An enzyme-linked immunosorbent assay for host cell protein contaminants in recombinant PEGylated staphylokinase mutant SY161.

Abstract

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.

• Authors: Min Wan, Yunjuan Wang, Susan Rabideau, Randall Moreadith, Jeff Schrimsher, Greg Conn
• Journal: Journal of pharmaceutical and biomedical analysis (J Pharm Biomed Anal) Vol. 28 Issue 5 Pg. 953-63 (Jun 01 2002) ISSN: 0731-7085 [Print] England
• PMID: 12039638 (Publication Type: Journal Article)

Chemical References

• Coloring Agents
• Indicators and Reagents
• Proteins
• Recombinant Proteins
• Polyethylene Glycols
• Alkaline Phosphatase
• Plasminogen Activators
• Metalloendopeptidases
• PEGylated staphylokinase SY161

Topics

• Alkaline Phosphatase (analysis)
• Animals
• Blotting, Western
• Coloring Agents
• Drug Contamination
• Electrophoresis, Polyacrylamide Gel
• Enzyme-Linked Immunosorbent Assay
• Escherichia coli (chemistry)
• Goats (immunology)
• Indicators and Reagents
• Metalloendopeptidases (analysis)
• Mutation
• Plasminogen Activators (analysis)
• Polyethylene Glycols (analysis)
• Proteins (analysis)
• Recombinant Proteins (analysis)
• Reference Standards
• Reproducibility of Results