On-line Monitoring Of Trastuzumab Charge Variants Separation By Cation Exchange Chromatography

Trastuzumab for injection is the primary targeted therapy for breast cancer with HER2 overexpression.In the separation of principal component and its charge variants by cation exchange chromatography(CEX),the charge variants’peaks overlap seriously with the main peak.In order to solve the problems of low recovery of principal component and insufficient control means,the effects of elution pH,elution gradient slope and elution velocity on chromatographic separation performance were studied,and the CEX process was optimized.An on-line monitoring method for the separation of charge variants by CEX technology based on Raman spectroscopy was established.A sample collection strategy based on spectral model was proposed and a collection scheme to meet production requirements was given.The main researches of this thesis are as follows.1.Optimization of the separation process of Trastuzumab charge variants by CEX.For elution pH,elution gradient slope and elution volume flow rate,Box-Behnken response surface experiment design was used.The recovery and purity of principal component and solvent consumption ratio were used as responses,and the three response indexes were fitted by quadratic polynomial model.The optimization results can be obtained by setting the constraint conditions within the experimental range and the expectation function containing three response indicators and their weights:The eluent pH was 5.665,the gradient slope was 5.280 CV,the volume flow rate was 1.300 mL/min,the recovery of principal component was about 63.448%,the highest purity was about 87.592%,and the solvent consumption ratio was about 1.697 mL/mg.2.Research on on-line monitoring method of CEX separation of charge variants based on Raman spectroscopy.Using experimental data and Raman spectroscopy of the whole process,partial least squares(PLS)quantitative models of total protein concentration,principal component concentration and acid variant content were established.The R~2 of the correction sets and prediction sets of the three models were all greater than 0.93,showing good fitting ability and high prediction ability.But the prediction of total protein concentration and principal component concentration at low concentration was poor.The prediction of acid variant content can reflect the change trend correctly,but the accuracy was poor.It was speculated that the reason affecting the prediction ability of Raman model was the change of eluent salt concentration.3.Research on sample collection strategies of CEX based on Raman spectroscopy. Based on the established rapid quantitative models of total protein concentration, principal component concentration and acid variant content,a strategy of first indentation and then expansion of sample collection was proposed to mix as many components as possible,and the acid variant content and principal component content of the mixed sample met the requirements.High performance liquid chromatography(HPLC)analysis results were used to verify the final combined set obtained by the strategy,which proved the feasibility of the sample collection strategy, but whether the strategy is accurate and effective depends on the prediction accuracy of the fast quantitative model.