Study On The Production Technology,quality Standard,stability And Immunity Of Biological Agents TPIN And TPKH

Objective In the early stage of our research group,two biological agents were constructed using the attenuated Salmonella Ty21a as a carrier:attenuated salmonella typhimurium carrying IL-2/hepatocyte growth factor antagonist?NK4??named TPIN?and attenuated salmonella typhimurium carrying keratinocyte growth factor?KGF?/hypoxia-inducible factor 1??HIF--1???named TPKH?and completed the pharmacodynamics and preliminary safety studies of TPIN and TPKH.In order to apply TPIN and TPKH to clinical practice successfully,this study will be conducted from four aspects,including the optimization of the production process,drafted quality standards,stability and immunity research.Methods?1?The optimization of the production process:the survival rate of TPIN and TPKH before and after vacuum freeze-drying was calculated by the plate count,the optimal protectant concentrations of skim milk powder,sucrose,gelatin,and DMSO was selected by single factor experiments,which further decided the formula of optimized the lyoprotectants,explore the vacuum freeze-drying process that meets TPIN and TPKH.What's more,pilot-scale production is carried out in this way to establish a reliable and effective production mode.?2?Drafted quality standards:physical properties of TPIN and TPKH powders were determined by physical inspection.The number of viable bacteria per gram of TPIN and TPKH was measured by plate count.The proliferating activity of human hepatocellular carcinomas cells?HepG2?was detected at different time points after transfect TPIN into HepG2 cells,the proliferating activity of intestinal crypt cells?IEC-6?was detected at different time points after transfect TPKH into IEC-6cells.The safety of TPIN and TPKH was tested by the abnormal toxicity of mice and guinea pigs study.Establishing safe and effective quality standards for biological agents.?3?Stability evaluation:TPIN and TPKH were subcultured for 40 generations in Luria-Bertani?ampicillin?medium?LA?and Luria-Bertani mrdium?LB?medium plate.The target gene fragment was amplified by PCR every 5 generations,colony and bacterial morphology,concentration of plasmid and the expression of the target protein in vitro were evaluated every 10 generations to evaluate its passage stability.TPIN and TPKH were placed in 5 different temperature?-80?,-20?,4?,room temperature and 37??for 31d,the plate count was used to detect the number of viable bacteria at different time points to evaluate the temperature storage stability.?4?Immunity study:BALB/c mice were randomly divided into 4 groups?6 mice per group?:Control group?10%NaHCO₃?,Ty21a group?10⁹ cfu Ty21a?,TPIN group?10⁹ cfu TPIN?and TPKH group?10⁹ cfu TPKH?.Each group received another gavage 7 days after the initial gavage.21 days after the second gavage,the concentration of serum IgG in each group of mice were detected by ELISA.The spleen of each group was collected aseptically,the spleen lymphocyte cells were isolated for proliferation test and cytokine IFN-??Th1 reaction?and IL-4?IL-10?Th2 reaction?detection.The spleen and thymus were weighted and stained for organ index and pathological examination.Results?1?The formula of lyoprotectant was optimized as follows:skim milk powder 15%,sucrose 10%,gelatin 1%,DMSO 0.5%.Under these conditions,skim milk powder,sucrose,gelatin and DMSO significantly affected the freeze-dried survival rates of TPIN and TPKH?P<0.05?.The survival rate of TPIN after freeze-drying could reach?94.553±0.027?%and the number of viable cells was?4.267±0.156?×10⁹ cfu/mL,the survival rate of TPKH after freeze-drying could reach?96.064±0.031?%and the number of viable cells was?2.430±0.006?×10⁹ cfu/mL.?2?The TPIN and TPKH freeze-dried powder obtained by the above method for pilot production?20 L for fermentation,20 L for vacuum freeze-drying?have a pale yellow loose body.The vaible counts of TPIN was?4.033±0.058?×10¹00 cfu/g,the vaible counts of TPKH was?2.500±0.066?×10⁷ cfu/g.TPIN was found significantly inhibited the proliferation of HepG2 cells at 12 h?P<0.01?after transfected into HepG2 cells,TPKH was found significantly promot the proliferation of IEC-6 cells at 48h?P<0.01?after transfected into IEC-6 cells.The abnormal toxicity test of mice and guinea pigs showed that,the weight of the control group,TPIN group and TPKH group were raised after 7 days of gavage.?3?The IL-2,NK4 of TPIN and KGF,HIF-1?bands amplified by colonies per 5generations were identical in size.The colony and bacterial morphology of TPIN and TPKH are consistent every 10 generations.The purity and concentration of the plasmids contained in TPIN and TPKH have no significant changes during the passage?P>0.05?.After TPIN and TPKH were transfected into HepG2 cells and IEC-6 cells respectively,there was no significant difference in the concentrations of NK4,IL-2 and HIF,KGF protein expressed in every 10 generations?P>0.05?.The TPIN and TPKH freeze-dried powder stored under the conditions of-80?and-20?did not decrease significantly in the viable count in 31 d?P>0.05?.?4?Compared with the control group and the Ty21a group,the concentration of serum IgG in the TPIN?2.044±0.196??g/mL and TPKH groups?1.333±0.152??g/mL was significantly increased?P<0.01?,TPIN and TPKH could significantly enhance the proliferation of splenic lymphocytes in mice?P<0.01?and induced the expression of IFN-?,IL-4 and IL-10?P<0.05?,the spleen index and thymus index of the TPIN group increased?P<0.05?while the TPKH group showed no significant changes?P>0.05?.Conclusions?1?TPIN and TPKH are biological agents with attenuated Salmonella Ty21a as carriers.The compound lyoprotectant optimizes the freeze-drying process of TPIN and TPKH through orthogonal experiment.?2?TPIN and TPKH were passaged for 40 times without loss of target genes and mutations,nor did they affect the expression of TPIN and TPKH in vitro,which demonstrate their stable expression characteristics.When stored at-80?for 31 d,TPIN and TPKH did not significantly reduce the number of viable cells.?3?TPIN and TPKH can cause cellular and humoral immunity in mice,which induce the body to respond to diseases,accelerate the treatment process and improve the treatment effect.