5 The presence of HCPs in a final drug preparation is referred to as a process‐related impurity, 6 and such impurities may potentially affect immunogenicity, the production of anti‐drug antibodies, and drug efficacy and/or safety. 4 During purification it is well established that small amounts of host cell protein (HCP), including intracellular proteins, will co‐purify with the production biopharmaceutical. 1 Biopharmaceuticals, including many antibody fragments that are currently in development, 2, 3 are primarily produced in non‐human host cells, including Escherichia coli, rodent cell lines and yeast. For example, in 2015 the world's top selling drug was AbbVie's monoclonal antibody Humira (adalimumab). Collectively these data reveal that DnaK has the potential to modify and enhance immunogenicity when associated with aggregated protein.īiopharmaceuticals, and in particular therapeutic antibodies, currently make up some of the highest global sales of pharmaceutical products. 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. DnaK was shown to be associated with the aggregated scFv by Western blot analysis. The addition of DnaK further enhanced IgG and IgG2a antibody responses, but only in the presence of aggregated protein. Aggregation alone enhanced IgM and IgG2a antibody responses, but had no significant effect on total IgG or IgG1 responses. Mice were immunized with monomeric and aggregated preparations, with and without 0♱% DnaK by mass. coli DnaK to mimic the process‐related impurity. Preparations of scFv lacking detectable DnaK were spiked with recombinant E. coli chaperone protein DnaK was identified as a key contaminant of scFv by mass spectrometric analysis. 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 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. 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 production of anti‐drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics.
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