title: 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.affiliation: Diosynth RTP Inc., 3000 Weston Parkway, NC 27513, USA. mindy.wan@diosynth-rtp.com
authors.forename: Min
authors.initials: M
authors.lastname: Wan
authors.forename: Yunjuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Susan
authors.initials: S
authors.lastname: Rabideau
authors.forename: Randall
authors.initials: R
authors.lastname: Moreadith
authors.forename: Jeff
authors.initials: J
authors.lastname: Schrimsher
authors.forename: Greg
authors.initials: G
authors.lastname: Conn
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D004396:Coloring Agents; D004340:Drug Contamination; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry; D006041:Goats / Q000276:immunology; D007202:Indicators and Reagents; D008666:Metalloendopeptidases / Q000032:analysis; D009154:Mutation; D010960:Plasminogen Activators / Q000032:analysis*; D011092:Polyethylene Glycols / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D004396:Coloring Agents; D007202:Indicators and Reagents; D011506:Proteins; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D000469:Alkaline Phosphatase; D010960:Plasminogen Activators; D008666:Metalloendopeptidases; C461653:PEGylated staphylokinase SY161