The Study On Impurities Related To MUC1-MBP Fusion Protein And Its Effect On Vaccine Activity

The MUC1-MBP fusion protein vaccine is a therapeutic anti-tumor vaccine developed by the research group in the early stage with MUC1 as the target.It exerts good anti-tumor effects by inducing MUC1 specific cellular and humoral immune responses in the body.The purity and impurity content of proteins are important quality control standards for products,as well as key influencing factors for vaccine activity and anti-tumor effects.The research group has established quality control standards for protein process impurities in the early stage.In order to conduct in-depth research on the related impurities in the MUC1-MBP fusion protein stock solution,this study intends to establish and optimize an analysis method suitable for MUC1-MBP fusion protein related impurities,clarify the impact of different types and contents of impurities on vaccine activity,and provide experimental basis for the quality control of related impurities and the formulation of limit standards.1.Study on impurities related to MUC1-MBP fusion proteinObjective:To clarify the types of impurities related to the MUC1-MBP fusion protein and establish a detection method for related impurities.Methods:The related impurity types of MUC1-MBP fusion protein were identified and analyzed by mass spectrometry and SDS-PAGE;Establish a SEC-HPLC method for detecting polymerization and degradation impurities by optimizing the pore size of the chromatographic column and conduct method validation;The RP-HPLC detection method for analytical chemistry modified impurities was established and validated by optimizing the chromatographic conditions of column packing,mobile phase system and mobile phase gradient.Result:MUC1-MBP fusion protein related impurities include polymerization/degradation impurities and chemical modification impurities;Successfully established SEC-HPLC and RP-HPLC impurity detection methods,which have been validated to have good stability and accuracy.2.Preparation and detection of impurities in MUC1-MBP fusion protein vaccineObjective:To establish a preparation method for aggregation and degradation impurities,prepare vaccines containing different levels of aggregation/degradation impurities,and provide samples for subsequent vaccine activity experiments.Method:Collect absorption peaks of polymeric impurities through UV monitoring,and obtain high content of polymeric impurities through ultrafiltration concentration;A preparation method for screening high content degradation impurities through acid,alkali,and 37℃ high temperature conditions;Detect the content of polymerization and degradation impurities in the prepared product using SEC-HPLC method;Vaccines containing polymerized/degraded impurities were prepared by adding untreated normal protein stock and adjuvants.Result:UV monitoring and collection of polymeric impurity absorption peaks were established as the preparation method for polymeric impurities;Establish 37℃ high temperature as the preparation method for degradation impurities;A MUC1-MBP fusion protein vaccine containing 2%polymeric impurities,0.1%,0.3%,and 1%degradation impurities was prepared.3.Effect of Different Content of Polymerization/Degradation Impurities on the Activity of MUC1-MBP Fusion Protein VaccineObjective:To evaluate the effects of different levels of polymeric/degradable impurities on the activity and anti-tumor effect of MUC1-MBP fusion protein vaccine,and to provide experimental basis for the formulation of quality control standards for related impurities.Methods:Mice were immunized with vaccines containing different levels of polymerization/degradation impurities,and B16 melanoma cells expressing human full length MUC1(B16-MUC1)were injected to establish tumor bearing mice models;Evaluate the impact of impurities on the anti-tumor effect of the vaccine by calculating its anti-tumor rate;Detection of Tc1(CD8+IFN+)in mouse splenic lymphocytes by flow cytometry.The proportion of cell subpopulations was detected by ELISA for MUC1 specific antibodies IgG,IgG1,IgG2c levels and IgG2c/IgG1 ratio in mouse serum.The impact of impurities on vaccine activity was evaluated by real-time unlabeled cell analysis(RTCA)for MUC1 specific CTL cytotoxicity.Result:2%polymeric impurities had no significant effect on the anti-tumor effect of the vaccine;2%polymeric impurities have no significant effect on vaccine induced specific humoral immune response,Thl type immune response level,and CTL killing level;1%degradation impurities significantly reduced the anti-tumor effect of the vaccine(P=0.009),while 1%degradation impurities significantly downregulated the proportion of Tc1 and Th1 cell subpopulations induced by the vaccine(P=0.0072,P=0.0089),IgG2c/IgG1 ratio(P=0.0096),and CTL killing level(P=0.0093).Conclusion:This study successfully established SEC-HPLC and RP-HPLC methods for analyzing the purity and related impurities of MUC1-MBP fusion protein;Established a preparation method for vaccine aggregation/degradation impurities;A preliminary quality control limit of 2%for vaccine aggregation impurities and 1%for degradation impurities has been established,providing experimental basis for the formulation of quality control standards for vaccine impurities.