title: Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography.
abstract: Staphylokinase variant SY161 is a recombinant mutant of the Staphylococcus aureus polypeptide staphylokinase (Sak), and is currently in human clinical trials as a thrombolytic agent. The 15 kDa single chain SY161 protein is expressed as a soluble cytoplasmic product in E. coli with a single cysteine inserted near the N-terminus. The protein as extracted from E. coli is a mixture of both monomeric and intermolecularly disulfide crosslinked species. To improve protein purification yields SY161 is sulfitolyzed during the early stages of production, preventing disulfide formation. The protein is later modified during manufacturing to incorporate a single 5 kDa polyethylene glycol group on the single sulfhydryl sidechain. We have developed and qualified a reverse-phase chromatographic method to quantitate SY161 during product manufacturing. We discuss the use of the assay during manufacturing development to monitor fermentation yields, the SY161 PEGylation reaction, and as an in-process manufacturing control assay. The assay has been applied as a product purity and identity release assay and is suitable for use in assessing product structural integrity during stability testing. The assay has a linear range of quantitation for SY161 from at least 0.15 to 16 micro g, and is-in addition capable of detecting and quantitating protein de-PEGylation events and host cell-derived protein contaminants.
authors.affiliation: Pharmaceutical Sciences, Diosynth-RTP, 3000 Weston Parkway, Cary, NC 27513, USA.
authors.forename: Catharine
authors.initials: C
authors.lastname: Johnson
authors.forename: Mabel
authors.initials: M
authors.lastname: Royal
authors.forename: Randall
authors.initials: R
authors.lastname: Moreadith
authors.forename: Frank
authors.initials: F
authors.lastname: Bedu-Addo
authors.forename: Siddharth
authors.initials: S
authors.lastname: Advant
authors.forename: Min
authors.initials: M
authors.lastname: Wan
authors.forename: Greg
authors.initials: G
authors.lastname: Conn
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D002853:Chromatography, Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004340:Drug Contamination; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005285:Fermentation; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D008970:Molecular Weight; D009154:Mutation; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000302:isolation & purification*; D017433:Protein Structure, Secondary; D011786:Quality Control; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D008666:Metalloendopeptidases; C461653:PEGylated staphylokinase SY161; D003545:Cysteine