A new product-related contaminant was discovered during the analysis of a human monoclonal IgG1 antibody produced in CHO cells.   The novel species contains three light chains and two heavy chains rather than two light and two heavy chains but is distinct from the recently described �Triple Light Chain Antibody� (Biotechnol Bioeng. 105(4 ):748-760).  The structure of this species was characterized by size exclusion chromatography, multiangle light scattering, capillary gel electrophoresis and amino terminal protein sequencing.  Antibody binding to its target was measured by ELISA and interferometry (Octet).  The novel species eluted between monomeric and dimeric antibody in size exclusion HPLC, and has a mass of 176 kDa.   Analysis of the novel species by reducing and nonreducing capillary SDS gel electrophoresis demonstrated the presence of a noncovalently bound subunit with the same molecular weight as light chain.  The stoichiometry was one noncovalently bound subunit per antibody monomer, and three light chains to two heavy chains.  Edman degradation sequencing confirmed that the extra subunit was human light chain.  Binding to target antigen in an ELISA was approximately 15% the level of purified monomeric antibody.  A similar difference was found in the dissociation constants measured by interferometry.  The three light chain contaminant is present early in CHO cell culture and copurified with monomer during protein A, anion and cation exchange chromatography.   The contaminant could be separated from monomer by hydrophobic interaction chromatography.

Characterization of a monoclonal antibody product related contaminant with three light chain subunits

243rd National Meeting (2012)

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