Godfrey W. Amphlett

Structural characterization of the maytansinoid–monoclonal antibody immunoconjugate, huN901–DM1, by mass spectrometry

Immunoconjugates are being explored as novel cancer therapies with the promise of target‐specific drug delivery. The immunoconjugate, huN901–DM1, composed of the humanized monoclonal IgG1 antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901–DM1 contains an average of three to four DM1 drug molecules per huN901 antibody molecule. The drug molecules are linked to huN901 through random modification of huN901 at ε‐amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901–DM1 by electrospray time‐of‐flight mass spectrometry(ESI‐TOFMS), which showed that one to six DM1 drug molecules were attached to an antibody molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp‐N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp‐N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.

Structural Characterization of a Recombinant Monoclonal Antibody by Electrospray Time-of-Flight Mass Spectrometry

PurposeThe aim of this study was to perform structural characterization of a recombinant monoclonal antibody (MAb), huN901, by electrospray time-of-flight mass spectrometry (ESI-TOFMS) using both “top-down” and “bottom-up” approaches.MethodsIn the top-down approach, the molecular masses of the deglycosylated huN901 and the light and heavy chains of the antibody were measured by direct infusion MS and liquid chromatography–mass spectrometry (LC–MS). In the bottom-up approach, trypsin and Asp-N protease were used to digest the separated, reduced and alkylated light and heavy chains followed by LC–MS analysis of the digests.ResultsThe primary structure and post-translational modifications of huN901 were characterized by both top-down and bottom-up MS approaches. Modifications of N-terminal pyroglutamate formation, cleavage of C-terminal lysine, glycosylation, and deamidation were identified in the antibody heavy chain by both protein mass measurement and peptide mapping. No modifications were found in the complementarity determining regions (CDRs) of both chains. Both trypsin and Asp-N protease digestion had an average sequence recovery of 97%, and generated complimentary mapping results with complete sequence recovery.ConclusionsESI-TOFMS is a superior tool to characterize MAb and other complex protein pharmaceuticals.