抗ErbB2嵌合抗体chA21的大规模纯化工艺的建立及质量研究
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摘要
ErbB2是具有酪氨酸激酶活性的跨膜糖蛋白,属于表皮生长因子受体家族,在多种癌细胞特别乳腺癌中有高表达,针对其的单克隆抗体具有潜在的治疗效果。本实验室自行研制的抗ErbB2嵌合抗体chA21具有抑制高表达ErbB2乳腺癌细胞生长的作用。本论文从chA21的大规模纯化生产工艺、质量研究以及工艺改进进行了研究:
1、以填充床生物反应器大规模培养CHO工程细胞株表达的上清为原料,采用亲和层析、凝胶过滤除盐、阳离子交换层析、分子筛等步骤,分离纯化嵌合抗体chA21,建立大规模纯化工艺。该工艺能有效解决抗体纯化过程形成的多聚体问题,去除内毒素和DNA残留;可以确保每批纯化10L～20L培养上清,蛋白总回收率大于50%,纯度可达98%。
2、按照国家对生物制品的质量要求,对大规模生产的chA21原液产品进行了全面鉴定和质量控制,主要包括含量鉴定、纯度和杂质污染物的检测、理化性质鉴定、生物学活性测定等。鉴定结果表明制备的chA21产品符合动物实验和临床试验的要求。
3、最后进一步对纯化工艺新策略摸索,探讨可以替代的纯化方案。
嵌合抗体大规模纯化工艺的确定和质量研究的初步摸索,为今后的药物申请奠定了基础。
The ErbB2 is a transmembrane glycoprotein with intrinsic tyrosine-kinase activities and belongs to epidermal growth factor receptor family. It overexpressed in several malignant human tumors like breast cancer. So using monoclonal antibodies to target overexpressed ErbB2 cancer for clinical therapy became a new strategy and powerful tool. Our libratory constructed single-chain Chimeric Anti-ErbB2 Antibody chA21 by assembling a single-chain Fv antibody and a human IgG1 Fc fragment. In my dissertation, we developed the purification procedure of chA21, identified the chA21 product and researched the new purification strategy, mainly including three chapters:
1、chA21 from engineered CHO cells were cultured in the packed bed bioreactor. To establish mass-scale purification technology, culture supernatant was collected and purified in a sequence of downstream processing steps, namely, affinity chromatography, desalting, ion exchange chromatography and molecular sieve chromatography. By the developed mass-scale procedure, formed aggregates could be removed effetely; endotoxins and residual DNA were also extracted. 10L~20L of culture supernatant could be treated in a single operation. The purify rate reached more than 98% and the total recovery rate was over 50%.
2、chA21 product was all analyzed according to the national standard of biologic product. The items included content, purity, foreigh material, contaminant, physoci-chemical property and biology activity. The results indicated that chA21 product was in line with the standard of injection medicine. So the purify rate of the produce technics was high and quality inspection methods were stability and reliable, which could be fit for in large-scale production of chA21.
3、Researched the new purification strategy through the other chromatograghy.
The study on purification and stability of chA21 may be the foundation for drug application.
1、以填充床生物反应器大规模培养CHO工程细胞株表达的上清为原料,采用亲和层析、凝胶过滤除盐、阳离子交换层析、分子筛等步骤,分离纯化嵌合抗体chA21,建立大规模纯化工艺。该工艺能有效解决抗体纯化过程形成的多聚体问题,去除内毒素和DNA残留;可以确保每批纯化10L～20L培养上清,蛋白总回收率大于50%,纯度可达98%。
2、按照国家对生物制品的质量要求,对大规模生产的chA21原液产品进行了全面鉴定和质量控制,主要包括含量鉴定、纯度和杂质污染物的检测、理化性质鉴定、生物学活性测定等。鉴定结果表明制备的chA21产品符合动物实验和临床试验的要求。
3、最后进一步对纯化工艺新策略摸索,探讨可以替代的纯化方案。
嵌合抗体大规模纯化工艺的确定和质量研究的初步摸索,为今后的药物申请奠定了基础。
The ErbB2 is a transmembrane glycoprotein with intrinsic tyrosine-kinase activities and belongs to epidermal growth factor receptor family. It overexpressed in several malignant human tumors like breast cancer. So using monoclonal antibodies to target overexpressed ErbB2 cancer for clinical therapy became a new strategy and powerful tool. Our libratory constructed single-chain Chimeric Anti-ErbB2 Antibody chA21 by assembling a single-chain Fv antibody and a human IgG1 Fc fragment. In my dissertation, we developed the purification procedure of chA21, identified the chA21 product and researched the new purification strategy, mainly including three chapters:
1、chA21 from engineered CHO cells were cultured in the packed bed bioreactor. To establish mass-scale purification technology, culture supernatant was collected and purified in a sequence of downstream processing steps, namely, affinity chromatography, desalting, ion exchange chromatography and molecular sieve chromatography. By the developed mass-scale procedure, formed aggregates could be removed effetely; endotoxins and residual DNA were also extracted. 10L~20L of culture supernatant could be treated in a single operation. The purify rate reached more than 98% and the total recovery rate was over 50%.
2、chA21 product was all analyzed according to the national standard of biologic product. The items included content, purity, foreigh material, contaminant, physoci-chemical property and biology activity. The results indicated that chA21 product was in line with the standard of injection medicine. So the purify rate of the produce technics was high and quality inspection methods were stability and reliable, which could be fit for in large-scale production of chA21.
3、Researched the new purification strategy through the other chromatograghy.
The study on purification and stability of chA21 may be the foundation for drug application.
引文
[1] Jukkola A, Bloigu R, Soini Y, et al. c-erbB-2 positivity is a factor for poor prognosis in breast cancer and poor response to hormonal or chemotherapy treatment in advanced disease. Eur J Cancer, 2001, 37(3):347~354
[2] Slamon D J, Godolphin W, Jones L A, et al. Studies of the HER2 /neu proto-oncogene in human breast and ovarian cancer. Science, 1989, 244 (4905): 707~712
[3] Drebin JA, Link VC, Stern DF, Weinberg RA, Greene MI. Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen. Proc. Natl. Acsd. Sci., 1986, 96: 9129-9133
[4] Doherty JK, Bond C, Jardim A, Adelman JP, Clinton GM. The Her-2/neu receptor tyrosine kinase gene encodes a secreted autoinhibitor. Proc. Natl. Acsd. Sci., 1999, 96(19): 10869-10874
[5] Toru Kumagai, James G. Davis, Takeo Horie, Donald M. O’Rourke, Mark I. Greene. The role of distinct p185neu extracellular subdomains for dimerization with the epidermal growth factor(EGF) receptor and EGF-mediated signaling. Proc Natl Acad Sci USA, 2001;10;5526-5331
[6] Yosef Yarden. Biology of HER2 and Its Importance in Breast Cancer. Oncology. 2001, 61(suppl 2):1–13
[7] Yoichi Nagata, et al.,PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumabresistance in patients . Cancer Cell, 2004. 15(6): 117-27
[8] Eldad Tzahar, Yosef Yarden. The ErbB-2rHER2 oncogenic receptor of adenocarcinomas: fromorphanhood to multiple stromal ligands. Biochimica et Biophysica Acta. 1998
[9] Pinkas-Kramarski, R., et al., Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. Embo J, 1996. 15(10): 2452-67.
[10] Yarden, Y. and M.X. Sliwkowski, Untangling the ErbB signalling network. Nat Rev Mol Cell Biol, 2001. 2(2): 127-37.
[11] Tanner, K.G. and J. Kyte, Dimerization of the extracellular domain of the receptor for epidermal growth factor containing the membrane-spanning segment in response to treatment with epidermal growth factor. J Biol Chem, 1999. 274(50): 35985-90.
[12] Andersen, T.I., et al., Detection of c-erbB-2 related protein in sera from breast cancer patients. Relationship to ERBB2 gene amplification and c-erbB-2 protein overexpression in tumour. Acta Oncol, 1995. 34(4): 499-504.
[13] Dankort, D., et al., Grb2 and Shc adapter proteins play distinct roles in Neu (ErbB-2)-induced mammary tumorigenesis: implications for human breast cancer. Mol Cell Biol, 2001. 21(5): 1540-51.
[14] Tsutsui, S., et al., Prognostic value of the combination of epidermal growth factor receptor and c-erbB-2 in breast cancer. Surgery, 2003. 133(2): 219-21.
[15] Zemzoum, I., et al., Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node-negative breast cancer. J Clin Oncol, 2003. 21(6): 1022-8.
[16] Neve, R.M., H.A. Lane, and N.E. Hynes, The role of overexpressed HER2 in transformation. Ann Oncol, 2001. 12 Suppl 1: S9-13.
[17] Horton, R.M., et al., Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene, 1989. 77(1): 61-8.
[18] Hoogenboom, H.R., et al., Antibody phage display technology and its applications. Immunotechnology, 1998. 4(1): 1-20.
[19] Le Gall, F., et al., Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett, 1999. 453(1-2): 164-8.
[20] De Jonge, J., et al., Production and characterization of bispecific single-chain antibody fragments. Mol Immunol, 1995. 32(17-18): 1405-12.
[21] Shak S. Overview of the trastuzumab (Herceptin) anti-HER2 monoclonal antibody clinical program in HER2-overexpressing metastatic breast cancer. Herceptin Multinational Investigator Study Group. Semin Oncol 1999. 26:71-77
[22] Dennis J. Slamon, Brian Leyland-Jones, Steven Shak, Hank Fuchs, Virginia Paton, Alex Bajamonde, Thomas Fleming, Wolfgang Eiermann, Janet Wolter, Mark Pegram, Jose Baselga, Larry Norton. N Engl J Med 2001.344:783-792
[23] Johann S. De Bono and Eric K. Rowinsky. The ErbB receptor family: a therapeutic target for cancer. Trends in Molecular Medicine 2002;Vol.8 No.4 (Suppl): 19-26
[24] Klapper LN, Waterman H, Sela M, Yarden Y. Tumor-inhibitory antibodies to HER-2/erbB-2 may act by recruiting c-cb1 and enhancing ubiquitination of HER-2. Cancer Res 2000; 60: 3384-3388
[25] Michael Yakes F, Wichai Chinratanalab, Christoph A. Ritter, Walter King, Steven Seelig, and Carlos L. Arteaga. Herceptin-induced Inhibition of Phosphatidylinositol-3 Kinase and Akt Is Required for Antibody-mediated Effects on p27, Cyclin D1, and Antitumor Action. Cancer Research 2002;62:4132-4141
[26] Lweis A. Chodosh.The reciprocal dance between cancer and development. The New England Journal of Medicine 2002;347(2) July 11:134-136
[27] A. J. E. Freebairn,A. T. J. Last,T. M. Illidge. Trastuzumab: Designer Drug or Fashionable Fad? Clinical Oncology 2001;13:427–433
[28] Sangkyou Lee, Wentao Yang, Keng-Hsueh Lan, Shankar Sellappan, Kristine Klos, Gabriel Hortobagyi, Mien-Chie Hung, and Dihua Yu. Enhanced Sensitization to Taxol-induced Apoptosis by Herceptin Pretreatment in ErbB2-overexpressing Breast Cancer Cells. Cancer Research 2002;62:5703-5710
[29] D. S. Leonard, A. D. K. Hill, L. Kelly, B. Dijkstra, E. McDermott, N. J. O'Higgins. Anti-human epidermal growth factor receptor 2 monoclonal antibody therapy for breast cancer. British Journal of Surgery 2002; 89:262-271
[30] Slamon DJ, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. New Engl J Med 2001; 344: 783-792
[31] Vogel C, Cobleigh M, Tripathy D, Harris L, Fehrenbacher L, Slamon D, Ash M, Novotny W, Stewart S, Shak S: First-line, nonhormonal, treatment of women with HER2 overexpressing metastatic breast cancer with herceptin (trastuzumab, humanised anti-HER2 antibody). Proc Am Soc Clin Oncol 2001, 20:71a
[32] David WM. Update on Her-2 as a target for cancer therapy Herceptin in the clinical setting. Breast Cancer Res., 2001, 3: 380-384
[33] Schaller G, Bangemann N, Becker C, Buhler H, Opri F, Weitzel HK. Therapy of metastatic breast cancer with humanized antibodies against the HER2 receptor protein. J Cancer Res Clin Oncol 1999;125:520
[34] Eiermann W, on behalf of the International Herceptin Study Group. Trastuzumab combined with chemotherapy for the treatment of HER2-positive metastatic breast cancer: Pivotal trial data.Annals of Oncology 2001;12 (Suppl. 1): S57-S62
[35] Cook-Bruns N. Retrospective Analysis of the Safety of Herceptin? Immunotherapy in Metastatic Breast Cancer. Oncology 2001;61(suppl 2):58–66
[36] Karlsson, D., et al., Model-based optimization of a preparative ion-exchange step for antibody purification. J Chromatogr A, 2004. 1055(1-2): p. 29-39.
[37]王军志,《生物技术药物研究开发和质量控制》,2002,p. 107
[38] Grunfeld, H. and P. Moore, Reduction of bovine immunoglobulin contamination from monoclonal antibodies by SOURCE 15PHE chromatography. J Immunol Methods, 1997. 201(2): p. 233-41.
[39] Felinger, A. and G. Guiochon, Comparing the optimum performance of the different modes of preparative liquid chromatography. J Chromatogr A, 1998. 796(1): p. 59-74.
[40] Chen Zhi-Nan, Y.X.-L.M.L.C., Comparison of Expended Bed Adsorption Chromatography and Packed Bed ChrmatogRaphy for Monoclonal Antibody Purification. China Biotechnology, 2003. 23(1): p. 61-64.
[41] Brorson, K., et al., Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use. J Chromatogr A, 2003. 989(1): p. 155-63.
[42] Amersham Biosciences, Long-term CIP of rProtein A media:a test for MabSelect, Downstream, 2003. 36: p. 12-14.
[1] Hahn R, Bauerhansl P, Shimahara K, et a1. Comparison of protein A affinity sorbents II. Mass transfer properties. J Chromatogr A, 2005, 1093(1-2): 98~110
[2] Hahn R, Shimahara K, Steindl F, et a1. Comparison of protein A affinity sorbents III. Life time study.J Chromatogr A, 2006, 1102(1-2): 224~231
[3] Wei W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm, 2005, 289(1-2): 1~30
[4] Shukla A A, Gupta P, Han X. Protein aggregation kinetics during Protein A chromatography. Case study for an Fc fusion protein. J Chromatogr A, 2007, 1171(1-2):22~28
[5] Pete Gagnon. Improved antibody aggregate removal by hydroxyapatite chromatography in the presence of polyethylene glycol. J Immunol Methods, 2008, 336 (2): 222~228
[6] Wan Y, Vasan S, Ghosh R,et al. Separation of monoclonal antibody alemtuzumab monomer and dimers using ultrafiltration. Biotechnol Bioeng, 2005, 90(4):422~432
[7] S. Aldington, J. Bonnerjea. Scale-up of monoclonal antibody purification processes. J Chromatogr B, 2007, 848(1): 64~78
[8] Baselga J, Norton L, et al. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res, 1998, 58(13):2825~2831
[2] Slamon D J, Godolphin W, Jones L A, et al. Studies of the HER2 /neu proto-oncogene in human breast and ovarian cancer. Science, 1989, 244 (4905): 707~712
[3] Drebin JA, Link VC, Stern DF, Weinberg RA, Greene MI. Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen. Proc. Natl. Acsd. Sci., 1986, 96: 9129-9133
[4] Doherty JK, Bond C, Jardim A, Adelman JP, Clinton GM. The Her-2/neu receptor tyrosine kinase gene encodes a secreted autoinhibitor. Proc. Natl. Acsd. Sci., 1999, 96(19): 10869-10874
[5] Toru Kumagai, James G. Davis, Takeo Horie, Donald M. O’Rourke, Mark I. Greene. The role of distinct p185neu extracellular subdomains for dimerization with the epidermal growth factor(EGF) receptor and EGF-mediated signaling. Proc Natl Acad Sci USA, 2001;10;5526-5331
[6] Yosef Yarden. Biology of HER2 and Its Importance in Breast Cancer. Oncology. 2001, 61(suppl 2):1–13
[7] Yoichi Nagata, et al.,PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumabresistance in patients . Cancer Cell, 2004. 15(6): 117-27
[8] Eldad Tzahar, Yosef Yarden. The ErbB-2rHER2 oncogenic receptor of adenocarcinomas: fromorphanhood to multiple stromal ligands. Biochimica et Biophysica Acta. 1998
[9] Pinkas-Kramarski, R., et al., Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. Embo J, 1996. 15(10): 2452-67.
[10] Yarden, Y. and M.X. Sliwkowski, Untangling the ErbB signalling network. Nat Rev Mol Cell Biol, 2001. 2(2): 127-37.
[11] Tanner, K.G. and J. Kyte, Dimerization of the extracellular domain of the receptor for epidermal growth factor containing the membrane-spanning segment in response to treatment with epidermal growth factor. J Biol Chem, 1999. 274(50): 35985-90.
[12] Andersen, T.I., et al., Detection of c-erbB-2 related protein in sera from breast cancer patients. Relationship to ERBB2 gene amplification and c-erbB-2 protein overexpression in tumour. Acta Oncol, 1995. 34(4): 499-504.
[13] Dankort, D., et al., Grb2 and Shc adapter proteins play distinct roles in Neu (ErbB-2)-induced mammary tumorigenesis: implications for human breast cancer. Mol Cell Biol, 2001. 21(5): 1540-51.
[14] Tsutsui, S., et al., Prognostic value of the combination of epidermal growth factor receptor and c-erbB-2 in breast cancer. Surgery, 2003. 133(2): 219-21.
[15] Zemzoum, I., et al., Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node-negative breast cancer. J Clin Oncol, 2003. 21(6): 1022-8.
[16] Neve, R.M., H.A. Lane, and N.E. Hynes, The role of overexpressed HER2 in transformation. Ann Oncol, 2001. 12 Suppl 1: S9-13.
[17] Horton, R.M., et al., Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene, 1989. 77(1): 61-8.
[18] Hoogenboom, H.R., et al., Antibody phage display technology and its applications. Immunotechnology, 1998. 4(1): 1-20.
[19] Le Gall, F., et al., Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett, 1999. 453(1-2): 164-8.
[20] De Jonge, J., et al., Production and characterization of bispecific single-chain antibody fragments. Mol Immunol, 1995. 32(17-18): 1405-12.
[21] Shak S. Overview of the trastuzumab (Herceptin) anti-HER2 monoclonal antibody clinical program in HER2-overexpressing metastatic breast cancer. Herceptin Multinational Investigator Study Group. Semin Oncol 1999. 26:71-77
[22] Dennis J. Slamon, Brian Leyland-Jones, Steven Shak, Hank Fuchs, Virginia Paton, Alex Bajamonde, Thomas Fleming, Wolfgang Eiermann, Janet Wolter, Mark Pegram, Jose Baselga, Larry Norton. N Engl J Med 2001.344:783-792
[23] Johann S. De Bono and Eric K. Rowinsky. The ErbB receptor family: a therapeutic target for cancer. Trends in Molecular Medicine 2002;Vol.8 No.4 (Suppl): 19-26
[24] Klapper LN, Waterman H, Sela M, Yarden Y. Tumor-inhibitory antibodies to HER-2/erbB-2 may act by recruiting c-cb1 and enhancing ubiquitination of HER-2. Cancer Res 2000; 60: 3384-3388
[25] Michael Yakes F, Wichai Chinratanalab, Christoph A. Ritter, Walter King, Steven Seelig, and Carlos L. Arteaga. Herceptin-induced Inhibition of Phosphatidylinositol-3 Kinase and Akt Is Required for Antibody-mediated Effects on p27, Cyclin D1, and Antitumor Action. Cancer Research 2002;62:4132-4141
[26] Lweis A. Chodosh.The reciprocal dance between cancer and development. The New England Journal of Medicine 2002;347(2) July 11:134-136
[27] A. J. E. Freebairn,A. T. J. Last,T. M. Illidge. Trastuzumab: Designer Drug or Fashionable Fad? Clinical Oncology 2001;13:427–433
[28] Sangkyou Lee, Wentao Yang, Keng-Hsueh Lan, Shankar Sellappan, Kristine Klos, Gabriel Hortobagyi, Mien-Chie Hung, and Dihua Yu. Enhanced Sensitization to Taxol-induced Apoptosis by Herceptin Pretreatment in ErbB2-overexpressing Breast Cancer Cells. Cancer Research 2002;62:5703-5710
[29] D. S. Leonard, A. D. K. Hill, L. Kelly, B. Dijkstra, E. McDermott, N. J. O'Higgins. Anti-human epidermal growth factor receptor 2 monoclonal antibody therapy for breast cancer. British Journal of Surgery 2002; 89:262-271
[30] Slamon DJ, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. New Engl J Med 2001; 344: 783-792
[31] Vogel C, Cobleigh M, Tripathy D, Harris L, Fehrenbacher L, Slamon D, Ash M, Novotny W, Stewart S, Shak S: First-line, nonhormonal, treatment of women with HER2 overexpressing metastatic breast cancer with herceptin (trastuzumab, humanised anti-HER2 antibody). Proc Am Soc Clin Oncol 2001, 20:71a
[32] David WM. Update on Her-2 as a target for cancer therapy Herceptin in the clinical setting. Breast Cancer Res., 2001, 3: 380-384
[33] Schaller G, Bangemann N, Becker C, Buhler H, Opri F, Weitzel HK. Therapy of metastatic breast cancer with humanized antibodies against the HER2 receptor protein. J Cancer Res Clin Oncol 1999;125:520
[34] Eiermann W, on behalf of the International Herceptin Study Group. Trastuzumab combined with chemotherapy for the treatment of HER2-positive metastatic breast cancer: Pivotal trial data.Annals of Oncology 2001;12 (Suppl. 1): S57-S62
[35] Cook-Bruns N. Retrospective Analysis of the Safety of Herceptin? Immunotherapy in Metastatic Breast Cancer. Oncology 2001;61(suppl 2):58–66
[36] Karlsson, D., et al., Model-based optimization of a preparative ion-exchange step for antibody purification. J Chromatogr A, 2004. 1055(1-2): p. 29-39.
[37]王军志,《生物技术药物研究开发和质量控制》,2002,p. 107
[38] Grunfeld, H. and P. Moore, Reduction of bovine immunoglobulin contamination from monoclonal antibodies by SOURCE 15PHE chromatography. J Immunol Methods, 1997. 201(2): p. 233-41.
[39] Felinger, A. and G. Guiochon, Comparing the optimum performance of the different modes of preparative liquid chromatography. J Chromatogr A, 1998. 796(1): p. 59-74.
[40] Chen Zhi-Nan, Y.X.-L.M.L.C., Comparison of Expended Bed Adsorption Chromatography and Packed Bed ChrmatogRaphy for Monoclonal Antibody Purification. China Biotechnology, 2003. 23(1): p. 61-64.
[41] Brorson, K., et al., Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use. J Chromatogr A, 2003. 989(1): p. 155-63.
[42] Amersham Biosciences, Long-term CIP of rProtein A media:a test for MabSelect, Downstream, 2003. 36: p. 12-14.
[1] Hahn R, Bauerhansl P, Shimahara K, et a1. Comparison of protein A affinity sorbents II. Mass transfer properties. J Chromatogr A, 2005, 1093(1-2): 98~110
[2] Hahn R, Shimahara K, Steindl F, et a1. Comparison of protein A affinity sorbents III. Life time study.J Chromatogr A, 2006, 1102(1-2): 224~231
[3] Wei W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm, 2005, 289(1-2): 1~30
[4] Shukla A A, Gupta P, Han X. Protein aggregation kinetics during Protein A chromatography. Case study for an Fc fusion protein. J Chromatogr A, 2007, 1171(1-2):22~28
[5] Pete Gagnon. Improved antibody aggregate removal by hydroxyapatite chromatography in the presence of polyethylene glycol. J Immunol Methods, 2008, 336 (2): 222~228
[6] Wan Y, Vasan S, Ghosh R,et al. Separation of monoclonal antibody alemtuzumab monomer and dimers using ultrafiltration. Biotechnol Bioeng, 2005, 90(4):422~432
[7] S. Aldington, J. Bonnerjea. Scale-up of monoclonal antibody purification processes. J Chromatogr B, 2007, 848(1): 64~78
[8] Baselga J, Norton L, et al. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res, 1998, 58(13):2825~2831