狂犬病毒单克隆抗体CHO细胞灌流培养中细胞特异性灌流速率的研究

Investigation of cell specific perfusion rate (CSPR) of perfusion cell culture of CHO engineering cell line expressing monoclonal antibody against rabies virus

目的 研究灌流培养中,不同的细胞特异性灌流速率(cell specific perfusion rate, CSPR)对狂犬病毒单克隆抗体CHO细胞生长及抗体蛋白表达的影响,摸索适合本细胞株灌流培养的CSPR。方法 在标号为CSPR 1~5[CSPR1;0.02 nL/(细胞·d)、CSPR2:0.03 nL/(细胞·d)、CSPR3:0.04 nL/(细胞·d)、CSPR4:0.05 nL/(细胞·d)、CSPR5:0.06 nL/(细胞·d),每组3个重复]的15个TPP管中按100万个/mL的初始密度接种相同的CHO种子细胞;摇床中,以转速225 r/min、CO_(2)浓度5.0%、湿度80%和温度37℃的条件培养细胞;以后每天取细胞样品,分别检测活细胞密度(viable cell density, VCD)、细胞活率、葡萄糖浓度、乳酸浓度和渗透压。接种3 d起,每天分别从CSPR 1~5的各管中,按0.02~0.06 nL/(细胞·d)的CSPR计算所需要更换的细胞悬液体积,将各管中的细胞悬液离心,取出相应体积的上清并补入相同体积的新鲜培养液重悬细胞后,继续培养。如细胞悬液中的葡萄糖质量浓度≤3 g/L时,用300 g/L的葡萄糖补至8 g/L。CHO细胞培养约19 d时,结束实验。根据CHO细胞生长、细胞代谢、抗体蛋白滴度(titer)、收获液总体积和抗体蛋白总收量,确定适合本细胞生长和表达的CSPR。结果 在CSPR 1~2[0.02~0.03 nL/(细胞·d)]的条件下灌流培养时,最高VCD分别为1 059万个/mL、1 298万个/mL,细胞活率在培养中后期下降较快,抗体蛋白总收量分别为98 mg和112 mg。CSPR3~5[0.04~0.06 nL/(细胞·d)]条件下的最高VCD分别为1 422万个/mL、1 523万个/mL和1538万个/mL,细胞活率维持较好,抗体蛋白总收量分别为119 mg、120 mg和107 mg。结论 当CSPR为0.04~0.05 nL/(细胞·d)时,细胞生长和抗体蛋白表达情况较好,为适合本细胞株灌流培养的CSPR。

Objective To investigate the effects of different cell specific perfusion rate(CSPR) on the growth and protein expression of CHO cells expressing monoclonal antibody against rabies virus in perfusion culture, and to explore the suitable CSPR for this cell line. Methods The same CHO seed cells were seeded in 15 TPP tubes labeled CSPR 1-5(CSPR1: 0.02 nL/cell/day, CSPR2: 0.03 nL/cell/day, CSPR3: 0.04 nL/cell/day, CSPR4: 0.05 nL/cell/day, CSPR5: 0.06 nL/cell/day, each group has three replicates)at an equal initial density of 1.0×10~6 cells/mL. In the shaker, the cells were cultured at a speed of 225 r/min, CO2 concentration of 5.0%, humidity of 80% and temperature of 37 ℃. Viable cell density(VCD), cell viability, glucose concentration, lactate concentration and osmolality were tested by daily sampling. After 3 days of inoculation, the volume of cell suspension liquid to be replaced was calculated according to the CSPR of 0.02-0.06 nL/cell/day from each tube labeled CSPR 1-5, respectively. The cell suspension in each tube was centrifuged, and the corresponding volume of supernatant was removed and the same volume of fresh culture medium was added to resuspend cells, and then the culture was continued. Once the glucose concentration in the cell suspension was less than or equal to 3 g/L, 300 g/L glucose was used to replenish to 8 g/L. The experiment was finished when CHO cells in all tubes were cultured for about 19 days. The CSPR suitable for the growth and expression of CHO cells was determined according to the cell growth, cell metabolism, titer of antibody protein, total volume of harvest fluid and total amount of antibody protein. Results Under the condition of CSPR1-2(0.02-0.03 nL/cell/day), the highest VCD was 10.59×10~(6 )and 12.98×10~6 cells/mL, respectively. The cell viability decreased rapidly in the middle and late stage of culture. The total amount of antibody protein was 98 and 112 mg, respectively. Under the condition of CSPR3-5(0.04-0.06 nL/cell/day), the maximum VCD was 14.22×10~6、15.23×10~6 and 15.38×10~6 cells/mL, respectively. The cell viability was well maintained, and the total amount of antibody protein was 119、120 and 107 mg, respectively. Conclusion The cell growth and antibody protein expression were better when CSPR was 0.04-0.05 nL/cell/day, which was the suitable CSPR for this cell line in perfusion culture.