A chromatographic method for the production of a human immunoglobulin G solution for intravenous use

Abstract

Immunoglobulin G (IgG) of excellent quality for intravenous use was obtained from the cryosupernatant of human plasma by a chromatographic method based on a mixture of ion-exchange, DEAE-Sepharose FF and arginine Sepharose 4B affinity chromatography and a final purification step by Sephacryl S-300 HR gel filtration. The yield of 10 experimental batches produced was 3.5 g IgG per liter of plasma. A solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1% Triton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates. Anticomplementary activity was 0.3 CH 50 /mg IgG and prekallikrein activator levels were less than 5 IU/ml. Stability at 37 o C for 30 days in the liquid state was satisfactory. IgG was stored in flasks (2.5 g/flask) at 4 to 8 o C. All the characteristics of the product were consistent with the requirements of the 1997 Pharmacopée Européenne.

Purification of human albumin by the combination of the method of Cohn with liquid chromatography

“…Ethanol (95%, w/v) at -30 o C was slowly added with constant stirring until a final concentration of 19% was obtained. The material was left to stand overnight at -5 o C and then centrifuged at the same temperature in a Westphalia BKA-2 centrifuge at a flow of 35 l/h in order to obtain the F-I + II + III precipitate which was used for the production of intravenous immunoglobulin G (IgG) (9,10).…”

Medicinal Chemistry Meets Proteomics: Fractionation of the Human Plasma Proteome

“…Another abundant plasma component, immunoglobulin G was separated from human plasma cryosupernatant by the combination of several methods such as ion exchange chromatography (DEAE-Sepharose), affinity chromatography (arginine Sepharose 4B) and size exclusion chromatography (Sephacryl S-300 HR) as the final purification tool obtaining a yield of 3.5 g IgG from one liter plasma [17].…”

High quality human immunoglobulin G purified from Cohn fractions by liquid chromatography

“…The final volume of the F-II+III solution was the same as that of the initial plasma (2 liters), and pH was adjusted to 6.0 with 0.5 M acetic acid. This solution was then immediately taken to a cold chamber at 4 o C, so that the euglobulin precipitate could be obtained and left to stand overnight at 4 o C. Euglobulin was removed by centrifugation at 4,000 rpm for 10 min at 4 o C. The protein solution had a volume of 1950 ml, pH 6.0, and conductivity of 1.4 mS/cm, ideal conditions for liquid chromatography (4,5).…”

“…The pH was then adjusted to 6.0 with 0.5 M acetic acid and the solution was immediately taken to a 4 o C cold chamber to precipitate euglobulin. The precipitated euglobulin was removed by centrifugation at 4,000 rpm for 10 min, at 4 o C. The supernatant was submitted to liquid chromatography (4,5). See flow diagram in Figure 1.…”

“…Chromatographic procedure (4,5): an XK-50/30 column (Pharmacia, Uppsala, Sweden) containing 320 ml of anion exchange Q-Sepharose FF gel was connected to another XK-50/30 column containing 160 ml cation exchange gel, CM-Sepharose FF. Both of them were previously equilibrated with 20 mM sodium acetate, pH 6.0, and buffer with 1.4 mS/cm conductivity.…”