Detection and reduction of protein A contamination in immobilized protein A purified monoclonal antibody preparations.
Protein A chromatography is an excellent technique for the purification of monoclonal antibodies. However, the presence of Protein A in therapeutic monoclonal antibody preparations due to leaching has been linked with toxicity in animals and humans. Two sandwich ELISAs were developed to monitor Protein A column leaching: (1) rabbit anti-Protein A for capture and anti-Protein A F(ab')2-HRP for detection; and (2) rabbit anti-Protein A for capture and anti-Protein A-biotin for detection. The biotin ELISA is sensitive to the subnanogram range. In addition, these assays were used to develop DEAE and gel filtration chromatography techniques that substantially reduce Protein A levels in monoclonal antibodies purified by Protein A chromatography.
Bloom
J W
JW
Protein Purification Department, Cutter Biological, Berkeley, CA 94710.
Wong
M F
MF
Mitra
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000592:standards; D001710:Biotin; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004340:Drug Contamination; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D006735:Horseradish Peroxidase; D007140:Immunoglobulin Fab Fragments; D011817:Rabbits; D013205:Staphylococcal Protein A / Q000032:analysis* / Q000276:immunology / Q000592:standards
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D013205:Staphylococcal Protein A; D001710:Biotin; D006735:Horseradish Peroxidase