IgM are immunogloublins secreted upon the body’s first contact with an antigen. They are the first type of immunoglobuilins released by plasmocytes. The presence of IgM in the blood indicates a current infection (Schmitthaeusler (US7771725).

IgM by nature is a vigorous activator of complement after the binding of antigens. Thus unspecific anti-complementary activity of denatured IgM molecules is far more dangerous to patients than denatured IgG molecules.

Initial Purification of IgM by Cohn Fractionation

The initial purification of human IgM solutions has been carried out by classical Cohn plasma fractionation methods or its well known modification. Using cold ethanol precipitation processes the IgM fraction is recovered in fraction III or fraction I/III (also called B or B+1). Starting from fraction III or I/III the following methods have been descirbed for purification of protein solution enriched in IgM. (Moeller US2013/0052208).

Isolation from Cohn Fraction III precipitate: Plasma IgM can be recovered from the byproducts of the production of IVIG. An example of such a byproduct is Cohn fraction III precipitate. The IgM is most easily solubilized from the Cohn fraction III precipitate by 20 mM sodium acetate. Other pleasma proteins are similarly solubilized along with the IgM. (Brown US 14.476,559)

Treatments with Various Agents

B-propiolactone

Pentaglobin® described in EP 13901 is a commerical example of an intravenously tolerable IgM preparation. This immunoglobulin preparation which is treated with 0.01 to 0.15% of beta-propiolactone to make it intravenously tolerable, contains, in addition to 10% of IgM, 80% of IgG and 10% of IgA. (Moller, US 5,190,752). 

Moller (US5,190,752) discloses an immunoglobulin preparation that contains at least 50% by weight of IgM, has low anticomplementary activity. The prepration is manufactured form an IgM containing fraction such as Cohn Fraction III obtained from plasma by treatment with an ion exchanger, eluting the exchanger with a saline gradient or pH gradient and gelfiltraiton where treatment with beta-propiolactone, precipitation with PEG 4000 and optionally heating are conducted either before or after chromatography. 

Beta-propiolactone is a well known chemical used in sterilization steps in order to inactivate viruses which are potentially present. However, it is very reactive which causes the chemical modification of proteins and there is also substantial loss of the anti-viral and anti-bacterial activities of the immunoglobulins. (Moeller US2013/0052208).

–Colloidal silica – AEX – Beta-Propiolactone: 

Stephan,( US4,318,902; EP0013901 equivalent in German) discloses a process for the preparation of IgM by treating an IgM containing fraction obtained by conventional fractionation (ie.., starting material can be Cohn fraction III  from blood plasma or serum with beta-propiolactone. Prior to the treatment, the IgM containing protein can be freed of lipids by treatment with colloidal silica gel and with crosslinked dextrans or diethylaminoethyl cellulose.  

–AEX-Beta-Propiolactone – UVC light irradiation: EP035255 described the preparation of an IgM concentrate for intravenous application with a reduced anti-complementary activity by using AEX, bet-propiolactone, UVC light irradiation and an incubation step at increased temperature. 

Octanoic acid (also known as Caprylate Acid)

–Octanoic acid (mixing with a vibrating agitator or stirring or vigorous mixing): 

Moeller (13/655,649 and US2013/0052208) describe a process for the preparation of an IgM immunoglobulin from plasma fraction by providing a plasma fraction adn mixing it with a C7 to C9 carboxylic acid such as octanoic acid with a vibrating agitator to precipitate containating proteins and then separating the precipitated proteins from the solution to yield the IgM containing immunoglobulin composition. 

Ng (Vox Sang 1993, 65: 81-86) discloses solubilization of a fraction III past with acetate buffer followed by stirring with 1.5 (v/w) octanoic acid, pH 4.66, for 4 h. Precipitate was then removed by centrifugation and supernatant was clarified by passed through filter.

Perosa (J. Immunological Methods, 128 (1990) 9-16) discloses a two step procedure for purification of IgG, IgA and IgM by deluting serum with acetate buffer and then (1) adding caprylic acid dropwise with vigorous mixing, centrigugation and then filtering the supernatant folowed by(2) addition of ammonium sulfate. The preciptiated Ig is collected by centrigutation.

–Octanoic acid – protease treatment: 

Rentsch (EP0835880, see also US 6,136,312) describes the preparation of an IgM containing protein solution for intravenous application by using octanoic acid precipitation followed by protease (pepsin) treatment ti redyce the anticomplementary activity. 

–Octanoic acid and AEX: 

The preparation of proteins solutions enriched in IgM without chemical modification by beta-propiolactone has been described.

Chtourou (US7,186,410) describes prepurification by precipitation of lipidic and proteic contamics using known agents such as octanoic acid, tricalcium phosphate or bentonite and a single AEX at alkaline pH thereby retaining immunoglobulins on the support. 

Miles (EP0345543) discloses starting with fraction III, suspending it in sodium acetate and adding caprylic acid. After aging at 5C for aobut 16 hours, the insoluble impurities are removed by centrifugation and filtration. The filtrate was was adjusted to 0.16 M sodium potassium phosphate at pH 6.5 and the IgM absorbed on an AEX such as Q Sepharose. 

Moller (US5,410,025) discloses  starting with a fraction such as Cohn II/III or III that contains immunoglobulin which can be dissolved in buffer and then eliminating most impurities by preciptiation with 0.5 to 5% octanoic acid at a pH of 4-6 and preferably 5 and then treating the solution at low conductivity with an AEX that binds most of the IgA and IgM. If the desired product is to contain IgA nand IgM, elution is carried out with a salt gradient that will elave about 10-20% of the IgM on the matrix. If the product is intended to contain IgA and IgM, the elution is carried out at a lower osmolarity and the IgM will remain absorbed onto th matrix. A pure IgM, extensively free of IgA, can be obtained by washing the AEX ahead of time with a buffer to elute the IgA before the IgM.

EP0413187 (the octanoic acid treatment is carried out by stirring for 15 in in order to remov lipids in the Cohn fraction III) and EP0413188 described subjecting a suitable protein solution to octanoic acid and AEX starting fro Cohn fraction III or II/III. 

–Octanoic Acid (by Stiring) – active charcoal – AEX – Beta-propiolactone:

Kotitschke (US 5,075,425) discloses a process for the preparation of immunogoglobulin suitable for intraenous administration from a human blood protein fraction containing IgG, IgA and IgM by adding octanoic acid, centrifuging, subjecting the supernatant to AEX (DEAE-Sephadex A), and filtering the solution. 

Moller (US 5,190,752) disclsoes preparation of IgM by starting with fraction that contains IgM such as Cohn Fraction III obtained by Cohn’s alcohol fractionation or the IgM fraction that occurs during the chromatographic isolation of IgG from blood is precipitated with 1-5% and preferably 2.5% caprylic acid. The supernatant that contains the IgM is applied to an AEX with DEAE, QAE or AMA groups for example, at a pH of 5.5-7.5. The IgM fraciton bound is eluted with a saline or pH gradient. Subjsequent to concnetraiton by UF, the IgM eluate is treated with 0.05 to 0.5 ml of beta-propiolactone per 100 ml of IgM solution. 

–Caprylic acid – PEG/AEX – virus inactivation: discloses preparation of globulin composition (56.8% IgM, 21.6% IgG and 21.7% IgA) from human plasma sarting with Fraction III from the Cohn/Oncley cold ethanol method or alternatively precipitate B from the method of Kistler and Nitschmann. The Fraction III paste is suspeneded in sodium acetate and caprylic acid is added. The filtrate which is adjusted with PEG, the IgM paste solubilized and virus inactivation. In a separate embodiment, AEX follows the caprylic acid treatmen

Miles (EP0345543) discloses starting with fraction III, suspending it in sodium acetate and adding caprylic acid. After aging at 5C for aobut 16 hours, the insoluble impurities are removed by centrifugation and filtration. The filtrate was adjusted to 7.5 PEG and the resulting IgM precipitate collected by centrifugation.

–Aerosil – Octoanoic acid beta-propiolactone -active charcoal:

Stephan,( US4,318,902; EP0013901 equivalent in German) teaches dissovling Cohn fraction III of human plasma, freeying it of lipids with 3#% aerosil, treating with DEAE-Sephadex A-50 and then treating with octanoic acid, then beta-orpiolactone. 

–Octanoic acid and Affinity chromatography:

EP0345543 describes a hihgly concentrated IgM preparation with at least 33% IgM for therapeutic use wehre octanoic acid precipitation is carried out by adding the actonoic acid and the isoagglutinins are removed by Synsorb affinity chromatography. 

Polyethylene glycol (PEG): It is known that IgM, IgA and alpha2 macroglobulin are present in Cohn fraction III. Precipitation of IgM is complete at 10% PEG and alpha2 macroglobulin at 20% while both IgA and IgG are partially soluble even at 20% PEG concentration. Based on this information, Wickerhauser (Vox Sang, 23: 119-125 (1972) prepared 2 sub-fractions: a 4-10% and 10-20% PEG fractions. IgM was present mainly in the first fraction and alpha2macroglobulin in the second fraction, while IgA was present in both. To prepare IgM from this, certain impurities were precipitated by zinc sulfate adn further purificaiton of the zinc sulfate supernatant was done by gel filtration on a Sephadex G-200 column. The IgM fraction was eluted about in the first quarter of the total protein eluate. Thsi fraction, recovered form the eluate by ammonium sulfate precipitation, contained 45% IgM at a yeild of 25%. It also contained about 22% IgA and a small amount of IgG, races of alpha2 macroglobulin of C-=3 complement and several other proteins.

Chromatography for the Purification of IgM

Affinity chromatography:

Brown (US 14.476,559) discloses where plasma IgM from a soublized Cohn fraction III precipitate (see above) is covalently bound to recombinant histidine tagged secretory component in vitro forming a secretory IgM within the protien mixture. The secretory IgM that is now tagged can be purified by affinity binding of the tag to an immobilized nickel or other divalent metal ion or other suitable binding moeity.

Corthesy (US 2015/0056180) disclosses preparation of human plasma IgM by affinity chromatography using CaptureSelect Human IgA resin using 3 different sources of plasma IgA; cryo-depleted plasma, resolubilised cold ethanol fractionation paste or a strip fraction from ACX. Secretory Ig was latter obtained by combining in vitro IgA with recombinant human secretory component.

AEX-heat treatment:

EP0413188B1 describes the preparation of an IgM enriched preparation for intravenous administration by using an anion exchange chromatography in order to reduce anti-complementary activity. Additionaly, a heat treatment at pH 4-4.5 at 40-60C was used to reduce the anticomplementary activity. 

Reduction of Non-specific Complementary Activity

Heat-treatment: Tsay (US 5,612,033 and EP0450412A1) disclsoes mild heat-treatment (40-62D)  in a solution having acid pH (preferablye 4-5) for at least about 10 minutes of IgM antibody concentrates diminishes non-specific complement activaiton without significant loss of normal immunologic effector functions. 

Commercial preparations

Pentaglobin: is a commercial immunoglobulin preparation which is enriced specifically for IgG (klingemann, Bone Marrow Transplant 1990 6(3) 199-202.

Purification of dimeric/polymeric IgA containing Secretory Component (SC)

Simon (US 15/205, 359, published as US 2016/0319039) discloses a method of purifying IgM with recombinant secretory component. According to the procedure a fraction III precipitate that is produced as a byproduct from production of IgM by ethanol fractionation of pooled human plasma is further purified by ion exchange adsorption purificaiton follwed by incubation with immobilized hydrolases to inactivate viruses and vasoactive substances. From 5-10% of plasma IgM is dimeric and polymeric IgA. The resulting dimeric IgAMis further coupled to recombinant secretory comonent that is produced by recombinant techniques. the coupling is accomplished by forming disulfide bonds under mildly oxidizing conditions. Dimeric IgM cotnaining both J chain and SC is again purified by ion-exchange and size exclusion chromatography and/or UF. The purified dimeric and polymeric IM  containing SC is iptionally stablized for example by the addition of human serum albumin to a final concentraiton of 5%. 

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

See also precipitation agents used,  See also Chromatography for removal of aggregates from plasma

Removal of Anti-complmentary Aggregates from IVIG Preparations

Isolated IgG preparation have marked anticomplementary activities (ACA). It has been shown that the components responsible for these activities are aggregates of IgG which form either spontaneously or as a result of the isolation procedure. These anticomplementary aggregates have been shown to be harmful such as anaphylactic shock. Accordingly varous solutions have been proposed to covercome the problem (Anderson, US 2003/0143222).

In Cohn’s plasma protein fractionation method, IgG is obtained from Cohn fraction II or II+III. However, the recovered IgG may contain a significant amount of aggregates because IgG tends to aggregate spontaneouly during storage of human blood plasma or tends to aggregate in contact with alcohol or other chemical during the fractionation procedure, or also tends to aggregate during lyophilization. If Igg containing aggregates is adminsitered intravenously, the aggregates may exert an anticomplement effect to cause serious anaphylactoid reacitons such as hypotension, chill and deverl. Methods used to remove aggregates from IgG include (1) the polyethyene glycol precipitation method (i.e., PEG is added to an aqueous IgG solution containing aggregates and the resulting precipitate of aggregates is separated by filtration), (2) the method of dissociating aggregates by reducing the pH of an aggregate contianing aqueous IgG solution to a low value such as 4 and allowing to stand so as to dissociate the aggregates, (3) or a combination of (2) above and addition of a slight amount of a proteolytic enzymes, (4) the removing aggregates by adsorption to an ion exchange resin, (5) adsorption to an adsorbent such as activated charcol 6) gel filgration method such as by using sephadex G-200 and (7) the membrane separation method such as by using a porous polymethyl methacrylate membrane which permits the passage of IgG monomer and dimer but blocks the passage of aggregates (Suzuki US 5,219,999). 

Four basic procedures exist for further processing of immunoglobulin obtained by Cohn fractioantion to prevent IgG aggregates. These are 1. enzymatic degradation by plasmin or pepsin, 2. chemical modification of the IgG molcule by beta-propiolactone or by cleavange of the interchain disulfide bridges by sulfonation or reduction and akylation, 3. slective elimination of aggregates by precipitation with PEG and hydroxyethyl starg (HES) and 4. adsorption of aggregates by DEAE gels such as Sephadex C50 Hou, EP0180766).

Enzymatic degradation

–Pepsin: US 3,966,906 describe treating a crude gamma globulin fraction with pepsin to aggregate IgG and reduce anticomplement activity.

See also (DE1148037), (US3966906, pepsin).

A drawback is that the immungolbulin Fc binding capacity is lower than for native immunoglobulins (Anderson, US 2003/0143222)

—-Stabilization of pepsin treated IgG preparation with PEG: has been disclosed (WO86/06993).

–plasmin: treatment of gamma globulin with human plasmin results in cleavage into 3 components of about 50k MW. When sufficiently low levels of plasmin are used, only about 15% of the molecules are cleaved, with 85% remianing as intact gamma globulin which show little anti-complement activity (J, Hinman, et al. Vox Sang 13:85 (1967).

Reduction and alkylation of SH groups: has also been disclosed to solve the problem of high ACA activity (US 3,902,262).

Precipitation with PEG: Prior patents directed to selective elimination of aggregates by precipitation with PEG in clude Coval (US 3,763,135),  (US 4,093,606) (US4,165,370) and Eibl (US4,276,283).

Acid Treatment:

–Caprylic (octanoic) acid: 

Kothe (US 5,164,487) discloses a method for manufacturing IVIG free of aggregates, vasoactive substances and proteolytic enzymes form a starting materials treated with 0.4-1.5% vy volume of octaonic acid and then chromatographed on an ion, CEX or hydrophobi matrix. 

–Low pH: 

When dissociation of IgG aggregates and denaturation of IgG mononmers contained in IgG preparation obtained using ethanol fractionation was examined at various pH values, treatment for 60 min at 28C at pH 3.8-4 was found to be optimum for obtaining such monomeric IgG. (Uemura, Tohoku J. Exp. Med. 1983, 141, 337-349. 

Other methods to remove aggregate removal

 DEAE Gels: Selective adsorption of aggregates by DEAE gels such as SephadexC50 after Cohn fractionation are reported by Curling, J.M et al. Vox Sang 33:97 (1997) and Suomela H., et al. Vox Sang 33:37 (1977), Ppier (US 3,664,994), Kandi (US4,136,094), Yokoo (US4,256,631) and Zuffi (US4,272,521).

Coppola (WO00/67789) discloses a method for producing an immunoglobulin preparation by subjecting an immunoglobulin solution to conditions which enhance the formation of immunoglobulin aggregates as by incubating the immunoglobulin solution at a pH of about 5.8 to about 8.0 at temperature of about 4-27 for at least 6 hours, then removing the aggregates as by filtration.

Reducing Amidolytic Content (e.g., FXI/FXIa)

Studies have shown that adminsitration of high levels of amidolytic activity may result in unwanted thromboembolic events. The EDQM (European Directorate on the Quality of Medicines & Health Care) provides for steps that have been shown to remove thrombosis generating agents. Emphasis is given to the identification of activated coagulation factors and their zymogens and process steps that may cause their activation. Serine proteases, generically known as coagulation factors, are integral components of both the contact activation and tissue factor pathways of the coagulation cascade. Upon a stimulus of the coagulation pathways, erine protease zymogens, which are inactive enzyme precursors, become activated proteases that catalyze the activaiton of the next protease zymogen, resulting in an activation cascade. Thsi coagulation cascade culminates in the activaiton of Thrombin (Factor IIa) and Factor XIIIa, which funciton to convert Fibrinnogen (Factor I) into Fibrin (Factor Ia) and cross-link fibrin to form a fibrin cot, respectively. The contact activation pathway, also known as the intrinsic coagulation pathway, begins with the activation of Kallikrein and Factor XIIa (FXIIa) from Prekallikrein and Factor XII, respectively. The activated serine proteiase FXIIa cleaves Factor XI (FXI), covnerting the zymogen into Factor XIa (FXI1), an active serine proteinase which participates in the subsequent activaiton of Factor Xa (FXa).  (Teschner (US2013/0058961)

Use of Cation Exchange to reduce levels of amidolytic activity: See Cation Exchange

For virus removal from plasma proteins by filtration see “filtration” and “particular components”

Taking into consideration how many blood donations are necessary for the production of IVIG, the prevention of the transmission of viruses is a key aspect in the development of such products. The state of hte art viral reduction or inactivation steps usually integrate into the purificaiton process the use o chemical agents as for instance during solvent/detergent (S/D) treatment, extreme physical conditions, such as low pH incubation or high termpature during pastuerization, ultraviolet light irradiation or exploiting the sieze difference to the target protein as for example in nanofiltration (Teschner “A new liquid, intravenous immunoglobulin product (IGIV 10%) highly purified by a state of the art process” VoxSanguinis 2007, 92, 42-55).

Kriel teaches 3 dedicated virus reduction steps (1) 3 component solvent detergent treatment, (2) a 35 nannofiltration step and (3) a prolonged incubation period at low pH and elvevated temperature (Kriel, J. Allergy Immunol., Februayr 2004, pS128 abstracts).

Treatments used Generally for Viral Inactivation

Low pH

Most proteins are damaged by exposure to the acidic conditions needed to kill viruses. For example, few viruses are killed at pH 5-5.5, a condition known to inactive factor VIII. Immune globulin solutions are an exception. Various studies have shown that low pH, such as pH 4 used in the preparation of immunogloublins, inactivates several enveloped viruses. (“Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, Wold Health Organization, WHO Technical Report, Series No. 924, 2004)

Low pH treatemtns at 37C for 20 h or more, initially intorduced to reduce anticomplementary activity, were later identified to inactivate >4 logs of several enveloped viruses including HIV and various model virruses. Incubation of IVIG at e.g., pH 4.25 for 3 weeks at 21C inactivates enveloped virsues such as HCV. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28.

Solvent/detergment (S/D) treatment

Organic solvent/detergent mixtures disrupt the lipid membrane of enveloped viruses. Once dsrupted, the virus can no longer bind to and infect cells. Non-enveloped viruses are not inactivated. The conditions typically used are 0.3% tri_n-butyl) phosphate (TNBP) and 1% nonionic detergent, either Tween 80 or Triton X-100, at 24C for a minimum of 4 hours with Triton X-100, or 6 hours with Tween 80. (“Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, Wold Health Organization, WHO Technical Report, Series No. 924, 2004)

S/D treatment has been used for viral inactivation of IVIG. The prcoess involves an incubation of a IVIG liquid intermediate in the presence of a solvent (tri-n-butylphosphate) adn generally one, sometimes two detergents (polysorbate 80, Triton X-100 and less frequently, sodium cholate). In most processes the incbuation lasts at least 1-6 h and is carried out at 20-35 C. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

–solvent/detergent treatment, 35-nm nanofiltration and low pH/elevated termpature incubation (Poelsler “A new liquid intravenous immunoglobulin with three dedicated virus reduction steps: virus and prion reduction capacity” Vax Sanguinis 2007). 

–(7655233, S/D treatment after fractionation and nanofiltration).

high temperature during pasteurization:

A problem with inactivating viruses with gamma globulins, particularly IgG, is that they have a poor heat stability. In this respect, stabilizers have been used. Hirao (EP0196761A2) discloses that the thermal stability of gamma globulin against heat treatment is markedly improved when at least one stabilizer such as a monosaccharide (glucose, mannose, galactose and fructose), a disaccharide (sucrose, maltose, lactose) or a sugar alcohol(mannitol, sorbitol, xylitol)  is added to an aqueous solution containing the gamma globulin prior to or at the time of the heat treatment. Thermal stability of the gama-globulin is further enhanced when at least one member slected from a neutral amino acid (glycine, alanine, valine, leucine, isoleucine), a neutral inorganic acid salt (halide of alkali metals or alkaline earch metals such as sodium chloride, potassium chloride and magnesium chloride), a surface active agent (nonionic surface active agents such as alkylphenyl-polyoxyethylene, anionic surrace active agents such as bile acid salts and cationic surface active agents such as benzalkonium chloride and polyhdric alcohols) and an organic carboxylic acid  (hydrocarbon residue such as an alkyl group or an aryle group (such as a phenyl group) and a carboxyl substitute attached), preferably about 3-15 carbon atoms,  is added to the solution in addition to the stabilizer. The kind of salts of the oranic carboxylic acids include alkali metal salsts such as sodium salts and potassium salts and alkaline earth metal satls such as calcium salts. Examples of the organic acid salts include alkali metal salts (sodium or potassium salt) of propanoic, butanoic, pentanoic, caprylic, caproic, malonic, succinic, tglutaric, adipic, citric and mandelic acid.

It is particularly suitable if prior to a virus inactivation treatment, the plasma protein recovered is further subjected to a dialyses against a medium with a low salt content (e.g., water) which may provide an additional stabilizing effect for the plasma protein because of the present of the mono- or dicarboxylate. Teschner (US8,709,492B2). 

Chen (US6,338,849) discloses process for preparing IVIG comprising dissolving Cohn’s component I in ice water, adjusting pH to between 3.5 and 5, filtering with an UF to remove alcohol and salt, sterilizing the immunoglobulin concentration at a temeprature between 59 and 6C for 10 hours and then filtering with an UF. 

Feldman (4,876,241) discloses inactivating pathogens in proteinaceous biological products by adding primary stabilizers selected from the group consisting of sugars and sugar alcohols and one or more secondary stabilizers selected form the group consisting of sodium acetate, potassium acetate, lithium acetate, magnesium acetate, ammonium acetate, barium acetate, sodium sulfate, ammonium sulfate, lithium sulfate, potassium sulfate and magnesium sulfate and subsecting the mixture ot a pathogen inactivating prcoess 

Precipitation: 

A very effective removal of viruses is accomplished by precipitation, especially where the precipitate is a waste fraction. Thus, the plasma protiens like albumin and IgG which are dervied after several subsequent ethanol precipitation steps are regarded as very safe.

Precipitation with ethanol is the single most widely used plasma fractionation tool worldwide. In addition to its use as a precipitant, ethanol is also a disinfectant. Unfortunately, it acts as a disinfectant mostly at room temperature or above, whereas plasma fractionation is carred out at a low temeprature to avoid protein denaturation. The contribution of ethanol ot viral safety through inactivation is, therefore, marginal at best. (“Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, Wold Health Organization, WHO Technical Report, Series No. 924, 2004)

Precipitation/filtration/diafiltration/dialysis: Starting Cohn Fraction II paste: is typically about 95% IgG and is comprised of the 4 IgG subtypes. Precipitation: Dissolve in a cold purified alcohol solution and impurities removed via precipitation and filtration. The IgG suspension can be dialyzed or diafiltrated (e.g., using ultrafiltration membranes have a nomimal MW of less than or equal to 100k daltons to remove the alchol. The solution can be concentrated or diluted to obtained the desired protein concentration and further purified (US20090148463).

Filtration:

Barrett (WO99/43362) discloses a method for eliminating pathogens from a solution containing plasma proteins by filtration. The solution is incubated in the presence of an inorganic, particulate, surfactant detergent and filtered in a deep filter and a clear solution is obtained. 

Van Holten (US6,096,872) discloses method for making virus free immunoglobulin formulation by missing an immunoglobuiln fraction isolated form human plasma with a high ionic strengh buffer containing a non-ionic detergent, performing nanofiltration using less than about 30 nm nanofile and collecting permeate of the nanofiltration. 

–Nanofiltration Filters: with nomimal poor sizes in the namometer range are well established tools for enchacing virus safety margins of plasma derived products, (Kreil, Transfusion, 46, 2006). Viral removal cn be chieved by nonfiltration where purified immunoglobulin solutions are passed through commerical filters of a controlled pore-size (typically 15, 20 or 35 nm) and defined design to retain viruses based on steric exclusion. The process has been shown to be robust and simple and contributes to the removal of both enveloped and non-enveloped viruses. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Irradiation with ultraviolet light (UVC):

ultraviolet irradiation, typicaly at a WL of 254 nm targets nucleic acid, thus a wide variety of viruses are inactiated, irrespective of the nature of their envelope. (“Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products, Wold Health Organization, WHO Technical Report, Series No. 924, 2004)

Caprylic aicd (Caprylate) (also known as octanoic acid): (see also fractionation -fatty acids)

Besides its role in IgG purification, caprylic acid in the non-inoniced form also acts as an agent that can robustly inactive lipid-enveloped viruses within a few minutes when used at pH < 6 and a concentraiton of >3.7 g/l. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Debart (US 15/27655, published as US 20170198009) disclsoses a method of preapring a solution of immunogloublin from an initial solution such as a fraction II+III in the presence of a polymer of glyocol such as polyethylene glycol (PEG) by adding caprylic acid to a concentration of 9-15 mM, adjusting the pH, incubating the solution for a time and temperature to inactviate enveloped virsues and then UF/DF. The us of cparylate at a lower concentraiton than described in the prior art and in the prresence of at least one polyether or polymer of glycol is disclosed as avoiding aggregates which forms as a result of cpaprylate at the concentraiton and pH (e.g., pH 5-5.2) in the prior art 

Lebing (US 5,886,154) teaches the use of caprylate for viral inactivation.

Seng (US4,939.176) reports a process for inactivating viruses by contacting solutions with caprylic acids with pH 4-8. 

Combination of viral reduction treatments: 

The newest generations of IVIG have unprecedented margin of viral saftety afforded by a combination of complementary dedicated viral reduction treatments, such as low pH treatment/nanofiltration, caprylic acid/low pH treatment, pasteurization/nanofiltration, S/D.nanofiltration/low pH treatment. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Anion Exchange with Viral Inactivation Steps

AEX-Viral Inactivation-CEX:  Burnouf-Radosevich (US 6,069,236) disclose a process for preparing immunoglobulin G from plasma which includes an anion exchange gel, a viral inactivation treatment and a cation exchange gel.

Precipitation-AEX-CEX-Virus inactivation-AEX-CEX: Laursen (6,281,336; WO99/64462) disclose a process for purifying IgG from crude plasma by adding a water soluble non-denaturating protein precipitant to preipitate a high proportion of non-immungolublin G proteins, recovering the clarified IgG supernatnat and applying it to an AEX and subsequently a CEX, eluting the IgG from the CEX and performing DF/UF, adding a virucidal amoung of virus inactivating agent, applying the IgG to AEX and subsequently to CEX, eluting the IgG from the CEX and performing DF/UF. 

precipitation (caprylate) – viral inactivation (sodium caprylate) -AEX:  Lebing, (US 5,886,154; EP0893450B1), disclose purification of antibodies from human plasma and other sources by suspending the antibodies at pH 3.8-4.5 followed by addition of caprylate acid and a pH shift to pH 5.0 to 5.2 to precipitate of contaminating proteins, lipids and caprylate forms is removed, while the majority of the antibodies remain in solution. Sodium caprylate is again added to a final concentration of not less than about 15 mM and incubated under conditions to reduce active virus (e.g., 1 hour at 25C). A precipitate (Mianly caprylate) is removed and the clear solution is diluted with water to reduce ionic strenght. Anion exchange is then used to obtain pure antibody prepration. (Lebing, Vox Sang 2003 (84):193-201) also disclose that caprylate is added to precipitate non-IgG proteins and to inactive enveloped viruses) (US20070244305, caprylic acid treatment, anion exchange, filtering).

Precipitation-AEX-UF-Virus Inactivation: (Ristol Debart (EP1225180B1)) discloses starting with a precipitate rich IgG obtained by ethanolic fractionation of human plasma, suspending the precipitate in an aqueous solution which contains a carbohydrate, preferably a sugar alcohol such as sorbiotol, precipitating the IgG accompanying globulins with PEG, filtration of the precipitate, bringing the filtrate to a pH of 5.7-6.3 and subjecting it to ion excahnge with anionic ligand, preferably those of DEAE. The liquid effluent is ultrafiltrated to reduce PEG contact and obtain a suitable concentration of protein to carry out subsequent steps of viral inactivation. 

AEX-Pastueurization-DF/UF: Aghaie (Human Antibodies 19 (2010) pp. 1-6) discloses redisolving Fraction II paste with glycine buffer and performing AEX on CM sepharose fast flow (FF). The eluate was then dia-ultrafiltered and sorbitol and glycine were added as stabilizers and the solution was pasteurized in water bath of 60C for 10 hours. The pastuerized solution was then filtered and then DF/UF. 

Cation Exchange with Viral Inactivation Steps

CEX-Virus Inactivation: Andersson (US2003/0143222; WO/2001/072844) discloses a method for producing IgG from plasma by removal of albumin, then adsorbing IgG to a CEX , collecting the adsorbed IgG fraction, adjusting pH to 4 (permits virus inactvation to be carried out around 30C) and virus inactivation of the Ig using chemicals such as a solvent/detergent (S/D) solution at 30C for at least 4 hours

Precipitation-Virus Inactivation-CEX-AEX: Sarno (EP0440483A2) discloses a multip step process for purifying an immune serum globuli fraction from crude plasma comprising precipitating non-serum globulin proteins from an aqueous supension of the crude plasma protein fraction, vrus inactivation, followed by absorbing the immune serum globulins onto a cation exchange followed by anion exchange to absort non-serum globulin contaminants.

CEX-Virus Inactivation-AEX: Andersson (US6835379) discloses a method for producing IgG from plams by removal of albumin resulting in an IgG fraction, absorbing Ig to a cation exchanger and collecting the absorbed IgG fraction, virus inactivation and and purifying plasma fraction obtained after remo

removal of euglobulins and albumin on an anion exchanger.  

CEX (strong)-Viral Inactivation-AEX (strong)-Virus Inactivation: Nardini (13/519368) discloses a process for the production of IVIG starting from human plasma or an intermediate plasma fraction enriched in IgG comprising capturing IgG on a strong CEX followed by viral inactivation performed by solvent-detergent treatment, elution with a buffer having 0.25-1M NaCL followed by a strong AEX where elution is performed using an increased ionic strenght or by means of pH variation. 

CEX-AEX-Nano/Dialysis-3 virus inactivation steps: Starting with separation of cryo-precipitate, optinal absorption of blood coagulation factors and antithrombin, subject supernatant to modified Cohn-Oncley cold ethanol fractional. Following method 6 of Cohn, fibrinogen prcipitated in factionation Iand raw immunoglobulin spearated form raw albumin by a II & III preciption. In traditional ethanol fractionation, the next step would be separation of fraction III form IgG (decrases IgG yeild by about 25%. This step is omitted. The II &III paste dissolved and filtered. Precipate y-globulin by 25% ethanol at neutral pH similar to Chon II precipitation (IgG purity 75%, impurities are claases A and M), dissolve paste and filter to obtain filtrate which is treated for 1st virus inactivation step (S/D treatment), carboxy methyl cation exchange used to separate S/D reagents, elute under high pH (8.5), adjust ph to 6, apply to anion exchange (impurities like IgA bound), nanfilter flow through, diafiltrate against .25 mol/l glycine and concetrate. (Tschner, Vox Sanguinis (2007), 92,42-55.

Virus inactivation (by detergent/solvent) – CEX: Aghaie (Human Antibodies 19 (2010) pp. 1-6) discloses taking a fraction II paste, redesolving it in glycine buffer, virus inactivation using TNBP as a solvent and Tween 80 as a detergent and then using CEX on CM Sepharose FF. The elute was then dia/ultrafiltered after adding stabilizers. 

DF-Stabilize-Pasteurize-CEX-DF: Debart (US 13/838424) discloses a method for obtaining a liuqid IgG composition from an IgG solution which has previously been purified from human plasma by DF to reduce below concentration undersirable components such as ETOH, stabilizing the solution by the addition of sorbitol, pastuerisation by heating treating the solution at a temeprature of between 55-63C for 1-24 hours, selectively adsorbing high molecular weight aggregates and/or polymers by CEX such as a strong CEX resin and diafiltering the flow through. Optionlly the process can include one or more viral inactivation/elimination treatments to complement the heat treatment of the solution. 

Virus inactivation (S/D treatment) – CEX-AEX – Virus inactivation (pH): Teschner (Vox Sanguinis 2007, 92, 42-55 (2006) discloses an IGIV 10% manufacturing process starting with separation of the cryo-precipitate, followed by the optional adsorption of blood coagulation factors and antithrombin. The supernatant of these steps is then the starting material for a modified Cohn-Oncely cold thenaol fractional in that fibrinogen is precipitated in fraction I (following method 6 of Cohn) and raw immunoglobulin is separated from raw albumin by a II+III precipitation. In traditional ethanol fractional as carried out in Gammagard SD process, the next step would be the seapration of fraction III from IgG which is known to decrease the IgG yeild by about 25%. Thus this step is omitted and instead the II+III paste is dissolved in buffer anf filtered through a depth filter for depletion of lipids and undissovled proteins. From this filtrate, gama-globulin is precipitated by 25% ETOH at neutral pH similar to the Cohn II precipitation. The resulting intermediate has an IgG purity of aobut 75%. The main impurities are IgA and IgM. The paste is dissolved and filtered to obtain a clear filtrate for the first virus inactivation step, the S/D treatment (1% polysorbate 80, 3% Triton X 100 3% TnBP) and separated from IgG using carboxy-methyl CEX. Elution of IgG is conducted by moderately increasing the conductivity under alkaline conditions (higher pH 8.5). The diluted euate is adjusted to pH 6 and applied to an AEX (DEAE – diethylaminoethyl) in flow thorugh mode (IgA is bound while IgG flows through). The flow through is nanofiltered. The nanofiltrate is diafiltrated against glycine and concentration to 10% protein w/v. After sterile filtration the liquid is incubated for virus inactivation at pH 4.4-4.9 for 21-22 days. 

The method of almost universal choice for large scale operation is “method 6” as described by Cohn. Briefly, in 1) crude fibrinogen is removed by centriguation from plasma by adding cold 53.3% ETOH to a final concentraiton of 8% ETOH, temp -2.5-3.  2) 53.3% ETOH is added to the supernatant to attain a concentraiton of about 25% ETOH, temp about ‘5 and pH about 6.9. The resulting precipitate (Fraction II+III) contains the immune globulins and other proteins which is removed by centrifugation at about -5. (3) a fraction consisting of most of the alpha globulins (and called IV-1) is precipitated by adding water to the 25% ETOH supernatant until a concentration of about 18% ETOH is reached, and the pH is adjusted to about 5.3, temp about -5. Fraction IV-1 is removed by centrifugation. (4) the ETOH concentraiton of the 18% ethanol supernatant is increased to 40 and pH adjusted to about 5.9. Under these conditions, another fraciton (IV-4) is precipitated and removed by centrifugation. (5) the 40% ETOH supernatant is clarified by UF at -5C and the albumin fraction precipitated therefrom by adjustment of the pH to 4.8 (with acetic acid-sodium acetate buffer). The precipitated albumin fraction is then removedd by centrifugation and further purified by dissolving in 10% ETOH and reprecipitating by adjusting the ETOH to about 40% at pH of 5.2.  from Tillman (US2,710,294)

 from Tillman (US2,710,294)

from Teschner (US8,921,520)

From Radowitz (US4,218,205)

From Liebing (WO2008/113589)

Frm McIntosh (US6,485,932) showing combined Fraction I, II and III precipitate

BayRho-D: (Bayer Biological)

Gamimune N (Bayer Biological)

Gammagard S/D: (Baxter healthcare)

IgAbulin (Immuno AG, Vienna, Austria) contains 90 mg of immunoglublin (of which 60 mg are IgA) per milliliter. (Tjellstrom (US 2002/0114802).

Intraglobulin (Biotest Pharma GmbH, Frankfurt, Germany). This product is made by beta-propriolactone treatment of gamma globulin. (Tenold, US4,499,073)

Polygam S/D (american Red Cross)

Sandoglobulin  (Novartis) contains 96% IgG with traves of IgA and IgM. (Tjellstrom (US 2002/0114802).

Venoglobulin (Alpha Therapeutic) (Green Cross Corporation of Japan) is prepared by treating gamma globulin with plasmin. It also contains 0.5 parts of a protein stabilizer (amino acetate) per part by weight of plasmin treated gamma globulin. (Tenold, US4,499,073)

VZIG (American Red Cross)

Sandoglobulin I.v. (Sandoz Pharmaceuticals)

Conditions/Parameters for CEX

Bind and Elute Mode:

If a CEX is used at pH 6.5-6.6, IgG binds to the resin whereas negatively charged protein fraction is unbound. An almost pure IgG can be isolated in one step this way. Falksveden “Ion exchange and Polyethylene Glycol Precipitaiton of Immunoglobulin G” ).

Hodler (WO/1995/018155) disclsoes a method of producing anti-D immunogolobulin of the IgG subclasses  1 and 3 from human plasma where plasma plasma from Rh-negative blood of donors sensitized to rhesus factor D or a plasma fraction containing anti-D IgG is subjection to CEX with carboxymethyl groups, the anti-D IgG being bound to the adsorbent, then washing with solution at a pH in the range of 5-8 and conductivity 2-4 mS/cm and subsequently eluting with pH 6-8 and conductivity 2-4 and then finally ocncentrating the anti-D IgG.

Teschner (US 2013/0058961) discloses methods for reducing the amidolytic activity (e.g.,FX1 and FX1a) and anti-complement activity (ACA) content of immunoglobulin composition through the use of CEX which includes eluting the IgG from the CEX with an elution buffer that includes a pH of at least 7.5 and a conductivity of at least 1.5 mS/cm to form and eluate that includes a leading porition and lagging protion and collecting the leading portion of a CEX eluate. The majority of the amidolytic activity present in the composition elutes off the resin in the lagging porition of the eluate. 

Flow through Mode:

Maneg (US 16/320,900, published as US 2019/0161533) discloses a prcoess for reducingthe properdin content of a properdin-containing IgG composition which includes CEX under conditions of pH and conductivity where properdin is bound to the column to obtain an IgG coomposition having a reduced preoprdin content. In one embodiment, the CEX is contacted withthe IgG composiiton at a pH in the range of 5-6 and a conductivity in the range of from 16-30 mS/cm and IgG is recoved in the flow-through.. 

Mintz (US2012/0294847) disclosea a method for remvoing a thrombogenic agent from an immunoglbulin solution by pvodiign the solution in the range of higehr than 3.81 to lower than 5.3, contacting with a support comprising immobilized negatively charged groups (CEX) and collecting the unbound fraction I (flow through).

CEX in Combination with other Purification Techniques

CEX-Virus Inactivation: Andersson (US6,835,379) teaches producing IgG from plasma by removal of albumin resulting in an IgG fraction and adsorbing IgG to a CEX and collecting the adsorbed IgG fraction which is subjected to virus inactivation. 

-Plasma-citrate-viral inactivation – CEX:

Zurlo (US 2007/0049733) discloses large scale alcohol free plasma fractionation which produces human IgG product. The process employs sodium citrate in two initial fractionation steps, followed by diafiltration to remove the sodium citrate. For the first fractionation, sodium citrate is added to plasma by gentle stiring such that the supernatant contains virtually of the IgG. For the second fractionation, additional soidum ictriate is added to the spernatant. The resulting supernatant contains virtually no IgG and virtually all the IG and other serum proteins are now found in the paste. Next remal of sodium citrate is done by diafiltration, viral inactivation is accomplished such as by solvent/detergent (S/D). Finally purificaiton is done by chromatographic purificaiton techniques. In one embodiment a weak CEX in bind and elute mode Toyopearl CM-60C resin is sued. 

CEX-AEX: 

Laursen (WO/1999/064462) discloses a process for preparating IgG from a crude immunoglobulin containing plasma protein fraction which includes the steps of preparing an aqueous suspension of crude immunoglublin containing plasma protein fraction, adding a water soluble protein precipitant to said suspension to cause precipitation of the non-immunoglobulin G proteins, applying a clarified IgG containing supernatant to CEX, washing out protein contaminants, eluting the IgG from the CEX, UF/DF, virus inactivation and then AEX and subsequently CEX, washing and leuting from the CEX and then UF/DF.

(Olas, Clinical Exp. Immun. 140, 2005, 478-490, human serum IgA prepared from plasma pool (Cohn fractions II and III) by ethanol precipitation and cation-exchange, followed by anion-exchange.)

Sarno (US 5,177,194) discloses a process for purifying an immune serum globulin fraction from a crude plasma protein fraction which includes precipitating non-serum globulin proteins from an aqueous suspension of the crude plasma protein faction, adding a virus inactivating agent, absorbing the immune serum globulins onto a CEX and subjecting the eluate to UF and then conctacting the concentrate with an AEX to absorbe non-serum globulin contaminants. 

PEG-CEX-PEG-AEX

Falksveden (US3,869,436) discloses a method of fractionating plasma proteins by removing blood corpuscles and cell fragments from the plasma by centrifugation, precipitating the globulins with PEG having a MW of about 6000 in a concentration of 10-15% by weight and at a pH of 6.5-8.0 at room temperature, centrifuging out the prcipitate which contains practically all the globulins (the solution contains albumin) and then dissolving the precipitate at pH 5.8 and centrifuging out undissolved fibrinogen, plasminogen and IgM, adsorbing the globulins in the clear solution on a carboxy methyl dextran CEX at pH 5.8, eluting the CEX to obtain the absorbed globulins including IgG, precipitate the eluate with additional PEG at pH 6.5-8.0, centrifuging and dissolving the precipitate and adsorbing all the globulins in the solution excluding IgG on an AEX at pH 6.6, cooling the IgG and precipitating with ethanol in a concentration of 25% by volume at -5 to -10C.

Lars-Gunnar (GB1344340) disclose a method of fractionating plasma proteins by precipitating the gloublins with PEG, dissolving the precipitate and centrifuging out undissolved fibrinogen, plasminogen and IgM, then adsorbing the globulins onto a cation exchanger and eluting with sodium chloride followed by precipitation with PEG and dissolving the prcipitate in buffer followed by adsorbing the globulins excluding IgG on an anion exchanger, cooling the IgG and precipitating with ethanol, separating the precipitate, followed by another ethanol step and finally dissolving in glycine.

Precipitation (ETOH)-Silicon Dioxide-CEX-AEX-NF-UF-DF:

Bruckschwaiger (US2013/0224183) disclsoes a method for preparing an enriched IgG composition by precipitating from 95-100% of IgG and A1pI from a Cohn pool in a first precipitation step by adding ethanol to a final concetnration of between 20 and 30% at a pH from 5-6 to form a first precipitate and first supernatant, suspending the first preciptiate to form a first suspension, treating the first suspension with finely divided silicon dioxide, separating the soluble porition of the suspension from the insoluble porition, where the soluble porition contains immunoglobulins and the insoluble contains A1pI, fibrinogen, Factor H and IaIp, binding the immunoglobuilins to CEX, eluting the Igs and contacting the eluate with an AEX and recvoering the IgGs that do not bind to the resin.

Bruckschwaiger (US2013/0224184) also disclose a method for prearing an enriched IgG from plasma by adjusting the pH of a cryopoor plasma fraction to about 7, the ETOH to about 25% at -7C–9C, spearating liquid from precipitate, resupsending precipitate, mixing finely divided SiO2 with the suspension, filtering the suspension, washing the filter press with at least 3 filter press dead volumes buffer, combining the filtrate with the wash and treating the solution with a detergent, adjusting the pH to about 7 and adding ETOH to about 25% , spearating the precipitate and disolving it in a solution containing a detergent for at least 60 minutes, passing the solution through a CEX and eluting, passeing the eluate through an AEX and passing this effluent through a nanofilter and then UF/DF.

Teschner (US12/789,345 and US20100330071; see also 13/949565) discloses a method of the following steps: 1) Separation of cryoprecipitates: separate liquid and precipitate form plasma by centrifugation, 2) Obtain Supernatant of Fractionation I: mix precooled ethanol with the liquid form (1) to form a mixture, (3) separate liquid and precipitate as by centrifugation (4) Precipitate of Fractionation II+III: adjust pH and ethanol concentraiton of liquid from (3) (5) separate liquid and precipitate from the mixture by centrifugation (6) Extraction from Fractionations II and III Precipitate: resuspend precipitate of (5) & Fumed Silica Treatment and Filtration: (7), mix silicon dioxide with suspension form (6) and obtain a filtrate by filtration, Fractionation of Precipitate G: (8) mix detergent and cold alcohol with the filtrate of (7) and obtain a precipitate by centrifugation, Suspension of Precipitate G: (9) dissolve precipitate Cation Exchange chromatography: (10) pass solution after (9) through a cation exchange chromatography column and elute proteins absorbed on the column Anion exchange chromatography: (11) and eluate from (1) through anion exchange to generate an effluent Nanofiltration: (12) pass effluent through a nanofilter Ultrafiltration and Diafiltration: (13) pass nanofiltrate through an ultrafiltration membrane Formulation: (14) diafiltrate the ultrafiltrate against a diafiltration buffer to generate a solution having a protein concentration of about 20% (w/v) (15) sterilize solution from 14 by filtering through a filter of 0.2 um or less.

Teschner (WO2013/033042A1) also discloses methods for reducing the amidolytic and anti-complement activity (ACA) contact of an immunoglublin composition by CEX where the Fraction II precipitate may be res-suspended in water or a low ionic strengh buffer and subjected to CEX. Typically, the pH of the re-suspended Fraction II precipitate is 5.2 and conductivity is low, ytpically no more than 1 mS/cm. The Fraction II suspension is then filtered to remove non-solubilized materail prior to loading onto a CEX. To elute the immunoglubilins, the CEX is then contacts with an elution buffer having a conductivity of at least 15 mS/cm (e.g., with a salt concentration of at least 150 mM NaCL) and a pH of greater than 7. In one embodiment, after loading a clarified Fraction II suspension onto the CEX, the resin is washed with a buffer have a sufficiently low conductivity such that the immunoglbuilinbs are not eluted form the resin and a Ph between 5.1-5.9. The pH of the elution buffer is greater than 75. and the ocnductivity of the eltuion buffer is at least 10 mS/cm.

Precipitation (PEG)-Virus inactivation -CEX-UF-AEX: Sarno (US5,177,194) disclsoes a multi step process for purifying an immune serum globulin fraction from a crude plasma prtoein fraction by adding a virus inactivating agetn to the clarified immuen serum globulin contining liquied, absorbing the immune serum globulins onto a CEX and subjecting the eluate to UF to concentrate the immune globulins and separate low MW species and contacting the concentrate with an AEX to absorb non-serum globulin contaminants. 

Disolving fraction I+II+III or II+III plasma fraction-Precipitation (caprylic acid)-AEX-Inactivate viruses (solvent/detergent) – CEX – DF-AEX-DF/UF

Park (US 15/123925, published as US 2017/0015732) discloses purification of an immunoglobulin by dissovling fraction I+II+III of II+III protein fraction followed by precipitation such as with carpylic acid, removing the precipitate and concentrating the supernatant followed by AEX in flow through mode, inactivating viruses as with solvent/detergent followed by CEX to remove the solvent/detergent, DF/UF and EX in flow through mode and then DF/UF.

Reduction of Particular Impurities

Amidolytic content (e.g., FXI/FXIa/proteases):

Teschner (US2013/0058961) discloses methods for reducing the amidolytic content of IgG immunoglobulin compositions (Factor XI and/or Factor XIa) by contacting a plasma derived immunoglobulin composition with a CEX under a first solution having a pH of no more than 6 and conductivity of no more than 11 mS/cm to bind the IgG and at least a fraction of the FXI and/or FXIa and eluting the IgGs from the resin by using an elution buffer having a pH of at least 7.5 and doncutivity of at least 15 mS/cm to form an eluate having a leading and lagging portion and collecting the elading porition of the eluate separately form the lagging porition of the eluate, whereas the leading porition of the eluate comprises no more than 80% of the eluate.

Anion Exchange

Almost pure IgG can be isolated in one step by AEX on DEAE ion exchangers at pH 6.5-6.6. Under these conditions, about 90% of the plasma proteins are negatively charged and consequently bound to the AEX. Positively charged, residual protein, mainly conisting of the IgG fraction is left unbound. (Falksveden “Ion exchange and Polyethylene Glycol Precipitaiton of Immunoglobulin G” , from “methods of plasma Protein Fractionation” by J.M. Curling, Academic Press 1980).  

Baumstart (Archives of Biochemistry and Biophysics 108, 414-522 (1964) dicloses separation of gamma globulin in high yield from undialyzed normal human serum by a rapid two stage procedure employing DEAE-Sephadex at pH 6.5. The chromatography on DEAE cellulose appears to be the method of choice for the samll scle or noncommercial production of gamma globuilin from human serum with Cohn alcohol precipitation being preferred for large scale. Web (Vox Sang 23: 279-290 (1972) also disclose a 30 minute preparative method for isolation of IgG from human serum with DEAE Sephadex. 

(US6093,324, subjecting plasma to macroporous anion exchange). (US7186410, purification and a single anion-exchange carried out at alkaline pH, thereby enabling the immnoglobulins to be retains on the support). (US7553938, caprylate and/or heptanoate ions and one or more anion exchange resins).

Bertolini (WO/1998/005686 discloses a method for the purification of immunoglobulins from plasma which comprises subjecting the plasma to macro-porous anion exchange. Preferred starting materials are plasma or plasma fractions obtained by the Cohn fractionation process such as Cohn Supernatant I (fibrinogen depleted plasma) or solubilised and clarified Fraction II + III. Where the starting material is plasma which contains lipoproteins, preferably a pretreatment step where lipoproteins are removed prio to the chromatographic factionation step.

Chtourou (US7,186,410) discloses praring immunoglobulin (IgG, IgA and IgM) from plasma by prepurifcation and a single AEX carried out at alkaline pH. 

Laursen, (US7138120, “process for producing immunoglobulins for intravenous administration and other immunoglobulin products”). Almost pure IgG can be isolated in one step by ion exchange chromatography on DEAE-ion exchangers at pH 6.5-6.6 and suitable ionic strength. Under these conditions, about 90% of the plasma proteins are negatively charged and consequently bound to the anion exchanger. Positively charged, residual protein, mainly consisting of the IgG fraction is left unbound. (If a cation exchange material is used instead, an opposite effect obtained, leaving the negatively charged protein faction unbound (Falksveden, “Ion exchange and polyethylene glycol precipitation of Immunoglobulin G, p. 95).

Moller (US5,410,025) discloses precipitation of a Cohn Fraction II/III or III in a buffer, elimination of impurities by precipitation with octanoic acid at pH 4-6, then treating at a low conductivity with an AEX, attaching most of the IgA and IgM Since the anticomplementary activity of the IgG fractio nnot attached to the AEX is low, the fraction can be employed in conjunction with the fractions taht contain IgA and/or IgM to prepare mixture that can be converted into IVIG preparations.

Plasma-citrate-viral inactivaiton -AEX:

Zurlo (US 2007/0049733) discloses large scale alcohol free plasma fractionation which produces human IgG product. The process employs sodium citrate in two initial fractionation steps, followed by diafiltration to remove the sodium citrate. For the first fractionation, sodium citrate is added to plasma by gentle stiring such that the supernatant contains virtually of the IgG. For the second fractionation, additional soidum ictriate is added to the spernatant. The resulting supernatant contains virtually no IgG and virtually all the IG and other serum proteins are now found in the paste. Next remal of sodium citrate is done by diafiltration, viral inactivation is accomplished such as by solvent/detergent (S/D). Finally purificaiton is done by chromatographic purificaiton techniques. A strong AEX Toyopearl AQE-550C resin is performed in flwo through mode

Anion Exchange in combination with other techniques

Cohn Fraction I+II+III – Glycine extraction + AEX: Levy (WO03/034982) teaches a method for purification of immune globulins from Cohn’s fraction I+II+III or II+III prepared from plsma by precipitation of the paste at 20% ethanol and pH 6.7-6.8, followed by glycine extraction and the AEX. 

–Cohn Fractionation – caprylate acid -AEX:   (see also fatty acids used as precipitation agent)

Alred (US20030152966) discloses a process for the purification of antibodies from human plasma by addition of caprylic acid and a pH shift to pH 5.0 to 5.2. A precipitate of contaminating proteins, lipids and cprylate forms is removed while antibodies remain in solution. Sodium caprylate is against added. Anion exchange is used to obtain an exceptionally pure IgG.

Buchacher (US 7,553,938) discloses preparing a preufied virus inactived antibody prepraration from a starting solution by adjusting the pH to about 4.6-4.95, adding caprylate, diafiltrating the filtered solution, applying the filtered solution with at least one AEX. 

Lebing (6,307,028) discloses the purification of antibodies from plasma by addition of caprylic acid where the precipitate of contamination proteins, lipids and caprylate forms are removed followed by anion exchange chromatography using two different resins to obtain a high yield of IgG.

Lebing (US5,886,154; see also Lebing, “Properties of a new intravenous immuhnoglobuiolin (IGIV-C, 10%) produced by virus inactivation with caprylate and column chromatography, Vox Sanguinis, 2003, 84, 193-201) discloses supsending a composition containing precipitated immunoglobulins such as a fraction II+III past, dissolving the immunogloublins into solution by lowering the pH 3.8-4.5, preferably 4.2 by addition of an acid like acetic acid, adding a source of caprylate ions and adjusting Ph 5.0-5.2, remove precipitated proteins, lipids and caprylate by filtration, addition of further caprylate and passing the solution through two AEX.

Parkkinen (WO2005073252) teaches a process for preparing a purified Ig preparation by subjecting a crude Ig solution to cprylic acid treatment, removing protein aggregates and viruses from the Ig solution and subjecting the Ig solution to AEX.

–PEG treatment – AEX: 

Hirao (US 6,159,471) discloses starting with a gamma globulin fraction and subjecting it to low concentration of PEG, recovering the supernatant, treating the suspension with PEG have a MW of from about 1 to 10k, centrifugation and then treating the supernatant thus separated with a high concentration of PEG, centrifugation and then disoolving the gamma-globulin containing fraction and contacting it with an AEX to recover the non-adsorbed fraction.

Anion-Cation exchange: 

(Bertolini (US2011009244) discloses Large scale separataion of alpha-1 proteinase; US7879800).

Burnouf-Radosevich (US6069236) using a series of chromatographic separation steps without ethanol precipitation to produce plasma derived IgG. In one embodiment, the process includes a deslating step, AEX, viral inactivation, CEX.

Mamidi (WO 00/76534) discloses producing IVIG substantially free of viruses by starting with a Cohn Fraction II + III or Cohn Fraction II paste or in the alternative the Fraction II + III paste can be subjected to a preliminary washing procedure for form Fraction II + IIIs (done by suspending Fraction II + III precipitate in cold water and adding sodium phosphate solution) heat treating a gamma globulin solution for viral inactivation, PEG fractionation, and then a second viral inactivation using solvent-detergent which can be carried out prior to or following AEX, inacitvation of viruses using a a solvent detergent solution containing a mixture of tri-n-buyl phosphate (TNB) and polysorbate 80, then CM Sepahdex C0-50 (weak CEX) was added to the solution which was mixed and filtered. The resin contining the adsorbed IgG was washed and eluted with 1.4 M sodium chloride and the eluate was clarified, concnetrated and diafiltered with cold water. D-sorbitol was added. 

Tanaka “High quality human immunoglobulin G purified form Cohn fractions by liquid chromatography” Brazilian J of Medical and Biological Research 2000, 33: 27-30) discloses using Q-Sepharose FF AEX followed by CM-Sepharose FF CEX and then gel filtration for purificaiton of intravenous IgG from F-I+III+III or F-II+III pastes preared by the Cohn method. 

–Precipitation (salt)-AEX-CEX (low capacity):

Zurlo (US Patent Application 17/560,163, published US 2022/0204556) discloses a method of isolating an antibody from a solution such as plasma from contaminants such Factor IX or Factor XI which includes the steps of adding a salt to the solution to generate a supernatant and a precipitate, dissolving the precipitate and appplying the dissolved precipitate to a frist ion exchange media such as AEX in flow-through mode and applying the first flow-through to a second IEX such as CEX also in flow through mode where second exchange media has a limited capacity such that greater than 70% of the content of the target protein is recvoed in the second flow-through. The size of thsi small or low capacity CEX is selected so that it is near or slightly greater than the amout or capacity for breakthrough of a contaminating protein found in the flow-through of the AEX. The inventors believed that the contaminating proteins displace any IgG that may temperarily bind the the CEX media. Careful selection of the amount/capacity of the CEX provides efficient removal of contaminating protein while also providing high yields of IgG in the flow-through fraction. 

–Fraction I+II+III or fraction II+III – precipitation (PEG/ caprylic acid or ammonium sulfate) -AEX- solvent/detergent – CEX-AEX

Maneg (US 16/320,900, published as US 2019/0161533) disclsoes preparing an immunogloublin composition from a plasma dervied fraction that includes Cohn fraction I/II/III or Kistler-Nitchmann fraction A+1 which includes the steps of  resuspending the Cohn fraction I/II/III fraction under conditions to adjust the conductivity to at least 1 mS/cm to obtain a suspension contining resolubilized IgG, IgM and IgA, subjecting the resolubilized immunogloublin to precipitation with as with octanoic acid and removing contaminating protection form the suspension as by filtration (the impurity depleted immunoglobulin composition typically has an IgG content 8.5-94% by weight, IgA content 3-9% by weight and IgM 3-9% by weight at this point), subjecting the impurity depleted immunoglobulin composition to AEX under conditions of pH and conductivity to substantially bind IgM and IgA and optionally IgG to the AEX (so IgG enriched immunoglobulin may be obtaiend int he flow-through fraction and/or by eluting the IgG), and then subjecting the IgG enriched immunoglobulin to CEX under conditions where properdin is bound and recovering the IgG in the flow-through fraction and/or by eluting the IgG. . Following the procedure the IgG content was at least 45 g/l, 95% by weight, a properdin content of not mroe than 001 ug/mg and a IgG polymer content of not more than 0.05%.  In a specific example, Maneg outlines the procedure as follows: To obtain the Cohn fraction I/II/III a cryopprecipitation step was first perfromed to separate factors such as Factor Viii, von Willebrand Factor and Fibrinogen by adjusting temperatrue under gentle stirring at 2 C such that the cryoprecipitate remains undissolved in the thawed plasma which could be separated by centrifugation, from the sueprnatn of the cryoprecipitation step the Cohn fraction I/II/III was precipitated by ethanol precipitated (temp -5C, 20% ETOH), the Cohn fraction I/II/III precipitate which includes all the immunoglobulins (IgG, IgA, IgM) was resuspended with sodium acetate buffer, pH 4.8 and treated with octanoic acid and calcium phosphate treatment, the filtrate was diafiltered and subjected to virus inactivation at pH 4.0 and then subjected to AEX using a macroporous POROS 50 HQ AEX with the IgG enirched composition obtained in the flow-through fraction (the IgM/IgA) enriched fraction could also be obtained by eluting by increasing conductivity). The IgG enirched flow through fraction was then subjected to CEX with the flow through and wash fraction collected and further processed by nanofiltration. 

Park, (US 15/123925, published as US 2017/0015732) discloses preparing an intravenous immunogolbulin by dissolving with distilled water an immunoglobulin containing plasma protein fraction I+II+III or fraction II+III followed by precipitation using polyethylene glycol (PEG), carpylic acid or ammonium sulfate, , removing the precipitate, filtering the supernatant and subjecting the concentrated filtrate to AEX such asdiethylaminoethyl (DEAE) in flow through mode to remove caprylate and other plasma proteins, treatinghte recovered fraction with asolvent/detergent to inactivate viruses, following by CEX and then dialyzing and/or concentrating the eluate from the CEX and subjeting the eluate to AEX in flothrough mode followed by filtering the recvoered fraction through a virus filter and dialyzing and/or concentrating the filtrate. 

–Fraction II Paste –dissolvaing –dialyzing/concentration –AEX –solvent/detergent –CEX –dialyzing/concentration

Son (US 15/123,869, published as US 2017/0022248) discloses a method for purifying an immunogloublin by dissolving a fraction II paste, followed by filtration to obtain a fraction II solution, dialyzing/concentration the fraction II solution and subjecting this to AEX in flow through mode, treating the recovered fraction with a solvent/detergent to inactivate viruses followed by subjecting the fraction to CEX to remove the solvent and/or the detergent and thrombotic substances, dialyzing/concentraiton the eluate from the CEX and then filtering.

–AEX-CEX-AEX-CEX (connected in series):

Laursen (US 2001/0051708; see also WO 99/64462) also discloses a process for purifying IgG from crude plasma by adding a water soluble precipitant to cause precipitation of non-immunoglobulin G proteins, applying the IgG to an anion exchange resin and subsequently a cation exchange resin, performing a dia-UF and performing a virus inactivation step. Laursen (US6281336) also disclose purifying IgG from a plasma fraction which includes adding a water soluble non-denaturating protein precipitant (e.g., PEG, caprylic acid and ammonium sulphate) to a crude immunoglobulin containing plasma protein fraction to precipitate non-immunoglobulin G proteins, recvovering the IgG containing supernatant and applying the supernatant to an anion exchange chromatography followed by cation exchange chromatography. Laursen (US7138120 and US2001/0051708) also teaches a process for purifying immunoglobulin G from a crude plasma protein fraction in which AEX operated in flow through mode and CEX operated in bind and elute mode are preferably connected in series. The use of two serially connected chromatography columns makes the operation more practical in that there is no need for intermediary steps of collecting the IgG containing fraction between the two IEX. Laursen (EP2272870) also discloses preaparing an aqueous suspension of crude immunoglobulin containing plasma protein fraction, adding a water soluble non-denaturating protein precipitate to cause precipitation of non-immunoglobulin G proteins, recovering the clarified IgG supernatant and applying it to AEX and subseqeuntly a CEX, washing the protein contaminants from the CEX with a buffer having pH and ionic strenght sufficient to remove the contaminats from the resin without causeing elution of IgG, eluting IgG from the CEX with buffer ahving a pH and ionic strenght sufficient to cause elution of the IgG, performing DF/UF and adding a stabilzing agent, virus inactviation, applying again to an AEX and then CEX.

In the 1970s, chromatography was also found to be useful in the separation and purification of plasma proteins, particularly for the final purification of gamma globulin after separation from the plasma by Cohn or modified Cohn methodologies. Immunoglobulin purified from normal human plasma has proved effective in the treatment of various diseases when adminsitered intravenously.

The ion-exchangers consist of ligands of strong anionic type (quaternary ammonia-ethyl: AQE) and weak anionic type (diethmlamino etyl: DEAE), or strong cationic (sulphopropyl: SP) and weak cationic (carboxymethyl: CM). The ligands are covalently immobilised on insoluble supports or matirices such as silica (ceramics), acrylic (polyacrylamides, polystyrene), carbohydrate (cellulose, dextran, agarose). Those formed by destran or agarose are most efficient and most used. (Ristol Debart (EP1225180B1))

Affinity Chromatography

Preparative steps can be used to enrich a particular isotype. For example, protein A chromatoaphy can be used to enrich a mixture of immunoglobulins for IgG (US5180810).

Protein A – AEX: Merlino (Giorn Batt. Virol. Immun. LXXVIII, 77-85, 1985) (abstract only) discloses loading serum on a Protein A sepharose column, IgG3 is recovered as they do not bind to the Protein A. IgG of the other subclasses are eluted, equilibrated at pH 6 and loaded on a AEX wehre IgG4 is retained and then recovered by lowering the pH wehreas IgG1 and IgG2 pass through the column and can be recoved.

Multi-column Immuno Affinity columns

Typically, protein A columan can only be loaded to about 30-50% of their maximum binding capacity, because at higher loads, product is lsot in the breakthrough. This means that much of the resin resmins unused, leading to a production cost increase. A straight forward solution is to capture teh mAb that is breaking through in a second column. This very simple principle is the basis of a novel multi-column chromatography capture process, teh Capture SMB or 2-column periodic countercurrent chromatography (2C-PCC) process. (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Bataille (US 15/125483, published as US 2017/0073396) discloses using a an IgSelect gel from GE Healthcare (ligand si from CAC, BioAffinityCompany) which specifically binds the Fc fragments of human IgGs and  Capture Select FcXL affinity gel from Life Technologies which specifically binds the CH3 domain of the 4 human IgG subclasses on 4 columns, controlled sequentially an an automated system followed by diafiltration concentraiton of the eluate. Elution with the IgSelect was carreid out with 0.1 M glycine solution, pH 3. 

Burton (US 8,198,407) discloses sequential protein isolation and purificaiton from a biological sample such as plasma by affinity chromatography which is conducted using ligands or ligand support complexes that selectively and specifically bind to proteins in the sample. The ligands/support complexes are contacted sequentally in a predetermeind order with the sample to sequentially bind a protein from the sample. For example, the target protein may be fibrinogen, immunoglobulins and albumin such that albumin is siolated from the plasma prior to or after the IgG. 

Naylor (US 2005/0042772) discloses methods for depletion of proteins suchas albumin, IgG and a third abundant protein such as fibrinogen from a plasma sample using affinity columns such as Protein A columns and anti-fibrinogen columns. The removal of abundant proteins facilitates the detection of lower conentraiton proteins. 

Ion Exchange Chromatography Generally

Generally: Cohn Fraction (I+)(II)+III ethanol precipitation (Tanaka, Braz J Med Biol Res 2000 (33)37-30) coupled to column chromatography.

Teschner (Vox Sang, 2007 (92):42-55) have described a method for production of a 10% IVIG product in which cryo-precipitate is first removed from pooled plasma and then a modified Cohn-Oncley cold ethanaol fractionation is performed, followed by S/D treatment of the intermediate, ion exchange chromatography, nanofiltatration, and optionally ultrafiltration/diafiltration.

Ion Exchange – Filtration (activated carbon) – Affinity: Hou (US4639513) discloses a method for producing IVIG comprising passing prefiltered human plasma through an ion exchange column (IgG passes through the column and other components remaining behind), the IgG stream then passes to an affintiy column. Hou (US 4,639,513 see also EP0180766) also discloses a process for isolation of IgG from plasma where after a first dilution step using deionized water to insolubilize lipis, plasma is subjected to filtration with preferred materials such as activated carbon to remove micron particles such as lipids and then the filtrate is passed to IEX where IgG passes through the column while other large molecule proteins remain absorbed to the column. Proteoytic enzymes are then removed from the IgG by affinity chromatography. 

Cohn Fractionation – CEX – AEX: Sarno (US 5, 177,194)  teaches starting with a plasma protein fraction such as the Fraction I+II+III precipitate from a Cohn fractionation proecdures (this is usually obtained by subjecting a conventional cyroprecipitate supernatant to cold ETAOH at pH 6.9. In addiition to the immune serum globulins, the Cohn Fraction I+II+III contains fibrinogen, various lipoproteins, several proteins involved in the hemostatic and fibrinolytic systems and numerous mino components) or in the alternative a Cohn Fraction II+III or Cohn II fraction. The first step involves suspending the plasma fraction in water at acidic pH, pH 4.5-5.5, precipitating out the non-serum globulin proteins from the suspension using a precipitant such as pEG and recovering the clarified immune gloublin containing liquid (as by centrifugation and filtration), inactivating viruses, contacting the solution with a CEX to remove the virus inactivating agent and other non-serum globulin contaminants, eluting the immune serum globulins, UF, and then contacting with AEX. 

Anion Exchange  See outline (also see virus inactivation)

Cation Exchange See outline (see also virus inactivation) 

Multi-Ion Exchange (in series)

Laursen (WO 99/64462) dcloses a process for purifying IgG from a crude IgG containing plasma protein fraction using AEX (DEAE Sepharose resin in flow through mode) and CEX (CM Sepharose in bind and elute mode) connected in series. The eluted IgG fraction from the CEX is desalted by UF/DV against a buffer that includes sorbitol. 

Hydrophobic Interaction Chromatography (HIC)

Kothe (US7,041,798) discloses fractionation of plasma or serum into at least one albumin fraction and one immunoglobuilin fraction by HIC using an incremental salt gradient such as ammonium sulfate buffer. The separation is based on the interaction of hydrophobic domains of the proteins with hydrophobic groups on the media. Under physiological conditions, the hydrophobic groups of the proteins are not freely accessible so that binding to the HIC does not take place. By adding the salt, the hydrate sheath of the proteins is decreased so that the hydrophobic domains are available for HIC. The first fraction from the HIC is an albumin fraction and is similar in composition to a supernatant II/III of the Cohn alcohol fractionation. It contains albumin, transferrin, antithrombin II and alpha-antitrypsin. All immunoglobuilins are contained in fraction 2 which is similar to the composition of a past II/II obtained by cold thenaol precipitation. A lipoprotein fraction remains bound to the media and can be eluted by reducing the ammonium sulfate concentration to 0 moles/L.  Fractions 1 and 2 obtained from the HIC can be processed further into therapeutically usable plasma protein solutions by known methods. For example, IVIG can be produced form the fraction 2 using anion exchange chromatography, optional virus filtration, treatment with actanoic acid, CEX and the usual concentrating, filtering and sterilizing steps. 

In combination with Expanded/fluidized Bed chromatography  See definitions for definition of EBA

Lihme (US2007/0299251) discloses a process for the isolation of protein(s) from a solution by applying the solution to eitehr a packed bed or expanded bed comprising an adsorbent which comprises a functionalised matrix polymer carrying a plurality of covalently attached funcitonal groups comprising an aromatic or heteroaromatic ring system and one or more acidic groups. The absorbent also comprises a particle density of at least 1.5 gml and a mean volume particle diameter of at most 150 um. EBA offers a robust process comprising fewer steps and can be scaled up to industrial scale without any significant considerations regarding increased back pressures or breakdown of the process due to clogging of the system which often is a problem when using packed bed columns. The source of the protein for the procedure maybe by croy-poor plasma, sueprnatant I, supernatant II+III, supernatant IV-1, supernatant Iv-4, resolubilised cryo-preipitate, resolubilised fraction 1, resolubilised fraction II_III, resolubilised frraciton IV-1, resolubilised fraction IV-4, resolubised fraction V. 

–Caprylic acid – HIC (on fluidized bed): 

(Chtourou, US14/131944) discloses a method for preapring human polyvalent immunoglobulins from blood plasma by removing protein contaminants with caprylic acid to obtain a solution free of proteases and a fluidized bed chromatography step which may be of of the ion exchange, affinity or mixed-mode type. 

Ultrafiltration/diafiltration: 

The last manufacturing steps of IVIG before the formulation is an ultra/diafiltration step or washing out unwanted salts present in the IgG preparation and for adjusting the desired protein concentration (Ahrer (J.Membrane Science, 274 (2006) 108-115).

In General:

Separation of Immune globuline (IgG) from blood plasma depends upon the early work by Edwin J. Cohn which is based on cold ethanol fractionation which co-precipitates groups of proteins based on their isoelectric points at given alcohol concentration. For this method the plasma is treated with ethanol at below 5C and by increasing the concentration of ethanol and reducing the pH, a succession of fractions are precipitated. The five most abundant proteins were enriched in Fractions I to V by sequential precipitation by increasing concentration of ethanol are described in fractionation. Applying Cohn’s fracionation, IgG can be obtained from fractions II+III. For example, Oncley used Cohn Fractions II+III as starting material with different combinations of cold ethanol, pH, temperature and protein concentrations to produce an active immune globuilin serum fraction. 

Variations to the Cohn Fractionation have been developed with the aim of improving polyvalent IgG recoveries. For example, Oncley, used Cohn Frations II+III as a starting material with different combinations of cold ethanol, pH, temperature and protein concentration to those described by Cohn, to produce an active immune globulin serum fraciton. Today, the Oncley method is the classic method used for proudction of polyvalent IgG. Neverthess, it is known that about 5% of gamma-globulins (antibody-rich porition) is co-preciptiated with Fraction I and about 15% of the total gamma-globulin present in plasma is lost by the Fraciton II+III step. The Kistler/Nitschmann method aimed to imporve IgG recvoered by reducgin the ethanol content of some of the precipitaiton steps (Precipitation B vs Fraction III). The increased yield, however, is at the expense of the purity. 

Despite the fact that many commerical plasma frationation processes still use the original Cohn process or a variation thereof (e.g., Kistler/Nitschmann), it is known that about 5% of gamma globuilins (anitbody rich fraction) is co-precipitated with Fraction I and about 15% of the total gamma globuilin present in plasma is lost by the Fraction II+III step. The Kistler/Nitschmann method aims to improve immunogoblulins recovery by reducing the ethanol content of some of the precipitation steps (Precipitation B vs Fraction III). The increased yield, however, is at the expense of the purity.

In the classical Cohn-Oncley process, fraction II (IgG) was further purified by at least 3 additional precipitaitons with IgG losses at each step. Some manufucatures thus limit IgG precipitation form plasma to a single cold ethanol precipitation step to produce what Cohn referred to as fraction I+II+III. IgG losses are minimized by using I+II+III (or II+III) if fraction I-fibrinogen is precipitated earlier) as the starting material for AEX and virus inactation and removal steps that have been incorproated into the process. (Hooper, Immunol Allergy Clin N Am 28 (2008) 765-778). 

Cohn-Oncley Ethanol Manurfacturing Schemes

Method 6 of Cohn is particulalry suited for application on an industrial scale and has become the starting material for the isolation of most IVIG processes. Method 6 is in industrial use in many countries, but is seldom carried out according to the original description. Many modifications exist. As an example, see J.G. Watt, Clinics in Haematology, 5, 95-112 (1976) and P. Kistler and H. Friedli in “Methods of Plasma Protein Fractionation”, edited by J.M. Curfing, Academic Press (1980), pages 3-15. Fractionation of Fraciton II+III may continue by the use of ethanol or by other precipitation agents like PEG or caprylate. Deutsch (1945) increased the yield of IgG in Fraction II by lowering the ionic strenght for precipitation of Fraction III. Oncley’s (1949) separation method 9 for 

separation of Fraction III from Fraction II -III was based on the work of Deutsch.

In Method 9, a supernatant liquid containmign albumin and a precipitate consisting of gamma globulin can be obtained by treating human blood plasma with ethanol, under conditions of pH 6.9 and thanol concentration 25%. (described in J. Amer. Chem. Soc., 71, 541-50 (1949). 

SCHEME 1: Fraction I Precipitation step -Fraction II+III preciptiation step — Fraction II preciptiation step:

In one purificaiton scheme, a Fraction I preciptiaiton step and a fraction II_III precipitaiton step prior to a Fraction II preciptiation step is used. Teschner (US 2013/0058961)

1. Fraction I Preciptation:

In one embodiemtn, a fraciton 1 preciptiation is foremd by adding ethanol to cryo-por plasma to a final concentraiton of from 6-10% (v/v) at a p 6.7-73. The mixture is tehn incubated while stirring at form -4-2 C. The resulting Fraciton 1 preciptiate can be separated form the Fraction I supernatnat by centrifugation or filtration. The majroity of the immunoglobulin content is present in the fraciton I superantant which can be further enriched. Teschner (US 2013/0058961)

2. Fraction II Precipitation (Fraction II+III precipitation):

To further enrich the IgG content, a a Fraction I superant is used as the material for a second preciptaiton step (Fraction II+III) preciptiation. Fraciton II+III preciptiaiton is performed by adding ethanol to the fraction I supernatnt to a final concentraiton of form 22-28% (v/v) at a pH between 6.7 and 7.3. The mixture is then incubated while stirring at between -10-4C. The resulting Fraction II+III preciptiate can be separated form Fraction II+III sueprnatant by centrifugation or filtration which is generaly perforemd in the cold. The majroty of the immunoglbouin is present in the Fraciton II+III precipitate which can be re-suspended and ruther enriched. Teschner (US 2013/0058961)

3. Fraction II Precipitation:

To further enrich the Ig G content a third alchohol precipitaiton step (Fraciton II precipitaiton) is pformed. Fraciton II preciptiaiton is perforemd by adjsuting the pH of a Fraction II_III preciptiate suspension between 6.7-75 and adding ehtanol to a final concentraiotn of 22-28% (v/v). Teh mixture is then incubated while stirring at between -13-2C. 

The mixture is then incubated while stirring at between -10-4C. The resulting Fraction II+III preciptiate can be separated form Fraction II_III sueprnatant by centrifugation or filtration which is generaly perforemd in the cold. The majroty of the immunoglbouin is present in the Fraciton II+III precipitate which can be re-suspended and further enriched. Teschner (US 2013/0058961)

Examples/schemes which Process Fraction II/III precipitate

Buchacher (Biotechnol. J. 2006, 1, 148-163) teaches ethanol fractionation for the production of IVIG which includes two separate precipitation steps with alcohol. 

Bruckshwaiger has a number of patent applications modifications over the Cohn process: (1) (US13/085056, now US 8,796,430) also teaches that at least one of the precipitation steps can be by spray addition of the alcohol. (2)  (US 13/653,332, nos US8,940,877) teaches a method for prearing an enriched IgG from plasma by conducting a series of precipitation steps with alcohol at various pHs. In one embodiment cryo-poor plasma fraction is first precipitated with from about 6-10 alcohol at a pH of from about 7-7.5, the supernatant is then precipitated with from about 23-27% alcohol at a pH of from about 6.7-7.1 at a temperature of from -7 to -9°C to form a second precipitate. This second precipitate is then resupended and precipitated with from 22-28% alcohol at a pH of from 6.7-7.3 to form a third precipitate which is resupended to form a suspension and spearating the soluble fraction from this supension to form an enriched IgG composition. (3)  Bruckschwaiger (US12/789365, published as US Patent 8,993,734) teaches a method for preparting IgG from plasma by a series of alcohol precipitation steps wherein the second precipitate is re-suspended with an extraction buffer at a pH between 4.5-5 and then treated with silicon dioxide (SiO2). (4) Bruckschwaiger (US13/776448) teaches a method for producing an enriched immunoglobulin composition from a Cohn pool by co-precipitating immunoglobulins and alpha-1-antitrypsin (A1PI) from a Cohn pool. (5) Bruckschwaiger (US13/830862, published as US 2013/0224184) also teaches that the addition of a pH modifying agent  during the first, secondor third alcohol precipitation steps is achieved after addition of the alcohol. (6) Bruckshwaiger (US14/309668) also teaches a method for preparing an enriched IgG from plasma by precipitating a cryo-poor plasmid fraction with alcohol in two separate precipitation steps using spray addition of the alcohol. 

Curling (“Methods of plasma protein fractionation” Academic Press, 1980, pp.12-13) shows method 6 from Cohns procedure where in a first precipitation step, plasma is subjected to ethyl alcohol 8%, pH 7.2 temperature -3C, the supernatant I is then subjected to ethyl alcohol at 25%, pH 6.9 temperature -5C to form a Supernatant II+III and Precipitate II+III. Then according to Oncley’s procedure method 9 the Precipitate II+III is subjected to ethyl alcohol 20%, pH 7.2 at temperature -5C. A subsequent series of precipitations with ehtyl alcohol results in gama-globulin fraction. 

Eibl (US 5,122,373) teaches an IgG containing fraction from human plasma comprising monomeric IgG as well as at least 70% gammaglobulins obtained by (1) a first precipitation step by mixing human blood plasma with 8% ETOH at pH 7.2, -2C, the precipitate is obtained and separated and (2) in a second precipitation step, the ethanol concentration of the supernatant is increased to 25% at -6C, pH 7, the precipitate obtained is separated and the immunogloublin contained therein is further purified by (3) extraction with a sodium acetate acetic acid buffer. Affer addition of 12% ETOH at -2C, the alpah and beta globulins are removed by centrifugation and discarded. In the supernatant, immunogloublins are present in a purifty of about 70%. (4) They are concentrated by precipitation with 25% ETOH at a pH 7, -6C, centrigued off and feeze drived for the removal of ethanol. 

Kimura (US 4,476,109) discloses a method of prearing gamma globulin suitable for intravenous administration compirsing adding ethanol to human blood plasma under conditions (e.g., ethanol 8-9%, Temperature 0 to -3C) effective to precipitate a first precipitate containing most of the fibrinogen while minimizing the amount of albumin and gamma globulin contained in the first precipitate, separating the first supernatant liquid from the first precipitate, then adding ethanol 30-35%, pH 5.5-6.5 to the first suerpnatant liquid to obtain a second supernatant liquid containing albumin and a second preciptiate consisting of gamma globulin and albumin. The second precipitate can then be further prcocessed by resuspending it in distilled water, adjusting pH to 4-5 and then adding ethanol to 16% to form a third precipitate and third supernatant. The third supernatant is then freeze dried to form the gamma globulin product.

Teschner (US 13/633697, now US 8,921,520) teaches a method for preapring an IgG composition via a series of alcohol precipitation steps at various pHs where the second precipitate which is resuspending is contacted with SiO2 under condition suitable to bind a serine protease or serine protease zymogen and then separating the SiO2 from the suspension to form a clarified suspension.

Teschner (Vox Sang, 92(1), 2007, 42-55) disclsoes an IGIV 10% manufacturing prcocess by separation of the cryo-recipitate, followed by the optional adsorption of blood coagulation factors and antithrombin. The supernatant is then (1) subject to an ethanol fractionation to precipitate  fibrinogen and (2) raw immunoglobulin is separated from raw albumin by a II+III precipitation. (3) the II+III past is dissovled in a phosphate acetate buffer and filtered through a depth filter for the depletion of lipids and undissolved proteins. (4) From this filtrate gamma-globulin is precipitated by 25% ethanol at nuetral pH (similar to the Cohn II precipitation). The resulting intermediate has an IgG purifty of about 75%. (5) The paste is dissolved and filtered through a depth filter to obtain a clear filtrate appropirate for a first virus inactivation step with S?D. Next the S/D reagents are separated form the IgG using carboxy-methyl CEX (IgG is bound to the resin and S/D reagents and other impurities are washed out). Elution is facilitated by moderately increasing the conductivity under alkaline conditions and loaded onto an AEX (DEAE for functional groups). Impurities, especially IgA are bound to the column while IgG is found in the flow through which is nanofiltered. The nanofiltrate is DF agaisnt glycine. After steril filtration, virus inactivation (pH 4.4). The formulation buffer consist of glycine. 

Weisbart (US 2002/0098182) discloses preparation of human Cohn Plasma Fraction II+III uisng a 1st precipitation step with cold ethanol to 8%, pH 7.2-73, Temperature -1C to -3C, removing the precipitate (Fraction I) by centrifugation, cooling the supernatant to -5C, adjusting pH to 6.7 to 6.9 with ctric acid, adding cold ethanol to 25%, resulting in a Fraction II+III precipitate that contains IgG, IgA and IgM and amounts of albumin, alpha and beta globulins. The Fraction II+III precipitate was then suspended and freezed. Then the fraction II+III precipitate is dissolved in water at -% to give a 1% protein concentration. In a third precipitation step, cold ethanol is added to 20-25%, pH 7.2, tempertature -5C. The precipitate (Fraction II) is removed from the filtrate by centrifugation. The supernatant produced contains Fraction III and is subjected to a 4th preciptiation with cold ethyl alcohol to 25%, pH 5.7, temperature -5C. The precipitate Fraction III is removed from the filtrate by centrifugation. Fraction II Fraction II and/or Fraction III is redissolved in water suitable for injection to give a solution that is 1-5% protein and about 15% glycine.

1st Precipitation (ETOH 8-9%, temp 0-3C — 2nd Precipitation on the supernatant (ETOH 30-35%, pH 5.5-6.5) –Add ETOH the 2nd Precipitate (contains the gamma aglobulin and albumin) to form 3rd Precipitate and 3rd Supernatant. The Third supernatant contains gamma globulin. 

(Kimura, 4,476,109) discloses a method of separating gamma globulin from human blood plasma by two precipitations. In the first, ETOH is added under 0-3 C to obtain a 1st precipitant and 1st supernatant. ETOH of 30-35% at pH 5.5-6.5 is added to the 1st supernatant to obtain a second precipitate and second supernatant. The mixture is centrifuged to separate the supernatant and second precipitate. The second precipitate is suspended in distilled water and the pH adjusted 4.05.00, and ETOH is added 10-16% until third precipitate is formed. The resulting third supernatant liquied is freeze dried to form the gamma globulin product. 

SCHEME2: Fraction I+II+III Precipitation (aka/ “Fraction I+II+III+IV-1 Precipitation”  or “Fraction I-IV-1 precipitation” or “Initial low pH, high alcohol precipitation”)

Bruckschwaiger (US 13/776448, published as US 2013/0224183) discloses recovery of IgG and A1PI from pooled plasma by removing the need for multiple initial preciptiation steps. Rather, a single initial precipitation step that captures all of the proteins normally precipitated in the Fraction I, Fraction II+III and Fraction IV-1 precipitates combined. This single precipitation step uses a low pH, (e.g., 5-6 pH) high alcohol (e.g, 20-30%) precipitation step as an initial step in the purification of IgG from cryo-poor plasma. The plasma or cryo-poor plasma is thus fractionated into a Fraction I-IV-1 precipitate and Fraction I-IV-1 supernatant. The Fraction I-IV-1 precipitate contains nearly all immunoglobulins (e.g., IgG, IgA, and IgM) as well as alpha 1 elastase inhibitor (A1pI) while the supernatant contains mainly albumin. 

Levy (“Chemical, clinical, and immunological studies on the products of human plasma fractional. XL. quatiative seapration and determination of the protein components in small amounts of normal human plasma, 195) discloses starting whith plasma and adding ethanol to a concentraiton of 195 at pH 5.8 to form a first precipitate (refereed to as “precipiate fraction I+II+III”) and a first supernatant (referred to as “filtrate -fraction IV+V+VI”). Lever further teaches extration of Fraction II from the fraction I+II+III, leaving fraction I+III as residue by suspending the Fraction I+III+III with buffer to form a suspension, removing teh residue representing Fraction I+II and recovering the Fraction II which contained the gamma-globulins in the filtrate. 

McIntosh (US 6,485,932, issued 9/26/2002) discloses that Fraction II can be isolated from a combined Fraciton I, II and III precipitate which is islated using the Fraction II and II precipitation conditions of Hink et al (Vox San. 2: 174-186, 1957). The technique of preparing a combined Fraciton I, II and III precipitate has been described also by Kistler and Nitschman (Vox Sang. 7: 414-424, 1962). See Figs. 

Fraction I+II+III Precipitation — Fraction A precipitation –Fraction B Preipitation. 

Teschner (US 2013/0058961) disclsoes a fraction I+II+III precipitation that is formed by adding ethanol to cryo-poor plasma at a concentration of 17-23% (v/v) at a pH between 6.5-73. The mixture is then incubated while stirring at between -8-2 C. The resulting Fraction I+II+III preciptiate can be separated form the Fraction (I+II+III) supernatant by contrifugation or filtration of the mixture. The majority of the immunoglobulin content is present in the Fraction I+II+III precpitate which can be re-suspended and furtehr enriched. For example, a Fraction I+II+III prepitate is re-suspended and a second precipitation step (Fraction A precipitation) can be performed by adding ethanol to teh Fraction I+II+III suspension to a ifnal concentraiton of 17-23% (v/v) at a pH 6.8-76. The mixture is then incucated while stirring at -8-2C. The resulting Fraciton A preciptiate can be seaprated form teh Fraciton A supernatant by centrifugation or filtartion. The majority of the immunoglobulin content is present in teh Fraction A preciptiate which can be re-suspended and further enriched. To further enrich the IgG content, the Fraction A preciptiate is re-suspended and a third preciptiation step (Fraction B precipitation) is performed by adding ethanol to teh Fraction A suspension to a final concentraiton of 14-20% (v/v) at a pH 5-5.8. The mixture is then incubated while stirringat -8-2C. The resulting Fraction B preipitate can be seaprated form teh Fraction B supernatnt by centrifugaiton. The majority of the immunoglobulin content is present in the Fraction B supernatant which can be further enriched. 

Fraction II Precipitation:

To further enrich the Ig G content a fourth (or third in the case of scheme 1 above) alchohol precipitaiton step (Fraciton II precipitaiton) is performed. Fraciton II preciptiaiton is performedd by adjsuting the pH of a Fa Fraction B filtration (fraction II+III preciptiate) suspension between 6.7-75 and adding ethanol to a final concentraiotn of 22-28% (v/v). The mixture is then incubated while stirring at between -13-2C. The resulting Fraction II precipitate can be seaprated from the Fraction B supernatnt by cnetrifugaion or filtratio. The majority of the immunoglobulin content is present in the Fraction II preciptiate which can be re-suspended and further enriched. 

Fraction II Paste

Son (US 15/123869, published as US 2017/0022248) discloses obtaining a fraction II paste. 1st, plasma is thawed to produce a cryopricipitate, 2nd a precipitation I step is performed using 96% ethanol pH 7.2, the precipitate is removed by centrifugation and the supernatant recovered. 2nd, a precipitation II+III step and filtration is performed on the supernatant using final concentraiton of ethanol 20% at -5C, pH adjusted to 6.9 and a filter aid added to the solution. The suerpnatant (“supernatant I+II+III 9or II+III) and the precipitate (“fraction I+II+IIIw (or II+IIIw” (w; wash) is next dissolved in cold distilled water, then 96% ethanol is added such that the final ethanol concentraiton is 18% at -5C, pH adjusted to 5.2 by addition of acetate buffer. The suprnatant is seaprated from the precipitate by means of a filter press. The superatant is called “filtrate I+III (or III)” and the precipitate named “fraction I+III (or III). Next a precipitation II step and filtration is performed using the filtrate I+III (or III) with 96% ethanol to final concentraiton 25% at -10C, adjusting pH to 7.4 by addition of 1M sodium bicarbonate. The suerpatant and precipitate are sepparated by means of a filter press. the precipitate was named “fraction II paste”. 

In combination with Ion Exchange  (See outline; these combinations will incorporate the alcohol precipitation steps above)

Alcohol Precipitation + Anion Exchange  See outline

Alcohol Precipitation + Cation Exchange See outline

In cominbation with Filtration (See outline; this combination will incorporate the alcohol precipiation steps above)