Production Process Reproducibility And Product Quality Consistency Of Transient Gene Expression

Background and aim There are a growing number of therapeutic biological molecule candidates in the pipeline waiting for preclinical and clinical evaluation.As the medical treatment is on its way to precision medicine,it will bring us much more candidates to evaluate in development stages.Traditionally,it usually takes 6 to 12 months to generate cell lines to express recombinant proteins,which is a time-and resource consuming process and can’t satisfy the need for biopharmaceuticals research and development.So,if one can establish a procedure to screen the candidates with a shorter period of time and lower cost,it would dramatically facilitate the development of innovative biological therapeutics.Over the last decade,transient gene expression(TGE)method has been actively pursued for this purpose,which only takes several weeks to set up a production system.A wide range of products from monoclonal antibody(m Ab),Fc fusion protein,to other recombinant proteins have been successfully manufactured by a TGE system with high product quality.The production of TGE has been scaled up to 100 L,and the expression level reached 1 g/L for monoclonal antibody.Technically,it is feasible now to produce 100 g of protein with reasonable similar glycan composition by TGE,which were demonstrated by several publications.However,TGE has not been accepted for manufacturing therapeutic materials for a preclinical toxicology study and early human clinical trials,except for vaccine products.Production process reproducibility of TGE and multiple batch product quality consistency have been the main concerns.In this research,we assess the overall cell growth,cell apoptotic status,and multiple batch product yield and quality profile through physicochemical and biological activity assays,as well as analysis of glycan composition.Methods The overall cell growth including cell density,cell viability and cell apoptotic status are vital for the reproducibility of different TGE batches,as cells are the factories for the production of proteins.In this study,we produced 9 batches of anti-PD1 antibody through TGE in HEK293 E cells under repeated cell culture conditions and parameters.Then we compared the cell density,cell viability and cell apoptotic status during the production process among the 9 batches of TGE and yield of different TGE batches to characterize the reproducibility of different TGE batches.According to the guideline of CFDA and European Medicine Agency,the PI,secondary structure,binding affinity,thermal stability,and glycan composition are all important profiles required to be compared among products from different batches to demonstrate the product quality consistency.So,we compared these quality attributes among the 9 batches of antibody to characterize the quality consistency of antibodies from different batches of production.Results Production process-related parameters such as cell density,cell viability,and cell apoptosis assessed during cultural time after TGE showed process reproducibility with narrow variation in overall cell growth,resulting in reproducible product expression.More critical aspects are product quality attributes such as PI,binding affinity,secondary structure,and thermal stability which were compared and showed excellent comparability among multiple lots.The most critical quality attributes,N-glycosylation of the Ig G antibody from different TGE batches,were also compared to demonstrate the comparability in post-translational modification.As the results shown,the N-glycan distribution of antibodies produced in different TGE batches was highly similar.