiencies, inflammatory and autoimmune diseases, and acute infectious diseases. Those IgG preparations may also be useful for treating multiple sclerosis (particularly, relapsing-remitting multiple sclerosis or RRMS), Alzheimer's disease, and Parkinson's disease. The purified IgG preparations of the present invention are suitable for those purposes as well as other clinically acceptable uses of IgG preparations.

[0134] The FDA has approved the use of IVIG to treat a variety of indications, including allogeneic bone marrow transplantation, chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP), pediatric HIV, primary immunodeficiency, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP), and kidney transplantation in highly antibody recipients or ABO-incompatible donors. In certain embodiments, the IVIG compositions provided herein are useful for the treatment or management of those diseases and conditions.

[0135] Off-label uses of IVIG are commonly provided to patients for the treatment or management of a variety of indications such as chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves ophthalmopathy, Guillain-Barré 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, kidney transplant rejection, spontaneous abortion / miscarriage, stiff person syndrome, opsoclonus myoclonus, severe sepsis and septic shock in critically ill adults, toxic epidermal necrolysis, chronic lymphocytic leukemia, multiple myeloma, X-linked agammaglobulinemia, and hypogammaglobulinemia. In certain embodiments, the IVIG compositions provided herein are useful for the treatment or management of those diseases and conditions.

[0136] Finally, the experimental use of IVIG for the treatment or management of diseases including primary immunodeficiency, RRMS, Alzheimer's disease, and Parkinson's disease has been proposed (U.S. Patent Application Publication No. 2009 / 0148463, which is hereby incorporated by reference in its entirety for all purposes). In certain embodiments, the IVIG compositions provided herein are useful for the treatment or management of primary immunodeficiency, RRMS, Alzheimer's disease, or Parkinson's disease. In certain embodiments, including daily administration, the effective amount to be administered to a subject can be determined by a physician taking into account age, weight, disease severity, route of administration (e.g., intravenous vs. subcutaneous), and individual differences in response to the therapy. In certain embodiments, the immunoglobulin preparation of the present invention can be administered to a subject at about 5 mg / kg to about 2000 mg / kg per day. In additional embodiments, the immunoglobulin preparation can be administered in an amount of at least about 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, or at least 50 mg / kg. In additional embodiments, the immunoglobulin preparation can be administered to a subject at a dose of up to about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 400 mg / kg per day. In other embodiments, the dose of the immunoglobulin preparation can be greater or less. Further, the immunoglobulin preparation can be administered in more than one dose per day. A clinician skilled in the diseases treated by the IgG preparation can determine the appropriate dose for a patient according to standards known in the art.

[0137] According to the present invention, the time required to complete a treatment course can be determined by a physician and can range from a short period of one day to over one month. In certain embodiments, the treatment course can be from 1 to 6 months.

[0138] An effective amount of the IVIG preparation is administered to the subject by intravenous means. The term "effective amount" refers to the amount of the IVIG preparation that results in an improvement or recovery of a disease or condition in the subject. The effective amount to be administered to the subject can be determined by a physician taking into account age, body weight, the disease or condition being treated, the severity of the disease, and individual differences in response to the therapy. In certain embodiments, the IVIG preparation may be administered to the subject at a dosage of about 5 mg / kg to about 2000 mg / kg per administration. In certain embodiments, the dosage may be at least about 5 mg / kg, or at least about 10 mg / kg, or at least about 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 350 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 650 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 850 mg / kg, 900 mg / kg, 950 mg / kg, 1000 mg / kg, 1100 mg / kg, 1200 mg / kg, 1300 mg / kg, 1400 mg / kg, 1500 mg / kg, 1600 mg / kg, 1700 mg / kg, 1800 mg / kg, 1900 mg / kg, or at least about 2000 mg / kg.

[0139] The dosage and frequency of IVIG treatment depend on several factors, particularly the disease or condition being treated and the severity of the disease or condition in the patient. Generally, for primary immunodeficiency, a dose of about 100 mg / kg body weight to about 400 mg / kg body weight is administered every about 3 to 4 weeks. For neurological and autoimmune diseases, up to 2 g / kg body weight is administered once a month for a 5-day course for 3 to 6 months. This is generally complemented by maintenance therapy that includes administration of about 100 mg / kg body weight to about 400 mg / kg body weight about once every 3 to 4 weeks. Generally, patients receive dosing or treatment about once every about 14 to 35 days, or about once every about 21 to 28 days. The frequency of treatment depends on several factors, particularly the disease or condition being treated and the severity of the disease or condition in the patient.

[0140] In a preferred embodiment, there is provided a method of treating immunodeficiency, autoimmune disease, or acute infection in a human in need thereof, the method comprising administering an IVIG pharmaceutical composition of the present invention. In related embodiments, the present invention provides an IVIG composition manufactured according to the methods provided herein for the treatment of immunodeficiency, autoimmune disease, or acute infection in a human in need thereof.

[0141] In certain embodiments, the immunodeficiency, autoimmune disease, or acute infectious disease is selected from allogeneic bone marrow transplantation, chronic lymphocytic leukemia, idiopathic thrombocytopenic purpura (ITP), pediatric HIV, primary immunodeficiency, Kawasaki disease, chronic inflammatory demyelinating polyneuropathy (CIDP), kidney transplantation with high antibody recipient or ABO-incompatible donor, chronic fatigue syndrome, Clostridium difficile colitis, dermatomyositis and polymyositis, Graves ophthalmopathy, Guillain-Barré 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, kidney transplant rejection, spontaneous abortion / miscarriage, stiff person syndrome, opsoclonus myoclonus, severe sepsis and septic shock in critically ill adults, toxic epidermal necrolysis, chronic lymphocytic leukemia, multiple myeloma, X-linked agammaglobulinemia, hypogammaglobulinemia, primary immunodeficiency, RRMS, Alzheimer's disease, and Parkinson's disease.

[0142] 15. Pharmaceutical Composition In another aspect, the present invention provides pharmaceutical compositions and formulations comprising purified IgG prepared by the methods provided herein. Generally, IgG pharmaceutical compositions and formulations prepared by the novel methods described herein have high IgG content and purity. For example, the IgG pharmaceutical compositions and formulations provided herein can have a protein concentration of at least about 7% (w / v) and an IgG content of greater than about 95% purity. Those high-purity IgG pharmaceutical compositions and formulations are suitable for therapeutic administration, for example, for IVIG therapy. In a preferred embodiment, the IgG pharmaceutical composition is formulated for intravenous administration (e.g., IVIG therapy).

[0143] In one embodiment, the pharmaceutical composition provided herein is prepared by formulating an aqueous IgG composition isolated using the methods provided herein. Generally, the formulated composition will be subjected to at least one, preferably at least two, and most preferably three steps of virus inactivation or removal. Non-limiting examples of steps of virus inactivation or removal that can be used with the methods provided herein include solvent detergent treatment (Horowitz et al., Blood Coagul Fibrinolysis 1994(5 Suppl 3):S21-S28 and Kreil et al., Transfusion 2003(43):1023-1028, which are hereby incorporated by reference in their entirety for all purposes), nanofiltration (Hamamoto et al., Vox Sang 1989(56)230-236 and Yuasa et al., J Gen Virol.1991(72(pt8)):2021-2024, which are hereby incorporated by reference in their entirety for all purposes), and low pH incubation at elevated temperature (Kempf et al., Transfusion 1991(31)423-427 and Louie et al., Biologicals 1994(22):13-19).

[0144] In certain embodiments, pharmaceutical formulations are provided having an IgG content of from about 80 g / L to about 220 g / L of IgG. Generally, those IVIG formulations are prepared by isolating the IgG composition from plasma using the methods described herein, concentrating the composition, and formulating the concentrated composition in a solution appropriate for intravenous administration. The IgG composition may be concentrated using any suitable method known to those of skill in the art. In one embodiment, the composition is concentrated by ultrafiltration / diafiltration. In some embodiments, the ultrafiltration device used to concentrate the composition employs an ultrafiltration membrane having a nominal molecular weight cut-off (NMWCO) of less than about 100 kDa, or less than about 90, 80, 70, 60, 50, 40, or 30 kDa. In a preferred embodiment, the ultrafiltration membrane has an NMWCO of 50 kDa or less. Buffer exchange can be accomplished using any suitable technique known to those of skill in the art. In certain embodiments, buffer exchange is accomplished by diafiltration.

[0145] In one particular embodiment, a pharmaceutical composition of IgG is provided, the IgG composition being purified from a C1-INH deficient supernatant fraction comprising IgG, the method comprising (a) contacting the C1-INH deficient supernatant fraction with heparin, thereby forming a heparin-added fraction; and (b) isolating IgG from the heparin-added fraction, thereby forming an IgG-enriched fraction.

[0146] ​In certain embodiments, a pharmaceutical composition of IgG is provided, and the IgG composition comprises: (a) in a first precipitation step, precipitating a heparin-containing fraction with about 6% to about 10% ethanol at a pH of about 7.0 to 7.5 to obtain a first precipitate and a first supernatant; (b) adjusting the ethanol concentration of the heparin-containing fraction from step (a) to about 25% (v / v) at a temperature of about -5°C to about -9°C, thereby forming a mixture; (c) separating the liquid and the precipitate from the mixture of step (b); (d) resuspending the precipitate of step (c) with a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted with 600 ml of glacial acetic acid per 1000 L of the buffer, thereby forming a suspension; (e) mixing finely divided silicon dioxide (SiO2) with the suspension from step (d) for at least about 30 minutes; (f) filtering the suspension with a filter press, thereby forming a filtrate; (g) washing the filter press with a buffer containing phosphate and acetate at least three filter press dead volumes, wherein the pH of the buffer is adjusted with 150 ml of glacial acetic acid per 1000 L of the buffer, thereby forming a washing solution; (h) combining the filtrate of step (f) with the washing solution of step (g), thereby forming a solution, and treating the solution with a detergent; (i) adjusting the pH of the solution of step (h) to about 7.Adjust to 0, add ethanol to a final concentration of about 25%, thereby forming a precipitate, and (j) separating the liquid and the precipitate from the mixture of step (i), and (k) dissolving the precipitate in an aqueous solution containing a solvent or a detergent and holding the solution for at least 60 minutes, and (l) passing the solution after step (k) through a cation exchange chromatography column and eluting the protein absorbed on the column into the eluate, and (m) passing the eluate from step (l) through an anion exchange chromatography column to produce an effluent, and (n) passing the effluent from step (m) through a nanofiltration membrane to produce a nanofiltered solution, and (o) passing the nanofiltered solution from step (n) through an ultrafiltration membrane to produce an ultrafiltered solution, and (p) diafiltrating the ultrafiltered solution from step (o) against a diafiltration buffer to produce a diafiltered solution having a protein concentration of about 8% (w / v) to about 12% (w / v), thereby obtaining a composition enriched in IgG. It is purified from the heparin-added fraction using a method comprising the steps.

[0147] In certain embodiments, a pharmaceutical composition of IgG is provided, and the IgG composition is prepared using the methods provided herein that include improvements in two or more of the fractionation process steps described above. For example, in certain embodiments, improvements can be found in the first precipitation step, the modified fraction II+III precipitation step, the modified fraction II+III dissolution step, and / or the modified fraction II+III suspension filtration step.

[0148] In certain embodiments, a pharmaceutical composition of IgG is provided, the IgG composition being prepared using the purification methods described herein, the methods including spray addition of one or more solutions that may be introduced into the plasma fraction by fluid addition. For example, in certain embodiments, the method includes introduction of alcohol (e.g., ethanol) into the plasma fraction by spraying. In other embodiments, solutions that can be added to the plasma fraction by spraying include, but are not limited to, pH adjustment solutions, solvent solutions, detergent solutions, dilution buffers, and conductivity adjustment solutions. In preferred embodiments, one or more alcohol precipitation steps are carried out by addition of alcohol to the plasma fraction by spraying. In a second preferred embodiment, one or more pH adjustment steps are carried out by addition of a pH adjustment solution to the plasma fraction by spraying.

[0149] In certain embodiments, a pharmaceutical composition of IgG is provided, the IgG composition being prepared by the purification methods described herein, the methods including adjusting the pH of the plasma fraction that is precipitated after or simultaneously with the addition of a precipitant (e.g., alcohol or polyethylene glycol). In some embodiments, continuous monitoring and adjustment of the pH provides a process improvement such that the pH of the plasma fraction being actively precipitated is maintained throughout the precipitation incubation or hold step. In preferred embodiments, the pH adjustment is carried out by spray addition of a pH adjustment solution.

[0150] In one embodiment, the present invention provides a pharmaceutical composition of IgG comprising a protein concentration of about 70 g / L to about 130 g / L. In certain embodiments, the protein concentration of the IgG composition is about 80 g / L to about 120 g / L, such as about 90 g / L to about 110 g / L, such as about 100 g / L, or any suitable concentration within those ranges, such as about 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, 105 g / L, 110 g / L, 115 g / L, 120 g / L, 125 g / L, or 130 g / L. In a preferred embodiment, a pharmaceutical composition having a protein concentration of 100 g / L or about 100 g / L is provided. In a particularly preferred embodiment, the pharmaceutical composition has a protein concentration of 102 g / L or about 102 g / L.

[0151] In another embodiment, the present invention provides a pharmaceutical composition of IgG comprising a protein concentration of about 170 g / L to about 230 g / L. In certain embodiments, the protein concentration of the IgG component is about 180 g / L to about 220 g / L, such as about 190 g / L to about 210 g / L, such as about 200 g / L, or any suitable concentration within those ranges, such as about 170 g / L, 175 g / L, 180 g / L, 185 g / L, 190 g / L, 195 g / L, 200 g / L, 205 g / L, 210 g / L, 215 g / L, 220 g / L, 225 g / L, or 230 g / L. In a preferred embodiment, a pharmaceutical composition having a protein concentration of 200 g / L or about 200 g / L is provided.

[0152] The methods provided herein enable the preparation of IgG pharmaceutical compositions having a very high level of purity. For example, in one embodiment, at least about 95% of the total protein within the compositions provided herein is IgG. In other embodiments, at least about 96% of the protein is IgG, or about 97%, 98%, 99%, or more than 99.5% of the total protein of the composition is IgG. In a preferred embodiment, at least 97% of the total protein of the composition is IgG. In another preferred embodiment, at least 98% of the total protein of the composition is IgG. In another preferred embodiment, at least 99% of the total protein of the composition is IgG.

[0153] Similarly, the methods provided herein enable the preparation of IgG pharmaceutical compositions containing extremely low levels of contaminants. For example, in certain embodiments, an IgG composition is provided wherein the IgA contained is less than about 100 mg / L. In other embodiments, the IgG composition has less than about 50 mg / L of IgA contained, preferably less than about 35 mg / L of IgA contained, and most preferably less than about 20 mg / L of IgA contained.

[0154] The pharmaceutical compositions provided herein typically include one or more buffering agents or pH stabilizers suitable for intravenous, subcutaneous, and / or intramuscular administration. Non-limiting examples of buffering agents suitable for formulating the IgG compositions provided herein include glycine, citrate, phosphate, acetate, glutamate, tartrate, benzoate, lactate, histidine or other amino acids, gluconate, malate, succinate, formate, propionate, carbonate, or any combination thereof adjusted to an appropriate pH. Generally, the buffering agent is sufficient to maintain an appropriate pH within the formulation over a long period of time. In a preferred embodiment, the buffering agent is glycine.

[0155] In some embodiments, the concentration of the buffer in the formulation is from about 100 mM to about 400 mM, such as from about 150 mM to about 350 mM, such as from about 200 mM to about 300 mM, such as 250 mM. In particularly preferred embodiments, the IVIG composition comprises from about 200 mM to about 300 mM of glycine, such as about 250 mM of glycine.

[0156] In certain embodiments, the pH of the formulation is from about 4.1 to about 5.6, such as from about 4.4 to about 5.3, such as from 4.6 to about 5.1. In specific embodiments, the pH of the formulation may be about 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or 5.6. In preferred embodiments, the pH of the formulation is from about 4.6 to about 5.1.

[0157] In some embodiments, the pharmaceutical compositions provided herein may optionally further comprise an agent for adjusting the osmolality of the composition. Non-limiting examples of osmolality adjusting agents include mannitol, sorbitol, glycerol, sucrose, glucose, dextrose, levulose, fructose, lactose, polyethylene glycol, phosphate, sodium chloride, potassium chloride, calcium chloride, calcium gluconoglucoheptonate, and dimethyl sulfone, among others.

[0158] Typically, the formulations provided herein have an osmolarity corresponding to physiological osmolarity, having an osmolarity of about 285-295 mOsmol / kg (Lacy et al., Drug Information Handbook - Lexi-Comp 1999:1254). In certain embodiments, the osmolarity of the formulation is from about 200 mOsmol / kg to about 350 mOsmol / kg, preferably from about 240 to about 300 mOsmol / kg. In specific embodiments, the osmolarity of the formulation is about 200 mOsmol / kg, or 210 mOsmol / kg, 220 mOsmol / kg, 230 mOsmol / kg, 240 mOsmol / kg, 245 mOsmol / kg, 250 mOsmol / kg, 255 mOsmol / kg, 260 mOsmol / kg, 265 mOsmol / kg, 270 mOsmol / kg, 275 mOsmol / kg, 280 mOsmol / kg, 285 mOsmol / kg, 290 mOsmol / kg, 295 mOsmol / kg, 300 mOsmol / kg, 310 mOsmol / kg, 320 mOsmol / kg, 330 mOsmol / kg, 340 mOsmol / kg, 340 mOsmol / kg, or 350 mOsmol / kg.

[0159] The IgG formulations provided herein are generally liquid and stable over a long period. In certain embodiments, the formulation is stable at room temperature for at least about 3 months, or at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. The formulation is also generally stable in the refrigerated state (typically about 2°C to about 8°C) for 6 or at least about 18 months, or at least about 21, 24, 27, 30, 33, 36, 39, 42, or 45 months in the refrigerated state.

Examples

[0160] The following examples are not intended to be limiting, but are provided for illustrative purposes only. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce essentially the same or similar results.

[0161] Abbreviations used: CAE, cellulose acetate membrane electrophoresis; CZE, capillary zone electrophoresis; FC, final container; NAPTT, non-activated partial thromboplastin time; NP, normal plasma; PKA, prekallikrein activity; PL-1, amidolytic activity measured by chromogenic substrate PL-1; PptG, precipitate G; TGA, thrombin generation assay, TP, total protein

[0162] Example 1 This example demonstrates that a large amount of fibrinogen, amidolytic activity, and prekallikrein activity can be removed from the PptG precipitate obtained from C1-INH-deficient plasma supernatant (DDCPP).

[0163] The fibrinogen content from the starting material to Supernatant I is reduced from 0.94 g / L DDCPP to 0.26 g / L DDCPP in the variant native (see Table 1), from 1.23 g / L DDCPP to 0.34 g / L DDCPP in the variant heparin (see Table 2), and from 1.4 g / L DDCPP to 0.37 g / L DDCPP in the variant NaCl (see Table 3). During aerosol treatment and filtration, a further reduction to 0.01 g / L DDCPP in the variant heparin (Table 2), and a further reduction to 0.02 g / L DDCPP in the other two variants (Table 1 and Table 3) occur. The fibrinogen in the dissolved PptG samples from 6 lots is 0.1% - 0.3% of the total protein (Table 4). The fibrinogen content at this step is equal to the content in the qualified lots produced from PptG (0.1% - 0.3% of the total protein). Fibrinogen was below the detection limit at the level in the final container (see Table 15).

[0164] Fractions II + III separate unpurified immunoglobulins (II + III precipitate) from unpurified albumin (II + III supernatant). Haptoglobin and transferrin are mainly retained in the II + III supernatant (see Tables 1, 2, and 3). Complement C3 is low in the starting Cohn pool and is removed during the Aerosil treatment and filtration steps from 0.03 - 0.05 g / L DDCPP to 0.004 g / L DDCPP in heparin and to 0.01 g / L DDCPP in the other two variants. FXI protein is reduced from approximately 1000 U / L DDCPP to 148 U / L DDCPP in heparin, to 383 U / L DDCPP in native, and to 461 U / L DDCPP in the NaCl variant. The Aerosil treatment and filtration steps reduce fibrinogen, haptoglobin, along with some of the IgA and IgM and FXI proteins.

[0165] In PptG, low molecular weight components are present in low content as measured by molecular size distribution (see Table 4). Transferrin and α2-macroglobulin remain in the PptG supernatant (Tables 1 - 3). The IgA content in the dissolved PptG (7.7% - 10.9% of total protein (TP), measured by ELISA) has a slightly lower range compared to the compliant lots in VIE (9.6% - 12.7% of TP). In the dissolved PptG, the α2-macroglobulin levels vary between 4.7% - 5.6% of TP (Table 4). The FXI protein is at a similar high level for all lots in the native and NaCl variants in PptG (37.5 - 41.9 U / g protein), but is lower in the lots with added heparin (12.1 - 12.4 U / g protein) (see Table 4). This is more strongly reflected by the g / L DDCPP values shown in Table 4.

[0166] (Table 1) Upstream intermediate results (from Cohn pool to PptG supernatant) - native TIFF0007717715000001.tif188158

[0167] (Table 2) Upstream Intermediate Results (from Cohn Pool to PptG Supernatant) - Variant Heparin TIFF0007717715000002.tif178162

[0168] (Table 3) Upstream Intermediate Results (from Cohn Pool to PptG Supernatant) - Variant NaCl TIFF0007717715000003.tif223150

[0169] (Table 4) Precipitate G Property Evaluation TIFF0007717715000004.tif215133

[0170] PKA in the dissolved PptG varies from below the limit of quantification up to a maximum of 9.4 U / mL. In bulk, PKA is below the limit of quantification for all process options (see Table 5). Kallikrein-like activity is high in the PptG dissolution step (490 - 733 nmol / mL*min), but may be greatly reduced by downstream processes, and by using 35 mM elution buffer in CM Sepharose chromatography, the level is below the limit of quantification (<10 nmol / mL*min). The activated partial thromboplastin time tested in FXI-deficient plasma is not shortened in the dissolved PptG for any of the options. The amidolytic activity measured by the chromogenic substrate PL-1 is high in the dissolved PptG (97.2 - 163.1 nmol / mL*min), but in most cases is reduced to levels below the limit of quantification (<10 nmol / mL*min) at the final bulk level. Thrombin generation was measured at the level of PptG dissolution for reference only. The tests vary, but the TGA measured at this step is lower for the lots where heparin was added to the DDCPP (113.14% and 103.33% of normal plasma) (limit of observation for FC was 132% of NP). In bulk (samples before low pH incubation), the TGA values exceed the limit of observation for the specified final container of 132% for all samples. The TGA values are 185% - 195% of normal plasma for implementation with 35 mM elution buffer, regardless of NaCl addition, heparin addition, or native. The FXIa values are below the limit of quantification in the dissolved PptG for the lots produced by the heparin variant. In the other two lots, the values are considerably higher (10.3 - 16.7 ng / g protein) compared to the other tests. The FXIa values were detected in the final bulk for all lots. Even in this case, the minimum value was confirmed for the lot where heparin was added to the DDCPP.

[0171] The high kallikrein-like activity in the dissolved PptG is also reflected by the amide hydrolysis activity profile. The substrates specifically measure kallikrein, FXIa, and FXIIa, and the measured values are 570 - 1780 nmol / ml*min. These values are reduced to 8 - 22 nmol / ml*min in the final bulk (see Table 5).

[0172] (Table 5) Results of PKA, procoagulant impurities, and amide hydrolysis activity in PptG and bulk TIFF0007717715000005.tif218146

[0173] Example 2 The purity in the Cohn pool, II + III supernatant (albumin), and intermediate PptG paste is determined by cellulose acetate membrane electrophoresis (see Table 6). According to the current Gammagard Liquid / KIOVIG specifications, the intermediate product, precipitate G, needs to meet the purity specification of ≥86% gamma globulin as measured by CAE electrophoresis or its equivalent means. The PptG paste obtained from DDCPP (plasma after C1 inhibitor adsorption) clearly meets the intermediate specification limit of >86% Gammagard Liquid / KIOVIG (see Table 6). The addition of heparin and sodium chloride increases the purity from 88% to 93%.

[0174] Also, the purity is measured by CZE in the PptG dissolution step and the final container. Ppt G has a γ-globulin purity of 92% - 93%, and the final container purity was 100% γ-globulin (see Table 7).

[0175] (Table 6) Purity of Cohn pool, II + III supernatant, and PptG measured by CAE TIFF0007717715000006.tif109162

[0176] (Table 7) Purity of PptG dissolution and final container (FC) measured by CZE TIFF0007717715000007.tif30128

[0177] Example 3 To confirm that the starting material is suitable for use in the production of IgG, the high IgG recovery and protein yield are determined. The protein and IgG yields are given in %, and g / L plasma, and indicate the efficiency of the process. The high IgG recovery from the Cohn pool to the bulk reflects a very good process efficiency. Recoveries of 68% - 75% based on the measurement of IgG were obtained (Tables 8 - 10). The addition of sodium chloride to the Cohn pool for conductivity adjustment results in a slightly lower overall recovery compared to the other two options (68% vs. over 70%).

[0178] (Table 8) Protein and IgG Recovery - Native using 35 mM CM elution buffer TIFF0007717715000008.tif165156 1) Measured by QC VIE

[0179] (Table 9) Protein and IgG Recovery - Variant Heparin using 35 mM CM elution buffer TIFF0007717715000009.tif169160 1) Measured by QC VIE

[0180] (Table 10) Protein and IgG Recovery - Variant NaCl using 35 mM CM elution buffer TIFF0007717715000010.tif164158 1) Measured by QC VIE 2) Measured by PSP / PSTO

[0181] Example 4 The final container release parameters were tested according to the manufacturing method of Gammagard Liquid / KIOVIG and summarized in Table 11 for implementation with 35 mM elution buffer. The results of the antibody titers of the release parameters are summarized in Tables 12 and 13.

[0182] (Table 11) Results of the final container specification test using 35 mM CM Sepharose elution buffer TIFF0007717715000011.tif206161TIFF0007717715000012.tif105160

[0183] Example 5 For the final container lot, release tests were conducted for anti-A / anti-B hemagglutinin and anti-D antibody, diphtheria (US only), HAV (EU only), HBsAg, measles (US only), parvovirus B19 (EU only), and antibody to polio (US only) (see Table 12, 35 mM elution buffer). All antibody tests met the requirements.

[0184] (Table 12) Antibody levels in FC (IU / g protein calculated relative to total protein) - 35 mM elution buffer TIFF0007717715000013.tif188147

[0185] Example 6 To determine the residual serine protease content and activity present in the plasma-derived protein composition, an amidolytic activity profile was determined for the IgG preparation from C1-INH-deficient plasma supernatant. Briefly, an amidolytic activity profile for the plasma-derived protein composition was determined. PL-1, amidolytic activity profile, TGA, NAPTT, FXIa, and FXI protein were tested and the results summarized in Table 13 (35 mM elution buffer). As shown in Table 13, the amidolytic activity measured by the chromogenic substrate PL-1 was below the limit of quantification for all lots, which demonstrates the high reduction capacity of the downstream process regardless of the phosphate concentration of the CM elution buffer. Amidolytic activity data generated using different chromogenic substrates also show very low values. NAPTT tested in FXI-deficient plasma is not shortened in the final container samples. FXIa is below the limit of quantification with the use of 35 mM CM-elution buffer when heparin is added. The FXI protein assay that detects not only FXI but also FXIa has very low values when heparin is added to DDCPP with the use of 35 mM CM elution buffer.

[0186] (Table 13) Amidolytic activity and procoagulant activity measured by FC using 35 mM elution buffer TIFF0007717715000014.tif91139

[0187] Example 7 The FXI protein assay is also an indicator throughout the manufacturing process. Table 14 summarizes the overall decrease in FXI protein from DDCPP to the final container. The FXI protein value in the starting material is set at 100%. The main decrease occurs during the Aerosil treatment and subsequent filtration. The downstream process further decreased the FXI protein content to a level of 0.01% of the initial value.

[0188] (Table 14) Overall decrease in FXI protein (%) recovery from DDCPP starting material to FC TIFF0007717715000015.tif87163

[0189] Example 8 Next, the levels of various protein impurities in the IgG preparation derived from C1-INH-deficient plasma supernatant were measured. As shown in Table 15, fibrinogen was below the detection limit (<0.03 μg / mL), and the complement C3 level (0.04 - 0.07 mg / dL) was far below the limit of observation (<19.4 mg / dL).

[0190] (Table 15) Trace protein content in the final container using 35 mM elution buffer TIFF0007717715000016.tif31160

[0191] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes contemplated thereby are intended to be suggested to those skilled in the art and should be within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method for preparing an IgG-concentrated fraction from a C1 inhibitor (C1-INH)-deficient supernatant fraction containing immunoglobulin G (IgG), comprising: (a) contacting the C1-INH-deficient supernatant fraction with heparin to thereby form a heparin-added fraction; (b) isolating IgG from the heparin-added fraction to thereby form an IgG-concentrated fraction. The method as described above.

2. The method according to claim 1, wherein the supernatant fraction is the supernatant after C1-INH adsorption.

3. The method according to claim 1 or 2, wherein the supernatant fraction is a plasma supernatant.

4. The method according to claim 3, wherein the plasma supernatant is C1-INH-deficient cryoprecipitate-free plasma.

5. The method according to any one of claims 1 to 4, wherein the supernatant fraction lacks one or more of other blood coagulation factors selected from factors II, VII, IX, and X, or a mixture thereof.

6. The method according to any one of claims 1 to 5, wherein the supernatant fraction is concentrated to the protein value of normal plasma before further treatment.

7. The method according to any one of claims 1 to 6, wherein the heparin is added in an amount of 1 to 20 units per 1 ml of the supernatant fraction.

8. The method according to claim 7, wherein the heparin is added in an amount of 5 units per 1 ml of the supernatant fraction.

9. The method according to claim 7, wherein the heparin is added in an amount of 10 units per 1 ml of the supernatant fraction.

10. Further comprising removing C1-INH from a cryoprecipitate-free plasma fraction containing C1-INH before step (a) to thereby form the C1-INH-deficient supernatant fraction. The method according to any one of claims 1 to 9.

11. The method according to any one of claims 1 to 10, wherein the IgG-concentrated fraction contains at least 50% of the IgG content found in the supernatant fraction.

12. The method according to any one of claims 1 to 11, wherein the purity of IgG in the IgG-concentrated fraction is at least 95%.

13. Isolating IgG from the heparin-added fraction in (b) is (i) precipitating the heparin-added fraction at a pH of 7.0 to 7.5 using 6% to 10% ethanol to obtain a fraction I precipitate and a fraction I supernatant; (ii) To form the fraction II + III precipitate, IgG is precipitated from the fraction I supernatant at a pH of 6.7 to 7.3 using 18% to 27% alcohol. The method according to any one of claims 1 to 12, comprising the above.

14. Isolating IgG from the heparin-added fraction in (b) (i) To form the fraction I + II + III precipitate, IgG is precipitated from the heparin-added fraction at a pH of 6.7 to 7.3 using 18% to 27% alcohol. The method according to any one of claims 1 to 12, comprising the above.

15. (iii) Suspending the precipitate of fraction II + III or fraction I + II + III in a suspension buffer, thereby forming an IgG suspension. (iv) Mixing the finely divided silicon dioxide (SiO ２ ) with the IgG suspension for at least 30 minutes, and (v) Filtering the IgG suspension, thereby forming a filtrate and a filter cake. The method according to claim 13 or 14, further comprising the above.

16. (vi) Washing the filter cake with a washing buffer having a pH of 4.9 to 5.3 and at least one filter press dead volume, thereby forming a washing solution. (vii) Combining the filtrate with the washing solution, thereby forming a solution, and treating the solution with a detergent. (viii) Adjusting the pH of the solution in step (vii) to 7.0 and adding ethanol to a final concentration of 20% to 30%, thereby forming a precipitate G precipitate. (ix) Dissolving the precipitate G precipitate in an aqueous solution containing one solvent and / or one or more detergents, and holding the solution for at least 60 minutes. (x) Passing the solution through a cation exchange chromatography column and eluting the protein absorbed on the column into the eluate. (xi) Passing the eluate through an anion exchange chromatography column to generate a flow-through effluent. (x) Passing the effluent through a nanofilter to generate a nanofiltrate. (xi) Concentrating the nanofiltrate by ultrafiltration to generate a first ultrafiltrate. (xii) Performing diafiltration on the first ultrafiltrate against a diafiltration buffer to generate a diafiltrate. (xiii) Concentrating the dialysis filtrate by ultrafiltration to produce a second ultrafiltrate having a protein concentration of 8% (w / v) to 22% (w / v), thereby forming an IgG concentrated fraction The method according to claim 15, further comprising. **Claim 17** (iv) adding SiO to a final concentration of 0.02 to 0.10 grams per gram of the precipitate of fraction II + III or fraction I + II + III ２ The method according to claim 15 or 16, comprising **Claim 18** (vi) The method according to claim 16 or 17, comprising washing the filter cake with at least two filter press dead volumes of washing buffer. **Claim 19** (x) The method according to any one of claims 16 to 18, comprising eluting the protein with at least 35 mM sodium dihydrogen phosphate dihydrate. **Claim 20** (xii) The dialysis filtration buffer in (xii) contains 200 mM to 300 mM glycine. The method according to any one of claims 16 to 19. **Claim 21** (vii) Treating the solution with one solvent and / or one or more detergents in (vii) includes at least one step of virus inactivation or removal. The method according to any one of claims 16 to 20. **Claim 22** The method according to claim 21, wherein the virus inactivation is a solvent / detergent (S / D) virus inactivation step. **Claim 23** 4. The method according to any one of claims 16 to 22, further comprising an incubation step at a low pH of 0 to 5.

2. **Claim 24** 4. The method according to any one of claims 16 to 22, further comprising an incubation step at a low pH of 4 to 4.

9. **Claim 25** The supernatant after C1-INH adsorption of the defrosted plasma fraction containing IgG, C1-INH of 30% or less of the total amount of C1-INH present in the defrosted plasma fraction before C1-INH adsorption 1 to 20 IU of heparin per ml of supernatant The supernatant containing.

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