重组蛋白药物的生产技术进展

doi:10.19586/j.2095-2341.2021.0130

生物技术进展 ›› 2021, Vol. 11 ›› Issue (6): 724-731.DOI: 10.19586/j.2095-2341.2021.0130

重组蛋白药物的生产技术进展

周娜娜¹^,²^,³(), 王小艳¹^,²^,³, 张媛¹^,²^,³, 王靖¹^,²^,³, 赵国淼¹^,²^,³, 魏超¹^,²^,³, 杨凯¹^,²^,³, 安泰¹^,²^,³()

^1.中粮营养健康研究院有限公司，北京 102209
^2.营养健康与食品安全北京市重点实验室，北京 102209
^3.老年营养食品研究北京市工程实验室，北京 102209

收稿日期:2021-07-06 接受日期:2021-10-08 出版日期:2021-11-25 发布日期:2021-11-26
通讯作者: 安泰
作者简介:周娜娜 E-mail：zhounana@cofco.com；
基金资助:
北京市科技新星计划项目(Z191100001119045)

Progress on the Production Technology of Recombinant Therapeutic Proteins

Nana ZHOU¹^,²^,³(), Xiaoyan WANG¹^,²^,³, Yuan ZHANG¹^,²^,³, Jing WANG¹^,²^,³, Guomiao ZHAO¹^,²^,³, Chao WEI¹^,²^,³, Kai YANG¹^,²^,³, Tai AN¹^,²^,³()

^1.Nutrition & Health Research Institute，COFCO Corporation，Beijing 102209，China
^2.Beijing Key Laboratory of Nutrition，Health and Food Safety，Beijing 102209，China
^3.Beijing Engineering Laboratory for Geriatric Nutrition Food Research，Beijing 102209，China

Received:2021-07-06 Accepted:2021-10-08 Online:2021-11-25 Published:2021-11-26
Contact: Tai AN

摘要/Abstract

摘要：

重组蛋白药物是生物药物中的核心产品，主要是通过基因工程菌来生产功能蛋白或其突变体，用于弥补体内蛋白的缺失，从而对疾病的治疗发挥关键作用。近年来，重组蛋白药物在疾病治疗中发挥作用越来越大，相关技术也发展迅速。通过综述重组蛋白药物的中上游生产流程，并重点分析了重组蛋白药物在表达系统、细胞培养、纯化和质量控制等环节的最新技术进展，展示了重组蛋白药物制备的技术提升水平，以期为国内重组蛋白药物的生产提供一定的参考依据。

关键词: 重组蛋白药物, 表达系统, 细胞培养, 分离纯化, 质量控制

Abstract:

Recombinant therapeutic proteins are the core products in biopharmaceuthc. They are mainly used to product functional proteins or their mutants by genethcally engineered bacteria to compensate for the loss of protenis in the body and play a key role in the treatment of diseases. In recent years， recombinant therapeutic proteins are playing an increasingly important role in the treatment of diseases， and related technologies are developing rapidly. The middle and upstream production process of recombinant therapeutic proteins was reviewed. The latest technological progress of recombinant therapeutic proteins in expression system， cell culture， purification and quality control was analyzed. The technological advances in the preparation of recombinant therapeutic proteins were demonstrated. It was expected to provide certain reference for domestic production of recombinant therapeutic proteins.

Key words: recombinant therapeutic protein, expression system, cell culture, separation and purification, quality control

中图分类号:

R915

周娜娜, 王小艳, 张媛, 王靖, 赵国淼, 魏超, 杨凯, 安泰. 重组蛋白药物的生产技术进展[J]. 生物技术进展, 2021, 11(6): 724-731.

Nana ZHOU, Xiaoyan WANG, Yuan ZHANG, Jing WANG, Guomiao ZHAO, Chao WEI, Kai YANG, Tai AN. Progress on the Production Technology of Recombinant Therapeutic Proteins[J]. Current Biotechnology, 2021, 11(6): 724-731.

图/表 4

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表达系统	优点	缺点
大肠杆菌	表达系统相对简单、易于操作、营养需求简单、易于遗传和发酵操作、周期短^[27]，多种表达质粒和宿主可供选择^[7]	缺乏翻译后修饰机制，内毒素的存在可能会对给患者的药物产生免疫反应^[28]
酵母系统	酿酒酵母系统：可以在无蛋白培养基中快速生长，存在翻译后修饰机制和细胞外分泌产物的能力^[29]，基因以单拷贝至多拷贝形式稳定地整合到宿主基因组中，可供大规模商业化蛋白的稳定生产	酿酒酵母：重组蛋白过表达可能导致在细胞内积累和产量下降^[30]，关于翻译后修饰，在细胞内往往导致过糖基化蛋白的产生^[31]
酵母系统	毕赤酵母系统：多种具有翻译后修饰功能的高表达菌株且可高密度生长^[32]，易于发酵放大，成本相比CHO细胞系便宜，具有较强的甲醇诱导型启动子（AOX）和组成型（pGAP）启动子、多种商业化的载体（pPIC9、pPICZa‑A、pPICZa‑B、pPICZa‑C等）和菌株（GS115、 KM71、 X33等）	毕赤酵母：在存在碳源如葡萄糖、甘油或乙醇时，启动子AOX1被强烈抑制^[33]
CHO细胞系	可以产生与人同源性最高的重组蛋白，保证正确的折叠与糖基化	需要营养更丰富的培养基，生长条件严苛，生长周期较长^[34]，基因导入和细胞发育比微生物系统更为繁琐

表达系统	优点	缺点
大肠杆菌	表达系统相对简单、易于操作、营养需求简单、易于遗传和发酵操作、周期短^[27]，多种表达质粒和宿主可供选择^[7]	缺乏翻译后修饰机制，内毒素的存在可能会对给患者的药物产生免疫反应^[28]
酵母系统	酿酒酵母系统：可以在无蛋白培养基中快速生长，存在翻译后修饰机制和细胞外分泌产物的能力^[29]，基因以单拷贝至多拷贝形式稳定地整合到宿主基因组中，可供大规模商业化蛋白的稳定生产	酿酒酵母：重组蛋白过表达可能导致在细胞内积累和产量下降^[30]，关于翻译后修饰，在细胞内往往导致过糖基化蛋白的产生^[31]
酵母系统	毕赤酵母系统：多种具有翻译后修饰功能的高表达菌株且可高密度生长^[32]，易于发酵放大，成本相比CHO细胞系便宜，具有较强的甲醇诱导型启动子（AOX）和组成型（pGAP）启动子、多种商业化的载体（pPIC9、pPICZa‑A、pPICZa‑B、pPICZa‑C等）和菌株（GS115、 KM71、 X33等）	毕赤酵母：在存在碳源如葡萄糖、甘油或乙醇时，启动子AOX1被强烈抑制^[33]
CHO细胞系	可以产生与人同源性最高的重组蛋白，保证正确的折叠与糖基化	需要营养更丰富的培养基，生长条件严苛，生长周期较长^[34]，基因导入和细胞发育比微生物系统更为繁琐

宿主	重组蛋白药物	产量	参考文献
毕赤酵母（P. pastoris）	乙型肝炎表面B抗原	7 g·L^-1	[35]
	人粒细胞⁃巨噬细胞集落刺激因子	285 mg·L^-1	[36]
	人血清蛋白	92.29 mg·L^-1	[37]
酿酒酵母（S. cerevisiae）	胰高血糖素样肽2	—	[38]
酿酒酵母（S. cerevisiae）	人表皮生长因子	5 mg·L^-1	[39]
解脂耶氏酵母（Yarrowia lipolytica）	人干扰素α2a（IFNα2a）	425 mg·L^-1	[40]
乳酸克鲁维酵母（Kluyveromyces lactis）	人干扰素β	—	[41]
汉森酵母（Hansenula polymorpha）	HPV 16型L1‑L2嵌合蛋白（SAF）	132.10 mg·L^-1	[42]

宿主	重组蛋白药物	产量	参考文献
毕赤酵母（P. pastoris）	乙型肝炎表面B抗原	7 g·L^-1	[35]
	人粒细胞⁃巨噬细胞集落刺激因子	285 mg·L^-1	[36]
	人血清蛋白	92.29 mg·L^-1	[37]
酿酒酵母（S. cerevisiae）	胰高血糖素样肽2	—	[38]
酿酒酵母（S. cerevisiae）	人表皮生长因子	5 mg·L^-1	[39]
解脂耶氏酵母（Yarrowia lipolytica）	人干扰素α2a（IFNα2a）	425 mg·L^-1	[40]
乳酸克鲁维酵母（Kluyveromyces lactis）	人干扰素β	—	[41]
汉森酵母（Hansenula polymorpha）	HPV 16型L1‑L2嵌合蛋白（SAF）	132.10 mg·L^-1	[42]

纯化方式		机理
超滤		目标蛋白和其他杂质基于尺寸大小而被分离
单柱色谱	反相液相色谱（RP‑LC）	根据分析物的疏水特性进行分离^[61]，C18 配体是 RP‑LC 中最常用的固定相，但偶尔在非疏水蛋白和多肽情况下，C8 甚至 C4 配体显示出更好的保留特性；此色谱可以轻松与质谱联用^[62]
	离子交换色谱（IEX）	基于分析物的电荷与固定相之间的静电相互作用分离；可鉴定通过 RP‑LC 难以检测的肽修饰，如脱酰胺或乙酰化，能够区分具有相似疏水性的分析物^[63]
	亲水相互作用色谱（HILIC）	正相色谱的一种变体，洗脱顺序与反相色谱相反^[64]
	混合模式	在同一固定相上结合两种配体，具备两种分离机制^[55]
多维色谱		连续应用两种或两种以上的混合模式色谱的组合
MCSGP技术		与单柱色谱分离原理相同，使用两个或更多相同的色谱柱，不纯组分可在内部循环进入系统，性能参数增加^[60]

重组蛋白药物的生产技术进展

Progress on the Production Technology of Recombinant Therapeutic Proteins

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