The Mechanism And Regulation Of Reactive Oxygen Species In CHO Cells Fed-batch Culture

The monoclonal antibody(mAb) drug has been one of the most indispensable cancer treatments due to its ability to target the cancer with fewer side effect and lasting treatment effect. It is reported that 21 antineoplastic mAb drugs for the treatment of solid tumors or hematological tumors have been approved worldwide up to February 2016. The technologies to improve mAb drugs production help ensure a sustainable supply to meet market demand and represent the core competitiveness of a company even a country.Chinese hamster ovary(CHO) cells have emerged as the most popular choice for mAb drugs production in the world. Apoptosis can lead to decrease of cell viability in large-scale culture, and reactive oxygen species(ROS), which is generated during aerobic metabolism, is reported to be closely related to cell apoptosis. This research was intended to study the effects and mechanism of ROS, and provide approaches to enhancement of mAb drug production.The ROS level of CHO cells in batch culture was detected via DCFH-DA probe and increased exponentially at early stage. The viable cell density(VCD) declined immediately as it reached the top, indicating the accumulation of ROS may be responsible for the decrease of VCD. The cells in batch culture were grouped and treated with 50, 100 and 200μM hydrogen peroxide respectively, and cell growth was inhibited. To to control ROS level efficiently, we designed the experiment in two aspects.In exogenous way, lipoic acid(LA) was supplemented in the medium during the culture. The cells in batch culture were grouped and treated with 25, 50, 100 and 200 μM lipoic acid respectively, and 100 μM group achieved the highest integrated viable cell density(IVCD), which was 54.65×106 cells/mL days. In the further research we set 100μM as proper concentration of lipoic acid. In fed-batch culture cells supplemented with100 μM lipoic acid performed better than control in VCD, viability and IVCD during the late stage. Meanwhile the titer of antibody in LA group was 1.04 g/L, approximately 18.0%higher than 0.88 g/L of control with significant difference(p0.05). As for main quality attributes, the reducing and non-reducing SDS-PAGE showed no differences between two groups in molecular weight and structures. According to the LC-MS analysis the glycosylation profiles of LA group was similar with control in both major oligose type and proportion. The ROS level on day 5-8 of LA group was significantly lower than that of the control after detection via DCFH-DA. Apoptosis analysis by Annexin V/PI showed thatapoptosis rate of LA group during day 8-10 was significantly lower than that of the control,and western blot experiment confirmed this by detection of cleaved Caspase-7. All results above indicated that the supplement of lipoic acid in fed-batch could inhibit cell apoptosis via reducing ROS level, and therefore help improve cell growth and antibody production.In endogenesis way human malic enzyme 1(ME-1), which is an oxidative decarboxylase generating NADPH, was overexpressed in CHO cell. The plasmid pcDNA3.0-ME-1 was constructed after cloning ME-1 gene by PCR, and then transfected in CHO cells. The cell pool expressing ME-1 stably was established after selection pressure of antibiotics, followed by the real-time PCR to detect ME-1 expression. In fed-batch culture ME-1 cells performed better than control in VCD, viability and IVCD during the late stage. Meanwhile the IVCD and titer increased about 33.0% and 16.9%respectively with significant difference(p0.01). In antibody main quality attributes, the reducing and non-reducing SDS-PAGE showed no differences between two groups in molecular weight and structures, and LC-MS analysis of glycosylation profiles of ME-1cell group was similar with control in both major oligose type and proportion. In both ROS level and cell apoptosis rate ME-1 group showed better performance than control after detection and western blot of cleaved Caspase-7.This study discovered that the accumulation of ROS could inhibit CHO cell growth and induce apoptosis in late stage of fed-culture. Both exogenous and endogenesis methods were established and evaluated to improbe cell IVCD and antibody production,maintaining the key quality attributes of mAb drugs. It would benefit the serum-free medium optimization, bioprocess development and facilitate the bio-pharmaceutical industry of our country.