新型耐碱蛋白A介质的制备与性能

doi:10.12034/j.issn.1009-606X.218246

过程工程学报 ›› 2019, Vol. 19 ›› Issue (3): 609-616.DOI: 10.12034/j.issn.1009-606X.218246

新型耐碱蛋白A介质的制备与性能

韦巍^1,2，黄永东^2*，赵岚²，吴学星²，朱天孝¹，李冬雪¹，靳海波¹，张荣月^1*，苏志国²，马光辉²

1. 北京石油化工学院化学工程学院，北京 102617 2. 中国科学院过程工程研究所生化工程国家重点实验室，北京 100190

收稿日期:2018-07-17 修回日期:2018-09-30 出版日期:2019-06-22 发布日期:2019-06-20
通讯作者: 黄永东 ydhuang@ipe.ac.cn
基金资助:
国家重点研发计划资助项目;中国科学院重点部署项目;北京市自然科学基金资助项目;北京市教委科技一般项目;北京市属高校高水平教师队伍建设支持计划高水平创新团队建设计划项目

Preparation and properties of a new alkali-resistant rProtein A chromatographic medium

Wei WEI^1,2, Yongdong HUANG^2*, Lan ZHAO², Xuexing WU², Tianxiao ZHU¹, Dongxue LI¹, Haibo JIN¹, Rongyue ZHANG^1*, Zhiguo SU², Guanghui MA²

1. Department of Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing 102617, China 2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

Received:2018-07-17 Revised:2018-09-30 Online:2019-06-22 Published:2019-06-20
Contact: HUANG YongDong ydhuang@ipe.ac.cn

摘要/Abstract

摘要： 以蛋白A结构中耐碱性更好的C区为基础，构建一种新型耐碱蛋白A，并偶联至琼脂糖基质，获得新型耐碱蛋白A亲和层析介质，分别用激光共聚焦显微镜和石英晶体微天平研究了人免疫球蛋白(hIgG)与蛋白A介质的结合过程。结果表明，该介质具有较商品配基更高的结合力和稳定的再生性能，hIgG动态载量达62.0 mg/mL，40次再生操作后载量为初始载量的84%。该介质在抗体纯化领域具有较好的应用前景。

关键词: 耐碱蛋白A, 层析介质, 亲和层析, 抗体纯化, 动态载量

Abstract: Monoclonal antibodies (mAb) have been applied for curing a wide range of diseases and is considered to be a major source of new therapies in the next decades. mAb?s production has been received worldwide attention and efficient purification strategies have been always explored. Protein A chromatography was one of the most popular methods for mAb purification. Cleaning-in-place has been widely used in protein A chromatography for meeting the demand of high quality, and therefore an alkali-resistant ligand is necessary. Since protein A from natural sources is not alkali-resistant, it should be genetically modified. In this study, a new alkali-resistant rProtein A was constructed based on C-region gene construction followed by being coupled to epoxy-activated agarose-based microspheres under optimized conditions, and a new rProtein A chromatographic medium was prepared. Both confocal laser scanning microscopy and quartz crystal microbalance were used for analyzing the binding of hIgG to rProtein A chromatographic medium. The results showed that it was transparent and full bright. It had a uniform particle size distribution with an average diameter of 84 μm. This medium had good hydraulics properties with a maximum flow rate of 1400 cm/h. This new rProtein A chromatographic medium had a higher hIgG dynamic binding capacity of 62.0 mg/mL than that of commercial ligand. Also, this medium had a better cleaning-in-place performance and the dynamic binding capacity was kept to 84% of the initial value after 40 cycles, as beneficial for its application on an industrial scale. At the beginning of adsorption to the medium, hIgG was bound quickly to the surface coupled with rProtein A, however, the binding rate decreased gradually due to mass transfer resistance from the inner part of the medium. The desorption rate had a similar tendency to that of the adsorption. The new rProtein A chromatographic medium had great prospects in mAb purification and gave a good basis for its application in the future.

Key words: Alkali-resistant rProtein A, Chromatographic medium, Affinity chromatography, mAb purification, Dynamic binding capacity

韦巍黄永东赵岚吴学星朱天孝李冬雪靳海波张荣月苏志国马光辉. 新型耐碱蛋白A介质的制备与性能[J]. 过程工程学报, 2019, 19(3): 609-616.

Wei WEI, Yongdong HUANG Lan ZHAO Xuexing WU Tianxiao ZHU Dongxue LI Haibo JIN Rongyue ZHANG Zhiguo SU Guanghui MA. Preparation and properties of a new alkali-resistant rProtein A chromatographic medium[J]. Chin. J. Process Eng., 2019, 19(3): 609-616.

参考文献

[1]Razinkov V I, Treuheit M J, Becker G W.Accelerated formulation development of monoclonal antibodies (mAbs) and mAb-based modalities: review of methods and tools[J].J. Biomol. Screen, 2015, 20(4):468-483 [2]Oldham R K, Dillman R O.Monoclonal antibodies in cancer therapy: 25 years of progress[J].J. Clin. Oncol., 2008, 26(11):1774-1777 [3]Schoofs T, Klein F, Braunschweig M, et al.HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1[J].Science, 2016, 352(6288):997-1001 [4] Grom M, Kozorog M, Caserman S, et al.Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling[J].J. Chromatogr. B, 2018, (1083):44-57 [5] Ghose S, Rajshekaran R, Labanca M, et al.Modeling on-column reduction of trisulfide bonds in monoclonal antibodies during protein A chromatography[J].J. Chromatogr. A, 2017, (1479):81-86 [6] Dutta A K, Tran T, Napadensky B, et al.Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography[J].J. Biotechnol., 2015, (213):54-64 [7]Pathak M, Rathore A S.Implementation of a fluorescence based PAT control for fouling of protein A chromatography resin[J].J. Chem. Technol. Biotechnol., 2017, 92(11):2799-2807 [8]Hober S, Nord K, Linhult M.Protein A chromatography for antibody purification[J].J. Chromatogr. B, 2007, 848(1):40-47 [9]Jiang C P, Liu J, Rubacha M, et al.A mechanistic study of Protein A chromatography resin lifetime[J].J. Chromatogr. A, 2009, 1216(31):5849-5855 [10]Pathak M, Rathore A S, Lintern K, et al.Protein A chromatography resin lifetime-impact of feed composition[J].Biotechnol. Prog., 2018, 34(2):412-419 [11]Lute S, Norling L, Hanson M, et al.Robustness of virus removal by protein A chromatography is independent of media lifetime[J].J. Chromatogr. A, 2008, 1205(1):17-25 [12]Nord K, Gunneriusson E, Ringdahl J, et al.Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain[J].Nat. Biotechnol., 1997, 15(8):772-777 [13]Jendeberg L, Tashiro M, Tejero R, et al.The mechanism of binding staphylococcal protein A to immunoglobin G does not involve helix unwinding[J].Biochemistry, 1996, 35(1):22-31 [14]Hilbold N J, Le Saout X, Valery E, et al.Evaluation of several protein A resins for application to multicolumn chromatography for the rapid purification of fed-batch bioreactors[J].Biotechnol. Prog., 2017, 33(4):941-953 [15]Linhult M, Gülich S, Gr?slund T, et al.Improving the tolerance of a protein A analogue to repeated alkaline exposures using a bypass mutagenesis approach[J].Proteins, 2004, 55(2):407-416 [16]Xia H F, Liang Z D, Wang S L, et al.Molecular modification of Protein A to improve the elution pH and alkali resistance in affinity chromatography[J].Appl. Biochem. Biotechnol., 2014, 172(8):4002-4012 [17]Zhao L, Zhu K, Huang Y D, et al.Enhanced binding by dextran-grafting to Protein A affinity chromatographic media[J].J. Sep. Sci., 2017, 40(7):1493-1499 [18]Van Sommeren A P G, Machielsen P A G M, Gribnau T C J.Comparison of three activated agaroses for use in affinity chromatography: Effects on coupling performance and ligand leakage[J].J. Chromatogr. A, 1993, 639(1):23-31 [19]Zhao L, Zhang J F, Huang Y D, et al.Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation[J].J. Sep. Sci., 2016, 39(6):1130-1136 [20]Zhao L, Liu Y D, Huang Y D, et al.Deliberate manipulation of the surface hydrophobicity of an adsorbent for an efficient purification of a giant molecule with multiple subunits[J].J. Sep. Sci., 2015, 34(22):3186-3193 [21]Ogi H, Motohisa K, Hatanaka K, et al.Concentration dependence of IgG-protein A affinity studied by wireless-electrodeless QCM[J].Biosens. Bioelectron., 2007, 22(12):3238-3242

新型耐碱蛋白A介质的制备与性能

Preparation and properties of a new alkali-resistant rProtein A chromatographic medium

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