Size exclusion HPLC analysis of a human monoclonal antibody showed the presence of a new MAb species (referred to as �shoulder�) that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species using analytical methods indicated that it was a MAb containing an extra light chain (molecular weight ~175 kDa). Interestingly, the amount of shoulder (typically 1-3% of the total MAb present) varies with the CHO cell line producing the MAb and to a lesser extent shoulder abundance is also influenced by the growth conditions.  Ion exchange chromatography (AEX, CEX) or hydrophobic interaction chromatography (HIC) are commonly used as polishing steps to remove contaminants from monomeric MAb preparations. Attempts to purify the high molecular weight species from our MAb using our AEX ? CEX platform was successful at removing dimer and higher molecular weight species, but not sufficient for removing the shoulder contaminant. It was determined that HIC could be used in place of CEX to exploit the subtle differences in hydrophobicity between monomer and shoulder. We have developed an antibody polishing process on Butyl Sepharose HP resin capable of removing the majority of high and low molecular weight contaminants yielding 99% pure MAb monomer with a step recovery of about 80%.

Studies on the formation and purification of a monoclonal antibody species containing a third light chain

243rd National Meeting (2012)

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