Study On Quality Control Of Lentiviral Vectors For Immunotherapy

Tumor immunotherapy has gradually developed into an effective and low-invasive treatment strategy,which can be applied to the treatment of various malignancies.Among them,lentiviral vectors(LVs)have become ideal tools for gene therapy due to their efficient gene integration and stable expression ability in target cell genes.Clinical applications have high quality requirements for the final product to ensure its purity,efficacy and safety.In order to ensure the production of high-quality LVs,this paper has carried out relevant researches on the quality control methods especially the quantitative detection methods of LVs,and characterzation of physicochemical properties of LVs and the existence of exosomes in LVs.This work provides a reference for the quality control of LVs products.The specific content includes:(1)The HPSEC-MALLS method was established for quantifying LVs particles.The influences of analytical column,concentration and flow rate on the detection results were investigated.Among them,the TSK-GEL G6000PWxl results showed a good linear relationship between the particle count of LVs and the concentration of LVs,which can be used for the quantitative detection of LVs,and the molecular weight and size distribution analyses are more accurate.The molecular weight of LVs was determined to be 6.0×10~7-9.0×10~7Da,the number of lentiviral particles is on the order of 10~8,and the average radius is 51-52 nm.Compared with traditional virus titer detection methods,HPSEC-MALLS has the advantages of rapid detection and comprehensive characterization.In addition,other physical and chemical properties of LVs were analyzed:the Tm of LVs was51.6±0.35℃measured by DSF;the average diameter of LVs was 128.7 nm measured by DLS,which is consistent with the results of HPSEC-MALLS;Zeta potential of LVs was 13.8±0.4 m V,and TEM detection found that there were two kinds of morphological structures in the particles.The above results provide a reference for the establishment of quality control methods for LVs.(2)Two extraction methods of ultracentrifugation and PS immunoaffinity were employed to extract exosomes from HEK 293T cell culture fluid.By comparing the analysis results of HPSEC-MALLS,DLS,TEM,etc.of the extracted exosomes,the production of exosomes was verified.It was found the yield of exosomes obtained by d UC is greater,while the purity obtained by PS affinity is higher.The molecular radius of the exosomes extracted by PS was mainly around 67-68 nm,and the molecular weight was 9.956×10~7(±33.176%)Da.The average radius of exosomes obtained by d UC was 34-35 nm,and the molecular weight was 1.303×10~8(±7.744%)Da.(3)The presence of exosomes in LVs was verified by Western blotting,and the exosomes in LVs can be completely separated by PS affinity extraction.HPSEC-MALLS analysis showed the molecular weight of LVs after exosome removal was 2.104×10~7(±3.977%)Da,and the average radius was 48-49 nm.According to the results of particle count analysis,exosomes accounted for about1/10 of the number of particles in LVs.The PS affinity extraction combined with HPSEC-MALLS can more accurately quantitatively analyze LVs and their size and molecular weight distribution.