title: Influence of Escherichia coli chaperone DnaK on protein immunogenicity.
abstract: The production of anti-drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics. It is known that various factors, including aggregation and the presence of process-related impurities, can modify and augment the immunogenic potential of proteins. The purpose of the investigations reported here was to characterize in mice the influence of aggregation and host cell protein impurities on the immunogenicity of a humanized single-chain antibody variable fragment (scFv), and mouse albumin. Host cell protein impurities within an scFv preparation purified from Escherichia coli displayed adjuvant-like activity for responses to the scFv in BALB/c strain mice. The 70 000 MW E. coli chaperone protein DnaK was identified as a key contaminant of scFv by mass spectrometric analysis. Preparations of scFv lacking detectable DnaK were spiked with recombinant E. coli DnaK to mimic the process-related impurity. Mice were immunized with monomeric and aggregated preparations, with and without 0·1% DnaK by mass. Aggregation alone enhanced IgM and IgG2a antibody responses, but had no significant effect on total IgG or IgG1 responses. The addition of DnaK further enhanced IgG and IgG2a antibody responses, but only in the presence of aggregated protein. DnaK was shown to be associated with the aggregated scFv by Western blot analysis. Experiments with mouse albumin showed an overall increase in immunogenicity with protein aggregation alone, and the presence of DnaK increased the vigour of the IgG2a antibody response further. Collectively these data reveal that DnaK has the potential to modify and enhance immunogenicity when associated with aggregated protein.
authors.affiliation: Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Manchester, UK.
authors.forename: Kirsty D
authors.initials: KD
authors.lastname: Ratanji
authors.affiliation: Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Manchester, UK.
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Derrick
authors.affiliation: Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Manchester, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Kimber
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.
authors.forename: Robin
authors.initials: R
authors.lastname: Thorpe
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.
authors.forename: Meenu
authors.initials: M
authors.lastname: Wadhwa
authors.affiliation: Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Manchester, UK.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Dearman
mesh_terms: D000251:Adenosine Triphosphatases / Q000276:immunology*; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000378:metabolism; D000917:Antibody Formation; D001688:Biological Products / Q000627:therapeutic use*; D001709:Biotechnology; D003001:Cloning, Molecular; D004345:Drug Industry; D004926:Escherichia coli / Q000276:immunology; D029968:Escherichia coli Proteins / Q000276:immunology*; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology*; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D055503:Protein Multimerization; D012709:Serum Albumin / Q000276:immunology; D057127:Single-Chain Antibodies / Q000378:metabolism
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D001688:Biological Products; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D012709:Serum Albumin; D057127:Single-Chain Antibodies; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli
keywords: DnaK; aggregation; host cell impurities; immunogenicity