色谱技术在抗体分离纯化中的应用进展

doi:10.3724/SP.J.1123.2023.12010

摘要/Abstract

摘要：

抗体作为一类重要的生物药物,在疾病诊断和治疗等方面拥有广阔的前景。抗体药物的需求逐年上升,生产规模不断扩大,下游抗体纯化已经成为抗体药物生产的瓶颈。色谱技术具有选择性好、分离效率高等优点,在抗体纯化领域占据主导地位。抗体分离纯化流程一般包括样品的粗提、粗提物精制和抗体精纯3个步骤,每个步骤都涉及色谱分离技术。目前已有多种色谱技术(如亲和色谱、离子交换色谱、疏水作用色谱、混合模式色谱以及新型的温敏色谱等)应用于抗体的分离纯化。本文介绍了近年来国内外各种色谱技术在抗体分离纯化方面所取得的研究进展,并对抗体色谱分离技术的未来发展趋势进行了展望。

关键词: 抗体, 色谱技术, 分离纯化, 亲和色谱, 混合模式色谱, 温敏色谱, 综述

Abstract:

Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.

Key words: antibody, chromatographic techniques, separation and purification, affinity chromatography, mixed-mode chromatography, temperature-responsive chromatography, review

中图分类号:

O658

刘家玮, 唐长伟, 夏一然, 白泉. 色谱技术在抗体分离纯化中的应用进展[J]. 色谱, 2024, 42(6): 533-543.

LIU Jiawei, TANG Changwei, XIA Yiran, BAI Quan. Recent progress of chromatographic techniques for antibody purification[J]. Chinese Journal of Chromatography, 2024, 42(6): 533-543.

图/表 4

参考文献

[1]	Roque A C A, Silva C S O, Taipa M A. J Chromatogr A, 2007, 1160(1/2): 44
[2]	Zhai D G, Chen L, Qian P. Biological Chemical Engineering, 2018, 4(3): 145
[2]	翟东改, 陈凌, 钱平. 生物化工, 2018, 4(3): 145
[3]	Arora S, Saxena V, Ayyar B V. Methods, 2017, 116: 64
[4]	Lu H L, Lin D Q, Yao S J. CIESC Journal, 2018, 69(1): 341
[4]	卢慧丽, 林东强, 姚善泾. 化工学报, 2018, 69(1): 341
[5]	Ayyar B V, Arora S, Murphy C, et al. Methods, 2012, 56(2): 116
[6]	Zhang H, Zhang J Y, Wu H R, et al. Membrane Science and Technology, 2023, 43(5): 150
[6]	张昊, 张君怡, 武浩然, 等. 膜科学与技术, 2023, 43(5): 150
[7]	Chahar D S, Selvan R, Pisal S S. Biologicals, 2020, 63: 1
[8]	Gagnon P. J Chromatogr A, 2012, 1221: 57
[9]	Zarrineh M, Mashhadi I S, Farhadpour M, et al. Anal Biochem, 2020, 609: 113909
[10]	Kangwa M, Yelemane V, Ponnurangam A, et al. Protein Expres Purif, 2019, 155: 27
[11]	Bailey L J, Sheehy K M, Hoey R J, et al. J Immunol Methods, 2014, 415: 24
[12]	Paloni M, Cavallotti C. ACS Omega, 2017, 2(10): 6464
[13]	von Roman M F, Berensmeier S. J Chromatogr A, 2014, 1347: 80
[14]	Tsukamoto M, Watanabe H, Ooishi A, et al. J Biol Eng, 2014, 8: 15
[15]	Koguma I, Yamashita S, Sato S, et al. J Chromatogr A, 2013, 1305: 149
[16]	Shi Q H, Sun Y. Chin J Chem Eng, 2021, 30: 194
[17]	Boschetti E. J Biochem Biophys Methods, 2001, 49(1-3): 361
[18]	Qian H, Li C J, Lin Z Y, et al. Colloid Surface B, 2010, 75(1): 342
[19]	Lin Z Y, Zhang Y X, Li C J, et al. J Immunol Methods, 2021, 489: 112914
[20]	Qian H. Colloid Surface B, 2013, 108: 72
[21]	Cheung R C F, Wong J H, Ng T B. Appl Microbiol Biotechnol, 2012, 96(6): 1411
[22]	Ino Y, Yamaoka Y, Tanaka K, et al. Proteomics, 2023, 23(11): 2200334
[23]	Maggi M, Scotti C. Data Brief, 2017, 12: 132
[24]	Yavuz H, Akgol S, Say R, et al. Int J Biol Macromol, 2006, 39(4/5): 303
[25]	Wongchuphan R, Tey B T, Tan W S, et al. Biochem Eng J, 2009, 45(3): 232
[26]	Gondim D R, Lima L P, de Souza M C M, et al. Adsorpt Sci Technol, 2012, 30(8/9): 701
[27]	Huseynli S, Baydemir G, Sari E, et al. Mater Sci Eng C, 2015, 46: 77
[28]	Li D J, Chen Y, Liu Z. Chem Soc Rev, 2015, 44(22): 8097
[29]	dos Santos R, Rosa S A S L, Aires-Barros M R, et al. J Chromatogr A, 2014, 1355: 115
[30]	Rosa S A S L, Wagner A, da Silva C L, et al. Biotechnol J, 2019, 14(10): 1800586
[31]	Lhota G, Sissolak B, Striedner G, et al. Biotechnol Progress, 2021, 37(3): e3124
[32]	An J, Kim S, Shrinidhi A, et al. Nat Biomed Eng, 2020, 4(11): 1044
[33]	Lu H L, Lin D Q, Yao S J. CIESC Journal, 2016, 67(9): 3523
[33]	卢慧丽, 林东强, 姚善泾. 化工学报, 2016, 67(9): 3523
[34]	Xue A Y, Fan S J. Biomolecules, 2022, 12(6): 821
[35]	Farzi-Khajeh H, Toraby S, Akbarzadeh-Khiavi M, et al. J Chromatogr A, 2022, 1684: 463559
[36]	El Khoury G, Lowe C R. J Mol Recognit, 2013, 26(4): 190
[37]	Fang Y M, Lin D Q, Yao S J. J Chromatogr A, 2018, 1571: 1
[38]	Fang Y M, Chen S G, Lin D Q, et al. J Chromatogr A, 2019, 1604: 460474
[39]	Fang M Y, Lin D Q, Yao S J. Biochem Eng J, 2021, 176: 108213
[40]	Fang Y M, Zhang Q L, Lin D Q, et al. J Chromatogr B, 2022, 1208: 123407
[41]	Huang H T, Dong X Y, Sun Y, et al. J Chromatogr A, 2023, 1707: 464305
[42]	Wang X Y, Xia D H, Han H, et al. Anal Chim Acta, 2018, 1017: 57
[43]	Sanchez-Reyes G, Graalfs H, Hafner M, et al. J Sep Sci, 2021, 44(4): 805
[44]	Hardin A M, Harinarayan C, Malmquist G, et al. J Chromatogr A, 2009, 1216(20): 4366
[45]	Saleh D, Hess R, Ahlers-Hesse M, et al. Biotechnol Bioeng, 2023, 120(1): 125
[46]	Shen H, Lin Q Q, Yi Y, et al. Pharmaceutical Biotechnology, 2012, 19(5): 401
[46]	沈泓, 林琼秋, 易喻, 等. 药物生物技术, 2012, 19(5): 401
[47]	Bresolin I R A P, Lingg N, Bresolin I T L, et al. J Biotechnol, 2020, 324(S): 100020
[48]	Müller E, Sevilla M, Endres P. J Sep Sci, 2020, 43(12): 2255
[49]	Fekete S, Veuthey J L, Beck A, et al. J Pharmaceut Biomed, 2016, 130(S1): 3
[50]	King C, Patel R, Ponniah G, et al. J Chromatogr B, 2018, 1085: 96
[51]	Ma Y Y, Zhang D, Li Y R, et al. Food Science, 2023, 44(9): 185
[51]	马月云, 张丹, 李盈柔, 等. 食品科学, 2023, 44(9): 185
[52]	Wang S J, Xue Y X, Jin H B, et al. Modern Chemical Research, 2022(4): 1
[52]	王仕杰, 薛彦晓, 靳海波, 等. 当代化工研究, 2022(4): 1
[53]	Li M T, Zhang Q L, Lin D Q, et al. J Chromatogr B, 2019, 1134/1135: 121850
[54]	Lin D Q, Tong H F, Wang H Y, et al. J Phys Chem B, 2012, 116(4): 1393
[55]	Lin D Q, Tong H F, Wang H Y, et al. J Chromatogr A, 2012, 1260: 143
[56]	Boschetti E. Trends Biotechnol, 2002, 20(8): 333
[57]	Wu M B, Zhang F F, Liang Y F, et al. J Dairy Sci, 2015, 98(5): 2973
[58]	Tong H F, Lin D Q, Pan Y, et al. Biochem Eng J, 2011, 56(3): 205
[59]	Luo Y D, Zhang Q L, Yao S J, et al. J Chromatogr A, 2018, 1533: 77
[60]	Ma N, Yao D X, Yang H, et al. Ind Eng Chem Res, 2018, 57(39): 13295
[61]	Shi Q H, Shen F F, Sun S. Biotechnol Prog, 2010, 26(1): 134
[62]	Zhao G F, Peng G Y, Li F Q, et al. J Chromatogr A, 2008, 1211(1/2): 90
[63]	Phottraithip W, Lin D Q, Shi F, et al. Biotechnol Bioprocess Eng, 2013, 18: 1169
[64]	Ren J, Xiang X, Ji F L, et al. J Chromatogr B, 2021, 1179: 122652
[65]	Tong H F, Lin D Q, Chu W N, et al. J Chromatogr A, 2016, 1429: 258
[66]	Zou X J, Zhang Q L, Lu H L, et al. Chem Eng J, 2019, 368: 678
[67]	Arakawa T, Tomioka Y, Nakagawa M, et al. Antibodies, 2023, 12(1): 15
[68]	Chu W N.[PhD Dissertation]. Hangzhou: Zhejiang University, 2019
[68]	褚文宁.[博士学位论文]. 杭州: 浙江大学, 2019
[69]	Kaleas K A, Tripodi M, Revelli S, et al. J Chromatogr B, 2014, 969: 256
[70]	Pezzini J, Cabanne C, Gantier R, et al. J Chromatogr B, 2015, 976/977: 68
[71]	Wang J, Zhou J X, Gowtham Y K, et al. Biotechnol Progress, 2017, 33(3): 658
[72]	Cabanne C. J Chromatogr A, 2015, 1393: 57
[73]	Wan Y, Zhang T, Wang Y M, et al. Protein Expres Purif, 2020, 175: 105712
[74]	Song M K, Yang H, Cao W T, et al. Process Biochem, 2023, 132: 110
[75]	Bernardo S C, Carapito R, Neves M C, et al. Molecules, 2022, 27(5): 1618
[76]	Liu H F, Chen J, Chen M L, et al. Anal Chim Acta, 2023, 1274: 341496
[77]	Capela E V, Bairos J, Pedro A Q, et al. Sep Purif Technol, 2023, 305: 122464
[78]	Chen J, Tetrault J, Zhang Y Y, et al. J Chromatogr A, 2010, 1217(2): 216
[79]	Nagase K, Kanazawa H. Anal Chim Acta, 2020, 1138: 191
[80]	Shaibie N A, Ramli N A, Faizal N D F M, et al. Macromol Chem Phys, 2023, 224(20): 2300157
[81]	Nagase K. Adv Colloid Interface Sci, 2021, 295: 102487
[82]	Nagase K, Ishii S, Ikeda S, et al. Sci Rep, 2020, 10(1): 11896
[83]	Nomoto D, Nagase K, Nakamura Y, et al. Colloid Surface B, 2021, 205: 111890
[84]	Rahman M, Wang J Z, Xia H J, et al. Chem Eng J, 2020, 391: 123561
[85]	Guo D M, Xia Y R, ur Rahman M, et al. Chinese Journal of Chromatography, 2023, 41(12): 1045
[85]	郭冬梅, 夏一然, Mujeeb ur Rahman, 等. 色谱, 2023, 41(12): 1045

Affinity binding protein	Molecular weight/kDa	Binding domains	Ig binding target
Protein A (SpA)	40-60	5 for IgG (most common form)	heavy chain constant region (Fc) of IgG (CH₂-CH₃ region)
Protein G (SpG)	40-65	1 to 2 for IgG, 0 to 2 for human	heavy chain constant region (Fc) of IgG (CH₂-CH₃ region)
		serum albumin (HSA)
Protein L (SpL)	76	5 for Ig	Kappa (κ) light chains of Igs (V_L-κ)

Affinity binding protein	Molecular weight/kDa	Binding domains	Ig binding target
Protein A (SpA)	40-60	5 for IgG (most common form)	heavy chain constant region (Fc) of IgG (CH₂-CH₃ region)
Protein G (SpG)	40-65	1 to 2 for IgG, 0 to 2 for human	heavy chain constant region (Fc) of IgG (CH₂-CH₃ region)
		serum albumin (HSA)
Protein L (SpL)	76	5 for Ig	Kappa (κ) light chains of Igs (V_L-κ)

Ligand	pK_a	Target	Feedstock	Purity/%	Recovery/%	Ref.
4-Mercaptoethyl-pyridine (MEP)	4.8	IgG	bovine colostrums	93.9	91.5	[57]
2-Mercapto-1-methylimidazole (MMI)	5.3	IgY (ΔFc)	goose blood	98.6	85.0	[58]
5-Aminobenzimidazol (ABI)	6.5	monoclonal IgM	cell culture supernatant	99	80.8	[59]
2-Mercaptobenzimidazole (MBI)	4.1	IgG	IgG/BSA mixture	~90	96	[60]
Histamine	6.04	lysozyme	protein solution	N.A.	N.A.	[61]
5-Aminoindole (AI)	3.9	lysozyme	protein solution	~90	~90	[62]
2-Mercaptoimidazole (MI)	6.5	IgG	porcine plasma	89.4	N.A.	[63]
Benzotriazole-5-carboxylic (BTA)	N.A.	mAb	cell culture supernatant	89.7	95.1	[64]

Ligand	pK_a	Target	Feedstock	Purity/%	Recovery/%	Ref.
4-Mercaptoethyl-pyridine (MEP)	4.8	IgG	bovine colostrums	93.9	91.5	[57]
2-Mercapto-1-methylimidazole (MMI)	5.3	IgY (ΔFc)	goose blood	98.6	85.0	[58]
5-Aminobenzimidazol (ABI)	6.5	monoclonal IgM	cell culture supernatant	99	80.8	[59]
2-Mercaptobenzimidazole (MBI)	4.1	IgG	IgG/BSA mixture	~90	96	[60]
Histamine	6.04	lysozyme	protein solution	N.A.	N.A.	[61]
5-Aminoindole (AI)	3.9	lysozyme	protein solution	~90	~90	[62]
2-Mercaptoimidazole (MI)	6.5	IgG	porcine plasma	89.4	N.A.	[63]
Benzotriazole-5-carboxylic (BTA)	N.A.	mAb	cell culture supernatant	89.7	95.1	[64]