Removal of monoclonal antibody aggregates with ion exchange chromatography by flow-through mode

doi:10.11949/0438-1157.20230666

Abstract

Abstract:

Two cation exchange media, Eshmuno CP-FT and Eshmuno CPX, with the same matrix and ligand but different ligand density and grafting methods, were compared in their ability to remove monoclonal antibody aggregates in flow-through mode. First, the static adsorption properties of monoclonal antibody (mAb) monomers and aggregates with two resins under different pH and conductivity were investigated. It was found that high monomer purity and yield could be achieved under low pH and high conductivity, or high pH and low conductivity conditions. The comprehensive performance of Eshmuno CP-FT was better than that of Eshmuno CPX. The breakthrough curves under different residence times were measured at the optimal adsorption conditions. With the target of mAb monomer purity higher than 99%, the loading capacity of Eshmuno CP-FT resin could reach 556 g/L, and the monomer yield was 83.2%. For Eshmuno CPX, the loading capacity was only 269 g/L with a yield of 60.4%. In addition, the productivity of Eshmuno CP-FT was twice that of Eshmuno CPX, and the buffer consumption was only about half. The adsorption kinetics results indicated that Eshmuno CP-FT showed faster adsorption of the aggregates and stronger displacement effect on the monomers. In general, to remove monoclonal antibody aggregates with flow-through mode, it is important to design the resins in a rational way. The optimization of ligand density and its distribution can adjust and balance the binding between monomers and aggregates, and promote the adsorption and separation of aggregates. The results in the present work provide some guidance for the development of new resins for flow-through chromatographic separation.

Key words: monoclonal antibody, flow-through mode, aggregates, bioseparation, ion exchange

摘要：

针对基质和配基相同、配基密度和接枝方式不同的两款阳离子交换介质Eshmuno CP-FT和Eshmuno CPX，比较它们在流穿模式下对单抗聚集体的去除能力。首先考察了不同pH和电导率下单抗单体和聚集体的静态吸附性能，发现在低pH和高电导、高pH和低电导条件下均能实现较高的单抗单体纯度和收率，综合性能Eshmuno CP-FT较优。进一步考察了最佳吸附条件下不同保留时间的穿透曲线，若控制单抗单体纯度99%，Eshmuno CP-FT介质载量高达556 g/L，单抗单体收率为83.2%；而Eshmuno CPX载量只有269 g/L，收率仅60.4%。此外，Eshmuno CP-FT的产率是Eshmuno CPX的2倍，缓冲液消耗仅为Eshmuno CPX的一半左右。吸附动力学结果表明，Eshmuno CP-FT吸附聚集体速度更快，对单体的顶替作用也更强。对于流穿模式去除单抗聚集体，合理的介质设计很重要，优化配基密度及其空间分布可以调整和平衡单抗单体和聚集体的结合，促进聚集体吸附分离，该结果可为研发新型流穿模式介质提供指导。

关键词: 单克隆抗体, 流穿模式, 聚集体, 生物分离, 离子交换

CLC Number:

TQ 028.8

Yaxin ZHAO, Xueqin ZHANG, Rongzhu WANG, Guo SUN, Shanjing YAO, Dongqiang LIN. Removal of monoclonal antibody aggregates with ion exchange chromatography by flow-through mode[J]. CIESC Journal, 2023, 74(9): 3879-3887.

赵亚欣, 张雪芹, 王荣柱, 孙国, 姚善泾, 林东强. 流穿模式离子交换层析去除单抗聚集体[J]. 化工学报, 2023, 74(9): 3879-3887.

Figures/Tables 10

Fig.1 Absorption properties of Eshmuno CP-FT under different conditions

Fig.2 Absorption properties of Eshmuno CPX under different conditions

Fig.3 Comparison of monomer purity and yield under different conditions

Fig.4 Comparison of different load conditions on Eshmuno CP-FT

Fig.5 Separation performance of Eshmuno CP-FT under different residence times

Table 1 Evaluation of separation performance with two resins

Resin	Residence time/min	Load/(g/L)	Yield/%	Productivity/(g/(L·h))	Buffer consumption/(L/g)
Eshmuno CP-FT	1	240	62.8	78.1	0.54
	3	580	82.4	92.4	0.24
	5	710	85.8	70.1	0.21
Eshmuno CPX	1	173	39.7	37.1	1.07
	3	269	61.3	46.6	0.50
	5	345	71.9	44.0	0.36

Fig.6 Separation performance of Eshmuno CPX under different residence time

Fig.7 Schematic diagram of absorption mechanism with two resins

Table 2 Verification of batch experiment on two resins

Resin	Residence time/min	Yield/%	Productivity/(g/(L·h))	Buffer consumption/(L/g)
Eshmuno CP-FT	3	83.2	90.9	0.25
Eshmuno CPX	3	60.4	43.8	0.54

Fig.8 Adsorption kinetic curves on Eshmuno CP-FT resin under different adsorption conditions

References 30

1	Swain S M, Shastry M, Hamilton E. Targeting HER2-positive breast cancer: advances and future directions[J]. Nature Reviews Drug Discovery, 2023, 22(2): 101-126.
2	Briani C, Visentin A. Therapeutic monoclonal antibody therapies in chronic autoimmune demyelinating neuropathies[J]. Neurotherapeutics: the Journal of the American Society for Experimental Neuro Therapeutics, 2022, 19(3): 874-884.
3	Saoudi Gonzalez N, López D, Gómez D, et al. Pharmacokinetics and pharmacodynamics of approved monoclonal antibody therapy for colorectal cancer[J]. Expert Opinion on Drug Metabolism & Toxicology, 2022, 18(11): 755-767.
4	Khanal O, Lenhoff A M. Developments and opportunities in continuous biopharmaceutical manufacturing[J]. mAbs, 2021, 13(1): 1903664.
5	史策, 虞骥, 高栋, 等. 单抗制备的过程模拟和经济性分析[J]. 化工学报, 2018, 69(7): 3198-3207.
	Shi C, Yu J, Gao D, et al. Process simulation and economic evaluation of monoclonal antibody production[J]. CIESC Journal, 2018, 69(7): 3198-3207.
6	卢慧丽, 林东强, 姚善泾. 抗体药物分离纯化中的层析技术及进展[J]. 化工学报, 2018, 69(1): 341-351.
	Lu H L, Lin D Q, Yao S J. Chromatographic technology in antibody purification and its progress[J]. CIESC Journal, 2018, 69(1): 341-351.
7	陈泉, 卓燕玲, 许爱娜, 等. 蛋白A亲和层析法纯化单克隆抗体工艺的优化[J]. 生物工程学报, 2016, 32(6): 807-818.
	Chen Q, Toh P, Hoi A, et al. Improved protein-A chromatography for monoclonal antibody purification[J]. Chinese Journal of Biotechnology, 2016, 32(6): 807-818.
8	鲁伟, 应国清, 杨晓明, 等. 单克隆抗体工业生产中蛋白A亲和层析步骤的成本分析[J]. 高校化学工程学报, 2023, 37(2): 276-284.
	Lu W, Ying G Q, Yang X M, et al. Cost analysis of protein A affinity chromatography in industrial production of monoclonal antibody[J]. Journal of Chemical Engineering of Chinese Universities, 2023, 37(2): 276-284.
9	荆淑莹, 史策, 姚善泾, 等. 连续流层析及用于抗体分离的新进展[J]. 高校化学工程学报, 2021, 35(1): 1-12.
	Jing S Y, Shi C, Yao S J, et al. Progress on continuous chromatography and its application in antibody separation[J]. Journal of Chemical Engineering of Chinese Universities, 2021, 35(1): 1-12.
10	Hari S B, Lau H, Razinkov V I, et al. Acid-induced aggregation of human monoclonal IgG1 and IgG2: molecular mechanism and the effect of solution composition[J]. Biochemistry, 2010, 49(43): 9328-9338.
11	Liu H F, Ma J F, Winter C, et al. Recovery and purification process development for monoclonal antibody production[J]. mAbs, 2010, 2(5): 480-499.
12	Arosio P, Barolo G, Müller-Späth T, et al. Aggregation stability of a monoclonal antibody during downstream processing[J]. Pharmaceutical Research, 2011, 28(8): 1884-1894.
13	Joshi V, Yadav N, Rathore A. Aggregation of monoclonal antibody products: formation and removal[J]. BioPharm International, 2013, 26(3): 40-45.
14	Rosenberg A S. Effects of protein aggregates: an immunologic perspective[J]. The AAPS Journal, 2006, 8(3): 59.
15	Cromwell M E M, Hilario E, Jacobson F. Protein aggregation and bioprocessing[J]. The AAPS Journal, 2006, 8(3): 66.
16	Moussa E M, Panchal J P, Moorthy B S, et al. Immunogenicity of therapeutic protein aggregates[J]. Journal of Pharmaceutical Sciences, 2016, 105(2): 417-430.
17	Vázquez-Rey M, Lang D A. Aggregates in monoclonal antibody manufacturing processes[J]. Biotechnology and Bioengineering, 2011, 108(7): 1494-1508.
18	Shukla A A, Hubbard B, Tressel T, et al. Downstream processing of monoclonal antibodies—application of platform approaches[J]. Journal of Chromatography B, 2007, 848(1): 28-39.
19	Lu Y F, Williamson B, Gillespie R. Recent advancement in application of hydrophobic interaction chromatography for aggregate removal in industrial purification process[J]. Current Pharmaceutical Biotechnology, 2009, 10(4): 427-433.
20	McCue J T. Chapter 25 theory and use of hydrophobic interaction chromatography in protein purification applications[M]//Burgess R R, Deutscher M P. Methods in Enzymology. 2nd ed. Amsterdam: Elsevier, 2009: 405-414.
21	McCue J T, Engel P, Ng A, et al. Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography[J]. Bioprocess and Biosystems Engineering, 2008, 31(3): 261-275.
22	Ichihara T, Ito T, Kurisu Y, et al. Integrated flow-through purification for therapeutic monoclonal antibodies processing[J]. mAbs, 2018, 10(2): 325-334.
23	Liu H F, McCooey B, Duarte T, et al. Exploration of overloaded cation exchange chromatography for monoclonal antibody purification[J]. Journal of Chromatography A, 2011, 1218(39): 6943-6952.
24	Reck J M, Pabst T M, Hunter A K, et al. Separation of antibody monomer-dimer mixtures by frontal analysis[J]. Journal of Chromatography A, 2017, 1500: 96-104.
25	Wollacott R, Roth L, Sears T, et al. The development of a flow-through mode cation exchange process for the purification of a monoclonal antibody[J]. BioProcessing Journal, 2015, 14(2): 5-13.
26	Stone M T, Cotoni K A, Stoner J L. Cation exchange frontal chromatography for the removal of monoclonal antibody aggregates[J]. Journal of Chromatography A, 2019, 1599: 152-160.
27	Vogg S, Pfeifer F, Ulmer N, et al. Process intensification by frontal chromatography: performance comparison of resin and membrane adsorber for monovalent antibody aggregate removal[J]. Biotechnology and Bioengineering, 2020, 117(3): 662-672.
28	Wolf M K F, Closet A, Bzowska M, et al. Improved performance in mammalian cell perfusion cultures by growth inhibition[J]. Biotechnology Journal, 2019, 14(2): 1700722.
29	Reck J M, Pabst T M, Hunter A K, et al. Adsorption equilibrium and kinetics of monomer-dimer monoclonal antibody mixtures on a cation exchange resin[J]. Journal of Chromatography A, 2015, 1402: 46-59.
30	Carta G, Jungbauer A. Downstream processing of biotechnology products[M]//Protein Chromatography. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA, 2010: 1-55.

[1]	Yan GAO, Peng WU, Chao SHANG, Zejun HU, Xiaodong CHEN. Preparation of magnetic agarose microspheres based on a two-fluid nozzle and their protein adsorption properties [J]. CIESC Journal, 2023, 74(8): 3457-3471.
[2]	Yali HU, Junyong HU, Suxia MA, Yukun SUN, Xueyi TAN, Jiaxin HUANG, Fengyuan YANG. Development of novel working fluid and study on electrochemical characteristics of reverse electrodialysis heat engine [J]. CIESC Journal, 2023, 74(8): 3513-3521.
[3]	Zhaoguang CHEN, Yuxiang JIA, Meng WANG. Modeling neutralization dialysis desalination driven by low concentration waste acid and its validation [J]. CIESC Journal, 2023, 74(6): 2486-2494.
[4]	Caihong LIN, Li WANG, Yu WU, Peng LIU, Jiangfeng YANG, Jinping LI. Effect of alkali cations in zeolites on adsorption and separation of CO₂/N₂O [J]. CIESC Journal, 2023, 74(5): 2013-2021.
[5]	Junying YAN, Huangying WANG, Ruirui LI, Rong FU, Chenxiao JIANG, Yaoming WANG, Tongwen XU. Selective electrodialysis: opportunities and challenges [J]. CIESC Journal, 2023, 74(1): 224-236.
[6]	Ruohan DU, Bo PANG, Ning WANG, Fujun CUI, Minggang GUO, Gaohong HE, Xuemei WU. Continuous covalent organic framework composite membrane with size-sieving effect for vanadium flow battery [J]. CIESC Journal, 2022, 73(9): 4163-4172.
[7]	Hongxin YANG, Xingya LI, Liang GE, Tongwen XU. Preparation of mono-/divalent anion permselective membranes with piperidinium-type long side-chain [J]. CIESC Journal, 2022, 73(8): 3739-3748.
[8]	Shanshan YANG, Yuyang YAO, Yundi DONG, Zhipeng XU, Shangshang GAO, Huimin RUAN, Jiangnan SHEN. Preparation and performance of ion exchange membrane with K⁺ selectivity based on dibenzo-18-crown-6 modification [J]. CIESC Journal, 2022, 73(4): 1781-1793.
[9]	FU Fengyan, XING Guang'en. Progress of polymer-based anion exchange membrane for alkaline fuel cell application [J]. CIESC Journal, 2021, 72(S1): 42-52.
[10]	LI Tengfei, MIAO Yun, YANG Liu, WANG Longyao, ZHU Huacheng. Microwave enhanced ion exchange technology of Y molecular sieve [J]. CIESC Journal, 2021, 72(S1): 406-412.
[11]	Zi'ang XU, Lei WAN, Kai LIU, Baoguo WANG. Recent progress of molecular design for highly stable alkaline anion exchange membranes [J]. CIESC Journal, 2021, 72(8): 3891-3906.
[12]	LIU Xuan, MA Yichang, ZHANG Qiugen, LIU Qinglin. Preparation of fullerene crosslinked quaternized polyphenylene oxide anion exchange membrane [J]. CIESC Journal, 2021, 72(7): 3849-3855.
[13]	NI Jia, SUN Xueyan, SHUI Ziyi, HE Feihong, HUI Xiaomin, ZHU Liangliang, CHEN Xi. Energy consumption and performance optimization of moisture swing sorbents for direct air capture of CO₂ [J]. CIESC Journal, 2021, 72(3): 1409-1418.
[14]	Chunhui FAN,Zongye GAO,Shanjing YAO,Dongqiang LIN. Quantitative characterization of non-specific adsorption on affinity chromatography resins [J]. CIESC Journal, 2021, 72(10): 5218-5225.
[15]	Shuping ZOU, Zhentao JIANG, Zhicai WANG, Zhiqiang LIU, Yuguo ZHENG. Synthesis of (R)-epichlorohydrin catalyzed by cross-linked cell aggregates of epoxide hydrolase [J]. CIESC Journal, 2020, 71(9): 4238-4245.