USOO910904.4B2

(12) United States Patent (10) Patent No.: US 9,109,044 B2 BairstOW et al. (45) Date of Patent: Aug. 18, 2015

(54) MANUFACTURE OF FACTOR H (FH) AND (52) U.S. Cl. FH-DERVATIVES FROM PLASMA CPC ...... C07K 14/472 (2013.01); A61K38/1709 (2013.01); A61 K38/1725 (2013.01); C07K (71) Applicants: Baxter International Inc., Deerfield, IL I/36 (2013.01) (US); Baxter Healthcare S.A., Glattpark (58) Field of Classification Search (Opfikon) (CH) None See application file for complete search history. (72) Inventors: Shawn F. Bairstow, Gurnee, IL (US); Richard Johnson, Mundelein, IL (US); (56) References Cited Sindhu Ramachandran, Lake Zurich, IL (US); Ruth Madlener, Vienna (AT): U.S. PATENT DOCUMENTS Wolfgang Teschner, Vienna (AT); 2007/0020647 A1 1/2007 Hageman et al. Hans-Peter Schwarz, Vienna (AT) 2008.0318841 A1 12/2008 ChtOurou et al. 2009.0118163 A1 5/2009 Gronksi et al. (73) Assignees: Baxalta Incorporated, Bannockburn, IL (US); Baxalta GmbH, Glattpark FOREIGN PATENT DOCUMENTS (Opfikon) (CH) EP O 222 611 A2 5, 1987 Notice: WO WO 2007/038995 A1 4/2007 (*) Subject to any disclaimer, the term of this WO WO 2007/066O17 A2 6, 2007 patent is extended or adjusted under 35 WO WO 2007/066O17 A3 6, 2007 U.S.C. 154(b) by 221 days. WO WO 2008, 113589 A1 9, 2008 (21) Appl. No.: 13/633,057 OTHER PUBLICATIONS International Search Report, Applin. No. PCT/US2010/043151. Int. (22) Filed: Oct. 1, 2012 Filing Date: Jul 23, 2010. Nitschmann, H., et al., “Vereinfachtes Verfahren Zur Gewinnung von (65) Prior Publication Data Humanem Albumin and Gamma-Globulin aus Blutplasma Mittels US 2013/O1 O2537 A1 Apr. 25, 2013 Alkoholfaellung.” Helvetica Chimica Acta, Verlag Helvetica Chimica Acta, vol. 37, Jan. 1, 1954, pp. 866-873. Primary Examiner — Suzanne M Noakes Related U.S. Application Data (74) Attorney, Agent, or Firm — Morgan, Lewis & Bockius (63) Continuation of application No. 12/842.944, filed on LLP Jul. 23, 2010, now Pat. No. 8,304,524. (57) ABSTRACT (60) Provisional application No. 61/227.968, filed on Jul. The present invention provides compositions and pharmaceu 23, 2009. tical formulations of Factor H derived from plasma. Also (51) Int. C. provided are methods for the manufacture of the Factor H C07K I4/47 (2006.01) compositions and formulations, as well as methods for the A6 IK38/17 (2006.01) treatment of diseases associated with Factor H dysfunction. C07K L/36 (2006.01) 17 Claims, 13 Drawing Sheets

U.S. Patent Aug. 18, 2015 Sheet 2 of 13 US 9,109,044 B2

Aerosii FC (DEAE-Sepharose) 140 12O 100 s FIG. 2A 60 40

O 10 20 30 40 50 6O 70 8O Raction if

Aerosil FC (Heparin Sepharose) 120 100 FIG. 2B 80 60 40 2O

O 10 2O 3O 40 50 60 70 80 Raction it

S388.88.33:38

*::::::::::::::: **3888:

3:33. &::s

FIG. 2C FIG. 2D U.S. Patent Aug. 18, 2015 Sheet 3 of 13 US 9,109,044 B2

7.5% SDS-PAGE

250 MW Standards 13 Factor H CompTech) O Factor HFraction I) 75 Factor H(Aerosil FC) - pig per lane each) non-reduced COOmassie

FIG. 3 U.S. Patent Aug. 18, 2015 Sheet 4 of 13 US 9,109,044 B2

Average Factor Has % of Total Protein

OO8.

3O8.

...&

OO8.

OO8.

8.

OOO8. Cohn Fract Fract Fract Fract Aero Precip Frecip Fract Fract Fract Fract Fract F. Sup Precip Hill HIll Fitt 3 Sup P.. 1 I P&4 Precip Sup Frecip Sup Frecip Slup Precip

FIG. 4 U.S. Patent Aug. 18, 2015 Sheet 5 of 13 US 9,109,044 B2

Factor Hinhibition of Alternative Complement Activation

3. E E

O Facto HCIcetation gill Suppleted into III plans

FIG. 5 U.S. Patent Aug. 18, 2015 Sheet 6 of 13 US 9,109,044 B2

Factor H: Factor Cofactor Activity O 35 -- Factor H (CompTech) 300-8- A&Psil Filtrate ... 250 -k- Fraction ...r : 2 SO

SO

-SO . O Facto' Hipg:TL

FIG. 6 U.S. Patent Aug. 18, 2015 Sheet 7 of 13 US 9,109,044 B2

FIG. 7 U.S. Patent Aug. 18, 2015 Sheet 8 of 13 US 9,109,044 B2

8. & 8.

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& 8 : 88: in: is i88&

FIG. 8 U.S. Patent Aug. 18, 2015 Sheet 9 of 13 US 9,109,044 B2

late 2 3 : 3 8 g : ; ; ; ; ; ; 3. SS

U.S. Patent Aug. 18, 2015 Sheet 11 of 13 US 9,109,044 B2

FIG. IOB

FIG. IOC U.S. Patent Aug. 18, 2015 Sheet 12 of 13 US 9,109,044 B2

$3......

isg: 38883 & 83.838: U.S. Patent Aug. 18, 2015 Sheet 13 of 13 US 9,109,044 B2

FIG. I. IB

FIG. I IC US 9,109,044 B2 1. 2 MANUFACTURE OF FACTOR H (FH) AND improvement of the overall IVIG yield. This requirement will FH-DERVATIVES FROM PLASMA limit the availability of plasma for the manufacture of new plasma-derived blood products. CROSS-REFERENCES TO RELATED Due to the lack of plasma available for the manufacture of APPLICATIONS new plasma-derived products, their manufacture must be integrated into the existing framework of the established The present application is a continuation of U.S. patent manufacturing processes for plasma-derived products such as application Ser. No. 12/842,944, filed Jul. 23, 2010, which immunoglobulins and albumin. Factor H, implicated as a claims the benefit of U.S. Provisional Application No. potential therapeutic for AMD, aHUS, and MPGN, among 61/227.968 filed Jul. 23, 2009, which are expressly incorpo 10 other conditions, is one Such plasma-derived blood product rated herein by reference in its entirety for all purposes. that is gaining the attention of physicians. However, due to the BACKGROUND OF THE INVENTION resources devoted to, for example, IgG gamma globulin manufacture, methods are needed for the manufacture of Unlike other biologics that are produced via recombinant 15 Factor H that can be introduced into the existing manufactur expression of DNA vectors in host cell lines, plasma-derived ing schemes. Several methods have been Suggested to achieve proteins are fractionated from human blood and plasma dona just this, however, many of these proposed solutions require tions. Thus, the Supply of these products cannot be increased modification of the existing manufacturing scheme for estab by simply increasing the Volume of production. Rather the lished products. Such changes will require new regulatory level of commercially available blood products is limited by approvals for the established products and may even result in the available supply of blood and plasma donations. This alterations of the characteristics of the established products. dynamic results in a shortage in the availability of raw human For example, WO 2007/066017 describes methods for the plasma for the manufacture of new plasma-derived blood production of Factor H preparations from the Supernatant of a factors that have lesser established commercial markets, cryoprecipitate. The disclosed method consists of preparing a including Complement Factor H(CFH). 25 Supernatant of a cryoprecipitate, Submitting the Supernatant Factor H (FH) is a member of the regulators of complement to anion exchange chromatography (AEC), Submitting the activation family and is a complement control protein. It is a flow through from the AEC to heparin affinity chromatogra large (155 kilodaltons), soluble glycoprotein that circulates in phy (HAC), submitting the relevant eluate from the HAC to human plasma (at a concentration of 500-800 micrograms per strong cation exchange chromatography (CEC), Submitting milliliter). Its main job is to regulate the Alternative Pathway 30 the relevant eluate from the CEC to strong anion exchange of the complement system, ensuring that the complement chromatography (SAEC) and eluting the Factor H from the system is directed towards pathogens and does not damage SAEC. Disadvantageously, cryoprecipitate supernatants are host tissue. Factor H regulates complement activation on self common intermediate fractions in the manufacturing pro cells by possessing both cofactor activity for Factor I medi cesses of many commercially important plasma-derived ated C3b cleavage, and decay accelerating activity against the 35 blood products, including IgG gamma globulins (IVIG and alternative pathway C3 convertase, C3bBb. Thus, Factor H Subcutaneous) and albumin. Submitting this fraction to chro protects self cells, but not foreign pathogens (e.g., bacteria, matography steps will alter the cryoprecipitate Supernatant protists, and viruses), from complementactivation by binding and would require that the manufacturing processes of the to glycosaminoglycans (GAGs) that are present on the Sur established downstream blood products be adapted in face of human cells, but not on the pathogenic cell Surfaces. 40 unknown fashions. In addition to requiring a complete revali Due to its regulatory role in complement activation, Factor dation and possible redesign of these manufacturing pro H has been implicated as a potential therapeutic agent for cesses, regulatory re-approval of the manufacturing proce several human disease States, including age-related macular dures from key regulatory agencies is needed. degeneration (AMD), hemolytic uremic syndrome (aHUS) Likewise, WO 2008/113589 describes methods for the pro and membranoproliferative glomerulonephritis (MPGN). 45 duction of Factor H preparations from human plasma. Spe While a causal relationship between the single nucleotide cifically, this publication describes the purification of Factor polymorphism (SNP) in complement control protein (CCP) H from three known plasma processing fractions, namely a module 7 of Factor H and age-related macular degeneration Cohn-Oncley Fraction I supernatant, a Cohn-Oncley Fraction (AMD) has been characterized, medicaments based on this III precipitate, and a Kistler/Nitschmann Precipitate B frac causal relationship have thus far not been identified. 50 tion. With respect to the first method, WO 2008/113589 dis Due in part to the increasing global demand and fluctua closes that Factor H can be removed from a Cohn-Oncley tions in the available supply of plasma-derived blood prod Fraction I supernatant by the addition of a heparin affinity ucts, such as immunoglobulin products, several countries, chromatography step. Disadvantageously, the Cohn-Oncley including Australia and England, have implemented demand Fraction I Supernatant is a common intermediate fraction in management programs to protect Supplies of these products 55 the manufacturing processes of many commercially impor for the highest demand patients during times of product short tant plasma-derived blood products, including IgG gamma ageS. globulins (IVIG and subcutaneous) and albumin. Similarly, For example, it has been reported that in 2007, 26.5 million many immunoglobulin (e.g., IgG, IVIG, etc.) manufacturing liters of plasma were fractionated, generating 75.2 metric processes do not rely on Cohn-Oncley Fraction III precipita tons of IVIG, with an average production yield of 2.8 grams 60 tion or Kistler/Nitschmann Precipitate B steps, for example per liter (Robert P. Supra). This same report estimated that Gammagard R. Liquid and Kiovig (Baxter International Inc.). global IVIG yields are expected to increase to about 3.43 The disadvantage of the introduction of additional steps. Such grams per liter by 2012. However, due to the continued as a heparin affinity chromatography, Fraction III precipita growth in global demand for IVIG, projected at between tion, or Precipitate B steps, into the manufacturing schemes of about 7% and 13% annually between now and 2015, more 65 established blood products, as outlined above, is that it raw plasma will need to be dedicated to immunoglobulin requires revalidation of the manufacturing procedure, regu purification to meet the demand in spite of the expected latory re-approval of the manufacturing procedures from key US 9,109,044 B2 3 4 regulatory agencies, and may further have unforeseen conse toid arthritis, IgA nephropathy, asthma, systemic lupus quences for the yield and/or purity of the otherwise estab erythematosus, and ischemia reperfusion injury. lished product. In one aspect, the present invention provides a composition As such, a need remains in the art for methods of manu of Factor H prepared by extracting Factor H from a Fraction facturing Factor H that do not require the use of additional I precipitate or Fraction II+III filter cake. In one embodiment input plasma or the redesign and regulatory re-approval of of the Factor H compositions provided herein, at least 90% of existing manufacturing processes for commercially impor the protein content of the composition is Factor H. In another tant plasma-derived blood products, such as albumin and IgG embodiment, at least 95% of the protein content of the com gamma globulins for intravenous (IVIG) or Subcutaneous position is Factor H. administration. Advantageously, the present invention fulfills 10 In one embodiment of the Factor H compositions provided these and other needs by providing methods of manufacturing herein, the composition is formulated for pharmaceutical Factor H that rely entirely on previously unused manufactur administration. In one embodiment of the pharmaceutical ing byproducts. Among other aspects, the present invention compositions provided herein, at least 90% of the protein also provides novel Factor H compositions and methods for content of the composition is Factor H. In another embodi treating Factor H and complement-related diseases and dis 15 ment, at least 95% of the protein content of the pharmaceu orders. tical composition is Factor H. In one embodiment of the pharmaceutical compositions provided herein, the concentra BRIEF SUMMARY OF THE INVENTION tion of Factor H is between about 1% and about 25%. In one embodiment, a pharmaceutical composition of Fac Among other aspects, the present invention provides meth tor H is formulated for intravenous, intravitreal, or subcuta ods for preparing enriched compositions of plasma-derived neous administration. In one specific embodiment, the com Factor H. Advantageously, the methods provided herein position is formulated for intravenous administration. In one allow for the industrial-scale preparation of Factor H compo embodiment, the composition is formulated for treatment of sitions from materials otherwise discarded during the prepa atypical haemolytic uremic syndrome (ahUS). In another ration of other commercially important blood products by 25 specific embodiment, the composition is formulated for intra plasma fractionation. vitreal administration. In one embodiment, the composition is In one aspect, the present invention provides a method for formulated for treatment of age-related macular degeneration preparing an enriched Factor H composition from plasma by (AMD). extracting Factor H from a Fraction II+III filter cake. In one In another aspect, the present invention provides a method embodiment, the method involves the capture of Factor H 30 for treating a disease associated with Factor H dysfunction in from a Fraction II+III Suspension by a solid phase and sepa a Subject in need thereof, the method comprising administer ration from the resulting Fraction II+III suspension. ing a therapeutically effective dose of a Factor H composition In a second aspect, the present invention provides a method according to a method provided herein. In one embodiment, for preparing an enriched Factor H composition from plasma the disease or condition associated with Factor H dysfunction by extracting Factor H from a Fraction I precipitate. 35 is selected from the group consisting of age-related macular In a third aspect, the present invention provides aqueous degeneration, hemolytic uremic syndrome, and membrano compositions of plasma-derived Factor H prepared from proliferative glomerulonephritis. materials otherwise discarded during the preparation of other In another aspect, the present invention provides a method commercially important blood products by plasma fraction for treating a disease associated with abnormal alternative ation, for example, IgG gamma globulins. 40 pathway complement activity in a subject in need thereof, the In a fourth aspect, the present invention provides pharma method comprising administering a therapeutically effective ceutical compositions of plasma-derived Factor H prepared dose of a Factor H composition according to a method pro from materials otherwise discarded during the preparation of vided herein. In one embodiment, the disease associated with other commercially important blood products by plasma frac abnormal alternative pathway complement activity is tionation, for example, IgG gamma globulins. 45 selected from the group consisting of an autoimmune disease, In a fifth aspect, the present invention provides methods for a renal disease, asthma, Alzheimer disease, adult macular treating a disease or disorder associated with Factor H dys degeneration, proximal nocturnal hemoglobinuria, abdomi function in a Subject in need thereof by administering athera nal aortic aneurism, ischemia reperfusion injury, and sepsis. peutically effective dose of a Factor H composition prepared In a specific embodiment, the autoimmune disease is selected from materials otherwise discarded during the preparation of 50 from the group consisting of rheumatoid arthritis, IgA neph other commercially important blood products by plasma frac ropathy, asthma, Systemic lupus erythematosus, multiple tionation. Diseases and disorders associated with Factor H sclerosis, Anti-Phospholipid syndrome, ANCA-associated dysfunction include, but are not limited to, Complement Fac vasculitis, pemphigus, uveitis, myathemia gravis, and Hash tor H(CFH) deficiency, atypical haemolytic uremic syndrome imoto's thyroiditis. (aFIUS), age-related macular degeneration (AMD), membra 55 noproliferative glomulonephritis type II, myocardial infarc BRIEF DESCRIPTION OF THE DRAWINGS tion, coronary heart disease/coronary artery disease (CAD/ CHD), and Alzheimer's disease FIG. 1. Overview of an exemplary plasma fractionation In a sixth aspect, the present invention provides methods scheme. for treating a disease or disorder associated with abnormal 60 FIG. 2. Chromatographs of (A) DEAE SepharoseTM and alternative pathway complement activity in a Subject in need (B) Heparin SepharoseTM enrichment steps of a Factor H thereof by administering a therapeutically effective dose of a manufacturing process utilizing a Fraction II+III filter cake as Factor H composition prepared from materials otherwise dis the starting material. SDS-PAGE and Western blot analysis of carded during the preparation of other commercially impor the (C) DEAE SepharoseTM and (D) Heparin SepharoseTM tant blood products by plasma fractionation. Diseases and 65 chromatography. disorders associated with abnormal alternative pathway FIG. 3. SDS-PAGE analysis comparing a commercial complement activity include, but are not limited to, rheuma preparation of Factor H (CompTech; lane 2) to Factor H US 9,109,044 B2 5 6 compositions prepared from Fraction I precipitate (lane 4) heparin load; lane 6 contains a sample of the 98 mMelution and Fraction II+III filter cake (lane 6). peak; lane 7 contains a sample of the 98 mMelution shoulder; FIG. 4. Amount of Factor H, expressed as a percentage of lane 8 contains a sample of the 204 mMelution peak; and lane total protein, found in various fractions of an exemplary 9 contains a commercial Factor H standard. plasma fractionation scheme. FIG. 5. Factor Hinhibition of alternative complement acti DETAILED DESCRIPTION OF THE INVENTION vation using Factor H recovered from a Fraction II+III filter cake. I. Introduction FIG. 6. Comparison of the Factor I cofactor activity of a commercial preparation of Factor H (CompTech: 0) to Factor 10 Factor H has been implicated as a potential therapeutic H compositions prepared from Fraction I precipitate (A) and agent for several human disease states, including age-related Fraction II+III filter cake (). macular degeneration, hemolytic uremic syndrome and FIG. 7. SDS-PAGE analysis of Factor Hextracted from a membranoproliferative glomerulonephritis (MPGN). Factor Fraction II+III filter cake using an EDTA extraction buffer H is a relatively abundant plasma protein (0.5 to 0.8 mg/mL (lane 3: 20 mM Tris (pH 8.0), 5 mM EDTA, 200 mM sodium 15 plasma), however, this protein is currently discarded from the chloride) or a phosphate extraction buffer (lane 4: 100 mM operations of various manufactures that specialize in the frac Sodium phosphate (pH 7.5), 150 mM sodium chloride). tionation of human plasma. Among other aspects, the present Samples were normalized through dilution to 500 g/ml Fac invention provides methods for the isolation of Factor H from tor H content (measured by Enzyme-linked Immunosorbent discard fractions of various fractionation processes, for Assay (ELISA) before loading to the gel (7.5% Mini-Pro example, Cohn, Oncley, Cohn-Oncley, Deutsch, tean RTGXTM Gel, Bio-RadR). Lane 1 contains a Factor H Nitschmann, Kistler, and similar fractionation processes, standard (1.05 mg/ml, 97% purity, CalbiochemR); total load without affecting, disrupting, or altering the normal or estab of 5 Lig) and lane 2 contains standard protein molecular lished processing of the plasma used for the manufacture of weight marker (Precision Plus ProteinTM Standards, Bio other plasma-derived products. Thus, in one aspect, the inven RadR). Both buffer systems extracted the Factor H effi 25 tion makes use of discarded plasma fractions for the produc ciently. tion of a useful medicament for the treatment of AMD and FIG. 8. Quantitation of the amount of Factor H (g FH/L other disorders. plasma, as measured by ELISA) extracted from a Fraction Accordingly, in one aspect, it is an object of the invention II+III filter cake by continuous recirculation of an extraction to produce a purified preparation of Factor H from a pooled buffer (phosphate/sodium chloride) through the filter for 30 plasma sample. In a related aspect, the invention involves the between 10 and 60 minutes. Total average yield for combined use of such a purified fraction for the manufacture of a medi filtrate and post-rinse fractions is shown as the last bar. cament for the treatment of AMD. FIG. 9. SDS-PAGE analysis of the resulting protein con In one aspect, the present invention provides new methods tents from precipitations performed on Factor H solutions for the preparation of Factor H from pooled plasma. In spe extracted from a Fraction II+III filter cake. Lane 1 contains 35 cific embodiments, the methods are for the preparation of standard protein molecular weight marker (Precision Plus Factor H from pooled human plasma and comprise the steps ProteinTM Standards, Bio-RadR); lane 2 contains a Factor H of performing Cohn fractionation to obtain Fraction II+III standard (1.05 mg/ml, 97% purity, CalbiochemR); total load precipitates and preparing a Suspension of the combined pre of 1 Jug); lanes 3, 5, 7, 9, and 11 contain dissolved precipitates cipitates; filtering the re-suspended Fraction II+III precipi from PEG precipitations (10%, 12.5%, 17.5%, 20%, and 40 tates to obtain the Aerosil R filter cake discard left behind after 12.5%+0.2% Tween, respectively); lanes 4, 6, 8, and 10 con filtration of the Fraction II+III suspension; extracting the filter tain supernatants from PEG precipitations (10%, 12.5%, cake according to a method comprising the steps of: (i) dis 17.5%, and 20%, respectively); lane 12 contains a superna solving the filter cake in a suitable buffer of appropriate ionic tant from a 15% ethanol precipitation performed at pH 8.0: strength for a time sufficient to dissolve the protein caked on and lane 13 contains a dissolved precipitate from a 25% 45 said filter cake; (ii) diluting said dissolved protein with addi ethanol precipitation performed at pH 6.0. Precipitation with tional buffer (iii) removing debris from the diluted protein; ethanol as well as with PEG are efficient. Only very small (iv) purifying Factor H protein from said diluted protein amounts of Factor H remained in the Supernatants. preparation by subjecting the diluted protein to ultrafiltration FIG. 10. (A) Chromatograph, (B) SDS-Page Analysis, and on a 0.45 um filter to produce a filtrate containing Factor H: (C) Western blot analysis of DEAE chromatography per 50 (v) Subjecting the filtrate containing Factor H to anion formed with step-wise elution of a Factor H solution extracted exchange chromatography using a NaCl gradient in the run from a Fraction II+III filter cake. Lane 1 contains standard ning buffer or a single step elution at a higher salt concentra protein molecular weight markers; lane 2 contains a sample of tion to produce a pooled crude Factor H fraction; and (vi) the Factor H solution loaded onto the DEAE resin; lanes 3 and purifying Factor H from said crude Factor H preparation by 4 contain samples of the flow through from the DEAE load; 55 Heparin SepharoseM chromatography using a gradient of lane 5 contains a sample of the 100 mMelution peak; lane 6 NaCl or a single step elution at a higher salt concentration. In contains a sample of the 100 mM elution shoulder; lane 7 other embodiments, Factor H may be recovered by using step contains a sample of the 155 mMelution peak; lane 8 contains elution off of a chromatographic resin, such as an anion a sample of the 230 mM elution peak; and lane 9 contains a exchange resin or heparin affinity resin. commercial Factor H standard. 60 While in specific embodiments, the anion exchange chro FIG. 11. (A) Chromatograph, (B) SDS-Page Analysis, and matography uses DEAE SepharoseTM as the anion exchange (C) Western blot analysis of Heparin SepharoseTM chroma resin, it should be understood that any anion exchange resin tography performed with step-wise elution of a peak Factor H can be used to perform the anion exchange chromatography. fraction enriched by DEAE chromatography. Lane 1 contains Using the method described herein the purified Factor H standard protein molecular weight markers; lane 2 contains a 65 produced is an active Factor H preparation that is composed sample of the Factor H solution loaded onto the heparin resin; of a mixture of Tyr-402 and His-402 isoforms in the range lanes 3, 4, and 5 contain samples of the flow through from the 50%+20% His-402: 50%+20% Tyr-402. US 9,109,044 B2 7 8 In certain purification steps, the method may further com eration. For example, the genetic marker is a polymorphism. prise addition of Factor I precipitate with the filter cake in step In one embodiment, the Subject is diagnosed as at risk for the (c)(1). development of AMD by the detection of a specific sequence In specific embodiments the buffer used is 25 mM Tris: 5 variant in the CFH gene. In some embodiments, the treatment mM EDTA; 50 mM NaCl having a pH of 8.0. method may be performed on a subject that has not been The Factor H produced by the method described may fur diagnosed with AMD. The treatment may be performed alone ther be lyophilized. or in combination with other treatments for AMD. The treat Another aspect of the invention is a substantially purified ment method may be administered to a Subject that has not Factor H preparation prepared according to a method previously been treated for AMD, and as such is “treatment described herein. More specifically, the Factor H preparation 10 naive'. is an active Factor H preparation that is composed of a mix Another aspect of the invention is a pharmaceutical prepa ture of Tyr-402 and His-402 isoforms in the range 50%-20% ration that comprises an active Factor H preparation prepared His-402: 50%+20% Tyr-402. from a method described herein and a pharmaceutically The invention further contemplates a method for limiting acceptable carrier. complement activation resulting in delayed progression or 15 onset of the development of age related macular degeneration II. Definitions (AMD) in a Subject, comprising administering a therapeuti cally or prophylactically effective amount of Factor H prepa As used herein, “Factor H” refers to a protein component of ration of the invention. The method of administration may be the alternative pathway of complement encoded by the carried out on a subject who does not have any symptoms of complement factor G gene (for example, CFH: NM000186; AMD but is of an age and history that Suggests that the Subject GeneID:3075; UniProt ID P08603; Ripoche et al., Biochem. is at risk of developing such symptoms. In some embodi J. 249:593-602 (1988)). Factor H is translated as a 1,213 ments, the Subject has drusen. The method also may be used amino acid precursor polypeptide which is processed by to effectively treat Subjects that display signs and/or symp removal of an 18 amino acid signal peptide, resulting in the toms of AMD. The subject may for example have been diag 25 mature Factor H protein (amino acids 19-1231). As used in nosed with AMD. the present invention, Factor Hencompasses any natural Vari In certain embodiments, the administration is intravenous. ants, alternative sequences, isoforms or mutant proteins that In other embodiments, the administration is through eye can be foundina plasma sample, for example a human plasma drops or other intravitreal route (e.g., intravitreal injection). sample. Examples of Factor H mutations found in the human In other embodiments, the invention provides methods for 30 population include, without limitation, Y402H; V62I: R78G: limiting complement activation otherwise resulting in a dis R127L: A224; Q400K; C431S: T493R; C536R; 1551T: ease or disorder associated with Factor H dysfunction, includ R567G C630W. C673S. C673Y, E850K; S8901; H893R; ing without limitation, Complement Factor H(CFH) defi C915S; E936D, Q950H; Y951H: T956M; C959Y; W978C; ciency, atypical haemolytic uremic syndrome (aHUS), age N997T V10071, V1007L A1010T, T1017I; Y1021 F; related macular degeneration (AMD), 35 C1043R; N1050Y; I1059T; Q1076R; R1078S: D1119G: membranoproliferative glomulonephritis type II, myocardial V1134G: Y1142D; Q1143E; W1157R; C1163 W: W1183Li: infarction, coronary heart diseasefcoronary artery disease W1183R; T1184R; L1189R; S1191L: G1 194D: V1197A: (CAD/CHD), and Alzheimer's disease, or a diseases or dis E1198A: F1199S; R1210C; R1215G; R1215Q: YPT order associated with abnormal alternative pathway comple CAKR1225:1231FQS; and P1226S. Many of the these muta ment activity, including without limitation, rheumatoid 40 tions have been found to be associated with a variety of arthritis, IgA nephropathy, asthma, Systemic lupus erythema diseases and disorders, including, atypical haemolytic uremic tosus, and ischemia reperfusion injury by administering a syndrome (aHUS), age-related macular degeneration therapeutically or prophylactically effective amount of a Fac (AMD), membranoproliferative glomulonephritis type II tor H preparation provided herein. (MPGNII), CFH deficiency, and basal laminar drusen. Factor Also contemplated is a method of treating a human Subject 45 H also includes proteins containing post-translational modi judged to be at risk for the development of a disease or fications. For example, Factor His believed to be modified by disorder associated with Factor H dysfunction, including N-acetylglucosamine (GlcNAc) at residues 529, 718, 802. without limitation, Complement Factor H(CFH) deficiency, 822, 882, 911, 1029, and 1095. atypical haemolytic uremic syndrome (aFIUS), age-related As used herein, "cryo-poor plasma’ refers to the Superna macular degeneration (AMD), membranoproliferative glo 50 tant created after the removal of cryo-precipitate formed by mulonephritis type II, myocardial infarction, coronary heart thawing plasma or pooled plasma attemperatures near freez disease/coronary artery disease (CAD/CHD), and Alzhe ing, e.g., at temperatures below about 10°C., preferably at a imer's disease, or a diseases or disorder associated with temperature no higher than about 6°C. In the context of the abnormal alternative pathway complementactivity, including present invention, plasma may refer interchangeably to without limitation, rheumatoid arthritis, IgA nephropathy, 55 recovered plasma (i.e., plasma that has been separated from asthma, systemic lupus erythematosus, and ischemia reper whole blood ex vivo) or source plasma (i.e., plasma collected fusion injury, comprising the step of administering to the via plasmapheresis). Cryo-precipitation is commonly per Subject a prophylactically ortherapeutically effective amount formed, for example, by thawing previously frozen pooled of a Factor H preparation of prepared according to the meth plasma, which has already been assayed for safety and quality ods described herein, and periodically repeating said admin 60 considerations, although fresh plasma may also be used. After istration. In some examples, the administration is repeated for complete thawing of the frozen plasma at low temperature, a time effective to delay the progression or onset of the devel separation of the Solid cryo-precipitates from the liquid opment of macular degeneration in said Subject. In specific Supernatant is performed in the cold (e.g., sGC.) by centrifu embodiments, the human Subject is judged to be at risk for the gation of filtration. development of age-related macular degeneration as identi 65 As used herein, a “Cohn pool” refers to the starting mate fied based on the presence of one or more genetic markers rial used for the fractionation of a plasma sample or pool of associated with development of age-related macular degen plasma samples. Cohn pools include whole plasma, cryo US 9,109,044 B2 9 10 poor plasma samples, and pools of cryo-poor plasma samples (2008)), myocardial infarction (Kardys et al., Journal of the that may or may not have been Subjected to a pre-processing American College of Cardiology 47, 1568-1575 (2006); step. In certain embodiments, a Cohn pool is a cryo-poor Mooijaart et al., Experimental Gerontology 42, 1116-1122 plasma sample from which one or more blood factor have (2007); Nicaud et al., Journal of Molecular Medicine 85, been removed in a pre-processing step, for example, adsorp 771-775 (2007): Paiet al., European Heart Journal 28, 1297 tion onto a solid phase (e.g., aluminum hydroxide, finely 1303 (2007); Stark et al., Clinical Science (Lond) 113, 213 divided silicon dioxide, etc.), or chromatographic step (e.g., 218 (2007)), coronary heart disease/coronary artery disease ion exchange or heparin affinity chromatography). Various (CAD/CHD; (Meng et al., BMC Medical Genetics 8, 62 blood factors, including but not limited to Factor Eight Inhibi (2007); Pulido et al., Mayo Clinic Proceedings 82,301-307 tor Bypass Activity (FEIBA), Factor IX-complex, Factor VII 10 (2007); Topol et al., Human Molecular Genetics 15 Spec No concentrate, or Antithrombin III-complex, may be isolated 2, R117-R123 (2006)), and Alzheimer's disease (Hamilton et from the cryo-poor plasma sample to form a Cohn pool. al., Neuromolecular Medicine 9, 331-334 (2007); Zetterberg As used herein, a "Fraction II+III filter cake” refers to a et al., American Journal of Ophthalmology 143, 1059-1060 solid phase recovered after the filtration or centrifugation of a (2007)). The disclosures of the forgoing references describ Cohn-Oncley or equivalent Fraction II+III paste Suspension. 15 ing the associations between mutations and polymorphisms In a preferred embodiment, a Fraction II+III suspension will in the CFH gene and diseases associated with Factor H dys be treated with an adsorptive material, for example, finely function are herein incorporated by reference in their entire divided silicon dioxide, to remove impurities such as lipids, ties for all purposes. fibrinogen, amidolytic activity, prekallikren activity, and As used herein, a “disease or disorder associated with lipoproteins. In another preferred embodiment, filter aid may abnormal alternative pathway complement activity” refers to be added to the Fraction II+III suspension prior to centrifu a disease, disorder, or condition that results from uncontrolled gation or filtration. In a most preferred embodiment, a Frac or aberrant activation of the alternative pathway of comple tion II+III suspension will be treated with both an adsorptive ment. Generally, uncontrolled or aberrant activation of the material and a filter aid prior to centrifugation or filtration. alternative pathway of complement can result in bystander Upon separation of the clarified Fraction II+III suspension 25 damage of host cells and tissues, as well as a depletion of C3 Supernatant, the recovered solid phase material is referred to and corresponding Susceptibility to pathogenic infections as the Fraction II+III filter cake. (e.g., fungal, bacterial, viral, and protistal). Examples of dis As used herein, “finely divided silicon dioxide’ or “finely eases and disorders associated with abnormal alternative divided silica” refers to an oxide of silicon having the formula pathway complement activity include, without limitation, SiO, manufactured in a fashion that allows for the adsorption 30 various autoimmune diseases (such as rheumatoid arthritis, of Factor H onto its surface. Exemplary forms of finely IgA nephropathy, asthma, systemic lupus erythematosus, divided silicon dioxide suitable for use in the methods of the multiple sclerosis, Anti-Phospholipid syndrome, ANCA-as present invention include, without limitation, fumed silica, Sociated vasculitis, pemphigus, uveitis, myathemia gravis, pyrogenic silica, Aerosil R., Cab-O SiltM, colloidal silica, Hashimoto's thyroiditis), Renal diseases (such as IgA neph diatomaceous earth, and the like. In a preferred embodiment, 35 ropathy, hemolytic uremic syndrome, membranoproliferative a commercial hydrophilic fumed silica product is used for the glomerulonephritis) other disease Such as asthma, Alzheimer methods provided herein. Non-limiting examples of these disease, adult macular degeneration, proximal nocturnal products include those marketed by Evonik Industries under hemoglobinuria, abdominal aortic aneurism, ischemia, and the trade name Aerosil R (e.g., Aerosil 90, Aerosil 130, Aero sepsis. sil 150, Aerosil 200, Aerosil 300, Aerosil 380, Aerosil OX50, 40 As used herein, the term “ultrafiltration (UF)' encom Aerosil EG 50, Aerosil TT 600, Aerosil 200 SP. Aerosil 300 passes a variety of membrane filtration methods in which SP, and Aerosil 300/30). hydrostatic pressure forces a liquid against a semi-permeable As used herein, a “disease or disorder associated with membrane. Suspended solids and solutes of high molecular Factor H dysfunction” refers to any disease, disorder, or con weight are retained, while water and low molecular weight dition in a subject that is caused by, characterized by, or 45 Solutes pass through the membrane. This separation process results in a reduced level of Factor H activity in the subject. is often used for purifying and concentrating macromolecular For purposes of the present invention, Factor Hactivity may (10-10 Da) solutions, especially protein solutions. A num refer to the ability of Factor H to bind a protein or ligand, for ber of ultrafiltration membranes are available depending on example, C3b, C3bBb, C3b2Bb, csbC3b, complement factor the size of the molecules they retain. Ultrafiltration is typi B (CFB), C-reactive protein, endothelial cells, glycosami 50 cally characterized by a membrane pore size between 1 and noglycans (GAGs), or alternatively, may refer to its Factor I 1000 kDa and operating pressures between 0.01 and 10 bar. cofactor activity or its ability to accelerate the irreversible As used herein, the term “diafiltration' is performed with dissociation of C3bBb and C3b2Bb. In one embodiment, a the same or a similar membrane as ultrafiltration and is typi disease or disorder associated with Factor H dysfunction cally performed in a tangential flow filtration mode. During results in a C3 deficiency and susceptibility to bacterial infec 55 diafiltration, buffer is introduced into the recycle tank while tions. In some instances, diseases or disorders associated with filtrate is removed from the unit operation. In processes where Factor H dysfunction include conditions that are caused by or the product is in the retentate (for example, Factor H), diafil linked to mutations and polymorphism in the CFH gene tration is particularly useful for separating protein from Small encoding Factor H (for review, see, Barlow et al., Adv Exp molecules like Sugars and salts. In certain cases, diafiltration Med Biol. 2008: 632:117-42, the disclosure of which is herein 60 can be used to exchange the solution, buffer, or individual incorporated by reference in its entirety for all purposes). components of a buffering system. Diseases that have been linked to mutations or polymor As used herein, the term “about denotes an approximate phisms in the CFH gene include, without limitation, Factor H range of plus or minus 10% from a specified value. For deficiency, atypical haemolytic uremic syndrome (aHUS), instance, the language 'about 20% encompasses a range of age-related macular degeneration (AMD), membranoprolif 65 18-22%. erative glomulonephritis type II (MPGNII; de Cordoba and As used herein, the term “mixing describes an act of de Jorge, Clinical and Experimental Immunology 151, 1-13 causing equal distribution of two or more distinct compounds US 9,109,044 B2 11 12 or Substances in a solution or Suspension by any form of manually or automatically to generate a fine mist from a agitation. Complete equal distribution of all ingredients in a liquid. Typically, spraying is performed while the system Solution or Suspension is not required as a result of “mixing receiving the liquid Substance is continuously stirred or oth as the term is used in this application. erwise mixed to ensure rapid and equal distribution of the As used herein, the term "solvent encompasses any liquid liquid within the system. Substance capable of dissolving or dispersing one or more other Substances. A solvent may be inorganic in nature. Such III. Methods for the Manufacture of Plasma-Derived as water, or it may be an organic liquid, Such as ethanol, Factor H acetone, methyl acetate, ethyl acetate, hexane, petrol ether, etc. As used in the term “solvent detergent treatment,” solvent 10 Having shown that the methods described herein are suit denotes an organic solvent (e.g., tri-N-butyl phosphate), able for the preparation and production of human Factor H which is part of the solvent detergent mixture used to inacti that retains activity, a specific object of the present invention vate lipid-enveloped viruses in Solution. is to provide a production procedure for a plasma-derived As used herein, the term “detergent' is used in this appli version of human Factor H for therapeutic use suitable for cation interchangeably with the term “surfactant” or “surface 15 large scale. Large scale with regard to the present invention acting agent. Surfactants are typically organic compounds means a production procedure based on at least 200 liters that are amphiphilic, i.e., containing both hydrophobic plasma, preferentially at least 500 liters, even more preferen groups (“tails”) and hydrophilic groups (“heads'), which ren tially at least 2000 liters human plasma. der Surfactants soluble in both organic solvents and water. A Regarding production, the claimed processes starting from surfactant can be classified by the presence of formally human plasma shall be based on the Sub-fractionation of charged groups in its head. A non-ionic Surfactant has no typical industrial intermediates obtained by, e.g., the frac charge groups in its head, whereas an ionic Surfactant carries tional precipitation by ethanol in the cold (reviewed in a net charge in its head. A Zwitterionic Surfactant contains a Schultze HE, Heremans J F: Molecular Biology of Human head with two oppositely charged groups. Some examples of Proteins. Volume I: Nature and Metabolism of Extracellular common Surfactants include: Anionic (based on Sulfate, Sul 25 Proteins 1966, Elsevier Publishing Company; p. 236-317). A fonate or carboxylate anions): perfluorooctanoate (PFOA or preferred embodiment of such purification is the purification PFO), perfluorooctanesulfonate (PFOS), sodium dodecyl of functional Factor H from side fractions of industrial scale sulfate (SDS), ammonium lauryl sulfate, and other alkyl sul plasma fractionation in Such a way that established and fate salts, sodium laureth Sulfate (also known as sodium lauryl licensed manufacturing processes of plasma products, which ether sulfate, or SLES), alkyl benzene sulfonate; cationic 30 are under control of pharmaceutical regulatory authorities, (based on quaternary ammonium cations): cetyltrimethylam like immunoglobulins, are not affected. For example, the monium bromide (CTAB) a.k.a. hexadecyl trimethyl ammo filter cake obtained after filtration of a Fraction II+III paste nium bromide, and other alkyltrimethylammonium salts, suspension (Teschner W et al., Vox Sang. 2007 January; cetylpyridinium chloride (CPC), polyethoxylated tallow 92(1):42–55), Precipitate III (Schultze H E, Heremans J F; amine (POEA), benzalkonium chloride (BAC), benzetho 35 Molecular Biology of Human Proteins. Volume I: Nature and nium chloride (BZT); Long chain fatty acids and their salts: Metabolism of Extracellular Proteins 1966, Elsevier Publish including caprylate, caprylic acid, heptanoat, hexanoic acid, ing Company; p. 236-317 at p. 253) and precipitate B (method heptanoic acid, nanoic acid, decanoic acid, and the like: Zwit of Kistler and Nitschmann; supra at p. 253) are examples of terionic (amphoteric): dodecyl betaine; cocamidopropyl such industrial sources for Factor H. Starting from those side betaine; coco amphoglycinate; nonionic: alkyl poly(ethylene 40 fractions, purification procedures known in the art can be oxide), alkylphenol poly(ethylene oxide), copolymers of poly used to purify Factor H. They may be based on precipitation (ethylene oxide) and poly(propylene oxide) (commercially with polyethylene glycol (Nagasawa S. Stroud R M: Mol known as Poloxamers or Poloxamines), alkyl polyglucosides, Immunol 1980; 17:1365-72), affinity chromatography via including octyl glucoside, decyl maltoside, fatty alcohols immobilized heparin (citation as before), ion exchange chro (e.g., cetyl alcohol and oleyl alcohol), cocamide MEA, coca 45 matography (Crossley L. G. Porter R R; Biochem J 1980; mide DEA, polysorbates (Tween 20, Tween 80, etc.), Triton 191: 173-82) and hydrophobic interaction chromatography detergents, and dodecyl dimethylamine oxide. (Ripoche J, Al Salihi A. Rousseaux J. Fontaine M. Biochem J As used herein, the term “therapeutically effective amount 1984; 221, 89-96). or dose” or “sufficient/effective amount or dose.” refers to a The starting material for the invention is prepared using dose that produces effects for which it is administered. The 50 Cohn fractions. This fractionation is a well known fraction exact dose will depend on the purpose of the treatment, and ation used for the preparation of immunoglobulin prepara will be ascertainable by one skilled in the art using known tions can be prepared from donor serum or monoclonal or techniques (see, e.g., Lieberman, Pharmaceutical Dosage recombinant immunoglobulins. In a typical example, blood is Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Tech collected from healthy donors. Usually, the blood is collected nology of Pharmaceutical Compounding (1999); Pickar, 55 from the same species of animal as the Subject to which the Dosage Calculations (1999); and Remington. The Science immunoglobulin preparation will be administered (typically and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., referred to as "homologous' immunoglobulins). The immu Lippincott, Williams & Wilkins; the disclosures of which are noglobulins are isolated from the blood by suitable proce herein incorporated by reference in their entireties for all dures, such as, for example, Cohn fractionation, ultracentrifu purposes). 60 gation, electrophoretic preparation, ion exchange As used in this application, the term "spraying refers to a chromatography, affinity chromatography, immunoaffinity means of delivering a liquid Substance into a system, e.g., chromatography, polyethylene glycol fractionation, or the during an alcohol precipitation step. Such as a Cohn fraction like. (See, e.g., Cohn et al., J. Am. Chem. Soc. 68:459–75 ation I or II+III precipitation step, in the form of fine droplets (1946): Oncley et al., J. Am. Chem. Soc. 71:541-50 (1949): or mist of the liquid Substance. Spraying may be achieved by 65 Barundern et al., Vox Sang. 7:157-74 (1962); Koblet et al., any pressurized device, such as a container (e.g., a spray Vox Sang. 13:93-102 (1967); U.S. Pat. Nos. 5,122,373 and bottle), that has a spray head or a nozzle and is operated 5,177, 194; the disclosures of which are incorporated herein US 9,109,044 B2 13 14 by reference in their entireties for all purposes.) The present filtering the Suspension through a filter press containing a invention uses the discarded fractions from the preparation of suitable filter. In one embodiment, Factor H can be extracted immunoglobulins. In particular, the present invention uses the by re-circulating an extraction buffer through a filter press fraction that is precipitated on the filtration cake once the containing a filter cake. Fraction II+III extract is filtered through an Aerosil R filter. In a second aspect, the present invention provides a method Generally, Factor H preparations according to the present for preparing an enriched Factor H composition from plasma invention can be prepared from any suitable starting materi by extracting Factor H from a Fraction I precipitate. als, for example, recovered plasma or source plasma. In a In a preferred embodiment, a method is provided for pre typical example, blood or plasma is collected from healthy paring an enriched Factor H composition from plasma, the donors. Usually, the blood is collected from the same species 10 method comprising the steps of: (a) precipitating proteins ofanimal as the subject to which the Factor H preparation will from a cryo-poor plasma fraction, in a first precipitation step, be administered (typically referred to as “homologous' Fac with between about 6% and about 10% alcohol at a pH of tor H). The Factor H is isolated from the blood or plasma by between about 7.0 and about 7.5 to obtain a first precipitate Suitable procedures, such as, for example, precipitation (alco and a first Supernatant; and (b) extracting Factor H from the hol fractionation or polyethylene glycol fractionation), chro 15 precipitate with a Factor H extraction buffer, thereby prepar matographic methods (ion exchange chromatography, affin ing an enriched Factor H composition. ity chromatography, immunoaffinity chromatography, etc.) In one aspect, a method is provided for preparing an ultracentrifugation, and electrophoretic preparation, and the enriched Factor H composition from plasma, by extracting like. (See, e.g., Cohn et al., J. Am. Chem. Soc. 68:459–75 Factor H from a pool of two or more manufacturing byprod (1946): Deutschet al., J. Biol. Chem. 164:109-118: Oncley et uct fractions created by a process designed to provide a sec al., J. Am. Chem. Soc. 71:541-50 (1949); Cohn et al., J. Am. ond blood protein, for example, IgG gamma globulins. In one Chem. Soc. 72:465-474 (1950); Cohn et al., Blood Cells and embodiment, the method comprises pooling a Fraction I pre Plasma Proteins. Their State in Nature (J. L. Tullis, ed), pp. cipitate and a Fraction II+III filter cake formed during the 1-58, Academic Press, NewYork and London (1953); manufacture of IgG gamma globulins (e.g., IVIG) and Nitschmann et al., Helv. Chim. Acta 37:866-873; Kistler and 25 extracting Factor H from the pooled fractions. Nitschmann, Vox Sang. 7:414-424 (1962); Barundern et al., In a related aspect, Factor H extracted from two or more Vox Sang. 7:157-74 (1962); Koblet et al., Vox Sang. 13:93 manufacturing byproduct fractions created by a process 102 (1967); U.S. Pat. Nos. 5,122,373 and 5,177, 194; the designed to provide a second blood protein, for example, IgG disclosures of which are hereby incorporated by reference in gamma globulins, is pooled and further purified. In one their entireties for all purposes). 30 embodiment, Factor Hextracted from a Fraction I precipitate In certain embodiments, Factor His recovered from mate and a Fraction II+III filter cake is pooled and subsequently rial otherwise discarded during the manufacture of other purified further. commercially important blood products by plasma fraction In certain embodiments, an enriched Factor H composition ation. For example, in an exemplary embodiment, Factor His may be further purified subsequent to extraction from a Frac extracted from a Fraction I precipitate and/or extracted from 35 tion I precipitate and/or Fraction II+III filter cake. Various a filter cake formed after centrifugation or filtration of a methods are available for further purifying Factor H, includ re-Suspended Fraction II+III paste. Advantageously, accord ing without limitation, additional precipitation steps or frac ing to the methods provided herein, industrial-scale prepara tionations, affinity chromatography, ion exchange chroma tion of Factor H can be achieved without the need for addi tography, hydrophobic interaction chromatography, size tional input plasma or the redesign and regulatory re-approval 40 exclusion chromatography, solvent/detergent (S/D) treat of existing manufacturing processes for other commercially ment, nanofiltration, ultrafiltration, diafiltration, and the like. important plasma-derived blood products, such as IgG In one embodiment, the method further comprises precipi gamma globulins for intravenous (IVIG) or Subcutaneous tating impurities from an enriched Factor H composition. In administration. certain embodiments, this step comprises precipitating at In one aspect, the present invention provides a method for 45 least one impurity, for example a lipid or protein, from the preparing an enriched Factor H composition from plasma by composition and then separating the precipitate from the extracting Factor H from a Fraction II+III filter cake. In a Supernatant containing Factor H. Optionally, Factor H can related embodiment, the method involves the adsorption of then be precipitated from the Supernatant in a separate pre Factor H from a suspended Fraction II+III precipitate and cipitation. separation from the Suspension. 50 Advantageously, precipitation and Subsequent re-suspen In a preferred embodiment, a method is provided for pre sion of Factor H from an enriched composition allows for the paring an enriched Factor H composition from plasma, the reduction of Volume prior to additional purification steps, method comprising the steps of: (a) precipitating proteins Such as chromatography or nanofiltration. In one embodi from a cryo-poor plasma fraction, in a first precipitation step, ment, the enriched Factor H composition may be further with between about 6% and about 10% alcohol at a pH of 55 purified Subsequent to extraction from a Fraction I precipitate between about 7.0 and about 7.5 to obtain a first precipitate or Fraction II+III filter cake by precipitating Factor H out of and a first Supernatant; (b) precipitating Factor H from the the enriched composition. In certain embodiments, an first Supernatant, in a second precipitation step, with between enriched Factor H composition may be subjected to a first about 20% and about 30% alcohol at a pH of between about precipitation step to remove at least one impurity from the 6.7 and about 7.3 to form a second precipitate; (c) re-suspend 60 composition, as described above, and then to a second pre ing the second precipitate to form a Suspension; (d) mixing cipitation step to precipitate and recover Factor H. finely divided silicon dioxide (SiO) with the suspension In one embodiment, a Factor H composition extracted from from step (c); (e) separating the Suspension to form a filter a Fraction I precipitate or Fraction II+III filter cake is further cake and a Supernatant; and (f) extracting Factor H from the enriched by precipitating impurities from the enriched Factor filter cake with a Factor Hextraction buffer, thereby preparing 65 H composition, in a second or third precipitation step, thereby an enriched Factor H composition. In a preferred embodi forming a Supernatant containing Factor H. Subsequently, ment, the filter cake is separated from the Supernatant by Factor H may further be enriched by precipitating Factor H, in US 9,109,044 B2 15 16 a third or fourth precipitation step. In one embodiment, Factor In one aspect, Factor H isolated from material otherwise H is precipitated with between about 20% and about 25% discarded during the manufacture of other commercially alcohol at a pH of between about 6.0 and about 8.0. important blood products by plasma fractionation, e.g., a In certain embodiments, the method for preparing an Fraction I precipitate or Fraction II+III filter cake, may be enriched Factor H composition further comprises at least one, further enriched by chromatography with a series of step preferably two, chromatographic steps to further enrich the elutions that are amenable to a large scale manufacturing purity of the composition. Generally, any Suitable chromato process. In one embodiment, DEAE SepharoseTM and Hep graphic method may be employed to further enrich the Factor arin SepharoseTM chromatography resins are used with a suit H composition extracted from a Fraction I precipitate or able buffer system, for example, one containing 25 mM Tris 10 (pH 8.0) and 5 mM EDTA. Fraction II+III filter cake. In certain embodiments, prior to The chromatographic enrichment of a Factor H composi chromatographic enrichment, the extracted Factor H compo tion can be modified to use buffer systems other than Tris/ sition will be subjected one or more additional precipitation EDTA at pH 8.0. These processes can be adapted for buffers steps, as described above, to reduce the impurities present in and Solutions commonly used in manufacturing of biophar the composition, reduce the load volume for the chromato 15 maceuticals. An example is a purification scheme using phos graphic step, and/or exchange the buffer of the composition. phate buffer at pH 7.0. The key parameter to successful puri In certain embodiments, a Factor H composition may be fication is manipulation of conductivity or ionic strength to further enriched by a chromatographic step comprising anion achieve separation of the desired compound. If pH of the exchange chromatography (AEC), cation exchange chroma buffer system is maintained at pH 8.0, the conductivity of the tography (CEC), heparinaffinity chromatography, hydropho elution buffers must be matched to the purification process bic exchange chromatography (HIC), hydroxyapatite chro described here. If the pH of the buffer system is changed, matography (HAP), immunoaffinity chromatography, size some adjustment of the ionic strength will be needed which exclusion chromatography (i.e., gel filtration), or other Suit can be done with standard techniques used in optimization of able chromatographic step. Chromatographic steps may be chromatographic processes. performed in either batch or column mode. 25 In one aspect, the present invention provides a method of In a preferred embodiment, the method comprises the use preparing Factor H from pooled human plasma comprising ofanion exchange chromatography and heparin affinity chro (a) performing Cohn fractionation to obtain Fraction II+III matography. precipitates and preparing a Suspension of the combined pre In certain embodiments, the methods provided herein for cipitates, (b) filtering the re-suspended Fraction II+III pre the preparation of an enriched Factor H composition will 30 cipitates to obtain the Aerosil R filter cake discard left behind further include at least one, preferably at least two, most after filtration of the Fraction II+III suspension; (c) extracting preferably at least three, viral inactivation or removal steps. the filter cake according to a method comprising the steps of: Non-limiting examples of viral inactivation or removal steps (i) dissolving the filter cake in a suitable buffer of appropriate that may be employed with the methods provided herein ionic strength for a time sufficient to dissolve the protein include, solvent detergent treatment (Horowitz et al., Blood 35 caked on said filter cake; (ii) diluting said dissolved protein Coagul Fibrinolysis 1994 (5 Suppl3): S21-S28 and Kreil et with additional buffer, (iii) removing debris from the diluted al., Transfusion 2003 (43):1023-1028, both of which are protein, (iv) purifying Factor H protein from said diluted herein expressly incorporated by reference in their entirety protein preparation by Subjecting the diluted protein to ultra for all purposes), nanofiltration (Hamamoto et al., Vox Sang filtration on a 0.45 um filter to produce a filtrate containing 1989 (56)230-236 and Yuasa et al., J Gen Virol. 1991 (72 (pt 40 Factor H; (v) subjecting the filtrate containing Factor H to 8)):2021-2024, both of which are herein expressly incorpo anion exchange chromatography using a NaCl gradient (50 rated by reference in their entirety for all purposes), low pH 500 mM) in the running buffer to produce a pooled crude incubation at high temperatures (Kempf et al., Transfitsion Factor H fraction; and (vi) purifying Factor H from said crude 1991 (31)423-427 and Louie et al., Biologicals 1994 (22): 13 Factor H preparation by Heparin SepharoseTM chromatogra 19), and heat treatment of lyophilized Factor H compositions 45 phy using a gradient of NaCl (50-500 mM). (Piszkiewicz et al., Thromb Res. 1987 Jul. 15:47(2):235-41: In one embodiment of the method of preparing Factor H. Piszkiewicz et al., Curr Stud Hematol Blood Transfits. 1989; the anion exchange chromatography uses DEAE (56):44-54; Epstein and Fricke, Arch Pathol Lab Med. 1990 SepharoseTM as the anion exchange resin. March; 114(3):335-40) In another embodiment of the method of preparing Factor In a preferred embodiment, the present invention provides 50 H, the purified Factor H is an active Factor H preparation that a method of preparing a virally safe enriched Factor H com is composed of a mixture of Tyr-402 and His-402 isoforms in position comprising (i) extracting Factor H from a Fraction the range 50%+20% His-402: 50%+20% Tyr-402. II+III filter cake, (ii) performing a first precipitation step to In another embodiment of the method of preparing Factor precipitate at least one impurity from the Factor H composi H, the method further comprises placing Factor I precipitate tion, (iii) performing a second precipitation step to precipitate 55 with the filter cake in step (c)(i). Factor H from the composition, and (iv) performing at least In another embodiment of the method of preparing Factor one viral inactivation or removal step, thereby preparing a H, the buffer in step (c)(v) and/or (c)(vi) is 25 mM TRIS/5 virally safe enriched Factor H composition. mM EDTA/50 mM. NaCl having a pH of 8.0. In another preferred embodiment, the invention provides a In another embodiment of the method of preparing Factor method of preparing a virally safe enriched Factor H compo 60 H, the method further comprises lyophilizing the Factor H sition comprising (i) extracting Factor H from a Fraction I produced in step (c)(vi). precipitate, (ii) performing a first precipitation step to pre In another embodiment of the method of preparing Factor cipitate at least one impurity from the Factor H composition, H, the anion exchange chromatography uses a first buffer (iii) performing a second precipitation step to precipitate having an ionic strength equal to a NaCl concentration of Factor H from the composition, and (iv) performing at least 65 about 50 mM to about 65 mM for bindingFactor H to the resin one viral inactivation or removal step, thereby preparing a and a second buffer having anionic strength equal to a sodium virally safe enriched Factor H composition. chloride concentration of at least about 100 mM for eluting US 9,109,044 B2 17 18 the Factor H from the resin. In a preferred embodiment, the In one aspect, a method is provided for preparing an elution buffer has an ionic strength equal to a sodium chloride enriched Factor H composition from plasma comprising the concentration of between about 100 mMandabout 120 mM. steps of: (a) extracting Factor H from a Fraction I precipitate Optionally, a Subsequent heparin affinity chromatography and/or a Fraction II+III filter cake, to form a Factor Hextract; step may then be performed to further enrich the Factor H and (b) precipitating impurities from the Factor H extract to composition. The heparin affinity step consists of adjusting form a Supernatant containing Factor H, thereby preparing an the ionic strength of the Factor H composition to an ionic enriched Factor H composition. strength equal to a NaCl concentration of about 50 mMorless A. Alcohol Precipitation and Chromatographic Fraction for binding Factor H to the resin, optionally washing the ation Methods column with a buffer having an ionic strength equal to a 10 In one aspect, the present invention provides methods for sodium chloride concentration of about 50 mMNaCl to about the preparation of enriched compositions of Factor H from 75 mM NaCl, preferably about 50 mM. NaCl or less, and material otherwise discarded during the manufacturing pro eluting Factor H from the resin with a buffer having an ionic cess of a second blood factor. In an exemplary embodiment, strength equal to a sodium chloride concentration of between Factor H can be recovered from fractions generated by the about 100 mM and about 250 mM NaCl. 15 manufacturing process for plasma-derived IgG compositions, An alternative purification process consists of taking the Such as IgG compositions formulated for intravenous (i.e., Fraction I precipitate or Fraction II+III filter cake extract at IVIG), Subcutaneous, and/or intramuscular administration. pH 8 and adjusting the salt concentration to an ionic strength In a preferred embodiment, a method for the preparation of equal that of an NaCl concentration of between about 120 and an enriched composition of Factor H is provided, the method 150 mM and then contacting the composition with an anion comprising (i) extracting Factor H from a Fraction I precipi exchange resin (e.g., DEAE SepharoseTM) to bind unwanted tate and/or Fraction II+III filter cake, (ii) optionally perform proteins in the extract. The Factor H does not bind to the resin ing a first precipitation step to precipitate at least one impurity and can be recovered in a flow through fraction, which is from the Factor H composition, (iii) optionally performing a collected for further processing. The ionic strength of the second precipitation step to precipitate Factor H from the Factor H pool is then adjusted to equal that of a sodium 25 composition, (iv) optionally performing at least one ion chloride concentration of no more than about 75 mM, pref exchange chromatography step, (v) optionally performing at erably no more than about 50 mM, to bind the Factor H to a least one heparin affinity chromatography step; and (vi) per heparin affinity resin. The resin is eluted to recover Factor H forming at least one viral inactivation or removal step. with a buffer having an ionic strength equal to a sodium In one embodiment, a method for the preparation of an chloride concentration of between about 90 mM and about 30 enriched composition of Factor H from material otherwise 250 mM, preferably between about 100 mM and about 200 discarded during an IgG manufacturing process comprises mM. Optionally, the ionic strength of the Factor Heluate may one or more of the following steps. be adjusted to equal a sodium chloride concentration of no 1. Preparation of Cryo-Poor Plasma more than about 75 mM, preferably no more than about 65 In certain embodiments, the starting material used for the mM, most preferably no more than about 50 mM to bind the 35 preparation of Factor Hand IgG compositions generally con Factor H to a second anion exchange resin, which can be the sists of either recovered plasma (i.e., plasma that has been same or a different resin as the first anion exchange resin, to separated from whole blood ex vivo) or source plasma (i.e., remove impurities from the pool that have low affinity for the plasma collected via plasmapheresis). The purification pro resin. The Factor His then eluted from the resin with a buffer cess typically starts with thawing previously frozen pooled having an ionic strength equal to at least about 100 mM 40 plasma, which has already been assayed for safety and quality sodium chloride, more preferably at least about 120 mM. considerations, although fresh plasma may also be used. In In one embodiment, a Factor H composition is further certain embodiments thawing is typically carried out at a enriched by (i) binding impurities to a an anion exchange temperature not higher than at or about 6°C. After complete resin under conditions such that Factor H does bind to the thawing of the frozen plasma at low temperature, centrifuga resin and is collected in the flow through fraction; (ii) binding 45 tion is performed in the cold (e.g., sGC.) to separate solid Factor H from the flow through fraction to a heparin affinity cryo-precipitates from the liquid Supernatant. Alternatively, resin; (iii) eluting Factor H from the heparin affinity resin to the separation step can be performed by filtration rather than form an eluate; (iv) binding Factor H from the eluate to an centrifugation. The liquid Supernatant (also referred to as anion exchange resin; and (V) eluting Factor H from the anion “cryo-poorplasma, after cold-insoluble proteins removed by exchange resin, thereby further purifying Factor H. 50 centrifugation from freshthawed plasma) is then processed in Purification from Fraction I or the Aerosil R filter cake the next step. Various additional steps can be taken at this could be performed by several alternative methods, including juncture for the isolation of other blood coagulation factors immunoaffinity purification, variant resins for anion and inhibitors, e.g., Factor Eight Inhibitor Bypass Activity exchange chromatography (e.g. DEAE SephacelTM, etc) and (FEIBA), Factor IX-complex, Factor VII, or Antithrombin inclusion of size exclusion chromatography (e.g., 55 III-complex. SephacrylTM S-300) as an optional polishing step, if neces 2. First Precipitation Step—Fraction I Precipitation sary. Hydrophobic interaction chromatography (e.g., Phenyl The cryo-poor plasma Solution is then typically cooled to SepharoseTM) may also be employed as part of the above about 0+1° C. and the pH is adjusted to between at or about purification scheme. These Factor H purification schemes 7.0 and at or about 7.5. In another embodiment, the pH is have all been described in the prior art. Additionally, the 60 adjusted to between at or about 7.1 and at or about 7.3. In one Aerosil R filter cake may be “washed in place with a Factor H embodiment, the pH of the cryo-poor plasma is adjusted to a eluting buffer of sufficientionic strength to dissolve Factor H pH of at or about 7.2. Pre-cooled ethanol is then added while from the filter cake while maintaining its functional activity the plasma is stirred to a target concentration of between at or (such as the 25 mM Tris; 200 mM. NaCl; 5 mM EDTA; pH 8 about 6% and at or about 10%. In a preferred embodiment, buffer used above). This material would then be processed via 65 ethanol is added to a target concentration of between at or downstream chromatography (such as that described above) about 7% and at or about 9%. In a more preferred embodi to purify the Factor H to homogeneity. ment, ethanol is added to a target concentration of at or about US 9,109,044 B2 19 20 8% (v/v). At the same time the temperature is further lowered compared to an analogous precipitation step in which the pH to between at or about -4° C. and at or about 0°C. In a of the solution is adjusted prior to but not after addition of the preferred embodiment, the temperature is lowered to at or precipitating alcohol. about -2°C., to precipitate components such as fibrinogen. In other certain embodiments, the precipitating alcohol Typically, the precipitation event will include a hold time of at 5 and/or the Solution used to adjust the pH is added by spraying, least about 1 hour, although shorter or longer hold times may rather than by fluent addition. As such, in certain embodi also be employed. Subsequently, the Supernatant (Superna ments, a reduced amount of blood factor is irreversibly lost tant I) is then separated from the precipitate (Fraction I pre during the first precipitation step due to protein denaturation, cipitate) by centrifugation, filtration, or another suitable as compared to an analogous precipitation step in which the method. 10 alcohol and/or solution used to adjust the pH is introduced by Typically, the Fraction I precipitation step is performed to fluent addition. remove fibrinogen and other impurities in the manufacturing In yet other embodiments, the pH of the solution is adjusted process of plasma-derived blood factors such as IgG and after addition of the precipitating alcohol and by adding the albumin. Advantageously, it was found that a significant frac precipitating alcohol and/or a solution used to adjust the pH tion of Factor H is present in this precipitate. For example, 15 by spraying, rather than by fluent addition. In a particular Example 1 demonstrates that more than 180grams of Factor embodiment, the pH of the solution is adjusted to at or about H. nearly 10% of the total content found in the cryo-poor 7.2 after addition of the precipitating alcohol and by adding plasma starting material (Cohn pool), is present in the Frac the precipitating alcohol and/or the Solution used to adjust the tion I precipitate of an industrial-scale plasma fractionation. pH by spraying, rather than by fluent addition. Accordingly, in one embodiment, Factor H is extracted from 3. Second Precipitation Step—Fraction II+III Precipitation the Fraction I precipitate. Suitable buffers and methods for the In order to enrich the content and purity of the relevant extraction of Factor H from the Factor I precipitate are pro blood factors present in the Fraction I Supernatant (e.g., Fac vided herein. tor H. IgG), the Fraction Supernatant I is subjected to a second As compared to conventional methods employed as a first precipitation step, which is a Cohn-Oncley Fraction II+III fractionation step for cryo-poor plasma (Cohn et al., Supra; 25 type fractionation. Generally, the pH of the solution is Oncley et al., Supra), the present invention provides, in several adjusted to a pH of between at or about 6.6 and at or about 7.2. embodiments, methods that result in improved yields of In a preferred embodiment, the pH of the solution is adjusted plasma factors (e.g., Factor H. IgG, etc.). In one embodiment, to between at or about 6.6 and at or about 6.8. In a more the precipitating alcohol is added in a fashion that finely preferred embodiment, the pH of the solution is adjusted to a disperses or that rapidly disperses the alcohol at the point of 30 pH of at or about 6.7. Alcohol, preferably ethanol, is then addition. In one embodiment, the alcohol is added by spray added to the solution while being stirred to a final concentra ing. In a second embodiment, the alcohol is added from below tion of between at or about 20% and at or about 30% (v/v) to or directly adjacent to a stirring apparatus, for example, a precipitate Factor H and IgG present in the fraction. In a propeller. Addition of alcohol by any of these mechanisms preferred embodiment, alcohol is added to a final concentra avoids local over-concentration of alcohol which occurs, for 35 tion of at or about 25% (v/v) to precipitate the IgG in the example, at the point of fluent addition and results in the fraction. irreversible denaturation of proteins and/or precipitation of Prior to or concomitant with alcohol addition, the solution proteins that would otherwise be recovered in the superna is further cooled to between at or about -5°C. and at or about tant. -9°C. In a preferred embodiment, the solution is cooled to a In another embodiment, one or more pH modifying agentis 40 temperature at or about -7°C. After completion of the alcohol added in a fashion that finely disperses or that rapidly dis addition, the pH of the solution is immediately adjusted to perses the pH modifying agent at the point of addition. In one between at or about 6.6 and at or about 7.2. In a preferred embodiment, the pH modifying agent is added by spraying. In embodiment, the pH of the solution is adjusted to between at a second embodiment, the pH modifying agent is added from or about 6.6 and at or about 6.8. In a particular embodiment, below or directly adjacent to a stirring apparatus, for example, 45 the pH of the solution is adjusted to at or about 6.9. Typically, a propeller. In a third embodiment, the pH modifying agent is the precipitation event will include a hold time of at least at or added by Sprinkling a solid pH modifying agent over a delo about 10 hours, although shorter or longer hold times may calized area. also be employed. Subsequently, the precipitate (Fraction In yet another embodiment, the pH of the solution is II+III), which contains the majority of the Factor H and IgG adjusted after addition of the alcohol. In a related embodi 50 content of the cryo-poor plasma, is separated from the Super ment, the pH of the solution is adjusted during the addition of natant by centrifugation, filtration, or another Suitable method the alcohol. In one embodiment, the pH of the solution is and collected. As compared to conventional methods maintained at the desired pH during the precipitation hold or employed as a second fractionation step for cryo-poor plasma incubation time by continuously adjusting the pH of the solu (Cohn et al., Supra: Oncley et al., Supra), the present invention tion. In a preferred embodiment, the alcohol is ethanol. 55 provides, in several embodiments, methods that result in In certain embodiments, the pH of the solution is adjusted improved blood factor yields in the Fraction II+III precipi to betweenator about 7.0 and about at or 7.5 after the addition tate. In a related embodiment, the present invention provides of the precipitating alcohol. In other embodiments, the pH of methods that result in a reduced loss of Factor H and IgG in the solution is adjusted to between at or about 7.1 and at or the Fraction II+III supernatant. about 7.3 after addition of the precipitating alcohol. In yet 60 As compared to conventional methods employed as a sec other embodiments, the pH of the solution is adjusted to at or ond fractionation step for cryo-poor plasma (Cohn et al., about 7.0 or at or about 7.1, 7.2, 7.3, 7.4, or 7.5 after addition Supra: Oncley et al., Supra), the present invention provides, in of the precipitating alcohol. In a particular embodiment, the several embodiments, methods that result in improved blood pH of the solution is adjusted to at or about 7.2 after addition factor yields in the Fraction II+III precipitate. In one embodi of the precipitating alcohol. As such, in certain embodiments, 65 ment, the precipitating alcohol is added in a fashion that finely a reduced amount of blood factor is irreversibly lost during disperses the alcohol or that rapidly disperses the alcohol at the first precipitation step due to protein denaturation, as the point of addition. In one embodiment, the alcohol is added US 9,109,044 B2 21 22 by spraying. In a second embodiment, the alcohol is added ages of the starting Factor H and/or IgG are present in the from below or directly adjacent to a stirring apparatus, for Fraction II+III precipitate. In certain embodiments, the pH of example, a propeller. the solution is adjusted to between at or about 6.6 and at or In another embodiment, one or more pH modifying agentis about 7.2 immediately after or during the addition of the added in a fashion that finely disperses or that rapidly dis precipitating alcohol. In another embodiment, the pH of the perses the pH modifying agent at the point of addition. In one solution is maintained between at or about 6.6 and at or about embodiment, the pH modifying agent is added by spraying. In 7.2 continuously during the precipitation incubation period. a second embodiment, the pH modifying agent is added from In other embodiments, the pH of the solution is adjusted to below or directly adjacent to a stirring apparatus, for example, between at or about 6.8 and at or about 7.0 immediately after a propeller. In a third embodiment, the pH modifying agent is 10 or during the addition of the precipitating alcohol, or to a pH added by Sprinkling a solid pH modifying agent over a delo of at or about 6.7, 6.8, 6.9, 7.0, or 7.1 immediately after or calized area. during the addition of the precipitating alcohol. In a particular In yet another embodiment, the pH of the solution is embodiment, the pH of the solution is adjusted to at or about adjusted after addition of the alcohol. In a related embodi 6.9 immediately after or during the addition of the precipitat ment, the pH of the solution is adjusted during addition of the 15 ing alcohol. In certain embodiments, the pH of the solution is alcohol. In one embodiment, the pH of the solution is main maintained at between at or about 6.8 and at or about 7.0 tained at the desired pH during the precipitation hold or continuously during the precipitation incubation period, or at incubation time by continuously adjusting the pH of the solu a pH of at or about 6.9 continuously during the precipitation tion. In a preferred embodiment, the alcohol is ethanol. incubation period. As such, in certain embodiments, a In one embodiment, the temperature of the Fraction II+III reduced amount of Factor H and/or IgG is lost in the super precipitation step is between at or about -7°C. and at or about natant fraction of the second precipitation step as compared to -9°C. In a related embodiment, the concentration of alcohol an analogous precipitation step in which the pH of the solu (e.g., ethanol) used in the Fraction II+III precipitation step is tion is adjusted prior to but not after addition of the precipi at or about 25% (v/v) and the temperature is between at or tating alcohol or to an analogous precipitation step in which about -7°C. and at or about -9°C. In comparison, both Cohn 25 the pH of the solution is not maintained during the entirety of et al. and Oncley et al. perform precipitation at -5°C. and the precipitation incubation period. Oncley et al. use 20% alcohol, in order to reduce the level of In another embodiment, both the precipitating alcohol and contaminants in the precipitate. Advantageously, the methods the Solution used to adjust the pH are added by spraying, provided herein allow for maximal Factor H and IgG yield rather than by fluent addition. As such, in certain embodi without high levels of contamination in the final product. 30 ments, a reduced amount of Factor Hand/or IgG is lost in the In another embodiment, the precipitation step is performed Supernatant fraction of the second precipitation step as com at a temperature between at or about -7°C. and at or about pared to an analogous precipitation step in which the alcohol -9°C. In one embodiment, the precipitation step is performed and/or solution used to adjust the pH is introduced by fluent at a temperature of at or about -7°C. In another embodiment, addition. the precipitation step is performed at a temperature of at or 35 In another embodiment, the pH of the solution is adjusted about -8°C. In another embodiment, the precipitation step is to between at or about 6.7 and at or about 7.1 immediately performed at a temperature of at or about -9°C. after or during the addition of the precipitating alcohol by In certain embodiments, the alcohol concentration of the spray addition of either or both the alcohol and pH modifying precipitation step is between at or about 20% and at or about agent. In another embodiment, the pH of the solution is 30%, preferably between at or about 23% and at or about 40 adjusted to at or about 6.9 immediately after or during the 27%. In a preferred embodiment, the alcohol concentration is addition of the precipitating alcohol by spray addition of between at or about 24% and at or about 26%. In another either or both the alcohol and pH modifying agent. In one preferred embodiment, the alcohol concentration is at or embodiment, the pH of the solution is maintained at or about about 25%. In other embodiments, the alcohol concentration between 6.7 and 7.1 by continuously adjusting the pH during may be at or about 20%, 21%. 22%, 23%, 24%, 25%, 26%, 45 the precipitation incubation period by adding the precipitat 27%, 28%, 29% or 30%. In a particular embodiment, the ing alcohol and/or the solution used to adjust the pH by second precipitation step is performed at a temperature of at spraying, rather than by fluent addition. In another embodi or about -7°C. with an alcohol concentration of at or about ment, the pH of the solution is maintained at or about 6.9 by 25%. In one embodiment, the alcohol is ethanol. continuously adjusting the pH during the precipitation incu It has been discovered that when the pH of the solution is 50 bation period by adding the precipitating alcohol and/or the adjusted to a pH of about 6.9 prior to addition of the precipi Solution used to adjust the pH by spraying, rather than by tating alcohol, the pH of the solution shifts from 6.9 to fluent addition. between about 7.4 and about 7.7, due in part to protein pre In another particular embodiment, the precipitation step is cipitation. As the pH of the solution shifts away from 6.9, performed at or about a temperature between -7°C. and -9 precipitation of IgG becomes less favorable and the precipi 55 C., orator about -7°C. with an alcohol concentration of at or tation of certain contaminants becomes more favorable. about between 23% and 27%, or at or about 25%. In one Advantageously, the inventors have found that by adjusting embodiment, Factor Hand IgG are precipitated at a tempera the pH of the solution after addition of the precipitating alco ture of at or about -7°C. with at or about 25% ethanol added hol, that higher percentages of IgG is recovered in the Frac by spraying, wherein the pH of the Solution is adjusted to at or tion II+III precipitate. In one embodiment, the pH of the 60 about 6.9 after addition of the precipitating alcohol. In yet Solution is maintained at the desired pH during the precipita another embodiment, the pH of the solution is maintained at tion hold or incubation time by continuously adjusting the pH or about 6.9 for the entirety of the precipitation incubation or of the solution. In a preferred embodiment, the alcohol is hold time. ethanol. 4. Extraction of the Fraction II+III Precipitate Accordingly, in one aspect, reduced amounts of Factor H 65 In order to solubilize the Factor H and IgG content of the and/or IgG are lost in the supernatant fraction of the Fraction Fraction II+III precipitate, a cold extraction buffer is used to II+III precipitation step. In other words, increased percent re-suspend the Fractionation II+III precipitate at a typical US 9,109,044 B2 23 24 ratio of at or about 1 part precipitate to 15 parts of extraction In one embodiment, the extraction buffer will containator buffer. In another aspect, a cold extraction buffer is used to about 5 mM monobasic sodium phosphate, 5 mMacetate, and re-Suspend the Fractionation II+III precipitate at a typical 0.051% to 0.06% glacial acetic acid (v/v). In a preferred ratio of at or about 1 part precipitate to 20 parts of extraction embodiment, the Fraction II+III precipitate is extracted with buffer. Other suitable re-suspension ratios may be used, for a paste to buffer ratio of at or about 1:15 at a pH of at or about example at a range of at or about between 1:4 and 1:40, or at 4.5.0.2. or about between 1:8 and 1:30, or at or about between 1:10 In one embodiment, the pH of the solution is maintained and 1:20, or at or about between 1:12 and 1:18, orator about for the duration of the extraction process. In one embodiment, between 1:13 and 1:17, orator about between 1:14 and 1:16. the pH of the solution is maintained at or about between 4.1 In certain embodiments, the re-suspension ratio may be at or 10 and 4.9 for the duration of the extraction process. In a pre about 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, ferred embodiment, the pH of the solution is maintained at or 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, about between 4.2 and 4.8 for the duration of the extraction 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, process. 1:37, 1:38, 1:39, 1:40, or higher. In a preferred embodiment, In a more preferred embodiment, the pH of the solution is the Fraction II+III paste is re-suspended at or about a ratio of 15 maintained at or about between 4.3 and 4.7 for the duration of 1 part precipitate to 15 parts extraction buffer. In another the extraction process. In another preferred embodiment, the preferred embodiment, the Fraction II+III paste is re-sus pH of the solution is maintained at or about between 4.4 and pended at or about a ratio of 1 part precipitate to 20 parts 4.6 for the duration of the extraction process. In yet another extraction buffer. preferred embodiment, the pH of the solution is maintained at Suitable solutions for the extraction of the II+III precipitate or about 4.5 for the duration of the extraction process. will generally have a pH at or about between 4.0 and 5.5. In 5. Pretreatment and Extraction of Factor H from the Frac certain embodiments, the solution will have a pH at or about tion H+III Suspension between 4.3 and 4.7, in other embodiments, the extraction Advantageously, it has been found that pretreatment of solution will have a pH of at or about 4.0, 4.1, 4.2, 4.3, 4.4. solubilized Fraction II+III precipitate with finely divided sili 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 25 con dioxide (SiO) significantly reduces impurities. Such as preferred embodiment, the pH of the extraction buffer will be lipids, fibrinogen, amidolytic activity, prekallikren activity, at or about 4.3. In another preferred embodiment, the pH of and lipoproteins, from the IgG manufacturing process. Unex the extraction buffer will be at or about 4.5. In another pre pectedly, the inventors have found that the majority of Factor ferred embodiment, the pH of the extraction buffer will be at H found in the Cohn pool staring material is drawn into the or about 4.7. Generally, these pH requirements can be met 30 Fraction II+III filter cake after treatment with silicon dioxide using a buffering agent selected from, for example, acetate, and filter aid. For example. Example 1 demonstrates that citrate, monobasic phosphate, dibasic phosphate, mixtures more than 1.74 kilograms of Factor H, more than 80% of the thereof, and the like. Suitable buffer concentrations typically total Factor H content of the cryo-poor plasma starting mate range from about 2.5 to about 100 mM, or from about 5 to rial (Cohn pool), is present in the Fraction II+III precipitate of about 50 mM, or about 2.5, 5, 10, 15, 20, 25, 30,35, 40, 45,50, 35 an industrial-scale plasma fractionation (about 3000 L), yet is 55, 60, 65,70, 75,80, 85,90, 95, or 100 mM buffering agent. absent completely in the filtrate after the Fraction II+III sus The extraction buffer will preferably have a conductivity of pension is treated with silicon dioxide and filter aid addition. at or about between 0.5 mS-cm and 2.0 mS-cm'. For Furthermore. Example 2 demonstrates that Factor H is not example, in certain embodiments, the conductivity of the irreversibly lost and can be extracted from the Fraction II+III extraction buffer will be at or about 0.5 mS cm, or at or 40 filter cake. Even more importantly, as demonstrated in 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, Example 4. Factor H extracted from the filter cake maintains 1.9, orator about 2.0 mS cm. One of ordinary skill in the art equivalent Factor I cofactor and C3/C5 convertase inhibition will know how to generate extraction buffers having an appro activities as compared to a commercial preparation of Factor priate conductivity. H (CompTech). In one particular embodiment, an exemplary extraction 45 Accordingly, in one embodiment, Factor H is extracted buffer may contain at or about 5 mM monobasic sodium from a filter cake after filtration or centrifugation of a Fraction phosphate and at or about 5 mMacetate at a pH of at or about II+III paste suspension in the presence of finely divided sili 4.5+0.2 and conductivity of at or about 0.7 to 0.9 mS/cm. con dioxide (e.g., Aerosil R) and/or filter aid. Generally, the extraction is performed at or about between In certain embodiments, Fraction II+III precipitate that has 0° C. and 20° C., preferably at or about between 2° C. and 8° 50 been extracted with a suitable dissolution buffer will be C. In certain embodiments, the extraction may be performed treated with at or about between 5 mg and 100 mg finely at or about 0°C., 1° C., 2°C., 3° C., 4°C., 5° C., 6°C., 7°C., divided silicon dioxide per gram of suspended Fraction II+III 80 C.9°C., 10°C., 11o C., 12° C., 13°C., 149 C., 15° C., 160 precipitate. In a preferred embodiment, the Fraction II+III C., 17°C., 18°C., 19°C., or 20° C. In a particular embodi suspension will be treated with at or about between 20 mg and ment, the extraction is performed at or about between 2°C. 55 80 mg finely divided silicon dioxide per gram of suspended and 10° C. Typically, the extraction process is performed Fraction II+III precipitate. In a more preferred embodiment, under continuous stirring until all soluble components of the the Fraction II+III suspension will be treated with at or about II+III paste are brought into solution. In certain embodi between 40 mg and 60 mg finely divided silicon dioxide per ments, the extraction will proceed for at or about between 60 gram of Suspended Fraction II+III precipitate. In another and 300 minutes, or forator between 120 and 240 min, or for 60 preferred embodiment, the Fraction II+III suspension will be at or about between 150 and 210 minutes, while the suspen treated with at or about 50 mg finely divided silicon dioxide sion is continuously stirred. In certain embodiments, the per gram of Suspended Fraction II+III precipitate. In certain extraction process will proceed for at or about 60, 70, 80.90, embodiments, finely divided silicon dioxide is added at a 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, concentration of at or about between 20 g/kg II+III paste and 220, 230, 240, 250, 260, 270, 280, 290, or at or about 300 65 100 g/kg II+III paste (i.e., for a Fraction II+III precipitate that minutes. In a preferred embodiment, the extraction process is extracted at a ratio of 1:15, finely divided silicon dioxide will proceed for at least 60 minutes with continuous stirring. should be added at a concentration at or about between 20 US 9,109,044 B2 25 26 g/16 kg II+III suspension and 100 g/16 kg II+III Suspension, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, or at a final concentration at or about between 0.125% (w/w) 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, and 0.625% (w/w)). In certain embodiments, the finely 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, or higher. divided silicon dioxide may be added at a concentration of at In a related embodiment, Factor H can be extracted from a or about 5 g/kg II+III paste, or at or about 10, 15, 20, 25, 30. 5 Fraction I precipitate by re-circulating a Factor H extraction 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85,90, 95, or 100 g/kg buffer through a Fraction I precipitate cake or pellet. In one II+III paste. In one specific embodiment, finely divided sili embodiment, a Factor H extraction buffer may be re-circu con dioxide is added to the Fraction II+III suspension to a lated through a Fraction I precipitate (e.g., a Fraction I filter final concentration of at or about 40 g/16 kg II+III Suspension. cake) at a ratio of 1 part precipitate to at or about between 25 In a preferred embodiment, the finely divided silicon dioxide 10 and 30 parts of extraction buffer. Other suitable re-suspension used is Aerosil R. 380 or an equivalent thereof. ratios may be used, for example at or about between 1:4 and Generally, the finely divided silicon dioxide treatment will 1:40, or at or about between 1:8 and 1:30, or at or about be performed at a temperature at or about between 0° C. and between 1:10 and 1:20, orator about between 1:12 and 1:18, 20° C., preferably at or about between 2° C. and 8°C. In or at or about between 1:13 and 1:17, or at or about between certain embodiments, the treatment may be performed at or 15 1:14 and 1:16. In certain embodiments, the ratio may beat or about 0°C., 1° C., 2°C.,3°C., 4°C.,5°C., 6°C., 7 C., 8°C., about 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 9°C., 10°C., 11o C., 12°C., 13°C., 140 C., 15°C., 16° C. 170 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, C., 18° C., 19°C., or 20° C. Typically, the Fraction II+III 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, suspension will be stirred with finely divided silicon dioxide 1:37, 1:38, 1:39, 1:40, or higher. In a preferred embodiment, for at least 15 minutes. In certain embodiments, the Fraction the Fraction I filter cake will remain in the filter press used to II+III suspension will be stirred with finely divided silicon filter the precipitate from the Supernatant during the extrac dioxide for at least 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, tion process. 110 minutes, or at least 1, 2, 3, 4, 5, 6, or more hours. In a After separation of a Fraction II+III filter cake from a preferred embodiment, the Fraction II+III suspension will be Fraction II+III suspension by centrifugation or filtration, Fac stirred with finely divided silicon dioxide for at or about 25 tor H can be extracted from the Fraction II+III filter cake by between 30 and 60 minutes. In another preferred embodi the addition of a Factor Hextraction buffer, which can be used ment, the Fraction II+III suspension will be stirred with finely to re-suspend the Fraction II+III filter cake at a ratio of 1 part divided silicon dioxide for at least 30 minutes. precipitate to at or about between 25 and 30 parts of extraction In certain embodiments, filter aid, for example Cellpure buffer. Other suitable re-suspension ratios may be used, for C300 (Celpure) or Hyflo-Supper-Cel (World Minerals), will 30 example at or about between 1:4 and 1:40, or at or about be added to facilitate depth filtration. Filter aid can be added between 1:8 and 1:30, or at or about between 1:10 and 1:20, at a final concentration of from about 0.1 kg/kg Fraction II+III or at or about between 1:12 and 1:18, or at or about between precipitate to about 0.7 kg/kg Fraction II+III precipitate, or 1:13 and 1:17, orator about between 1:14 and 1:16. In certain from about 0.2 kg/kg Fraction II+III precipitate to about 0.6 embodiments, the ratio may be at or about 1:4, 1:5, 1:6, 1:7, kg/kg Fraction II+III precipitate, or from about 0.3 kg/kg 35 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, Fraction II+III precipitate to about 0.5 kg/kg Fraction II+III 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, precipitate. In certain embodiments, the filter aid will be 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, added at a final concentration of about 0.1 kg/kg Fraction or higher. II+III precipitate, or about 0.2,0.3, 0.4,0.5,0.6, or 0.7 kg/kg In a related embodiment, Factor H can be extracted from a Fraction II+III precipitate. 40 Fraction II+III filter cake by re-circulating a Factor Hextrac In order to remove the non-solubilized and adsorbed frac tion buffer through a Fraction II+III filter cake or pellet. In one tion of the Fraction II+III precipitate (i.e., the Fraction II+III embodiment, a Factor H extraction buffer may be re-circu filter cake) after silicon dioxide treatment, the Suspension is lated through a Fraction II+III filter cake at a ratio of 1 part filtered, typically using depth filtration. Depth filters that may precipitate to at or about between 25 and 30 parts of extraction be employed in the methods provided herein include, metal 45 buffer. Other suitable re-suspension ratios may be used, for lic, glass, ceramic, organic (Such as diatomaceous earth) example at or about between 1:4 and 1:40, or at or about depth filters, and the like. Example of suitable filters include, between 1:8 and 1:30, or at or about between 1:10 and 1:20, without limitation, Cuno 50SA, Cuno 90SA, and Cuno VR06 or at or about between 1:12 and 1:18, or at or about between filters (Cuno). Alternatively, the separation step can be per 1:13 and 1:17, orator about between 1:14 and 1:16. In certain formed by centrifugation rather than filtration. In a preferred 50 embodiments, the ratio may be at or about 1:4, 1:5, 1:6, 1:7, embodiment, the finely divided silicon dioxide treated Frac 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, tion II+III paste suspension is filtered through a depth filter 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, situated in a filter press. 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 6. Extraction of Factor H from Fraction I Precipitate and or higher. In a preferred embodiment, the Fraction I filter cake Fraction II+III Filter Cake 55 will remain in the filter press used to filter the precipitate from After separation of a Fraction I precipitate from a Fraction the Supernatant during the extraction process. I supernatant by centrifugation or filtration, Factor H can be In a preferred embodiment, the step of extracting Factor H extracted from the Fraction I precipitate by the addition of a from a filter cake or precipitate comprises recirculation of a Factor H extraction buffer, which can be used to re-suspend Factor Hextraction buffer through a filterpress containing the the Fraction I precipitate at a ratio of 1 part precipitate to at or 60 Factor H filter cake for at least 10 minutes. In other embodi about between 25 and 30 parts of extraction buffer. Other ments, the step of extracting Factor H from a filter cake or Suitable re-suspension ratios may be used, for example at or precipitate comprises recirculation of a Factor H extraction about between 1:4 and 1:40, or at or about between 1:8 and buffer through a filter press containing the Factor H filter cake 1:30, or at or about between 1:10 and 1:20, or at or about for at or about between 10 and 60 minutes. In a preferred between 1:12 and 1:18, orator about between 1:13 and 1:17, 65 embodiment, the step of extracting Factor H from a filter cake orator about between 1:14 and 1:16. In certain embodiments, or precipitate comprises recirculation of a Factor Hextraction the ratio may be at or about 1:4, 1:5, 1:6, 1:7, 1:8. 1:9, 1:10, buffer through a filter press containing the Factor H filter cake US 9,109,044 B2 27 28 for at or about between 20 and 40 minutes. In yet other salts (e.g., ammonium phosphate, ammonium sulfate, sodium embodiments, the step of extracting Factor H from a filter citrate, etc.), short chain fatty acids (e.g., hexanoic acid, hep cake or precipitate comprises recirculation of a Factor H tanoic acid, caprylic acid, nanoic acid, decanoic acid, etc.), extraction buffer through a filterpress containing the Factor H and the like. In certain embodiments, precipitation may be filter cake for at least 10 minutes or at least 15, 20, 25, 30, 35, 5 facilitated by matching the pH of the solution to the isoelec 40, 45, 50, 55, 60, 65,70, 75, 80, 85, or 90 minutes, or longer tric point of a component of interest, i.e., isoelectric point (e.g., at least about 2, 3, 4, 5, or 6 hours). precipitation. Generally, the extraction is performed at or about between In a preferred embodiment, the method comprises the step 0° C. and 20° C., preferably at or about between 2° C. and 8° of precipitating at least one impurity from an enriched Factor C. In certain embodiments, the extraction may be performed 10 H composition with at or about between 10% and 20% etha at or about 0°C., 1° C., 2°C., 3° C., 4°C., 5° C., 6°C., 7°C., nol at a pH at or about between 7.0 and 9.0. In a preferred 8°C., 9° C., or 10°C., 11° C., 12°C., 13°C., 14°C., 15° C., embodiment, the method comprises the step of precipitating 16° C., 17° C., 18° C., 19° C., or 20° C. In a particular at least one impurity from an enriched Factor H composition embodiment, the extraction is performed at or about between with at or about between 12% and 18% ethanol at a pH at or 2° C. and 10° C. 15 about between 7.3 and 8.7. In a more preferred embodiment, Any suitable buffer may be used for the extraction of Factor the method comprises the step of precipitating at least one H from a Fraction I precipitate or Fraction II+III filter cake. impurity from an enriched Factor H composition with at or Typical extractions buffers will contain at least a buffering about between 14% and 16% ethanol at a pH at or about agent and a salt. In certain embodiments, the extraction buffer between 7.5 and 8.5. In a more preferred embodiment, the will containator about between 10 and 250 mMofa buffering method comprises the step of precipitating at least one impu agent. In certain embodiments, the buffering agent will be rity from an enriched Factor H composition with at or about presentata concentration of at or about 10 mM, or 20, 30, 40, between 14% and 16% ethanolata pH at or about between 7.8 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, and 8.2. In a most preferred embodiment, the method com 190, 200, 210, 220, 230, 240, 250 mM or more buffering prises the step of precipitating at least one impurity from an agent. In certain embodiments, the extraction buffer will have 25 enriched Factor H composition with at or about 15% ethanol an ionic strength of at or about between 5 and 100 mS/cm. In and at or about a pH of 8.0. specific embodiments the extraction buffer will contain at or The concentration of the precipitant, e.g., ethanol, may be about between 50 and 500 mM salt. In certain embodiments, adjusted to maximize the precipitation of one or more impu the salt will be presentata concentration of at or about 50 mM rities and/or minimize the precipitation of Factor H. In certain orator about 75, 100, 125, 150, 175, 200, 225, 250, 300, 350, 30 embodiments, the precipitation may be performed by the 400, 450, 500 mM or more salt. addition of at or about 10%, 11%, 12%, 13%, 14%, 15%, Factor Hextraction buffers will generally have a pH ofator 16%, 17%, 18%, 19% or 20% ethanol. In a preferred embodi about between 6.0 and 9.0. In certain embodiments, a Factor ment, the precipitation is performed by the addition of at or H extraction buffer will have a pH of at or about 6.0, or at or about between 12% and 18% ethanol. In a more preferred about 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, 35 embodiment, the precipitation is performed with at or about 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, between 13% and 17% ethanol. In a more preferred embodi 8.8, 8.9, or 9.0. In a preferred embodiment, the Factor H ment, the precipitation is performed with at or about between buffer will have a pH at or about between 7.0 and 8.0. In a 14% and 16% ethanol. In a most preferred embodiment, the specific embodiment, the extraction buffer will have a pH of precipitation is performed with at or about 15% ethanol. at or about 7.0. In another specific embodiment, the extraction 40 The pH of the solution may be adjusted to maximize the buffer will have a pH of at or about 7.5. In another specific precipitation of one or more impurities and/or to minimize the embodiment, the extraction buffer will have a pH of at or precipitation of Factor H. In certain embodiments, the pH of about 8.0. Non-limiting examples of buffering agents that the solution is adjusted to at or about 7.0. 7.1, 7.2.7.3, 7.4, 7.5, may be used for the formulation of a Factor H extraction 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, buffer include potassium phosphate, sodium phosphate, 45 or 9.0. In a preferred embodiment, the pH of the solution is Sodium acetate, sodium citrate, ammonium acetate, cacodylic adjusted to at or about between 7.2 and 8.8. In another pre acid, imidazole, boric acid, bicine, ACES, BES, BIS-Tris, ferred embodiment, the pH of the solution is adjusted to at or BIS-Tris-propane, CAPS, CHES, glycine amide, glycylgly about between 7.3 and 8.7. In another preferred embodiment, cine, MES, MOPS, PIPES, HEPES, TAPS, TES, tricine, tri the pH of the solution is adjusted to at or about between 7.4 ethanolamine, and Tris. 50 and 8.6. In a more preferred embodiment, the pH of the In one embodiment, the Factor H extraction buffer will solution is adjusted to at or about between 7.5 and 8.5. In a include or consist of 25 mM Tris; 5 mM EDTA; 200 mM more preferred embodiment, the pH of the solution is NaCl; pH 8.0. In another embodiment, the Factor H extrac adjusted to at or about between 7.6 and 8.4. In a more pre tion buffer will include or consist of 100 mM sodium phos ferred embodiment, the pH of the solution is adjusted to at or phate: 150 mM sodium chloride; pH 7.5. 55 about between 7.7 and 8.3. In a more preferred embodiment, 7. Third Precipitation Step—Removal of Impurities the pH of the solution is adjusted to at or about between 7.8 In one embodiment, the method further comprises precipi and 8.2. In a more preferred embodiment, the pH of the tating impurities from an enriched Factor H composition to solution is adjusted to at or about between 7.9 and 8.1. In a form a precipitate (herein referred to as “Precipitate 3’) and a most preferred embodiment, the pH of the solution is adjusted supernatant (herein referred to as “Supernatant 3). In certain 60 to at or about 8.0. embodiments, this step comprises precipitating at least one 8. Fourth Precipitation Step—Factor H Precipitation impurity, for example a lipid or protein, from the composition In one embodiment, the enriched Factor H composition and then separating the precipitate from the Supernatant con may be further purified subsequent to extraction from a Frac taining Factor H. Precipitants suitable for precipitating impu tion I precipitate, Fraction II+III Suspension, or Fraction rities from a plasma-derived fraction are well known in the art 65 II+III filter cake by precipitating Factor Hout of the enriched and include, without limitation, alcohol (e.g., ethanol, metha composition to form a precipitate (herein referred to as “Pre nol, etc.), water soluble polymers (e.g., PEG, dextrans, etc.), cipitate 4') and a supernatant (herein referred to as "Super US 9,109,044 B2 29 30 natant 4''). In certain embodiments, an enriched Factor H tration), or other Suitable chromatographic step. Chromato composition may be subjected to a third precipitation step, as graphic steps may be performed in either batch or column described above, to remove at least one impurity from the mode. composition, as described above, and then to a fourth precipi In a preferred embodiment, the method comprises the steps tation step to precipitate and recover Factor H. of binding Factor H to an anion exchange resin; eluting In a preferred embodiment, precipitating Factor H from an Factor H from the anion exchange resin with an elution buffer, enriched composition comprises the addition of between thereby forming a first eluate containing Factor H. In a more about 20% and about 30% ethanol at a pH between about 5.0 preferred embodiment, the method further comprises the and about 7.0. In a preferred embodiment, the method com steps of binding Factor H from the anion exchange eluate to a prises the step of precipitating Factor H with between about 10 heparin affinity resin; and eluting the Factor H from the hep 22% to about 28% ethanol at a pH between about 5.5 and arin affinity resin with an elution buffer, thereby forming a about 6.5. In a more preferred embodiment, the method com second eluate containing Factor H. In certain embodiments, prises the step of precipitating Factor H with between about the chromatographic methods may further comprise wash 24% to about 26% ethanol at a pH between about 5.8 and 15 steps to remove loosely bound impurities from the chromato about 6.2. In a most preferred embodiment, the method com graphic resin prior to the elution of Factor H. In certain prises the step of precipitating Factor H with at or about 25% embodiments, Factor H may be eluted from a chromatogra ethanol and at or about a pH of 6.0. phy resin by gradient elution, e.g., with a salt gradient, or by The concentration of the precipitant, e.g., ethanol, may be step elution, e.g., with buffers having increasing ionic adjusted to maximize the precipitation Factor Hand/or mini strength. mize the precipitation of one or more impurities. In certain Generally, the conductivity of the Factor H solution is embodiments, the precipitation may be performed by the adjusted to an appropriate ionic strength prior to binding addition of at or about 20%, 21%, 22%, 23%, 24%, 25%, Factor H onto a chromatographic resin. The ionic strength 26%. 27%, 28%, 29% or 30% ethanol. In a preferred embodi should be low enough to promote the interaction between ment, the precipitation is performed by the addition at or 25 Factor H and the resin, yet high enough to maintain the about between 22% and 28% ethanol. In a more preferred stability of the protein in solution. The requirements for the embodiment, the precipitation is performed with at or about ionic strength of the solution will vary dependent upon factors between 23% and 27% ethanol. With a more preferred Such as the identity of the resin used (e.g., strong VS. weak embodiment, the precipitation is performed with at or about anion exchange resin) and the starting purity of the solution. between 24% and 26% ethanol. In a most preferred embodi 30 Various methods may be employed for reducing the ionic ment, the precipitation is performed with at or about 25% strength of a Factor H composition, including without limi ethanol. tation, dilution of the composition with a solution having a The pH of the solution may be adjusted to maximize the low ionic strength, precipitating Factor H from the starting precipitation Factor H and/or minimize the precipitation of composition and re-suspending in a buffer having lower ionic one or more impurities. In certain embodiments, the pH of the 35 strength, ultrafiltration/diafiltration, desalting and/or buffer solution is adjusted to at or about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, exchange chromatography, dialysis, and the like. 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, Any Suitable anion exchange resin may be used in the or 7.0. In a preferred embodiment, the pH of the solution is methods provided herein. Non-limiting examples of anion adjusted to at or about between 5.2 and 6.8. In another pre exchange resins Suitable for use include, diethylaminoethyl ferred embodiment, the pH of the solution is adjusted to at or 40 (DEAE), quaternary aminoethyl (QAE), and quaternary about between 5.3 and 6.7. In another preferred embodiment, ammonium (Q) resins. In a preferred embodiment, the anion the pH of the solution is adjusted to at or about between 5.4 exchange resin used is DEAE SepharoseTM (diethylaminoet and 6.6. In a more preferred embodiment, the pH of the hyl-sepharose). In certain embodiments, the Factor H com solution is adjusted to at or about between 5.5 and 6.5. In a position recovered from a Fraction I precipitate or a filter cake more preferred embodiment, the pH of the solution is 45 formed after the filtration or centrifugation of a Fraction II+III adjusted to at or about between 5.6 and 6.4. In a more pre paste Suspension in the presence of finely divided silicon ferred embodiment, the pH of the solution is adjusted to at or dioxide and/or filter aid, as described above. about between 5.7 and 6.3. In a more preferred embodiment, In a preferred embodiment, Factor H is bound to a DEAE the pH of the solution is adjusted to at or about between 5.8 SepharoseTM resin in the presence of a low ionic strength and 6.2. In a more preferred embodiment, the pH of the 50 loading buffer. Typically, the column will be equilibrated with solution is adjusted to at or about between 5.9 and 6.1. In a the same loading buffer or a compatible buffer with an ionic most preferred embodiment, the pH of the solution is adjusted strength similar to the loading buffer. In certain embodiments, to at or about 6.0. the loading and/or equilibration buffer will have an ionic 9. Chromatographic Methods strength of less than at or about 12 mS/cm. In a preferred In certain embodiments, the method for preparing an 55 embodiment, the loading and/or equilibration buffer will have enriched Factor H composition further comprises at least one, an ionic strength of less than at or about 10 mS/cm. In a most preferably two or more, chromatographic steps to further preferred embodiment, the loading and/or equilibration enrich the purity of the composition. Generally, any Suitable buffer will have an ionic strength of at or about 9 mS/cm. In a chromatographic method may be employed to further enrich preferred embodiment, the loading and/or equilibration the Factor H composition extracted from a Fraction I precipi 60 buffer will have a salt concentration of less than at or about tate or Fraction II+III filter cake. 100 mM NaCl, or ionic strength corresponding to less than In certain embodiments, the chromatographic step may that of a 100 mM NaCl solution. In a more preferred embodi comprise anion exchange chromatography (AEC), cation ment, the loading and/or equilibration buffer will have a salt exchange chromatography (CEC), heparin affinity chroma concentration, or corresponding ionic strength, of less than at tography, hydrophobic exchange chromatography (HIC), 65 or about 75 mM NaCl. In a more preferred embodiment, the hydroxyapatite chromatography (HAP), immuno-affinity salt concentration, or corresponding ionic strength, will be at chromatography, size exclusion chromatography (i.e., gel fil or about between 30 and 70 mM NaCl. In a most preferred US 9,109,044 B2 31 32 embodiment, the salt concentration, or corresponding ionic less thanator about 20 mMNaCl, or less than at or about 25, strength, will be at or about 50 mM NaCl. 30, 40, 50, 60, 70, 80, 90, or 100 mM. NaCl. Optionally, after binding Factor H, the anion exchange Optionally, after binding Factor H, the heparin affinity resin may be washed with one or more buffers having ionic resin may be washed with one or more buffers having ionic strengths intermediate of the loading buffer and the elution strengths intermediate of the loading buffer and the elution buffer. In certain embodiments, a wash buffer may have an buffer. In certain embodiments, a wash buffer may have an ionic strength at or about between 9 mS/cm and 12.5 mS/cm. ionic strength at or about between 3 mS/cm and 12.5 mS/cm. In a preferred embodiment, the washbuffer may have anionic In a preferred embodiment, the washbuffer may have anionic strength at or about between 5 mS/cm and 10 mS/cm. In strength at or about between 5 mS/cm and 10 mS/cm. In certain embodiments, the washbuffer will have a salt concen 10 certain embodiments, the washbuffer will have a salt concen tration, or ionic strength corresponding to, at or about tration, or ionic strength corresponding to, at or about between 30 and 100 mM NaCl. In a preferred embodiment, between 30 and 100 mM NaCl. In a preferred embodiment, the wash buffer will have a salt concentration, or ionic the wash buffer will have a salt concentration, or ionic strength corresponding to, at or about between 40 and about strength corresponding to, at or about between 30 and 80 mM 70 mM NaCl. In certain embodiments, the wash buffer will 15 NaCl. In certain embodiments, the washbuffer will have a salt have a salt concentration, or ionic strength corresponding to, concentration, or ionic strength corresponding to, at or about at or about 30, 35, 40, 45, 50,55, 60, 65,70, 75,80, 85,90, 95, 30, 35, 40, 45, 50,55, 60, 65,70, 75,80, 85,90, 95, or 100 mM or 100 mM. NaCl. NaCl. In certain embodiments, Factor His eluted from the anion In certain embodiments, Factor H is eluted from the hep exchange resin (e.g., DEAE SepharoseTM) with an elution arin affinity resin (e.g., DEAE SepharoseTM) with an elution buffer having suitable ionic strength to disrupt the interaction buffer having suitable ionic strength to disrupt the interaction between the resin and Factor H. In some embodiments, the between the resin and Factor H. In some embodiments, the elution buffer will not have a suitable ionic strength to disrupt elution buffer will not have a suitable ionic strength to disrupt the interaction between the resin and a contaminant that binds the interaction between the resin and a contaminant that binds the resin with higher affinity than does Factor H. In certain 25 the resin with higher affinity than does Factor H. In certain embodiments, the elution buffer will have an ionic strength of embodiments, the elution buffer will have an ionic strength of at least at or about 12 mS/cm. In a preferred embodiment, the at least at or about 12 mS/cm. In certain embodiments, the elution buffer will have an ionic strength of at or about 13 elution buffer will have a salt concentration, or ionic strength mS/cm. In another preferred embodiment, the elution buffer corresponding to, at least at or about 100 mM NaCl. In will have an ionic strength of at or about 14 mS/cm. In certain 30 another embodiment, the elution buffer will have a salt con embodiments, the elution buffer will have a salt concentra centration, or ionic strength corresponding to, at least at or tion, or ionic strength corresponding to, at least at or about about 150 mMNaCl. In yet another embodiment, the elution 100 mM. NaCl, preferably at least at or about 120 mM. In buffer will have a salt concentration, or ionic strength corre certain embodiments, the elution buffer will have a salt con sponding to, at least at or about 200 mM NaCl. In certain centration, or ionic strength corresponding to, at least at or 35 embodiments, the elution buffer will have a salt concentra about 90 mM. NaCl or at least at or about 95, 100, 105, 110, tion, or ionic strength corresponding to, at least at or about 90 115, 120, 125, 130, 140, 150 mM. NaCl, or more. mM NaCl or at least at or about 95, 100, 105, 110, 115, 120, Any suitable heparin affinity resin may be used in the 125, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 mM methods provided herein, for example, resins conjugated to a NaCl, or more. heparin ligand, derivative or mimetic of a heparin ligand, or 40 An alternative purification process consists of taking an heparin-like ligand (e.g., a Sulfated glycosaminoglycan). In a enriched Factor H composition (e.g., extracted from a Frac preferred embodiment, the heparin affinity resin used is Hep tion I precipitate or Fraction II+III filter cake) at a pH at or arin SepharoseTM about between 7.0 and 9.0, preferably at or about 8.0, and In one embodiment, Factor His further purified by heparin adjusting the ionic strength such that Factor H will not bind to affinity chromatography. In a preferred embodiment, Factor 45 an anion exchange resin, for example to an ionic strength H from the eluate of an anion exchange chromatography step equal to at or about between 120 and 150 mM NaCl, and then is further purified by heparin affinity chromatography, e.g., a contacting the solution with an anion exchange resin (e.g., a Heparin SepharoseTM resin. In one embodiment, the ionic DEAE Sepharose column) to bind unwanted proteins in the strength of the Factor H eluate is reduced by a suitable extract. The Factor H will not bind to the column and can be method, e.g., dilution, buffer exchange, dialysis, etc., and 50 collected as a flow through fraction for further processing. Factor H is bound to a heparin affinity resin. In certain The Factor Hpool is then adjusted to an ionic strength equiva embodiments, the ionic strength of the anion exchange eluate lent to no more than at or about 65 mM NaCl, preferably no is reduced to less than at or about 12 mS/cm. In a preferred more than at or about 50 mM NaCl and bound to a heparin embodiment, the ionic strength is reduced to less than at or affinity resin (e.g., a Heparin SepharoseTM column). A buffer about 10 mS/cm. In a more preferred embodiment, the ionic 55 having an ionic strength suitable to elute the Factor H from the strength is reduced to less thanator about 8 mS/cm. In certain resin is then added to recover the Factor H. In a preferred embodiments, the ionic strength may be reduced to less than embodiment, the elution buffer will have an ionic strength at or about 4 mS/cm, or less than at or about 5, 6, 7, 8, 9, 10, equivalent to a sodium chloride concentration of at or about 11, or 12 mS/cm. In certain embodiments, the salt concentra between 90 and 250 mM. NaCl, preferably at or about tion of the anion exchange eluate, or ionic strength corre 60 between 100 and 200 mM. NaCl. The ionic strength of the sponding to, is reduced less than at or about 100 mMNaCl. In Factor H eluate can then be lowered to a level suitable for a preferred embodiment, the salt concentration, or ionic binding Factor H to an anion exchange resin, for example to strength corresponding to, is reduced less than at or about 75 an ionic strength equivalent to no more than at or about 65 mM NaCl. In a more preferred embodiment, the salt concen mM NaCl, preferably no more than about 50 mM. NaCl. tration, or ionic strength corresponding to, is reduced to less 65 Factor H is then bound to the anion exchange resin (e.g., a thanator about 50 mMNaCl. In certain embodiments, the salt DEAE SepharoseTM column) to remove impurities from the concentration, or ionic strength corresponding to, is reduced pool that do not bind the resin under these conditions. The US 9,109,044 B2 33 34 Factor H is eluted with a buffer having a suitable ionic natant 3). In a preferred embodiment the Precipitation 3 strength, for example anionic strength equivalent to at least at supernatant is subjected to solvent and detergent (S/D) treat or about 100 mMNaCl, preferably at least at or about 120mM ment. NaCl. In a third embodiment, a viral inactivation or removal step Any suitable cation exchange resin may be used in the 5 is performed on the Precipitate 4 suspension. In a preferred methods provided herein. Non-limiting examples of cation embodiment the Precipitate 4 suspension is subjected to sol exchange resins suitable for use include, carboxymethyl vent and detergent (S/D) treatment. (CM), sulfopropyl (SP), methyl sulfonate (S) resins. In a fourth embodiment, a viral inactivation or removal step Any suitable hydroxyapatite or other calcium-based resin is performed on the anion exchange eluate. In a preferred 10 embodiment the anion exchange eluate is Subjected to solvent may be used in the methods provided herein. Non-limiting and detergent (S/D) treatment. In another preferred embodi examples of Suitable resins include hydroxyapatite resins, ment, the anion exchange eluate is Subjected to nanofiltration. fluorapatite resins, fluorhydroxyapatite resins, and the like. In a fifth embodiment, a viral inactivation or removal step Any suitable hydrophobic interaction chromatography is performed on the heparin affinity eluate. In a preferred resin may be used in the methods provided herein. Non 15 embodiment the heparin affinity eluate is subjected to solvent limiting examples of Suitable resins include phenyl-resins, and detergent (S/D) treatment. In another preferred embodi methyl-resins, butyl-resins, octyl-resins, and the like. ment, the heparin affinity eluate is subjected to nanofiltration. In certain embodiments, Factor H may be further enriched In a sixth embodiment, a viral inactivation or removal step by immuno-affinity chromatography, for example with resins is performed on an enriched Factor H bulk solution. In a conjugated to an antibody, aptamer, or other binding mol- 20 preferred embodiment, the enriched Factor H bulk solution is ecule highly specific for Factor H. subjected to nanofiltration. In another preferred embodiment, In certain embodiments, individual or all chromatographic the enriched Factor H bulk solution is subjected to incubation steps will rely on a common buffer system, in which only the at low pH and/or high temperatures. salt concentration varies between the equilibration, wash, and In a seventh embodiment, a lyophilized Factor H compo elution buffers. Any suitable buffer may be used, e.g., a Tris 25 sition is heat treated to inactivate viruses. buffer, a phosphate buffer, a citrate buffer, etc. The pH of the In one embodiment, a manufacturing process for plasma loading buffer will range at or about between 6.0 and 9.0. In derived Factor H is provided that contains two viral inactiva a preferred embodiment, the pH of the buffer system is at or tion or removal steps. In a preferred embodiment, the process about between 7.0 and 9.0. In a more preferred embodiment, contains both solvent and detergent treatment and nanofiltra 30 tion steps for the inactivation and removal of viral particles. In the pH of the buffer system is at or about between 7.5 and 8.5. one preferred embodiment, the manufacturing process com In a most preferred embodiment, the pH of the buffer system prises subjecting the Precipitate 3 supernatant to S/D treat will be at or about 8.0. ment and the heparin eluate to nanofiltration. 10. Virus Inactivation and Removal In another preferred embodiment, the manufacturing pro In certain embodiments, the methods provided herein for 35 cess comprises Subjecting the Precipitate 4 Suspension or a the preparation of an enriched Factor H composition will clarified filtrate thereof to S/D treatment and the heparin further include at least one, preferably at least two, most eluate to nanofiltration. In another preferred embodiment, the preferably at least three, viral inactivation or removal steps. manufacturing process further comprises a viral inactivation Non-limiting examples of viral inactivation or removal steps step comprising incubating a final bulk Factor H composition that may be employed with the methods provided herein 40 at low pH for an extended period of time. include, solvent detergent treatment (Horowitz et al., Blood a) Solvent and Detergent (S/D) Treatment Coagul Fibrinolysis 1994 (5 Suppl3): S21-S28 and Kreil et In order to inactivate various viral contaminants which al., Transfusion 2003 (43):1023-1028, the disclosures of may be present in plasma-derived products, one or more which are expressly incorporated by reference herein in their Factor Hintermediate solutions may be subjected to a solvent entireties for all purposes), nanofiltration (Hamamoto et al., 45 detergent (S/D) treatment. Methods for the detergent treat Vox Sang 1989 (56)230-236 and Yuasa et al., J Gen Virol. ment of plasma-derived fractions are well known in the art 1991 (72 (pt 8)):2021-2024, the disclosures of which are (for review see, Pelletier J Pet al., Best Pract Res Clin Hae expressly incorporated by reference herein in their entireties matol. 2006; 19(1):205-42, the disclosure of which is for all purposes), and low pH incubation at high temperatures expressly incorporated by reference herein in its entirety for (Kempfet al., Transfusion 1991 (31)423-427 and Louie et al., 50 all purposes). Generally, any standard S/D treatment may be Biologicals 1994 (22):13-19, the disclosure of which is used in conjunction with the methods provided herein. For expressly incorporated by reference herein in its entirety for example, an exemplary protocol for an S/D treatment is pro all purposes). vided below. Viral inactivation or removal steps may be performed on In one embodiment, Triton X-100. Tween-20, and tri(n- any intermediate Factor H fractions generated during the 55 butyl)phosphate (TNBP) are added to a Factor Hintermediate manufacturing process. For example, in one embodiment, a solution at final concentrations of at or about 1.0%, 0.3%, and viral inactivation or removal step may be performed on a 0.3%, respectively. The mixture is then stirred at a tempera Fraction I Supernatant, Fraction II+III Suspension, Fraction ture at or about between 18°C. and 25°C. for at least about an II+III filter cake extract, Supernatant 3, Precipitate 4 suspen hour. Sion, anion exchange eluate, heparin affinity eluate, and the 60 In one embodiment, the S/D reagents (e.g., Triton X-100, like. Tween-20, and TNBP) are added by spraying rather than by In one embodiment, a viral inactivation or removal step is fluent addition. In other embodiments, the detergent reagents performed on the Fraction II+III filter cake extract. In a pre may be added as solids to the Factor Hintermediate solution, ferred embodiment the Fraction II+III filter cake extract is which is being mixed to ensure rapid distribution of the S/D subjected to solvent and detergent (S/D) treatment. 65 components. In certain embodiments, it is preferable to add In a second embodiment, a viral inactivation or removal Solid reagents by sprinkling the Solids over a delocalized step is performed on the Precipitation 3 supernatant (Super surface area of the filtrate such that local overconcentration US 9,109,044 B2 35 36 does not occur, such as influent addition. In another embodi than at or about 5.5 prior to incubation. In a more preferred ment, the Factor H containing solution is pumped into a tank embodiment, the pH of the solution is lowered to less than at where the SD-reagents are already present either in concen or about 5.0 prior to incubation. In certain embodiments, the trated or diluted form. pH of the solution is lowered to less thanator about 6.0 or less b) Nanofiltration and Ultra/Diafiltration than at or about 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, In order to further reduce the viral load of the Factor H 4.9, 4.8, 4.7, 4.6, 4.5, 4.4.4.3, 4.2, 4.1, 4.0, or lower prior to composition provided herein, a Factor H fraction, for incubation. example the heparin affinity eluate, may be nanofiltered using In certain embodiments, the solution is then incubated for a suitable nanofiltration device. In certain embodiments, the at least at or about one week, or at least at or about 2, 3, 4, or nanofiltration device will have a mean pore size of at or about 10 more weeks, or for at least at or about 1, 2, 3, or more months. between 15 nm and 200 nm. Examples of nanofilters suitable In preferred embodiments, the composition is incubated at a for this use include, without limitation, DVD, DV 50, DV 20 temperature at or about above 20°C., orator about above 25° (Pall), Viresolve NFP, Viresolve NFR (Millipore), Planova C., or at or about above 30°C. In particular embodiments, the 15N, 2ON, 35N, and 75N (Planova). In a specific embodi composition is incubated at a temperature of at or about 20° ment, the nanofilter may have a mean pore size of at or about 15 C., orator about 21°C., 22°C., 23°C., 24°C., 25°C., 26°C., between 15 and 72 nm, orator about between 19 and 35 nm, 27 C., 280 C., 29° C., 30° C., 31° C., 32° C., 33° C., 34°C., or of at or about 15 nm, 19 nm, 35 nm, or 72 nm. In a preferred 35° C., 36° C., 37° C., 38°C., 39° C., 40° C., or higher. embodiment, the nanofilter will have a mean pore size of at or d) Lyophilization and Heat Treatment about 35 nm, such as an Asahi PLANOVA 35N filter or In yet other embodiments, the present invention provided equivalent thereof. lyophilized Factor H compositions. The viral activity of these Optionally, ultrafiltration/diafiltration may performed to lyophilized composition, which may have previously been further concentrate the nanofiltrate. In one embodiment, an Subjected to other viral inactivation or removal steps such as open channel membrane is used with a specifically designed S/D treatment or nanofiltration, may be further reduced by post-wash and formulation near the end the production pro heat treatment of the lyophilized composition (i.e., Factor H cess resulting in a Factor H composition of high concentra 25 lyo cake). Heat treatments for the inactivation of viral loads in tion. blood factors are well known in the art (for example, see, Subsequent to nanofiltration, the filtrate may be further Piszkiewicz et al., Thromb Res. 1987 Jul. 15:47(2):235-41: concentrated by ultrafiltration and/or the buffer composition Piszkiewicz et al., Curr Stud Hematol Blood Transfits. 1989; adjusted by diafiltration. In one embodiment, the nanofiltrate (56):44-54; Epstein and Fricke, Arch Pathol Lab Med. 1990 may be concentrated by ultrafiltration to a protein concentra 30 March; 114(3):335-40). tion of at or about between 0.5% and 10% (w/v). In certain 11. Formulation embodiments, the ultrafiltration is carried out in a cassette Upon completion of the Factor Henrichment process, e.g., with an open channel Screen and the ultrafiltration membrane after a final diafiltration step, the protein concentration of the has a nominal molecular weight cut off (NMWCO) of less solution is adjusted to with a buffer, e.g., the diafiltration thanator about 150kDa or less thanator about 140, 130, 120, 35 buffer, to a final concentration of at or about between 0.5% 100, 90, 80, 70, 60, 50, 40, 30, or fewer kDa. In a preferred and 20% (w/v), or at or about between 1% and 25% (w/v), or embodiment, the ultrafiltration membrane has a NMWCO of at or about between 2% and 20% (w/v), orator about between no more than 50 kDa. 3% and 15% (w/v), orator about between 5% and 10% (w/v), Upon completion of the ultrafiltration step, buffer or at or about between 9% and 12%, or at or about between exchange may be performed by diafiltration against a solution 40 3% and 7% (w/v), orator about between 8% and 14% (w/v), Suitable for intravenous, intramuscular, intraocular, Subcuta orator about between 4% and 6%, or to a final concentration neous, or other appropriate administration. In certain embodi of at or about 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, ments, the diafiltration solution may comprise a stabilizing 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, and/or buffering agent, for example, salts, Sugars, and/or a 18%, 19%, 20%, or higher. non-ionic detergent (e.g., PolySorbate 80). 45 In certain embodiments, the formulated bulk solution may Typically, the minimum exchange Volume is at least at or be further sterilized by filtering through a membrane filter about 3 times the original concentrate Volume or at least at or with an absolute pore size of no more than about 0.22 micron, about 4, 5, 6, 7, 8, 9, or more times the original concentrate for example about 0.1 or 0.2 micron. In certain embodiments, volume. The Factor H solution may be concentrated to a final the solution may be aseptically dispensed into final containers protein concentration of at or about between 0.5% and 25% 50 for proper sealing, with samples taken for testing. (w/v), orator about between 1% and 25% (w/v), orator about B. Alcohol Addition between 2% and 20% (w/v), or at or about between 3% and Advantageously, it has been found that, for purposes of 15% (w/v), orator about between 5% and 10% (w/v), orator fractionating blood products (e.g., Factor H. IgG, Albumin) about between 9% and 12%, or at or about between 3% and from plasma, addition of alcohol by a method that finely 7% (w/v), or at or about between 8% and 14% (w/v), or at or 55 disperses or that rapidly disperses the alcohol at the point of about between 4% and 6%, or to a final concentration of at or addition results in reduced loss of blood product yields. With about 0.1%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 6%, 7%, out being bound by theory, during fluent addition to a plasma 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, fraction, transient local overconcentration of alcohol at the 19%. 20%, 21%. 22%, 23%, 24%, 25% or higher. fluid ingress may lead to protein denaturation and irreversible c) Incubation at Low pH 60 loss and/or precipitation of a blood factor during steps in In certain embodiments, a Factor H containing Solution which the blood factor should remain in the supernatant. may be treated to reduce or inactivate the viral load of the Furthermore, these effects may by amplified when large Vol composition. In one embodiment, this is achieved by adjust umes of alcohol need to be added. Such as in industrial scale ing the pH of the of the composition to low pH, for example, purifications involving the fractionation of at least 100 L of less than at or about 6.0, and incubating for at least about a 65 pooled plasma. week prior to releasing the composition. In a preferred In one embodiment, alcohol is added in one or more pre embodiment, the pH of the bulk solution is adjusted to less cipitation steps by a method that finely disperses the alcohol US 9,109,044 B2 37 38 over a delocalized area. For example, alcohol can be added to more plasma fractionation steps by a method that finely dis a fractionation step by spraying onto the Surface of the vessel perses or that rapidly disperses the alcohol at the point of or tank containing the plasma fraction. Accordingly, in one addition. aspect of the methods provided herein, one or more precipi In one embodiment, a pH modifying agent is added in one tation steps are performed by the spray addition of alcohol. In or more steps by a method that finely disperses the pH modi certain embodiments, spray addition may be performed by fying agent over a delocalized area. For example, the pH using any pressurized device. Such as a container (e.g., a spray modifying agent can be added to a step by spraying onto the bottle), that has a spray head or a nozzle and is operated Surface of the plasma fraction contained within a vessel or manually or automatically to generate a fine mist from a tank. In another embodiment of the methods provided herein, liquid. In certain embodiments, spray addition is performed 10 the pH of a plasma fraction or precipitation step may be while the system is continuously stirred or otherwise mixed to adjusted by spray addition of a pH modifying agent. In certain ensure rapid and equal distribution of the liquid within the embodiments, spray addition may be performed by using any system. pressurized device, such as a container (e.g., a spray bottle), In another embodiment, alcohol is added in one or more that has a spray head or a nozzle and is operated manually or precipitation steps by a method that rapidly disperses the 15 automatically to generate a fine mist from a liquid. In certain alcohol at the point of addition. For example, alcohol can be embodiments, spray addition is performed while the system is added from below the vessel or tank containing the plasma continuously stirred or otherwise mixed to ensure rapid and fraction, directly adjacent to a stirring apparatus (e.g., a pro equal distribution of the liquid within the system. peller). In certain embodiments, fluent addition at a ingress In another embodiment, a pH modifying agent is added in directly adjacent to a stirring apparatus is performed while the one or more steps by a method that rapidly disperses the pH system is continuously stirred or otherwise mixed to ensure modifying agent at the point of addition. For example, a pH rapid and equal distribution of the liquid within the system. modifying agent can be added from below the vessel or tank C. Adjustment of the pH containing the plasma fraction, directly adjacent to a stirring The protein precipitation profiles of plasma fractions is apparatus (e.g., a propeller). In certain embodiments, fluent highly dependent upon the pH of the solution from which the 25 additionata ingress directly adjacent to a stirring apparatus is plasma proteins are being precipitated. This fact has been performed while the system is continuously stirred or other exploited by Scientists fractionating plasma proteins since the wise mixed to ensure rapid and equal distribution of the liquid introduction of the Cohn and Oncley methods in 1946 and within the system. 1949, respectively. Traditionally, the pH of a plasma fraction In yet another embodiment, a pH modifying agent is added is adjusted prior to alcohol addition to facilitate the highest 30 in one or more steps by sprinkling a solid pH modifying agent recovery yields for the component(s) of interest. Advanta over a delocalized area on the Surface of the plasma fraction. geously, it has now been found that by adjusting the pH of the In certain embodiments, addition by this means is performed solution directly after addition of alcohol or concomitant with while the system is continuously stirred or otherwise mixed to alcohol addition results in a more defined and reproducible ensure rapid and equal distribution of the pH modifying agent precipitation. It was found that ethanol addition to plasma 35 within the system. fractions results in fluctuations in the pH of the solution, generally by raising the pH of the Solution. As such, by IV. Factor H Compositions adjusting the pH of a plasma fraction to a predetermined pH before but not after alcohol addition, the precipitation reac Factor H compositions have been described for the treat tion will occur at a non-optimal pH. 40 ment of certain complement related disorders. (See, for Likewise, precipitation of proteins from a plasma fraction example, U.S. Patent Publication No. US 2009/0118163 and will effect the electrostatic environment and will thus alter the European Patent Application No. EPO 222 611 A2, the dis pH of the solution. Accordingly, as a precipitation event is closures of which are hereinincorporated by reference in their allowed to progress, the pH of the solution will begin to entireties for all purposes.) diverge from the predetermined pH value that allows for 45 In one aspect, the present invention provides Factor H maximal recovery of the protein species of interest. This is compositions prepared according to a method described especially true for precipitation events in which a large frac herein. In one embodiment, Factor H is prepared from mate tion of the protein is being precipitated, precipitation events in rials otherwise discarded during the manufacture of a com which a high alcohol content is used, and precipitation events mercial plasma-derived blood product, for example IgG or that require a long incubation period. 50 Albumin. In one embodiment, a Factor H composition is Accordingly, in one aspect of the methods provided herein, provided, wherein Factor H is extracted from a Fraction I the pH of a plasma fraction is adjusted directly after the precipitate and/or a Fraction II+III filter cake. addition of alcohol. In related embodiments, the pH may be Factor Has described in this invention can be formulated adjusted before and after alcohol addition, or during and after into pharmaceutical preparations for therapeutic use. The alcohol addition, or before, during, and after alcohol addition. 55 purified protein may be dissolved in conventional physiologi In a related embodiment, the pH of a solution is continuously cally compatible aqueous buffer solutions to which there may adjusted during one or more alcohol precipitation events or be added, optionally, pharmaceutical excipients to provide incubations. In certain embodiments, the pH of a solution is pharmaceutical preparations. continuously adjusted or maintained while the system is con Such pharmaceutical carriers and excipients as well as tinuously stirred or otherwise mixed to ensure rapid and equal 60 suitable pharmaceutical formulations are well known in the distribution of the pH modifying agent within the system. art (see for example “Pharmaceutical Formulation Develop Similar to the case of fluent alcohol addition, it has now ment of Peptides and Proteins’. Frokjaer et al., Taylor & been found that the fluent addition of large volumes of a pH Francis (2000) or “Handbook of Pharmaceutical Excipients', modifying agent may cause transient local pH variations, 3" edition, Kibbe et al., Pharmaceutical Press (2000)). In resulting in unwanted protein denaturation or precipitation. 65 particular, the pharmaceutical composition comprising the Accordingly, in one embodiment of the methods provided polypeptide variant of the invention may be formulated in herein, pH modifying agents may be introduced into one or lyophilized or stable soluble form. The polypeptide variant US 9,109,044 B2 39 40 may be lyophilized by a variety of procedures known in the provided that is prepared by a method comprising the steps of art. Lyophilized formulations are reconstituted prior to use by (i) extracting Factor H from a Fraction II+III filter cake, (ii) the addition of one or more pharmaceutically acceptable dilu optionally performing a first precipitation step to precipitate ents such as sterile water for injection or sterile physiological at least one impurity from the Factor H composition, (iii) saline Solution. optionally performing a second precipitation step to precipi Formulations of the composition are delivered to the indi tate Factor H from the composition, (iv) optionally perform vidual by any pharmaceutically suitable means of adminis ing at least one ion exchange chromatography step, (v) tration. Various delivery systems are known and can be used optionally performing at least one heparin affinity chroma to administer the composition by any convenient route. Pref tography step; and (vi) optionally performing at least one erentially the compositions of the invention are administered 10 viral inactivation or removal step, thereby preparing an aque systemically. For systemic use, Factor H of the invention is ous Factor H composition. formulated for parenteral (e.g. intravenous, Subcutaneous, In a preferred embodiment, a Factor H composition is intramuscular, intraperitoneal, intracerebral, intrapulmonar, provided that is prepared by a method comprising the steps of intranasal, intravitreal, or transdermal) or enteral (e.g., oral, (a) precipitating proteins from a cryo-poor plasma fraction, in vaginal or rectal) delivery according to conventional meth 15 a first precipitation step, with between about 6% and about ods. The formulations can be administered continuously by 10% alcohol at a pH of between about 7.0 and about 7.5 to infusion or by bolus injection. Some formulations encompass obtain a first precipitate and a first Supernatant; (b) precipi slow release systems. Preferred routes of administration will tating Factor H from the first Supernatant, in a second precipi depend upon the indication being treated, managed, or pre tation step, with between about 20% and about 25% alcohol at vented. For example, in one embodiment, wherein Factor His a pH of between about 6.7 and about 7.3 to form a second administered for the treatment of AMD, the preferred route of precipitate; (c) re-suspending the second precipitate to form a administration will be intravitreal. In a second embodiment, Suspension; (d) mixing finely divided silicon dioxide (SiO2) wherein Factor H is being administered for the treatment or with the Suspension from step (c); (e) filtering the Suspension management of aHUS, the preferred route of administration with a filter press, thereby forming a filter cake and a Super will be intravenous. A skilled physician will readily be able to 25 natant; and (f) extracting Factor H from the filter cake with a determine the preferred route of administration for the par Factor H extraction buffer, thereby preparing an aqueous ticular affliction being treated, managed, or prevented. composition of Factor H. In another aspect, the present invention provides a Substan In certain embodiments, Factor H is extracted from the tially purified Factor H preparation prepared according to a filter cake by re-circulation of an extraction buffer through a method provided herein. In one embodiment, the method 30 filterpress containing the filter cake. Generally, the extraction comprises (a) performing Cohn fractionation to obtaina Frac buffer will be re-circulated through the filter cake for at or tion II+III precipitate and preparing a suspension of the com about between 5 minutes and 2 hours. In a preferred embodi bined precipitates, (b) filtering the re-suspended Fraction ment, the extraction buffer will be re-circulated through the II+III precipitate to obtain a filter cake discard left behind filter cake for at or about between 10 and 60 minutes. In after filtration of the Fraction II+III suspension; (c) extracting 35 another preferred embodiment, the extraction buffer will be the filter cake according to a method comprising the steps of re-circulated through the filter cake forator about between 20 (i) dissolving the filter cake in a suitable buffer of appropriate and 40 minutes. In another preferred embodiment, the extrac ionic strength for a time sufficient to dissolve the protein tion buffer will be re-circulated through the filter cake forator caked on said filter cake; (ii) diluting said dissolved protein about 30 minutes. In other embodiments, the extraction buffer with additional buffer, (iii) removing debris from the diluted 40 will be re-circulated through the filter cake for at least at or protein, (iv) purifying Factor H protein from said diluted about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, protein preparation by Subjecting the diluted protein to ultra 80, 85,90, 95, 100, 105, 110, 115, 120, or more minutes. filtration on a 0.45um filter to produce a filtrate containing In another embodiment, aqueous compositions of Factor H Factor H; (v) subjecting the filtrate containing Factor H to are provided that are prepared from a Fraction I precipitate. In anion exchange chromatography using a NaCl gradient (50 45 one embodiment, an aqueous composition of Factor H is 500 mM) in the running buffer to produce a pooled crude provided that is prepared by a method comprising the steps of Factor H fraction; and (vi) purifying Factor H from said crude (i) extracting Factor H from a Fraction I precipitate, (ii) Factor H preparation by Heparin SepharoseTM chromatogra optionally performing a first precipitation step to precipitate phy using a gradient of NaCl (50-500 mM). at least one impurity from the Factor H composition, (iii) In one aspect of the substantially purified Factor H prepa 50 optionally performing a second precipitation step to precipi ration, the Factor H preparation is an active Factor H prepa tate Factor H from the composition, (iv) optionally perform ration that is composed of a mixture of Tyr-402 and His-402 ing at least one ion exchange chromatography step, (v) isoforms in the range 50%+20% His-402: 50%+20% Tyr optionally performing at least one heparin affinity chroma 402. tography step; and (vi) performing at least one viral inactiva A. Aqueous Compositions 55 tion or removal step, thereby preparing an aqueous Factor H In one aspect, the present invention provides aqueous com composition. positions of plasma-derived Factor Hprepared from materials In a preferred embodiment, aqueous compositions of Fac otherwise discarded during the preparation of other commer tor Hare provided that are prepared from a Fraction I precipi cially important blood products by plasma fractionation. tate. In a particularly preferred embodiment, a Factor H com Aqueous Factor H compositions prepared by the methods 60 position is provided that is prepared by a method comprising provided herein will have high Factor H content and purity. the steps of: (a) precipitating proteins from a cryo-poor For example, Factor compositions provided herein may have plasma fraction, in a first precipitation step, with between a protein concentration of at least at or about 3% (w/v) and a about 6% and about 10% alcohol at a pH of between about 7.0 Factor H content of greater than at or about 90% purity. and about 7.5 to obtain a first precipitate and a first superna In one embodiment, aqueous compositions of Factor Hare 65 tant; and (b) extracting Factor H from the precipitate with a provided that are prepared from a Fraction II+III filter cake. In Factor H extraction buffer, thereby preparing an aqueous one embodiment, an aqueous composition of Factor H is composition of Factor H. US 9,109,044 B2 41 42 In certain embodiments, Factor H is extracted from the In one embodiment, the present invention provides aque Fraction I precipitate by re-circulation of an extraction buffer ous Factor H compositions comprising a protein concentra through a filter press containing the Fraction I precipitate. tion of at or about between 10 g/L and 250 g/L. In certain Generally, the extraction buffer will be re-circulated through embodiments, the protein concentration of the Factor H com the precipitate for at or about between 5 minutes and 2 hours. position is at or about between 50 g/L and 200 g/L, or at or In a preferred embodiment, the extraction buffer will be re about between 70 g/L and 150 g/L, or at or about between 90 circulated through the precipitate for at or about between 10 g/L and 120 g/L, or at or about between 30 g/L and 70 g/L, or and 60 minutes. In another preferred embodiment, the extrac at or about between 40 g/L and 60 g/L or any suitable con tion buffer will be re-circulated through the precipitate for at centration within these ranges, for example at or about 10 g/L. or about between 20 and 40 minutes. In another preferred 10 or at or about 15 g/L, 20 g/L, 25g/L, 30 g/L, 35 g/L, 40 g/L, embodiment, the extraction buffer will be re-circulated 45g/L, 50 g/L, 55 g/L, 60 g/L, 65g/L, 70 g/L, 75 g/L, 80 g/L, through the precipitate for at or about 30 minutes. In other 85 g/L. 90 g/L. 95 g/L. 100 g/L. 105 g/L, 110 g/L, 115 g/L, embodiments, the extraction buffer will be re-circulated 120 g/L, 125 g/L, 130g/L, 13.5g/L, 140g/L, 145g/L, 150 g/L, through the precipitate for at least at or about 5, 10, 15, 20, 25, 155 g/L, 160 g/L, 165g/L, 170 g/L, 175g/L, 180g/L, 185g/L, 30,35, 40, 45, 50,55, 60, 65,70, 75, 80, 85,90, 95, 100,105, 15 190 g/L, 195g/L, 200g/L,205g/L, 210 g/L, 215 g/L, 220 g/L, 110, 115, 120, or more minutes. 225 g/L, 230 g/L, 235 g/L, 240 g/L, 245 g/L. 250 g/L, or In certain embodiments, Factor H is extracted from the higher. In a preferred embodiment, Factor H compositions Fraction I precipitate or Fraction II+III filter cake by the having high protein concentrations will also high levels of addition of a Factor Hextraction buffer, which can be used to purity. In one embodiment, at least 90% of the protein in the re-Suspend the Fraction I precipitate at a ratio of 1 part pre composition will be Factor H. In a preferred embodiment, at cipitate to at or about between 25 and 30 parts of extraction least 95% of the protein in the composition will be Factor H. buffer. In other embodiments, the re-suspension ratio is at or The methods provided herein allow for the preparation of about between 1:4 and 1:40, or at or about between 1:8 and Factor H compositions having very high levels of purity. In 1:30, or at or about between 1:10 and 1:20, or at or about one embodiment, at least 90% of the total protein in a com between 1:20 and 1:30, orator about between 1:12 and 1:18, 25 position provided herein will be Factor H. In a preferred or at or about between 1:13 and 1:17, or at or about between embodiment, at least 95% of the total protein in a composition 1:14 and 1:16. In one embodiment, the re-suspension ratio is provided herein will be Factor H. In other embodiments, at at or about 1:25. In another embodiment, the re-suspension least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, ratio is at or about 1:30. In certain embodiments, the ratio may 99%, 99.5%, or more of the total protein of the composition beator about 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 30 will be Factor H. In one preferred embodiment, at least 96% 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, of the total protein of the composition will be Factor H. In a 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, preferred embodiment, at least 97% of the total protein of the 1:36, 1:37, 1:38, 1:39, 1:40, or higher. In a preferred embodi composition will be Factor H. In another preferred embodi ment, Factor His extracted by re-circulation of the extraction ment, at least 98% of the total protein of the composition will buffer through a filter or filter press containing the Fraction I 35 be Factor H. In another preferred embodiment, at least 99% of precipitate or Fraction II+III filter cake. the total protein of the composition will be Factor H. In certain embodiments, an aqueous composition of Factor B. Pharmaceutical Compositions His provided, wherein the Factor H composition is prepared In one aspect, the present invention provides pharmaceu using a purification method described herein, wherein the tical compositions of plasma-derived Factor H prepared from method comprises the addition of one or more solutions, that 40 materials otherwise discarded during the preparation of other would otherwise be introduced into a plasma fraction by commercially important blood products by plasma fraction fluent addition, by a method that finely disperses or that ation. Pharmaceutical Factor H compositions prepared by the rapidly disperses the solution at the point of addition. For methods provided herein will have high Factor H content and example, in certain embodiments the method will comprise purity. For example, Factor H compositions provided herein the introduction of alcohol (e.g., ethanol) into a plasma frac 45 may have a protein concentration of at least at or about 1% tion by spraying. In other embodiments, solutions that may be (w/v) and a Factor H content of greater than at or about 90% added to a plasma fraction by spraying include, without limi purity. These high purity Factor H pharmaceutical composi tation, a pH modifying Solution, a solvent solution, a deter tions and formulations are Suitable for therapeutic adminis gent Solution, a dilution buffer, a conductivity modifying tration. In certain embodiments, the pharmaceutical compo Solution, and the like. In one embodiment, one or more alco 50 sitions provided herein will consist of aqueous formulations hol precipitation steps is performed by the addition of alcohol of Factor H formulated for therapeutic administration. In to a plasma fraction by spraying. In a second preferred other embodiments, the pharmaceutical compositions pro embodiment, one or more pH adjustment steps is performed vided herein will consist of lyophilized formulations of Fac by the addition of a pH modifying solution to a plasma frac tor H formulated for therapeutic administration after recon tion by spraying. 55 stitution with water for injection (WFI) or a biologically In certain embodiments, an aqueous Factor H composition compatible liquid, e.g., a saline or buffered solution. is provided that is prepared by a purification method In one embodiment, the pharmaceutical compositions pro described herein, wherein the method comprises adjusting vided hereinare prepared by formulating an aqueous Factor H the pH of a plasma fraction being precipitated after and/or composition isolated using a method provided herein. Gen concomitant with the addition of the precipitating agent (e.g., 60 erally, the manufacturing pathway to the formulated compo alcohol or polyethylene glycol). In some embodiments, a sition will include at least one, preferably at least two, most process improvement is provided in which the pH of a plasma preferably at least three, viral inactivation or removal steps. fraction being actively precipitated is maintained throughout Non-limiting examples of viral inactivation or removal steps the entire precipitation incubation or hold step by continuous that may be employed with the methods provided herein monitoring and adjustment of the pH. In preferred embodi 65 include, solvent detergent treatment (Horowitz et al., Blood ments the adjustment of the pH is performed by the spray Coagul Fibrinolysis 1994 (5 Suppl3): S21-S28 and Kreil et addition of a pH modifying Solution. al., Transfusion 2003 (43):1023-1028, the disclosures of US 9,109,044 B2 43 44 which are incorporated by reference herein in their entireties (v) optionally performing at least one heparin affinity chro for all purposes), nanofiltration (Hamamoto et al., Vox Sang matography step; (vi) performing at least one viral inactiva 1989 (56)230-236 and Yuasa et al., J Gen Virol. 1991 (72 (pt tion or removal step; and (vii) optionally lyophilizing the 8)):2021-2024, the disclosures of which are incorporated by Factor H composition, thereby preparing a pharmaceutical reference herein in their entireties for all purposes), and low Factor H composition. pH incubation at high temperatures (Kempfet al., Transfit In a preferred embodiment, pharmaceutical compositions Sion 1991 (31)423-427 and Louie et al., Biologicals 1994 of Factor H are provided that are prepared from a Fraction I (22): 13-19, the disclosure of which is incorporated herein by precipitate. In a particularly preferred embodiment, a Factor reference in its entirety for all purposes). H composition is provided that is prepared by a method In one embodiment, pharmaceutical compositions of Fac 10 comprising the steps of: (a) precipitating proteins from a tor H are provided that are prepared from a Fraction II+III cryo-poor plasma fraction, in a first precipitation step, with filter cake. In one embodiment, an aqueous composition of between about 6% and about 10% alcohol at a pH of between Factor H is provided that is prepared by a method comprising about 7.0 and about 7.5 to obtain a first precipitate and a first the steps of: (i) extracting Factor H from a Fraction II+III filter Supernatant; and (b) extracting Factor H from the precipitate cake, (ii) optionally performing a first precipitation step to 15 with a Factor H extraction buffer; and (c) performing at least precipitate at least one impurity from the Factor H composi one viral inactivation or removal step, thereby preparing a tion, (iii) optionally performing a second precipitation step to pharmaceutical composition of Factor H. precipitate Factor H from the composition, (iv) optionally In certain embodiments, Factor H is extracted from the performing at least one ion exchange chromatography step, Fraction I precipitate by re-circulation of an extraction buffer (v) optionally performing at least one heparin affinity chro through a filter press containing the Fraction I precipitate. matography step; (vi) performing at least one viral inactiva Generally, the extraction buffer will be re-circulated through tion or removal step; and (vii) optionally lyophilizing the the precipitate for at or about between 5 minutes and 2 hours. Factor H composition, thereby preparing a pharmaceutical In a preferred embodiment, the extraction buffer will be re Factor H composition. circulated through the precipitate for at or about between 10 In a preferred embodiment, a pharmaceutical Factor H 25 and 60 minutes. In another preferred embodiment, the extrac composition is provided that is prepared by a method com tion buffer will be re-circulated through the precipitate for at prising the steps of: (a) precipitating proteins from a cryo or about between 20 and 40 minutes. In another preferred poor plasma fraction, in a first precipitation step, with embodiment, the extraction buffer will be re-circulated between about 6% and about 10% alcohol at a pH of between through the precipitate for at or about 30 minutes. In other about 7.0 and about 7.5 to obtain a first precipitate and a first 30 embodiments, the extraction buffer will be re-circulated Supernatant; (b) precipitating Factor H from the first Super through the precipitate for at least at or about 5, 10, 15, 20, 25, natant, in a second precipitation step, with between about 30, 35, 40, 45, 50,55, 60, 65,70, 75,80, 85,90, 95, 100,105, 20% and about 25% alcohol at a pH of between about 6.7 and 110, 115, 120, or more minutes. about 7.3 to form a second precipitate; (c) re-suspending the In certain embodiments, Factor His extracted from a Frac second precipitate to form a suspension; (d) mixing finely 35 tion I precipitate or Fraction II+III filter cake by the addition divided silicon dioxide (SiO2) with the suspension from step of a Factor H extraction buffer, which can be used to re (c); (e) filtering the suspension with a filter press, thereby suspend the Fraction I precipitate or Fraction II+III filter cake forming a filter cake and a Supernatant, (f) extracting Factor H at a typical ratio of 1 part precipitate to at or about between 25 from the filter cake with a Factor H extraction buffer, (g) and 30 parts of extraction buffer. In other embodiments, the performing at least one viral inactivation or removal step; and 40 re-suspension ratio is at or about between 1:4 and 1:40, or at (h) optionally lyophilizing the composition, thereby prepar or about between 1:8 and 1:30, or at or about between 1:10 ing an aqueous composition of Factor H. and 1:20, orator about between 1:12 and 1:18, orator about In certain embodiments, Factor H is extracted from the between 1:13 and 1:17, orator about between 1:14 and 1:16. filter cake by re-circulation of an extraction buffer through a In one embodiment, the re-suspension ratio is at or about filterpress containing the filter cake. Generally, the extraction 45 1:25. In another embodiment, the re-suspension ratio is at or buffer will be re-circulated through the filter cake for at or about 1:30. In certain embodiments, the ratio may be at or about between 5 minutes and 2 hours. In a preferred embodi about 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, ment, the extraction buffer will be re-circulated through the 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, filter cake for at or about between 10 and 60 minutes. In a 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, more preferred embodiment, the extraction buffer will be 50 1:37, 1:38, 1:39, 1:40, or higher. In a preferred embodiment, re-circulated through the filter cake for at or about between 20 Factor His extracted by re-circulation of the extraction buffer and 40 minutes. In another preferred embodiment, the extrac through a filter or filter press containing the Fraction I pre tion buffer will be re-circulated through the filter cake forator cipitate or Fraction II+III filter cake. about 30 minutes. In other embodiments, the extraction buffer In certain embodiments, a pharmaceutical composition of will be re-circulated through the filter cake for at least at or 55 Factor H is provided, wherein the Factor H composition is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, prepared using a purification method described herein, 80, 85,90, 95, 100, 105, 110, 115, 120, or more minutes. wherein the method comprises the addition of one or more In another embodiment, pharmaceutical compositions of solutions that would otherwise be introduced into a plasma Factor H are provided that are prepared from a Fraction I fraction by fluent addition, by a method that finely disperses precipitate. In one embodiment, an aqueous composition of 60 or that rapidly disperses the solution at the point of addition. Factor H is provided that is prepared by a method comprising For example, in certain embodiments the method will com the steps of: (i) extracting Factor H from a Fraction I precipi prise the introduction of alcohol (e.g., ethanol) into a plasma tate, (ii) optionally performing a first precipitation step to fraction by spraying. In other embodiments, solutions that precipitate at least one impurity from the Factor H composi may be added to a plasma fraction by spraying include, with tion, (iii) optionally performing a second precipitation step to 65 out limitation, a pH modifying solution, a solvent solution, a precipitate Factor H from the composition, (iv) optionally detergent solution, a dilution buffer, a conductivity modifying performing at least one ion exchange chromatography step, Solution, and the like. In a preferred embodiment, one or more US 9,109,044 B2 45 46 alcohol precipitation steps is performed by the addition of tion by subjecting the diluted protein to ultrafiltration on a alcohol to a plasma fraction by spraying. In a second pre 0.45 um filter to produce a filtrate containing Factor H; (v) ferred embodiment, one or more pH adjustment steps is per Subjecting the filtrate containing Factor H to anion exchange formed by the addition of a pH modifying Solution to a plasma chromatography using a NaCl gradient (50-500 mM) in the fraction by spraying. 5 running buffer to produce a pooled crude Factor H fraction; In certain embodiments, an aqueous Factor H composition and (vi) purifying Factor H from said crude Factor H prepa is provided that is prepared by a purification method ration by Heparin Sepharose M chromatography using a gra described herein, wherein the method comprises adjusting dient of NaCl (50-500 mM). the pH of a plasma fraction being precipitated after and/or The pharmaceutical compositions provided herein will concomitant with the addition of the precipitating agent (e.g., 10 typically comprise one or more buffering agents or pH stabi alcohol or polyethylene glycol). In some embodiments, a lizing agents suitable for intravenous, intravitreal, Subcutane process improvement is provided in which the pH of a plasma ous, and/or intramuscular administration. Non-limiting fraction being actively precipitated is maintained throughout examples of buffering agents suitable for formulating a Fac the entire precipitation incubation or hold step by continuous tor H composition provided herein include glycine, histidine, monitoring and adjustment of the pH. In preferred embodi 15 or other amino acids, salts like citrate, phosphate, acetate, ments the adjustment of the pH is performed by the spray glutamate, tartrate, benzoate, lactate, gluconate, malate. Suc addition of a pH modifying Solution. cinate, formate, propionate, carbonate, or any combination In one embodiment, the present invention provides phar thereof adjusted to an appropriate pH. Generally, the buffer maceutical Factor H compositions comprising a protein con ing agent will be sufficient to maintain a suitable pH in the centration of between about 10 g/L and about 250 g/L. In formulation for an extended period of time. certain embodiments, the protein concentration of the Factor In some embodiments, the concentration of buffering agent H composition is at or about between 50 g/L and 200 g/L, or in the formulation will beat or about between 5 mM and 500 at or about between 70 g/L and 150 g/L, orator about between mM. In certain embodiments, the concentration of the buff 90 g/L and 120 g/L, or at or about between 30 g/L and 70 g/L, ering agent in the formulation will be at or about 5 mM, 10 or at or about between 40 g/L and 60 g/L or any suitable 25 mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 concentration within these ranges, for example at or about 10 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 g/L, or at or about 15 g/L, 20 g/L, 25g/L, 30 g/L, 35 g/L, 40 mM, 300 mM, 325 mM, 350 mM, 375 mM, 400 mM, 425 g/L, 45g/L, 50 g/L, 55 g/L, 60 g/L, 65g/L, 70 g/L, 75 g/L, 80 mM, 450 mM, 475 mM, 500 mM or higher. g/L, 85g/L, 90 g/L. 95g/L, 100 g/L. 105 g/L, 110 g/L, 115 In certain embodiments, the pH of the formulation will be g/L, 120 g/L, 125 g/L, 130g/L, 135 g/L, 140 g/L, 145g/L, 150 30 at or about between about pH 4.0 and pH 8.0. g/L, 155g/L, 160 g/L, 165g/L, 170 g/L, 175g/L, 180g/L, 185 In some embodiments, the pharmaceutical compositions g/L, 190g/L, 195g/L, 200g/L,205g/L,210 g/L,215 g/L,220 provided herein may optionally further comprise an agent for g/L, 225 g/L, 230 g/L, 235 g/L, 240 g/L, 245 g/L, 250 g/L, or adjusting the osmolarity of the composition. Non-limiting higher. In a preferred embodiment, Factor H compositions examples of osmolarity agents include mannitol, Sorbitol, having high protein concentrations will also high levels of 35 glycerol. Sucrose, glucose, dextrose, levulose, fructose, lac purity. In one embodiment, at least 90% of the protein in the tose, polyethylene glycols, phosphates, Sodium chloride, composition will be Factor H. In a preferred embodiment, at potassium chloride, calcium chloride, calcium gluconoglu least 95% of the protein in the composition will be Factor H. coheptonate, dimethyl Sulfone, and the like. In one embodiment, at least 90% of the total protein in a In some embodiments, the formulations provided herein composition provided herein will be Factor H. In a preferred 40 will have osmolarities that are comparable to physiologic embodiment, at least 95% of the total protein in a composition osmolarity, about 285 to 295 mCosmol/kg (Lacy et al., Drug provided herein will be Factor H. In other embodiments, at Information Handbook Lexi-Comp 1999:1254). In certain least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, embodiments, the osmolarity of the formulation will be at or 99%, 99.5%, or more of the total protein of the composition about between 200 and 350 mOsmol/kg, preferably at or will be Factor H. In one preferred embodiment, at least 96% 45 about between 240 and 300 mOsmol/kg. In particular of the total protein of the composition will be Factor H. In a embodiments, the osmolarity of the formulation will be at or preferred embodiment, at least 97% of the total protein of the about 200 mOsmol/kg, or 210 mCSmol/kg, 220 mOsmol/kg, composition will be Factor H. In another preferred embodi 230 mOsmol/kg, 240 mOsmol/kg, 245 mOsmol/kg, 250 ment, at least 98% of the total protein of the composition will mOsmol/kg, 255 mOsmol/kg, 260 mOsmol/kg, 265 mOs be Factor H. In another preferred embodiment, at least 99% of 50 mol/kg, 270 mOsmol/kg, 275 mOsmol/kg, 280 mOsmol/kg, the total protein of the composition will be Factor H. 285 mCosmol/kg, 290 mOsmol/kg, 295 mCosmol/kg, 300 In one embodiment, the present invention provides a phar mOsmol/kg, 310 mCSmol/kg, 320 mOsmol/kg, 330 mOs maceutical preparation comprising as active ingredient a mol/kg, 340 mOsmol/kg, 340 mOsmol/kg, or 350 mOsmol/ preparation of Factor Has provided herein and a pharmaceu kg. In yet other embodiments, the osmolarity of the formula tically acceptable carrier. In one embodiment, the Factor H 55 tion will be higher, for example at or about between 200 and preparation is prepared by a method comprising (a) perform 1000 mOsmol/kg, or at or about 400 mOsmol/kg, 450 mOs ing Cohn fractionation to obtain Fraction II+III precipitate mol/kg, 500 mOsmol/kg, 550 mOsmol/kg, 600 mOsmol/kg, and preparing a suspension of the combined precipitates, (b) 650 mOsmol/kg, 700 mOsmol/kg, 750 mOsmol/kg, 800 filtering the re-suspended Fraction II+III precipitates to mOsmol/kg, 850 mOsmol/kg, 900 mOsmol/kg, 950 mOs obtain the filter cake discard left behind after filtration of the 60 mol/kg, 1000 mOsmol/kg, or higher. Fraction II+III Suspension; (c) extracting the filter cake The Factor H formulations provided herein are generally according to a method comprising the steps of: (i) dissolving stable in liquid form for an extended period of time. In certain the filter cake in a suitable buffer of appropriate ionic strength embodiments, the formulations are stable for at least at or for a time sufficient to dissolve the protein caked on said filter about 3 months at room temperature, or at least at or about 4. cake; (ii) diluting said dissolved protein with additional 65 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, buffer, (iii) removing debris from the diluted protein, (iv) 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37,38, 39, purifying Factor H protein from said diluted protein prepara 40, 41, 42, 43, 44, 45, 46, 47, 48 months or more at room US 9,109,044 B2 47 48 temperature. The formulation will also generally be stable for In one embodiment, a method is provided for treating a at least at or about 18 months under refrigerated conditions disease or disorder associated with a Factor H dysfunction or (typically between about 2°C. and about 8°C.), or for at least abnormal alternative pathway complement activity in a Sub at or about 21, 24, 27, 30,33, 36,39, 42, 45, 48,51,54, 57, 60 ject in need thereof by administering to the Subject a thera months or more under refrigerated conditions. 5 peutically effective dose of a Factor H composition prepared In other embodiments, the Factor H formulations provided by a method comprising the steps of: (i) extracting Factor H herein are generally stable in lyophilized form for an from a Fraction II+III filter cake, (ii) optionally performing a extended period of time. In certain embodiments, the formu first precipitation step to precipitate at least one impurity from lations are stable for at least at or about 3 months at room the Factor H composition, (iii) optionally performing a sec temperature, or at least at or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 10 ond precipitation step to precipitate Factor H from the com 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, position, (iv) optionally performing at least one ion exchange 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41,42, 43,44, 45,46, chromatography step, (v) optionally performing at least one 47, or 48 months at room temperature. The formulation will heparin affinity chromatography step; (vi) performing at least also generally be stable for at least at or about 18 months one viral inactivation or removal step; and (vii) optionally under refrigerated conditions (typically between about 2°C. 15 lyophilizing the Factor H composition, thereby preparing a and about 8°C.), or for at least at or about 21, 24, 27, 30, 33, composition of Factor H Suitable for use in treating a disease 36,39, 42, 45, 48,51,54, 57, or 60 months under refrigerated or disorder associated with a Factor H dysfunction or abnor conditions. mal alternative pathway complement activity. In one aspect, the present invention is also directed to In a preferred embodiment, a method is provided for treat compositions containing Factor H purified according to the ing a disease or disorder associated with a Factor H dysfunc present invention and a pharmaceutically acceptable carrier tion or abnormal alternative pathway complement activity in for systemic or local ocular administration is administered to a Subject in need thereof by administering to the Subject a a mammal in need thereof. The compositions may further therapeutically effective dose of a Factor H composition pre comprise additional therapeutic compounds that may be use pared by a method comprising the steps of: (a) precipitating ful in the treatment of AMD, such as for example VEGF 25 proteins from a cryo-poor plasma fraction, in a first precipi inhibitors, complement factor Dinhibitor, such as BCX-1470 tation step, with between about 6% and about 10% alcohol at and the like. The therapeutic compositions are formulated in a pH of between about 7.0 and about 7.5 to obtain a first accordance with methods known in the art for the particular precipitate and a first Supernatant; (b) precipitating Factor H route of administration desired. from the first Supernatant, in a second precipitation step, with According to the methods of the present invention, a com 30 between about 20% and about 25% alcohol at a pH of position comprising a Factor H purified according to the between about 6.7 and about 7.3 to form a second precipitate; methods described herein and a pharmaceutically acceptable (c) re-suspending the second precipitate to form a suspension; carrier for systemic or local administration is administered to (d) mixing finely divided silicon dioxide (SiO) with the a mammal in need thereof. Suspension from step (c); (e) filtering the Suspension with a The compositions administered according to the present 35 filter press, thereby forming a filter cake and a Supernatant; invention comprise a pharmaceutically effective amount, or and (f) extracting Factor H from the filter cake with a Factor therapeutically effective amount, of Factor Heither alone or H extraction buffer, (g) performing at least one viral inacti in combination with another therapeutic agent. As used Vation or removal step; and (h) optionally lyophilizing the herein, a “pharmaceutically effective amount’ or “therapeu composition, thereby preparing a composition of Factor H tically effective amount is an amount of active agent that is 40 Suitable for use in treating a disease or disorder associated Sufficient to reduce complement activation and limit the pro with a Factor H dysfunction or abnormal alternative pathway gression of pathology in various settings such as AMD. HUS, complement activity. MPGN and others. In some aspects, the therapeutically effec In another embodiment, a method is provided for treating a tive amount is reduces or prevents AMD and/or loss of visual disease or disorder associated with a Factor H dysfunction or acuity associated with AMD. Generally, for compositions 45 abnormal alternative pathway complement activity in a Sub intended to be administered systemically for the treatment of ject in need thereof by administering to the Subject a thera AMD, the total amount of Factor H will be about 0.01-100 peutically effective dose of a Factor H composition prepared mg/kg. For local administration, the preferred concentration by a method comprising the steps of: (i) extracting Factor H of Factor H in the composition will be from about 0.01% to from a Fraction I precipitate, (ii) optionally performing a first about 10% (w/v). 50 precipitation step to precipitate at least one impurity from the Factor H composition, (iii) optionally performing a second V. Methods of Treatment precipitation step to precipitate Factor H from the composi tion, (iv) optionally performing at least one ion exchange In one aspect, the present invention provides methods for chromatography step, (v) optionally performing at least one treating a disease or disorder associated with a Factor H 55 heparin affinity chromatography step; (vi) performing at least dysfunction or abnormal alternative pathway complement one viral inactivation or removal step; and (vii) optionally activity in a Subject in need thereof by administering a thera lyophilizing the Factor H composition, thereby preparing a peutically effective dose of a Factor H composition prepared composition of Factor H Suitable for use in treating a disease from materials otherwise discarded during the preparation of or disorder associated with a Factor H dysfunction or abnor other commercially important blood products by plasma frac 60 mal alternative pathway complement activity. tionation. In a preferred embodiment, a method is provided for treat In one embodiment, a method is provided for treating a ing a disease or disorder associated with a Factor H dysfunc disease or disorder associated with a Factor H dysfunction or tion or abnormal alternative pathway complement activity in abnormal alternative pathway complement activity in a Sub a Subject in need thereof by administering to the Subject a ject in need thereof by administering a therapeutically effec 65 therapeutically effective dose of a Factor H composition pre tive dose of a Factor H composition prepared from a Fraction pared by a method comprising the steps of: (a) precipitating II+III filter cake or a Fraction I precipitate. proteins from a cryo-poor plasma fraction, in a first precipi US 9,109,044 B2 49 50 tation step, with between about 6% and about 10% alcohol at physician and may range from as short as one day to more a pH of between about 7.0 and about 7.5 to obtain a first than a month. In certain embodiments, a course of treatment precipitate and a first Supernatant; (b) extracting Factor H can be from 1 to 6 months. from the precipitate with a Factor H extraction buffer, (c) An effective amount of a Factor H preparation is adminis performing at least one viral inactivation or removal step; and tered to the subject by any suitable means to treat the disease (d) optionally lyophilizing the composition, thereby prepar or disorder. For example, in certain embodiments, Factor H ing a composition of Factor H suitable for use in treating a may be administered by intravenous, intraocular, Subcutane disease or disorder associated with a Factor H dysfunction or ous, and/or intramuscular means. In a preferred embodiment, abnormal alternative pathway complement activity. a method for treating age-related macular degeneration in a In one embodiment, the present invention provides athera 10 Subject in need thereof is provided comprising the intraocular peutically effective dose of a Factor H composition prepared administration of a Factor H composition to the patient. by a method disclosed herein for use in a method for treating In certain embodiments, the Factor H compositions pro a disease associated with Factor H dysfunction in a Subject in vided herein can be administered either systemically or need thereof. In one embodiment, the Factor H composition is 15 locally. Systemic administration includes: oral, transdermal, prepared by extracting Factor H from a Fraction I precipitate. Subdermal, intraperitioneal. Subcutaneous, transnasal, Sub In another embodiment, the Factor H composition is prepared lingual, or rectal. The most preferred systemic route of by extracting Factor H from a Fraction II+III filter cake. In administration is oral. Local administration for ocular admin certain embodiments, a method is provided for treating a istration includes: topical, intravitreal, periocular, transcleral, disease or disorder associated with a Factor H dysfunction in retrobulbar, juxtascleral, Sub-tenon, or via an intraocular a Subject in need thereof by administering to the Subject a device. Preferred methods for local delivery include transs therapeutically effective dose of a Factor H composition pro cleral delivery to the macula by posterior juxtascleral admin vided herein. In certain embodiments, the disease or disorder istration; via intravitreal injection; or via cannula, Such as that associated with a Factor H dysfunction is selected from atypi described in U.S. Pat. No. 6,413,245, the disclosure of which cal haemolytic uremic syndrome (aHUS), age-related macu 25 is incorporated by reference herein in its entirety for all pur lar degeneration (AMD), membranoproliferative glomulone poses. Alternatively, the inhibitors may be delivered via a phritis type II (MPGNII), myocardial infarction, coronary sustained delivery device implanted intravitreally or transs heart disease/coronary artery disease (CAD/CHD), and clerally, or by other known means of local ocular delivery. Alzheimer's disease. In one particular embodiment, the dis In certain embodiments, the term “effective amount” refers ease is atypical haemolytic uremic syndrome (aHUS). In 30 to an amount of a Factor H preparation that results in an another particular embodiment, the disease is age-related improvement or remediation of disease or condition in the macular degeneration (AMD). In yet another particular subject. An effective amount to be administered to the subject embodiment, the disease is membranoproliferative glomulo can be determined by a physician with consideration of indi nephritis type II (MPGNII). vidual differences in age, weight, the disease or condition In one embodiment, the present invention provides athera 35 being treated, disease severity and response to the therapy. In peutically effective dose of a Factor H composition prepared certain embodiments, an Factor H preparation can be admin by a method disclosed herein for use in a method for treating istered to a subject at dose of at or about between 5 mg/kilo a disease associated with abnormal alternative pathway gram and 2000 mg/kilogram per administration. In certain complement activity in a Subject in need thereof. In one embodiments, the dose may be at least at or about 5 mg/kg, or embodiment, the Factor H composition is prepared by 40 at least at or about 10 mg/kg, or at least at or about 20 mg/kg, extracting Factor H from a Fraction I precipitate. In another 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 embodiment, the Factor H composition is prepared by mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 extracting Factor H from a Fraction II+III filter cake. In mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 certain embodiments, a method is provided for treating a mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 disease or disorder associated with a abnormal alternative 45 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 pathway complement activity in a subject in need thereof by mg/kg, 950 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200 mg/kg, administering to the Subject a therapeutically effective dose 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 of a Factor H composition provided herein. In certain mg/kg, 1800 mg/kg, 1900 mg/kg, or 2000 mg/kg. The dosage embodiments, the disease or disorder associated with abnor and frequency of Factor H treatment will depend upon, mal alternative pathway complement activity is selected from 50 among other factors, the disease or condition being treated an autoimmune disease (such as rheumatoid arthritis, IgA and the severity of the disease or condition in the patient. nephropathy, asthma, systemic lupus erythematosus, mul B. Age-Related Macular Degeneration (AMD) tiple sclerosis, Anti-Phospholipid syndrome, ANCA-associ In a preferred embodiment, the present invention provides ated vasculitis, pemphigus, uveitis, myathemia gravis, Hash a method of treating age-related macular degeneration in a imoto's thyroiditis), a renal disease (such as IgA 55 Subject in need thereof by administering to the Subject a nephropathy, hemolytic uremic syndrome, membranoprolif therapeutically effective dose of a Factor H composition pro erative glomerulonephritis) asthma, Alzheimer disease, adult vided herein. macular degeneration, proximal nocturnal hemoglobinuria, Age-related macular degeneration (AMD) is the number abdominal aortic aneurism, ischemia reperfusion injury, and one cause of blindness for the elderly population over 60 sepsis. 60 years of age. Today, it is estimated that 35-40% of those over The pharmaceutical compositions provided by the inven 75 years of age have some degree of AMD. It has been tion may be administered alone or in conjunction with other estimated that approximately 50 million people are affected therapeutic agents. These agents may be incorporated as part world-wide, with 10 million in the US alone. Currently, about of the same pharmaceutical. 155,000 new diagnoses of AMD are made every year. As the A. Administration 65 worldwide population continues to age, the number of annual In accordance with the present invention, the time needed diagnoses are expected to triple by the year 2020. It is a to complete a course of the treatment can be determined by a devastating disease that destroys central vision in the affected US 9,109,044 B2 51 52 individuals, robbing them of their ability to perform activities prepared by a method comprising (a) performing Cohn frac necessary for everyday life Such as reading and driving. tionation to obtain Fraction II+III precipitates and preparing AMD is a slow, progressive disease that involves cells of a Suspension of the combined precipitates, (b) filtering the the outer retinal layers (including photoreceptors and the re-suspended Fraction II+III precipitates to obtain the filter retinal pigment epithelial (RPE) cells that support the photo cake discard left behind after filtration of the Fraction II+III receptors), as well as cells in the adjacent vascular layer of the Suspension; (c) extracting the filter cake according to a eye known as the choroid. Macular degeneration is charac method comprising the steps of: (i) dissolving the filter cake terized by the breakdown of the macula, a small portion of the in a suitable buffer of appropriate ionic strength for a time central retina (about 2 mm in diameter) responsible for high sufficient to dissolve the protein caked on said filter cake; (ii) acuity vision. Late-onset macular degeneration (i.e., AMD) is 10 diluting said dissolved protein with additional buffer, (iii) generally defined as either “dry” or “wet.” The wet (“exuda removing debris from the diluted protein, (iv) purifying Fac tive') neovascular form of AMD affects approximately 10% tor H protein from said diluted protein preparation by sub of those with the disease, and is characterized by abnormal jecting the diluted protein to ultrafiltration on a 0.45um filter blood vessels growing from the choriocapillaris through the to produce a filtrate containing Factor H., (v) Subjecting the RPE. typically resulting in hemorrhage, exudation, Scarring, 15 filtrate containing Factor H to anion exchange chromatogra and/or serous retinal detachment. Approximately 90% of phy using a NaCl gradient (50-500 mM) in the running buffer patients with AMD have the non-neovascular, or dry form of to produce a pooled crude Factor H fraction; and (vi) purify the disease, which is characterized by atrophy of the RPE and ing Factor H from said crude Factor H preparation by Heparin loss of macular photoreceptors. SepharoseTM chromatography using a gradient of NaCl (50 AMD is characterized by the presence of deposits of 500 mM). debris-like material, termed "drusen,” that accumulate on In one embodiment of a method for limiting complement Bruch's membrane, a multilayered composite of extracellular activation resulting in delayed progression or onset of the matrix components separating the RPE (the outermost layer development of age related macular degeneration (AMD) in a of the retina) from the underlying choroid. Drusen can be subject, the subject does not have any symptoms of AMD. observed by funduscopic eye examination. These deposits 25 In another embodiment of a method for limiting comple have been extensively characterized in microscopic studies of ment activation resulting in delayed progression or onset of donor eyes from patients with AMD. The deposits observed in the development of age related macular degeneration (AMD) the living eye upon clinical examination are classified as in a subject, the Subject has drusen. either soft drusen or hard drusen, according to several criteria In another embodiment of a method for limiting comple including relative size, abundance, and shape of the deposits. 30 ment activation resulting in delayed progression or onset of Histochemical and immunocytochemical studies have shown the development of age related macular degeneration (AMD) that drusen contain a variety of lipids, polysaccharides, gly in a subject, the subject is at increased risk of developing cosaminoglycans and proteins. AMD. Presently, there no known cure for AMD, although several In another embodiment of a method for limiting comple types of treatments has been shown to be effective at manag 35 ment activation resulting in delayed progression or onset of ing the disease. Laser photocoagulation of abnormal vessels the development of age related macular degeneration (AMD) in the wet form of the disease is the standard treatment. This in a subject, the administration is intravenous. treatment is limited by the fact that only well-delineated In another embodiment of a method for limiting comple neovascular lesions can be treated in this way and that 50% of ment activation resulting in delayed progression or onset of patients will suffer recurrence of the leakage from the vessels 40 the development of age related macular degeneration (AMD) (Fine et al., 2000). Because of the energy of the laser required in a subject, the method further comprises treating a subject for this treatment, the photoreceptors in the treated area will having signs and/or symptoms of AMD. also die, and the patient will also often suffer central blindness In another embodiment of a method for limiting comple immediately after the treatment. New neovascular lesions ment activation resulting in delayed progression or onset of will eventually develop, requiring repeated treatments. Other 45 the development of age related macular degeneration (AMD) interventions include changing lifestyles by cessation of in a subject, the subject has been diagnosed with AMD. Smoking and beginning therapy with antioxidants. Antiangio In another aspect, the present invention provides a method genic treatments using VEGF inhibitors e.g., intravitrial of treating a human Subject judged to be at risk for the devel injection of ranibizumab or bevacizumab also have been Sug opment of age related macular degeneration, comprising the gested. 50 step of administering to the Subject a prophylactically or Recently it was discovered that about 35% of individuals therapeutically effective amount of a Factor H preparation carry at an at-risk single nucleotide polymorphism (SNP) in provided herein, and periodically repeating said administra one or both copies of their Factor H gene. Homozygous tion. In one embodiment, the Factor H preparation is prepared individuals have an approximately sevenfold increased by a method comprising (a) performing Cohn fractionation to chance of developing age-related macular degeneration, 55 obtain Fraction II+III precipitates and preparing a suspension while heterozygotes have a two-to-threefold increased likeli of the combined precipitates, (b) filtering the re-suspended hood of developing the disease. This SNP located in CCP Fraction II+III precipitates to obtain the filter cake discard left module 7 of Factor H, has been shown to affect the interac behind after filtration of the Fraction II+III suspension; (c) tions between Factor Hand both C-reactive protein and hep extracting the filter cake according to a method comprising arin indicating a causal relationship between the SNP and 60 the steps of: (i) dissolving the filter cake in a suitable buffer of disease. The polymorphism is a Y420H polymorphism. appropriate ionic strength for a time Sufficient to dissolve the In one aspect, the present invention provides a method for protein caked on said filter cake; (ii) diluting said dissolved limiting complement activation resulting in delayed progres protein with additional buffer, (iii) removing debris from the sion or onset of the development of age related macular diluted protein, (iv) purifying Factor H protein from said degeneration (AMD) in a subject, comprising administering a 65 diluted protein preparation by Subjecting the diluted protein therapeutically effective amount of Factor H preparation pro to ultrafiltration on a 0.45 um filter to produce a filtrate con vided herein. In one embodiment, the Factor H preparation is taining Factor H., (v) Subjecting the filtrate containing Factor US 9,109,044 B2 53 54 H to anion exchange chromatography using a NaCl gradient Factor H in the industrial fractionation process was followed (50-500 mM) in the running buffer to produce a pooled crude by ELISA using an antibody specific for the full-length vari Factor H fraction; and (vi) purifying Factor H from said crude ant of Factor H. The amount of Factor H determined to be in Factor H preparation by Heparin SepharoseTM chromatogra eachfraction is provided in Table 1 and is Summarized graphi phy using a gradient of NaCl (50-500 mM). cally in FIG. 4. TABLE 1. Relative amounts of Factor H found in each of the major fractions of an industrial scale plasma fractionation. Ppt 9% Protein Total Factor Liquid Solid Protein Factor H % of wtfwt H Fraction L kg mg/mL g/mL Total Correction* kg Cohn Pool 3210 SO.1 66.3.3 1.323 2.13 Fraction I Sup 3480 45.2 592.9 1312 2.06 Fraction I Ppt 41.1 4.1 107.7 2.6O2 O.17 O.18 Fraction II + 388O O.OO)4 II Sup Fraction II + 1245 21.2 689 3.257 O.43 1.74 II Ppt Filtrate (post 2340 O.O2 Aerosil (R) Ppt G 58.6 O.OS3 Ppt G Sup 3140 O.OO8 Fraction IV-1 Sup 3800 O.OO1 Fraction IV-1 Ppt 76.8 O.O31 Fraction IV-4 Sup 5990 O Fraction IV-4 Ppt O Crude V Ppt 229.4 O.OO2 *Determined by freeze drying 30 In one embodiment of a method of treating a human subject As seen in Table 1, the 3210 L Cohn pool of pooled human judged to beat risk for the development of age related macular plasma used contained about 2.13 kg of Factor H, as deter degeneration, the administration is repeated for a time effec mined by an ELISA assay. The majority of Factor His present tive to delay the progression or onset of the development of 35 in the Fraction II+III precipitate (1.74 kg), however, insignifi macular degeneration in said subject. cant amount are found in the clarified Fraction II+III filtrate, In another embodiment of a method of treating a human i.e., post precipitate dissolution and Aerosil(R) treatment. This subject judged to be at risk for the development of age related result Suggests that a large portion of the Factor H present in macular degeneration, the human Subject is judged to be at the Cohn pool starting material is captured in the Fraction risk for the development of age-related macular degeneration 40 II+III filter cake. A second significant fraction of Factor H as identified based on the presence of one or more genetic (180g) is also captured in the Fraction I precipitate, as seen in markers associated with development of age-related macular Table 1. degeneration. In another embodiment of a method of treating a human Example 2 subject judged to be at risk for the development of age related 45 macular degeneration, the genetic marker is a polymorphism. The present example describes experiments performed to In another embodiment of a method for limiting comple determine the feasibility of extracting Factor H from a Frac ment activation resulting in delayed progression or onset of tion II+III filter cake. Briefly, the Fraction II+III filter cake the development of age related macular degeneration (AMD) from the plasma fractionation performed in Example 1 was in a subject, the subject is not diagnosed with AMD. 50 dissolved in a Factor H extraction buffer (25 mM Tris (pH 8.0): 5 mM EDTA; 200 mM. NaCl) at a ratio of 25:1 (mL VI. Examples buffer per g filter cake). The conductivity of the resulting Suspension was then adjusted by diluting the solution 3:1 with The following examples are provided by way of illustration low salt extraction buffer (25 mM Tris (pH 8.0): 5 mM only and not by way of limitation. Those of skill in the art will 55 EDTA). Un-dissolved material was cleared from the suspen readily recognize a variety of non-critical parameters that sion by centrifugation followed by filtration through a 0.45 could be changed or modified to yield essentially the same or um filter. similar results. The clarified filter cake suspension was then loaded onto a Example 1 60 DEAE SepharoseTM chromatograph column equilibrated with a low saltbuffer (25 mM Tris (pH 8.0): 5 mM EDTA; 50 To determine an economically beneficial scheme for the mM NaCl). A linear gradient from 50 mM. NaCl to 500 mM manufacture of Factor H from a plasma sample, which allows NaCl (25 mM Tris (pH 8.0): 5 mM EDTA; NaCl) was then for the recovery of additional blood factors from the same used to elute the Factor H from the DEAE SepharoseTM plasma sample, a 3000 L lot of pooled human plasma was 65 column, the eluate of which was collected fractionally as Subjected to industrial fractionation according to the scheme shown by the chromatograph in FIG. 2A. Samples of the outlined in the flow-diagram shown in FIG. 1. The fate of eluate fractions were analyzed by SDS-PAGE and Western US 9,109,044 B2 55 56 blot analysis (FIG. 2C) to determine that Factor Heluted off reactions were incubated at 37° C., denatured in Laemmli of the anion exchange column in the first of three major peaks. sample buffer and resolved via SDS-PAGE. The intensities of The first elution peak from the DEAE SepharoseTM chro the C40 fragment of C3b were quantified via densitometry matography elution, containing primarily Factor H, was and the resulting activity curves are shown in FIG. 6. As pooled based on the chromatograph and gel analysis per shown, the activity of the purified Factor H, both derived from formed, concentrated, and the conductivity was reduced. The Fraction I precipitate and from Fraction II+III filter cake, is Factor H solution was then loaded onto a Heparin comparable to a commercial preparation of Factor H. In this SepharoseTM chromatography column equilibrated with a regard, the Factor H preparations from Example 2 and 3 low salt buffer (25 mM Tris (pH 8.0): 5 mM EDTA; 50 mM appear to have similar specific activities as the commercial NaCl). A linear gradient from 50 mM. NaCl to 500 mM. NaCl 10 Factor H preparation. (25 mM Tris (pH 8.0): 5 mM EDTA; NaCl) was then used to To further characterize the activity of the purified Factor H elute the Factor H from the DEAE SepharoseTM column, the compositions, a second activity assay was employed to deter eluate of which was collected fractionally as shown by the mine the C3 () and C5 (0) convertase inhibition activity of chromatograph in FIG. 2B. Samples of the eluate fractions the preparation. Briefly, an alternative pathway stimulator were analyzed by SDS-PAGE and Western blot analysis. As 15 (Sepharose 4B) was incubated with heparnized plasma can be seen in FIG. 2B, Factor H eluted from the Heparin spiked with increasing amounts of the Factor H composition SepharoseTM column in a single peak, which provides a pure purified from Fraction II+III filter cake (Example 2). After Factor H composition (FIG. 2D). incubation, complement was quenched with EDTA and the levels of C3a and C5a were determined via ELISA. As shown Example 3 in FIG. 5, the Fraction II+III filter cake Factor H preparation demonstrates inhibitory activity against C3a and C5a conver The present example describes experiments performed to S1O. determine the feasibility of extracting Factor H from a Frac The experimental results provided above in examples 1 to tion I precipitate. Briefly, the Fraction I precipitate from the 4 demonstrate that Factor H can be purified from both Frac plasma fractionation performed in Example 1 was dissolved tion I precipitate and Fraction II+III filter cake to yield Factor in a Factor H extraction buffer (25 mM Tris; 5 mM EDTA; 25 H compositions having purity and enzymatic activities com 200mMNaCl; pH 8.0) at a ratio of 25:1 (mL buffer perg filter parable to currently available commercial preparations cake). The conductivity of the resulting Suspension was then (CompTech). adjusted by diluting the solution 3:1 with low salt extraction buffer (25 mM Tris (pH 8.0): 5 mM EDTA). Un-dissolved Example 5 material was cleared from the Suspension by centrifugation 30 followed by filtration through a 0.45 um filter. Since the Factor H was purified from a large plasma pool, The clarified Fraction I precipitate suspension was then this preparation contains many variants (isoforms) that con loaded onto a DEAE SepharoseTM chromatograph column tribute to the overall activity. An unanticipated finding relates equilibrated with a low salt buffer (25 mM Tris (pH 8.0): 5 to the percent of His-402 variant in the final preparation of mM EDTA; 50 mM. NaCl). A linear gradient from 50 mM 35 Factor Hpurified from a Fraction II+III filter cake, which was NaCl to 500 mM NaCl (25 mM Tris (pH 8.0): 5 mM EDTA; determined to be approximately 50% via LC-MS (Table 2). NaCl) was then used to elute the Factor H from the DEAE The incidence of the His-402 FH isoform in the general SepharoseTM column, the eluate of which was collected frac population has been reported to range from 7% to 34% tionally. (Grassi et al. Human Mutation (2006) 27:921–925), suggest The Factor H peak from the DEAE SepharoseTM chroma ing that the method used above in examples 1 to 3, for pre tography elution was pooled, concentrated, and the conduc 40 paring Factor H compositions, yields a distinct population of tivity was reduced by buffer exchange. The Factor H solution Factor H variants. It was also observed that Factor H. purified was then loaded onto a Heparin SepharoseTM chromatogra in the manner described above, eluted as two sequential peaks phy column equilibrated with a low salt buffer (25 mM Tris (FIG. 2). These two peaks appeared to have differences in (pH 8.0): 5 mM EDTA; 50 mMNaCl). A linear gradient from their affinity for heparin (due to the difference in their elution 50 mM NaCl to 500 mM NaCl (25 mM Tris (pH 8.0): 5 mM 45 time in the linear salt gradient) but a somewhat similar dis EDTA; NaCl) was then used to elute the Factor H from the tribution of H403 versus Y402 variants (as shown in Table 2). Heparin SepharoseTM column, the eluate of which was col lected fractionally. Factor H containing fractions were then TABLE 2 pooled and the final composition was analyzed by SDS 50 Relative amounts of H4O2 and Y402 Factor Hvariants found in a PAGE analysis (FIG. 3). commercial preparation (CompTech) and the Fraction II + III filter cake Example 4 purification of example 2. Commercial The purity and activity of Factor H prepared from the Preparation Factor-H (peak 1) Factor-H (peak 2 Fraction I precipitate (Example 3) and Fraction II+III filter 55 cake (Example 2) fractions from the plasma fractionation H4O2 SO.0% 49.9% 52.3% performed in example 1 was investigated. As can be seen from Y402 SO.0% SO.1% 47.7% FIG.3, Factor H prepared from the Fraction I precipitate (lane 4) and Fraction II+III filter cake (lane 6) was at least as pure as a commercial preparation of Factor H (CompTech; lane 2). Example 6 The activity of the purified Factor H compositions was 60 assessed via two methods. First, a Factor I cofactor activity In order to further explore conditions under which Factor H was examined. C3b and Factor I used in the assay were may be extracted from a Factor II+III filter cake, pooled obtained commercially from CompTech. Briefly, C3b was human plasma samples were fractionated as described in combined with Factor I and various concentrations of Factor Example 1. After suspension and silicon dioxide (SiO) treat H derived from either a commercial source (CompTech; 0), 65 ment of the Fraction II+III precipitate, the Fraction II+III purified from Fraction I precipitate (Example 3; A) or puri filter cake was collected in a filterpress, effectively separating fied from Fraction II+III filter cake (Example 2: ). The it from the corresponding clarified Fraction II+III suspension. US 9,109,044 B2 57 58 The filter cake, still located within the filter press, was first Example 8 pre-rinsed with a Factor H extraction buffer outlined below until the pH of the rinsed solution reached 7.0 to displace In order to reduce the volume and potentially remove residual filtrate and to fill the filter press with the appropriate impurities from the Factor Hextract recovered from Fraction extraction buffer. This is an optional operation to fully recover 5 II+III filter cake, precipitation experiments were performed the filtrate which does not affect Factor H yield in the extract. to investigate conditions that may be used. Briefly, polyeth Factor H was extracted from the filter cake by continuous ylene glycol 4000 (PEG 4K) or ethanol was added at varying recirculation of one of two Factor H extraction buffers, concentrations to samples of a Factor H filter cake extract, extraction buffer A (20 mM Tris (pH 8.0): 5 mM EDTA; 200 prepared as described in Example 7, and the pH of the solu mMNaCl) or extraction buffer B (100 mM sodium phosphate 10 tion was adjusted to either 6.0 or 8.0 for the ethanol samples (pH 7.5): 150 mM sodium chloride) through the filter press and 7.0 for the PEG samples. The samples containing ethanol for one hour. were then stirred overnight at -10°C. and the samples con Analysis of the resulting Fraction II+III filter cake extracts taining PEG were stirred overnight at 4°C. The resulting revealed that extraction with buffer A resulted in recovery of precipitates and Supernatants were then separated and the 0.39 g Factor H/L plasma and examination of the filter cake precipitates dissolved in extraction buffer. As seen in Table 3, revealed that 0.04 g Factor H/L plasma was retained in the 15 more protein remained in the Supernatant of the 25% ethanol filter cake after a one hour extraction. Similarly, extraction precipitation than in the 12.5%, 17.5%, and 20% PEG pre with buffer Bresulted in recovery of 0.44 g Factor H/L plasma cipitations. Consistent with this, the dissolved PEG precipi and examination of the filter cake revealed that 0.03 g Factor tates contained more protein than did the ethanol precipitate, H/L plasma was retained in the filter cake after a one hour resulting in cloudier Suspensions that may require additional extraction. Consistent with this, SDS-PAGE analysis of the treatment prior to further purification of Factor H by chro extracted fraction shows that the distribution of proteins and matographic methods. TABLE 3 Factor H contents of precipitates and Supernatants resulting from overnight ethanol and PEG precipitation experiments. Protein in the Appearance Protein in the Amount of Appearance of Supernatant of the Precipitate Precipitate the Dissolved Precipitant (mg/ml) Supernatant (mg/ml) (gg filtrate) Precipitate 12.5% PEG 1.11 mg/ml Clear 10.0 mg/ml 0.034 gig Very Cloudy filtrate 17.5% PEG 0.5 mg/ml Clear 9.3 mg/ml 0.045 gig Very Cloudy filtrate 20% PEG 0.5 mg/ml Clear 9.0 mg/ml 0.048 gig Very Cloudy filtrate 25% Ethanol 1.5 mg/ml Cloudy 5.3 mg/ml 0.070 gig Slightly Cloudy (pH 7.0) filtrate relative amounts of Factor H in both extractions was similar The Factor H content of each sample was then determined (FIG.7; lane 1=Factor H standard (CompTech); lane2=stan 40 by ELISA analysis, values for which are given in Table 4. To dard protein MW markers; lane 3-buffer A extract; lane further analyze the resulting fractions, samples of each Super 4-buffer B extract). These results suggest that a wide range of natant and dissolved precipitate were subjected to SDS buffers may be effective for the extraction of Factor H from a PAGE analysis (FIG. 9). As seen in Table 5, the majority of Fraction II+III filter cake. Factor H is present in the supernatant of the 15% ethanol 45 precipitation performed at pH 8.0, while Factor H is precipi Example 7 tated with 25% ethanol at a pH of 6.0. In one embodiment, a fractional precipitation scheme may be used wherein some To investigate the extraction time required for efficient impurities are first removed by precipitating the Factor H recovery of Factor H from the Fraction II+III filter cake, extract using 15% ethanol and then recovering the Factor H in pooled human plasma samples were fractionated as described 50 a second precipitation using 25% ethanol. in Example 1. After suspension and silicon dioxide (SiO2) treatment of the Fraction II+III precipitate, the Fraction II+III TABLE 4 filter cake was collected in a filterpress, effectively separating Factor H contents of precipitates and Supernatants resulting from overnight it from the corresponding clarified Fraction II+III suspension. ethanol and PEG precipitation experiments. The filter cake, still located within the filter press, was first 55 pre-rinsed with a Factor Hextraction buffer (100 mM sodium Lane # gFactor H phosphate (pH 7.5): 150 mM sodium chloride) until the pH of (FIG.9) Sample L Plasma the rinsed solution reached 7.0. Factor H was then extracted Lane 1 size marker from the filter cake by continuous recirculation of the Factor Lane 2 Factor H Standard H extraction buffer for between 10 and 60 minutes. The 60 Lane 3 PEG 10% precipitate dissolved O.38 Factor H content was then determined for each of the extrac Lane 4 PEG 10% supernatant Lane 5 PEG 12.5% precipitate dissolved O43 tions by ELISA analysis. As seen in FIG. 8, no significant Lane 6 PEG 12.5% supernatant difference in the amount of Factor Hextracted from the filter Lane 7 PEG 17.5% precipitate dissolved O41 cakes could be seen for extraction times ranging from 10 to 60 Lane 8 PEG 17.5% supernatant minutes. However, post-washing the filter press with one void 65 Lane 9 PEG 20% precipitate dissolved O42 Volume of extraction buffer resulted in an increase in the final Lane 10 PEG 20% supernatant Factor H recovery from the filter cake. US 9,109,044 B2 59 60 TABLE 4-continued through fractions contain very little Factor Has shown by the Western blot results in FIG. 11C. Factor H contents of precipitates and Supernatants resulting from overnight In a first step elution, the salt concentration of the buffer (25 ethanol and PEG precipitation experiments. mM Tris (pH 8.0): 5 mM EDTA; NaCl) was increased to 98 Lane # gFactor H mM NaCl to elute Factor H from the column. SDS-PAGE (FIG.9) Sample L Plasma (FIG. 11B) and Western blot (FIG. 11C) analysis shows that Lane 11 PEG 12.5% + 0.2% Tween, precipitate dissolved 0.4 the resulting Factor H pool is quite pure. There are some low Lane 12 15% EtOH, pH = 8, Supernatant O.39 molecular weight impurities that may be removed by a size Lane 13 25% EtOH, pH = 6, precipitate dissolved O43 exclusion polishing or equivalent step. A second elution step 10 was performed with buffer containing 204 mM NaCl and another Factor H peak was eluted off the column. This frac tion did not have any detected impurities. Subsequent elution TABLE 5 steps at 288 mM and 1 M NaCl did not show any additional Fate of Factor H in ethanol precipitations performed at 15% ethanol protein peaks (FIG. 10A), indicating all Factor Heluted from pH 8.0 and 25% ethanol pH 6.0. 15 the column at the elution steps containing 98 mM and 204 mM NaCl. This method can be modified to optimize the Yield 96 of Factor H in filter cake process. In one embodiment, all of the Factor H may be eluted Recycled filter cake (filter press) in a single step, for example with a single elution with buffer EtOH Precipitation 15% pH = 8.0 containing greater than 98 mMNaCl. The load and wash may EtOH Supernatant 89 still be performed at 50 mM NaCl, and Factor Helution may EtOH Precipitate dissolved 7 be done, for example, with buffer containing 204 mM NaCl. EtOH Precipitation 25% pH = 6.0 An extended wash at 50 mMNaCl orata salt concentration of below 98 mM NaCl may be added after loading in an attempt EtOH Supernatant 2 EtOH Precipitate dissolved to remove more weakly bound impurities from the Factor H 25 pool. The above chromatography steps can be modified to use buffer systems other than Tris/EDTA at pH 8. These processes Example 9 can be adapted for buffers and solutions commonly used in manufacturing of biopharmaceuticals. An example is a puri In order to aid with the industrial scale-up for the Factor H 30 fication scheme using phosphate buffer at about pH 7.5. The purification after extraction, an alternate purification scheme key parameter to Successful purification is manipulation of was devised that replaces the salt gradient elution of the conductivity or ionic strength to achieve separation of the chromatography columns with a series of step elutions that desired compound. If the pH of the buffer system is changed, are more amenable to a large scale manufacturing process. some adjustment of the ionic strength will be needed which Briefly, a Factor H extraction was then loaded onto a DEAE 35 can be done with standard techniques used in optimization of SepharoseTM chromatography column equilibrated with a chromatographic processes. low salt buffer (25 mM Tris; 5 mM EDTA; 65 mM NaCl; pH 8.0). The conductivity of the load was similar to that of the Example 10 equilibration buffer (about 9 mS/cm). After the load, the column was washed with buffer containing 65 mM NaCl for 40 In order to demonstrate that plasma-derived Factor H is 5 column volumes (CV) to remove the unbound protein impu efficacious in vivo, an experiment was performed using the rities. The flow-through fractions contain very little Factor H Choriodial Neovascularization (CNV) model in mice (a as shown by the Western blot results in FIG. 10C. model of AMD in humans). Human FH was isolated to homo In a first step elution, the salt concentration of the buffer (25 geneity as described above (Example 2) and shown to have mM Tris (pH 8.0): 5 mM EDTA; 65 mMNaCl) was increased 45 low endotoxin levels. CNV was induced by laser photoco to 100 mMNaCl (conductivity 12.6 mS/cm) for 5 CV to elute agulation in C57/B1 male mice with an argon laser (50 um Factor H bound to the column. The Factor H came off the size spots, 0.05s duration, 250 mW) as described (N. S. Bora, column in a sharp peak followed by a shoulder, as seen in the S. Kaliappan, P. Jha, Q. Xu, B. Sivasankar, C. L. Harris, B. P. chromatograph provided in FIG. 10A. The corresponding Morgan and P. S. Bora. J. Immunol. 178:1783-1790 (2007)). Coomassie stained SDS-PAGE gel (FIG. 10B) and Western 50 Two groups of 8 animals were treated, Group 1 (controls) blot (FIG. 10C) show the majority of the Factor H in the peak. received 2 ul intravitreal injection of PBS within 1 hour of the Analysis of further step elutions performed with buffers con laser treatment while Group 2 (test) received 2 ul intravitreal taining 155 mM NaCl, 230 mM NaCl, and 1 M NaCl dem injection of human FH within 1 hour of the laser treatment. onstrate that very little Factor H remains bound to the DEAE Animals were sacrificed 7 days after laser treatment. Before SepharoseTM resin after the 100 mM NaCl elution (FIG. 10). 55 sacrifice, the mice were perfused through the heart with 0.5 The major Factor H fractions from the 100 mM NaCl elution mL of FITC-dextran (Sigma, molecular weight approx. were pooled together. 2x10'). The eyes were fixed in normal buffered formalin for To reduce the salt concentration of the pooled Factor H 2 hours at 25°C., and the posterior part of the eyes (with the fractions, the sample was dialyzed against low salt buffer to spots) were dissected and additionally stained for elastin reduce the conductivity to about 8 mS/cm (around 50 mM 60 using immunohistochemistry. Primary goat polyclonal Abs NaCl). The sample was then passed through a 0.45um filter to against elastin (C-21, SC-1751, Loti.J0505, Santa Cruz) in remove any particulates. The filtered sample was then loaded working dilution 1:200 and secondary donkey anti goat Alexa onto a Heparin SepharoseTM chromatography column equili Fluor 594 conjugated Abs (Invitrogen) in working dilution brated with a low salt buffer (25 mM Tris (pH 8.0): 5 mM 1:400 were used. EDTA; 50 mMNaCl). After the load, the column was washed 65 Samples were flat mounted in ProLong antifade reagent with buffer containing 50 mM NaCl for 5 column volumes (Invitrogen). Laser confocal microscopy was performed (CV) to remove the unbound protein impurities. The flow using LSM 510 Zeiss microscope. Single image of CNV in US 9,109,044 B2 61 62 each laser injured area were captured and spots with hemor (c) precipitating Factor H from the Supernatant formed in rhagic changes were excluded from the investigation. The step (b) using a final alcohol concentration of from 24% area of CNV in um was measured using Image.J program to 26% at a pH of from 5.8 to 6.2. (NIH). Area of laser spots (area of interest, AOI) was manu 6. The method of claim 2, wherein the method further ally determined and area of green fluorescence (threshold comprises the step of: values 33-255) in area of interest was measured. Mean value (c) precipitating Factor H from the Supernatant formed in of CNV area and standard error for each group was calculated step (b) using a final alcohol concentration of from 20% using EXEL program. to 30% at a pH of from 5.0 to 7.0. The results of this analysis are show below in Table 6. 7. The method of claim 2, wherein the method further 10 comprises the step of: Treatment of the mice with Factor H, following laserablation (c) precipitating Factor H from the Supernatant formed in of the back of the eye, significantly reduced the level of step (b) using a final alcohol concentration of from 24% neovascular response measured on day 7 post treatment to 26% at a pH of from 5.8 to 6.2. (p=0.04). 8. The method of claim 3, wherein the method further 15 comprises the step of: TABLE 6 (c) precipitating Factor H from the Supernatant formed in Effect of Factor H on the Size of FITC-Dextran Perfused Wessels in step (b) using a final alcohol concentration of from 20% Murine CNV Model to 30% at a pH of from 5.0 to 7.0. 9. The method of claim 3, wherein the method further Group Number of Spots Area of CNV Percent of Control comprises the step of: 1 (PBS) 47 3618561 (c) precipitating Factor H from the Supernatant formed in 2 (Factor H) 43 2298 316 63.5 step (b) using a final alcohol concentration of from 24% to 26% at a pH of from 5.8 to 6.2. It is understood that the examples and embodiments 10. The method of claim 1, wherein the method further 25 comprises the steps of described herein are for illustrative purposes only and that (c) binding Factor H to an anion exchange resin; and various modifications or changes in light thereofwill be Sug (d) eluting Factor H from the anion exchange resin with an gested to persons skilled in the art and are to be included elution buffer, thereby forming a first eluate containing within the spirit and purview of this application and scope of Factor H. the appended claims. All publications, patents, and patent 30 11. The method of claim 10, further comprising the steps applications cited herein are hereby incorporated by refer of: ence in their entirety for all purposes. (e) binding Factor H from the first eluate to a heparin affinity resin; and What is claimed is: (f) eluting the Factor H from the heparin affinity resin with 1. A method for preparing an enriched Factor H composi 35 an elution buffer, thereby forming a second eluate con tion from plasma, the method comprising the steps of: taining Factor H. (a) extracting Factor H from a Fraction I precipitate and/or 12. The method of claim 1, further comprising the steps of: a Fraction II+III precipitate with an extraction buffer (i) binding an impurity to an anion exchange resin under Selected from the group consisting of a solution com conditions such that Factor H does bind to the resin and prising from about 10 mM to about 250 mM of a buff 40 is collected in the flow through fraction; ering agent, a conductivity of from about 5 mS/cm to (ii) binding Factor H from the flow through fraction to a about 100 mS/cm, and a pH from about 6.0 to about 9.0, heparin affinity resin; and a solution comprising from about 2.5 mM to about (iii) eluting Factor H from the heparin affinity resin to form 100 mM of a buffering agent, a conductivity of from an eluate; about 0.5 mS/cm to about 2.0 mS/cm, and a pH from 45 (iv) binding Factor H from the eluate to an anion exchange about 4.0 to about 5.5, to form a Factor H extract; and resin; and (b) precipitating an impurity from the Factor H extract to (V) eluting Factor H from the anion exchange resin, thereby form a Supernatant containing Factor H, thereby prepar further purifying Factor H. ing an enriched Factor H composition. 13. The method of claim 6, wherein the method further 2. The method of claim 1, wherein precipitation step (b) 50 comprises the steps of comprises precipitating an impurity from the Factor Hextract (d) binding Factor H to an anion exchange resin; and using a final alcohol concentration of from 10% to 20% at a (e) eluting Factor H from the anion exchange resin with an pH of from 7.0 to 9.0. elution buffer, thereby forming a first eluate containing 3. The method of claim 1, wherein precipitation step (b) Factor H. comprises precipitating an impurity from the Factor Hextract 55 14. The method of claim 13, further comprising the steps using a final alcohol concentration of from 14% to 16% at a of: pH of from 7.5 to 8.5. (f) binding Factor H from the first eluate to a heparin 4. The method of claim 1, wherein the method further affinity resin; and comprises the step of: (g) eluting the Factor H from the heparin affinity resin with (c) precipitating Factor H from the Supernatant formed in 60 an elution buffer, thereby forming a second eluate con step (b) using a final alcohol concentration of from 20% taining Factor H. to 30% at a pH of from 5.0 to 7.0, 15. The method of claim 6, further comprising the steps of: (c) precipitating Factor H from the Supernatant formed in (i) binding an impurity to an anion exchange resin under step (b) using a final alcohol concentration of from 20% conditions such that Factor H does bind to the resin and to 30% at a pH of from 5.0 to 7.0. 65 is collected in the flow through fraction; 5. The method of claim 1, wherein the method further (ii) binding Factor H from the flow through fraction to a comprises the step of: heparin affinity resin; US 9,109,044 B2 63 64 (iii) eluting Factor H from the heparin affinity resin to form an eluate; (iv) binding Factor H from the eluate to an anion exchange resin; and (V) eluting Factor H from the anion exchange resin, thereby further purifying Factor H. 16. The method of claim 1, further comprising at least one viral inactivation step. 17. A method for treating a disease associated with Factor H dysfunction in a subject in need thereof, the method com 10 prising administering a therapeutically effective dose of a pharmaceutical composition of Factor H prepared according to the method of claim 1.

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