Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NSO cells

被引:106
作者
Beck, A
Bussat, MC
Zorn, N
Robillard, V
Klinguer-Hamour, C
Chenu, S
Goetsch, L
Corvaïa, N
Van Dorsselaer, A
Haeuw, JF
机构
[1] Ctr Immunol Pierre Fabre, F-74160 St Julien En Genevois, France
[2] Univ Strasbourg 1, CNRS, Ecole Chim Polymeres & Mat, Lab Spectromet Masse Bioorgan,UMR 7509, F-67087 Strasbourg, France
来源
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2005年 / 819卷 / 02期
关键词
CHO cells; NSO cells; peptide mapping; glycopeptide mapping; recombinant monoclonal antibodies; insulin-like growth factor-1 receptor;
D O I
10.1016/j.jchromb.2004.06.052
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (c) 2005 Elsevier B.V. All rights reserved.
引用
收藏
页码:203 / 218
页数:16
相关论文
共 62 条
[1]   Letter: The use of electrospray ionization mass spectrometry to distinguish the lot-to-lot heterogeneity of an antigen specific monoclonal antibody from a specific cellular clone [J].
Adamczyk, M ;
Gebler, JC ;
Harrington, CA ;
Sequeira, AF .
EUROPEAN MASS SPECTROMETRY, 1999, 5 (03) :165-168
[2]   MONITORING OF IGG ANTIBODY THERMAL-STABILITY BY MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY AND MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS-SPECTROMETRY [J].
ALEXANDER, AJ ;
HUGHES, DE .
ANALYTICAL CHEMISTRY, 1995, 67 (20) :3626-3632
[3]   MASS-SPECTROMETRY OF THE HUMANIZED MONOCLONAL-ANTIBODY CAMPATH 1H [J].
ASHTON, DS ;
BEDDELL, CR ;
COOPER, DJ ;
CRAIG, SJ ;
LINES, AC ;
OLIVER, RWA ;
SMITH, MA .
ANALYTICAL CHEMISTRY, 1995, 67 (05) :835-842
[4]   Characterization of the stability of recombinant protein production in the GS-NS0 expression system [J].
Barnes, LM ;
Bentley, CM ;
Dickson, AJ .
BIOTECHNOLOGY AND BIOENGINEERING, 2001, 73 (04) :261-270
[5]   Stability and CTL-activity of P40/ELA melanoma vaccine candidate [J].
Beck, A ;
Goetsch, L ;
Champion, T ;
Bussat, MC ;
Aubry, JP ;
Klinguer-Hamour, C ;
Haeuw, JF ;
Bonnefoy, JY ;
Corvaïa, N .
BIOLOGICALS, 2001, 29 (3-4) :293-298
[6]   Synthesis and characterization of Respiratory Syncytial Virus protein G related peptides containing two disulfide bridges [J].
Beck, A ;
Zorn, N ;
Bussat, MC ;
Haeuw, JF ;
Corvaïa, N ;
Nguyen, TN ;
Bonnefoy, JY ;
Van Dorsselaer, A .
JOURNAL OF PEPTIDE RESEARCH, 2000, 55 (01) :24-35
[7]   Stability and CTL activity of N-terminal glutamic acid containing peptides [J].
Beck, A ;
Bussat, MC ;
Klinguer-Hamour, C ;
Goetsch, L ;
Aubry, JP ;
Champion, T ;
Julien, E ;
Haeuw, JF ;
Bonnefoy, JY ;
Corvaia, N .
JOURNAL OF PEPTIDE RESEARCH, 2001, 57 (06) :528-538
[8]   Therapeutic applications of monoclonal antibodies [J].
Berger, M ;
Shankar, V ;
Vafai, A .
AMERICAN JOURNAL OF THE MEDICAL SCIENCES, 2002, 324 (01) :14-30
[9]  
Brass J M, 1996, Pharm Acta Helv, V71, P395, DOI 10.1016/S0031-6865(96)00051-9
[10]   Therapeutic antibodies for human diseases at the dawn of the twenty-first century [J].
Brekke, OH ;
Sandlie, I .
NATURE REVIEWS DRUG DISCOVERY, 2003, 2 (01) :52-62