Description:
AbstractThe covalent immobilization of antibodies to solid supports for immunoaffinity (IA) purification is widely used in the biological sciences. However, relative immobilization yields, immobilization stability, and antigen‐binding capacity vary significantly with the antibody and protocol used. A systematic study was conducted to determine the most versatile antibody immobilization method for use in on‐line and off‐line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We evaluated a method to survey optimal elution conditions and estimated immobilization yields, matrix stability, antigen binding capacities, and antigen recovery of different IA matrices. Some mAbs were sensitive to aminogroup‐based immobilization, i.e., losing antigen binding capabilities after immobilization especially using epoxy chemistry. In general, the most optimal covalent antibody immobilization for on‐line IA‐LC‐MS was achieved using aminogroup immobilization of intact antibodies by epoxy‐ or aldehyde‐activated POROS R20‐matrices and in some cases by chemical crosslinking to Protein G‐POROS. Protein G‐based matrices are very stable showing essentially no decline in performance after 50 application–wash–elution–reequilibration cycles and being easily prepared within 2–3 h of working time with a typical antibody coupling yield of above 80%. In off‐line applications where constant flow conditions are not used, covalent crosslinking onto Protein G‐POROS or Protein G‐agarose is to be recommended.