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%; 14,9297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 15,2878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 15,4459 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.
Beck
Alain
A
Centre d'Immunologie Pierre Fabre, 5 Avenue Napoléon III, 74160 Saint-Julien-en-Genevois, France. alain.beck@pierre-fabre.com
Bussat
Marie-Claire
MC
Zorn
Nathalie
N
Robillard
Virginie
V
Klinguer-Hamour
Christine
C
Chenu
Stéphane
S
Goetsch
Liliane
L
Corvaïa
Nathalie
N
Van Dorsselaer
Alain
A
Haeuw
Jean-François
JF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D001483:Base Sequence; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007334:Insulin-Like Growth Factor I / Q000276:immunology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000911:Antibodies, Monoclonal; D007334:Insulin-Like Growth Factor I