Detecting low level sequence variants in recombinant monoclonal antibodies
| Autor: | Boyan Zhang, Hongbin Liu, Viswanatham Katta, Kathleen Francissen, Amy Shen, Charlene Li, Yi Yang, Alex Strahan, Jun Ouyang |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
clone (Java method)
Receptor ErbB-2 Ultraviolet Rays medicine.drug_class Molecular Sequence Data Immunology Peptide CHO Cells Computational biology Biology Monoclonal antibody Peptide Mapping Sensitivity and Specificity Mass Spectrometry law.invention Cricetulus Protein sequencing law Cricetinae medicine Animals Humans Immunology and Allergy Amino Acid Sequence Peptide sequence Chromatography High Pressure Liquid Sequence (medicine) chemistry.chemical_classification Chinese hamster ovary cell Antibodies Monoclonal Molecular biology Recombinant Proteins chemistry Recombinant DNA Reports |
| Zdroj: | mAbs. 2:285-298 |
| ISSN: | 1942-0870 1942-0862 |
| Popis: | A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5-5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach's application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274T at 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins. |
| Databáze: | OpenAIRE |
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