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.
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]. The Chinese Journal of Process Engineering, 2019
, 19(3)
: 609
-616
.
DOI: 10.12034/j.issn.1009-606X.218246
[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
