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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (8): 74-83    DOI: 10.13523/j.cb.2004004
综述     
工程原核生物和酵母菌中生产单克隆抗体和抗体片段研究进展 *
赵妍淑,张金华,宋浩()
天津大学化工学院 系统生物工程教育部重点实验室 天津 300072
Advances in Production of Monoclonal Antibody and Antibody Fragments in Engineered Prokaryotes and Yeast
ZHAO Yan-shu,ZHANG Jin-hua,SONG Hao()
Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering amd Technology,Tianjin University, Tianjin 300072, China
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摘要：

抗体药物和抗体片段药物在药物市场占据了重要的地位,主要通过哺乳动物细胞系统进行生产,操作复杂并且成本高。为了能够克服哺乳动物细胞系统生产抗体药物的弊端,越来越多的抗体及抗体片段在原核细胞及酵母菌中生产,但是产率往往不高并且没有糖基化。从基因转录和翻译的优化、分子伴侣的共表达和抑制蛋白水解降解等方面概述了在原核生物表达系统及酵母菌中提高单克隆抗体和抗体片段产量的研究进展,为未来利用原核生物和酵母菌实现工业化生产单克隆抗体及抗体片段奠定基础。
关键词： 单克隆抗体;  抗体片段;  原核生物;  酵母菌;  工业化生产    
Abstract:

Monoclonal antibodies and antibody fragments play important roles in the pharmaceutical market. They are mainly produced in mammalian cell systems, which have several limitations such as complex manipulation and high costs. For purpose of using cheap drugs, monoclonal antibodies and antibody fragments have been produced in prokaryotes and yeasts. However, the lack of glycosylation and the low yield of antibodies prevent their development. The progress has been made recently in the prokaryotes and yeasts to enhance the antibodies production via optimization of transcription and translation, co-expressing chaperones and inhibiting proteolytic degradation, etc. This will lay the foundation for the industrialization production of antibodies in prokaryotes and yeasts.
Key words: Monoclonal antibody    Antibody fragments    Prokaryotes    Yeast    Industrial production
收稿日期: 2020-04-02 出版日期: 2020-09-10
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(21621004)
通讯作者: 宋浩     E-mail: hsong@tju.edu.cn
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赵妍淑,张金华,宋浩. 工程原核生物和酵母菌中生产单克隆抗体和抗体片段研究进展 *[J]. 中国生物工程杂志, 2020, 40(8): 74-83.

ZHAO Yan-shu,ZHANG Jin-hua,SONG Hao. Advances in Production of Monoclonal Antibody and Antibody Fragments in Engineered Prokaryotes and Yeast. China Biotechnology, 2020, 40(8): 74-83.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2004004    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I8/74

宿主菌 形式 产量 备注 参考文献
大肠杆菌 scFv 0.9 mg/g① 阿拉伯糖诱导剂 [20]
大肠杆菌 scFv >40 mg/L PelB信号肽及优化的工艺条件 [23]
大肠杆菌 scFv 3.1 g/L 细胞质中产生 [23]
大肠杆菌 scFv 325 mg/L 共表达分子伴侣提高溶解度 [16]
大肠杆菌 Fab 529 mg/L 氮供应量及温度对产量的影响 [22]
大肠杆菌 Fab 10 mg/L phoA启动子;STⅡ信号肽 [24]
大肠杆菌 Fab n.d.② 不同信号肽对产量的影响 [25]
大肠杆菌 Fab 3.3 g/L 过表达DsbC [27]
大肠杆菌 Fab 7.4 mg/g③ 无质粒表达系统 [30]
大肠杆菌 Fab' 2.4 g/L 共表达DsbC的Tsp缺陷型菌株 [28]
大肠杆菌 F(ab)2 2.45 g/L 缺乏Prc蛋白酶 [4]
大肠杆菌 Fv/Fab 1 g/L 无细胞蛋白合成系统 [31]
大肠杆菌 IgG(无糖基化) 150 mg/L 大肠杆菌首次生产IgG [32]
大肠杆菌 IgG(无糖基化) 1~4 mg/L 双顺反子表达系统 [33]
大肠杆菌 IgG(无糖基化) 1.3 g/L IgG产量最高 [34]
大肠杆菌 IgG(无糖基化) 65 mg/L SRP分泌系统;共表达DsbC [36]
大肠杆菌 IgG(无糖基化) n.d.[2] 共表达DsbA;增强信号肽疏水性 [35]
大肠杆菌 IgG(无糖基化) >130 mg/L RpoD突变体;共表达分子伴侣 [37]
大肠杆菌 IgG(无糖基化) 150 mg/L 大肠杆菌丙酮酸代谢路径对产量的影响 [38]
大肠杆菌 IgG变体(无糖基化) 40~50 mg/L 首个不糖基化但能结合FcγRs的IgG变体 [40]
大肠杆菌 IgG(糖基化) 50 mg/L 大肠杆菌首个糖基化IgG抗体 [41]
大肠杆菌 IgG(糖基化) n.d.[2] 表达设计的糖基化序列 [42]
Table 1  Expression of full-length antibodies and antibody fragments in E. coli
宿主菌 形式 产量 备注 溶解性 参考文献
枯草芽孢杆菌 scFv 130 mg/L 革兰阳性菌分泌scFv片段的最高水平 可溶 [43]
地衣芽孢杆菌 scFv 12~17 mg/L 可溶 [43]
巨大芽孢杆菌 scFv 14 mg/L 优化的木糖诱导型启动子 可溶 [43]
短芽孢杆菌 Fab 1.25 g/L 革兰阳性菌分泌Fab片段的最高水平 可溶 [45]
谷氨酸棒杆菌 scFv n.d.① 首次在谷氨酸棒杆菌中生产抗体片段 可溶 [47]
谷氨酸棒杆菌 scFv 68 mg/L 首次在谷氨酸棒杆菌中采用分批补料培养生产抗体片段 可溶 [15]
谷氨酸棒杆菌 VHH 1.57 g/L Cg1514信号肽 可溶 [48]
谷氨酸棒杆菌 Fab 57.6 mg/L 首次在谷氨酸棒杆菌中生产Fab片段 可溶 [49]
表2  抗体片段在芽孢杆菌、谷氨酸棒杆菌中的表达
宿主菌 形式 产量 备注 溶解性 参考文献
毕赤酵母 scFv >100 mg/L 毕赤酵母首次生产抗体片段 可溶 [62]
毕赤酵母 scFv/IgG n.d.① 工程化信号肽MFa1PP 可溶 [66]
毕赤酵母 scFv 8 g/L 共表达BiP和PDI 可溶 [68]
毕赤酵母 scFv 4.88 g/L 0.5%(v/v)甲醇,较低的pH 可溶 [69]
毕赤酵母 Fab 420~458 mg/L 可溶 [63]
毕赤酵母 Fab 20 mg/L 引入设计的链间二硫键 可溶 [59]
毕赤酵母 Fab ≈25 mg/L 碳源对产量的影响 可溶 [70]
毕赤酵母 F(ab)2 2~8 mg/L 共表达BiP,使用Fos和Jun拉链 可溶 [66]
毕赤酵母 Fc 207 mg/L N-糖基化位点突变 可溶 [65]
毕赤酵母 Fc 635 mg/L 分批补料培养条件优化 可溶 [71]
毕赤酵母 IgG(人源化糖基化) >1 g/L 优化pH、温度、DO浓度和甲醇进料率 可溶 [74]
毕赤酵母 IgG(人源化糖基化) 1.4 g/L 控制甲醇进料速度研究表达动力学 可溶 [76]
毕赤酵母 IgG(人源化糖基化) >1.6 g/L 工业规模(1200L)生产商用IgG 可溶 [77]
毕赤酵母 IgG(人源化糖基化) n.d.[1] 去糖基化之后转糖基化 可溶 [78]
毕赤酵母 IgG(糖基化) 3.05 mg/L 鼠IgG1信号肽 可溶 [79]
毕赤酵母 IgG(糖基化) 6.5 mg/L 鼠IgG1信号肽融合至轻链时可获得最大产量 可溶 [80]
酿酒酵母 scFv n.d.[1] 改造内质网提高转运能力 可溶 [83]
酿酒酵母 IgG(糖基化) n.d.[1] 删除OPI1,共表达CPR5 可溶 [81]
酿酒酵母 IgG(糖基化) n.d.[1] 共表达BiP或GRP或FKBP2 可溶 [82]
解脂耶氏酵母 scFv 20 mg/L 自身信号肽,Kex2p型内切酶 可溶 [86]
乳酸克鲁维酵母 scFv 10 mg/L 自身信号肽,Kex2p型内切酶 可溶 [86]
乳酸克鲁维酵母 IgG(糖基化) 140 mg/L 可溶 [87]
表3  全长抗体及抗体片段在酵母菌中的表达
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