An enzyme-linked immunosorbent assay for host cell protein contaminants in recombinant PEGylated staphylokinase mutant SY161
| Autor: | Jeff Schrimsher, Greg Conn, Susan Rabideau, Min Wan, Yunjuan Wang, Randall Moreadith |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Blotting
Western Clinical Biochemistry Pharmaceutical Science Enzyme-Linked Immunosorbent Assay medicine.disease_cause Polyethylene Glycols Analytical Chemistry law.invention Plasminogen Activators law Drug Discovery Escherichia coli medicine Animals Coloring Agents Spectroscopy chemistry.chemical_classification biology Goats Immunogenicity Metalloendopeptidases Proteins Reproducibility of Results Staphylokinase Reference Standards Alkaline Phosphatase Molecular biology Recombinant Proteins Enzyme Biochemistry chemistry Polyclonal antibodies Mutation biology.protein Recombinant DNA Electrophoresis Polyacrylamide Gel Indicators and Reagents Antibody Drug Contamination Plasminogen activator |
| Zdroj: | Journal of Pharmaceutical and Biomedical Analysis. 28:953-963 |
| ISSN: | 0731-7085 |
| Popis: | Staphylokinase, a bacterially-derived protein which functions as a plasminogen activator, has potential utility as a human therapeutic for thrombotic disorders. A recombinant version of this protein, SY161, contains 13 amino acid substitutions designed to decrease immunogenicity, and has been covalently modified by crosslinking a 5 kDa polyethyleneglycol (PEG) group to the N-terminal region to prolong the drug circulating half-life. The recombinant PEG-modified SY161 staphylokinase is currently in phase II clinical trials as a treatment for acute myocardial infarction. We have developed a sensitive product specific host cell protein (HCP) assay in the ELISA format to monitor in process host-derived contaminant clearance and final drug product purity. The assay is based upon use of goat polyclonal antibodies raised against E. coli host strain cell proteins from a null cell line, extracted by the same manufacturing process used to produce SY161. The identification and clearance of HCP contaminants was confirmed during drug product production using SDS-PAGE and Western blotting utilizing the same polyclonal HCP antibodies. The assay is specific for E. coli host cell strain proteins with a useful detection range from 1 to 100 ng/ml, and is not affected by product level. The level of residual HCPs in the clinical product produced by our manufacturing process was determined to be less than 1 ng/ml at a product concentration of 1 mg/ml. |
| Databáze: | OpenAIRE |
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