Screening and optimization of process parameters of SARS-CoV-2 monoclonal antibody F61 in affinity chromatography stage using design of experiment
Objective To select and optimize the process parameters of SARS-CoV-2 F61 affinity chromatography by the screening and optimization experiment of Design of Experiment(DoE),in order to obtain the optimal process conditions.Methods Eight process parameters that may affect the experimental response results in affinity chromatography were sele-cted and their level ranges were determined.DoE screening test was used to perform 8 factor 2 level screening tests on the sele-cted process parameters.The response values were detected and the mathematical model was fitted by statistical soft-ware.Three key process parameters significantly affecting the key quality attributes were obtained by Pareto diagram analysis(P＜0.05).Then DoE response surface method(RSM)was selected to optimize the key process parameters.First,the full factorial experiment design was completed,the response results were detected to fit the mathematical model,and the bending term P value was analyzed to judge the range of significant factors in the optimal range(bending P＜0.05),then according to the sequential complement of the central composite face-centered design(CCF)experiment,through the detection of the response results to fit the response surface model,the range of optimal conditions was obtained,and the stability of the opti-mal parameters was finally verified by repeated experiments.Results In the screening experiment,it was found that the sig-nificant factors affecting F61 in the affinity chromatography stage were elution buffer pH,elution buffer salt concentration,leaching buffer salt concentration and equilibrium buffer salt concentration.The optimal conditions of key process parame-ters in affinity chromatography were obtained by CCF.When the pH of elution buffer was 3.2,the elution buffer salt concen-tration was 0.07 mol/L NaCl,and the leaching buffer salt concentration was 0.31 mol/L NaCl,the yield of F61 reached 95.25％,the residual amount of host cell protein(HCP)was 97.33 ppm,and the monomer purity of the sample was 98.51％at the affinity chromatography stage.Conclusion Different types of DoE methods were used to screen and optimize the process parameters of F61 affinity chromatography stage,and the optimal process conditions were obtained,which lays a foundation for the esta-blishment of F61 purification process.
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