See also Antibody precipitation under Antibody purification   See also IVIG for use of ethanol as fractionation agent

The main fractionation agent used to isolate immunoglobulins from donor plasma is ethanol fractionation based on the Cohn procedure and covered in a separate section. However, even with the Cohn procedure, it is necessary to further process the immunoglobulins due to the anti-complementary activity caused by IgG aggregates formed during the fracxtionation process. Four basic procedures to do this include 1. enzymatic degradation by plasmin or pepsin, 2. chemical modification of the IgG by beta-propiolactone or by cleavage of the interchain disulfide bridges by sulfonation or reduction and alkylation, 3. selective elimination of aggregates by precipitation with PEG ands hydroxyethyl starge (HES) or by treatment at pH with traces of pepsin and 4. adsorption of aggregates by DEAE gels such as Sephadex C50® (Hou, US 4,639,513). 

Acidic/pH Fractionation/Preciption

Low pH followed by pH shift:

Menyawi (US 14/900499 published as US 2016-0368970) disclsoes a purificaiton process from a solution comprising IgG such as paslam-derived antibodies which includes (a) providing an acidic solution of the IgG solution at pH 3.5-5.2 and a total protein concentraiton of at least 10 g/l, (b) adjusting the pH to 5.2-6.2 while minatinaing conductivity below 1.5 mS/cm, (c) incubating and (d) removing precipitate such as by depth filtration. The adjustment of pH is advantageously done using a multi-hydroxylated amine compound with or without carboxyl groups such as with Tris. In one exemplification, pH was lowered to pH 4 and then adjusted ot pH 5.8 using 1M Tris buffer. The precipitate foremd was then removed by filtraiton. 

Takeda (US2006/0142549) discloses a method for remvoing impurities from a protein/antibody containing smaple which includes low conductivity at a pH below the isoelectric point of the antibody and then removing the resulting particles. In one embodiment elution fractions from Protein A column were adjusted to pH 3.2 with hydrochlorid acid for 30 inutes and each fraction was subsequently adjusted to pH 7.2 with a 300 mmol/L Tris solution.

–Low pH – Caprylic Acid -pH shift  (for precipitation with caprylic acid see outline)

Lebing (US2002/0177693) discloses a process for the purificaiton of antibodies form human plasma whihc includes suspension at pH 3.8-4.5 followed by addition of caprylic acid and a pH shift to pH 5.0-5.2. The precipitate of contaminating proteins, lipids and caprylate forms are removed while the majority of anitobdies remain in solution. 

Caprylate acid see outline 

Polyacids:

Polyacid + Salt: 

Van Alstein (US 2014/0343253) discloses providing a sample of blood plasma and adding a polyacid and a slat cuasing a first protein precipitate and first supernatant and adding a polyacid and/or a salt to said first supernatant causing formation of a second protein precipitate and a second supernatant. Thyl cellulose. The salt is slected form sodium pohsophate, potassium phosphates, ammonium phosphates, sodium citrates, potassium citrates, ammonium citrates, sodium sulphates, potassium sulphates, ammonium sulphates, sodium acetate, potassium acetate, ammonium acetate. rene-sulfonic acid, carboxymethl-dextran and carboxymethe first protein is fibrinogen and the second protin is an immunoglobulin such as IgG. Polyacids are slected from polyacrylic acid, poly-methacrylic acid, polyvinylsulfonic acid, polysty

Ethacridine lactate: 

Ethacridine lactate is a highly aromatic cationic dye shown to recover antibodies.

Glycine: 

Levy (WO/2003/034982) discloses a method of purification of immune gloubilins from human plasma from an immune globulin source such as Cohn’s fraction using glycine extraction.

Organic solvents (other than Ethanol): 

Organic solvents include emthanol which has the disadavantage of toxicity, acetone which has not been shown to be of value as a fractionating agent for proteins, ether which has been employed in England for extraction and separation of human plasma proteins,

Metal Ions:

Zinc ions have been used in the fractionation of plasma proteins.

Polyanions: 

Nucleic acids, when used at weakly acid pH, will precipitate proteins wihtout denaturing them. Polyacrylic acid is suitable for the fractionation of human serum.

Polymers: 

Polson et al have proposed to use linear polymers of high molecular weight for the fractionation of complex protein mixtures. Among the agents used are polyetheylene glycol, dextran and polyvinylpyrrolidone. In contrast with organic solvents, the agents do not cause denaturation of the proteins when employed at ordinary room termpature, nor is the selectivity of their action influenced by changes in ionic strenght of the mediu. A disadvantage of these methods is that the PEG must be removed form the end products such as through a column of DEAE or CM cellulose.

polyethylene glycol: 

Polyols, particularly polyethylene glycol (PEG) have for many years attracted the itnerest of the blood derivative industry. Curling (1980) presented a plasma fractionation method using PEG as a precipitating agent and concluded that PEG has considerable advantage of use compared to ethanol. PEG has also been used in human plasma fractionation for the production of a crude fraction of immunoglobuilin and then treated with two stage IEX by Teshner in 2007. (Lucena, “A new methodology for polyvalent intravenous immunoglobuilin solution production with a two-stage process of vrial inactivation” Brazilian J. Pharm. Sciences, 46(4), 2010, 777-783)

The fractionation with polyethyelne glycol or pluronics is believed to be the most desirable means to recover immunoglobulin. The procedure was disclosed by Polson and Coval (JP46,814/1975, 91,321/1976 and 20,415/1978).  Lars-Gunnar (Swededn 1344340) also disclose a method of fractionating plasma proteins, by removing blood corpuscles and cell fragments form the plasma in the blood, precipitating the globulins in the plasma with polyethylene glycol of average MW 6000, centriguging out all the precipitate, dissolving the precipitate, adsorbing the globulins on a cation exhcanger and eluting, precipitating with polyetheylene glycol and absorbing the globulins on an anion exchanger.  

Numerous variations have been disclosed where improvements consistent, as for example, improved yield. For example Uemura (US4371520) described using a starting material consiting of Cohn’s plasma fractions I+II+III, II+III, II, and III obtained by precipitation with cold ethanol and subjecting the starting material with an acid and adding to the resulting mateiral an alkyl-ene oxide polymer or copolymer haivng a MW of 2k-20k. (see also US4276283, ammonium sulfate and polyethyleneglycol), (Polyethylene glycol associated to single stage precipitation by ethanol rather than cohn-Oncley process which employs cold alcohol as the precipitating agent in a 3 stage process Lucena, “A new methodology for polyvalent intravenous immunoglobulin solution production with a two-stage process of viral inactivation” Brazilian J. P.S. 2103.1, 46(4), 777-78) (Fractionation with ethanol, PEG, anion resin, ultrafiltration, US 6875848).

(Falksveden “Ion exchange and Polyethylene Glycol Precipitaiton of Immunoglobulin G” , from “methods of plasma Protein Fractionation” by J.M. Curling, Academic Press 1980) discloses precipitating the main part of the globulins at neutral pH with PEG 600 under conditions that ost of the albumin remains in solution. The precipitate is separated and the IgG fraction extracted at pH 5.8 in such a way that most of the IgM, fibrinogen and plasminogen are left as an unextracted residue. Positively charged IgG, at pH 5.8, is then bound ato a CEX. 

 –Ethanol Precipitation to Fraction II/III – PEG:

Radowitz (US 4,216,205, IDS Ref. of 10/16/2012) teaches that it was known to apply PEG on the precipitate gamma-globulin fraction II/III of a standard Cohn procedure. According to an embodiment, (1) a first preciptiation step where crude fibringoen is removed from plasma by adding ETOH up to 8%, temperature -2.5 to -3C. S1 is further prcoessed (2) in 2nd precipitation with 18-25% ETOH, temperature -5C, pH 5.8. P2 (fraction II/III or gamma-globulin fraction) is removed by centrifuging. Now, Radowitz does different fractionations (see diagrams on right hand column, 1st diagram) where to obtain immunoglobulin, the Fraction II/III P2 is resuspended and 3% of PEG 4,000 or 2.5% of PEG 6,000 are added. Upon agitation for up to 4 hours the mixture is centrifuged whereby supernatant S5 and fraction III-1 (precipitate P5) are formed. Supernatant S5 is against reacted with PEG and fractionated. Fraction III-2 (precipitate P6) and the supernatant S6 are fored. S6 contains primarily IgG.

The S2 from the above can also be further processed (3) in a 3rd precipitation step with ETOH 40%, temp -7C and pH 5.8. P3 (fraction IV) contains alpha and beta globulines), S3 is further processed (4) in 4th preciptiation step with 40% ETOH. P4 (crude albumin).  The gama-globulin fraction II/III is further process by the following steps: (1) P2 is supended in a citrate phosphate buffer at pH of from 7-7.4, temp 15C, 3% of PEG are added. Supernatant S5 and fraction III-1 )precipitate P5) are formed. S5 is against reated with PEG and fractionated. Fraction III-2 (precipitate P 6) and S6 which contains IgG are formed. The globulins of fractions III-1 and III-2 are enriched or concentrated with the immune globulins IgG, IgA and IgM. Further, alpha1-antitripsin, alpha2-haptoglobulin, coeruloplasmin, transferrin and haemopexin are present in concentrated form.

 –PEG + caprylic acid: 

Debart (US 15/276544) discloses a method for prefaring immunogobulins from an initial solution such as a fraction II+III by adding caprylic acid to a solution having a polyether or polymer of glycol such as polyetheyle glycol (PEG), incubating the solution for a time to inactivate enveloped ciruses and then performing a step of UF/DF. In some embodiments the caprylic acid is added to a concentration of 9-15 mM.  In some embodiments the UF/DF is carried out using a membrane of 100k and is carried out in two phases; a first phase in which pH is adjsuted to 5-6 or irder to reduce or eliminate most of the caprylate and a 2nd pahse in which the pH is adjsuted to less than 5 in order to reduce or eliminate most of the polyether or polymer of glycol. 

Hansen (US 4,164,495) teaches that immunoglobulins or gammaglobulins can be recovered by fractionated precipitation of blood plasma with a polycondensed di or polyol, such as PEG, in the present of a mono or polyalkanoic acid have 4-12 carbon atoms such as caprylic acid.

Lucena, (“A new methodology for polyvalent intravenous immunoglobuilin solution production with a two-stage process of vrial inactivation” Brazilian J. Pharm. Sciences, 46(4), 2010, 777-783) discloses starting with fresh frozen plasma, thawing at 4C, clarification by centrifugation at 12,000 rpm. The cryoprecipitate paste recovered is then discarded and the sueprnatant treated with PEG, pH 4.1. After 6 hours under slow homogenization, the precipitate was discrarded and the supernatant suchjected to a 2nd precipitation with PEG 12%, pH 7.4. The sueprnatant was discarded and the paste was dissolved in water and then subjected to carpylic acid as the first viral inactivation step. PEG 3% was added and the supertant subjected again to PEG 25%, pH 7.4-7.6. 

Parkkinen (WO 2005/073252) disclose a process for preparing immunoglobulin by subjected a crude immunogloublin solution to caprylic acid treatment, remove the precipitatnt and subject to the supernatant to PEG precipitation, then AEX and vinally virus removal. 

 –PEG + Ethanol: 

Lucena (“A new methodology for polyvalent intravenous immunoglobulin solution production with a two-stage process of viral inactivation” Brazilian J. of Pharmaceutical Sciences, 46(4), 2010) discloses use of PEG for crude purifciation followed by ethanol in 25% concentration to produce IgG which had 95% purity.

–Silicon dioxide: see outline

Salts: 

The oldest method employed for precipitating plasma proteins is the use of neutral salts, especially ammonium sulfate. The salting out processs is a system comprising for variables: salt concentration, protein concentration, pH and temperature. Included in the salt precipitation are large amounts of neutral salts which must be removed by dialysis (Falksveden, US3,869,436). 

Ammonium sulphate: has a long history as a useful precipitatnt for the production of crude protein fractions.

Eibl (US 4,276,283) discloses a method of preparing an intravenously adminsitrable immune globulin preparation containing antibodies in yethylene glycol. which the immune globulin containing fraction is subjected in a first purificaiton step prior to polyethylene glycol precipitation to a treamtent with an aqeuous solution of a salt of an inorganic acid, particular ammonium sulphate. Suitably, the immune globulin containig fraction is gained form human blood plasma according to the Cohn method. First, the immunoglobulin fraction is subjected to a treatment with an ammonium sulfate solution. The precipitate is separated and rejection and in a subsequent precipitaotn step a precipitate containing immune globulin is precipitated from the remaining solution by treatment with an ammonium sulfate solution.  Then the rmaining immune globulin containing solution in a secon purificaiton step is subjected to treatment with pol

Sodium citrate: 

Bruckschwaiger (WO 2013/126904) discloses an initial low pH, high alcohol precipitation of blood plasma for production of IVIG and A1PI. However, they also disclose that that the precipitation can be carried out by salting out using ammonium phosphate, ammonium sulfate or sodium citrate. Next, the first precipitate is suspending to form a first suspension, removing the A1Pi from the suspension and recovering the soluble fraction thereby forming an enriched immunoglobulin composition. 

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 citrate is added to the supernatant. 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.

Zurlo (US 2019/0055282) also discloses methods in which one can produce a protein product from a blood based material which includes the steps of adding a salt to the material to produce a first sueprnatant and a first past, adding a salt at higher concentration to the first supernatant to rpodcue a second sueprnatant and second paste and subjecting the second supernatant to a suitable chromatogrpahy method such as ion exchange. With respect to the salt added to the blood based material, a side range of salts can be used including for example citrates, acetates, gluconates and/or caprylates. 

Sodium chloride: was once believed to precipitate gloublins as well as fibrinogen but most of the gloublin faction actually remained in solution even at the saturation point.

Magnesium sulfate: is probably the oldest of all protein precipitatns of the neutral salt class. At full startation it precipitates only the gloublin fraction, while the albumins remain in solution.