Continuous Chromatography For Antibody Charge Variants Separation And Model-assisted Process Development

Monoclonal antibody(mAb)is one of the most important biotechnological drugs.The mAb charge variants would be formed during cell culture and production process,resulting in slight differences on the number of charged residues and charge distribution on protein surface.Considering that charge variants may affect the efficacy and pharmacokinetics,it is necessary to separate and characterize mAb charge variants using ion exchange chromatography(IEC).However,due to the similar molecular structures and surface charge properties,the peaks of main mAb and charge variants show strong overlap,and it is difficult to separate target mAb with high purity and recovery together.Currenlty,continuous chromatography is a promising trend in biopharmaceutical industry,which has the potential to improve the separation performance.The continuous chromatography process is complicated with many operating parameters and it is hard for process optimization.Focusing on the separation of mAb charge variants,this thesis would compare traditional batch chromatography and novel twin-column continuous chromatography,and optimize the separation conditions.In addition,IEC model woule be constructed and the model-assisted process evaluaiton method would be developed for the optimization of key process parameters.Some guidance would be obtained to improve the continuous chromatography process development for hard-to-separate components.Firstly,the separation of mAb charge variants with traditional batch cation exchange chromatography(CEX)was studied.The resin particle diameter,flow rates,elution modes and gradients were investigated and optimized.The results showed that resin particle diameter had a crucial impact on the separation performance.Near 100%purity of main mAb could be obtained with the recovery of 56.5%using the small-particle-diameter resin(15 μm)under the gradient of 0.05 pH/CV and the flow rate of 100 cm/h.Both salt gradient and pH gradient elutions could be used for charge variants separation.The trade-off between purity and recovery could be improved with the shallower gradients and slower flow rates.Moreover,the interactive influences between elution gradient slopes and flow rates were found.The elution gradient slopes had a significant influence on recovery,while flow rate on process productivity.In general,shallower elution gradient and faster flow rate could improve the process efficiency for mAb charge variants separation with CEX.Secondly,twin-column continuous chromatography was applied for mAb charge variants separation.The multicolumn counter-current solvent gradient purification(MCSGP)mode was used with the recycling of overlapped components,which could improve the recovery at the given purity of target protein.The results demonstrated that the recovery of main protein was enhanced for both model protein system and mAb sample.The design of normal MCSGP was based on the batch elution chromatogram.However,the change of elution flow rates and proportion of loading protein during MCSGP procdure could cause some differences of the elution profiles.Thus the working window of normal MCSGP was not reasonable,which could influence the purity and recovery of target protein.To improve the separation performance,a strategy of improved MCSGP process design was proposed,called reMCSGP,which determined the working window based on the elution profiles of normal MCSGP process.For model protein system,reMCSGP could improve the recovery from 83.6%to 97.8%at the given purity more than 95%.For mAb sample,the recovery of reMCSGP was improved to 93.9%,which was 15.4%and 20.7%higher than two normal MCSGP processes at the given purity more than 84%.For better understanding on MCSGP process,mechanism model was introduced,and IEC model was established with equilibrium dispersive model(EDM)and steric mass-action model(SMA).Then MCSGP model was constructed with the process parameters of continuous chromatography.The results indicated that the model had the good prediction ability for protein elution profiles of MCSGP process.The rationality and necessity of reMCSGP process were verified.In addition,the key parameters of MCSGP process were evaluated systematically with MCSGP model.The retention time would be reduced with the increase of protein load,causing the decrease of target protein recovery seriously.The determination of the start point of dilution(Line A)and the start point of collection region(Line C)are very important.To increase the recovery of target protein,the conductivity of Line A should not be too high,in case of some protein washed earlier,which could reduce the recovery.Line C should move to the left properly to enlarge the collection region for the protein sample with relatively low overlap.Finally,to accumulate and characterize the molecular structures of mAb charge variants,batch chromatography and novel twin-column N-rich continuous chromatography were studied and compared.For batch process,the column was scaled up from 4.15 mL to 88.20 mL,and 1.19 mg acidic variants could be obained with the recovery of 59.2%.For continuous N-rich process,two 3.93 mL columns were used,and 1.33 mg acidic variants could be separated with the recovery of 86.2%after 22-cycle accumulation.With the same load amount,twin-column N-rich continuous chromatography showed a high potential to enrich minor compounds from the complex mixture with higher recovery,smaller column and lower resin cost.In addition,molecular structures of acidic variants were measured using LC-MS/MS and some useful structural information was obtained.To conclude,this thesis focused on the separation and accumulation of mAb charge variants,and traditional batch chromatography and novel twin-column continuous chromatography were compared.The results demonstrated that continuous chromatography could improve the separation performance and process productivity.Due to the complexity of continuous chromatography process,mechanism model is useful to evaluate the influences of key parameters,strengthen the understanding of process and assist the process analysis and optimization of continuous chromatography.