Experimental Validation of a Parenteral Permitted Daily Exposure Value for Cleaning-Induced Degradants from Recombinant Therapeutic Proteins with In Vitro Immunogenicity Assays.

Multiproduct manufacturing of biotherapeutic proteins generate cleaning-induced protein degradants because of extreme pH and temperature conditions during the cleaning process. Cleaning Acceptance limits are calculated based on the maximum allowable carryover (MAC) assessment of the previously manufactured active pharmaceutical ingredient (API) - or drug product - based on the permitted daily exposure (PDE) of the previously manufactured API into the dose of subsequent product. In this study, we tested a previously determined PDE value for cleaning-induced protein degradants of 650 µg/dose. A bench-scale cleaning method was used to generate cleaning induced degradants from both a half-life extension (HLE) BiTE® molecule and a mAb product. For this investigation degradants of HLE BiTE®-A and mAb-1 were characterized either alone or degradants of HLE BiTE®-A and mAb-1 spiked into mAb-1 at 650 µg. These samples were characterized by endotoxin testing, size exclusion chromatography (SEC), light obscuration by HIAC, and micro-fluidic imaging (MFI). These results suggest that significant degradation of the molecule occurs because of the cleaning procedure, and it is no longer in the intact form or active state. The biological impact was assessed using a cell line assay to assess immune activation, and a human Peripheral Blood Mononuclear Cell (PBMC) assay to assess T cell activation, T cell proliferation, and cytokine release after 20 hours and 7 days. Findings from the various in vitro cell-based assays suggest that the presence of 650 µg of carryover of degradants either alone or spiked into the same or a cross-product do not increase immunogenicity risk in cell-based assays - suggesting that the current PDE of 650 µg/dose for cleaning-induced degradant carryover does not have a risk of immunogenicity in patients.