Construction Of CHO Engineering Cell Line For The Production Of Anti-PD-1 Antibody And Development Of Cell Culture Process Based On QbD Concept

Among antibody drugs,anti-PD-1 antibodies have become the biggest hot spot in tumor immunotherapy in recent years due to their broad spectrum of malignant tumors,low side effects and good patient acceptance.In this paper,an engineered cell line for the production of anti-PD-1 antibodies was constructed,and under the guidance of the concept of quality by design(Qb D),a small-scale test process that could stably produce anti-PD-1antibodies was developed,with the hope that it would have industrial production value for subsequent development.The cell culture process provided a certain theoretical basis.The specific research contents were as follows:(1)The minipool cells were evaluated in 96?24?6-well plates,and the 6 minipool cells obtained by screening were evaluated in batches,and 3 minipool cells were selected for monoclonal screening.Using a round of limiting dilution method(less than 0.5cell/well)combined with imaging and photography to determine its monoclonal origin,and after evaluation by 96?24?6 well plates,a total of 9 monoclonal cells were Fed in TPP tubes.The experimental results showed that four monoclonal cells were identified through preliminary screening,namely 66-2G7,66-3D9,183-14C2 and 183-15F9.The antibody expression levels on the 14 th day of culture were 2361.47 mg/L,2306.34 mg,2567.53 mg/L and 2688.34 mg/L,respectively.The stability evaluation results showed that the Fed-batch yield of the 60 th generation 66-3D9 was 1594 mg/L,which was 32.45%lower than the 2306 mg/L of the P0 generation,indicating that it may not be stable enough.Shear resistance evaluation showed that 66-3D9 and 183-14C2 showed growth retardation or stagnation at 180 rpm and 200 rpm,respectively.Therefore,after considered the above results,the monoclonal strain was finally determined to be 183-15F9,and thesource report of monoclonal strain was issued.(2)On the basis of the best single clones obtained by screening,the best combination was determined through basic + feed medium screening,and then the key amino acids in the culture process were determined by amino acid metabolism analysis,and a new medium combination was optimized.The experimental results showed that: through the evaluation of 12 basic media and the combined screening with the feed medium,the final selection confirmed that the combination of the base + feed medium of the monoclonal strain 183-15F9 was determined to be B2(OPM-CHO CD052)+ F4(Irvine-Balan CD CHO Feed4).And through amino acid metabolism analysis,it was determined that the key amino acids in the basis of the monoclonal strain 183-15F9 and the optimal addition concentration were Asn: 1.04 mmol/L and Pro: 2.76 mmol/L,and the key amino acids in the feed medium and the optimal addition concentration For His: 40 mmol/L,Asn: 25mmol/L,Lys: 40 mmol/L,Tyr 40 mmol/L,Cys 30 mmol/L,and prepare a new base and feed medium accordingly to prepare a new culture Based on OPM-CHO CD052 plus +Irvine-Balan CD CHO Feed4 plus,after 3L bioreactor verification days,the expression level of antibody reached 4788.87 mg/L on the 14 th day,which was 26.11% higher than the original process.(3)Finally,on the basis of the combination of the obtained optimal basis and the feed medium,the key process parameters were determined through partial factorial design,and then the optimal process conditions were determined by the response surface design,Using minitab 19 to carry out the experimental design of some factors,temperature,dissolved oxygen,and p Hwere determined as the key process parameters.On this basis,surface design response was carried out with aggregate(%),acid,alkaline peak(%)and yield(g/L)as independent variables.Thebest culture process for determining the monoclonal strain 183-15F9 was: temperature: 36.5 ?,p H=7.0±0.1,and dissolved oxygen=45%.Finally,the optimized optimal process was verified on a 3L bioreactor through the obtained optimal conditions.The data showed that there was no significant difference in the parameters of the three parallel bioreactors.The antibody expression levels of the monoclonal strain 183-15F9 were 4788.96 mg/ L,4890.27 mg/L,4860.56mg/L.It showed that this study had established a small-scale cell culture process that can stably produce anti-PD-1 antibodies.