治疗性肝癌单克隆抗体HAb18生产用种子细胞的质量控制

目的进行治疗性肝癌单克隆抗体HAb18生产用种子细胞的质量控制研究。方法用细胞计数、ELISA和FCM分别检测细胞生长、抗体生成速度和抗原结合活性;冻存细胞复苏后及体外传代3个月,检测抗体分泌稳定性和克隆阳性率;进行无菌检查、细胞核型和同工酶谱分析,了解细胞遗传稳定性、外源因子污染和细胞间交叉污染情况。结果肝癌单克隆抗体HAb18生产用种子细胞的细胞生长和抗体生成速度分别为0.047~0.052/h和1.1~1.4 pg/cell.h,与靶细胞FHCC98结合的阳性率达99%,平均荧光强度在200以上。冻存细胞复苏后及体外连续传代3个月后,其特异性抗体生成速度维持在1.0 pg/cell.h以上,培养上清抗体效价仍为1∶1 000。不同批次的生成细胞核型均符合杂交瘤细胞特征,外源因子检查阴性,无细胞间交叉污染。结论HAb18生产用种子细胞的细胞生长和抗体生成速率、抗体稳定性和特异性均符合已建立的规模化放大培养的种子细胞质控标准。

Quality Control of Seed Cells for Production of Therapeutic Hepatocellular Carcinoma McAb HAb18

Objective To control the quality of seed cells for production of therapeutic hepatocellular McAb HAb18.Methods The cell growth,antibody production rate and antigen binding activity were determined by cell counting,ELISA and FCM respectively.The seed cells stored frozen were determined for antibody-secreting stability and cloning positive rate after resuscitation and 3 months after subculture in vitro separately.The cells were tested for karyotype,sterility,contamination with exogenous agents and cross contamination,and analyzed for genetic stability by isoenzyme map.Results The cell growth and antibody production rate were 0.047-0.052/h and 1.1-1.4 pg/cell·h respectively.The binding rate of the seed cells to target cell FHCC98 was 99%,and the mean fluorescence intensity of conjugate was above 200.Both the antibody production rates of seed cells after resuscitation and 3 months after subculture were maintained at above 1.0 pg/cell·h.The amtibody titer in cell supernatant was 1∶1 000.The karyotypes of seed cells of different batches were consistent with those of hybridoma cells.No contamination with exogenous agents or cross contamination among cells was observed.Conclusion The cell growth,antibody production rate,antibody-secreting stability and antibody specificity met the quality control standard for seed cells for large-scale culture.