Enhanced Production Of Anti-PD1 Antibody In Cho Cells Through Transient Co-transfection With Anti-apoptotic Genes

Background and aimProduction of biopharmaceuticals have increased due to improvements in optimized media and bioprocess design in mammalian cell lines.However,much more effort improving productivity is still needed to meet rapidly growing market demand for many biological products.According to the latest updates of the DrugBank,there were over 5,028 experimental drugs and 4,775 non-redundant protein(i.e.drug target/enzyme/transporter/carrier)sequences linked to these drug entries.Thus,fast development of innovative biological therapeutics would be further appreciated,if the process is capable of producing biopharmaceuticals in a short period with low cost.Transient gene expression(TGE)is gaining popularity as a cost-effective process to produce recombinant protein products.The protein of interest is expressed usually within 16-96 h using TGE technology.And the quality of protein obtained from TGE meets the standard of preclinical assessment.It would be necessary to optimize mammalian cell culture in TGE for cost-saving production of biopharmaceuticals.One of critical impact factors on productivity would be apoptosis during cell culture process,that is undergoing programmed cell death.When cells are exposed to stresses caused by various factors containing mechanical agitation,hypoxia,nutrient depletion,waste byproduct accumulation and viral infection etc.It was reported that apoptotic process accounted for approximately 80%cell death during cell culture,which affected cell survival state and the quality of protein production.Several methods had been evaluated to limit the onset of apoptosis and to extend cell life by inhibiting or delaying activation of cell death,which would produce more recombinant proteins for biopharmaceutical applications.One successful approach to extend cell lifetime was overexpressing anti-apoptotic genes to manipulate intracellular biochemistry.It was reported that anti-apoptotic genes could maintain the mitochondria membrane potential,and then prevent the release of cytochrome c during apoptosis.Bcl-xL was shown to prevent apoptosis in several production host cell lines including CHO,BHK and Hybridoma.It could be utilized to improve productivity through increasing cell viability and reduction of apoptosis.Previous reports related to overexpressing anti-apoptotic genes were mainly in stable cell lines,which consumed time and resource,due to requirement of screening cell lines.Mcl-1,another important anti-apoptotic gene,was previously published as it improved productivity in stable transfections with Mcl-1 by Betenbaugh's group.However,it has not been thoroughly examined for its effects on productivity in TGE before.It was unique that Mcl-1 did not suppress apoptosis induced by overexpression of either Bax or Bak like Bcl-xL,but did bind to them.Additionally,Mcl-1was related to maintenance of cell viability but not stimulation of proliferation.Autophagy was defined as being the destructive process by which part of the cytoplasm,organelles and proteins that must be degraded are enveloped by a single or double membrane,resulting in an organelle-“autophagosome”.Then the inside materials are broken down once the autophagosome fuses with a lysosome.Eventually,the degradation breakdown products are released and recycled into metabolic and biosynthetic pathways.Recently,autophagy has been focused on an anti-cell death engineering target in addition to apoptosis in the Chinese hamster ovary(CHO)cell engineering field.Rapamycin inhibits mTOR by blocking the mTOR complex 1(mTORC1).It has been widely used as a chemical activator in vitro and vivo research.Anti-PD1 antibody has been a great successful inhibitor of immune checkpoint,currently being widely used in approved clinical applications for the treatment of 6 different tumors.Besides of three companies were approved by FDA to sale anti-PD1 antibody on the market,many other followers are still in various development stages.Optimization of the production process or exploration of new approaches would be particular interested in biopharmaceutical industry.Here,we described an alternative approach to increase viability of production cells,delay apoptosis onset and enhance transient expression of anti-PD1 antibody through co-transfection with anti-apoptotic genes Bcl-xL or Mcl-1 without stable cell line screening.In addition,a new method demonstrated that it could enhance cell life through co-working of Bcl-xL and rapamycin.Methods(1)To determine the optimal conditions of PEI used in transfection of CHO-s cell line,DNA concentrations of 2.5-4.0?g/mL,cell densities of1-3×10~6 cells/mL and DNA/PEI ratio of 1:2-1:3(w/w)were evaluated by using pEGFP as a reporter protein.Using the optimal transfection conditions as described above,CHO cells were transfected by 3.0?g/mL of Bcl-xL or Mcl-1 respectively,in order to determine whether over-expression of those two anti-apoptotic genes could affect apoptosis of host cells.(2)To examine the optimal amount of anti-apoptotic genes Bcl-xL and Mcl-1,CHO cells were co-transfected 5-75%(w/w)anti-apoptotic genes in total 3.0?g/mL DNA with plasmids coding anti-PD1 antibody sequence.The amount of PEI,total DNA concentration and cell density in all groups were ongoing as same.(3)We co-transfected plasmids coding anti-PD1 antibody with 10%Bcl-xL or 50%Mcl-1respectively under optimized conditions described above,and productivity of anti-PD1 antibody was assessed.Cells in control group were transfected with 3.0?g/mL plasmids,in which containing anti-PD1 antibody genes only.Supernatants were taken every day and analyzed by ELISA.Considered that low cell viability had negative impact on target protein quality,the products were harvested immediately when the cell viability was below 50%.(4)To examine whether co-working of Bcl-xL and rapamycin prolong cell growth time or not,100 nM rapamycin was added after 24 h co-transfected with 10%Bcl-xL and anti-PD1 antibody.Access cell density and viability,apoptosis,cell cycle and productivity during cell culture.Results(1)The optimal transfection conditions as below:transfecting 3×10~6cells/mL with 3.0?g/mL DNA at a ratio of DNA:PEI=1:3(w/w).Bcl-xL and Mcl-1 could have positive impact on product yield through delaying apoptosis onset.(2)Co-transfection with 10%Bcl-xL or 50%Mcl-1 is the optimal condition.(3)After co-transfection with 10%Bcl-xL or 50%Mcl-1,it showed reduced levels of apoptosis and improved cell viability after co-transfected with Bcl-xL or Mcl-1.The overall production yield of the antibody anti-PD1 increased approximately 82%in CHO cells co-transfected with Bcl-xL,and 34%in CHO cells co-transfected with Mcl-1.(4)The group added rapamycin after co-transfected with 10%Bcl-xL showed increased cell viability,reduced levels of apoptosis,arrested G1 phase cell cycle and enhanced overall productivity.