Identification of multiple sources of charge heterogeneity in a recombinant antibody

Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both ligh...

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Bibliographic Details
Published in:Journal of chromatography. B, Biomedical sciences and applications Biomedical sciences and applications, 2001-03, Vol.752 (2), p.233-245
Main Authors: Harris, Reed J., Kabakoff, Bruce, Macchi, Frank D., Shen, Felicity J., Kwong, May, Andya, James D., Shire, Steven J., Bjork, Nancy, Totpal, Klara, Chen, Anthony B.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Antibodies - chemistry
Antibodies - immunology
Chromatography, High Pressure Liquid
Chromatography, Ion Exchange
Isoelectric Focusing
Molecular Sequence Data
Peptide Mapping
Receptor, ErbB-2 - immunology
Recombinant antibody
Recombinant Proteins - chemistry
Recombinant Proteins - immunology
Trypsin - chemistry
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Summary:Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.
ISSN:0378-4347
1387-2273
DOI:10.1016/S0378-4347(00)00548-X