6 research outputs found
Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells
7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced
in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods
based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with
A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation),
mass spectrometry (MALDI–TOF, ES–TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The
light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other
recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine
297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding
to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a
mixture of three families of antibodies corresponding (i) to the expected structure (17%; 149 297 Da; 1330 amino acids), (ii) a variant with a
translated intron in one heavy chains (33%; 152 878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%;
154 459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal
antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also
identified by peptide mapping, mass spectrometry and microsequencing