Abstract
A hyperglycosylated recombinant human interferon-β (rhIFN-β) R27T mutant was established to improve relapsing-remitting multiple sclerosis (RRMS) in our previous study. We focused on the stability of the R27T mutant throughout its production lifetime, including culture, purification, and storage before formulation prior to clinical use. Herein, we address the stability of this protein during optimized culture and purification processes. Additionally, we employed artificial stress conditions during culture and purification to characterize R27T instability. Although, among total R27T, relative native R27T ratio displayed transiently low even under optimized production process, the ratio was recovered by the end of the overall production process, suggesting that culture and purification processes are optimized. Artificial stress during culture and purification processes resulted in degradation of R27T acidic and basic variants, and mismatched disulfide bonds in no-aggregated forms as well as in the aggregated form. The presence of disulfide bond exchange without aggregation in the unfolded/misfolded state could be a novel finding for rhIFN-β products. The results provide meaningful information for the comprehensive evaluation of the stability of the R27T variant.
Highlights
Maintaining protein stability is a critical issue throughout all development, culture, refolding, purification, sterilization, shipping, and storage processes related to therapeutic recombinant protein products[1]
We found that the values (IU) from the enzyme-linked immunosorbent assay (ELISA) and cytopathic effect (CPE) was almost same when we conducted those assays with fully active substance such as Rebif and our in-house reference protein, R27T with 1.2 times small difference
We investigated R27T stability during culture and purification processes to better understand the process-related instability of R27T under artificial stress conditions, to characterize the vulnerability of the protein to instability, and to define the critical instability state during the production process
Summary
Maintaining protein stability is a critical issue throughout all development, culture, refolding, purification, sterilization, shipping, and storage processes related to therapeutic recombinant protein products[1] Several factors such as pH, temperature, shear force, protein and salt concentration, hydrophobic forces, and others may be responsible for protein instability[2], potentially resulting in protein degradation, chemical modification such as oxidation and deamidation, disulfide bond formation or exchange, and aggregation. Several studies revealed various types of instability for IFN-β, including oxidation, deamidation, and disulfide changes, their direct clinical relevance has not been confirmed[21,22] These aggregated or unfolded/misfolded products can be induced during the production process and the product storage period due to the undesirable physicochemical properties of IFN-β. We observed free thiol and disulfide bonding exchange without aggregation, revealing a new type of unfolded state, as well as aggregation and degradation, as occurs for rhIFN-β
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
