Influence of column type and chromatographic conditions on the ion-exchange chromatography of immunoglobulins.
Immunoglobulins are often purified by affinity chromatography. However, this technique is costly, can result in poor resolution for subclasses (or is only group specific), and leads to possible leaching of contaminants into the purified products. Ion-exchange chromatography has shown great potential and has found an increased usage in the purification of immunoglobulins. The aim of this study is to further understand the separation mechanism with emphasis on the influence of column type and chromatographic conditions on the peak shape, selectivity and changes in the elution patterns. Included are strong cation-exchange, strong anion-exchange and weak anion-exchange columns. Five immunoglobulin G antibodies were used as test probes. Some sera and ascites were also used in the study. Among the chromatographic conditions examined were mobile phase pH, buffer type, buffer concentration, gradient rate, and column temperature. Significant differences in the chromatographic behavior (elution pattern, peak shape and selectivity) of the test samples are discussed in regard to the column type and the chromatographic conditions.
Yang
Y B
YB
Separations Group, Inc., Hesperia, CA 92345, USA.
Harrison
K
K
D000818:Animals; D000837:Anion Exchange Resins / Q000737:chemistry*; D001202:Ascitic Fluid / Q000276:immunology; D001798:Blood Proteins / Q000032:analysis; D002021:Buffers; D002411:Cation Exchange Resins / Q000737:chemistry*; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006531:HEPES / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification*; D051379:Mice; D009994:Osmolar Concentration; D011817:Rabbits; D051381:Rats; D012710:Serum Albumin, Bovine / Q000032:analysis; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D013696:Temperature; D014325:Tromethamine / Q000737:chemistry
D000837:Anion Exchange Resins; D001798:Blood Proteins; D002021:Buffers; D002411:Cation Exchange Resins; D007074:Immunoglobulin G; D014325:Tromethamine; D012710:Serum Albumin, Bovine; D006531:HEPES