Reversed-phase high-pressure liquid chromatographic tryptic peptide mapping for the comparison and study of monoclonal antibodies
| Autor: | D. C. Anderson, Geoffrey F. Lee |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Iodoacetic acid
medicine.drug_class Biomedical Engineering Pharmaceutical Science Iodoacetates Bioengineering Peptide Monoclonal antibody Peptide Mapping High-performance liquid chromatography Immunoglobulin Fab Fragments chemistry.chemical_compound Papain medicine Trypsin Cysteine Chromatography High Pressure Liquid Pharmacology chemistry.chemical_classification Chromatography Organic Chemistry Antibodies Monoclonal Iodoacetic Acid chemistry Iodoacetamide Biotechnology |
| Zdroj: | Bioconjugate Chemistry. 2:367-374 |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/bc00011a012 |
| Popis: | We have examined and optimized several parameters to generate efficient, high-resolution, high-information tryptic peptide maps of monoclonal antibodies and their Fab fragments, without separating the H and L chains, using reversed-phase high-pressure liquid chromatography. Use of a high protease-to-substrate ratio with optimized digestion time and HPLC gradient conditions led to a reproducible mapping of the reduced, carboxymethylated Fab fragments of two antibodies. The technique was then used to screen Fab lots for batch-to-batch consistency, and for examining the effect of 10 mM cysteine on papain cleavage of whole antibody. The technique was modified by labeling cysteine with chromophoric analogues of iodoacetamide instead of radiolabeled iodoacetamide, resulting in a three-dimensional peptide map. With multiwavelength detection, this consisted of simultaneous observation of all chromophores at 214 nm, those with aromatic residues at 280 nm, and those with cysteine at 422 nm, without collecting and counting each peak to identify cysteine-containing peptides. |
| Databáze: | OpenAIRE |
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