Antibodies have been extensively used as essential components in diagnostic immunoassays. Polyclonal antibodies (pAbs) represent a population of antibodies collected from multiple B cell clones that have been activated by the immune response, in contrast to monoclonal antibodies (mAbs) produced from a single B cell. PAbs recognize multiple epitopes within the antigen and are therefore less susceptible to the epitope structural modifications as compared to mAbs. Analytical characterization of pAbs presents a substantial challenge due to the complexity of multiple antibody species contained in the pAbs. Mass spectrometry (MS) has been demonstrated as a powerful analytical tool for profiling complex biologics including antibodies. We have utilized electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) to characterize various pAbs. Purified polyclonal intact, reduced and the corresponding fragment F(ab')2 samples were characterized by these MS methods. ESI-MS analysis showed un-resolvable spectra from the pAb heavy chains, which was due to the antibody post-translational modifications (PTMs). In order to decrease the sample heterogeneity for optimal MS analysis, N-linked glycans were removed from the Fc region for further protein characterization. ESI-MS molecular mass profile of light chains demonstrated �fingerprints� for the species contained in pAbs. We have demonstrated that mass spectrometry approaches are useful for profiling pAbs and assessing unique pAb characteristics.

Exploring characterization of polyclonal antibodies with mass spectrometry

247th National Meeting (2014)

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