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Analysis of the composition of immunoconjugates using size‐exclusion chromatography coupled to mass spectrometry
Author(s) -
Lazar Alexandru C.,
Wang Lintao,
Blättler Walter A.,
Amphlett Godfrey,
Lambert John M.,
Zhang Wei
Publication year - 2005
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1987
Subject(s) - chemistry , chromatography , mass spectrometry , size exclusion chromatography , conjugate , high performance liquid chromatography , electrospray ionization , electrospray , reversed phase chromatography , conjugated system , organic chemistry , polymer , mathematical analysis , mathematics , enzyme
Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi‐chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI‐MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI‐MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed‐phase high‐performance liquid chromatography (HPLC), and size‐exclusion HPLC were evaluated for sample desalting. Size‐exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI‐MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non‐conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species. Copyright © 2005 John Wiley & Sons, Ltd.