Removal of serine proteases by treatment with finely divided silicon dioxide

Abstract

Abstract The present invention provides novel methods for reducing the serine protease and / or serine protease zymogen content of a plasma-derived protein composition. Also provided are methods for manufacturing plasma-derived protein compositions having reduced seine protease and\or seine protease zymogen content. Among yet other aspects, the present invention provides aqueous and lyophilized compositions of plasma-derived proteins having reduced serine protease and / or serine protease zymogen content. Yet other aspects include methods for treating, managing, and / or preventing a disease comprising the administration of a plasma-derived protein composition having a reduced serine protease or seine protease zymogen content.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to AU Patent Application No. 2010202125, filed May 26, 5   2010 and issued as Australian Patent No. 2010202125, U.S. Patent Application Serial No. 12 / 789,365, filed May 27, 2010, and U.S. Patent Application Serial No. 12 / 842,944, filed July 23, 2010, the disclosures of which are hereby incorporated herein by reference in their entireties for all purposes. BACKGROUND OF THE INVENTION 10

[0002] The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that such art forms part of the common general knowledge. Plasma-derived blood products are used to treat not only a variety of blood disorders, but diseases of other origin. For example, immune globulin (IgG) products from human plasma were first used in 1952 to treat immune deficiency. Since then, IgG preparations 15   have found widespread use in at least three main categories of medical conditions: (1) immune deficiencies such as X-linked agammaglobulinemia, hypogammaglobulinemia (primary immune deficiencies), and acquired compromised immunity conditions (secondary immune deficiencies), featuring low antibody levels; (2) inflammatory and autoimmune diseases; and (3) acute infections. 20

[0003] Likewise, Factor H has been implicated as a potential therapeutic agent for several human disease states, including age-related macular degeneration (AMD), hemolytic uremic syndrome (aHUS) and membranoproliferative glomerulonephritis (MPGN). Specifically, a causal relationship between the single nucleotide polymorphism (SNP) in complement control protein (CCP) module 7 of Factor H and age-related macular degeneration (AMD) has been 25    characterized.

[0004] Studies have shown correlations between decreased plasma levels of Inter-alphaInhibitor proteins (IaIp) and mortality in patients with severe sepsis (Lim et al., J Infect Dis. (2003) Sep 15;188(6):919-26 and Opal et al., Crit Care Med. (2007) Feb;35(2):387-92). Furthermore, several studies have shown that the administration of IaIp reduces mortality 30   associated with sepsis and septic shock (Jourdain et al., Am J Respir Crit Care Med. (1997) 2024204779   11 Jul 2024 Dec; 156(6): 1825-33; Yang et al., Crit Care Med. (2002) Mar;30(3):617-22; Lim et al., J Infect Dis. (2003) Sep 15; 188(6):919-26; and Wu et al., Crit Care Med. (2004) Aug;32(8): 1747-52; the disclosures of which are incorporated by reference herein in their entireties for all purposes).

[0005] Various safety precautions must be taken into consideration when manufacturing and 5 formulating plasma-derived biologic therapies. These include methods for removing and / or inactivating blood borne pathogens (e.g., viral and bacterial pathogens), anticomplement activity, and other unwanted contaminants arising from the use of donated plasma. Studies have suggested that administration of high levels of amidolytic activity may result in unwanted thromboembolic events (Wolberg AS et al., Coagulation factor XI is a contaminant in 10 intravenous immunoglobulin preparations. Am J Hematol 2000;65:30-34; 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; the disclosures of which are hereby incorporated by reference in their entireties for all purposes). Highlighting this concern was the recent voluntary withdrawal of octagam® (Octapharma) in the US and suspension of marketing 15 authorization for octagam® and octagam 10% by the European Commission following increased reports of thromboembolic events. It is likely that the increased thrombolic events were caused by high levels of amidolytic activity in the biologic, caused by serine protease and serine protease zymogen impurities, such as Factor XI, Factor Xia, Factor XII and Factor Xlla (FDA Notice: Voluntary Market Withdrawal - September 23,2010 Octagam [Immune Globulin 20 Intravenous (Human)] 5% Liquid Preparation; Octagam 50 mg / ml, solution pour perfusion -Octapharma France - Mise en quarantaine de tous les lots, published online September 9, 2010 by the AFSSAPS; and Questions and answers on the suspension of the marketing authorisations for Octagam (human normal immunoglobulin 5% and 10%), published online September 23, 2010 by the European Medicines Agency). 25

[0006] Dedicated serine proteases, generically known as coagulation factors, are integral components of both the contact activation and tissue factor pathways of the coagulation cascade. Upon a stimulus of the coagulation pathways, serine protease zymogens, which are inactive enzyme precursors, become activated proteases that catalyze the activation of the next protease zymogen, resulting in an activation cascade. This coagulation cascade culminates in the 30 activation of Thrombin (Factor Ila) and Factor Xllla, which function to convert Fibrinogen (Factor I) into Fibrin (Factor la) and cross-link fibrin to form a fibrin clot, respectively. 2024204779   11 Jul 2024

[0007] The contact activation pathway, also known as the intrinsic coagulation pathway, begins with the activation of Kallikrein and Factor Xlla (FXIIa) from Prekallikrein and Factor XII, respectively. The activated serine protease FXIIa cleaves Factor XI (FXI), converting the zymogen into Factor Xia (FXIa), an active serine protease which participates in the subsequent 5 activation of Factor Xa (FXa).

[0008] Due to rising concerns over the presence of serine protease and serine protease zymogens in plasma-derived protein compositions, there remains a need in the art for methods for reducing the levels of these contaminants, and particularly FXI, FXIa, FXII, and FXIIa. The present invention fulfils these and other needs by providing such methods and plasma-derived 10 protein compositions with reduced levels of serine protease and serine protease zymogen. BRIEF SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention is based on the surprising finding that serine proteases and serine protease zymogens, and specifically, FXI, FXIa, FXII, and FXIIa, can be 15 removed from plasma-derived protein compositions by treatment with finely divided silicon dioxide (SiO2). In this fashion the present invention provides methods for reducing the serine protease activity, serine protease content, and serine protease zymogen content of plasma-derived protein compositions. Also provided are therapeutic plasma-derived protein compositions having reduced serine protease activity, serine protease content, and serine 20 protease zymogen content, as well as methods for treating or preventing disease by the administration of the same.

[0010] In a first aspect, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived target protein composition, the method comprising the steps of: (a) contacting the composition with finely divided silicon 25 dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (b) separating the SiO2 from the composition to remove the bound serine protease. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), or Factor XII (FXII).

[0011] In certain embodiments, the method described above further comprises the step of 30 performing a first target protein enrichment step to form a first enriched composition, prior to contacting the composition with finely divided silicon dioxide (SiO2). In one embodiment, the 2024204779   11 Jul 2024 first target protein enrichment step is a protein precipitation step. In a specific embodiment, the protein precipitation step is an alcohol fractionation step. In another embodiment, the first target protein enrichment step is an ultrafiltration / diafiltration step.

[0012] In other embodiments of the methods described above, the method further comprises 5 the step of performing a second target protein enrichment step prior to contacting the enriched composition with finely divided silicon dioxide (SiO2). In one embodiment, the second target protein enrichment step is a protein precipitation step. In a specific embodiment, the protein precipitation step is an alcohol fractionation step. In another embodiment, the second target protein enrichment step is an ultrafiltration / diafiltration step. In yet another embodiment, the 10 second target protein enrichment step is a chromatographic enrichment step.

[0013] In other embodiments of the methods described above, the method further comprises the step of performing a third target protein enrichment step after contacting the composition with finely divided silicon dioxide (SiO2). In one embodiment, the third target protein enrichment step is a protein precipitation step. In a specific embodiment, the protein 15 precipitation step is an alcohol fractionation step. In another embodiment, the third target protein enrichment step is an ultrafiltration / diafiltration step. In yet another embodiment, the third target protein enrichment step is a chromatographic enrichment step.

[0014] In certain embodiments of the methods described above, the chromatographic enrichment step comprises the sub-steps of: (i) contacting the plasma-derived target protein 20 composition with a chromatographic resin under conditions suitable to bind the plasma-derived target protein; and (ii) eluting the plasma-derived target protein from the chromatographic resin. In a specific embodiment, the impurity does not bind to the chromatographic resin in sub-step (i). In another specific embodiment, the impurity binds to the chromatographic resin in sub-step (i), but is not eluted from the chromatographic resin in sub-step (ii). 25

[0015] In other certain embodiments of the methods described above, the chromatographic enrichment step comprises the sub-steps of: (i) contacting the first enriched plasma-derived target protein composition with a chromatographic resin under conditions suitable to bind at least one impurity; and (ii) separating the resin from the plasma-derived protein composition, wherein the plasma-derived target protein does not bind to the chromatographic resin in sub-step (i). 2024204779   11 Jul 2024

[0016] In certain embodiments of the methods comprising a chromatographic enrichment step described above, the chromatographic resin is selected from the group consisting of an anion exchange resin, a cation exchange resin, a hydrophobic interaction resin, a mixed mode resin, a hydroxyapatite resin, a ligand affinity resin, an immunoaffinity resin, and a size exclusion resin. 5

[0017] In other certain embodiments of the methods comprising a chromatographic enrichment step described above, the chromatographic enrichment step comprises separating at least one impurity from the target protein by size and / or shape using size exclusion chromatography.

[0018] In certain embodiments of the methods described above, the plasma-derived target protein is selected from an immunoglobulin (Ig), albumin, alpha-1-antitrypsin (AlPI), 10 butyrylcholinesterase, a protein of the complement system, and an inter-alpha-trypsin inhibitor (lai). In a specific embodiment, the protein of the complement system is selected from the group consisting of Factor H (FH), Factor D, complement protein C3, and C4 binding protein.

[0019] In yet another embodiment of the methods described above, the plasma-derived target protein composition is a manufacturing intermediate. 15

[0020] In a second aspect, the present invention provides a method for preparing a plasma- derived Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H and at least one serine protease or serine 20 protease zymogen; (b) separating the SiO2 from the composition; (c) eluting the serine protease or serine protease zymogen from the SiO2 under a solution condition in which the Factor H remains bound; and (d) eluting the Factor H from the SiO2.

[0021] In certain embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and the Factor H 25 remains bound comprises a pH greater than about 6.0. In another embodiment, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and the Factor H remains bound comprises a pH greater than about 6.5. In another embodiment, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and the Factor H remains bound comprises a pH greater than about 7.0. In yet another 2024204779   11 Jul 2024 embodiment, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and the Factor H remains bound comprises a pH of at least about 7.5.

[0022] In certain embodiment of the methods described above, the solution condition comprises a pH of no more than 11.0. In another embodiment, the solution condition comprises 5   a pH of no more than 10.0. In another embodiment, the solution condition comprises a pH of no more than 9.0.

[0023] In certain embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and the Factor H remains bound comprises a conductivity of greater than about 10 mS / cm. In another 10 embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and the Factor H remains bound comprises a conductivity of greater than about 20 mS / cm. In another embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and the Factor H remains bound comprises a 15   conductivity of between about 10 mS / cm and about 50 mS / cm. In yet another embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and the Factor H remains bound comprises a conductivity between about 20 mS / cm and about 50 mS / cm.

[0024] In a third aspect, the present invention provides a method for preparing a Factor H 20 composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H and at least one serine protease; (b) separating the SiO2 from the composition; and (c) eluting the Factor H from the SiO2 under conditions in which the 25 serine protease or serine protease zymogen remains bound to the SiO2.

[0025] In certain embodiment of the methods described above, the solution condition under which the Factor H is eluted from the SiO2 and the serine protease or serine protease zymogen remains bound comprises a pH greater than about 6.0. In another embodiment, the solution condition under which the Factor H is eluted from the SiO2 and the serine protease or serine 30 protease zymogen remains bound comprises a pH greater than about 6.5. In another embodiment, the solution condition under which the Factor H is eluted from the SiO2 and the 2024204779   11 Jul 2024 serine protease or serine protease zymogen remains bound comprises a pH greater than about 7.0. In yet another embodiment, the solution condition under which the Factor H is eluted from the SiO2 and the serine protease or serine protease zymogen remains bound comprises a pH of at least about 7.5. 5

[0026] In certain embodiment of the methods described above, the solution condition comprises a pH of no more than 11.0. In another embodiment, the solution condition comprises a pH of no more than 10.0. In another embodiment, the solution condition comprises a pH of no more than 9.0.

[0027] In certain embodiment of the methods described above, the solution condition under 10 which the Factor H is eluted from the SiO2 and the serine protease or serine protease zymogen remains bound comprises a conductivity of less than about 20 mS / cm. In another embodiment, the solution condition under which the Factor H is eluted from the SiO2 and the serine protease or serine protease zymogen remains bound comprises a conductivity of less than about 10 mS / cm. In another embodiment of the methods described above, the solution condition under 15 which the Factor H is eluted from the SiO2 and the serine protease or serine protease zymogen remains bound comprises a conductivity between about 2 mS / cm and about 20 mS / cm. In yet another embodiment of the methods described above, the solution condition under which the Factor H is eluted from the SiO2 and the serine protease or serine protease zymogen remains bound comprises a conductivity between about 2 mS / cm and about 10 mS / cm. 20

[0028] In a fourth aspect, the present invention provides a method for preparing a Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H but not the at least one serine protease or serine protease 25 zymogen; (b) separating the SiO2 from the composition; and (c) eluting the Factor H from the SiO2.

[0029] In certain embodiment of the methods described above, the solution condition under which the Factor H binds to the SiO2 and the serine protease or serine protease zymogen does not comprises a pH greater than about 6.0. In another embodiment, the solution condition under 30 which the Factor H binds to the SiO2 and the serine protease or serine protease zymogen does not comprises a pH greater than about 6.5. In another embodiment, the solution condition under 2024204779   11 Jul 2024 which the Factor H binds to the SiO2 and the serine protease or serine protease zymogen does not comprises a pH greater than about 7.0. In yet another embodiment, the solution condition under which the Factor H binds to the SiO2 and the serine protease or serine protease zymogen does not comprises a pH of at least about 7.5. 5

[0030] In certain embodiment of the methods described above, the solution condition comprises a pH of no more than 11.0. In another embodiment, the solution condition comprises a pH of no more than 10.0. In another embodiment, the solution condition comprises a pH of no more than 9.0.

[0031] In certain embodiment of the methods described above, the solution condition under 10 which the Factor H binds to the SiO2 and the serine protease or serine protease zymogen does not comprises a conductivity of greater than about 10 mS / cm. In another embodiment of the methods described above, the solution condition under which the Factor H binds to the SiO2 and the serine protease or serine protease zymogen does not comprises a conductivity of greater than about 20 mS / cm. In another embodiment of the methods described above, the solution condition 15 under which the Factor H binds to the SiO2 and the serine protease or serine protease zymogen does not comprises a conductivity between about 10 mS / cm and about 50 mS / cm. In yet another embodiment of the methods described above, the solution condition under which the Factor H binds to the SiO2 and the serine protease or serine protease zymogen does not comprises a conductivity between about 20 mS / cm and about 50 mS / cm. 20

[0032] In a fifth aspect, the present invention provides a method for preparing a Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the at least one serine protease or serine protease zymogen but not the 25 Factor H and (b) separating the SiO2 from the composition.

[0033] In certain embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and the Factor H does not comprises a pH greater than about 6.0. In another embodiment, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and the Factor H does not 30 comprises a pH greater than about 6.5. In another embodiment, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and the Factor H does not 2024204779   11 Jul 2024 comprises a pH greater than about 7.0. In yet another embodiment, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and the Factor H does not comprises a pH of at least about 7.5.

[0034] In certain embodiment of the methods described above, the solution condition 5 comprises a pH of no more than 11.0. In another embodiment, the solution condition comprises a pH of no more than 10.0. In another embodiment, the solution condition comprises a pH of no more than 9.0.

[0035] In certain embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and the Factor H does not 10 comprises a conductivity of less than about 20 mS / cm. In another embodiment, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and the Factor H does not comprises a conductivity of less than about 10 mS / cm. In another embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and the Factor H does not comprises a 15 conductivity between about 2 mS / cm and about 20 mS / cm. In yet another embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and the Factor H does not comprises a conductivity between about 2 mS / cm and about 10 mS / cm.

[0036] In a sixth aspect, the present invention provides a method for preparing an inter-alpha-20 trypsin inhibitor (lai) composition having reduced serine protease activity, the method comprising the steps of: (a) contacting a solution containing lai and at least one serine protease with finely divided silicon dioxide (SiO2) under conditions suitable to bind the lai and at least one serine protease; (b) separating the SiO2 from the composition; (c) eluting the serine protease from the SiO2 under conditions in which the lai remains bound; and (d) eluting the lai from the 25 SiO2.

[0037] In a seventh aspect, the present invention provides a method for preparing an interalpha-trypsin inhibitor (lai) composition having reduced serine protease activity, the method comprising the steps of: (a) contacting a solution containing lai and at least one serine protease with finely divided silicon dioxide (SiO2) under conditions suitable to bind the lai and at least 30 one serine protease; (b) separating the SiO2 from the composition; and (c) eluting the lai from the SiO2 under conditions in which the serine protease remains bound to the SiO2. 2024204779   11 Jul 2024

[0038] In an eighth aspect, the present invention provides a method for preparing an interalpha-trypsin inhibitor (lai) composition having reduced serine protease activity, the method comprising the steps of: (a) contacting a solution containing lai and at least one serine protease with finely divided silicon dioxide (SiOs) under conditions suitable to bind the lai but not the 5 least one serine protease; (b) separating the SiO2 from the composition; and (c) eluting the lai from the SiO2.

[0039] In a ninth aspect, the present invention provides a method for preparing a inter-alphatrypsin inhibitor (lai) composition having reduced serine protease activity, the method comprising the steps of: (a) contacting a solution containing inter-alpha-trypsin inhibitor (lai) 10 and at least one serine protease with finely divided silicon dioxide (SiO2) under conditions suitable to bind the serine protease but not the inter-alpha-trypsin inhibitor (lai); and (b) separating the SiO2 from the composition.

[0040] In certain embodiments of the aspects described above, the composition is contacted with SiO2 at a final concentration of at least 1 g SiO2 / g protein. In another embodiment of the 15 aspects described above, the composition is contacted with SiO2 at a final concentration of at least 2 g SiO2 / g protein. In another embodiment of the aspects described above, the composition is contacted with SiO2 at a final concentration of at least 2.5 g SiO2 / g protein.

[0041] In certain embodiments of the aspects described above, the serine protease or serine protease zymogen is Factor XI. In another embodiment of the aspects described above, the 20 serine protease or serine protease zymogen is Factor Xia. In another embodiment of the aspects described above, the serine protease or serine protease zymogen is Factor XII. In yet another embodiment of the aspects described above, the serine protease or serine protease zymogen is Factor Xlla.

[0042] In a tenth aspect, the present invention provides a plasma-derived protein composition 25 prepared by a process comprising a method for reducing serine protease activity according to any one of the aspects described above. In one embodiment, the composition is formulated for administration to a subject. In a specific embodiment, the composition is formulated for intravenous, intramuscular, or subcutaneous administration. In one embodiment, the composition is aqueous. In another embodiment, the composition is lyophilized. 2024204779   11 Jul 2024

[0043] In an eleventh aspect, the present invention provides a method for treating a disease associated with aberrant activity of a plasma protein in a subject in need thereof, the method comprising administering a plasma-derived protein composition according to the aspect outlined above. In certain embodiments, the composition comprises a plasma-derived protein is selected 5 from an immunoglobulin (Ig), albumin, alpha-1-antitrypsin (AlPI), butyrylcholinesterase, a protein of the complement system, and an inter-alpha-trypsin inhibitor (lai).

[0044] In a twelfth aspect, the present invention provides a method for preparing a Factor H composition comprising the steps of (a) contacting a suspended plasma precipitate fraction containing Factor H with finely divided silicon dioxide (SiCh), (b) washing the SiO2 with a wash 10 buffer comprising a low pH and a low conductivity, and (c) eluting Factor H from the SiO2 with an elution buffer comprising a pH between 7.0 and 8.0 and a conductivity of at least 10 mS / cm. In specific embodiments, the plasma precipitate fraction containing Factor H is a Cohn fraction II+III precipitate, a Cohn fraction 1+11+111 precipitate, a Kistler / Nitschmann Precipitate A, or a Kistler / Nitschmann Precipitate B. In certain embodiments, the methods further comprises one or 15 more additional steps selected from (d) precipitating and removing at least one impurity from the Factor H elution, (e) precipitating and recovering Factor H from the enriched composition, (f) further enriching Factor H by anion exchange chromatography, (g) further enriching Factor H by heparin affinity chromatography, (h) a dedicated viral inactivation step, and (i) concentrating the enriched Factor H composition by ultrafiltration / diafiltration. 20

[0045] In one embodiment, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived target protein composition, the method comprising the steps of: (a) contacting the composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (b) separating the SiO2 from the composition to remove the bound serine 25 protease, wherein the at least one serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), or Factor XII (FXII).

[0046] In a specific embodiment of the methods described above, the method further comprises the step of performing a first target protein enrichment step to form a first enriched composition, prior to contacting the composition with finely divided silicon dioxide (SiO2). In 30 one embodiment, the first target protein enrichment step is a protein precipitation step. In a 2024204779   11 Jul 2024 specific embodiment, the protein precipitation step is an alcohol fractionation step. In another specific embodiment, the first target protein enrichment step is an ultrafiltration / diafiltration step.

[0047] In a specific embodiment of the methods described above, the method further comprises the step of performing a second target protein enrichment step prior to contacting the 5 enriched composition with finely divided silicon dioxide (SiO2). In one embodiment, the second target protein enrichment step is a protein precipitation step. In a specific embodiment, the protein precipitation step is an alcohol fractionation step. In one embodiment, the second target protein enrichment step is an ultrafiltration / diafiltration step. In one embodiment, the second target protein enrichment step is a chromatographic enrichment step. 10

[0048] In a specific embodiment of the methods described above, the method further comprises the step of performing a third target protein enrichment step after contacting the composition with finely divided silicon dioxide (SiO2). In one embodiment, the third target protein enrichment step is a protein precipitation step. In a specific embodiment, the protein precipitation step is an alcohol fractionation step. In one embodiment, the third target protein 15 enrichment step is an ultrafiltration / diafiltration step. In one embodiment, the third target protein enrichment step is a chromatographic enrichment step.

[0049] In a specific embodiment of the methods described above, the chromatographic enrichment step comprises the sub-steps of: (i) contacting the plasma-derived target protein composition with a chromatographic resin under conditions suitable to bind the plasma-derived 20 target protein; and (ii) eluting the plasma-derived target protein from the chromatographic resin. In one specific embodiment, at least one impurity does not bind to the chromatographic resin in sub-step (i). In another specific embodiment, at least one impurity binds to the chromatographic resin in sub-step (i), but is not eluted from the chromatographic resin in sub-step (ii).

[0050] In a specific embodiment of the methods described above, the chromatographic 25 enrichment step comprises the sub-steps of: (i) contacting the first enriched plasma-derived target protein composition with a chromatographic resin under conditions suitable to bind at least one impurity; and (ii) separating the resin from the plasma-derived protein composition, wherein the plasma-derived target protein does not bind to the chromatographic resin in sub-step (i).

[0051] In a specific embodiment of the methods described above, the chromatographic resin is 30 selected from the group consisting of an anion exchange resin, a cation exchange resin, a 2024204779   11 Jul 2024 hydrophobic interaction resin, a mixed mode resin, a hydroxyapatite resin, a ligand affinity resin, an immunoaffinity resin, and a size exclusion resin. In one embodiment, the chromatographic resin is an anion exchange resin. In one embodiment, the chromatographic resin is a cation exchange resin. In one embodiment, the chromatographic resin is a hydrophobic interaction 5 resin. In one embodiment, the chromatographic resin is a mixed mode resin. In one embodiment, the chromatographic resin is a hydroxyapatite resin. In one embodiment, the chromatographic resin is a ligand affinity resin. In one embodiment, the chromatographic resin is an immunoaffinity resin. In one embodiment, the chromatographic enrichment step comprises separating at least one impurity from the target protein by size and / or shape using size exclusion 10 chromatography.

[0052] In a specific embodiment of the methods described above, the plasma-derived target protein is selected from an immunoglobulin (Ig), albumin, alpha-1-antitrypsin (AlPI), butyrylcholinesterase, a protein of the complement system, and an inter-alpha-trypsin inhibitor (lai). In one embodiment, the plasma-derived protein is an immunoglobulin (Ig). In one 15 embodiment, the plasma-derived protein is albumin. In one embodiment, the plasma-derived protein is alpha-1-antitrypsin. In one embodiment, the plasma-derived protein is butyrylcholinesterase. In one embodiment, the plasma-derived protein is a protein of the complement system. In one embodiment, the protein of the complement system is selected from the group consisting of Factor H (FH), Factor D, complement protein C3, and C4 binding 20 protein. In one embodiment, the plasma-derived protein is an inter-alpha-trypsin inhibitor.

[0053] In a specific embodiment of the methods described above, the plasma-derived target protein composition is a manufacturing intermediate.

[0054] In one embodiment, the present invention provides a method for preparing a plasma-derived Factor H composition having a reduced amount of a serine protease or a serine protease 25 zymogen, the method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H and at least one serine protease or serine protease zymogen; (b) separating the SiO2 from the composition; (c) eluting the serine protease or serine protease zymogen from the SiO2 under a solution condition in which a substantial 30 fraction of Factor H remains bound; and (d) eluting the Factor H from the SiO2. 2024204779   11 Jul 2024

[0055] In a specific embodiment of the methods described above, the solution condition under which the Factor H and at least one serine protease or serine protease zymogen bind the SiO2 comprises a pH below 7.0 and a conductivity of less than 11 mS / cm.

[0056] In a specific embodiment of the methods described above, the solution condition under 5 which the Factor H and at least one serine protease or serine protease zymogen bind the SiO2 comprises a pH between 4.5 and 6.5 and a conductivity of less than 6 mS / cm.

[0057] In a specific embodiment of the methods described above, the solution condition under which the Factor H and at least one serine protease or serine protease zymogen bind the SiO2 comprises a pH between 4.5 and 6.5 and a conductivity between 0.5 mS / cm and 5 mS / cm. 10

[0058] In a specific embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and a substantial fraction of the Factor H remains bound comprises a pH below 7.0 and a conductivity of less than 11 mS / cm.

[0059] In a specific embodiment of the methods described above, the solution condition under 15 which the serine protease or serine protease zymogen is eluted from the SiO2 and a substantial fraction of the Factor H remains bound comprises a pH between 4.5 and 6.5 and a conductivity of less than 6 mS / cm.

[0060] In a specific embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and a substantial 20 fraction of the Factor H remains bound comprises a pH between 5.0 and 6.5 and a conductivity between 0.5 mS / cm and 5 mS / cm.

[0061] In a specific embodiment of the methods described above, the solution condition under which the Factor H is eluted from the SiO2 comprises an ionic strength of at least 6 mS / cm.

[0062] In a specific embodiment of the methods described above, the solution condition under 25 which the Factor H is eluted from the SiO2 comprises an ionic strength of at least 11 mS / cm.

[0063] In a specific embodiment of the methods described above, the solution condition under which the Factor H is eluted from the SiO2 comprises a pH between 5.0 and 7.0. 2024204779   11 Jul 2024

[0064] In a specific embodiment of the methods described above, the solution condition under which the Factor H is eluted from the SiO2 comprises a pH between 7.0 and 8.0 and an ionic strength of between 4 mS / cm and 7 mS / cm.

[0065] In one embodiment, the present invention provides a method for preparing a Factor H 5   composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H and at least one serine protease; (b) separating the SiO2 from the composition; and (c) eluting the Factor H from the SiO2 under conditions in which a 10 substantial fraction of the serine protease or serine protease zymogen remains bound to the SiO2.

[0066] In a specific embodiment of the methods described above, the solution condition under which the Factor H and at least one serine protease or serine protease zymogen bind the SiO2 comprises a pH below 7.0 and a conductivity of less than 11 mS / cm.

[0067] In a specific embodiment of the methods described above, the solution condition under 15 which the serine protease or serine protease zymogen is eluted from the SiO2 and a substantial fraction of the Factor H remains bound comprises a pH between 4.5 and 6.5 and a conductivity of less than 6 mS / cm.

[0068] In a specific embodiment of the methods described above, the solution condition suitable to bind the Factor H and at least one serine protease or serine protease zymogen 20 comprises a pH between 5.0 and 6.0 and a conductivity of between 0.5 mS / cm and 5.0 mS / cm.

[0069] In a specific embodiment of the methods described above, the solution condition under which the Factor H is eluted from the SiO2 and a substantial fraction of the serine protease or serine protease zymogen remains bound comprises a pH between 5.0 and 7.0 and a conductivity of at least 11 mS / cm. 25

[0070] In a specific embodiment of the methods described above, the solution condition under which the Factor H is eluted from the SiO2 and a substantial fraction of the serine protease or serine protease zymogen remains bound comprises a pH between 7.0 and 8.0 and a conductivity of between 2 mS / cm and 10 mS / cm.

[0071] In a specific embodiment of the methods described above, the conductivity of the 30 solution condition is between 4 mS / cm and 7 mS / cm. 2024204779   11 Jul 2024

[0072] In a specific embodiment of the methods described above, the conductivity of the solution condition is between 5 mS / cm and 6 mS / cm.

[0073] In one embodiment, the present invention provides method for preparing a Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the 5 method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H but not a substantial fraction of the at least one serine protease or serine protease zymogen; (b) separating the SiO2 from the composition; and (c) eluting the Factor H from the SiO2. 10

[0074] In a specific embodiment of the methods described above, the solution condition under which the Factor H binds to the SiO2 and a substantial fraction of the serine protease or serine protease zymogen does not bind to the SiO2 comprises a pH of between 5.0 and 7.0 and a conductivity of no more than 14 mS / cm.

[0075] In a specific embodiment of the methods described above, the conductivity of the 15 solution condition is between 9 mS / cm and 14 mS / cm.

[0076] In one embodiment, the present invention provides a method for preparing a Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under 20 conditions suitable to bind the at least one serine protease or serine protease zymogen but not a substantial fraction of the Factor H; and (b) separating the SiO2 from the composition.

[0077] In a specific embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and a substantial fraction of the Factor H does not bind to the SiO2 comprises a pH between 5.0 and 7.0 and a conductivity 25 of at least 11 mS / cm.

[0078] In a specific embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen binds to the SiO2 and a substantial fraction of the Factor H does not bind to the SiO2 comprises a pH between 7.0 and 8.0 and a conductivity of between 2 mS / cm and 10 mS / cm. 2024204779   11 Jul 2024

[0079] In a specific embodiment of the methods described above, the conductivity of the solution condition is between 4 mS / cm and 7 mS / cm.

[0080] In a specific embodiment of the methods described above, the conductivity of the 5 solution condition is between 5 mS / cm and 6 mS / cm.

[0081] In one embodiment, the present invention provides a method for preparing a Factor H composition, the method comprising the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H; (b) washing the SiO2 with a solution 10 comprising a pH between 5.0 and 7.0 and a conductivity of less than 4 mS / cm; and (c) eluting Factor H from the SiO2 with a solution comprising a pH between 7.0 and 8.0 and a conductivity greater than 10 mS / cm.

[0082] In a specific embodiment of the methods described above, the solution used to wash the SiO2 comprises a pH between 5.5 and 6.5. 15

[0083] In a specific embodiment of the methods described above, the solution used to wash the SiO2 comprises a pH between of 6.0±0.2.

[0084] In a specific embodiment of the methods described above, the solution used to elute Factor H comprises a conductivity of at least 20 mS / cm.

[0085] In a specific embodiment of the methods described above, the solution used to elute 20 Factor H comprises a conductivity of between 25 mS / cm and 40 mS / cm.

[0086] In a specific embodiment of the methods described above, the starting composition containing Factor H is a suspended Cohn fraction II+III precipitate, or equivalent fraction thereof.

[0087] In a specific embodiment of the methods described above, the starting composition 25 containing Factor H is a suspended Kistler / Nitschmann Precipitate A, or equivalent fraction thereof. 2024204779   11 Jul 2024

[0088] In a specific embodiment of the methods described above, the starting composition containing Factor H is a suspended Kistler / Nitschmann Precipitate B, or equivalent fraction thereof.

[0089] In a specific embodiment of the methods described above, the method further 5 comprises the step of precipitating at least one impurity from the recovered Factor H solution, wherein Factor H is not precipitated.

[0090] In a specific embodiment of the methods described above, the precipitation step is PEG precipitation.

[0091] In a specific embodiment of the methods described above, the PEG precipitation 10 comprises precipitation with PEG 4000 at a final concentration between 3% and 7%.

[0092] In a specific embodiment of the methods described above, the final concentration of PEG 4000 is 5±0.5%.

[0093] In a specific embodiment of the methods described above, the method further comprises the step of precipitating Factor H from the recovered Factor H solution. 15

[0094] In a specific embodiment of the methods described above, the precipitation step is PEG precipitation.

[0095] In a specific embodiment of the methods described above, the PEG precipitation comprises precipitation with PEG 4000 at a final concentration between 10% and 15%.

[0096] In a specific embodiment of the methods described above, the final concentration of 20 PEG 4000 is 12±0.5%.

[0097] In a specific embodiment of the methods described above, the method further comprises a step of enriching Factor H by chromatography.

[0098] In a specific embodiment of the methods described above, the chromatographic enrichment step comprises anion exchange chromatography. 25

[0099] In a specific embodiment of the methods described above, the chromatographic enrichment step comprises heparin affinity chromatography. 2024204779   11 Jul 2024

[0100] In a specific embodiment of the methods described above, the chromatographic enrichment step comprises anion exchange chromatography followed by heparin affinity chromatography.

[0101] In a specific embodiment of the methods described above, the method further 5 comprises at least one dedicated viral removal or inactivation step.

[0102] In a specific embodiment of the methods described above, the method comprises a nanofiltration step.

[0103] In a specific embodiment of the methods described above, the method further comprises a step of concentrating the Factor H composition comprising 10 ultrafiltration / diafiltration.

[0104] In one embodiment, the present invention provides a method for preparing an interalpha-trypsin inhibitor (lai) composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a solution containing lai and at least one serine protease with finely divided silicon dioxide (SiO2) under 15 conditions suitable to bind the lai and at least one serine protease; (b) separating the SiO2 from the composition; (c) eluting the serine protease or serine protease zymogen from the SiO2 under conditions in which a substantial fraction of the lai remains bound; and (d) eluting the lai from the SiO2.

[0105] In one embodiment, the present invention provides a method for preparing an inter-20 alpha-trypsin inhibitor (lai) composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a solution containing lai and at least one serine protease with finely divided silicon dioxide (SiO2) under conditions suitable to bind the lai and at least one serine protease; (b) separating the SiO2 from the composition; and (c) eluting the lai from the SiO2 under conditions in which a substantial 25 fraction of the serine protease or serine protease zymogen remains bound to the SiO2.

[0106] In one embodiment, the present invention provides a method for preparing an interalpha-trypsin inhibitor (lai) composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) contacting a solution containing lai and at least one serine protease with finely divided silicon dioxide (SiO2) under 30   conditions suitable to bind the lai but not a substantial fraction of the least one serine protease or 2024204779   11 Jul 2024 serine protease zymogen; (b) separating the SiO2 from the composition; and (c) eluting the lai from the SiO2.

[0107] In one embodiment, the present invention provides a method for preparing a interalpha-trypsin inhibitor (lai) composition having a reduced amount of a serine protease or a 5 serine protease zymogen, the method comprising the steps of: (a) contacting a solution containing inter-alpha-trypsin inhibitor (lai) and at least one serine protease with finely divided silicon dioxide (SiO2) under conditions suitable to bind the serine protease or serine protease zymogen but not the inter-alpha-trypsin inhibitor (lai); and (b) separating the SiO2 from the composition. 10

[0108] In one embodiment, the present invention provides a method for preparing an Immunoglobulin G (IgG) composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising the steps of: (a) precipitating a cryo-poor plasmid fraction, in a first precipitation step, with between about 6% and about 10% alcohol at a pH of between about 7.0 and about 7.5 to obtain a first precipitate and a first supernatant; (b) 15 precipitating IgG from the first supernatant, in a second precipitation step, with between about 20% and about 25% alcohol at a pH of between about 6.7 and about 7.3 to form a second precipitate; (c) re-suspending the second precipitate to form a suspension; (d) contacting the suspension with finely divided silicon dioxide (SiO2) under a solution condition suitable to bind a serine protease or serine protease zymogen; and (e) separating the SiO2 from the suspension to 20 form a clarified suspension.

[0109] In a specific embodiment of the methods described above, the method further comprises the steps of: (f) precipitating IgG from the clarified suspension formed in step (e), in a third precipitation step, with between about 22% and about 28% alcohol at a pH of between about 6.7 and about 7.3 to form a third precipitate; (g) re-suspending the third precipitate to form 25 a suspension; and (h) separating the soluble fraction from the suspension formed in step (e), thereby forming an enriched IgG composition.

[0110] In a specific embodiment of the methods described above, the method further comprises an anion exchange chromatography enrichment step. [OlH] In a specific embodiment of the methods described above, the method further 30 comprises a cation exchange chromatography enrichment step. 2024204779   11 Jul 2024

[0112] In a specific embodiment of the methods described above, the method further comprises at least one dedicated viral inactivation or removal step.

[0113] In a specific embodiment of the methods described above, the method comprises a solvent / detergent (S / D) viral inactivation step. 5

[0114] In a specific embodiment of the methods described above, the method comprises a nanofiltration step.

[0115] In a specific embodiment of the methods described above, the method comprises an incubation step at low pH.

[0116] In a specific embodiment of the methods described above, step (b) comprises adjusting 10   the ethanol concentration of the first supernatant formed in step (a) to about 25% (v / v) at a temperature between about -7°C and about -9°C.

[0117] In a specific embodiment of the methods described above, the temperature is about -9°C.

[0118] In a specific embodiment of the methods described above, step (c) comprises re-15 suspending the precipitate of step (b) with a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted with between 300 mL and 700 mL of glacial acetic acid per 1000 L of buffer.

[0119] In a specific embodiment of the methods described above, step (d) comprises the addition SiO2 to a final concentration of between about 0.02 grams per gram precipitate formed 20 in step (b) and about 0.06 grams per gram precipitate formed in step (b).

[0120] In a specific embodiment of the methods described above, the solution condition suitable to bind a serine protease or serine protease zymogen comprises a pH between 4.5 and 6.0 and a conductivity of between 0.1 mS / cm and 3 mS / cm.

[0121] In a specific embodiment of the methods described above, the pH is between 4.9 and 25    5.3.

[0122] In a specific embodiment of the methods described above, the conductivity is between 0.5 mS / cm and 2 mS / cm. 2024204779   11 Jul 2024

[0123] In a specific embodiment of the methods described above, step (e) comprises the substeps of: (i) washing the filter press with at least 3 filter press dead volumes of a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted with between 50 mL and 200 mL of glacial acetic acid per lOOOL of buffer, thereby forming a wash solution; and (ii) 5 combining the filtrate of step (f) with the wash solution of step (g), thereby forming a solution.

[0124] In a specific embodiment of the methods described above, further comprising the substep of: (iii) treating the solution with a detergent.

[0125] In a specific embodiment of the methods described above, step (h) further comprises solvent and detergent (S / D) treatment of the enriched IgG composition. 10

[0126] In a specific embodiment of the methods described above, the enriched IgG composition obtained in step (h) contains at least 85% of the IgG content found in the cryo-poor plasma fraction used in step (a).

[0127] In a specific embodiment of the methods described above, the enriched IgG composition obtained in step (h) contains at least 90% of the IgG content found in the cryo-poor 15 plasma fraction used in step (a).

[0128] In a specific embodiment of the methods described above, the amount of a serine protease or a serine protease zymogen has been reduced by at least 90%.

[0129] In a specific embodiment of the methods described above, the amount of a serine protease or a serine protease zymogen has been reduced by at least 95%. 20

[0130] In a specific embodiment of the methods described above, the amount of a serine protease or a serine protease zymogen has been reduced by at least 98%.

[0131] In a specific embodiment of the methods described above, the amount of a serine protease or a serine protease zymogen has been reduced by at least 99%.

[0132] In a specific embodiment of the methods described above, the serine protease or a 25 serine protease zymogen is FXIa.

[0133] In a specific embodiment of the methods described above, the composition is contacted with SiO2 at a final concentration of at least 1 g SiO2 / g protein. 2024204779   11 Jul 2024

[0134] In a specific embodiment of the methods described above, the composition is contacted with SiO2 at a final concentration of at least 2 g SiO2 / g protein.

[0135] In a specific embodiment of the methods described above, the composition is contacted with SiO2 at a final concentration of at least 2.5 g SiO2 / g protein. 5

[0136] In a specific embodiment of the methods described above, the serine protease or serine protease zymogen is Factor XI.

[0137] In a specific embodiment of the methods described above, the serine protease or serine protease zymogen is Factor XII.

[0138] In a specific embodiment of the methods described above, the serine protease or serine 10 protease zymogen is Factor Xia.

[0139] In a specific embodiment of the methods described above, the serine protease or serine protease zymogen is Factor Xlla.

[0140] In one embodiment, the present invention provides a plasma-derived protein composition prepared by a process comprising a method for reducing serine protease activity 15   according to any one of the preceding claims.

[0141] In a specific embodiment of the compositions described above, the composition is formulated for administration to a subject.

[0142] In a specific embodiment of the compositions described above, the composition is formulated for intravenous, intramuscular, or subcutaneous administration. 20

[0143] In a specific embodiment of the compositions described above, the composition is aqueous.

[0144] In a specific embodiment of the compositions described above, the composition is lyophilized.

[0145] In one embodiment, the present invention provides a method for treating a disease 25 associated with aberrant activity of a plasma protein in a subject in need thereof, the method comprising administering a plasma-derived protein composition according to any one of claims 124 to 128. In one embodiment, the composition comprises a plasma-derived protein is selected 2024204779   11 Jul 2024 from an immunoglobulin (Ig), albumin, alpha-1-antitrypsin (AlPI), butyrylcholinesterase, a protein of the complement system, and an inter-alpha-trypsin inhibitor (lai). BRIEF DESCRIPTION OF THE DRAWINGS 5

[0146] Figure 1. Overview of an exemplary plasma fractionation scheme.

[0147] Figure 2. Factor H content in select fractions of an industrial-scale plasma protein fractionation, as measured by ELISA

[0148] Figure 3. Illustration of Factor H and amidolytic activity (as measured using substrate CS2166) eluted from SiO2 under solution conditions with varying conductivities at pH 7.5. 10 DETAILED DESCRIPTION OF THE INVENTION I. Introduction

[0149] Given the broad use of therapeutic plasma-derived blood protein compositions, such as immune globulin compositions, blood coagulation factors, coagulation factor inhibitors, and 15 proteins of the complement system, ensuring the safety of these compositions is of paramount importance. Recent concerns over the amidolytic content of these compositions paired with the occurrence of thromboembolic events in patients being administered plasma-derived protein compositions, has highlighted a need in the art for method for reducing serine proteases (e.g., FXIa and FXIIa) and serine protease zymogens (e.g., FXI and FXII) during the manufacturing of 20 these biologies. Advantageously, the present invention is based at least in part on the surprising finding that finely divided silicon dioxide (SiCh) can be used to bind serine proteases and serine protease zymogens present in plasma-derived protein compositions. As such, methods are provided herein for reducing the concentration of serine proteases and serine protease zymogens during the manufacture of plasma-derived protein compositions. 25

[0150] In certain aspects, the present invention provides manufacturing methods based on the surprising finding that finely divided silicon dioxide (SiO2) can be used to remove significant amounts of serine protease (e.g., FXIa and FXIIa) and serine protease zymogen (e.g., FXI and FXII) from plasma-derived protein solutions. As such, the methods provided herein may be easily integrated into existing manufacturing procedures, for example, the fractionation of pooled 30 plasma samples, preferably human plasma samples, by ethanol in the cold (reviewed in Schultze 2024204779   11 Jul 2024 H E, Heremans J F; Molecular Biology of Human Proteins. Volume I: Nature and Metabolism of Extracellular Proteins 1966, Elsevier Publishing Company; p. 236-317). However, the methods provided herein are in no way limited in their use to manufacturing methods including ethanol fractionation. Other methodologies for the purification of plasma-derived proteins are also 5 compatible with the methods provided herein, for example, polymer (e.g., PEG) fractionation and chromatographic methodologies (e.g., anion and / or cation exchange chromatography, affinity chromatography, immuno-affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, mixed mode chromatography, and the like).

[0151] Furthermore, unlike other biologies that are produced via recombinant expression of 10 DNA vectors in host cell lines, plasma-derived proteins are fractionated from human blood and plasma donations. Thus, the supply of these products cannot be increased by simply increasing the volume of production. Rather the level of commercially available blood products is limited by the available supply of blood and plasma donations. This dynamic results in a shortage in the availability of raw human plasma for the manufacture of new plasma-derived blood factors that 15 have lesser established commercial markets, including Complement Factor H (CFH) and interalpha-trypsin inhibitor proteins (lalp).

[0152] Due to the lack of plasma available for the manufacture of new plasma-derived products, their manufacture must be integrated into the existing framework of the established manufacturing processes for plasma-derived products such as immunoglobulins and albumin. 20 Factor H, implicated as a potential therapeutic for AMD, aHUS, and MPGN, among other conditions, is one such plasma-derived blood product that is gaining the attention of physicians. However, due to the resources devoted to, for example, IgG gamma globulin manufacture, methods are needed for the manufacture of Factor H that can be introduced into the existing manufacturing schemes. Several methods have been suggested to achieve just this, however, 25 many of these proposed solutions require modification of the existing manufacturing scheme for established products. Such changes will require new regulatory approvals for the established products and may even result in alterations of the characteristics of the established products.

[0153] For example, WO 2007 / 066017 describes methods for the production of Factor H preparations from the supernatant of a cryoprecipitate. The disclosed method consists of 30 preparing a supernatant of a cryoprecipitate, submitting the supernatant to anion exchange chromatography (AEC), submitting the flow through from the AEC to heparin affinity 2024204779   11 Jul 2024 chromatography (HAC), submitting the relevant eluate from the HAC to strong cation exchange chromatography (CEC), submitting the relevant eluate from the CEC to strong anion exchange chromatography (sAEC) and eluting the Factor H from the sAEC. Disadvantageous^, cryoprecipitate supernatants are common intermediate fractions in the manufacturing processes 5 of many commercially important plasma-derived blood products, including IgG gamma globulins (IVIG and subcutaneous) and albumin. Submitting this fraction to chromatography steps will alter the cryoprecipitate supernatant and would require that the manufacturing processes of the established downstream blood products be adapted in unknown fashions. In addition to requiring a complete revalidation and possible redesign of these manufacturing 10 processes, regulatory re-approval of the manufacturing procedures from key regulatory agencies is needed.

[0154] Likewise, WO 2008 / 113589 describes methods for the production of Factor H preparations from human plasma. Specifically, this publication describes the purification of Factor H from three known plasma processing fractions, namely a Cohn-Oncley Fraction I 15 supernatant, a Cohn-Oncley Fraction III precipitate, and a Kistler / Nitschmann Precipitate B fraction. With respect to the first method, WO 2008 / 113589 discloses that Factor H can be removed from a Cohn-Oncley Fraction I supernatant by the addition of a heparin affinity chromatography step. Disadvantageously, the Cohn-Oncley Fraction I supernatant is a common intermediate fraction in the manufacturing processes of many commercially important plasma- 20 derived blood products, including IgG gamma globulins (IVIG and subcutaneous) and albumin. Similarly, many immunoglobulin (e.g., IgG, IVIG, etc.) manufacturing processes do not rely on Cohn-Oncley Fraction III precipitation or Kistler / Nitschmann Precipitate B steps, for example Gammagard® Liquid and Kiovig (Baxter International Inc.). The disadvantage of the introduction of additional steps, such as a heparin affinity chromatography, Fraction III 25 precipitation, or Precipitate B steps, into the manufacturing schemes of established blood products, as outlined above, is that it requires revalidation of the manufacturing procedure, regulatory re-approval of the manufacturing procedures from key regulatory agencies, and may further have unforeseen consequences for the yield and / or purity of the otherwise established product. 30

[0155] As such, a need remains in the art for methods of manufacturing Factor H that do not require the use of additional input plasma or the redesign and regulatory re-approval of existing manufacturing processes for commercially important plasma-derived blood products, such as 2024204779   11 Jul 2024 albumin and IgG gamma globulins for intravenous (IVIG) or subcutaneous administration. Advantageously, the present invention is based at least in part on the surprising discovery that Factor H, serine proteases, and serine protease zymogens can be simultaneously bound to finely divided silicon dioxide (SiO2) thereby separating serine proteases and serine protease zymogen 5 from a first protein of interest not bound to (e.g., IgG) and then separated by differentially eluting Factor H and the serine protease and serine protease zymogens from the SiO2. Similarly, the present invention is based at least in part on the surprising discovery that lalp, serine proteases, and serine protease zymogens can be simultaneously bound to finely divided silicon dioxide (SiO2) and then separated by differentially eluting lalp and the serine protease and serine 10 protease zymogens from the SiO2. II. Definitions

[0156] As used herein, “Factor H” refers to a protein component of the alternative pathway of complement encoded by the complement factor H gene (for example, CFH; NM000186; GeneID:3075; UniProt ID P08603; Ripoche et al., Biochem. J. 249:593-602(1988)). Factor H is 15 translated as a 1,213 amino acid precursor polypeptide which is processed by removal of an 18 amino acid signal peptide, resulting in the mature Factor H protein (amino acids 19-1231). As used in the present invention, Factor H encompasses any natural variants, alternative sequences, isoforms or mutant proteins that can be found in a plasma sample, for example a human plasma sample. Examples of Factor H mutations found in the human population include, without 20 limitation, Y402H; V62I; R78G; R127L; A224; Q400K; C431S; T493R; C536R; 155IT; R567G; C630W; C673S; C673Y; E850K; S890I; H893R; C915S; E936D; Q950H; Y951H; T956M; C959Y; W978C; N997T; V1007I; V1007L; A1010T; T1017I; Y1021F; C1043R; N1050Y; 11059T; Q1076R; R1078S; DI 119G; VI134G; Y1142D; QI 143E; W1157R; Cl 163W; W1183L; W1183R; T1184R; L1189R; S1191L; G1194D; V1197A; E1198A; F1199S; R1210C; 25   R1215G; R1215Q; YPTCAKR1225:1231FQS; and P1226S. Many of the these mutations have been found to be associated with a variety of diseases and disorders, including, atypical haemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD), membranoproliferative glomulonephritis type II (MPGNII), CFH deficiency, and basal laminar drusen. Factor H also includes proteins containing post-translational modifications. For 30 example, Factor H is believed to be modified by N-acetylglucosamine (GlcNAc) at residues 529, 718, 802, 822, 882, 911, 1029, and 1095. 2024204779   11 Jul 2024

[0157] As used herein, “Inter-alpha-Inhibitor proteins” or “lalp” refers to a family of plasma protease inhibitors comprised of polypeptides encoded by one or more of the Alpha-1-microglobulin / bikunin precursor gene (AMBP; UniGene ID:231948, bikunin polypeptide), Interalpha (globulin) inhibitor Hl gene (ITIH1; UniGene ID:224173, Hl polypeptide), Inter-alpha 5 (globulin) inhibitor H2 gene (ITIH2; Unigene ID: 139782, H2 polypeptide), Inter-alpha (globulin) inhibitor H3 gene (ITIH3; UniGene ID: 140017, H3 polypeptide), or Inter-alpha (globulin) inhibitor H4 (plasma Kallikrein-sensitive glycoprotein, H4 polypeptide) gene (ITIH4; UniGene ID:3321613). Exemplary lalp protease inhibitors include, without limitation, lai (bikunin, Hl, and H2 polypeptides); Pal (bikunin and H3 polypeptides), laLI (bikunin and H2 10 polypeptides), IaIH4P (H4 polypeptide), and bikunin (Salier, J, et al., supra).

[0158] As used herein, “cryo-poor plasma” refers to the supernatant created after the removal of cryo-precipitate formed by thawing plasma or pooled plasma at temperatures near freezing, e.g., at temperatures below about 10°C, preferably at a temperature no higher than about 6°C. In the context of the present invention, plasma may refer interchangeably to recovered plasma (i.e., 15 plasma that has been separated from whole blood ex vivo) or source plasma (i.e., plasma collected via plasmapheresis). Cryo-precipitation is commonly performed, for example, by thawing previously frozen pooled plasma, which has already been assayed for safety and quality considerations, although fresh plasma may also be used. After complete thawing of the frozen plasma at low temperature, separation of the solid cryo-precipitates from the liquid supernatant is 20 performed in the cold (e.g., < 6°C) by centrifugation of filtration.

[0159] As used herein, a “Cohn pool” refers to the starting material used for the fractionation of a plasma sample or pool of plasma samples. Cohn pools include whole plasma, cryo-poor plasma samples, and pools of cryo-poor plasma samples that may or may not have been subjected to a pre-processing step. In certain embodiments, a Cohn pool is a cryo-poor plasma 25 sample from which one or more blood factor have been removed in a pre-processing step, for example, adsorption onto a solid phase (e.g., aluminum hydroxide, finely divided silicon dioxide, etc.), or chromatographic step (e.g., ion exchange or heparin affinity chromatography). Various blood factors, including but not limited to Factor Eight Inhibitor Bypass Activity (FEIBA), Factor IX-complex, Factor Vll-concentrate, or Antithrombin Ill-complex, may be isolated from 30 the cryo-poor plasma sample to form a Cohn pool. 2024204779   11 Jul 2024

[0160] As used herein, a “Fraction II+III filter cake” refers to a solid phase recovered after the filtration or centrifugation of a Cohn-Oncley or equivalent Fraction II+III paste suspension. In a preferred embodiment, a Fraction II+III suspension will be treated with an adsorptive material, for example, finely divided silicon dioxide, to remove impurities such as lipids, fibrinogen, 5 amidolytic activity, prekallikren activity, and lipoproteins. In another preferred embodiment, filter aid may be added to the Fraction II+III suspension prior to centrifugation or filtration. In a most preferred embodiment, a Fraction II+III suspension will be treated with both an adsorptive material and a filter aid prior to centrifugation or filtration. Upon separation of the clarified Fraction II+III suspension supernatant, the recovered solid phase material is referred to as the 10 Fraction II+III filter cake.

[0161] As used herein, “finely divided silicon dioxide” or “finely divided silica” refers to an oxide of silicon having the formula SiO2, manufactured in a fashion that allows for the adsorption of Factor H onto its surface. Exemplary forms of finely divided silicon dioxide suitable for use in the methods of the present invention include, without limitation, fumed silica, 15 pyrogenic silica, Aerosil®, Cab-O-Sil™, colloidal silica, diatomaceous earth, and the like. In a preferred embodiment, a commercial hydrophilic fumed silica product is used for the methods provided herein. Non-limiting examples of these products include those marketed by Evonik Industries under the trade name Aerosil® (e.g., Aerosil 90, Aerosil 130, Aerosil 150, Aerosil 200, Aerosil 300, Aerosil 380, Aerosil OX 50, Aerosil EG 50, Aerosil TT 600, Aerosil 200 SP, 20 Aerosil 300 SP, and Aerosil 300 / 30).

[0162] As used herein, a “disease or disorder associated with Factor H dysfunction” refers to any disease, disorder, or condition in a subject that is caused by, characterized by, or results in a reduced level of Factor H activity in the subject. For purposes of the present invention, Factor H activity may refer to the ability of Factor H to bind a protein or ligand, for example, C3b, C3bBb, 25 C3b2Bb, csbC3b, complement factor B (CFB), C-reactive protein, endothelial cells, glycosaminoglycans (GAGs), or alternatively, may refer to its Factor I cofactor activity or its ability to accelerate the irreversible dissociation of C3bBb and C3b2Bb. In one embodiment, a disease or disorder associated with Factor H dysfunction results in a C3 deficiency and susceptibility to bacterial infections. In some instances, diseases or disorders associated with 30 Factor H dysfunction include conditions that are caused by or linked to mutations and polymorphism in the CFH gene encoding Factor H (for review, see, Barlow et al., Adv Exp Med Biol. 2008;632:l 17-42, the disclosure of which is herein incorporated by reference in its entirety 2024204779   11 Jul 2024 for all purposes). Diseases that have been linked to mutations or polymorphisms in the CFH gene include, without limitation, Factor H deficiency, atypical haemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD), membranoproliferative glomulonephritis type II (MPGNII; de Cordoba and de Jorge, Clinical and Experimental Immunology 151, 1-13 5   (2008)), myocardial infarction (Kardys et al., Journal of the American College of Cardiology 47, 1568-1575 (2006); Mooijaart et al., Experimental Gerontology 42, 1116-1122 (2007); Nicaud et al., Journal of Molecular Medicine 85, 771-775 (2007); Pai et al., European Heart Journal 28, 1297-1303 (2007); Stark et al., Clinical Science (Lond) 113, 213-218 (2007)), coronary heart disease / coronary artery disease (CAD / CHD; (Meng et al., BMC Medical Genetics 8, 62 (2007); 10 Pulido et al., Mayo Clinic Proceedings 82, 301-307 (2007); Topol et aL, Human Molecular Genetics 15 Spec No 2, RI 17-R123 (2006)), and Alzheimer’s disease (Hamilton et aL, Neuromolecular Medicine 9, 331-334 (2007); Zetterberg et al., American Journal of Ophthalmology 143, 1059-1060 (2007)). The disclosures of the forgoing references describing the associations between mutations and polymorphisms in the CFH gene and diseases associated 15 with Factor H dysfunction are herein incorporated by reference in their entireties for all purposes.

[0163] As used herein, a “disease or disorder associated with abnormal alternative pathway complement activity” refers to a disease, disorder, or condition that results from uncontrolled or aberrant activation of the alternative pathway of complement. Generally, uncontrolled or 20 aberrant activation of the alternative pathway of complement can result in bystander damage of host cells and tissues, as well as a depletion of C3 and corresponding susceptibility to pathogenic infections (e.g., fungal, bacterial, viral, and protistal). Examples of diseases and disorders associated with abnormal alternative pathway complement activity include, without limitation, various autoimmune diseases (such as rheumatoid arthritis, IgA nephropathy, asthma, systemic 25 lupus erythematosus, multiple sclerosis, Anti-Phospholipid syndrome, ANCA-associated vasculitis, pemphigus, uveitis, myathemia gravis, Hashimoto’s thyroiditis), Renal diseases (such as IgA nephropathy, hemolytic uremic syndrome, membranoproliferative glomerulonephritis) other disease such as asthma, Alzheimer disease, adult macular degeneration, proximal nocturnal hemoglobinuria, abdominal aortic aneurism, ischemia, and sepsis. 30

[0164] As used herein, the term “ultrafiltration (UF)” encompasses a variety of membrane filtration methods in which hydrostatic pressure forces a liquid against a semi-permeable membrane. Suspended solids and solutes of high molecular weight are retained, while water and 2024204779   11 Jul 2024 low molecular weight solutes pass through the membrane. This separation process is often used for purifying and concentrating macromolecular (103 - 106 Da) solutions, especially protein solutions. A number of ultrafiltration membranes are available depending on the size of the molecules they retain. Ultrafiltration is typically characterized by a membrane pore size between 5   1 and 1000 kDa and operating pressures between 0.01 and 10 bar.

[0165] As used herein, the term "diafiltration" is performed with the same or a similar membrane as ultrafiltration and is typically performed in a tangential flow filtration mode. During diafiltration, buffer is introduced into the recycle tank while filtrate is removed from the unit operation. In processes where the product is in the retentate (for example, Factor H), 10 diafiltration is particularly useful for separating protein from small molecules like sugars and salts. In certain cases, diafiltration can be used to exchange the solution, buffer, or individual components of a buffering system.

[0166] As used herein, the term "mixing" describes an act of causing equal distribution of two or more distinct compounds or substances in a solution or suspension by any form of agitation. 15 Complete equal distribution of all ingredients in a solution or suspension is not required as a result of “mixing” as the term is used in this application.

[0167] As used herein, the term "solvent" encompasses any liquid substance capable of dissolving or dispersing one or more other substances. A solvent may be inorganic in nature, such as water, or it may be an organic liquid, such as ethanol, acetone, methyl acetate, ethyl 20 acetate, hexane, petrol ether, etc. As used in the term "solvent detergent treatment," solvent denotes an organic solvent (e.g., tri-N-butyl phosphate), which is part of the solvent detergent mixture used to inactivate lipid-enveloped viruses in solution.

[0168] As used herein, the term "detergent" is used in this application interchangeably with the term "surfactant" or "surface acting agent." Surfactants are typically organic compounds that are 25 amphiphilic, i.e., containing both hydrophobic groups ("tails") and hydrophilic groups ("heads"), which render surfactants soluble in both organic solvents and water. A surfactant can be classified by the presence of formally charged groups in its head. A non-ionic surfactant has no charge groups in its head, whereas an ionic surfactant carries a net charge in its head. A zwitterionic surfactant contains a head with two oppositely charged groups. Some examples of 30 common surfactants include: Anionic (based on sulfate, sulfonate or carboxylate anions): perfluorooctanoate (PFOA or PFO), perfluorooctanesulfonate (PFOS), sodium dodecyl sulfate 2024204779   11 Jul 2024 (SDS), ammonium lauryl sulfate, and other alkyl sulfate salts, sodium laureth sulfate (also known as sodium lauryl ether sulfate, or SLES), alkyl benzene sulfonate; cationic (based on quaternary ammonium cations): cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide, and other alkyltrimethylammonium salts, cetylpyridinium 5 chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT); Long chain fatty acids and their salts: including caprylate, caprylic acid, heptanoat, hexanoic acid, heptanoic acid, nanoic acid, decanoic acid, and the like; Zwitterionic (amphoteric): dodecyl betaine; cocamidopropyl betaine; coco ampho glycinate; nonionic: alkyl poly(ethylene oxide), alkylphenol poly(ethylene oxide), copolymers of 10 poly(ethylene oxide) and polypropylene oxide) (commercially known as Poloxamers or Poloxamines), alkyl polyglucosides, including octyl glucoside, decyl maltoside, fatty alcohols (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates (Tween 20, Tween 80, etc.), Triton detergents, and dodecyl dimethylamine oxide.

[0169] As used herein, the term "therapeutically effective amount or dose" or 15 "sufficient / effective amount or dose," refers to a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3,1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The 20   Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins; the disclosures of which are herein incorporated by reference in their entireties for all purposes).

[0170] As used in this application, the term "spraying" refers to a means of delivering a liquid substance into a system, e.g., during an alcohol precipitation step, such as a Cohn fractionation I 25 or 11+111 precipitation step, in the form of fine droplets or mist of the liquid substance. Spraying may be achieved by any pressurized device, such as a container (e.g., a spray bottle), that has a spray head or a nozzle and is operated manually or automatically to generate a fine mist from a liquid. Typically, spraying is performed while the system receiving the liquid substance is continuously stirred or otherwise mixed to ensure rapid and equal distribution of the liquid 30 within the system. 2024204779   11 Jul 2024

[0171] As used herein, the term “about” denotes an approximate range of plus or minus 10% from a specified value. For instance, the language “about 20%” encompasses a range of 18-22%. As used herein, about also includes the exact amount. Hence “about 20%” means “about 20%” and also “20%.” As used herein, “about” refers to a range of at or about the specified value. 5

[0172] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill in the art will recognize that the dose can be modified depending on the above factors or 10 based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.

[0173] As used herein, the term “prevent” refers to a decreased likelihood or reduced frequency of symptoms arising from a condition associated with the lack of function or 15   disfunction of a blood protein.

[0174] As used herein, the term “therapy,” “treatment,” and “amelioration” refer to any reduction in the severity of symptoms arising from a condition associated with the lack of function or disfunction of a blood protein. As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. Treatment can refer to any delay in onset, amelioration of 20 symptoms, improvement in patient survival, increase in survival time or rate, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment.

[0175] As used herein, the term “substantial fraction” refers to at least 10% of the population of a particular protein in a composition. For example, when referring to a substantial fraction of a serine protease in a composition, a substantial fraction of the serine protease corresponds to at 25 least 10% of the serine protease present in the composition. In one embodiment, a substantial fraction refers to at least 25% of the population of a particular protein in a composition. In another embodiment, a substantial fraction refers to at least 50% of the population of a particular protein in a composition. In another embodiment, a substantial fraction refers to at least 75% of the population of a particular protein in a composition. In yet other embodiments, a substantial 30 fraction refers to at least 10% of the population of a particular protein in a composition, or at 2024204779   11 Jul 2024 least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more of the population of a particular protein in a composition. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to 5    imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 2024204779   11 Jul 2024 III. Reduction of Serine Protease and Serine Protease Zymogen Content

[0176] In a first aspect, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived target protein composition by 5    binding the serine protease and / or serine protease zymogen to finely divided silicon dioxide (SiO2) and separating the SiO2 from the composition.

[0177] In one embodiment, the method comprises the steps of: (a) contacting the composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (b) separating the SiO2 from the composition to 10   remove the bound serine protease or serine protease zymogen. In a preferred embodiment, the serine protease or serine protease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa), Factor XI (FXI), and / or Factor XII (FXII).

[0178] Accordingly, in one embodiment, the invention provides a method for reducing the amount of Factor XI in a plasma-derived protein composition, the method comprising the steps 15    of: (a) contacting the composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind Factor XI; and (b) separating the SiO2 from the composition to remove the bound Factor XI.

[0179] In another embodiment, the invention provides a method for reducing the amount of Factor XIa in a plasma-derived protein composition, the method comprising the steps of: (a) 20    contacting the composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind Factor XIa; and (b) separating the SiO2 from the composition to remove the bound Factor XIa.

[0180] In another embodiment, the invention provides a method for reducing the amount of Factor XII in a plasma-derived protein composition, the method comprising the steps of: (a) 25    contacting the composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind Factor XII; and (b) separating the SiO2 from the composition to remove the bound Factor XII. 2024204779   11 Jul 2024

[0181] In yet another embodiment, the invention provides a method for reducing the amount of Factor Xlla in a plasma-derived protein composition, the method comprising the steps of: (a) contacting the composition with finely divided silicon dioxide (SiOs) under conditions suitable to bind Factor Xlla; and (b) separating the SiO2 from the composition to remove the bound Factor 5 Xlla.

[0182] In certain embodiments, the method described above further comprises the step of performing a first target protein enrichment step to form a first enriched composition, prior to contacting the composition with finely divided silicon dioxide (SiO2). In certain embodiments, the first target protein enrichment step is selected from a protein precipitation step (e.g., an 10 alcohol fractionation step), an ultrafiltration / diafiltration step, and a chromatographic step.

[0183] Accordingly, in one embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived target protein the method comprises the steps of: (a) forming a first enriched plasma-derived target protein composition by partially precipitating protein in a starting material derived from pooled plasma; 15   (b) contacting the first enriched composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (c) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen. In one embodiment, the partial precipitation is achieved using alcohol. In a preferred embodiment, the alcohol is ethanol. In another preferred embodiment, the serine protease or 20 serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII).

[0184] In another embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived target protein the method comprises the steps of: (a) forming a first enriched plasma-derived target protein composition by 25 ultrafiltering and / or diafiltering a starting material derived from pooled plasma; (b) contacting the first enriched composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (c) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), 30 Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). 2024204779   11 Jul 2024

[0185] In yet another embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived target protein the method comprises the steps of: (a) forming a first enriched plasma-derived target protein composition by contacting a starting material derived from pooled plasma with a chromatographic resin; (b) 5 contacting the first enriched composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (c) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen. In certain embodiments, the chromatographic resin is selected from an anion exchange resin, a cation exchange resin, a hydrophobic interaction resin, a mixed mode resin, a 10 hydroxyapatite resin, a ligand affinity resin, an immunoaffinity resin, and a size exclusion resin. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII).

[0186] In certain embodiments, the methods described above further comprises the step of performing a second target protein enrichment step to form a second enriched composition, prior 15 to contacting the composition with finely divided silicon dioxide (SiO2). In certain embodiments, the first target protein enrichment step is selected from a protein precipitation step (e.g., an alcohol fractionation step), an ultrafiltration / diafiltration step, and a chromatographic step.

[0187] Accordingly, in one embodiment, the invention provides a method for reducing the 20 amount of a serine protease or a serine protease zymogen in a plasma-derived target protein the method comprises the steps of: (a) performing a first target protein enrichment step to form a first enriched plasma-derived target protein composition; (b) performing a second target protein enrichment step to form a second enriched plasma-derived target protein composition; (c) contacting the second enriched composition with finely divided silicon dioxide (SiO2) under 25 conditions suitable to bind at least one serine protease or serine protease zymogen; and (d) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In certain embodiments, the combination of first and second enrichment steps is selected from any one of 30 variations Var. 1 to Var. 100, found in Table 1. Table 1. Exemplary embodiments for the combination of first and second enrichment steps. 2024204779   11 Jul 2024 First Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Second Enrichment Step Ppt Var. 1 Var. 11 Var. 21 Var. 31 Var. 41 Var. 51 Var. 61 Var. 71 Var. 81 Var. 91 UF / DF Var. 2 Var. 12 Var. 22 Var. 32 Var. 42 Var. 52 Var. 62 Var. 72 Var. 82 Var. 92 AEC Var. 3 Var. 13 Var. 23 Var. 33 Var. 43 Var. 53 Var. 63 Var. 73 Var. 83 Var. 93 CEC Var. 4 Var. 14 Var. 24 Var. 34 Var. 44 Var. 54 Var. 64 Var. 74 Var. 84 Var. 94 HIC Var. 5 Var. 15 Var. 25 Var. 35 Var. 45 Var. 55 Var. 65 Var. 75 Var. 85 Var. 95 HAC Var. 6 Var. 16 Var. 26 Var. 36 Var. 46 Var. 56 Var. 66 Var. 76 Var. 86 Var. 96 MMC Var. 7 Var. 17 Var. 27 Var. 37 Var. 47 Var. 57 Var. 67 Var. 77 Var. 87 Var. 97 LAC Var. 8 Var. 18 Var. 28 Var. 38 Var. 48 Var. 58 Var. 68 Var. 78 Var. 88 Var. 98 LAC Var. 9 Var. 19 Var. 29 Var. 39 Var. 49 Var. 59 Var. 69 Var. 79 Var. 89 Var. 99 SEC Var. 10 Var. 20 Var. 30 Var. 40 Var. 50 Var. 60 Var. 70 Var. 80 Var. 90 Var. 100 * Ppt: Precipitation UF / DF: Ultrafiltration / Diafiltration AEC: Anion Exchange Chromatography CEC: Cation Exchange Chromatography 5 HIC: Hydrophobic Interaction Chromatography HAC: Hydroxyapatite Chromatography MMC: Mixed Mode Chromatography LAC: Ligand Affinity Chromatography IAC: Immuno-Affinity Chromatography 10 SEC: Size Exclusion Chromatography

[0188] In certain embodiments, the methods described above further comprises the step of performing a target protein enrichment step after contacting the composition with finely divided silicon dioxide (SiO2). In certain embodiments, the target protein enrichment step is selected from a protein precipitation step (e.g., an alcohol fractionation step), an 15 ultrafiltration / diafiltration step, and a chromatographic step.

[0189] Accordingly, in one embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived target protein the method comprises the steps of: (a) performing a first target protein enrichment step to form a first enriched plasma-derived target protein composition; (b) contacting the first enriched 20 composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; (c) separating the SiO2 from the composition to 2024204779   11 Jul 2024 remove the bound serine protease or serine protease zymogen; and (d) performing a second target protein enrichment step to form a second enriched plasma-derived target protein composition. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In certain 5 embodiments, the combination of first and second enrichment steps is selected from any one of variations Var. 1 to Var. 100, found in Table 1.

[0190] Likewise, in one embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived target protein the method comprises the steps of: (a) performing a first target protein enrichment step to form a first 10 enriched plasma-derived target protein composition; (b) performing a second target protein enrichment step to form a second enriched plasma-derived target protein composition; (c) contacting the second enriched composition with finely divided silicon dioxide (SiOz) under conditions suitable to bind at least one serine protease or serine protease zymogen; (d) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen; 15 and (e) performing a third target protein enrichment step to form a third enriched plasma-derived target protein composition. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In certain embodiments, the combination of first and second enrichment steps is selected from any one of variations Var. 101 to Var. 1100, found in Table 2, Table 3, Table 4, Table 5, Table 6, 20 Table 7, Table 8, Table 9, Table 10, or Table 11. Table 2. Exemplary embodiments for the combination of a first precipitation enrichment step, a second, and a third enrichment step.____________________________________________________________ Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Ppt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. & 101 Ill 121 131 141 151 161 171 181 191 •w UF / DF Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 102 112 122 132 142 152 162 172 182 192 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. fl X 103 113 123 133 143 153 163 173 183 193 Q CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. fl Fvl 104 114 124 134 144 154 164 174 184 194 MM FQ Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. MIC 105 115 125 135 145 155 165 175 185 195 XS HAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 106 116 126 136 146 156 166 176 186 196 2024204779   11 Jul 2024 MMC Var. 107 Var. 117 Var. 127 Var. 137 Var. 147 Var. 157 Var. 167 Var. 177 Var. 187 Var. 197 T AC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 108 118 128 138 148 158 168 178 188 198 T AP Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 109 119 129 139 149 159 169 179 189 199 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 110 120 130 140 150 160 170 180 190 200 * As per Table 1. Table 3. Exemplary embodiments for the combination of a first Ultrafiltration / Diafiltration step, a second, and a third enrichment step. Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Ppt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 201 211 221 231 241 251 261 271 281 291 UF / DF Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 202 212 222 232 242 252 262 272 282 292 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. a 203 213 223 233 243 253 263 273 283 293 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. •w c 204 214 224 234 244 254 264 274 284 294 g Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. X 205 215 225 235 245 255 265 275 285 295 u • RM MAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 206 216 226 236 246 256 266 276 286 296 -e MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 207 217 227 237 247 257 267 277 287 297 T AC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 208 218 228 238 248 258 268 278 288 298 TAP Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 209 219 229 239 249 259 269 279 289 299 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 210 220 230 240 250 260 270 280 290 300 * As per Table 1. 5 Table 4. Exemplary embodiments for the combination of a first Anion Exchange Chromatography step, a second, and a third enrichment step.____________________ Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Third Enrichment Sten Ppt Var. 301 Var. 311 Var. 321 Var. 331 Var. 341 Var. 351 Var. 361 Var. 371 Var. 381 Var. 391 UF / DF Var. 302 Var. 312 Var. 322 Var. 332 Var. 342 Var. 352 Var. 362 Var. 372 Var. 382 Var. 392 AEC Var. 303 Var. 313 Var. 323 Var. 333 Var. 343 Var. 353 Var. 363 Var. 373 Var. 383 Var. 393 CEC Var. 304 Var. 314 Var. 324 Var. 334 Var. 344 Var. 354 Var. 364 Var. 374 Var. 384 Var. 394 HIC Var. 305 Var. 315 Var. 325 Var. 335 Var. 345 Var. 355 Var. 365 Var. 375 Var. 385 Var. 395 2024204779   11 Jul 2024 HAC Var. 306 Var. 316 Var. 326 Var. 336 Var. 346 Var. 356 Var. 366 Var. 376 Var. 386 Var. 396 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 307 317 327 337 347 357 367 377 387 397 LAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 308 318 328 338 348 358 368 378 388 398 IAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 309 319 329 339 349 359 369 379 389 399 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 310 320 330 340 350 360 370 380 390 400 * As per Table 1. Table 5. Exemplary embodiments for the combination of a first Cation Exchange Chromatography step, a second, and a third enrichment step. Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Pnt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. rpi 401 411 421 431 441 451 461 471 481 491 UF / DF Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 402 412 422 432 442 452 462 472 482 492 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. a o 403 413 423 433 443 453 463 473 483 493 +* CZ) CPC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. -w S ULlU 404 414 424 434 444 454 464 474 484 494 Q g pnc Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. JS TTlVz 405 415 425 435 445 455 465 475 485 495 UA C Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 406 416 426 436 446 456 466 476 486 496 A / IA / fC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 407 417 427 437 447 457 467 477 487 497 T AC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 408 418 428 438 448 458 468 478 488 498 TA C Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 409 419 429 439 449 459 469 479 489 499 err Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 012*^ 410 420 430 440 450 460 470 480 490 500 * As per Table 1. 5 Table 6. Exemplary embodiments for the combination of a first Hydrophobic Interaction Chromatography step, a second, and a third enrichment step.___________________________ Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Ppt Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 501 511 521 531 541 551 561 571 581 591 cs UF / DF Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. $ E 502 512 522 532 542 552 562 572 582 592 •C ' APC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. s all- 503 513 523 533 543 553 563 573 583 593 CEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 504 514 524 534 544 554 564 574 584 594 2024204779   11 Jul 2024 HIC Var. 505 Var. 515 Var. 525 Var. 535 Var. 545 Var. 555 Var. 565 Var. 575 Var. 585 Var. 595 HAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 506 516 526 536 546 556 566 576 586 596 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 507 517 527 537 547 557 567 577 587 597 T AC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 508 518 528 538 548 558 568 578 588 598 IAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 509 519 529 539 549 559 569 579 589 599 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 510 520 530 540 550 560 570 580 590 600 * As per Table 1. Table 7. Exemplary embodiments for the combination of a first Hydroxyapatite Chromatography step, a second, and a third enrichment step. Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Third Enrichment Step Ppt Var. 601 Var. 611 Var. 621 Var. 631 Var. 641 Var. 651 Var. 661 Var. 671 Var. 681 Var. 691 UF / DF Var. 602 Var. 612 Var. 622 Var. 632 Var. 642 Var. 652 Var. 662 Var. 672 Var. 682 Var. 692 AEC Var. 603 Var. 613 Var. 623 Var. 633 Var. 643 Var. 653 Var. 663 Var. 673 Var. 683 Var. 693 CEC Var. 604 Var. 614 Var. 624 Var. 634 Var. 644 Var. 654 Var. 664 Var. 674 Var. 684 Var. 694 HIC Var. 605 Var. 615 Var. 625 Var. 635 Var. 645 Var. 655 Var. 665 Var. 675 Var. 685 Var. 695 HAC Var. 606 Var. 616 Var. 626 Var. 636 Var. 646 Var. 656 Var. 666 Var. 676 Var. 686 Var. 696 MMC Var. 607 Var. 617 Var. 627 Var. 637 Var. 647 Var. 657 Var. 667 Var. 677 Var. 687 Var. 697 LAC Var. 608 Var. 618 Var. 628 Var. 638 Var. 648 Var. 658 Var. 668 Var. 678 Var. 688 Var. 698 IAC Var. 609 Var. 619 Var. 629 Var. 639 Var. 649 Var. 659 Var. 669 Var. 679 Var. 689 Var. 699 SEC Var. 610 Var. 620 Var. 630 Var. 640 Var. 650 Var. 660 Var. 670 Var. 680 Var. 690 Var. 700 * As per Table 1. 5 Table 8. Exemplary embodiments for the combination of a first Mixed Mode Chromatography step, a second, and a third enrichment step.___________________________________________________ Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Ppt 701 711 721 731 741 751 761 771 781 791 > s : -c . UF / DF Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. i < 702 712 722 732 742 752 762 772 782 792 s Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 703 713 723 733 743 753 763 773 783 793 2024204779   11 Jul 2024 CEC Var. 704 Var. 714 Var. 724 Var. 734 Var. 744 Var. 754 Var. 764 Var. 774 Var. 784 Var. 794 HIC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 705 715 725 735 745 755 765 775 785 795 HAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 706 716 726 736 746 756 766 776 786 796 MMC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 707 717 727 737 747 757 767 777 787 797 T AC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 708 718 728 738 748 758 768 778 788 798 IAC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 709 719 729 739 749 759 769 779 789 799 SEC Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 710 720 730 740 750 760 770 780 790 800 * As per Table 1. Table 9. Exemplary embodiments for the combination of a first Ligand Affinity Chromatography step, a second, and a third enrichment step. Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Third Enrichment Step Ppt Var. 801 Var. 811 Var. 821 Var. 831 Var. 841 Var. 851 Var. 861 Var. 871 Var. 881 Var. 891 UF / DF Var. 802 Var. 812 Var. 822 Var. 832 Var. 842 Var. 852 Var. 862 Var. 872 Var. 882 Var. 892 AEC Var. 803 Var. 813 Var. 823 Var. 833 Var. 843 Var. 853 Var. 863 Var. 873 Var. 883 Var. 893 CEC Var. 804 Var. 814 Var. 824 Var. 834 Var. 844 Var. 854 Var. 864 Var. 874 Var. 884 Var. 894 HIC Var. 805 Var. 815 Var. 825 Var. 835 Var. 845 Var. 855 Var. 865 Var. 875 Var. 885 Var. 895 HAC Var. 806 Var. 816 Var. 826 Var. 836 Var. 846 Var. 856 Var. 866 Var. 876 Var. 886 Var. 896 MMC Var. 807 Var. 817 Var. 827 Var. 837 Var. 847 Var. 857 Var. 867 Var. 877 Var. 887 Var. 897 LAC Var. 808 Var. 818 Var. 828 Var. 838 Var. 848 Var. 858 Var. 868 Var. 878 Var. 888 Var. 898 LAC Var. 809 Var. 819 Var. 829 Var. 839 Var. 849 Var. 859 Var. 869 Var. 879 Var. 889 Var. 899 SEC Var. 810 Var. 820 Var. 830 Var. 840 Var. 850 Var. 860 Var. 870 Var. 880 Var. 890 Var. 900 * As per Table 1. 5 Table 10. Exemplary embodiments for the combination of a first Immuno-Affinity Chromatography step, a second, and a third enrichment step._____________________ Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Enrich ment Ppt Var. 901 Var. 911 Var. 921 Var. 931 Var. 941 Var. 951 Var. 961 Var. 971 Var. 981 Var. 991 UF / DF Var. 902 Var. 912 Var. 922 Var. 932 Var. 942 Var. 952 Var. 962 Var. 972 Var. 982 Var. 992 2024204779   11 Jul 2024 AEC Var. 903 Var. 913 Var. 923 Var. 933 Var. 943 Var. 953 Var. 963 Var. 973 Var. 983 Var. 993 CEC Var. 904 Var. 914 Var. 924 Var. 934 Var. 944 Var. 954 Var. 964 Var. 974 Var. 984 Var. 994 HIC Var. 905 Var. 915 Var. 925 Var. 935 Var. 945 Var. 955 Var. 965 Var. 975 Var. 985 Var. 995 HAC Var. 906 Var. 916 Var. 926 Var. 936 Var. 946 Var. 956 Var. 966 Var. 976 Var. 986 Var. 996 MMC Var. 907 Var. 917 Var. 927 Var. 937 Var. 947 Var. 957 Var. 967 Var. 977 Var. 987 Var. 997 LAC Var. 908 Var. 918 Var. 928 Var. 938 Var. 948 Var. 958 Var. 968 Var. 978 Var. 988 Var. 998 IAC Var. 909 Var. 919 Var. 929 Var. 939 Var. 949 Var. 959 Var. 969 Var. 979 Var. 989 Var. 999 SEC Var. 910 Var. 920 Var. 930 Var. 940 Var. 950 Var. 960 Var. 970 Var. 980 Var. 990 Var. 1000 * As per Table 1. Table 11. Exemplary embodiments for the combination of a first Size Exclusion Chromatography step, a second, and a third enrichment step. Second Enrichment Step* Ppt UF / DF AEC CEC HIC HAC MMC LAC IAC SEC Third Enrichment Step Ppt Var. 1001 Var. 1011 Var. 1021 Var. 1031 Var. 1041 Var. 1051 Var. 1061 Var. 1071 Var. 1081 Var. 1091 UF / DF Var. 1002 Var. 1012 Var. 1022 Var. 1032 Var. 1042 Var. 1052 Var. 1062 Var. 1072 Var. 1082 Var. 1092 AEC Var. 1003 Var. 1013 Var. 1023 Var. 1033 Var. 1043 Var. 1053 Var. 1063 Var. 1073 Var. 1083 Var. 1093 CEC Var. 1004 Var. 1014 Var. 1024 Var. 1034 Var. 1044 Var. 1054 Var. 1064 Var. 1074 Var. 1084 Var. 1094 HIC Var. 1005 Var. 1015 Var. 1025 Var. 1035 Var. 1045 Var. 1055 Var. 1065 Var. 1075 Var. 1085 Var. 1095 HAC Var. 1006 Var. 1016 Var. 1026 Var. 1036 Var. 1046 Var. 1056 Var. 1066 Var. 1076 Var. 1086 Var. 1096 MMC Var. 1007 Var. 1017 Var. 1027 Var. 1037 Var. 1047 Var. 1057 Var. 1067 Var. 1077 Var. 1087 Var. 1097 LAC Var. 1008 Var. 1018 Var. 1028 Var. 1038 Var. 1048 Var. 1058 Var. 1068 Var. 1078 Var. 1088 Var. 1098 IAC Var. 1009 Var. 1019 Var. 1029 Var. 1039 Var. 1049 Var. 1059 Var. 1069 Var. 1079 Var. 1089 Var. 1099 SEC Var. 1010 Var. 1020 Var. 1030 Var. 1040 Var. 1050 Var. 1060 Var. 1070 Var. 1080 Var. 1090 Var. 1100 * As per Table 1. 5

[0191] In certain embodiments of the methods described above, a chromatographic enrichment step comprises the sub-steps of: (i) contacting the plasma-derived target protein composition with a chromatographic resin under conditions suitable to bind the plasma-derived target protein; and (ii) eluting the plasma-derived target protein from the chromatographic resin. In one specific embodiment, the impurity does not bind to the chromatographic resin in sub-step (i). In another 2024204779   11 Jul 2024 specific embodiment, the impurity binds to the chromatographic resin in sub-step (i), but is not eluted from the chromatographic resin in sub-step (ii).

[0192] In other certain embodiments of the methods described above, a chromatographic enrichment step comprises the sub-steps of: (i) contacting the first enriched plasma-derived 5 target protein composition with a chromatographic resin under conditions suitable to bind at least one impurity; and (ii) separating the resin from the plasma-derived protein composition, wherein the plasma-derived target protein does not bind to the chromatographic resin in sub-step (i).

[0193] In certain embodiments of the methods described above, the plasma-derived target protein is selected from an immunoglobulin (Ig), albumin, alpha-1-antitrypsin (AlPI), 10 butyrylcholinesterase, a protein of the complement system (e.g., Factor H), and an inter-alphatrypsin inhibitor (lai). In a specific embodiment, the protein of the complement system is selected from the group consisting of Factor H (FH), Factor D, complement protein C3, and C4 binding protein. In a preferred embodiment, the protein composition is a manufacturing intermediate. 15

[0194] In certain embodiments of the methods provided herein, the amount of a particular serine protease or serine protease zymogen is reduced by at least 10%. In another embodiment, the amount of a particular serine protease or serine protease zymogen is reduced by at least 25%. In another embodiment, the amount of a particular serine protease or serine protease zymogen is reduced by at least 50%. In another embodiment, the amount of a particular serine protease or 20 serine protease zymogen is reduced by at least 75%. In another embodiment, the amount of a particular serine protease or serine protease zymogen is reduced by at least 90%. In yet other embodiments, the amount of a particular serine protease or serine protease zymogen is reduced by at least 5%, or by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or to 25 levels below the detection limit of the test system.

[0195] Generally, the amount of finely divided silicon dioxide (SiO2) required for the methods described herein will vary dependent on several factors, including without limitation, the total amount of protein present in the composition, the concentration of serine protease and serine protease zymogen (e.g., FXI, FXIa, FXII, and FXIIa) in the composition, the target protein, and 30 the solution conditions (e.g., pH, conductivity, etc.). For example, SiOj may be added to a target composition at a concentration between about 0.01 g / g protein and about 10 g / g protein. In 2024204779   11 Jul 2024 another embodiment, SiO2 may be added to a target composition at a concentration between about 1 g / g protein and about 5 g / g protein. In another embodiment, SiO2 may be added to a target composition at a concentration between about 2 g / g protein and about 4 g / g protein. In one embodiment, SiO2 is added at a final concentration of at least 1 g per gram total protein. In 5 another specific embodiment, fumed silica is added at a concentration of at least 2 g per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 2.5 g per gram total protein. In another embodiment, SiO2 may be added to a target composition at a concentration between about 0.01 g / g protein and about 5 g / g protein. In another embodiment, SiO2 may be added to a target composition at a concentration between about 0.02 10 g / g protein and about 4 g / g protein. In one embodiment, SiO2 is added at a final concentration of at least 0.1 g per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 0.2 g per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 0.25 g per gram total protein. In yet other specific embodiments, finely divided silicon dioxide is added at a concentration of at least 0.01 g / g total 15 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 more g / g total protein.

[0196] In certain embodiments in which a target protein is extracted from a suspended plasma precipitate fraction, filter aid, for example Celpure C300 (Celpure) or Hyflo-Supper-Cel (World 20 Minerals), will be added after the silica dioxide treatment, to facilitate depth filtration. Filter aid can be added at a final concentration of from about 0.01 kg / kg precipitate to about 1.0 kg / kg precipitate, or from about 0.02 kg / kg precipitate to about 0.8 kg / kg precipitate, or from about 0.03 kg / kg precipitate to about 0.7 kg / kg precipitate. In other embodiments, filter aid can be added at a final concentration of from about 0.01 kg / kg precipitate to about 0.07 kg / kg 25 precipitate, or from about 0.02 kg / kg precipitate to about 0.06 kg / kg precipitate, or from about 0.03 kg / kg precipitate to about 0.05 kg / kg precipitate. In certain embodiments, the filter aid will be added at a final concentration of about 0.01 kg / kg precipitate, 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 precipitate. A. Immunoglobulins 30

[0197] In one embodiment, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived immunoglobulin (Ig) 2024204779   11 Jul 2024 composition. In one specific embodiment, the method comprises the steps of: (a) contacting the Ig composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (b) separating the SiO2 from the Ig composition to remove the bound serine protease or serine protease zymogen. In a preferred 5 embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In one embodiment, the Ig composition is an IgG composition. In other embodiments, the Ig composition is an IgA, IgM, IgG, or mixed composition thereof.

[0198] In one embodiment, the method further comprises the step of performing a first Ig 10 protein enrichment step to form a first enriched Ig composition, prior to contacting the composition with finely divided silicon dioxide (SiO2). In certain embodiments, the first Ig protein enrichment step is selected from a protein precipitation step (e.g., an alcohol fractionation step), an ultrafiltration / diafiltration step, and a chromatographic step. In one embodiment, the Ig composition is an IgG composition. In other embodiments, the Ig composition is an IgA, IgM, 15 IgG, or mixed composition thereof.

[0199] In certain embodiments, the methods described above further comprises the step of performing a second Ig protein enrichment step to form a second enriched Ig composition, prior to contacting the composition with finely divided silicon dioxide (SiO2). In certain embodiments, the first Ig protein enrichment step is selected from a protein precipitation step 20   (e.g., an alcohol fractionation step), an ultrafiltration / diafiltration step, and a chromatographic step.

[0200] Accordingly, in one embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived Ig composition, the method comprises the steps of: (a) performing a first Ig enrichment step to form a first 25 enriched plasma-derived Ig composition; (b) performing a second Ig enrichment step to form a second enriched plasma-derived Ig composition; (c) contacting the second enriched composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (d) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen. In a preferred embodiment, the 30 serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI 2024204779   11 Jul 2024 (FXI), and / or Factor XII (FXII). In certain embodiments, the combination of first and second enrichment steps is selected from any one of variations Var. 1 to Var. 100, found in Table 1.

[0201] In certain embodiments, the methods described above further comprises the step of performing an Ig enrichment step after contacting the composition with finely divided silicon 5 dioxide (SiOz). In certain embodiments, the Ig enrichment step is selected from a protein precipitation step (e.g., an alcohol fractionation step), an ultrafiltration / diafiltration step, and a chromatographic step.

[0202] Accordingly, in one embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived Ig composition the 10 method comprises the steps of: (a) performing a first Ig enrichment step to form a first enriched plasma-derived Ig composition; (b) contacting the first enriched composition with finely divided silicon dioxide (SiOz) under conditions suitable to bind at least one serine protease or serine protease zymogen; (c) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen; and (d) performing a second Ig enrichment step to form a 15 second enriched plasma-derived Ig composition. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In certain embodiments, the combination of first and second enrichment steps is selected from any one of variations Var. 1 to Var. 100, found in Table 1.

[0203] Likewise, in one embodiment, the invention provides a method for reducing the amount 20 of a serine protease or a serine protease zymogen in a plasma-derived Ig composition, the method comprising the steps of: (a) performing a first Ig enrichment step to form a first enriched plasma-derived Ig composition; (b) performing a second Ig enrichment step to form a second enriched plasma-derived Ig composition; (c) contacting the second enriched composition with finely divided silicon dioxide (SiOz) under conditions suitable to bind at least one serine protease 25 or serine protease zymogen; (d) separating the SiOz from the composition to remove the bound serine protease or serine protease zymogen; and (e) performing a third Ig enrichment step to form a third enriched plasma-derived Ig composition. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In certain embodiments, the combination of first and second 30 enrichment steps is selected from any one of variations Var. 101 to Var. 1100, found in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, or Table 11. 2024204779   11 Jul 2024

[0204] In a particular embodiment, the Ig composition is a manufacturing intermediate. For example, in certain embodiments, the Ig composition is an IgG manufacturing intermediate from a Cohn fractionation procedure (J. Am. Chern. Soc., 1946, 68(3): 459-475; J. Am. Chern. Soc. 72:465-474 (1950)), an Oncley fractionation procedure (J. Am. Chern. Soc., 1949, 71(2): 5415   550), a Deutsch purification procedure (J. Biol. Chem. 164:109-118), a Hoppe purification procedure (Munch Med Wochenschr 1967 (34): 1749-1752), a Falksveden purification procedure (Swedish Patent No. 348942), a Falksveden and Lundblad purification procedure (Methods of Plasma Protein Fractionation 1980), a Lebing purification procedure (Vox Sang 2003 (84):193-201), a Tanaka purification procedure (Braz J Med Biol Res 2000 (33)37-30)), a 10 Teschner purification procedure (Vox Sang, 2007 (92):42-55), a Nitschmann fractionation procedure (Helv. Chim. Acta 37:866-873), a Kistler / Nitschmann fractionation procedure (Vox Sang. 7:414-424 (1962)), a Barundem purification procedure (Vox Sang. 7:157-74 (1962)), a Koblet purification procedure (Vox Sang. 13:93-102 (1967)) a purification procedure disclosed in U.S. Patent Nos. 5,122,373 or 5,177,194, modified procedures thereof, and similar or 15 equivalent purification procedures known in the art.

[0205] In one particular embodiment, the IgG composition is a cryo-poor Cohn pool. In another particular embodiment, the IgG composition is a Cohn Fraction I supernatant or equivalent fraction thereof. In another particular embodiment, the IgG composition is a resuspended Cohn Fraction III precipitate, or equivalent fraction thereof. In another particular 20 embodiment, the IgG composition is a re-suspended Cohn Fraction II+III precipitate, or equivalent fraction thereof. In another particular embodiment, the IgG composition is a resuspended Cohn Fraction I+II+III precipitate, or equivalent fraction thereof. In another particular embodiment, the IgG composition is a re-suspended Precipitate G precipitate, or equivalent fraction thereof. In another particular embodiment, the IgG composition is a re- 25 suspended Kistler / Nitschmann Precipitate B precipitate, or equivalent fraction thereof.

[0206] In a specific embodiment, the present invention provides a method for reducing the amount of serine protease and / or serine protease zymogen in a re-suspended IgG Fraction II+III precipitate. Advantageously, it has been found that the levels of Factor XI, Factor XII, Factor Xia, and / or Factor Xlla in a re-suspended IgG Fraction II+III precipitate can be greatly reduced 30 by the addition of a pretreatment step prior to filtration / centrifugation. In one embodiment, this pretreatment step comprises addition of finely divided silica dioxide particles (e.g., fumed silica, Aerosil®) followed by a 40 to 80 minute incubation period during which the suspension is 2024204779   11 Jul 2024 constantly mixed. In certain embodiments, the incubation period will be between about 50 minutes and about 70 minutes, or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more minutes. Generally, the treatment will be performed at between about 0°C and about 10°C, or between about 2°C and about 8°C. In certain embodiments, the treatment may be performed at 5 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 performed at between about 2°C and about 10°C.

[0207] The effect of the fumed silica treatment is exemplified by the results found in Examples 3, 6, and 7. In these examples, Fraction II+III precipitates are re-suspended and treated with varying amounts of finely divided silicon dioxide. As can be seen in Table 22, Table 27, Table 10   28, and Table 29, Factor XI and XII serine protease activity and zymogen content can be reduced at least 90% by treating the suspension with SiO2.

[0208] In certain embodiments, fumed silica is added at a concentration of between about 20 g / kg II+III paste and about 100 g / kg II+III paste (i.e., for a Modified Fraction II+III precipitate 15 that is extracted at a ratio of 1:15, fumed silica should be added at a concentration from about 20 g / 16 kg II+III suspension to about 100 g / 16 kg II+III suspension, or at a final concentration of about 0.125% (w / w) to about 0.625% (w / w)). In certain embodiments, the fumed silica may be added at a concentration of about 20 g / kg II+III paste, or about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / kg II+III paste. In one specific embodiment, filmed silica (e.g., 20 Aerosil 380 or equivalent) is added to the Modified Fraction II+III suspension to a final concentration of about 40 g / 16 kg II+III. Mixing takes place at about 2 to 8°C for at least 50 to 70 minutes.

[0209] In certain embodiments, SiO2 is added to a an IgG composition at a concentration between about 0.01 g / g protein and about 10 g / g protein. In another embodiment, SiO2 is added 25 to a an IgG composition at a concentration between about 0.01 g / g protein and about 5 g / g protein. In another embodiment, SiO2 is added to an IgG composition at a concentration between about 0.02 g / g protein and about 4 g / g protein. In one embodiment, SiO2 is added at a final concentration of at least 0.1g per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 0.2 g per gram total protein. In another 30 specific embodiment, fumed silica is added at a concentration of at least 0.25 g per gram total protein. In other specific embodiments, fumed silica is added at a concentration of at least 1 g 2024204779   11 Jul 2024 per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 2 g per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 2.5 g per gram 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 5 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 more per gram total protein.

[0210] In certain embodiments, filter aid, for example Celpure C300 (Celpure) or Hyflo-Supper-Cel (World Minerals), will be added after the silica dioxide treatment, to facilitate depth 10 filtration. Filter aid can be added at a final concentration of from about 0.01 kg / kg II+III paste to about 1.0 kg / kg II+III paste, or from about 0.02 kg / kg II+III paste to about 0.8 kg / kg II+III paste, or from about 0.03 kg / kg II+III paste to about 0.7 kg / kg II+III paste. In other embodiments, filter aid can be added at a final concentration of from about 0.01 kg / kg II+III paste to about 0.07 kg / kg II+III paste, or from about 0.02 kg / kg II+III paste to about 0.06 kg / kg II+III paste, or from 15 about 0.03 kg / kg II+III paste to about 0.05 kg / kg II+III paste. In certain embodiments, the filter aid will be added at a final concentration of about 0.01 kg / kg 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 II+III paste.

[0211] In one embodiment, the process improvements are realized by inclusion of a fumed 20 silica treatment prior to filtration or centrifugal clarification of a Fraction II+III suspension. In certain embodiments, the fumed silica treatment will include addition of from about 0.01 kg / kg II+III paste to about 0.07 kg / kg II+III paste, or from about 0.02 kg / kg II+III paste to about 0.06 kg / kg II+III paste, or from about 0.03 kg / kg II+III paste to about 0.05 kg / kg II+III paste, or about 0.02 kg / kg II+III paste, 0.03 kg / kg II+III paste, 0.04 kg / kg II+III paste, 0.05 kg / kg II+III paste, 25   0.06 kg / kg II+III paste, 0.07 kg / kg II+III paste, 0.08 kg / kg II+III paste, 0.09 kg / kg II+III paste, or 0.1 kg / kg II+III paste, and the mixture will be incubated for between about 50 minutes and about 70 minutes, or about 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, or more minutes at a temperature between about 2°C and about 8°C. In another embodiment, the process improvements are realized by inclusion of a fumed silica treatment which reduced the levels of 30 residual fibrinogen, amidolytic activity, and / or prekallikrein activator activity. In a specific embodiment, the process improvements are realized by inclusion of a fumed silica treatment, which reduces the levels of FXI, FXIa, FXII, and FXIIa in the immunoglobulin preparation. 2024204779   11 Jul 2024

[0212] Generally, serine protease and / or serine protease zymogen removal from immunoglobulin compositions can be achieved by treating the immunoglobulin-containing solution with finely divided silicon dioxide (SiO2) under pH and conductivity solution conditions in which the serine protease and / or serine protease zymogen binds to the SiO2. As shown in the 5 examples, suitable conditions include low pH and low conductivity.

[0213] Accordingly, in one embodiment, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived immunoglobulin composition, the method comprising contacting the composition with SiO2 at a pH between about 4.0 and about 7.0 to bind a serine protease or a serine protease zymogen and 10 removing the SiO2 from the composition. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.0 and about 6.5. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.0 and about 6.0. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.0 and about 5.5. In another embodiment, the method 15 comprises contacting the composition with SiO2 at a pH between about 4.0 and about 5.0. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.5 and about 7.0. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.5 and about 6.5. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.5 and about 20   6.0. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.5 and about 5.5. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.5 and about 5.0. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 5.0 and about 7.0. In another embodiment, the method comprises contacting the composition with SiO2 at a pH 25 between about 5.0 and about 6.5. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 5.0 and about 6.0. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 5.0 and about 5.5. In yet another embodiment, the method comprises contacting the composition with SiCh at a pH between about 4.6 and about 5.6. In another embodiment, the method comprises contacting 30 the composition with SiO2 at a pH between about 4.7 and about 5.5. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.8 and about 5.4. In another embodiment, the method comprises contacting the composition with SiO2 at a pH 2024204779   11 Jul 2024 between about 4.9 and about 5.3. In another embodiment, the method comprises contacting the composition with SiO2 at a pH between about 5.0 and about 5.2. In another embodiment, the method comprises contacting the composition with SiO2 at a pH of about 5.1. In other embodiments, the method comprises contacting the composition with SiO2 at a pH of about 4.0 5 or 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, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or no more than 7.0. In yet other embodiments, the method comprises contacting the composition with SiO2 at a pH of no more than 4.0 or no more than 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, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or no more than 7.0. 10

[0214] In one embodiment, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived immunoglobulin composition, the method comprising contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 3.0 mS / cm to bind a serine protease or a serine protease zymogen and removing the SiO2 from the composition. In another embodiment, the method 15 comprises contacting the composition with SiO2 at an ionic strength between about 0.5 mS / cm and about 2.0 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 1.3 mS / cm and about 1.7 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.9 mS / cm. In another embodiment, the method comprises 20   contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.8 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.7 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.6 mS / cm. In another embodiment, the method comprises contacting the 25 composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.5 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.4 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.3 mS / cm. In another embodiment, the method comprises contacting the composition 30 with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.2 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.1 mS / cm. In another embodiment, the method comprises 2024204779   11 Jul 2024 contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 1.0 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm and about 0.9 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 5 mS / cm and about 0.8 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.2 mS / cm and about 1.0 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.3 mS / cm and about 1.0 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.1 mS / cm 10 and about 0.4 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.5 mS / cm and about 1.0 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.6 mS / cm and about 1.0 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength between about 0.7 mS / cm and about 15   0.9 mS / cm. In another embodiment, the method comprises contacting the composition with SiO2 at an ionic strength of about 0.8 mS / cm. In other embodiments, the method comprises contacting the composition with SiO2 at an ionic strength of about 0.1 mS / cm or no more than 0.2 mS / cm, 0.3 mS / cm, 0.4 mS / cm, 0.5 mS / cm, 0.6 mS / cm, 0.7 mS / cm, 0.8 mS / cm, 0.9 mS / cm, 1.0 mS / cm, 1.1 mS / cm, 1.2 mS / cm, 1.3 mS / cm, 1.4 mS / cm, 1.5 mS / cm, 1.6 mS / cm, 1.7 mS / cm, 1.8 mS / cm, 20   1.9 mS / cm, 2.0 mS / cm, 2.1 mS / cm, 2.2 mS / cm, 2.3 mS / cm, 2.4 mS / cm, 2.5 mS / cm, 2.6 mS / cm, 2.7 mS / cm, 2.8 mS / cm, 2.9 mS / cm, or 3.0 mS / cm. In yet other embodiments, the method comprises contacting the composition with SiO2 at an ionic strength of no more than 0.1 mS / cm or no more than 0.2 mS / cm, 0.3 mS / cm, 0.4 mS / cm, 0.5 mS / cm, 0.6 mS / cm, 0.7 mS / cm, 0.8 mS / cm, 0.9 mS / cm, 1.0 mS / cm, 1.1 mS / cm, 1.2 mS / cm, 1.3 mS / cm, 1.4 mS / cm, 1.5 mS / cm, 1.6 25 mS / cm, 1.7 mS / cm, 1.8 mS / cm, 1.9 mS / cm, 2.0 mS / cm, 2.1 mS / cm, 2.2 mS / cm, 2.3 mS / cm, 2.4 mS / cm, 2.5 mS / cm, 2.6 mS / cm, 2.7 mS / cm, 2.8 mS / cm, 2.9 mS / cm, or 3.0 mS / cm.

[0215] In certain embodiments, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived immunoglobulin composition, the method comprising contacting the composition with SiO2 at a low pH and low 30 ionic strength to bind a serine protease or a serine protease zymogen and removing the SiO2 from the composition. In a particular embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.8 and about 5.4 at an ionic strength between about 0.6 mS / cm 2024204779   11 Jul 2024 and about 1.0 mS / cm. In a more particular embodiment, the method comprises contacting the composition with SiO2 at a pH between about 4.9 and about 5.3 at an ionic strength between about 0.7 mS / cm and about 0.9 mS / cm. In a yet more particular embodiment, the method comprises contacting the composition with SiO2 at a pH between about 5.0 and about 5.2 at an 5 ionic strength of about 0.8 mS / cm. In yet other embodiments, the method comprises contacting the composition with SiO2 at a pH and ionic strength according to any one of variations Var. 1222 to 3041, as presented in Table 12, Table 13, Table 14, and Table 15. Table 12. Exemplary embodiments of solution conditions useful for binding serine proteases and / or serine protease zymogens to SiO2. _______________________________pH 4.0-7.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 4.5-5.0 Ionic Strength (mS / cm) 0.1-2.0 Var. 1222 Var. 1638 Var. 1638 Var. 1638 Var. 1638 Var. 1638 Var. 1638 Var. 1638 Var. 1638 0.1-1.9 Var. 1223 Var. 1639 Var. 1639 Var. 1639 Var. 1639 Var. 1639 Var. 1639 Var. 1639 Var. 1639 0.1-1.8 Var. 1224 Var. 1640 Var. 1640 Var. 1640 Var. 1640 Var. 1640 Var. 1640 Var. 1640 Var. 1640 0.1-1.7 Var. 1225 Var. 1641 Var. 1641 Var. 1641 Var. 1641 Var. 1641 Var. 1641 Var. 1641 Var. 1641 0.1-1.6 Var. 1226 Var. 1642 Var. 1642 Var. 1642 Var. 1642 Var. 1642 Var. 1642 Var. 1642 Var. 1642 0.1-1.5 Var. 1227 Var. 1643 Var. 1643 Var. 1643 Var. 1643 Var. 1643 Var. 1643 Var. 1643 Var. 1643 0.1-1.4 Var. 1228 Var. 1644 Var. 1644 Var. 1644 Var. 1644 Var. 1644 Var. 1644 Var. 1644 Var. 1644 0.1-13 Var. 1229 Var. 1645 Var. 1645 Var. 1645 Var. 1645 Var. 1645 Var. 1645 Var. 1645 Var. 1645 0.1-1.2 Var. 1230 Var. 1646 Var. 1646 Var. 1646 Var. 1646 Var. 1646 Var. 1646 Var. 1646 Var. 1646 0.1-1.1 Var. 1231 Var. 1647 Var. 1647 Var. 1647 Var. 1647 Var. 1647 Var. 1647 Var. 1647 Var. 1647 0.1-1.0 Var. 1232 Var. 1648 Var. 1648 Var. 1648 Var. 1648 Var. 1648 Var. 1648 Var. 1648 Var. 1648 0.1-0.9 Var. 1233 Var. 1649 Var. 1649 Var. 1649 Var. 1649 Var. 1649 Var. 1649 Var. 1649 Var. 1649 0.1-0.8 Var. 1234 Var. 1650 Var. 1650 Var. 1650 Var. 1650 Var. 1650 Var. 1650 Var. 1650 Var. 1650 0.2-2.0 Var. 1235 Var. 1651 Var. 1651 Var. 1651 Var. 1651 Var. 1651 Var. 1651 Var. 1651 Var. 1651 0.2-1.5 Var. 1236 Var. 1652 Var. 1652 Var. 1652 Var. 1652 Var. 1652 Var. 1652 Var. 1652 Var. 1652 0.2-1.0 Var. 1237 Var. 1653 Var. 1653 Var. 1653 Var. 1653 Var. 1653 Var. 1653 Var. 1653 Var. 1653 0.2-0.9 Var. 1238 Var. 1654 Var. 1654 Var. 1654 Var. 1654 Var. 1654 Var. 1654 Var. 1654 Var. 1654 0.2-0.8 Var. 1239 Var. 1655 Var. 1655 Var. 1655 Var. 1655 Var. 1655 Var. 1655 Var. 1655 Var. 1655 0.3-1.0 Var. 1240 Var. 1656 Var. 1656 Var. 1656 Var. 1656 Var. 1656 Var. 1656 Var. 1656 Var. 1656 0.3-0.9 Var. 1241 Var. 1657 Var. 1657 Var. 1657 Var. 1657 Var. 1657 Var. 1657 Var. 1657 Var. 1657 0.3-0.8 Var. 1242 Var. 1658 Var. 1658 Var. 1658 Var. 1658 Var. 1658 Var. 1658 Var. 1658 Var. 1658 0.4-1.0 Var. 1243 Var. 1659 Var. 1659 Var. 1659 Var. 1659 Var. 1659 Var. 1659 Var. 1659 Var. 1659 0.4-0.9 Var. 1244 Var. 1660 Var. 1660 Var. 1660 Var. 1660 Var. 1660 Var. 1660 Var. 1660 Var. 1660 0.4-0.8 Var. 1245 Var. 1661 Var. 1661 Var. 1661 Var. 1661 Var. 1661 Var. 1661 Var. 1661 Var. 1661 0.5-1.0 Var. 1246 Var. 1662 Var. 1662 Var. 1662 Var. 1662 Var. 1662 Var. 1662 Var. 1662 Var. 1662 0.5-0.9 Var. 1247 Var. 1663 Var. 1663 Var. 1663 Var. 1663 Var. 1663 Var. 1663 Var. 1663 Var. 1663 0.5-0.8 Var. 1248 Var. 1664 Var. 1664 Var. 1664 Var. 1664 Var. 1664 Var. 1664 Var. 1664 Var. 1664 0.6-1.0 Var. 1249 Var. 1665 Var. 1665 Var. 1665 Var. 1665 Var. 1665 Var. 1665 Var. 1665 Var. 1665 0.6-0.9 Var. 1250 Var. 1666 Var. 1666 Var. 1666 Var. 1666 Var. 1666 Var. 1666 Var. 1666 Var. 1666 0.6-0.8 Var. 1251 Var. 1667 Var. 1667 Var. 1667 Var. 1667 Var. 1667 Var. 1667 Var. 1667 Var. 1667 0.7-1.0 Var. 1252 Var. 1668 Var. 1668 Var. 1668 Var. 1668 Var. 1668 Var. 1668 Var. 1668 Var. 1668 0.7-0.9 Var. 1253 Var. 1669 Var. 1669 Var. 1669 Var. 1669 Var. 1669 Var. 1669 Var. 1669 Var. 1669 0.1 Var. 1254 Var. 1670 Var. 1670 Var. 1670 Var. 1670 Var. 1670 Var. 1670 Var. 1670 Var. 1670 0.2 Var. 1255 Var. 1671 Var. 1671 Var. 1671 Var. 1671 Var. 1671 Var. 1671 Var. 1671 Var. 1671 0.3 Var. 1256 Var. 1672 Var. 1672 Var. 1672 Var. 1672 Var. 1672 Var. 1672 Var. 1672 Var. 1672 0.4 Var. 1257 Var. 1673 Var. 1673 Var. 1673 Var. 1673 Var. 1673 Var. 1673 Var. 1673 Var. 1673 0.5 Var. 1258 Var. 1674 Var. 1674 Var. 1674 Var. 1674 Var. 1674 Var. 1674 Var. 1674 Var. 1674 0.6 Var. 1259 Var. 1675 Var. 1675 Var. 1675 Var. 1675 Var. 1675 Var. 1675 Var. 1675 Var. 1675 0.7 Var. 1260 Var. 1676 Var. 1676 Var. 1676 Var. 1676 Var. 1676 Var. 1676 Var. 1676 Var. 1676 0.8 Var. 1261 Var. 1677 Var. 1677 Var. 1677 Var. 1677 Var. 1677 Var. 1677 Var. 1677 Var. 1677 0.9 Var. 1262 Var. 1678 Var. 1678 Var. 1678 Var. 1678 Var. 1678 Var. 1678 Var. 1678 Var. 1678 1 Var. 1263 Var. 1679 Var. 1679 Var. 1679 Var. 1679 Var. 1679 Var. 1679 Var. 1679 Var. 1679 1.1 Var. 1264 Var. 1680 Var. 1680 Var. 1680 Var. 1680 Var. 1680 Var. 1680 Var. 1680 Var. 1680 1.2 Var. 1265 Var. 1681 Var. 1681 Var. 1681 Var. 1681 Var. 1681 Var. 1681 Var. 1681 Var. 1681 1.3 Var. 1266 Var. 1682 Var. 1682 Var. 1682 Var. 1682 Var. 1682 Var. 1682 Var. 1682 Var. 1682 2024204779   11 Jul 2024 1.4 Var. 1267 Var. 1683 Var. 1683 Var. 1683 Var. 1683 Var. 1683 Var. 1683 Var. 1683 Var. 1683 1.5 Var. 1268 Var. 1684 Var. 1684 Var. 1684 Var. 1684 Var. 1684 Var. 1684 Var. 1684 Var. 1684 1.6 Var. 1269 Var. 1685 Var. 1685 Var. 1685 Var. 1685 Var. 1685 Var. 1685 Var. 1685 Var. 1685 1.7 Var. 1270 Var. 1686 Var. 1686 Var. 1686 Var. 1686 Var. 1686 Var. 1686 Var. 1686 Var. 1686 1.8 Var. 1271 Var. 1687 Var. 1687 Var. 1687 Var. 1687 Var. 1687 Var. 1687 Var. 1687 Var. 1687 1.9 Var. 1272 Var. 1688 Var. 1688 Var. 1688 Var. 1688 Var. 1688 Var. 1688 Var. 1688 Var. 1688 2 Var. 1273 Var. 1689 Var. 1689 Var. 1689 Var. 1689 Var. 1689 Var. 1689 Var. 1689 Var. 1689 Table 13. Exemplary embodiments of solution conditions useful for binding serine proteases and / or serine protease zymogens to SiO;. ______________________________PH 5.0-7.0 5.0-6.5 5.0-6.0 5.0-5.5 4.6-5.6 4.7-5.5 4.8-5.4 4.9-5.3 5.0-5.2 Ionic Strength (mS / cm) 0.1-2.0 Var. 1690 Var. 1742 Var. 1794 Var. 1846 Var. 1898 Var. 1950 Var. 2002 Var. 2054 Var. 2106 0.1-1.9 Var. 1691 Var. 1743 Var. 1795 Var. 1847 Var. 1899 Var. 1951 Var. 2003 Var. 2055 Var. 2107 0.1-1.8 Var. 1692 Var. 1744 Var. 1796 Var. 1848 Var. 1900 Var. 1952 Var. 2004 Var. 2056 Var. 2108 0.1-1.7 Var. 1693 Var. 1745 Var. 1797 Var. 1849 Var. 1901 Var. 1953 Var. 2005 Var. 2057 Var. 2109 0.1-1.6 Var. 1694 Var. 1746 Var. 1798 Var. 1850 Var. 1902 Var. 1954 Var. 2006 Var. 2058 Var. 2110 0.1-1.5 Var. 1695 Var. 1747 Var. 1799 Var. 1851 Var. 1903 Var. 1955 Var. 2007 Var. 2059 Var. 2111 0.1-1.4 Var. 1696 Var. 1748 Var. 1800 Var. 1852 Var. 1904 Var. 1956 Var. 2008 Var. 2060 Var. 2112 0.1-1.3 Var. 1697 Var. 1749 Var. 1801 Var. 1853 Var. 1905 Var. 1957 Var. 2009 Var. 2061 Var. 2113 0.1-1.2 Var. 1698 Var. 1750 Var. 1802 Var. 1854 Var. 1906 Var. 1958 Var. 2010 Var. 2062 Var. 2114 0.1-1.1 Var. 1699 Var. 1751 Var. 1803 Var. 1855 Var. 1907 Var. 1959 Var. 2011 Var. 2063 Var. 2115 0.1-1.0 Var. 1700 Var. 1752 Var. 1804 Var. 1856 Var. 1908 Var. 1960 Var. 2012 Var. 2064 Var. 2116 0.1-0.9 Var. 1701 Var. 1753 Var. 1805 Var. 1857 Var. 1909 Var. 1961 Var. 2013 Var. 2065 Var. 2117 0.1-0.8 Var. 1702 Var. 1754 Var. 1806 Var. 1858 Var. 1910 Var. 1962 Var. 2014 Var. 2066 Var. 2118 0.2-2.0 Var. 1703 Var. 1755 Var. 1807 Var. 1859 Var. 1911 Var. 1963 Var. 2015 Var. 2067 Var. 2119 0.2-1.5 Var. 1704 Var. 1756 Var. 1808 Var. 1860 Var. 1912 Var. 1964 Var. 2016 Var. 2068 Var. 2120 0.2-1.0 Var. 1705 Var. 1757 Var. 1809 Var. 1861 Var. 1913 Var. 1965 Var. 2017 Var. 2069 Var. 2121 0.2-0.9 Var. 1706 Var. 1758 Var. 1810 Var. 1862 Var. 1914 Var. 1966 Var. 2018 Var. 2070 Var. 2122 0.2-0.8 Var. 1707 Var. 1759 Var. 1811 Var. 1863 Var. 1915 Var. 1967 Var. 2019 Var. 2071 Var. 2123 03-1.0 Var. 1708 Var. 1760 Var. 1812 Var. 1864 Var. 1916 Var. 1968 Var. 2020 Var. 2072 Var. 2124 03-0.9 Var. 1709 Var. 1761 Var. 1813 Var. 1865 Var. 1917 Var. 1969 Var. 2021 Var. 2073 Var. 2125 03-0.8 Var. 1710 Var. 1762 Var. 1814 Var. 1866 Var. 1918 Var. 1970 Var. 2022 Var. 2074 Var. 2126 0.4-1.0 Var. 1711 Var. 1763 Var. 1815 Var. 1867 Var. 1919 Var. 1971 Var. 2023 Var. 2075 Var. 2127 0.4-0.9 Var. 1712 Var. 1764 Var. 1816 Var. 1868 Var. 1920 Var. 1972 Var. 2024 Var. 2076 Var. 2128 0.4-0.8 Var. 1713 Var. 1765 Var. 1817 Var. 1869 Var. 1921 Var. 1973 Var. 2025 Var. 2077 Var. 2129 0.5-1.0 Var. 1714 Var. 1766 Var. 1818 Var. 1870 Var. 1922 Var. 1974 Var. 2026 Var. 2078 Var. 2130 0.5-0.9 Var. 1715 Var. 1767 Var. 1819 Var. 1871 Var. 1923 Var. 1975 Var. 2027 Var. 2079 Var. 2131 0.5-0.8 Var. 1716 Var. 1768 Var. 1820 Var. 1872 Var. 1924 Var. 1976 Var. 2028 Var. 2080 Var. 2132 0.6-1.0 Var. 1717 Var. 1769 Var. 1821 Var. 1873 Var. 1925 Var. 1977 Var. 2029 Var. 2081 Var. 2133 0.6-0.9 Var. 1718 Var. 1770 Var. 1822 Var. 1874 Var. 1926 Var. 1978 Var. 2030 Var. 2082 Var. 2134 0.6-0.8 Var. 1719 Var. 1771 Var. 1823 Var. 1875 Var. 1927 Var. 1979 Var. 2031 Var. 2083 Var. 2135 0.7-1.0 Var. 1720 Var. 1772 Var. 1824 Var. 1876 Var. 1928 Var. 1980 Var. 2032 Var. 2084 Var. 2136 0.7-0.9 Var. 1721 Var. 1773 Var. 1825 Var. 1877 Var. 1929 Var. 1981 Var. 2033 Var. 2085 Var. 2137 0.1 Var. 1722 Var. 1774 Var. 1826 Var. 1878 Var. 1930 Var. 1982 Var. 2034 Var. 2086 Var. 2138 0.2 Var. 1723 Var. 1775 Var. 1827 Var. 1879 Var. 1931 Var. 1983 Var. 2035 Var. 2087 Var. 2139 03 Var. 1724 Var. 1776 Var. 1828 Var. 1880 Var. 1932 Var. 1984 Var. 2036 Var. 2088 Var. 2140 0.4 Var. 1725 Var. 1777 Var. 1829 Var. 1881 Var. 1933 Var. 1985 Var. 2037 Var. 2089 Var. 2141 0.5 Var. 1726 Var. 1778 Var. 1830 Var. 1882 Var. 1934 Var. 1986 Var. 2038 Var. 2090 Var. 2142 0.6 Var. 1727 Var. 1779 Var. 1831 Var. 1883 Var. 1935 Var. 1987 Var. 2039 Var. 2091 Var. 2143 0.7 Var. 1728 Var. 1780 Var. 1832 Var. 1884 Var. 1936 Var. 1988 Var. 2040 Var. 2092 Var. 2144 0.8 Var. 1729 Var. 1781 Var. 1833 Var. 1885 Var. 1937 Var. 1989 Var. 2041 Var. 2093 Var. 2145 0.9 Var. 1730 Var. 1782 Var. 1834 Var. 1886 Var. 1938 Var. 1990 Var. 2042 Var. 2094 Var. 2146 1 Var. 1731 Var. 1783 Var. 1835 Var. 1887 Var. 1939 Var. 1991 Var. 2043 Var. 2095 Var. 2147 1.1 Var. 1732 Var. 1784 Var. 1836 Var. 1888 Var. 1940 Var. 1992 Var. 2044 Var. 2096 Var. 2148 1.2 Var. 1733 Var. 1785 Var. 1837 Var. 1889 Var. 1941 Var. 1993 Var. 2045 Var. 2097 Var. 2149 1.3 Var. 1734 Var. 1786 Var. 1838 Var. 1890 Var. 1942 Var. 1994 Var. 2046 Var. 2098 Var. 2150 1.4 Var. 1735 Var. 1787 Var. 1839 Var. 1891 Var. 1943 Var. 1995 Var. 2047 Var. 2099 Var. 2151 1.5 Var. 1736 Var. 1788 Var. 1840 Var. 1892 Var. 1944 Var. 1996 Var. 2048 Var. 2100 Var. 2152 1.6 Var. 1737 Var. 1789 Var. 1841 Var. 1893 Var. 1945 Var. 1997 Var. 2049 Var. 2101 Var. 2153 1.7 Var. 1738 Var. 1790 Var. 1842 Var. 1894 Var. 1946 Var. 1998 Var. 2050 Var. 2102 Var. 2154 1.8 Var. 1739 Var. 1791 Var. 1843 Var. 1895 Var. 1947 Var. 1999 Var. 2051 Var. 2103 Var. 2155 1.9 Var. 1740 Var. 1792 Var. 1844 Var. 1896 Var. 1948 Var. 2000 Var. 2052 Var. 2104 Var. 2156 2 Var. 1741 Var. 1793 Var. 1845 Var. 1897 Var. 1949 Var. 2001 Var. 2053 Var. 2105 Var. 2157 2024204779   11 Jul 2024 Tai anc t>le 14. Exemplary embodiments of solution conditions useful for binding serine proteases / or serine protease zymogens to SiO2. _______________________________Pl____ 5.1 NMT 4.0 NMT 4.2 NMT 4.4 NMT 4.6 NMT 4.8 NMT 5.0 NMT 5.2 NMT 5.4 Ionic Strength (mS / cm) 0.1-2.0 Var. 2158 Var. 2210 Var. 2262 Var. 2314 Var. 2366 Var. 2418 Var. 2470 Var. 2522 Var. 2574 0.1-1.9 Var. 2159 Var. 2211 Var. 2263 Var. 2315 Var. 2367 Var. 2419 Var. 2471 Var. 2523 Var. 2575 0.1-1.8 Var. 2160 Var. 2212 Var. 2264 Var. 2316 Var. 2368 Var. 2420 Var. 2472 Var. 2524 Var. 2576 0.1-1.7 Var. 2161 Var. 2213 Var. 2265 Var. 2317 Var. 2369 Var. 2421 Var. 2473 Var. 2525 Var. 2577 0.1-1.6 Var. 2162 Var. 2214 Var. 2266 Var. 2318 Var. 2370 Var. 2422 Var. 2474 Var. 2526 Var. 2578 0.1-1.5 Var. 2163 Var. 2215 Var. 2267 Var. 2319 Var. 2371 Var. 2423 Var. 2475 Var. 2527 Var. 2579 0.1-1.4 Var. 2164 Var. 2216 Var. 2268 Var. 2320 Var. 2372 Var. 2424 Var. 2476 Var. 2528 Var. 2580 0.1-1.3 Var. 2165 Var. 2217 Var. 2269 Var. 2321 Var. 2373 Var. 2425 Var. 2477 Var. 2529 Var. 2581 0.1-1.2 Var. 2166 Var. 2218 Var. 2270 Var. 2322 Var. 2374 Var. 2426 Var. 2478 Var. 2530 Var. 2582 0.1-1.1 Var. 2167 Var. 2219 Var. 2271 Var. 2323 Var. 2375 Var. 2427 Var. 2479 Var. 2531 Var. 2583 0.1-1.0 Var. 2168 Var. 2220 Var. 2272 Var. 2324 Var. 2376 Var. 2428 Var. 2480 Var. 2532 Var. 2584 0.1-0.9 Var. 2169 Var. 2221 Var. 2273 Var. 2325 Var. 2377 Var. 2429 Var. 2481 Var. 2533 Var. 2585 0.1-0.8 Var. 2170 Var. 2222 Var. 2274 Var. 2326 Var. 2378 Var. 2430 Var. 2482 Var. 2534 Var. 2586 0.2-2.0 Var. 2171 Var. 2223 Var. 2275 Var. 2327 Var. 2379 Var. 2431 Var. 2483 Var. 2535 Var. 2587 0.2-1.5 Var. 2172 Var. 2224 Var. 2276 Var. 2328 Var. 2380 Var. 2432 Var. 2484 Var. 2536 Var. 2588 0.2-1.0 Var. 2173 Var. 2225 Var. 2277 Var. 2329 Var. 2381 Var. 2433 Var. 2485 Var. 2537 Var. 2589 0.2-0.9 Var. 2174 Var. 2226 Var. 2278 Var. 2330 Var. 2382 Var. 2434 Var. 2486 Var. 2538 Var. 2590 0.2-0.8 Var. 2175 Var. 2227 Var. 2279 Var. 2331 Var. 2383 Var. 2435 Var. 2487 Var. 2539 Var. 2591 0.3-1.0 Var. 2176 Var. 2228 Var. 2280 Var. 2332 Var. 2384 Var. 2436 Var. 2488 Var. 2540 Var. 2592 0.3-0.9 Var. 2177 Var. 2229 Var. 2281 Var. 2333 Var. 2385 Var. 2437 Var. 2489 Var. 2541 Var. 2593 0.3-0.8 Var. 2178 Var. 2230 Var. 2282 Var. 2334 Var. 2386 Var. 2438 Var. 2490 Var. 2542 Var. 2594 0.4-1.0 Var. 2179 Var. 2231 Var. 2283 Var. 2335 Var. 2387 Var. 2439 Var. 2491 Var. 2543 Var. 2595 0.4-0.9 Var. 2180 Var. 2232 Var. 2284 Var. 2336 Var. 2388 Var. 2440 Var. 2492 Var. 2544 Var. 2596 0.4-0.8 Var. 2181 Var. 2233 Var. 2285 Var. 2337 Var. 2389 Var. 2441 Var. 2493 Var. 2545 Var. 2597 0.5-1.0 Var. 2182 Var. 2234 Var. 2286 Var. 2338 Var. 2390 Var. 2442 Var. 2494 Var. 2546 Var. 2598 0.5-0.9 Var. 2183 Var. 2235 Var. 2287 Var. 2339 Var. 2391 Var. 2443 Var. 2495 Var. 2547 Var. 2599 0.5-0.8 Var. 2184 Var. 2236 Var. 2288 Var. 2340 Var. 2392 Var. 2444 Var. 2496 Var. 2548 Var. 2600 0.6-1.0 Var. 2185 Var. 2237 Var. 2289 Var. 2341 Var. 2393 Var. 2445 Var. 2497 Var. 2549 Var. 2601 0.6-0.9 Var. 2186 Var. 2238 Var. 2290 Var. 2342 Var. 2394 Var. 2446 Var. 2498 Var. 2550 Var. 2602 0.6-0.8 Var. 2187 Var. 2239 Var. 2291 Var. 2343 Var. 2395 Var. 2447 Var. 2499 Var. 2551 Var. 2603 0.7-1.0 Var. 2188 Var. 2240 Var. 2292 Var. 2344 Var. 2396 Var. 2448 Var. 2500 Var. 2552 Var. 2604 0.7-0.9 Var. 2189 Var. 2241 Var. 2293 Var. 2345 Var. 2397 Var. 2449 Var. 2501 Var. 2553 Var. 2605 0.1 Var. 2190 Var. 2242 Var. 2294 Var. 2346 Var. 2398 Var. 2450 Var. 2502 Var. 2554 Var. 2606 0.2 Var. 2191 Var. 2243 Var. 2295 Var. 2347 Var. 2399 Var. 2451 Var. 2503 Var. 2555 Var. 2607 0.3 Var. 2192 Var. 2244 Var. 2296 Var. 2348 Var. 2400 Var. 2452 Var. 2504 Var. 2556 Var. 2608 0.4 Var. 2193 Var. 2245 Var. 2297 Var. 2349 Var. 2401 Var. 2453 Var. 2505 Var. 2557 Var. 2609 0.5 Var. 2194 Var. 2246 Var. 2298 Var. 2350 Var. 2402 Var. 2454 Var. 2506 Var. 2558 Var. 2610 0.6 Var. 2195 Var. 2247 Var. 2299 Var. 2351 Var. 2403 Var. 2455 Var. 2507 Var. 2559 Var. 2611 0.7 Var. 2196 Var. 2248 Var. 2300 Var. 2352 Var. 2404 Var. 2456 Var. 2508 Var. 2560 Var. 2612 0.8 Var. 2197 Var. 2249 Var. 2301 Var. 2353 Var. 2405 Var. 2457 Var. 2509 Var. 2561 Var. 2613 0.9 Var. 2198 Var. 2250 Var. 2302 Var. 2354 Var. 2406 Var. 2458 Var. 2510 Var. 2562 Var. 2614 1 Var. 2199 Var. 2251 Var. 2303 Var. 2355 Var. 2407 Var. 2459 Var. 2511 Var. 2563 Var. 2615 1.1 Var. 2200 Var. 2252 Var. 2304 Var. 2356 Var. 2408 Var. 2460 Var. 2512 Var. 2564 Var. 2616 1.2 Var. 2201 Var. 2253 Var. 2305 Var. 2357 Var. 2409 Var. 2461 Var. 2513 Var. 2565 Var. 2617 1.3 Var. 2202 Var. 2254 Var. 2306 Var. 2358 Var. 2410 Var. 2462 Var. 2514 Var. 2566 Var. 2618 1.4 Var. 2203 Var. 2255 Var. 2307 Var. 2359 Var. 2411 Var. 2463 Var. 2515 Var. 2567 Var. 2619 1.5 Var. 2204 Var. 2256 Var. 2308 Var. 2360 Var. 2412 Var. 2464 Var. 2516 Var. 2568 Var. 2620 1.6 Var. 2205 Var. 2257 Var. 2309 Var. 2361 Var. 2413 Var. 2465 Var. 2517 Var. 2569 Var. 2621 1.7 Var. 2206 Var. 2258 Var. 2310 Var. 2362 Var. 2414 Var. 2466 Var. 2518 Var. 2570 Var. 2622 1.8 Var. 2207 Var. 2259 Var. 2311 Var. 2363 Var. 2415 Var. 2467 Var. 2519 Var. 2571 Var. 2623 1.9 Var. 2208 Var. 2260 Var. 2312 Var. 2364 Var. 2416 Var. 2468 Var. 2520 Var. 2572 Var. 2624 2 Var. 2209 Var. 2261 Var. 2313 Var. 2365 Var. 2417 Var. 2469 Var. 2521 Var. 2573 Var. 2625 NMT = No More Than Table 15. Exemplary embodiments of solution conditions useful for binding serine proteases 5 and / or serine protease zymogens to SiO2. 2024204779   11 Jul 2024 _______________________________pH NMT 5.6 NMT 5.8 NMT 6.0 NMT 6.2 NMT 6.4 NMT 6.6 NMT 6.8 NMT 7.0 Ionic Strength (mS / cm) 0.1-2.0 Var. 2626 Var. 2678 Var. 2730 Var. 2782 Var. 2834 Var. 2886 Var. 2938 Var. 2990 0.1-1.9 Var. 2627 Var. 2679 Var. 2731 Var. 2783 Var. 2835 Var. 2887 Var. 2939 Var. 2991 0.1-1.8 Var. 2628 Var. 2680 Var. 2732 Var. 2784 Var. 2836 Var. 2888 Var. 2940 Var. 2992 0.1-1.7 Var. 2629 Var. 2681 Var. 2733 Var. 2785 Var. 2837 Var. 2889 Var. 2941 Var. 2993 0.1-1.6 Var. 2630 Var. 2682 Var. 2734 Var. 2786 Var. 2838 Var. 2890 Var. 2942 Var. 2994 0.1-1.5 Var. 2631 Var. 2683 Var. 2735 Var. 2787 Var. 2839 Var. 2891 Var. 2943 Var. 2995 0.1-1.4 Var. 2632 Var. 2684 Var. 2736 Var. 2788 Var. 2840 Var. 2892 Var. 2944 Var. 2996 0.1-1.3 Var. 2633 Var. 2685 Var. 2737 Var. 2789 Var. 2841 Var. 2893 Var. 2945 Var. 2997 0.1-1.2 Var. 2634 Var. 2686 Var. 2738 Var. 2790 Var. 2842 Var. 2894 Var. 2946 Var. 2998 0.1-1.1 Var. 2635 Var. 2687 Var. 2739 Var. 2791 Var. 2843 Var. 2895 Var. 2947 Var. 2999 0.1-1.0 Var. 2636 Var. 2688 Var. 2740 Var. 2792 Var. 2844 Var. 2896 Var. 2948 Var. 3000 0.1-0.9 Var. 2637 Var. 2689 Var. 2741 Var. 2793 Var. 2845 Var. 2897 Var. 2949 Var. 3001 0.1-0.8 Var. 2638 Var. 2690 Var. 2742 Var. 2794 Var. 2846 Var. 2898 Var. 2950 Var. 3002 0.2-2.0 Var. 2639 Var. 2691 Var. 2743 Var. 2795 Var. 2847 Var. 2899 Var. 2951 Var. 3003 0.2-1.5 Var. 2640 Var. 2692 Var. 2744 Var. 2796 Var. 2848 Var. 2900 Var. 2952 Var. 3004 0.2-1.0 Var. 2641 Var. 2693 Var. 2745 Var. 2797 Var. 2849 Var. 2901 Var. 2953 Var. 3005 0.2-0.9 Var. 2642 Var. 2694 Var. 2746 Var. 2798 Var. 2850 Var. 2902 Var. 2954 Var. 3006 0.2-0.8 Var. 2643 Var. 2695 Var. 2747 Var. 2799 Var. 2851 Var. 2903 Var. 2955 Var. 3007 0.3-1.0 Var. 2644 Var. 2696 Var. 2748 Var. 2800 Var. 2852 Var. 2904 Var. 2956 Var. 3008 0.3-0.9 Var. 2645 Var. 2697 Var. 2749 Var. 2801 Var. 2853 Var. 2905 Var. 2957 Var. 3009 0.3-0.8 Var. 2646 Var. 2698 Var. 2750 Var. 2802 Var. 2854 Var. 2906 Var. 2958 Var. 3010 0.4-1.0 Var. 2647 Var. 2699 Var. 2751 Var. 2803 Var. 2855 Var. 2907 Var. 2959 Var. 3011 0.4-0.9 Var. 2648 Var. 2700 Var. 2752 Var. 2804 Var. 2856 Var. 2908 Var. 2960 Var. 3012 0.4-0.8 Var. 2649 Var. 2701 Var. 2753 Var. 2805 Var. 2857 Var. 2909 Var. 2961 Var. 3013 0.5-1.0 Var. 2650 Var. 2702 Var. 2754 Var. 2806 Var. 2858 Var. 2910 Var. 2962 Var. 3014 0.5-0.9 Var. 2651 Var. 2703 Var. 2755 Var. 2807 Var. 2859 Var. 2911 Var. 2963 Var. 3015 0.5-0.8 Var. 2652 Var. 2704 Var. 2756 Var. 2808 Var. 2860 Var. 2912 Var. 2964 Var. 3016 0.6-1.0 Var. 2653 Var. 2705 Var. 2757 Var. 2809 Var. 2861 Var. 2913 Var. 2965 Var. 3017 0.6-0.9 Var. 2654 Var. 2706 Var. 2758 Var. 2810 Var. 2862 Var. 2914 Var. 2966 Var. 3018 0.6-0.8 Var. 2655 Var. 2707 Var. 2759 Var. 2811 Var. 2863 Var. 2915 Var. 2967 Var. 3019 0.7-1.0 Var. 2656 Var. 2708 Var. 2760 Var. 2812 Var. 2864 Var. 2916 Var. 2968 Var. 3020 0.7-0.9 Var. 2657 Var. 2709 Var. 2761 Var. 2813 Var. 2865 Var. 2917 Var. 2969 Var. 3021 0.1 Var. 2658 Var. 2710 Var. 2762 Var. 2814 Var. 2866 Var. 2918 Var. 2970 Var. 3022 0.2 Var. 2659 Var. 2711 Var. 2763 Var. 2815 Var. 2867 Var. 2919 Var. 2971 Var. 3023 0.3 Var. 2660 Var. 2712 Var. 2764 Var. 2816 Var. 2868 Var. 2920 Var. 2972 Var. 3024 0.4 Var. 2661 Var. 2713 Var. 2765 Var. 2817 Var. 2869 Var. 2921 Var. 2973 Var. 3025 0.5 Var. 2662 Var. 2714 Var. 2766 Var. 2818 Var. 2870 Var. 2922 Var. 2974 Var. 3026 0.6 Var. 2663 Var. 2715 Var. 2767 Var. 2819 Var. 2871 Var. 2923 Var. 2975 Var. 3027 0.7 Var. 2664 Var. 2716 Var. 2768 Var. 2820 Var. 2872 Var. 2924 Var. 2976 Var. 3028 0.8 Var. 2665 Var. 2717 Var. 2769 Var. 2821 Var. 2873 Var. 2925 Var. 2977 Var. 3029 0.9 Var. 2666 Var. 2718 Var. 2770 Var. 2822 Var. 2874 Var. 2926 Var. 2978 Var. 3030 1 Var. 2667 Var. 2719 Var. 2771 Var. 2823 Var. 2875 Var. 2927 Var. 2979 Var. 3031 1.1 Var. 2668 Var. 2720 Var. 2772 Var. 2824 Var. 2876 Var. 2928 Var. 2980 Var. 3032 1.2 Var. 2669 Var. 2721 Var. 2773 Var. 2825 Var. 2877 Var. 2929 Var. 2981 Var. 3033 1.3 Var. 2670 Var. 2722 Var. 2774 Var. 2826 Var. 2878 Var. 2930 Var. 2982 Var. 3034 1.4 Var. 2671 Var. 2723 Var. 2775 Var. 2827 Var. 2879 Var. 2931 Var. 2983 Var. 3035 1.5 Var. 2672 Var. 2724 Var. 2776 Var. 2828 Var. 2880 Var. 2932 Var. 2984 Var. 3036 1.6 Var. 2673 Var. 2725 Var. 2777 Var. 2829 Var. 2881 Var. 2933 Var. 2985 Var. 3037 1.7 Var. 2674 Var. 2726 Var. 2778 Var. 2830 Var. 2882 Var. 2934 Var. 2986 Var. 3038 1.8 Var. 2675 Var. 2727 Var. 2779 Var. 2831 Var. 2883 Var. 2935 Var. 2987 Var. 3039 1.9 Var. 2676 Var. 2728 Var. 2780 Var. 2832 Var. 2884 Var. 2936 Var. 2988 Var. 3040 2 Var. 2677 Var. 2729 Var. 2781 Var. 2833 Var. 2885 Var. 2937 Var. 2989 Var. 3041 NM T = No More Than 2024204779   11 Jul 2024 A. Modified Alcohol Precipitation / Ion Exchange Chromatography Fractionation Methods

[0216] In one aspect, the present invention provides improved methods for the manufacture of 5 IgG compositions suitable for use in IVIG therapy. Generally, these methods provide IgG preparations having higher yields and comparable if not higher purity than current methods employed for the production of commercial IVIG products.

[0217] In one specific aspect, the present invention provides a method for preparing a composition of concentrated IgG from plasma, e.g., 10% IVIG, the method comprising 10 performing at least one alcohol precipitation step and at least one ion exchange chromatography step. In particular, several steps in the improved upstream process are different from prior processes, e.g., the use of 25% ethanol at lower temperatures, ethanol addition by spraying, pH adjustment by spraying, and the use of finely divided silica particles.

[0218] In a certain embodiment, the method comprises the steps of (a) precipitating a cryo-15 poor plasmid fraction, in a first precipitation step, with between about 6% and about 10% alcohol at a pH of between about 6.7 and about 7.3 to obtain a supernatant enriched in IgG, (b) precipitating IgG from the supernatant with between about 20% and about 30% alcohol at a lower temperature and at a pH of between about 6.7 and about 7.3 to form a first precipitate, (c) re-suspending the first precipitate formed in step (b) to form a suspension, (d) treating the 20 suspension formed in step (c) with a detergent, (e) precipitating IgG from the suspension with between about 20% and about 30% alcohol at a pH of between about 6.7 and about 7.3 to form a second precipitate, (f) re-suspending the second precipitate formed in step (e) to form a suspension, (g) treating the suspension formed in step (f) with a solvent and / or detergent, and (h) performing at least one ion exchange chromatography fractionation thereby preparing a 25 composition of concentrated IgG. In one embodiment, the method further comprises treating the suspension formed in step (c) with finely divided silica dioxide (SiO2) and filtering the solution prior to step (d).

[0219] In one embodiment, a method for preparing a concentrated IgG composition from plasma is provided, the method comprising the steps of (a) adjusting the pH of a cryo-poor 30 plasma fraction to about 7.0, (b) adjusting the ethanol concentration of the cryo-poor plasma 2024204779   11 Jul 2024 fraction of step (a) to at or about 25% (v / v) at a temperature between about -5°C and about -9°C, thereby forming a mixture, wherein the ethanol concentration may be adjusted by spraying, (c) separating liquid and precipitate from the mixture of step (b), (d) re-suspending the precipitate of step (c) with a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted 5 with between about 400 and about 700 ml of glacial acetic acid per 1000L of 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 at least 3 filter press dead volumes of a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted with about 150 ml of 10 glacial acetic acid per WOOL of buffer, thereby forming a wash solution, (h) combining the filtrate of step (f) with the wash 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 7.0 and adding ethanol to a final concentration of at or about 25%, thereby forming a precipitate, wherein the ethanol concentration and / or pH may be adjusted by spraying (j) separating liquid and precipitate 15 from the mixture of step (i), (k) dissolving the precipitate in an aqueous solution comprising a solvent or detergent and maintaining the solution for at least 60 minutes, (1) passing the solution after step (k) through a cation exchange chromatography column and eluting proteins absorbed on the column in an eluate, (m) passing the eluate from step (1) through an anion exchange chromatography column to generate an effluent (i.e., flow-through), (n) passing the effluent from 20 step (m) through a nanofilter to generate a nanofiltrate, (o) passing the nanofiltrate from step (n) through an ultrafiltration membrane to generate an ultrafiltrate, and (p) diafiltrating the ultrafiltrate from step (o) against a diafiltration buffer to generate a diafiltrate having a protein concentration between about 8% (w / v) and about 22% (w / v), thereby obtaining a composition of concentrated IgG. In one embodiment, the temperature of step (b) is at or about -7°C. In one 25 specific embodiment, the suspension buffer in step (d) is adjusted with about 600 mL glacial acetic acid.

[0220] In certain embodiments, the diafiltrate will have a protein concentration between about 8% and about 12%, for example, about 8%, or about 9%, 10%, 11%, or 12%. In a preferred embodiment, the diafiltrate will have a protein concentration of at or about 10%. In another 30 preferred embodiment, the diafiltrate will have a protein concentration of at or about 11%. In yet another preferred embodiment, the diafiltrate will have a protein concentration of at or about 12%. In other embodiments, the diafiltrate will have a protein concentration between about 13% 2024204779   11 Jul 2024 and about 17%, for example, about 13%, or about 14%, 15%, 16%, or 17%. In yet other embodiments, the diafiltrate will have a protein concentration between about 18% and about 22%, for example, about 18%, or about 19%, 20%, 21%, or 22%. In a preferred embodiment, the diafiltrate will have a protein concentration of at or about 20%. In another preferred 5 embodiment, the diafiltrate will have a protein concentration of at or about 21%. In yet another preferred embodiment, the diafiltrate will have a protein concentration of at or about 22%.

[0221] In certain embodiments of the present invention, the methods provided herein may comprise improvements in two or more of the fractionation process steps described above. For example, embodiments may include improvements in the first precipitation step, the Modified 10 Fraction II+III precipitation step, the Modified Fraction II+III dissolution step, and / or the Modified Fraction II+III suspension filtration step.

[0222] In one embodiment, the improvement made in the first precipitation step is the addition of alcohol by spraying. In another embodiment, the improvement made in the first precipitation step is the addition of a pH modifying agent by spraying. In yet embodiment, the improvement 15 made in the first precipitation step is the adjustment of the pH of the solution after addition of the alcohol. In a related embodiment, the improvement made in the first precipitation step is the maintenance of the pH during the addition of the alcohol. In another related embodiment, the improvement made in the first precipitation step is the maintenance of the pH during the precipitation incubation time by continuously adjusting the pH of the solution. In certain 20 embodiments, the first precipitation step may be improved by implementing more than one of these improvements. Further improvements that may be realized in this step will be evident from the section provided below discussing the first precipitation step - Modified Fractionation I. By implementing one or more of the improvements described above, a reduced amount of IgG is lost in the precipitate fraction of the first precipitation step and / or a reduced fraction of IgG is 25 irreversibly denatured during the precipitation step.

[0223] In one embodiment, the improvement made in the Modified Fraction II+III precipitation step is the addition of alcohol by spraying. In another embodiment, the improvement made in the Modified Fraction II+III precipitation step is the addition of a pH modifying agent by spraying. In yet embodiment, the improvement made in the Modified 30 Fraction II+III precipitation step is the adjustment of the pH of the solution after addition of the alcohol. In a related embodiment, the improvement made in the Modified Fraction II+III 2024204779   11 Jul 2024 precipitation step is the maintenance of the pH during the addition of the alcohol. In another related embodiment, the improvement made in the Modified Fraction II+III precipitation step is the maintenance of the pH during the precipitation incubation time by continuously adjusting the pH of the solution. In another aspect, the Modified Fraction II+III precipitation step is improved 5 by increasing the concentration of alcohol to at or about 25%. In yet another embodiment, the Modified Fraction II+III precipitation step is improved by lowering the incubation temperature to between about -7°C and -9°C. In certain embodiments, the Modified Fraction II+III precipitation step may be improved by implementing more than one of these improvements. Further improvements that may be realized in this step will be evident from the section provided 10 below discussing the second precipitation step - Modified Fractionation II+III.. By implementing one or more of the improvements described above, a reduced amount of IgG is lost in the supernatant fraction of the Modified Fraction II+III precipitation step and / or a reduced fraction of IgG is irreversibly denatured during the precipitation step.

[0224] In one embodiment, the improvement made in the Modified Fraction II+III dissolution 15   step is achieved by increasing the glacial acetic acid content of the dissolution buffer to about 0.06%. In another embodiment, the improvement made in the Modified Fraction II+III dissolution step is achieved by maintaining the pH of the solution during the dissolution incubation time by continuously adjusting the pH of the solution. In another embodiment, the improvement made in the Modified Fraction II+III dissolution step is achieved by mixing finely 20 divided silicon dioxide (SiO2) with the Fraction II+III suspension prior to filtration. In certain embodiments, the Modified Fraction II+III dissolution step may be improved by implementing more than one of these improvements. Further improvements that may be realized in this step will be evident from the section provided below discussing the Modified Fraction II+III dissolution step - Extraction of the Modified Fraction II+III Precipitate. By implementing one 25 or more of the improvements described above, an increased amount of IgG is recovered in the Fraction II+III suspension and / or the amount of impurities is reduced in the Fraction II+III suspension.

[0225] An exemplary improvement made in the Modified Fraction II+III suspension filtration step is realized by post-washing the filter with at least about 3.6 dead volumes of dissolution 30 buffer containing at or about 150 mL glacial acetic acid per 1000 L. Further improvements that may be realized in this step will be evident from the section provided below discussing the Modified Fraction II+III suspension filtration step - Pretreatment and Filtration of the Modified 2024204779   11 Jul 2024 Fraction II+III Suspension. By implementing one or more of the improvements described above, a reduced amount of IgG is lost during the Modified Fraction II+III suspension filtration step.

[0226] In one embodiment, the method may comprise an improvement in the first precipitation step and the Modified Fraction II+III precipitation step. 5

[0227] In another embodiment, the method may comprise an improvement in the first precipitation step and the Modified Fraction II+III dissolution step.

[0228] In another embodiment, the method may comprise an improvement in the first precipitation step and the Modified Fraction II+III suspension filtration step.

[0229] In another embodiment, the method may comprise an improvement in the Modified 10 Fraction II+III precipitation step and the Modified Fraction II+III dissolution step.

[0230] In another embodiment, the method may comprise an improvement in the Modified Fraction II+III precipitation step and the Modified Fraction II+III suspension filtration step.

[0231] In another embodiment, the method may comprise an improvement in the Modified Fraction II+III dissolution step and the Modified Fraction II+III suspension filtration step. 15

[0232] In another embodiment, the method may comprise an improvement in the first precipitation step, the Modified Fraction II+III precipitation step, and the Modified Fraction II+III dissolution step.

[0233] In another embodiment, the method may comprise an improvement in the first precipitation step, the Modified Fraction II+III precipitation step, and the Modified Fraction 20 II+III suspension filtration step.

[0234] In another embodiment, the method may comprise an improvement in the first precipitation step, the Modified Fraction II+III dissolution step, and the Modified Fraction II+III suspension filtration step.

[0235] In another embodiment, the method may comprise an improvement in the Modified 25 Fraction II+III precipitation step, the Modified Fraction II+III dissolution step, and the Modified Fraction II+III suspension filtration step. 2024204779   11 Jul 2024

[0236] In another embodiment, the method may comprise an improvement in all of the first precipitation step, the Modified Fraction II+III precipitation step, the Modified Fraction 11+111 dissolution step, and the Modified Fraction II+III suspension filtration step.

[0237] In certain embodiments, one process improvement in the IgG purification methods 5 provided herein comprises the spray addition of one or more solutions that would otherwise be introduced into a plasma fraction by fluent addition. For example, in certain embodiments the process improvement comprises the addition of alcohol (e.g., ethanol) into a plasma fraction for the purposes of precipitation of one or more protein species by spraying. In other embodiments, solutions that may be added to a plasma fraction by spraying include, without limitation, a pH 10 modifying solution, a solvent solution, a detergent solution, a dilution buffer, a conductivity modifying solution, and the like. In a preferred embodiment, one or more alcohol precipitation steps is performed by the addition of alcohol to a plasma fraction by spraying. In a second preferred embodiment, one or more pH adjustment steps is performed by the addition of a pH modifying solution to a plasma fraction by spraying. 15

[0238] In certain embodiments, another process improvement, which may be combined with any other process improvement, comprises the adjustment of the pH of a plasma fraction being precipitated after and / or concomitant with the addition of the precipitating agent (e.g., alcohol or polyethelene glycol). In some embodiments, a process improvement is provided in which the pH of a plasma fraction being actively precipitated is maintained throughout the entire precipitation 20 incubation or hold step by continuous monitoring and adjustment of the pH. In preferred embodiments the adjustment of the pH is performed by the spray addition of a pH modifying solution.

[0239] In other embodiments, another process improvement, which may be combined with any other process improvement, comprises the use of a finely divided silica treatment step to remove 25 impurities. 1. Preparation of Cryo-poor Plasma

[0240] The starting material used for the preparation of concentrated IgG compositions generally consists of either recovered plasma (i.e., plasma that has been separated from whole blood ex vivo) or source plasma (i.e., plasma collected via plasmapheresis). The purification 30 process typically starts with thawing previously frozen pooled plasma, which has already been 2024204779   11 Jul 2024 assayed for safety and quality considerations. Thawing is typically carried out at a temperature no higher than 6°C. After complete thawing of the frozen plasma at low temperature, centrifugation is performed in the cold (e.g., < 6°C) to separate solid cryo-precipitates from the liquid supernatant. Alternatively, the separation step can be performed by filtration rather than 5 centrifugation. The liquid supernatant (also referred to as “cryo-poor plasma,” after coldinsoluble proteins removed by centrifugation from fresh thawed plasma) is then processed in the next step. Various additional steps can be taken at this juncture for the isolation of factor eight inhibitor bypass activity (FEIBA), Factor IX-complex, Factor Vll-concentrate, or Antithrombin Ill-complex. 10                2. First Precipitation Event - Modified Fractionation I

[0241] In this step, cryo-poor plasma is typically cooled to about 0 ± 1°C and the pH is adjusted to between about 7.0 and about 7.5, preferably between about 7.1 and about 7.3, most preferably about 7.2. In one embodiment, the pH of the cryo-poor plasma is adjusted to a pH of at or about 7.2. Pre-cooled ethanol is then added while the plasma is stirred to a target 15 concentration of ethanol at or about 8% v / v. At the same time the temperature is further lowered to between about -4 and about 0°C. In a preferred embodiment, the temperature is lowered to at or about -2°C, to precipitate contaminants such as a2-macroglobulin, Pia- and Pic-globulin, fibrinogen, and Factor VIII. Typically, the precipitation event will include a hold time of at least about 1 hour, although shorter or longer hold times may also be employed. Subsequently, the 20 supernatant (Supernatant I), ideally containing the entirety of the IgG content present in the cryopoor plasma, is then collected by centrifugation, filtration, or another suitable method.

[0242] As compared to conventional methods employed as a first fractionation step for cryopoor plasma (Cohn et al., supra', Oncley et al., supra), the present invention provides, in several embodiments, methods that result in improved IgG yields in the Supernatant I fraction. In one 25 embodiment, the improved IgG yield is achieved by adding the alcohol by spraying. In another embodiment, the improved IgG yield is achieved by adding a pH modifying agent by spraying. In yet another embodiment, the improved IgG yield is achieved by adjusting the pH of the solution after addition of the alcohol. In a related embodiment, the improved IgG yield is achieved by adjusting the pH of the solution during the addition of the alcohol. 30

[0243] In one specific aspect, the improvement relates to a method in which a reduced amount of IgG is lost in the precipitate fraction of the first precipitation step. For example, in certain 2024204779   11 Jul 2024 embodiments, a reduced amount of IgG is lost in the precipitate fraction of the first precipitation step as compared to the amount of IgG lost in the first precipitation step of the Cohn method 6 protocol.

[0244] In certain embodiments, the process improvement is realized by adjusting the pH of the 5 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 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 addition of the precipitating alcohol. In a particular embodiment, the pH of the solution is adjusted to about 7.2 after addition of the 10 precipitating alcohol. As such, in certain embodiments, a reduced amount of IgG is lost in the precipitate fraction of the first precipitation step as compared to an analogous precipitation step in which the pH of the solution is adjusted prior to but not after 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 15 alcohol is ethanol.

[0245] In other certain embodiments, the process improvement is realized by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying, rather than by fluent addition. As such, in certain embodiments, a reduced amount of IgG is lost in the precipitate fraction of the first precipitation step as compared to an analogous precipitation step in which the 20 alcohol and / or solution used to adjust the pH is introduced by fluent addition. In one embodiment, the alcohol is ethanol.

[0246] In yet other certain embodiments, the improvement is realized by adjusting the pH of the solution to between about 7.0 and about 7.5. In a preferred embodiment, the pH of the solution is adjusted to between about 7.1 and about 7.3. In other embodiments, the pH of the 25 solution is adjusted to at or about 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 after the addition of the precipitating alcohol and by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying, rather than by fluent addition. In a particular embodiment, the pH of the solution is adjusted to at or about 7.2 after addition of the precipitating alcohol and by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying, rather than by fluent 30 addition. In one embodiment, the alcohol is ethanol. 2024204779   11 Jul 2024 3. Second Precipitation Event - Modified Fractionation II+III

[0247] To further enrich the IgG content and purity of the fractionation, Supernatant I is subjected to a second precipitation step, which is a modified Cohn-Oncley Fraction II+III fractionation. Generally, the pH of the solution is adjusted to a pH of between about 6.6 and 5 about 6.8. In a preferred embodiment, the pH of the solution is adjusted to at or about 6.7. Alcohol, preferably ethanol, is then added to the solution while being stirred to a final concentration of between about 20% and about 25% (v / v) to precipitate the IgG in the fraction. In a preferred embodiment, alcohol is added to a final concentration of at or about 25% (v / v) to precipitate the IgG in the fraction. Generally, contaminants such as a i-lipoprotein, ai- 10 antitrypsin, Gc-globulins, aix-glycoprotin, haptoglobulin, ceruloplasmin, transferrin, hemopexin, a fraction of the Christmas factor, thyroxin binding globulin, cholinesterase, hypertensinogen, and albumin will not be precipitated by these conditions.

[0248] Prior to or concomitant with alcohol addition, the solution is further cooled to between about -7°C and about -9°C. In a preferred embodiment, the solution is cooled to a temperature at 15   or about -7°C. After completion of the alcohol addition, the pH of the solution is immediately adjusted to between about 6.8 and about 7.0. In a preferred embodiment, the pH of the solution is adjusted to at or about 6.9. Typically, the precipitation event will include a hold time of at least about 10 hours, although shorter or longer hold times may also be employed. Subsequently, the precipitate (Modified Fraction II+III), which ideally contains at least about 85%, preferably 20 at least about 90%, more preferably at least about 95%, of the IgG content present in the cryopoor plasma, is separated from the supernatant by centrifugation, filtration, or another suitable method and collected. As compared to conventional methods employed as a second fractionation step for cryo-poor plasma (Cohn et al., supra-, Oncley et al., supra}, the present invention provides, in several embodiments, methods that result in improved IgG yields in the 25 Modified Fraction II+III precipitate. In a related embodiment, the present invention provides methods that result in a reduced loss of IgG in the Modified II+III supernatant.

[0249] As compared to conventional methods employed as a second fractionation step for cryo-poor plasma (Cohn et al., supra-, Oncley et al., supra}, the present invention provides, in several embodiments, methods that result in improved IgG yields in the Modified Fraction II+III 30 precipitate. In one embodiment, the improvement is realized by the addition of alcohol by spraying. In another embodiment, the improvement is realized by the addition of a pH 2024204779   11 Jul 2024 modifying agent by spraying. In another embodiment, the improvement is realized by adjusting the pH of the solution after addition of the alcohol. In a related embodiment, the improvement is realized by adjusting the pH of the solution during addition of the alcohol. In another embodiment, the improvement is realized by increasing the concentration of alcohol (e.g., 5 ethanol) to about 25% (v / v). In another embodiment, the improvement is realized by lowering the temperature of the precipitation step to between about -7°C and -9°C. In a preferred embodiment, the improvement is realized by increasing the concentration of alcohol (e.g., ethanol) to about 25% (v / v) and lowing the temperature to between about -7°C and -9°C. In comparison, both Cohn et al. and Oncley et al. perform precipitation at -5°C and Oncley et al. 10 use 20% alcohol, in order to reduce the level of contaminants in the precipitate. Advantageously, the methods provided herein allow for maximal IgG yield without high levels of contamination in the final product.

[0250] It has been discovered that when the pH of the solution is adjusted to a pH of about 6.9 prior to addition of the precipitating alcohol, the pH of the solution shift from 6.9 to between 15 about 7.4 and about 7.7, due in part to protein precipitation (see, Figure 8). As the pH of the solution shifts away from 6.9, precipitation of IgG becomes less favorable and the precipitation of certain contaminants becomes more favorable. Advantageously, the inventors have found that by adjusting the pH of the solution after addition of the precipitating alcohol, that a higher percentage of IgG is recovered in the Fraction II+III precipitate. 20

[0251] Accordingly, in one aspect, the improvement relates to a method in which a reduced amount of IgG is lost in the supernatant fraction of the modified Fraction II+III precipitation step. In other words, an increased percentage of the starting IgG is present in the Fraction II+III precipitate. In certain embodiments, the process improvement is 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 25 precipitating alcohol. In another embodiment, the process improvement is realized by maintaining the pH of the solution to between about 6.7 and about 7.1 continuously during the precipitation incubation period. In other 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 30 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 maintained at between about 6.8 to about 7.0 2024204779   11 Jul 2024 continuously during the precipitation incubation period, or at a pH of about 6.9 continuously during the precipitation incubation period. As such, in certain embodiments, a reduced amount of IgG is lost in the supernatant fraction of the second precipitation step as compared to an analogous precipitation step in which the pH of the solution is adjusted prior to but not after 5 addition of the precipitating alcohol or to an analogous precipitation step in which the pH of the solution is not maintained during the entirety of 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. 10

[0252] In another embodiment, the process improvement is realized by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying, rather than by fluent addition. As such, in certain embodiments, a reduced amount of IgG is lost in the supernatant fraction of the second precipitation step as compared to an analogous precipitation step in which the alcohol and / or solution used to adjust the pH is introduced by fluent addition. In one embodiment, the 15 alcohol is ethanol.

[0253] In another embodiment, the process improvement is realized by performing the precipitation step at a temperature between about -7°C and about -9°C. In one embodiment, the precipitation step is performed at a temperature of at or about -7°C. In another embodiment, the precipitation step is performed at a temperature of at or about -8°C. In another embodiment, the 20 precipitation step is performed at a temperature of at or about -9°C. In certain embodiments, the alcohol concentration of the precipitation step is between about 23% and about 27%. In a preferred embodiment, the alcohol concentration is between about 24% and about 26%. In another preferred embodiment, the alcohol concentration is at or about 25%. In other embodiments, the alcohol concentration may be at or about 23%, 24%, 25%, 26%, or 27%. In a 25 particular embodiment, the second precipitation step is performed at a temperature of at or about -7°C with an alcohol concentration of at or about 25%. In one embodiment, the alcohol is ethanol.

[0254] The effect of increasing the alcohol concentration of the second precipitation from 20%, as used in Oncley et al., supra, to 25% and lowering the temperature of the incubation 30 from -5°C, as used in the Cohn and Oncley methods, to at or about -7°C is a 5 %to 6% increase in the IgG content of the modified Fraction II+III precipitate. 2024204779   11 Jul 2024

[0255] In another embodiment, the process improvement is realized by adjusting the pH of the solution to between about 6.7 and about 7.1, preferably at or about 6.9, immediately after or during the addition of the precipitating alcohol, maintaining the pH of the solution at a pH of between about 6.7 and about 7.1, preferably at or about 6.9, by continuously adjusting the pH 5 during the precipitation incubation period, and by adding the precipitating alcohol and / or the solution used to adjust the pH by spraying, rather than by fluent addition. In another particular embodiment, the process improvement is realized by performing the precipitation step at a temperature between about -7°C and about -9°C, preferably at or about -7°C and by precipitating the IgG with an alcohol concentration of between about 23% and about 27%, preferably at or 10 about 25%. In yet another particular embodiment, the process improvement is realized by incorporating all of the Modified Fraction II+III improvements provided above. In a preferred embodiment, the process improvement is realized by precipitating IgG at a temperature of at or about -7°C with at or about 25% ethanol added by spraying and then adjusting the pH of the solution to at or about 6.9 after addition of the precipitating alcohol. In yet another preferred 15 embodiment, the pH of the solution is maintained at or about 6.9 for the entirety of the precipitation incubation or hold time. 4. Extraction of the Modified Fraction II+III Precipitate

[0256] In order to solubilize the IgG content of the modified Fraction II+III precipitate, a cold extraction buffer is used to re-suspend the Fractionation II+III precipitate at a typical ratio of 1 20 part precipitate to 15 parts of extraction buffer. Other suitable re-suspension ratios may be used, for example from about 1:8 to about 1:30, or from about 1:10 to about 1:20, or from about 1:12 to about 1:18, or from about 1:13 to about 1:17, or from about 1:14 to about 1:16. In certain embodiments, the re-suspension 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. 25

[0257] Suitable solutions for the extraction of the modified II+III precipitate will generally have a pH between about 4.0 and about 5.5. In certain embodiments, the solution will have a pH between about 4.5 and about 5.0, in other embodiments, the extraction solution will have 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 a preferred embodiment, the pH of the extraction buffer will be at or about 4.5. In another preferred 30 embodiment, the pH of the extraction buffer will be at or about 4.7. In another preferred embodiment, the pH of the extraction buffer will be at or about 4.9. Generally, these pH 2024204779   11 Jul 2024 requirements can be met using a buffering agent selected from, for example, acetate, citrate, monobasic phosphate, dibasic phosphate, mixtures thereof, and the like. Suitable buffer concentrations typically range from about 5 to about 100 mM, or from about 10 to about 50 mM, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM 5 buffering agent.

[0258] The extraction buffer will preferably have 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 will be about 0.5 mS-cm1, 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. One of ordinary skill in the art will know how to generate 10 extraction buffers having an appropriate conductivity.

[0259] In one particular embodiment, an exemplary extraction buffer may contain at or about 5 mM monobasic sodium phosphate and at or about 5 mM acetate at a pH of at or about 4.5 ± 0.2 and conductivity of at or about 0.7 to 0.9 mS / cm.

[0260] Generally, the extraction is performed at between about 0°C and about 10°C, or 15 between about 2°C and about 8°C. In certain embodiments, the 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 a particular embodiment, the extraction is performed at between about 2°C and about 10°C. Typically, the extraction process will proceed for between about 60 and about 300 minutes, or for between about 120 and 240 min, or for between about 150 and 210 minutes, while the suspension is continuously stirred. 20 In certain embodiments, the extraction process will proceed 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 process will proceed for at least 160 minutes with continuous stirring.

[0261] It has been found that employing an extraction buffer containing 5 mM monobasic 25 sodium phosphate, 5 mM acetate, and 0.051% to 0.06% glacial acetic acid (v / v), a substantial increase in the yield increase in the final IgG composition can be obtained without jeopardizing the purity of the final product. The correlation of amount of acetic acid and extraction buffer pH is demonstrated in Figure 9. In a preferred embodiment, the Fraction II+III precipitate is extracted with a paste to buffer ration of at or about 1:15 at a pH of at or about 4.5 ± 0.2. 2024204779   11 Jul 2024

[0262] Advantageously, it has been found that compared to the current manufacturing process for GAMMAGARD® LIQUID (Baxter Healthcare), which employs an extraction buffer containing 5 mM monobasic sodium phosphate, 5 mM acetate, and 0.051% glacial acetic acid (v / v), that by increasing the glacial acetic acid content to at or about 0.06% (v / v), a substantial 5 increase in the yield increase in the final IgG composition can be obtained. As compared to methods previously employed for the extraction of the precipitate formed by the second precipitation step (GAMMAGARD® LIQUID), the present invention provides, in several embodiments, methods that result in improved IgG yields in the Modified Fraction II+III suspension. 10

[0263] In one aspect, the improvement relates to a method in which a reduced amount of IgG is lost in the non-solubilized fraction of the Modified Fraction II+III precipitate. In one embodiment, the process improvement is realized by extracting the Modified Fraction II+III precipitate at a ratio of 1:15 (precipitate to buffer) with a solution containing 5 mM monobasic 15 sodium phosphate, 5 mM acetate, and 0.06% glacial acetic acid (v / v). In another embodiment, the improvement is realized by maintaining the pH of the solution during the duration of the extraction process. In one embodiment, the pH of the solution is maintained at between about 4.1 and about 4.9 for the duration of the extraction process. In a preferred embodiment, the pH of the solution is maintained at between about 4.2 and about 4.8 for the duration of the extraction 20 process. In a more preferred embodiment, the pH of the solution is maintained at between about 4.3 and about 4.7 for the duration of the extraction process. In another preferred embodiment, the pH of the solution is maintained at between about 4.4 and about 4.6 for the duration of the extraction process. In yet another preferred embodiment, the pH of the solution is maintained at or at about 4.5 for the duration of the extraction process. 25

[0264] In another aspect, the improvement relates to a method in which an increased amount of IgG is solubilized from the Fraction II+III precipitate in the Fraction II+III dissolution step. In one embodiment, the process improvement is realized by solubilizing the Fraction II+III precipitate in a dissolution buffer containing 600 mL glacial acetic acid per 1000 L. In another embodiment, the improvement relates to a method in which impurities are reduced after the IgG 30 in the Fraction II+III precipitate is solubilized. In one embodiment, the process improvement is 2024204779   11 Jul 2024 realized by mixing finely divided silicon dioxide (SiO2) with the Fraction II+III suspension for at least about 30 minutes. 5. Pretreatment and Filtration of the Modified Fraction II+III Suspension 5

[0265] In order to remove the non-solubilized fraction of the Modified Fraction II+III precipitate (i.e., the Modified Fraction II+III filter cake), the suspension is filtered, typically using depth filtration. Depth filters that may be employed in the methods provided herein include, metallic, glass, ceramic, organic (such as diatomaceous earth) depth filters, and the like. Example of suitable filters include, without limitation, Cuno 50SA, Cuno 90SA, and Cuno VR06 10 filters (Cuno). Alternatively, the separation step can be performed by centrifugation rather than filtration.

[0266] Although the manufacturing process improvements described above minimize IgG losses in the initial steps of the purification process, critical impurities, including PKA activity, amidolytic activity, and fibrinogen content, are much higher when, for example, the II+III paste 15 is extracted at pH 4.5 or 4.6, as compared to when the extraction occurs at a pH around 4.9 to 5.0 (see, Examples 2 to 5).

[0267] In order to counter act the impurities extracted in the methods provided herein, it has now been found that the purity of the IgG composition can be greatly enhanced by the addition of a pretreatment step prior to filtration / centrifugation. In one embodiment, this pretreatment 20 step comprises addition of finely divided silica dioxide particles (e.g., fumed silica, Aerosil®) followed by a 40 to 80 minute incubation period during which the suspension is constantly mixed. In certain embodiments, the incubation period will be between about 50 minutes and about 70 minutes, or about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more minutes. Generally, the treatment will be performed at between about 0°C and about 10°C, or between about 2°C and 25 about 8°C. In certain embodiments, the treatment 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 a particular embodiment, the treatment is performed at between about 2°C and about 10°C.

[0268] The effect of the fumed silica treatment is exemplified by the results found in Example 17. In this example, a Fraction II+III precipitate is suspended and split into two samples, one of 30 which is clarified with filter aid only prior to filtration (Figure 7A) and one of which is treated 2024204779   11 Jul 2024 with fumed silica prior to addition Of the filter aid and filtration (Figure 7B). As can be seen in the chromatographs and in the quantitated data, the filtrate sample pretreated with fumed silica had a much higher IgG purity than the sample only treated with filter aid (68.8% vs. 55.7%; compare Tables 17 and 18, respectively). 5

[0269] In certain embodiments, fumed silica is added at a concentration of between about 20 g / kg II+III paste and about 100 g / kg II+III paste (i.e., for a Modified Fraction II+III precipitate that is extracted at a ratio of 1:15, fumed silica should be added at a concentration from about 20 g / 16 kg II+III suspension to about 100 g / 16 kg II+III suspension, or at a final concentration of about 0.125% (w / w) to about 0.625% (w / w)). In certain embodiments, the fumed silica may be 10 added at a concentration of about 20 g / kg II+III paste, or about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / kg II+III paste. In one specific embodiment, fumed silica (e.g., Aerosil 380 or equivalent) is added to the Modified Fraction II+III suspension to a final concentration of about 40 g / 16 kg II+III. Mixing takes place at about 2 to 8°C for at least 50 to 70 minutes. 15

[0270] In certain embodiments, SiO2 is added to a an IgG composition at a concentration between about 0.01 g / g protein and about 10 g / g protein. In another embodiment, SiO2 is added to a an IgG composition at a concentration between about 0.01 g / g protein and about 5 g / g protein. In another embodiment, SiO2 is added to an IgG composition at a concentration between about 0.02 g / g protein and about 4 g / g protein. In one embodiment, SiO2 is added at a 20 final concentration of at least 0.1 g per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 0.2 g per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 0.25 g per gram total protein. In other specific embodiments, fumed silica is added at a concentration of at least 1 g per gram total protein. In another specific embodiment, fumed silica is added at a concentration 25 of at least 2 g per gram total protein. In another specific embodiment, fumed silica is added at a concentration of at least 2.5 g per gram 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, 30   7.5 g, 8.0 g, 8.5 g, 9.0 g, 9.5 g, 10.0 g, or more per gram total protein. 2024204779   11 Jul 2024

[0271] In certain embodiments, filter aid, for example Celpure C300 (Celpure) or Hyflo-Supper-Cel (World Minerals), will be added after the silica dioxide treatment, to facilitate depth filtration. Filter aid can be added at a final concentration of from about 0.01 kg / kg II+III paste to about 1.0 kg / kg II+III paste, or from about 0.02 kg / kg II+III paste to about 0.8 kg / kg II+III paste, 5 or from about 0.03 kg / kg II+III paste to about 0.7 kg / kg II+III paste. In other embodiments, filter aid can be added at a final concentration of from about 0.01 kg / kg II+III paste to about 0.07 kg / kg II+III paste, or from about 0.02 kg / kg II+III paste to about 0.06 kg / kg II+III paste, or from about 0.03 kg / kg II+III paste to about 0.05 kg / kg II+III paste. In certain embodiments, the filter aid will be added at a final concentration of about 0.01 kg / kg II+III paste, or about 0.02, 0.03, 10    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 II+III paste.

[0272] A significant fraction of IgG was being lost during the filtration step of the GAMMAGARD® LIQUID manufacturing process. It was found that the current methods of post-filtration wash, using 1.8 dead volumes of suspension buffer to purge the filter press frames 15 and lines, were insufficient for maximal recovery of IgG at this step. Surprisingly, it was found that at least 3.0 dead volumes, preferably 3.6 dead volumes, of suspension buffer were required in order for efficient recovery of total IgG in the Modified Fraction II+III clarified suspension (see, Example 12 and Figure 1). In certain embodiments, the filter press may be washed with any suitable suspension buffer. In a particular embodiment, the wash buffer will comprise, for 20 example, 5 mM monobasic sodium phosphate, 5 mM acetate, and 0.015% glacial acetic acid (v / v).

[0273] In one aspect, the improvement relates to a method in which a reduced amount of IgG is lost during the Fraction II+III suspension filtration step. In one embodiment, the process improvement is realized by post-washing the filter with at least about 3.6 dead volumes of 25 dissolution buffer containing 150 ml glacial acetic acid per 1000 L. The relationship between the amount of glacial acetic acid and pH in the post-wash buffer is shown in Figure 10. In one embodiment, the pH of the post-wash extraction buffer is between about 4.6 and about 5.3. In a preferred embodiment, the pH of the post-wash buffer is between about 4.7 and about 5.2. In another preferred embodiment, the pH of the post-wash buffer is between about 4.8 and about 30   5.1. In yet another preferred embodiment, the pH of the post-wash buffer is between about 4.9 and about 5.0. 2024204779   11 Jul 2024

[0274] As compared to methods previously employed for the clarification of the suspension formed from the second precipitation step (GAMMAGARD® LIQUID), the present invention provides, in several embodiments, methods that result in improved IgG yields and purity in the clarified Fraction 11+111 suspension. In one aspect, the improvement relates to a method in which 5 a reduced amount of IgG is lost in the Modified Fraction II+III filter cake. In other aspect, the improvement relates to a method in which a reduced amount of an impurity is found in the clarified Fraction II+III suspension.

[0275] In one embodiment, the process improvements are realized by inclusion of a fumed silica treatment prior to filtration or centrifugal clarification of a Fraction II+III suspension. In 10 certain embodiments, the fumed silica treatment will include addition of from about 0.01 kg / kg II+III paste to about 0.07 kg / kg II+III paste, or from about 0.02 kg / kg II+III paste to about 0.06 kg / kg II+III paste, or from about 0.03 kg / kg II+III paste to about 0.05 kg / kg II+III paste, or about 0.02 kg / kg II+III paste, 0.03 kg / kg II+III paste, 0.04 kg / kg II+III paste, 0.05 kg / kg II+III paste, 0.06 kg / kg II+III paste, 0.07 kg / kg II+III paste, 0.08 kg / kg II+III paste, 0.09 kg / kg II+III paste, 15 or 0.1 kg / kg II+III paste, and the mixture will be incubated for between about 50 minutes and about 70 minutes, or about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more minutes at a temperature between about 2°C and about 8°C. In another embodiment, the process improvements are realized by inclusion of a fumed silica treatment which reduced the levels of residual fibrinogen, amidolytic activity, and / or prekallikrein activator activity. In a specific 20 embodiment, the process improvements are realized by inclusion of a fumed silica treatment, which reduces the levels of FXI, FXIa, FXII, and FXIIa in the immunoglobulin preparation.

[0276] In another embodiment, the process improvements are realized by washing the depth filter with between about 3 and about 5 volumes of the filter dead volume after completing the Modified Fraction II+III suspension filtration step. In certain embodiments, the filter will be 25 washed with between about 3.5 volumes and 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 of the filter dead volume. In a particular embodiment, the filter press will be washed with at least about 3.6 dead volumes of suspension buffer. 6. Detergent Treatment 30

[0277] In order to remove additional contaminants from the Modified Fraction II+III filtrate, the sample is next subjected to a detergent treatment. Methods for the detergent treatment of 2024204779   11 Jul 2024 plasma derived fractions are well known in the art. Generally, any standard non-ionic detergent treatment may be used in conjunction with the methods provided herein. For example, an exemplary protocol for a detergent treatment is provided below.

[0278] Briefly, polysorbate-80 is added to the Modified Fraction II+III filtrate at a final 5 concentration of about 0.2% (w / v) with stirring and the sample is incubated for at least 30 minutes at a temperature between about 2 to 8°C. Sodium citrate dehydrate is then mixed into the solution at a final concentration of about 8 g / L and the sample is incubated for an additional 30 minutes, with continuous of stirring at a temperature between about 2 to 8°C.

[0279] In certain embodiments, any suitable non-ionic detergent can be used. Examples of 10 suitable non-ionic detergents include, without limitation, Octylglucoside, Digitonin, C12E8, Lubrol, Triton X-100, Nonidet P-40, Tween-20 (i.e., polysorbate-20), Tween-80 (i.e., polysorbate-80), an alkyl poly(ethylene oxide), a Brij detergent, an alkylphenol poly(ethylene oxide), a poloxamer, octyl glucoside, decyl maltoside, and the like.

[0280] In one embodiment, a process improvement is realized by adding the detergent reagents 15   (e.g; polysorbate-80 and sodium citrate dehydrate) by spraying rather than by fluent addition. In other embodiments, the detergent reagents may be added as solids to the Modified Fraction II+III filtrate while the sample is being mixed to ensure rapid distribution of the additives. In certain embodiments, it is preferable to add solid reagents by sprinkling the solids over a delocalized surface area of the filtrate such that local overconcentration does not occur, such as in fluent 20 addition. 7. Third Precipitation Event - Precipitation G

[0281] In order to remove several residual small proteins, such as albumin and transferrin, a third precipitation is performed at a concentration of 25% alcohol. Briefly, the pH of the detergent treated II+III filtrate is adjusted to between about 6.8 and 7.2, preferably between 25 about 6.9 and about 7.1, most preferably about 7.0 with a suitable pH modifying solution (e.g., IM sodium hydroxide or IM acetic acid). Cold alcohol is then added to the solution to a final concentration of about 25% (v / v) and the mixture is incubated while stirring at between about -6°C to about -10°C for at least 1 hour to form a third precipitate (i.e., precipitate G). In one embodiment, the mixture is incubated for at lease 2 hours, or at least 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 30   13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more hours. In a preferred embodiment, the 2024204779   11 Jul 2024 mixture is incubated for at least 2 hours. In a more preferred embodiment, the mixture is incubated for at least 4 hours. In an even more preferred embodiment, the mixture is incubated for at least 8 hours.

[0282] In one aspect, a process improvement relates to a method in which a reduced amount of 5 IgG is lost in the supernatant fraction of the third precipitation step. In certain embodiments, the process improvement is realized by adjusting the pH of the solution to between about 6.8 and about 7.2 immediately after or during the addition of the precipitating alcohol. In another embodiment, the process improvement is realized by maintaining the pH of the solution to between about 6.8 and about 7.2 continuously during the precipitation incubation period. In 10 other embodiments, the pH of the solution is adjusted to between about 6.9 and 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 15 maintained at between about 6.9 to about 7.1 continuously during the precipitation incubation period, or at a pH of about 7.0 continuously during the precipitation incubation period. As such, in certain embodiments, a reduced amount of IgG is lost in the supernatant fraction of the third precipitation step as compared to an analogous precipitation step in which the pH of the solution is adjusted prior to but not after addition of the precipitating alcohol or to an analogous 20 precipitation step in which the pH of the solution is not maintained during the entirety of 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.

[0283] In another embodiment, the process improvement is realized by adding the precipitating 25 alcohol and / or the solution used to adjust the pH by spraying, rather than by fluent addition. As such, in certain embodiments, a reduced amount of IgG is lost in the supernatant fraction of the third precipitation step as compared to an analogous precipitation step in which the alcohol and / or solution used to adjust the pH is introduced by fluent addition. In one embodiment, the alcohol is ethanol. 2024204779   11 Jul 2024 8. Suspension and Filtration of Precipitate G (PptG)

[0284] In order to solubilize the IgG content of the precipitate G, a cold extraction buffer is used to re-suspend the PptG. Briefly, the precipitate G is dissolved 1 to 3.5 in Water for Injection (WFI) at between about 0°C and about 8°C to achieve an AU280-320 value of between 5 about 40 to 95. The final pH of the solution, which is stirred for at least 2 hours, is then adjusted to at or about 5.2 ± 0.2. In one embodiment, this pH adjustment is performed with IM acetic acid. To increase the solubility of IgG, the conductivity of the suspension is increased to between about 2.5 and 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 with a suitable 10 depth filter having a nominal pore size of between about 0.1 pm and about 0.4 pm in order to remove any undissolved particles. In one embodiment, the nominal pore size of the depth filter is about 0.2 pm (e.g., Cuno VR06 filter or equivalent) to obtain a clarified filtrate. In another embodiment, the suspended PptG solution is centrifuged to recover a clarified supernatant. Postwash of the filter is performed using a sodium chloride solution with a conductivity of between 15 about 2.5 and about 6.0 mS / cm. Typically, suitable solutions for the extraction of precipitate G include, WFI and low conductivity buffers. In one embodiment, a 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 20 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. 9. Solvent Detergent Treatment

[0285] In order to inactivate various viral contaminants which may be present in plasma- 25 derived products, the clarified PptG filtrate is next subjected to a solvent detergent (S / D) treatment. Methods for the detergent treatment of plasma derived fractions are well known in the art (for review see, Pelletier JP et al., Best Pract Res Clin Haematol. 2006; 19(1):205-42). Generally, any standard S / D treatment may be used in conjunction with the methods provided herein. For example, an exemplary protocol for an S / D treatment is provided below. 2024204779   11 Jul 2024

[0286] 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 between about 18°C and about 25°C for at least about an hour. 5

[0287] In one embodiment, a process improvement is realized by adding the S / D reagents (e.g., Triton X-100, Tween-20, and TNBP) by spraying rather than by fluent addition. In other embodiments, the detergent reagents may be added as solids to the clarified PptG filtrate, which is being mixed to ensure rapid distribution of the S / D components. In certain embodiments, it is preferable to add solid reagents by sprinkling the solids over a delocalized surface area of the 10 filtrate such that local overconcentration does not occur, such as in fluent addition. 10. Ion Exchange Chromatography

[0288] In order to further purify and concentrate IgG from the S / D treated PptG filtrate, cation exchange and / or anion exchange chromatography can be employed. Methods for purifying and concentrating IgG using ion exchange chromatography are well known in the art. For example, 15 U.S. Patent No. 5,886,154 describes a method in which a Fraction II+III precipitate is extracted at low pH (between about 3.8 and 4.5), followed by precipitation of IgG using caprylic acid, and finally implementation of two anion exchange chromatography steps. U.S. Patent No. 6,069,236 describes a chromatographic IgG purification scheme that does not rely on alcohol precipitation at all. PCT Publication No. WO 2005 / 073252 describes an IgG purification method involving 20 the extraction of a 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 the extraction of either a Fraction 1+11+111 or a 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 the extraction of either a Fraction I+II+III or a 25 Fraction II+III precipitate, caprylate treatment, and either one or two anion exchange chromatography steps. U.S. Patent No. 6,093,324 describes a purification method comprising the use of a macroporous anion exchange resin operated at a pH between about 6.0 and about 6.6. U.S. Patent No. 6,835,379 describes a purification method that relies on cation exchange chromatography in the absence of alcohol fractionation. The disclosures of the above 30 publications are hereby incorporated by reference in their entireties for all purposes 2024204779   11 Jul 2024

[0289] In one embodiment of the methods 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 is passed through a cation exchange column, which binds the IgG in the solution. The S / D reagents can 5 then be washed away from the absorbed IgG, which is subsequently eluted off of the column with a high pH elution buffer having a pH between about 8.0 and 9.0. In this fashion, the cation exchange chromatography step can be used to remove the S / D reagents from the preparation, concentrate the IgG containing solution, or both. In certain embodiments, the pH elution buffer may have a pH between about 8.2 and about 8.8, or between about 8.4 and about 8.6, or a pH of 10 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.

[0290] In certain embodiments, the eluate from the cation exchange column may be adjusted to a lower pH, for example between about 5.5 and about 6.5, and diluted with an appropriate buffer such that the conductivity of the solution is reduced. In certain embodiments, the pH of the 15 cation exchange eluate may be adjusted to a pH between about 5.7 and about 6.3, or between about 5.9 and 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 several contaminants found in the preparation. The column flow through, containing the IgG fraction, is collected during column 20 loading and washing. In certain embodiments, the ion exchange chromatographic steps of the present invention can be performed in column mode, batch mode, or in a combination of the two.

[0291] In certain embodiments, a process improvement is realized by adding the solution used to adjust the pH by spraying, rather than by fluent addition. 11. Nanofiltration and Ultra / Diafiltration 25

[0292] In order to further reduce the viral load of the IgG composition provided herein, the anion exchange column effluent may be nanofiltered using a suitable nanofiltration device. In certain embodiments, the nanofiltration device will have a mean pore size of between about 15 nm and about 200 nm. Examples of nanofilters suitable for this use include, without limitation, DVD, DV 50, DV 20 (Pall), Viresolve NFP, Viresolve NFR (Millipore), Planova 15N, 20N, 30   35N, and 75N (Planova). In a specific embodiment, the nanofilter may have a mean pore size of between about 15 nm and about 72 nm, or between about 19 nm and about 35 nm, or of about 15 2024204779   11 Jul 2024 nm, 19nm, 35nm, or 72 nm. In a preferred embodiment, the nanofilter will have a mean pore size of about 35 nm, such as an Asahi PLANOVA 35N filter or equivalent thereof.

[0293] Optionally, ultrafiltration / diafiltration may performed to further concentrate the nanofiltrate. In one embodiment, an open channel membrane is used with a specifically designed 5 post-wash and formulation near the end the production process render the resulting IgG compositions about twice as high in protein concentration (200mg / mL) compared to state of the art IVIGs (e.g., GAMMAGARD® LIQUID) without affecting yield and storage stability. With most of the commercial available ultrafiltration membranes a concentration of 200mg / mL IgG cannot be reached without major protein losses. These membranes will be blocked early and 10 therefore adequate post-wash is difficult to achieve. Therefore open channel membrane configurations have to be used. Even with open channel membranes, a specifically designed post-wash procedure has to be used to obtain the required concentration without significant protein loss (less than 2% loss). Even more surprising is the fact that the higher protein concentration of 200mg / mL does not effect the virus inactivation capacity of the low pH storage 15 step.

[0294] Subsequent to nanofiltration, the filtrate may be further concentrated by ultrafiltration / diafiltration. In one embodiment, the nanofiltrate may be concentrated by ultrafiltration to a protein concentration of between about 2% and about 10% (w / v). In certain embodiments, the ultrafiltration is carried out in a cassette with an open channel screen and the 20 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, 30, or fewer kDa. In a preferred embodiment, the ultrafiltration membrane has a NMWCO of no more than 50 kDa.

[0295] Upon completion of the ultrafiltration step, the concentrate may further be concentrated via diafiltration against a solution suitable for intravenous or intramuscular administration. In 25 certain embodiments, the diafiltration solution may comprise a stabilizing and / or buffering agent. In a preferred embodiment, the stabilizing and buffering agent is glycine at an appropriate concentration, for example between about 0.20 M and about 0.30M, or between about 0.22M and about 0.28M, or between about 0.24M and about 0.26 mM, or at a concentration 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 apreferred embodiment, the diafiltration buffer 30 contains at or about 0.25 M glycine. 2024204779   11 Jul 2024

[0296] Typically, the minimum exchange volume is at least about 3 times the original concentrate volume or at least about 4, 5, 6, 7, 8, 9, or more times the original concentrate volume. The IgG solution may be concentrated to a final protein concentration of between about 5% and about 25% (w / v), or between about 6% and about 18% (w / v), or between about 7% and 5 about 16% (w / v), or between about 8% and about 14% (w / v), or between about 9% and 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%, 25% or higher. In one embodiment, a final protein concentration of at least about 23% is achieved without adding the post-wash fraction to the concentrated solution. In another embodiment, a final protein concentration of at 10 least about 24% is achieved without adding the post-wash fraction to the concentrated solution. a final protein concentration of at least about 25% is achieved without adding the post-wash fraction to the concentrated solution. Typically, at the end of the concentration process, the pH of the solution will be between about 4.6 to 5.1.

[0297] In an exemplary embodiment, the pH of the IgG composition is adjusted to about 4.5 15 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 Polyether sulfon membrane). The concentrate is diafiltered against ten volumes of 0.25 M glycine solution, pH 4.5 ± 0.2. Throughout the ultra-diafiltration operation the solution is maintained at a temperature of between about 2°C to about 20   8°C. After diafiltration, the solution is concentrated to a protein concentration of at least 11 % (w / v). 12. Formulation

[0298] Upon completion of the diafiltration step, the protein concentration of the solution is adjusted to with the diafiltration buffer to a final concentration of between about 5% and about 25   20% (w / v), or between about 6% and about 18% (w / v), or between about 7% and about 16% (w / v), or between about 8% and about 14% (w / v), or between about 9% and 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%, or 20%. In a preferred embodiment, the final protein concentration of the solution is between about 9% and about 11%, more preferably about 10%. 30

[0299] The formulated bulk solution is further sterilized by filtering through a membrane filter with an absolute pore size of no more than about 0.22 micron, for example about 0.2 micron. 2024204779   11 Jul 2024 Then the solution is aseptically dispensed into final containers for proper sealing, with samples taken for testing.

[0300] In one embodiment, the IgG composition is further adjusted to a concentration of about 10.2 ± 0.2% (w / v) with diafiltration buffer. The pH is adjusted to about 4.4 to about 4.9 if 5 necessary. Finally, the solution is sterile filtered and incubated for three weeks at or about 30°C. B. Factor H

[0301] In one embodiment, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived Factor H composition. In one specific embodiment, the method comprises the steps of: (a) contacting the Factor H 10 composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (b) separating the SiO2 from the Factor H composition to remove the bound serine protease or serine protease zymogen. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). 15

[0302] In one embodiment, the method further comprises the step of performing a first Factor H protein enrichment step to form a first enriched Factor H composition, prior to contacting the composition with finely divided silicon dioxide (SiO2). In certain embodiments, the first Factor H protein enrichment step is selected from a protein precipitation step (e.g., an alcohol fractionation step), an ultrafiltration / diafiltration step, and a chromatographic step. 20

[0303] In certain embodiments, the methods described above further comprises the step of performing a second Factor H protein enrichment step to form a second enriched Factor H composition, prior to contacting the composition with finely divided silicon dioxide (SiO2). In certain embodiments, the first Factor H protein enrichment step is selected from a protein precipitation step (e.g., an alcohol fractionation step), an ultrafiltration / diafiltration step, and a 25 chromatographic step.

[0304] Accordingly, in one embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived Factor H composition, the method comprises the steps of: (a) performing a first Factor H enrichment step to form a first enriched plasma-derived Factor H composition; (b) performing a second Factor H 30 enrichment step to form a second enriched plasma-derived Factor H composition; (c) contacting 2024204779   11 Jul 2024 the second enriched composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and (d) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), 5 Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In certain embodiments, the combination of first and second enrichment steps is selected from any one of variations Var. 1 to Var. 100, found in Table 1.

[0305] In certain embodiments, the methods described above further comprises the step of performing an Factor H enrichment step after contacting the composition with finely divided 10 silicon dioxide (SiO2). In certain embodiments, the Factor H enrichment step is selected from a protein precipitation step (e.g., an alcohol fractionation step), an ultrafiltration / diafiltration step, and a chromatographic step.

[0306] Accordingly, in one embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived Factor H 15 composition the method comprises the steps of: (a) performing a first Factor H enrichment step to form a first enriched plasma-derived Factor H composition; (b) contacting the first enriched composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; (c) separating the SiO2 from the composition to remove the bound serine protease or serine protease zymogen; and (d) performing a second 20 Factor H enrichment step to form a second enriched plasma-derived Factor H composition. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In certain embodiments, the combination of first and second enrichment steps is selected from any one of variations Var. 1 to Var. 100, found in Table 1. 25

[0307] Likewise, in one embodiment, the invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived Factor H composition, the method comprising the steps of: (a) performing a first Factor H enrichment step to form a first enriched plasma-derived Factor H composition; (b) performing a second Factor H enrichment step to form a second enriched plasma-derived Factor H composition; (c) contacting the second 30 enriched composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; (d) separating the SiO2 from the 2024204779   11 Jul 2024 composition to remove the bound serine protease or serine protease zymogen; and (e) performing a third Factor H enrichment step to form a third enriched plasma-derived Factor H composition. In a preferred embodiment, the serine protease or serine protease zymogen is Factor Xia (FXIa), Factor Xlla (FXIIa), Factor XI (FXI), and / or Factor XII (FXII). In certain embodiments, the 5 combination of first and second enrichment steps is selected from any one of variations Var. 101 to Var. 1100, found in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, or Table 11. 1. Methods for the Manufacture of Plasma-Derived Factor H

[0308] Regarding production, the claimed processes starting from human plasma shall be 10 based on the sub-fractionation of typical industrial intermediates obtained by, e.g., the fractional precipitation by ethanol in the cold (reviewed in Schultze H E, Heremans J F; Molecular Biology of Human Proteins. Volume I: Nature and Metabolism of Extracellular Proteins 1966, Elsevier Publishing Company; p. 236-317). A preferred embodiment of such purification is the purification of functional Factor H from side fractions of industrial scale plasma fractionation in 15 such a way that established and licensed manufacturing processes of plasma products, which are under control of pharmaceutical regulatory authorities, like immunoglobulins, are not affected. For example, the filter cake obtained after filtration of a Fraction II+III paste suspension (Teschner W et al., Vox Sang. 2007 Jan;92(l):42-55), Fraction I precipitate (Cohn et al., (1946) supra), Precipitate III (Schultze H E, Heremans J F; Molecular Biology of Human Proteins. 20 Volume I: Nature and Metabolism of Extracellular Proteins 1966, Elsevier Publishing Company; p. 236-317 at p. 253) and precipitate B (method of Kistler and Nitschmann; supra at p. 253) are examples of such industrial sources for Factor H. Starting from those side fractions, purification procedures known in the art can be used to purify Factor H. They may be based on precipitation with polyethylene glycol (Nagasawa S, Stroud R M; Mol Immunol 1980; 17:1365-72), affinity 25 chromatography via immobilized heparin (citation as before), ion exchange chromatography (Crossley L G, Porter R R; Biochem J 1980; 191:173-82) and hydrophobic interaction chromatography (Ripoche J, Al Salihi A, Rousseaux J, Fontaine M; Biochem J 1984; 221, 8996).

[0309] In one embodiment, the starting material for the invention is prepared using Cohn 30 fractions. This fractionation is a well known fractionation used for the preparation of immunoglobulin preparations can be prepared from donor serum or monoclonal or recombinant 2024204779   11 Jul 2024 immunoglobulins. In a typical example, blood is collected from healthy donors. Usually, the blood is collected from the same species of animal as the subject to which the immunoglobulin preparation will be administered (typically referred to as "homologous" immunoglobulins). The immunoglobulins are isolated from the blood by suitable procedures, such as, for example, Cohn 5 fractionation, ultracentrifugation, electrophoretic preparation, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, polyethylene glycol fractionation, or the like. (See, e.g., Cohn et al., J. Am. Chern. Soc. 68:459-75 (1946); Oncley et al., J. Am. Chern. Soc. 71:541-50 (1949); Barundem et al., Vox Sang. 7:157-74 (1962); Koblet et al., Vox Sang. 13:93-102 (1967); U.S. Pat. Nos. 5,122,373 and 5,177,194; the disclosures of which are 10 incorporated herein by reference in their entireties for all purposes.) In one embodiment, the present invention uses the discarded fractions from the preparation of immunoglobulins. In a particular embodiment, the present invention uses the fraction that is found in a SiO2 filtration cake once the Fraction II+III extract is filtered.

[0310] Generally, Factor H preparations according to the present invention can be prepared 15 from any suitable starting materials, for example, recovered plasma or source plasma. In a typical example, blood or plasma is collected from healthy donors. Usually, the blood is collected from the same species of animal as the subject to which the Factor H preparation will be administered (typically referred to as “homologous” Factor H). The Factor H is isolated from the blood or plasma by suitable procedures, such as, for example, precipitation (alcohol 20 fractionation or polyethylene glycol fractionation), chromatographic methods (ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, etc.) ultracentrifiigation, and electrophoretic preparation, and the like. (See, e.g., Cohn et al., J. Am. Chern. Soc. 68:459-75 (1946); Deutsch et al., J. Biol. Chern. 164:109-118; Oncley et al., J. Am. Chern. Soc. 71:541-50 (1949); Cohn et al., J. Am. Chern. Soc. 72:465-474 (1950); Cohn et al., 25 Blood Cells and Plasma Proteins: Their State in Nature (J.L. Tullis, ed), pp. 1-58, Academic Press, NewYork and London (1953); Nitschmann et al., Helv. Chim. Acta 37:866-873; Kistler and Nitschmann, Vox Sang. 7:414-424 (1962); Barundem et al., Vox Sang. 7:157-74 (1962); Koblet et al., Vox Sang. 13:93-102 (1967); U.S. Patent Nos. 5,122,373 and 5,177,194; the disclosures of which are hereby incorporated by reference in their entireties for all purposes). 30

[0311] In certain embodiments, Factor H is recovered from material otherwise discarded during the manufacture of other commercially important blood products by plasma fractionation. For example, in an exemplary embodiment, Factor H is extracted from a Fraction I precipitate 2024204779   11 Jul 2024 and / or extracted from a filter cake formed after centrifugation or filtration of a re-suspended Fraction II+III paste. Advantageously, according to the methods provided herein, industrialscale preparation of Factor H can be achieved without the need for additional input plasma or the redesign and regulatory re-approval of existing manufacturing processes for other commercially 5 important plasma-derived blood products, such as IgG gamma globulins for intravenous (IVIG) or subcutaneous administration.

[0312] In one aspect, the present invention provides a method for preparing an enriched Factor H composition having reduced serine protease and / or serine protease zymogen content from plasma by extracting Factor H from a plasma fraction and reducing the FXI, FXIa, FXII, and / or 10 FXIIa content with a SiO2 treatment method provided herein.

[0313] In one embodiment, a method is provided for preparing an enriched Factor H composition from plasma, the method comprising the steps of: (a) precipitating proteins from a cryo-poor plasma fraction, in a first precipitation step, with between about 6% and about 10% alcohol at a pH of between about 7.0 and about 7.5 to obtain a first precipitate and a first 15 supernatant; (b) precipitating Factor H from the first supernatant, in a second precipitation step, with between about 20% and about 30% alcohol at a pH of between about 6.7 and about 7.3 to form a second precipitate; (c) re-suspending the second precipitate to form a suspension; (d) mixing finely divided silicon dioxide (SiO2) with the suspension from step (c); (e) separating the suspension to form a filter cake and a supernatant; and (f) extracting Factor H from the SiO2 20 filter cake under solution conditions that reduce the level of a serine protease or serine protease zymogen in the final composition. In a preferred embodiment, the filter cake is separated from the supernatant by filtering the suspension through a filter press containing a suitable filter. In one embodiment, Factor H can be extracted by re-circulating an extraction buffer through a filter press containing a filter cake. 25

[0314] In a second aspect, the present invention provides a method for preparing an enriched Factor H composition with reduced serine protease and / or serine protease zymogen content from plasma by extracting Factor H from a Fraction I precipitate.

[0315] In a preferred embodiment, a method is provided for preparing an enriched Factor H composition from plasma, the method comprising the steps of: (a) precipitating proteins from a 30 cryo-poor plasma fraction, in a first precipitation step, with between about 6% and about 10% alcohol at a pH of between about 7.0 and about 7.5 to obtain a first precipitate and a first 2024204779   11 Jul 2024 supernatant; (b) extracting Factor H from the precipitate with a Factor H extraction buffer, and (c) reducing the level of a serine protease or serine protease zymogen by treating the composition with SiCh, using a suitable method provided herein.

[0316] In one aspect, a method is provided for preparing an enriched Factor H composition 5 from plasma, by extracting Factor H from a pool of two or more manufacturing byproduct fractions created by a process designed to provide a second blood protein, for example, IgG gamma globulins. In one embodiment, the method comprises pooling a Fraction I precipitate and a Fraction II+III filter cake formed during the manufacture of IgG gamma globulins (e.g., IVIG) and extracting Factor H from the pooled fractions. 10

[0317] In certain embodiments, an enriched Factor H composition having reduced serine protease and / or serine protease zymogen content may be further purified subsequent to extraction from a Fraction I precipitate and / or Fraction II+III filter cake. Various methods are available for further purifying Factor H, including without limitation, additional precipitation steps or fractionations, affinity chromatography, ion exchange chromatography, hydrophobic interaction 15 chromatography, size exclusion chromatography, solvent / detergent (SZD) treatment, nanofiltration, ultrafiltration, diafiltration, and the like.

[0318] In one embodiment, the method further comprises precipitating impurities from an enriched Factor H composition. In certain embodiments, this step comprises precipitating at least one impurity, for example a lipid or protein, from the composition and then separating the 20 precipitate from the supernatant containing Factor H. Optionally, Factor H can then be precipitated from the supernatant in a separate precipitation.

[0319] In a specific embodiment, a Factor H composition extracted from a plasma fraction (e.g., fraction I precipitate, fraction II+III precipitate, Precipitate B precipitate, etc.) is further enriched by precipitating at least one impurity out of the solution using PEG at a final 25 concentration of between about 2.5% and about 7.5%. In another embodiment, PEG is used at a final concentration of between about 3% and about 7%. In another embodiment, PEG is used at a final concentration of between about 4% and about 6%. In yet another embodiment, PEG is used at a final concentration of about 5%.

[0320] In another specific embodiment, a Factor H composition extracted from a plasma 30 fraction (e.g., fraction I precipitate, fraction II+III precipitate, Precipitate B precipitate, etc.) is 2024204779   11 Jul 2024 further enriched by precipitating Factor H out of the solution using PEG at a final concentration of between about 9% and about 15%. In another embodiment, PEG is used at a final concentration of between about 10% and about 14%. In another embodiment, PEG is used at a final concentration of between about 11% and about 13%. In yet another embodiment, PEG is 5 used at a final concentration of about 12%.

[0321] In another specific embodiment, a Factor H composition extracted from a plasma fraction (e.g, fraction I precipitate, fraction II+III precipitate, Precipitate B precipitate, etc.) is further enriched by (a) precipitating at least one impurity out of the solution; (b) precipitating Factor H out of the solution; and (c) recovering the precipitate containing Factor H. In certain 10 embodiments, the precipitation steps are performed with alcohol (e.g., methanol or ethanol), PEG, or a combination thereof. In a particular embodiment, the precipitation steps are performed with PEG. In certain embodiments, the PEG concentration of the first precipitation step is between about 2.5% and about 7.5% and the PEG concentration of the second precipitation step is between about 9% and about 15%. In a specific embodiment, the PEG 15 concentration of the first step is between about 4% and about 6% and the PEG concentration of the second step is between about 11% and about 13%. In a more specific embodiment, the PEG concentration of the first precipitation step is about 5% and the PEG concentration of the second precipitation step is about 12%. In yet other embodiments, the PEG concentration of the first and second precipitation steps is selected from variations Var. 1101 and Var. 1221 listed in 20 Table 16. Table 16. PEG concentrations for enrichment of Factor H compositions. PEG Concentration - First Precipitation 2.5% 3.0% 3.5% 4.0% 4.5% 5.0% 5.5% 6.0% 6.5% 7.0% 7.5% ration -citation 2.5% Var. 1101 Var. 1112 Var. 1123 Var. 1134 Var. 1145 Var. 1156 Var. 1167 Var. 1178 Var. 1189 Var. 1200 Var. 1211 3.0% Var. 1102 Var. 1113 Var. 1124 Var. 1135 Var. 1146 Var. 1157 Var. 1168 Var. 1179 Var. 1190 Var. 1201 Var. 1212 PEG Concent Second Precii 3.5% Var. 1103 Var. 1114 Var. 1125 Var. 1136 Var. 1147 Var. 1158 Var. 1169 Var. 1180 Var. 1191 Var. 1202 Var. 1213 4.0% Var. 1104 Var. 1115 Var. 1126 Var. 1137 Var. 1148 Var. 1159 Var. 1170 Var. 1181 Var. 1192 Var. 1203 Var. 1214 4.5% Var. 1105 Var. 1116 Var. 1127 Var. 1138 Var. 1149 Var. 1160 Var. 1171 Var. 1182 Var. 1193 Var. 1204 Var. 1215 2024204779   11 Jul 2024 5.0% Var. 1106 Var. 1117 Var. 1128 Var. 1139 Var. 1150 Var. 1161 Var. 1172 Var. 1183 Var. 1194 Var. 1205 Var. 1216 5.5% Var. 1107 Var. 1118 Var. 1129 Var. 1140 Var. 1151 Var. 1162 Var. 1173 Var. 1184 Var. 1195 Var. 1206 Var. 1217 6.0% Var. 1108 Var. 1119 Var. 1130 Var. 1141 Var. 1152 Var. 1163 Var. 1174 Var. 1185 Var. 1196 Var. 1207 Var. 1218 6.5% Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1109 1120 1131 1142 1153 1164 1175 1186 1197 1208 1219 7.0% Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1110 1121 1132 1143 1154 1165 1176 1187 1198 1209 1220 7.5% Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1111 1122 1133 1144 1155 1166 1177 1188 1199 1210 1221

[0322] In certain embodiments, the method for preparing an enriched Factor H composition further comprises at least one, preferably two, chromatographic steps to further enrich the purity of the composition. Generally, any suitable chromatographic method may be employed to further enrich the Factor H composition, for example, extracted from a Fraction I precipitate or 5 Fraction II+III filter cake. In certain embodiments, prior to chromatographic enrichment, the extracted Factor H composition will be subjected one or more additional precipitation steps, as described above, to reduce the impurities present in the composition, reduce the load volume for the chromatographic step, and / or exchange the buffer of the composition.

[0323] In certain embodiments, a Factor H composition may be further enriched by a 10 chromatographic step comprising anion exchange chromatography (AEC), cation exchange chromatography (CEC), heparin affinity chromatography, hydrophobic exchange chromatography (HIC), hydroxyapatite chromatography (HAP), immunoaffinity chromatography, size exclusion chromatography (i.e., gel filtration), or other suitable chromatographic step. Chromatographic steps may be performed in either batch or column 15 mode.

[0324] In a preferred embodiment, the method comprises the use of anion exchange chromatography and heparin affinity chromatography.

[0325] In certain embodiments, the methods provided herein for the preparation of an enriched Factor H composition will further include at least one, preferably at least two, most preferably at 20 least three, viral inactivation or removal steps. Non-limiting examples of viral inactivation or removal steps that may be employed 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., 2024204779   11 Jul 2024 Transfusion 2003 (43):1023-1028, both of which are herein expressly 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 (pt 8)):2021-2024, both of which are herein expressly incorporated by reference in their entirety for all purposes), low pH incubation at high 5   temperatures (Kempf et al., Transfusion 1991 (31)423-427 and Louie et al., Biologicals 1994 (22):13-19), and heat treatment of lyophilized Factor H compositions (Piszkiewicz et al., Thromb Res. 1987 Jul 15;47(2):235-41; Piszkiewicz et al., Curr Stud Hematol Blood Transfus. 1989;(56):44-54; Epstein and Fricke, Arch Pathol Lab Med. 1990 Mar; 114(3):335-40).

[0326] In a preferred embodiment, the present invention provides a method of preparing a 10 virally safe enriched Factor H composition having reduced serine protease and / or serine protease zymogen content comprising (i) extracting Factor H from a Fraction II+III filter cake using SiO2, (ii) performing a first precipitation step to precipitate at least one impurity from the Factor H composition, (iii) performing a second precipitation step to precipitate Factor H from the composition, and (iv) performing at least one viral inactivation or removal step, thereby 15 preparing a virally safe enriched Factor H composition. In one embodiment, the precipitation steps comprise PEG precipitation. In a specific embodiment, the PEG concentration of the first and second precipitation steps is selected from variations Var. 1101 and Var. 1221 listed in Table 16. 2. Co-Binding and Differential Elution 20

[0327] In one aspect, the present invention provides a method for preparing a plasma-derived Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising co-extracting Factor H and a serine protease and / or serine protease zymogen from a composition derived from pooled plasma by binding the proteins to finely divided silicon dioxide (SiO2), eluting the serine protease and / or serine protease zymogen 25 from the SiO2 under a first solution condition, and subsequently eluting Factor H from the SiO2 under a second solution condition. In a preferred embodiment, the starting composition is a resuspended Fraction II+III precipitate or equivalent precipitate thereof.

[0328] In a specific embodiment, the method comprises the steps of: (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen 30 with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H and at least one serine protease or serine protease zymogen; (b) separating the SiO2 from the 2024204779   11 Jul 2024 composition; (c) eluting the serine protease or serine protease zymogen from the SiO2 under a solution condition in which the Factor H remains bound; and (d) eluting the Factor H from the SiO2.

[0329] In certain embodiments, a solution condition in which the Factor H remains bound 5 refers to a condition that preferentially elutes the serine protease or serine protease zymogen, while a substantial fraction of Factor H remains bound to the SiO2. In one embodiment, a substantial fraction refers to at least 10% of the Factor H bound to the SiO2. In another embodiment, a substantial fraction refers to at least 25% of the Factor H bound to the SiO2. In another embodiment, a substantial fraction refers to at least 50% of the Factor H bound to the 10 SiO2. In another embodiment, a substantial fraction refers to at least 75% of the Factor H bound to the SiO2. In yet other embodiments, a substantial fraction refers to at least 10% of the Factor H bound to the SiO2, or at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more of the Factor H bound to the SiO2.

[0330] In certain embodiments, differential elution of the serine protease or serine protease 15 zymogen and Factor H is achieved by sequentially contacting (i.e., step-wise elution) the SiO2 with a first solution condition (e.g., a first elution buffer) suitable to elute the majority of the serine protease or serine protease zymogen but not a substantial fraction of the bound Factor H, and a second solution condition (e.g., a second elution buffer) suitable to elute the substantial fraction of bound Factor H from the SiO2. 20

[0331] In other embodiments, differential elution of the serine protease or serine protease zymogen and Factor H is achieved by gradually changing the solution conditions (i.e., with an elution gradient) from a first solution condition suitable to elute the majority of the serine protease or serine protease zymogen but not a substantial fraction of the bound Factor H to a second solution condition suitable to elute the substantial fraction of bound Factor H from the 25 SiO2. In this fashion, the serine protease or serine protease zymogen and Factor H content eluted off of the SiO2 may be partially overlapping. By fractionating the elution and characterizing the individual fractions, a Factor H pool may be created from fractions having high Factor H content and low serine protease or serine protease zymogen content.

[0332] Solution conditions that may be varied to achieve a desired result from a method 30 described above include, without limitation, the pH of the solution, the conductivity of the solution, the temperature of the solution, the concentration of Factor H in the composition, and 2024204779   11 Jul 2024 the concentration of SiO2 used in the method. Generally, suitable pH ranges for methods of reducing serine protease and / or serine protease zymogen content in a Factor H enriched composition range from about 3 to about 11. Suitable conductivities for the methods described above range from about 0.1 mS / cm to about 100 mS / cm. Suitable temperatures for performing 5 the methods described above range from about -10°C to about 90°C. Finely divided silicon dioxide may be used at a final concentration ranging from about 0.01 g / g protein to about 10 g / g protein. Finally, Factor H compositions may vary in concentration from about 0.001 mg / mL to about 100 mg / mL.

[0333] In one embodiment, the solution condition under which the serine protease or serine 10 protease zymogen is eluted from the SiO2 and a significant fraction of the Factor H remains bound comprises a pH between about 5.0 and about 11.0. In another embodiment, the pH is between about 6.0 and about 1.0. In another embodiment, the pH is between about 7.0 and about 9.0. In another embodiment, the pH is between about 7.5 and about 8.5. In yet another embodiment, the pH is between about 7.0 and about 8.0. 15

[0334] In a particular embodiment ,the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and a significant fraction of the Factor H remains bound comprises a pH of about 7.0. In another specific embodiment, the pH is about 7.5. In another embodiment, the pH is about 8.0. In yet other embodiments, the pH is about 3.0 or about 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, 20    5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0.

[0335] In one embodiment, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and a significant fraction of the Factor H remains 25 bound comprises a pH of at least 6.0. In another embodiment, the pH is at least 6.5. In another embodiment, the pH is at least 7.0. In yet another embodiment, the pH is at least 7.5. In yet other embodiments, the pH of the solution is at least 3.0 or at least 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, or higher.

[0336] In another embodiment, of any of the methods described above, the solution condition 30 under which the serine protease or serine protease zymogen is eluted from the SiO2 and a significant fraction of the Factor H remains bound comprises a pH of no greater than about 11.0. 2024204779   11 Jul 2024 In another embodiment, the pH is no greater about 10.0. In another embodiment, the pH is no greater about 9.0. In another embodiment, the pH is no greater about 8.0. In yet other embodiments, the pH is no greater than about 11.0, or 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5,4.0, 3.5, or lower. 5

[0337] In one embodiment, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and a significant fraction of the Factor H remains bound comprises a conductivity of at least 10 mS / cm. In another embodiment, the conductivity is at least 20 mS / cm. In yet other embodiments, the conductivity of the solution condition is at least 2 mS / cm, or at least 3 mS / cm, 4 mS / cm, 5 mS / cm, 6 mS / cm, 7 mS / cm, 8 mS / cm, 9 mS / cm, 10   10 mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 mS / cm, 17 mS / cm, 18 mS / cm, 19 mS / cm, 20 mS / cm, 21 mS / cm, 22 mS / cm, 23 mS / cm, 24 mS / cm, 25 mS / cm, 26 mS / cm, 27 mS / cm, 28 mS / cm, 29 mS / cm, 30 mS / cm, 31 mS / cm, 32 mS / cm, 33 mS / cm, 34 mS / cm, 35 mS / cm, 36 mS / cm, 37 mS / cm, 38 mS / cm, 39 mS / cm, 40 mS / cm, 41 mS / cm, 42 mS / cm, 43 mS / cm, 44 mS / cm, 45 mS / cm, 46 mS / cm, 47 mS / cm, 48 mS / cm, 49 mS / cm, 50 15   mS / cm, 55 mS / cm, 60 mS / cm, 65 mS / cm, 70 mS / cm, 75 mS / cm, 80 mS / cm, 85 mS / cm, 90 mS / cm, 95 mS / cm, 100 mS / cm, or greater.

[0338] In one embodiment, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and a significant fraction of the Factor H remains bound comprises a conductivity between about 10 mS / cm and about 100 mS / cm. In another 20 embodiment, the conductivity is between about 10 mS / cm and about 50 mS / cm. In another embodiment, the conductivity is between about 20 mS / cm and about 100 mS / cm. In yet another embodiment, the conductivity is between about 20 mS / cm and about 50 mS / cm.

[0339] As shown in Example 5 and illustrated in Figure 3, it was found that the use of solution conditions having a pH greater than 6.0 (e.g, 7.5) and increasing conductivity (e.g., greater than 25   6.0 mS / cm), results in increased elution of serine proteases and / or serine protease zymogens from SiO2, and decreased elution of Factor H from SiO2. Advantageously, these findings can be used to provide methods for reducing the levels of serine protease and serine protease zymogen present in Factor H compositions. In a particular embodiment of the methods described above, the solution condition under which the serine protease or serine protease zymogen is eluted from 30 the SiO2 and a significant fraction of the Factor H remains bound comprises a conductivity of at least about 10 mS / cm and a pH of at least 7.0. In another particular embodiment, the solution 2024204779   11 Jul 2024 condition comprises a conductivity of at least 10 mS / cm and a pH of at least 7.5. In another embodiment, the solution condition comprises a conductivity of at least 20 mS / cm and a pH of at least 7.0. In yet another embodiment, the solution condition comprises a conductivity of at least 20 mS / cm and a pH of at least 7.5. 5                3. Co-Binding and Preferential Factor H Elution

[0340] In one aspect, the present invention provides a method for preparing a plasma-derived Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising co-extracting Factor H and a serine protease and / or serine protease zymogen from a composition derived from pooled plasma by binding the proteins to 10 finely divided silicon dioxide (SiO2), and eluting the Factor H from the SiO2 under conditions in which a substantial fraction of the bound serine protease and / or serine protease zymogen remains bound to the SiO2. In a preferred embodiment, the starting composition is a re-suspended Fraction II+III precipitate or equivalent precipitate thereof.

[0341] In a specific embodiment, the method comprises the steps of: (a) contacting a 15 composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H and at least one serine protease or serine protease zymogen; (b) separating the SiO2 from the composition; and (c) eluting the Factor H from the SiO2 under a solution condition in which the serine protease or serine protease zymogen remains bound. 20

[0342] In certain embodiments, a solution condition in which the serine protease or serine protease zymogen remains bound refers to a condition that preferentially elutes the Factor H, while a substantial fraction of the serine protease or serine protease zymogen remains bound to the SiO2. In one embodiment, a substantial fraction refers to at least 10% of the serine protease or serine protease zymogen bound to the SiO2. In another embodiment, a substantial fraction 25 refers to at least 25% of the serine protease or serine protease zymogen bound to the SiO2. In another embodiment, a substantial fraction refers to at least 50% of the serine protease or serine protease zymogen bound to the SiO2. In another embodiment, a substantial fraction refers to at least 75% of the serine protease or serine protease zymogen bound to the SiO2. In yet other embodiments, a substantial fraction refers to at least 10% of the serine protease or serine protease 30 zymogen bound to the SiO2, or at least 15,20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 2024204779   11 Jul 2024 90, 95, 96, 97, 98, 99, or more of the serine protease or serine protease zymogen bound to the SiO2.

[0343] Solution conditions that may be varied to achieve a desired result from a method described above include, without limitation, the pH of the solution, the conductivity of the 5 solution, the temperature of the solution, the concentration of Factor H in the composition, and the concentration of SiO2 used in the method. Generally, suitable pH ranges for methods of reducing serine protease and / or serine protease zymogen content in a Factor H enriched composition range from about 3 to about 11. Suitable conductivities for the methods described above range from about 0.1 mS / cm to about 100 mS / cm. Suitable temperatures for performing 10 the methods described above range from about -10°C to about 90°C. Finely divided silicon dioxide may be used at a final concentration ranging from about 0.01 g / g protein to about 10 g / g protein. Finally, Factor H compositions may vary in concentration from about 0.001 mg / mL to about 100 mg / mL.

[0344] In one embodiment, the solution condition under which the Factor H is eluted from the 15   SiO2 and a significant fraction of the serine protease or serine protease zymogen remains bound comprises a pH between about 5.0 and about 11.0. In another embodiment, the pH is between about 6.0 and about 1.0. In another embodiment, the pH is between about 7.0 and about 9.0. In another embodiment, the pH is between about 7.5 and about 8.5. In yet another embodiment, the pH is between about 7.0 and about 8.0. 20

[0345] In a particular embodiment ,the solution condition under which the Factor H is eluted from the SiO2 and a significant fraction of the serine protease or serine protease zymogen remains bound comprises a pH of about 7.0. In another specific embodiment, the pH is about 7.5. In another embodiment, the pH is about 8.0. In yet other embodiments, the pH is about 3.0 or about 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, 25    5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0.

[0346] In one embodiment, the solution condition under which the Factor H is eluted from the SiO2 and a significant fraction of the serine protease or serine protease zymogen remains bound 30 comprises a pH of at least 6.0. In another embodiment, the pH is at least 6.5. In another embodiment, the pH is at least 7.0. In yet another embodiment, the pH is at least 7.5. In yet 2024204779   11 Jul 2024 other embodiments, the pH of the solution is at least 3.0 or at least 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, or higher.

[0347] In another embodiment, of any of the methods described above, the solution condition under which the Factor H is eluted from the SiOs and a significant fraction of the serine protease 5 or serine protease zymogen remains bound comprises a pH of no greater than about 11.0. In another embodiment, the pH is no greater about 10.0. In another embodiment, the pH is no greater about 9.0. In another embodiment, the pH is no greater about 8.0. In yet other embodiments, the pH is no greater than about 11.0, or 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, or lower. 10

[0348] In one embodiment, the solution condition under which the Factor H is eluted from the SiO2 and a significant fraction of the serine protease or serine protease zymogen remains bound comprises a conductivity of no more than about 20 mS / cm. In another embodiment, the conductivity is no more than about 10 mS / cm. In yet other embodiments, the conductivity of the solution condition is no more than about 20 mS / cm, or no more than about 19 mS / cm, 18 mS / cm, 15   17 mS / cm, 16 mS / cm, 15 mS / cm, 14 mS / cm, 13 mS / cm, 12 mS / cm, 11 mS / cm, 10 mS / cm, 9 mS / cm, 8 mS / cm, 7 mS / cm, 6 mS / cm, 5 mS / cm, 4 mS / cm, 3 mS / cm, 2 mS / cm, or less.

[0349] In one embodiment, the solution condition under which the serine protease or serine protease zymogen is eluted from the SiO2 and a significant fraction of the Factor H remains bound comprises a conductivity between about 2 mS / cm and about 20 mS / cm. In another 20 embodiment, the conductivity is between about 2 mS / cm and about 10 mS / cm. In another embodiment, the conductivity is between about 20 mS / cm and about 6 mS / cm. In yet another embodiment, the conductivity is between about 10 mS / cm and about 6 mS / cm.

[0350] As shown in Example 5 and illustrated in Figure 3, it was found that the use of solution conditions having a pH greater than 6.0 (e.g., 7.5) and decreasing conductivity (e.g., less than 20 25 mS / cm), results in increased elution of Factor H from SiO2, and decreased elution of serine proteases and / or serine protease zymogens from SiO2. Advantageously, these findings can be used to provide methods for reducing the levels of serine protease and serine protease zymogen present in Factor H compositions. In a particular embodiment of the methods described above, the solution condition under which the Factor H is eluted from the SiO2 and a significant fraction 30   of the serine protease or serine protease zymogen remains bound comprises a conductivity of at no more than about 20 mS / cm and a pH of at least 7.0. In another particular embodiment, the 2024204779   11 Jul 2024 solution condition comprises a conductivity of no more than about 10 mS / cm and a pH of at least 7.5. In another embodiment, the solution condition comprises a conductivity between about 10 mS / cm and about 2 mS / cm and a pH of at least 7.0. In yet another embodiment, the solution condition comprises a conductivity between about 10 mS / cm and about 2 mS / cm and a pH of at 5 least 7.5. 4. Preferential binding of Factor H

[0351] In one aspect, the present invention provides a method for preparing a plasma-derived Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising (a) contacting a composition containing Factor H and at least 10 one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H but not the at least one serine protease or serine protease zymogen; (b) separating the SiO2 from the composition; and (c) eluting the Factor H from the SiO2.

[0352] In certain embodiments, a solution condition in which the serine protease or serine 15 protease zymogen does not bind to the SiO2 refers to a condition that preferentially allows Factor H binding to the SiO2, while a substantial fraction of the serine protease or serine protease zymogen remains unbound in the solution. In one embodiment, a substantial fraction refers to at least 10% of the serine protease or serine protease zymogen in the starting composition. In another embodiment, a substantial fraction refers to at least 25% of the serine protease or serine 20 protease zymogen in the starting composition. In another embodiment, a substantial fraction refers to at least 50% of the serine protease or serine protease zymogen in the starting composition. In another embodiment, a substantial fraction refers to at least 75% of the serine protease or serine protease zymogen in the starting composition. In yet other embodiments, a substantial fraction refers to at least 10% of the serine protease or serine protease zymogen in the 25 starting composition, or at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more of the serine protease or serine protease zymogen in the starting composition.

[0353] Solution conditions that may be varied to achieve a desired result from a method described above include, without limitation, the pH of the solution, the conductivity of the 30 solution, the temperature of the solution, the concentration of Factor H in the composition, and the concentration of SiO2 used in the method. Generally, suitable pH ranges for methods of 2024204779   11 Jul 2024 reducing serine protease and / or serine protease zymogen content in a Factor H enriched composition range from about 3 to about 11. Suitable conductivities for the methods described above range from about 0.1 mS / cm to about 100 mS / cm. Suitable temperatures for performing the methods described above range from about -10°C to about 90°C. Finely divided silicon 5 dioxide may be used at a final concentration ranging from about 0.01 g / g protein to about 10 g / g protein. Finally, Factor H compositions may vary in concentration from about 0.001 mg / mL to about 100 mg / mL.

[0354] In one embodiment, the solution condition under which Factor H binds to SiO2 and a significant fraction of the serine protease or serine protease zymogen does not bind comprises a 10 pH between about 5.0 and about 11.0. In another embodiment, the pH is between about 6.0 and about 1.0. In another embodiment, the pH is between about 7.0 and about 9.0. In another embodiment, the pH is between about 7.5 and about 8.5. In yet another embodiment, the pH is between about 7.0 and about 8.0.

[0355] In a particular embodiment ,the solution condition under which Factor H binds to SiO2 15   and a significant fraction of the serine protease or serine protease zymogen does not bind comprises a pH of about 7.0. In another specific embodiment, the pH is about 7.5. In another embodiment, the pH is about 8.0. In yet other embodiments, the pH is about 3.0 or about 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, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 20    7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0.

[0356] In one embodiment, the solution condition under which Factor H binds to SiO2 and a significant fraction of the serine protease or serine protease zymogen does not bind comprises a pH of at least 6.0. In another embodiment, the pH is at least 6.5. In another embodiment, the pH 25 is at least 7.0. In yet another embodiment, the pH is at least 7.5. In yet other embodiments, the pH of the solution is at least 3.0 or at least 3.5,4.0,4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,10.5, or higher.

[0357] In another embodiment, of any of the methods described above, the solution condition under which Factor H binds to SiO2 and a significant fraction of the serine protease or serine 30 protease zymogen does not bind comprises a pH of no greater than about 11.0. In another embodiment, the pH is no greater about 10.0. In another embodiment, the pH is no greater about 2024204779   11 Jul 2024 9.0. In another embodiment, the pH is no greater about 8.0. In yet other embodiments, the pH is no greater than about 11.0, or 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, or lower.

[0358] In one embodiment, the solution condition under which Factor H binds to SiO2 and a 5 significant fraction of the serine protease or serine protease zymogen does not bind comprises a conductivity of at least 10 mS / cm. In another embodiment, the conductivity is at least 20 mS / cm. In yet other embodiments, the conductivity of the solution condition is at least 2 mS / cm, or at least 3 mS / cm, 4 mS / cm, 5 mS / cm, 6 mS / cm, 7 mS / cm, 8 mS / cm, 9 mS / cm, 10 mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 mS / cm, 17 mS / cm, 18 mS / cm, 19 10 mS / cm, 20 mS / cm, 21 mS / cm, 22 mS / cm, 23 mS / cm, 24 mS / cm, 25 mS / cm, 26 mS / cm, 27 mS / cm, 28 mS / cm, 29 mS / cm, 30 mS / cm, 31 mS / cm, 32 mS / cm, 33 mS / cm, 34 mS / cm, 35 mS / cm, 36 mS / cm, 37 mS / cm, 38 mS / cm, 39 mS / cm, 40 mS / cm, 41 mS / cm, 42 mS / cm, 43 mS / cm, 44 mS / cm, 45 mS / cm, 46 mS / cm, 47 mS / cm, 48 mS / cm, 49 mS / cm, 50 mS / cm, 55 mS / cm, 60 mS / cm, 65 mS / cm, 70 mS / cm, 75 mS / cm, 80 mS / cm, 85 mS / cm, 90 mS / cm, 95 15 mS / cm, 100 mS / cm, or greater.

[0359] In one embodiment, the solution condition under which Factor H binds to SiO2 and a significant fraction of the serine protease or serine protease zymogen does not bind comprises a conductivity between about 10 mS / cm and about 100 mS / cm. In another embodiment, the conductivity is between about 10 mS / cm and about 50 mS / cm. In another embodiment, the 20 conductivity is between about 20 mS / cm and about 100 mS / cm. In yet another embodiment, the conductivity is between about 20 mS / cm and about 50 mS / cm.

[0360] As shown in Example 5 and illustrated in Figure 3, it was found that the use of solution conditions having a pH greater than 6.0 (e.g., 7.5) and increasing conductivity (e.g., greater than 6.0 mS / cm), results in a decreased affinity of serine proteases and / or serine protease zymogens 25 for SiO2, and increased affinity of Factor H for SiO2. Advantageously, these findings can be used to provide methods for reducing the levels of serine protease and serine protease zymogen present in Factor H compositions. In a particular embodiment of the methods described above, the solution condition under which Factor H binds to SiO2 and a significant fraction of the serine protease or serine protease zymogen does not bind comprises a conductivity of at least about 10 30 mS / cm and a pH of at least 7.0. In another particular embodiment, the solution condition comprises a conductivity of at least 10 mS / cm and a pH of at least 7.5. In another embodiment, 2024204779   11 Jul 2024 the solution condition comprises a conductivity of at least 20 mS / cm and a pH of at least 7.0. In yet another embodiment, the solution condition comprises a conductivity of at least 20 mS / cm and a pH of at least 7.5. 5. Preferential binding of Serine Protease or Serine Protease Zymogen 5

[0361] In one aspect, the present invention provides a method for preparing a plasma-derived Factor H composition having a reduced amount of a serine protease or a serine protease zymogen, the method comprising (a) contacting a composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the serine protease and / or serine protease zymogen but not the Factor 10 H; and (b) separating the SiO2 from the composition.

[0362] In certain embodiments, a solution condition in which the Factor H does not bind to the SiO2 refers to a condition that preferentially allows serine protease or serine protease zymogen binding to the SiO2, while a substantial fraction of the Factor H remains unbound in the solution. In one embodiment, a substantial fraction refers to at least 10% of the Factor H in the starting 15 composition. In another embodiment, a substantial fraction refers to at least 25% of the Factor H in the starting composition. In another embodiment, a substantial fraction refers to at least 50% of the Factor H in the starting composition. In another embodiment, a substantial fraction refers to at least 75% of the Factor H in the starting composition. In yet other embodiments, a substantial fraction refers to at least 10% of the Factor H in the starting composition, or at least 20    15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more of the Factor H in the starting composition.

[0363] Solution conditions that may be varied to achieve a desired result from a method described above include, without limitation, the pH of the solution, the conductivity of the solution, the temperature of the solution, the concentration of Factor H in the composition, and 25 the concentration of SiO2 used in the method. Generally, suitable pH ranges for methods of reducing serine protease and / or serine protease zymogen content in a Factor H enriched composition range from about 3 to about 11. Suitable conductivities for the methods described above range from about 0.1 mS / cm to about 100 mS / cm. Suitable temperatures for performing the methods described above range from about -10°C to about 90°C. Finely divided silicon 30 dioxide may be used at a final concentration ranging from about 0.01 g / g protein to about 10 g / g 2024204779   11 Jul 2024 protein. Finally, Factor H compositions may vary in concentration from about 0.001 mg / mL to about 100 mg / mL.

[0364] In one embodiment, the solution condition under which the serine protease or serine protease zymogen binds to SiO2 and a significant fraction of the Factor H does not bind 5 comprises a pH between about 5.0 and about 11.0. In another embodiment, the pH is between about 6.0 and about 1.0. In another embodiment, the pH is between about 7.0 and about 9.0. In another embodiment, the pH is between about 7.5 and about 8.5. In yet another embodiment, the pH is between about 7.0 and about 8.0.

[0365] In a particular embodiment ,the solution condition under which the serine protease or 10 serine protease zymogen binds to SiO2 and a significant fraction of the Factor H does not bind comprises a pH of about 7.0. In another specific embodiment, the pH is about 7.5. In another embodiment, the pH is about 8.0. In yet other embodiments, the pH is about 3.0 or about 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, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 15    7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0.

[0366] In one embodiment, the solution condition under which the serine protease or serine protease zymogen binds to SiO2 and a significant fraction of the Factor H does not bind comprises a pH of at least 6.0. In another embodiment, the pH is at least 6.5. In another 20 embodiment, the pH is at least 7.0. In yet another embodiment, the pH is at least 7.5. In yet other embodiments, the pH of the solution is at least 3.0 or at least 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, or higher.

[0367] In another embodiment, of any of the methods described above, the solution condition under which the serine protease or serine protease zymogen binds to SiO2 and a significant 25 fraction of the Factor H does not bind comprises a pH of no greater than about 11.0. In another embodiment, the pH is no greater about 10.0. In another embodiment, the pH is no greater about 9.0. In another embodiment, the pH is no greater about 8.0. In yet other embodiments, the pH is no greater than about 11.0, or 10.5, 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0,4.5, 4.0, 3.5, or lower. 2024204779   11 Jul 2024

[0368] In one embodiment, the solution condition under which the serine protease or serine protease zymogen binds to SiO2 and a significant fraction of the Factor H does not bind comprises a conductivity of no more than about 20 mS / cm. In another embodiment, the conductivity is no more than about 10 mS / cm. In yet other embodiments, the conductivity of the 5 solution condition is no more than about 20 mS / cm, or no more than about 19 mS / cm, 18 mS / cm, 17 mS / cm, 16 mS / cm, 15 mS / cm, 14 mS / cm, 13 mS / cm, 12 mS / cm, 11 mS / cm, 10 mS / cm, 9 mS / cm, 8 mS / cm, 7 mS / cm, 6 mS / cm, 5 mS / cm, 4 mS / cm, 3 mS / cm, 2 mS / cm, or less.

[0369] In one embodiment, the solution condition under which the serine protease or serine protease zymogen binds to SiO2 and a significant fraction of the Factor H does not bind 10 comprises a conductivity between about 2 mS / cm and about 20 mS / cm. In another embodiment, the conductivity is between about 2 mS / cm and about 10 mS / cm. In another embodiment, the conductivity is between about 20 mS / cm and about 6 mS / cm. In yet another embodiment, the conductivity is between about 10 mS / cm and about 6 mS / cm.

[0370] As shown in Example 5 and illustrated in Figure 3, it was found that the use of solution 15 conditions having a pH greater than 6.0 (e.g., 7.5) and decreasing conductivity (e.g., less than 20 mS / cm), results in increased affinity of Factor H for SiO2, and decreased affinity of serine proteases and / or serine protease zymogens from SiO2. Advantageously, these findings can be used to provide methods for reducing the levels of serine protease and serine protease zymogen present in Factor H compositions. In a particular embodiment of the methods described above, 20 the solution condition under which the serine protease or serine protease zymogen binds to SiO2 and a significant fraction of the Factor H does not bind comprises a conductivity of at no more than about 20 mS / cm and a pH of at least 7.0. In another particular embodiment, the solution condition comprises a conductivity of no more than about 10 mS / cm and a pH of at least 7.5. In another embodiment, the solution condition comprises a conductivity between about 10 mS / cm 25 and about 2 mS / cm and a pH of at least 7.0. In yet another embodiment, the solution condition comprises a conductivity between about 10 mS / cm and about 2 mS / cm and a pH of at least 7.5. 6. Method for Factor H Extraction from a Plasma Precipitate

[0371] In one aspect, the present invention provides a method for preparing a Factor H composition, the method comprising the steps of: (a) contacting a suspended plasma precipitate 30 composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H, (b) 2024204779   11 Jul 2024 washing the SiO2 with a solution comprising a pH between 5.0 and 7.0 and a conductivity of less than 4 mS / cm, and (c) eluting the Factor H from the SiO2 with a solution comprising a pH between 7.0 and 8.0 and a conductivity greater than 10 mS / cm, thereby providing an enriched Factor H composition. In a preferred embodiment, the serine protease or serine protease 5 zymogen is one or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, the plasma precipitate is a Cohn fraction I precipitate, a Cohn fraction II+III precipitate, a Cohn fraction 1+11+111 precipitate, a Kistler / Nitschmann Precipitate A, a Kistler / Nitschmann Precipitate B, or an equivalent fraction thereof. In one embodiment, the solution used to wash the SiO2 comprises a pH between 5.5 and 6.5. In a specific embodiment, the solution used to wash the SiO2 10 comprises a pH of 6.0±0.2. In one embodiment, the solution used to elute Factor H comprises a conductivity of at least 20 mS / cm. In a specific embodiment, the solution used to elute Factor H comprises a conductivity of between 25 mS / cm and 40 mS / cm.

[0372] In certain embodiments, the method described above further comprises an enrichment step comprising precipitating at least one impurity from the enriched Factor H composition, 15 wherein Factor H is not co-precipitated. In a specific embodiment, the method comprises the steps of (a) contacting a suspended plasma precipitate composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H, (b) washing the SiO2 with a solution comprising a pH between 5.0 and 7.0 and a conductivity of less than 4 mS / cm, (c) eluting the Factor H from 20 the SiO2 with a solution comprising a pH between 7.0 and 8.0 and a conductivity greater than 10 mS / cm, and (d) precipitating at least one impurity from the Factor H elution, wherein Factor H is not precipitated, thereby providing an enriched Factor H composition. In a preferred embodiment, the serine protease or serine protease zymogen is one or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, the plasma precipitate is a Cohn fraction I precipitate, a 25 Cohn fraction II+III precipitate, a Cohn fraction I+II+III precipitate, a Kistler / Nitschmann Precipitate A, a Kistler / Nitschmann Precipitate B, or an equivalent fraction thereof. In one embodiment, the solution used to wash the SiO2 comprises a pH between 5.5 and 6.5. In a specific embodiment, the solution used to wash the SiO2 comprises a pH of 6.0+0.2. In one embodiment, the solution used to elute Factor H comprises a conductivity of at least 20 mS / cm. 30 In a specific embodiment, the solution used to elute Factor H comprises a conductivity of between 25 mS / cm and 40 mS / cm. In one embodiment, the impurity precipitation step is PEG precipitation. In a specific embodiment, the impurity PEG precipitation comprises precipitation 2024204779   11 Jul 2024 with PEG 4000 at a final concentration between 3% and 7%. In a more specific embodiment, the final concentration of PEG 4000 in the impurity precipitation step is 5±0.5%.

[0373] In certain embodiments, the methods described above further comprises an enrichment step comprising precipitating Factor H from an enriched Factor H composition. In a specific 5 embodiment, the method comprises the steps of (a) contacting a suspended plasma precipitate composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H, (b) washing the SiO2 with a solution comprising a pH between 5.0 and 7.0 and a conductivity of less than 4 mS / cm, (c) eluting the Factor H from the SiO2 with a solution comprising a pH between 10   7.0 and 8.0 and a conductivity greater than 10 mS / cm, (d) precipitating at least one impurity from the Factor H elution, to form a supernatant comprising Factor H, and (e) precipitating Factor H from the supernatant, thereby providing an enriched Factor H composition. In a preferred embodiment, the serine protease or serine protease zymogen is one or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, the plasma precipitate is a Cohn fraction I 15 precipitate, a Cohn fraction II+III precipitate, a Cohn fraction 1+11+111 precipitate, a Kistler / Nitschmann Precipitate A, a Kistler / Nitschmann Precipitate B, or an equivalent fraction thereof. In one embodiment, the solution used to wash the SiO2 comprises a pH between 5.5 and 6.5. In a specific embodiment, the solution used to wash the SiO2 comprises a pH of 6.0±0.2. In one embodiment, the solution used to elute Factor H comprises a conductivity of at least 20 20 mS / cm. In a specific embodiment, the solution used to elute Factor H comprises a conductivity of between 25 mS / cm and 40 mS / cm. In one embodiment, the impurity precipitation step is PEG precipitation. In a specific embodiment, the impurity PEG precipitation comprises precipitation with PEG 4000 at a final concentration between 3% and 7%. In a more specific embodiment, the final concentration of PEG 4000 in the impurity precipitation step is 5±0.5%. In one 25 embodiment, the Factor H precipitation step is PEG precipitation. In a specific embodiment, the Factor H PEG precipitation comprises precipitation with PEG 4000 at a final concentration between 10% and 15%. In a more specific embodiment, the final concentration of PEG 4000 is 12±0.5% in the Factor H precipitation step.

[0374] In certain embodiments, the methods described above further comprises an enrichment 30 step comprising performing anion exchange chromatography with an enriched Factor H composition. In a specific embodiment, the method comprises the steps of (a) contacting a suspended plasma precipitate composition containing Factor H and at least one serine protease or 2024204779   11 Jul 2024 serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H, (b) washing the SiO2 with a solution comprising a pH between 5.0 and 7.0 and a conductivity of less than 4 mS / cm, (c) eluting the Factor H from the SiO2 with a solution comprising a pH between 7.0 and 8.0 and a conductivity greater than 10 mS / cm, (d) precipitating 5 at least one impurity from the Factor H elution, to form a supernatant comprising Factor H, (e) precipitating Factor H from the supernatant, (f) re-suspending the precipitate comprising Factor H, (g) binding Factor H present in the re-suspended precipitate to an anion exchange resin, and (h) eluting Factor H from the anion exchange resin, thereby providing an enriched Factor H composition. In a preferred embodiment, the serine protease or serine protease zymogen is one 10 or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, the plasma precipitate is a Cohn fraction I precipitate, a Cohn fraction 11+111 precipitate, a Cohn fraction I+II+III precipitate, a Kistler / Nitschmann Precipitate A, a Kistler / Nitschmann Precipitate B, or an equivalent fraction thereof. In one embodiment, the solution used to wash the SiO2 comprises a pH between 5.5 and 6.5. In a specific embodiment, the solution used to wash the SiO2 comprises 15 a pH of 6.0±0.2. In one embodiment, the solution used to elute Factor H comprises a conductivity of at least 20 mS / cm. In a specific embodiment, the solution used to elute Factor H comprises a conductivity of between 25 mS / cm and 40 mS / cm. In one embodiment, the impurity precipitation step is PEG precipitation. In a specific embodiment, the impurity PEG precipitation comprises precipitation with PEG 4000 at a final concentration between 3% and 20   7%. In a more specific embodiment, the final concentration of PEG 4000 in the impurity precipitation step is 5±0.5%. In one embodiment, the Factor H precipitation step is PEG precipitation. In a specific embodiment, the Factor H PEG precipitation comprises precipitation with PEG 4000 at a final concentration between 10% and 15%. In a more specific embodiment, the final concentration of PEG 4000 is 12±0.5% in the Factor H precipitation step. 25

[0375] In certain embodiments, the methods described above further comprises an enrichment step comprising performing heparin affinity chromatography with an enriched Factor H composition. In a specific embodiment, the method comprises the steps of (a) contacting a suspended plasma precipitate composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to 30 bind the Factor H, (b) washing the SiO2 with a solution comprising a pH between 5.0 and 7.0 and a conductivity of less than 4 mS / cm, (c) eluting the Factor H from the SiO2 with a solution comprising a pH between 7.0 and 8.0 and a conductivity greater than 10 mS / cm, (d) precipitating 2024204779   11 Jul 2024 at least one impurity from the Factor H elution, to form a supernatant comprising Factor H, (e) precipitating Factor H from the supernatant, (f) re-suspending the precipitate comprising Factor H, (g) binding Factor H present in the re-suspended precipitate to an anion exchange resin, (h) eluting Factor H from the anion exchange resin, (i) binding Factor H present in the anion 5 exchange eluate to a heparin affinity resin, and (j) eluting Factor H from the heparin affinity resin, thereby providing an enriched Factor H composition. In a preferred embodiment, the serine protease or serine protease zymogen is one or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, the plasma precipitate is a Cohn fraction I precipitate, a Cohn fraction II+III precipitate, a Cohn fraction 1+11+111 precipitate, a Kistler / Nitschmann Precipitate A, a 10 Kistler / Nitschmann Precipitate B, or an equivalent fraction thereof. In one embodiment, the solution used to wash the SiO2 comprises a pH between 5.5 and 6.5. In a specific embodiment, the solution used to wash the SiO2 comprises a pH of 6.0±0.2. In one embodiment, the solution used to elute Factor H comprises a conductivity of at least 20 mS / cm. In a specific embodiment, the solution used to elute Factor H comprises a conductivity of between 25 mS / cm and 40 15 mS / cm. In one embodiment, the impurity precipitation step is PEG precipitation. In a specific embodiment, the impurity PEG precipitation comprises precipitation with PEG 4000 at a final concentration between 3% and 7%. In a more specific embodiment, the final concentration of PEG 4000 in the impurity precipitation step is 5±0.5%. In one embodiment, the Factor H precipitation step is PEG precipitation. In a specific embodiment, the Factor H PEG 20 precipitation comprises precipitation with PEG 4000 at a final concentration between 10% and 15%. In a more specific embodiment, the final concentration of PEG 4000 is 12±0.5% in the Factor H precipitation step.

[0376] In certain embodiments, the methods described above further comprises subjecting a Factor H composition to a dedicated viral removal and / or inactivation step. In a specific 25 embodiment, the method comprises the steps of (a) contacting a suspended plasma precipitate composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H, (b) washing the SiO2 with a solution comprising a pH between 5.0 and 7.0 and a conductivity of less than 4 mS / cm, (c) eluting the Factor H from the SiO2 with a solution comprising a pH between 30   7.0 and 8.0 and a conductivity greater than 10 mS / cm, (d) precipitating at least one impurity from the Factor H elution, to form a supernatant comprising Factor H, (e) precipitating Factor H from the supernatant, (f) re-suspending the precipitate comprising Factor H, (g) binding Factor H 2024204779   11 Jul 2024 present in the re-suspended precipitate to an anion exchange resin, (h) eluting Factor H from the anion exchange resin, (i) binding Factor H present in the anion exchange eluate to a heparin affinity resin, (j) eluting Factor H from the heparin affinity resin, and (k) performing a dedicated viral removal and / or inactivation step selected from nanofiltration, solvent / detergent (S / D) 5 treatment, heat treatment, and incubation at low pH, thereby providing an enriched Factor H composition. In a preferred embodiment, the serine protease or serine protease zymogen is one or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, the plasma precipitate is a Cohn fraction I precipitate, a Cohn fraction II+III precipitate, a Cohn fraction I+II+III precipitate, a Kistler / Nitschmann Precipitate A, a Kistler / Nitschmann Precipitate B, or an 10 equivalent fraction thereof. In one embodiment, the solution used to wash the SiO2 comprises a pH between 5.5 and 6.5. In a specific embodiment, the solution used to wash the SiO2 comprises a pH of 6.0±0.2. In one embodiment, the solution used to elute Factor H comprises a conductivity of at least 20 mS / cm. In a specific embodiment, the solution used to elute Factor H comprises a conductivity of between 25 mS / cm and 40 mS / cm. In one embodiment, the impurity 15 precipitation step is PEG precipitation. In a specific embodiment, the impurity PEG precipitation comprises precipitation with PEG 4000 at a final concentration between 3% and 7%. In a more specific embodiment, the final concentration of PEG 4000 in the impurity precipitation step is 5±0.5%. In one embodiment, the Factor H precipitation step is PEG precipitation. In a specific embodiment, the Factor H PEG precipitation comprises precipitation 20 with PEG 4000 at a final concentration between 10% and 15%. In a more specific embodiment, the final concentration of PEG 4000 is 12±0.5% in the Factor H precipitation step.

[0377] In certain embodiments, the methods described above further comprises a step of concentrating an enriched Factor H composition by ultrafiltration / diafiltration. In a specific embodiment, the method comprises the steps of (a) contacting a suspended plasma precipitate 25 composition containing Factor H and at least one serine protease or serine protease zymogen with finely divided silicon dioxide (SiO2) under conditions suitable to bind the Factor H, (b) washing the SiO2 with a solution comprising a pH between 5.0 and 7.0 and a conductivity of less than 4 mS / cm, (c) eluting the Factor H from the SiO2 with a solution comprising a pH between 7.0 and 8.0 and a conductivity greater than 10 mS / cm, (d) precipitating at least one impurity 30 from the Factor H elution, to form a supernatant comprising Factor H, (e) precipitating Factor H from the supernatant, (f) re-suspending the precipitate comprising Factor H, (g) binding Factor H present in the re-suspended precipitate to an anion exchange resin, (h) eluting Factor H from the 2024204779   11 Jul 2024 anion exchange resin, (i) binding Factor H present in the anion exchange eluate to a heparin affinity resin, (j) eluting Factor H from the heparin affinity resin, (k) performing a dedicated viral removal and / or inactivation step selected from nanofiltration, solvent / detergent (S / D) treatment, heat treatment, and incubation at low pH, and (1) concentrating Factor H by 5 ultrafiltration / diafiltration, thereby providing an enriched Factor H composition. In a preferred embodiment, the serine protease or serine protease zymogen is one or more of FXI, FXIa, FXII, and FXIIa. In certain embodiments, the plasma precipitate is a Cohn fraction I precipitate, a Cohn fraction II+III precipitate, a Cohn fraction 1+11+111 precipitate, a Kistler / Nitschmann Precipitate A, a Kistler / Nitschmann Precipitate B, or an equivalent fraction thereof. In one 10 embodiment, the solution used to wash the SiO2 comprises a pH between 5.5 and 6.5. In a specific embodiment, the solution used to wash the SiO2 comprises a pH of 6.0±0.2. In one embodiment, the solution used to elute Factor H comprises a conductivity of at least 20 mS / cm. In a specific embodiment, the solution used to elute Factor H comprises a conductivity of between 25 mS / cm and 40 mS / cm. In one embodiment, the impurity precipitation step is PEG 15 precipitation. In a specific embodiment, the impurity PEG precipitation comprises precipitation with PEG 4000 at a final concentration between 3% and 7%. In a more specific embodiment, the final concentration of PEG 4000 in the impurity precipitation step is 5±0.5%. In one embodiment, the Factor H precipitation step is PEG precipitation. In a specific embodiment, the Factor H PEG precipitation comprises precipitation with PEG 4000 at a final concentration 20 between 10% and 15%. In a more specific embodiment, the final concentration of PEG 4000 is 12±0.5% in the Factor H precipitation step. C. Inter-alpha-Trypsin Inhibitor (lai)

[0378] In one embodiment, the present invention provides a method for reducing the amount of a serine protease or a serine protease zymogen in a plasma-derived lai composition. In one 25 specifi...

Claims

1. A plasma-derived immunoglobulin G (IgG) composition comprising an IgG concentration of at least 10% and a serine protease concentration that induces hydrolysis of the substrate S-2266 at a rate of less than 5 nmol / ml x min, and wherein said composition is prepared by a method comprising the steps of:(a) performing a first target enrichment step to form a first enriched composition;(b) contacting the composition with finely divided silicon dioxide (SiO2) under conditions suitable to bind at least one serine protease or serine protease zymogen; and(c) separating the SiO2 from the composition to remove the bound serine protease,wherein the at least one serine protease or serine protease zymogen is Factor XIa (FXIa), Factor XIIa (FXIIa), Factor XI (FXI), or Factor XII (FXII),wherein the first target protein enrichment step is a protein precipitation step,wherein the plasma-derived target protein is immunoglobulin G (IgG),wherein the method further comprises the step of performing a second target protein enrichment step prior to contacting the enriched composition with finely divided silicon dioxide (SiO2),wherein the method further comprises the step of performing a third target protein enrichment step after contacting the composition with finely divided silicon dioxide (SiO2)wherein the protein precipitation step and the third target protein enrichment step is an alcohol fractionation step, andwherein contacting the composition with finely divided silicon dioxide (SiO2) comprises the addition SiO2 to a final concentration of between about 0.02 grams per gram precipitate formed in the second precipitation step and about 0.06 grams per gram precipitate formed in the second precipitation step.

2. The plasma-derived immunoglobulin G (IgG) composition of claim 1, wherein the first target enrichment step comprises precipitating a cryo-poor plasmid2024204779   27 Aug 2026fraction, in a first precipitation step, with between about 6% and about 10% alcohol at a pH of between about 7.0 and about 7.5 to obtain a first precipitate and a first supernatant.

3. The plasma-derived immunoglobulin G (IgG) composition of claim 1, wherein the second target enrichment step comprises precipitating IgG from the first supernatant, in a second precipitation step, with between about 20% and about 30% alcohol at a pH of between about 6.7 and about 7.3 to form a second precipitate and suspending the second precipitate to form a first suspension.

4. The plasma-derived immunoglobulin G (IgG) composition of claim 3, wherein the second target protein enrichment step is:(i) a protein precipitation step;(ii) an ultrafiltration / diafiltration step; or(iii) a chromatographic enrichment step.

5. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 4, wherein the plasma-derived target protein composition is a manufacturing intermediate.

6. The plasma-derived immunoglobulin G (IgG) composition of any one of claims 1 to 5, wherein the method further comprises an anion exchange chromatography enrichment step and / or a cation exchange chromatography enrichment step.

7. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 6, wherein the second precipitation step comprises adjusting the ethanol concentration of the first supernatant formed in step (a) to about 25% (v / v) at a temperature between about -7°C and about -9°C.

8. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 7, wherein suspending the second precipitate comprises suspending the second precipitate with a buffer containing phosphate and acetate, wherein the pH of the buffer is adjusted with between 300 mL and 700 mL of glacial acetic acid per 1000 L of buffer.

9. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 8, wherein the solution condition suitable to bind a serine2024204779   27 Aug 2026protease or serine protease zymogen comprises a pH between 4.5 and 6.0 and a conductivity of between 0.1 mS / cm and 3 mS / cm.

10. The plasma-derived immunoglobulin G (IgG) composition of claim 9, wherein the pH is between 4.9 and 5.3.

11. The plasma-derived immunoglobulin G (IgG) composition of claim 9 or 10, wherein the conductivity is between 0.5 mS / cm and 2 mS / cm.

12. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 11, wherein the serine protease or a serine protease zymogen is FXIa.

13. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 12, wherein the composition is contacted with SiO2 at a final concentration of at least 1 g SiO2 / g protein.

14. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 13, wherein the second precipitation step comprises adjusting the alcohol concentration of the first supernatant to from about 24% to about 26%.

15. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 14, wherein the second precipitation step is performed at a temperature of from about -7°C to about -9°C.

16. The plasma-derived immunoglobulin G (IgG) composition according to any one of claims 1 to 15, wherein the finely divided silicon dioxide (SiO2) is fumed silica.

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