See also Virus inactivation (caprylic acid can be used as an agent to do this)

See also adjustment of pH with various agents under Agents used for fractionation See also AEX for the purification of immunoglobulin

In General

Chauntun (Archives of Biochemistry and Biophysics 89, 218-220 (196) discloses the precipiation of proteins from plama by caproic, htpylic, caprylic, pelargonic, capric and lauric acids. At low concentrsitons (0.04 M) these fatty acids form insoluble complexes, particulalry with alpah and beta globulins. At higher concentraiton (0.1M) albumin and all globulins are precipitated in appreciable amounts. 

Caprylate acid (also called octanoic acid) precipitation: 

The use of caprylic (octanoic) acid for the purificaiton of monoclonal or polyclonal antibodies such as antivenoms and human immunoglobulin is well established. The use of CA for the purificaiton of human immunoglobulin obtained from ethanol fractiona of plama also efficiently inactivates enveloped viruses. A CA final concentration of 3.5% is optimal to obtain immunoglobulins essentially free of albumin. It is proposed that CA binds to specific sites of the protein, thereby inducing partial unfolding of the protein, which exposes additional binding sites. The interfacial protein surface becomes hihgly hydrophobic and increases protein-protein attrraction, cusing association and precipitaiton of the macromolecular complexes. (Morales, Biotechnology and Applied Biochemistry, 59(1), 2012, pp. 50-54). 

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

Chanutin (Archives of Biochemistry and Biophysics 89, 218-220 (1960) discloses precipitation of proteins from plasma by caproic, heptylic, caprylic, pelorgonic, capric and lauric acids. At low concentration (0.4 M) these fatty acids form insoluble complexes, particularly with alpha and beta globulins. At higher concentraiton (0.1M) albumin and all globulins are precipitated in appreciable amounts. It is also known that a considerable portion of the globulins of plasma is precipitated in the presence of caprylic acid at pH 4.2. 

Habeeb (Preparative Biochemistry, 14(1), 1-17 1984) discloses that carpylic acid can be used to precipitate nonimmuogloulin proteins from human plasma. In one embodiemnt, to 200 ml of plasma add 400 ml of 0.06 M acetate buffer pH 4, add caprylic acid, centrigue and filter if necessary to remove finely suspended particles and adjust H 6.2 with 1 N sodium hydroxide. The solution was then dialized against sodium acetate pH 5.7 and then mixed with 20 gm DEAE cellulose. 

Steinbuch (Archives of Biochemistry and Biophysic 134, 279-284 (1969) discloses that it is possible to botain IgG from plasma in good yeild with at least 90% purity using caprylic acid. The remaining impurities can be removed on DEAE-cellulose. In one embodiment, the pH of human plasma prtoein is adjusted to pH 4.8 with acetate buffer to form a precipitate and the supernatant solution is pH adjsuted to 4.7, centrifuged with the sueprnatant solution cotnaing pure IgG. 

Caprylic Acid -pH shift:

Lebing (WP 0893450) discloses a method for the purificaiton of Ig from human plasma which includes suspending the antibodies at pH 3.8-4.5 followed by addition of caprylic acid (or other source of caprylate) and a pH sift to pH 5-5.2 A precipitate of contaminating protins forms and is removed, while the majority of antibodies remain in solution. Sodium cpayrlate is again added to a final concentration of not less than about 15 mM and incubated udner conditions sufficient to reduce the titer of active virus (e.g., 1 hour at 25C). A precipitate (mainly caprylate) is removed and the clear solution is diluted with purified water to reduce ionic strengh. Anion exchange is then used. 

Source for Caprylic Acid

Parkkinen (WO2005/073252) disclsoes caprylic acid precipitation by adding caprylic acid as a free acid instead of adding it in the form of a salt, such as sodium caprylate as in US Patents Nos: 5,886,154 and 6,307,028. Caprylic acid slighly lowers the pH of the solution in contrast to sodium caprylate, which increases the pH. Accordingly, no pH shift to pH 5.0-5.2 takes place and virus inactivaiton is carried out at a lower pH. This is beneficial since at low pH the propotion of the non-ioniced form of carpylic acid is higher, and it is the non-ionized form of caprylic acid, which is effective in virus inaction. 

Caprylate acid -AEX

Alred (US 6,955,917 and US2003/0152966) discloses purification of antibodies form human plasma which involves suspension of the antibodies at pH 3.8 – 4.5 followed by additionl of caprylic acid and a pH shift to pH 5.0 to 5.2. A precipitate of contaminating proteins, lipids and caprylate forms is removed while the majority of the antibodies remain in solution. Sodium caparylate is again added, the precipitate removed and the clearl solution applied to AEX to obtain a high yeild of IgG. 

Buchacher (US7,553,938) discloses pararing antibody by adjusting pH of the starting solution to about 4.6-4.95, adding carpylate and/or heptanoate ions such that precipitate is formed and the antibodies are essentially present in the supernatant, incubating the supernatant under conditions of caprylate and/or heptanoate ion concentration, time, pH and termpature, optionally concentrating and DF, applying the filtered solution with at least one AEX such that contaminatns bind the resin while antiboides pass through and then virus inactivation. 

Lebing (US5,886,154) discloses that during human immunoglobulin preparation caprylic acid is generally recognized as an effective precipitating agent for most plasma proteins at pH 4.8, so long as parameters such as temperature and ionic strenght are optimized.  Lebing discloses suspending antibodies at pH 3.8 to 4.5 followed by addition of caprylic acid and a pH shift to pH 5.0-5.2. A prcipitate of contaminating proteins, lipids and caprylate forms and is removed while the majority of the antibodies remain in solution. Sodium caprylate is again added, the precipitate (mainly caprylate) is removed and the clear solution diluted with water to reduce ionic strenght. AEX is subsequently used to obtain IgG. ( See also caprylate in combination with other methods below and chromatography).   

Menyawi (US 14/900499, published as US 2016/0368970) discloses a process for the purificiton of IgG form plasma by providing an acidic solution comprising IgG  with a between 3.5-5.2, adjusting the pH to 5.2-6.2 while maintaining conductivity below 1.5 mScm using a multi-hydroxylated amino compound with or without carboxyl group , incubating for at least 15 mintues and removing any precipitate as by filtration, followed by ANEX, virus filtraiton and UF/DF. . 

Parkkinen (WO2005/073252) discloses preparing immunoglobulin by subjecting crude immunoglobulin solution to caprylic acid to remove protein aggregates and viruses, then IEX to purify the immunoglobulin, filtering to remove virus. 

Steinbuch and Audran (Arch. Biochem.Biophys. 134, 279-294, 1969) disclose a purificaiton for IgG with caprylate (i.e., octanoate, a C8 saturated fatty acid) as a precipitating agent. Non-immunoglublins were precipitated form human plasma after diltuion with an acetate buffer to reach a final pH of 4.8. After addition of caprylate under vigorous stirring an IgG enriched solution was obtained. Batch absorption of the supernatant on DEAE cellulose was used to clear additional impurities from the isolated fraction. 

Zhang discloses a method for preparing intravenous cytomegalovirus human immune globulin which uses caprylic acid precipitation and AEX for replacing the step of ethanol precipitation in the conventional cold ethanol method. 

—-Caprylic Acid – PEG –AEX — Virus remal

Parkkinen (WO2005/073252) discloses a process of subjecting a crude immunoglobulin solution to capylic acid treatment at a pH below 5 and then subjecting the sueprnatant solution to a protein precipitation such as  poleythylene glycol (PEG) to a concentraiton of 10-50 g/l or adsorbent such as fumed silica at a pH in excess of 5.0 to remove protein aggregates followed by AEX.