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)