Behaviour of human immunoglobulin G subclasses on thiophilic gels: comparison with hydrophobic interaction chromatography.
We have used thiophilic and hydrophobic interaction chromatography in an attempt to obtain enriched human immunoglobulin G (IgG) subclasses from a therapeutic immunoglobulin preparation. Proteins were adsorbed on a thiophilic gel and on Phenyl-, Butyl-, or Octyl-Sepharose in 1 M ammonium sulphate. Elution with a decreasing salt gradient produced no marked subclass selectivity, except with Octyl-Sepharose, which yielded a poorly adsorbed fraction somewhat enriched in IgG2, representing ca. 20% of the total initial protein. Neither thiophilic nor hydrophobic interaction chromatography appear suitable for an efficient enrichment in subclasses, which all show a broad heterogeneity in their affinity for these columns. The influence of the starting salt concentration was also studied. With thiophilic gels, in the absence of ammonium sulphate, ca. 30% of the initial load was not absorbed, and was found to be enriched in IgG2. At 2.5 and 5% ammonium sulphate, practically no adsorption occurred. At 7.5% ammonium sulphate, the non-adsorbed fraction was enriched in IgG3. With Phenyl-Sepharose, adsorption increased smoothly with the salt concentration. It is concluded that different forces come into play for absorption on thiophilic gels at low and high salt concentration.
Centre National de Transfusion Sanguine, Les Ulis, France.
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Bridonneau
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Lederer
D000327:Adsorption; D000645:Ammonium Sulfate; D002021:Buffers; D002845:Chromatography; D004797:Enzyme-Linked Immunosorbent Assay; D005782:Gels; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D012965:Sodium Chloride; D013438:Sulfhydryl Compounds / Q000737:chemistry
D002021:Buffers; D005782:Gels; D007074:Immunoglobulin G; D013438:Sulfhydryl Compounds; D012965:Sodium Chloride; D000645:Ammonium Sulfate