Factor H has been implicated as a potential therapeutic for a variety of diseases including AMD and is a plasma derived blood product that is gaining the attention of physicians. However, due to the resources devoted to IgG gamma globulin manufacture, fewer methods are patented for the manufacture of Factor H. However, various purifications have been patented which introdce methods for the purificaiton of Factor H into existing manufacturing schemes.

Liebing (WO2008/113589) discloses a method for purification of complement Factor H by providing a fraction of Factor H obtained by large scale fractional precipitation of human plasma or serum with ethanol and then further purifying with at least one method selected from heparin affinity chromatography, HIC, AEX, CEX, HA or immunoaffinity chromatography. The source fraction can be a Factor H rich wash from Heparin affinity chromatography out of 8% suerpnatant from a Cohn/Oncley fractionation, or redissolved Fraction III from a Cohn/Oncley fractionation, or redissolved precipitate B from a kistler/Nitschmann fractionation. 

Alcohol precipitation

Single initial precipitation step (Fraction I+II+III+IV-1 preciptiation or Fraction I-IV-1 precipitation or initial low pH, high alcohol precipitation):

Bruckschwaiger (US13/776448) discloses an initial purificaiton step that copresicipates IgG and A1PI followed by a solubization step of this precipitate which leaves Factor H in the insoluble portion and the immunoglobulins in the soluble portion. The method includes the steps of precipitating immunoglobulins, A1PI, Factor H and IaIp in a first precipitation step by adding ethanol to a Cohn pool to 20-30% at a pH 5-6 to form a first precipitate and first supernatant, separating the first precipitate (containing the immunoglobulins, A1PI, IaIp, albumin, Factor H) from the supernatant. Next the first precipitate is suspended to form a first supension which is then treated as with silicon dioxide. The soluble porition of the supsnsion contains immunoglobulins while the insoluble porition contains A1pI, fibrinogen, Factor H and IaIP.

Fration I Precipitate or Fraction II/III Filter Cake:

Bairstow (US 2011/0021432) discloses purifying Factor H from a plasma sample by performing Cohn fractionation to obtain Faction II_III prcipitates and preparing a suspension of the precipitate, filtering the re-suspended Fraction II_III recipitate to obtain a filter cake left behind after filtraiton of the Fraction II_III suspension and extracting the Factor H from this filter cake using a buffer of appropirate ionic strenght to dissolve the coke and purifying Factor H by UF to produce a filtrate containing Factor H, subjecting the filtrate containing Factor H to AEX. Alternatively, Factor H can be obtained from the Fraction I precipitateand extracting Fraction H from the precipitate with an extraction buffer. 

Affinity-chromatography

Heparin affinity chromatography (HAC): WO2008/113589 teaches a method for the production of Factor H preparations form human plasma. In one method, purification of Factor H from a Cohn-Oncley Fraction I supernatant is described by the addition of a HAC step. A cited disadvantage is that the Cohn-Oncley Fraction I supernatant is a common intermediate fraction in the manufacturing processes of many commercially important plasma derived blood products, including IgG gamma globulines (IVIG and subcutaneous) and albumin and additional steps such as HAC into the manfuacturing schemes requires revalidation of the manufacturing procedure.

Heparin Affinity Chromatography/ HIC/AEX/CEC or HA: Liebing (WO2008/113589) discloses a method of purification of complement Factor H by large scale fractional precipitaiton of humnan plasma or serum with ethanol and further purifying Factor H by at least one purificaiton method selected from the group of Heparin affinity chromatography/HIC/AEX/CEX/HA.

Anion Exchange as a Preceeding Step

Anion exchange chromatography (AEC)-heparin affinity chromatography (HAC)-Cation exchange chromatography (CEC)-strong anion exchange chromatography (sAEC):  WO2007/066017 teaches methods for the production of Factor H preparations from the supernatant of a cryoprecipitate consisting of submitting the supernatant to AEC, submiting the flow through to HAC, submitting the eluate to strong CEC and then the eluate to sAEC. One cited disadvantage is that cryoprecipitate supernatants are common intermediate fractions in the manfuacturing processes of many commercially important plasma derived blood products, including IgG gamma globulins (IVIG and subcutaneous) and albumin and submiting this fraction to chromatogrpahy steps will alter the cryoproecipitate supernatant and would require the manufacturing processes of the established downstream blood products be adapted in unkown fashions. In addition to requiring a complete revalidation and possible redesign of these manufacturin processes, regulatory reapproval of the manufacturing procedures form key regulatory agencies is needed.

Anion Exchange-Affinity chromatography (heraprin sepharose FF type)-CEX-Vial inactivation-AEX:

Chtourou (US2008/0318841) discloses unfreezing human frozen plasma, the sueprnatant of the cryoprecipitate is separated from the insoluble fraction by centrifugation and subjected to AEX, the non-retained plasma supernatant fraction (fraction A) is then subjected to affinity chromatography7 in order to separate antitrhombin III form this fraction A by reatining antithrombin III on the resin, the pH of this non-retained fraction A (fraction B) is adjusted to 5.5-6.5 and subjected to chromatography having ligands of the heparin type. The eluted fraction containing Factor H (fraction C) is diluted and submited to sCEX. The Factor H retained on the gel is then eluted (fraction D) and submitted to a viral inactivation step.

Use of Cation Exchange as preceeding Step

CEX-AEX-HA-UF:

discloses a method for purificaiton of complement Factor H from blood or plasma such as a caprylate precipitate of a Factor H containing source using CEX as a first chromatographic step, AEX, HA and then UL/DF of the complement Factor H.

Use of discarded fractions from the preparation of immunoglobulins: 

Bairstow (US patent 8304524) teaches recovering Factor H from material otherwise discarded druing the manufacture of other commercially important blood products by plasma fractionation, as for example, from a Fraction I precipitate and/or extracted from a filter cake formed after centrifugation or filtration of a resuspended fraction II and III paste. Advantageously, the preparation of Factor H can be achieved without the need for additional input plasma or the redesign and regulatory reapproval of existing manufacturing processes for other commercially important plasma derived blood products such as IgG gamma globulins for IVIG or subcutaentous administration. In an exemplary embodiment, cryo-poor plasma is cooled to about 01C, pH 7-7.5, ethanol 6-10% to remove fibrinogen and other impurities in the manufacturing prcoess of IgG and albumin. A significant fraction of Factor H is present in this precipitate. Advantageously, the alcohol is added in a way that rapidly disperses the alcohol such as fine spraying and pH modifying agent is added by spraying.  In a second precipitation step, the Fraction Suerpnatant I is subjected to a Cohn-Oncley Fraction II+III type fractionation with pH around 6.6, acohol about 25%. Subsequently, the precipitate (Fraction II+III) which contains the majority of the Factor H and IgG content of the cryo-poor palsma is seaprated from the supernatant. In order to solubilize teh Factor H and IgG content of the Fraction II+III precipitate, a cold extraction buffer is used to re-suspend the Fractionation II_III precipitate. In one advantageous embodiment the pH is adjusted after addition of the precipitating acohol. In one embodiment, the method further comprises precipitating impurities from an enirched Factor H composition to form a third precipitate and supernatant with 10-20 ethanol at pH about 7-9. 

Precipitation with SiO2: 

Teschner (US 13/830815) also teaches a method for preparing a Factor H composition by first contacting a first comosition containing Factor H and at least one serine protease or serine protease zymogen with SiO2 under conditions suitable to bind the Factor H, washing the SiO2 and eluting Facto