表達(dá)TNFR-Fc融合蛋白的GS-CHO細(xì)胞動態(tài)流加培養(yǎng)過程的設(shè)計

范里，趙亮，孫亞婷，寇天賜，譚文松

華東理工大學(xué) 生物反應(yīng)器工程國家重點實驗室，上海 200237

表達(dá)TNFR-Fc融合蛋白的GS-CHO細(xì)胞動態(tài)流加培養(yǎng)過程的設(shè)計

范里，趙亮，孫亞婷，寇天賜，譚文松

華東理工大學(xué) 生物反應(yīng)器工程國家重點實驗室，上海 200237

人腫瘤壞死因子受體Ⅱ-Fc融合蛋白在治療風(fēng)濕性、類風(fēng)濕性關(guān)節(jié)炎方面擁有廣闊的市場前景和巨大的經(jīng)濟(jì)價值。本實驗以表達(dá)TNFR-Fc融合蛋白的GS-CHO細(xì)胞為研究對象，結(jié)合細(xì)胞生長代謝特性和動力學(xué)參數(shù)分析，以葡萄糖為關(guān)鍵控制參數(shù)，通過測定培養(yǎng)上清的葡萄糖濃度對培養(yǎng)過程中的葡萄糖消耗進(jìn)行及時的預(yù)測，調(diào)整流加速率，形成了以滿足細(xì)胞生長代謝需要為基本原則的動態(tài)流加培養(yǎng)過程設(shè)計模型。在此控制模型指導(dǎo)下，建立了高效的流加培養(yǎng)過程。使最大活細(xì)胞密度和最大融合蛋白濃度分別達(dá)9.4×106cells/mL和207 mg/L，較批次培養(yǎng)分別提高了3.4倍和3倍。本研究所采用的研究方法和控制策略為優(yōu)化GS-CHO細(xì)胞培養(yǎng)過程和TNFR-Fc融合蛋白成功邁向產(chǎn)業(yè)化奠定了基礎(chǔ)。

GS-CHO細(xì)胞，流加培養(yǎng)，TNFR-Fc融合蛋白，葡萄糖，動力學(xué)

Abstract:TNFR-Fc is an important fusion protein that has great potential in therapeutic and diagnostic applications.We developed an efficient fed-batch process for GS-CHO cells to produce TNFR-Fc.The rationale of this fed-batch process relies on the supply of sufficient nutrients to meet the requirements of cell metabolism.The optimal feed medium was designed through ration design.A metabolically responsive feeding strategy was designed and dynamically adjusted based on the residual glucose concentration determined off-line.In this process, the maximal viable cell density and antibody concentration reached above 9.4×106cells/mL and 207 mg/L, respectively.Compared with the batch process, the newly developed fed-batch process increased the cell yield by 3.4 fold and the final antibody concentration by 3 fold.This fed-batch process would therefore facilitate the production of therapeutic antibody by GS-CHO cells.

Keywords:GS-CHO cell, fed-batch, TNFR-Fc, glucose, dynamic model

隨著嵌合化、人源化和全人源化抗體融合蛋白藥物的設(shè)計技術(shù)的突破，抗體類藥物成為生物醫(yī)藥產(chǎn)業(yè)發(fā)展的主要方向。與微生物宿主相比，動物細(xì)胞具有更完備的蛋白質(zhì)后期修飾及加工能力，因此利用哺乳動物細(xì)胞生產(chǎn)抗體類藥物得到越來越廣泛的應(yīng)用。中國倉鼠卵巢細(xì)胞(CHO)表達(dá)的蛋白在分子結(jié)構(gòu)和生物學(xué)功能方面最接近天然蛋白分子，因此成為生產(chǎn)抗體類藥物的首選宿主細(xì)胞[1]。……