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.
Bridonneau
P
P
Centre National de Transfusion Sanguine, Les Ulis, France.
Lederer
F
F
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