Characterization of Humanized Anti-TAC, an Antibody Directed Against the Interleukin 2 Receptor, Using Electrospray Ionization Mass Spectrometry by Direct Infusion, LC/MS, and MS/MS

Characterization of a humanized monoclonal antibody (Hu-anti-TAC) directed against a surface protein expressed on T-lymphocytes was performed with an electrospray mass spectrometer. Capillary reversed-phase liquid chromatography (LC)/mass spectrometry (MS) and direct infusion MS were utilized along...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1994-03, Vol.66 (5), p.585-595
Main Authors: Lewis, Derf A, Guzzetta, Andrew W, Hancock, William S, Costello, Maureen
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Antibodies, immunoglobulins
Antibodies, Monoclonal - chemistry
Biological and medical sciences
Chemistry
Chromatography, High Pressure Liquid - methods
Deoxyribonucleic acid
DNA
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Genetic engineering
Humans
Mass Spectrometry - methods
Molecular immunology
Molecular Sequence Data
Monoclonal antibodies
Peptide Mapping
Proteins
Receptors, Interleukin-2 - immunology
Recombinant Proteins - chemistry
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Summary:Characterization of a humanized monoclonal antibody (Hu-anti-TAC) directed against a surface protein expressed on T-lymphocytes was performed with an electrospray mass spectrometer. Capillary reversed-phase liquid chromatography (LC)/mass spectrometry (MS) and direct infusion MS were utilized along with tandem MS/MS analysis to confirm the sequence and to determine the sources of heterogeneity in Hu-anti-TAC. The MS analysis was performed on disulfide-reduced and trypsin-digested samples of the antibody. Two forms of diantennary carbohydrate structures were identified and found to be consistent with those reported for the human IgG1 framework. The analysis demonstrated that the N-terminus was modified by conversion of a glutamine residue to pyroglutamic acid. Another source of heterogeneity was the partial removal of the C-terminal lysine residue and was confirmed by mass calculations of tryptic peptides followed by MS/MS sequencing. This study demonstrates that the high sensitivity of electrospray mass spectrometry when combined with capillary chromatography can allow detailed characterization of microgram samples of high molecular weight proteins such as antibodies.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac00077a003