人血小板生成素重组细胞的培养工艺研究
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摘要
本研究的内容主要是建立高密度、高表达培养TPO重组细胞的工艺。进
行了包括构建重组细胞株、确立重组细胞培养方法、建立TPO体外活性测定
方法、纯化和鉴定表达产物等一系列实验。
首先将带有二氢叶酸还原酶（DHFR）基因的TPO表达质粒pTcho以电
穿孔法转入二氢叶酸还原酶缺陷的CHO（DHFR）细胞，通过条件培养基选
择及氨甲碟呤加压筛选，获得能够高效、稳定地表达rhTPO的细胞株，命名
为STPO-1。经细胞稳定性等多项指标的鉴定，证实该细胞可以作为生产细胞
株。
为了实现高表达细胞培养，通过实验选择了合适的培养基、pH值和表达
刺激物质等，确定了STPO-1细胞在瓶中的培养及表达方法。使细胞在瓶培养
时的TPO表达量达到2000U/ml（相当于7mg/ml）。为了进一步实现重组细
胞的高密度培养，利用生物反应器进行了大规模连续培养，摸索出了STPO-1
细胞在生物反应器中培养时合适的pH值、溶解氧值、转数、通气流量、葡萄
糖浓度和流加速度等，确定了STPO-1细胞在生物反应器中的生长及表达条件，
在体积为5L的生物反应器中，利用无蛋白培养基培养，细胞密度达6×10~6
个/ml，TPO表达量达12000U/ml（相当于40mg/ml），细胞密度和表达量均
达到了瓶培养的6倍，表达时间达24天，收获量达100L，收获液经纯化后可
得TPO蛋白650mg，并且在表达期间细胞代谢和表达量比较稳定。
为验证表达产物的结构，进一步对表达产物进行纯化，获得了均一的rhTPO
组分。经鉴定，产品的纯度大于99％，分子量为70-120KD，N端17个氨基
酸序列依次为Ser-Pro-Ala-Pro-Pro-Ala-Cys-Asp-Leu-Arg-Val-Leu-Ser-Lys-Leu-
Leu-Arg，等电点小于5，免疫印迹为单一条带。
通过实验获得了能高效、稳定地表达重组TPO的细胞株，并建立起一套
完整有效的细胞培养及产物纯化技术。对大规模产业化生产和广泛的临床应用
打下了坚实的基础。
在研制过程中，确定了TPO在体外定量测定的方法，该方法简便、灵敏
沈阳药科大学硕士论文 人血小板生成素重组细胞的培养工艺研究
度高、重复性好，可做为常规方法推广使用。
In the current study, a method of high-density cell culture for producing rhTPO was developed. These research included construction of a CHO cell line stably transfected with rhTPO eDNA, selection of the cell line that highly expressed rhTPO protein, development of an assay for measuring the in-vitro activity of rhTPO, development of a method of cell culture using bioreactor, and finally, the purification and characterization of rhTPO.
In brief, a mammalian expression plasmid containing TPO eDNA and a selectable marker was first introduced into CHO cells. A cell line, which was named STP0-l, was obtained following the standard selection and amplification procedures. This STP0-l cell line could stably express rhTPO at a high level.
The optimization of cell culture was then performed and the resulting level of expression reached 2,000 units/mi (7 mglml) of rhTPO when the ST1抩-l cells were cultured in flasks. The high-density cell culture was studied using method of packed-bed bioreactor with continuous perfusion culture. Following optimization of the bioreaction condition, such as medium, pH, dissolved oxygen (DO2), agitation speed, glucose concentration, and perfusion rate, the cell density could reach 6 X 106/ml and level of TPO expression could reach 12,000 units/mI (40 mg/mi) by using a specific protein-free medium. As a result, an average of 100 liters of conditioned medium could be harvested using a bioreactor with a 5-liter vessel. A total of 650 mg of rhTPO protein could be obtained following the purification process, which included a series of filtration and chromatography steps.
The characterization of purified rhTPO showed 99% purity by High-
Performance-Liquid Chromatography (HPLC) and SDS-Polyacrylamide Gel
Electrophoresis (PAGE), 70 -120 kD of the apparent molecular weight, multiple Iso-
Electric Focusing (IEF) bands with p1 <5, and a single smeared band on a Western
Blot analysis. The N-terminal amino acid analysis showed correct sequence as
3
deduced from TPO genetic code.
In summary, a cell line that could highly express rhTPO was constructed, an efficient in-vitro activity assay of rhTPO and a complete process for production and purification of rhTPO was developed. These results provided a solid ground for large-scale manufacture process development as well as eventual clinical application of rhTPO.
行了包括构建重组细胞株、确立重组细胞培养方法、建立TPO体外活性测定
方法、纯化和鉴定表达产物等一系列实验。
首先将带有二氢叶酸还原酶（DHFR）基因的TPO表达质粒pTcho以电
穿孔法转入二氢叶酸还原酶缺陷的CHO（DHFR）细胞，通过条件培养基选
择及氨甲碟呤加压筛选，获得能够高效、稳定地表达rhTPO的细胞株，命名
为STPO-1。经细胞稳定性等多项指标的鉴定，证实该细胞可以作为生产细胞
株。
为了实现高表达细胞培养，通过实验选择了合适的培养基、pH值和表达
刺激物质等，确定了STPO-1细胞在瓶中的培养及表达方法。使细胞在瓶培养
时的TPO表达量达到2000U/ml（相当于7mg/ml）。为了进一步实现重组细
胞的高密度培养，利用生物反应器进行了大规模连续培养，摸索出了STPO-1
细胞在生物反应器中培养时合适的pH值、溶解氧值、转数、通气流量、葡萄
糖浓度和流加速度等，确定了STPO-1细胞在生物反应器中的生长及表达条件，
在体积为5L的生物反应器中，利用无蛋白培养基培养，细胞密度达6×10~6
个/ml，TPO表达量达12000U/ml（相当于40mg/ml），细胞密度和表达量均
达到了瓶培养的6倍，表达时间达24天，收获量达100L，收获液经纯化后可
得TPO蛋白650mg，并且在表达期间细胞代谢和表达量比较稳定。
为验证表达产物的结构，进一步对表达产物进行纯化，获得了均一的rhTPO
组分。经鉴定，产品的纯度大于99％，分子量为70-120KD，N端17个氨基
酸序列依次为Ser-Pro-Ala-Pro-Pro-Ala-Cys-Asp-Leu-Arg-Val-Leu-Ser-Lys-Leu-
Leu-Arg，等电点小于5，免疫印迹为单一条带。
通过实验获得了能高效、稳定地表达重组TPO的细胞株，并建立起一套
完整有效的细胞培养及产物纯化技术。对大规模产业化生产和广泛的临床应用
打下了坚实的基础。
在研制过程中，确定了TPO在体外定量测定的方法，该方法简便、灵敏
沈阳药科大学硕士论文 人血小板生成素重组细胞的培养工艺研究
度高、重复性好，可做为常规方法推广使用。
In the current study, a method of high-density cell culture for producing rhTPO was developed. These research included construction of a CHO cell line stably transfected with rhTPO eDNA, selection of the cell line that highly expressed rhTPO protein, development of an assay for measuring the in-vitro activity of rhTPO, development of a method of cell culture using bioreactor, and finally, the purification and characterization of rhTPO.
In brief, a mammalian expression plasmid containing TPO eDNA and a selectable marker was first introduced into CHO cells. A cell line, which was named STP0-l, was obtained following the standard selection and amplification procedures. This STP0-l cell line could stably express rhTPO at a high level.
The optimization of cell culture was then performed and the resulting level of expression reached 2,000 units/mi (7 mglml) of rhTPO when the ST1抩-l cells were cultured in flasks. The high-density cell culture was studied using method of packed-bed bioreactor with continuous perfusion culture. Following optimization of the bioreaction condition, such as medium, pH, dissolved oxygen (DO2), agitation speed, glucose concentration, and perfusion rate, the cell density could reach 6 X 106/ml and level of TPO expression could reach 12,000 units/mI (40 mg/mi) by using a specific protein-free medium. As a result, an average of 100 liters of conditioned medium could be harvested using a bioreactor with a 5-liter vessel. A total of 650 mg of rhTPO protein could be obtained following the purification process, which included a series of filtration and chromatography steps.
The characterization of purified rhTPO showed 99% purity by High-
Performance-Liquid Chromatography (HPLC) and SDS-Polyacrylamide Gel
Electrophoresis (PAGE), 70 -120 kD of the apparent molecular weight, multiple Iso-
Electric Focusing (IEF) bands with p1 <5, and a single smeared band on a Western
Blot analysis. The N-terminal amino acid analysis showed correct sequence as
3
deduced from TPO genetic code.
In summary, a cell line that could highly express rhTPO was constructed, an efficient in-vitro activity assay of rhTPO and a complete process for production and purification of rhTPO was developed. These results provided a solid ground for large-scale manufacture process development as well as eventual clinical application of rhTPO.
引文
1． Keleman E, Cserhati I ,Tanos B. Demonstration and some properties of human thrombopoietin in thrombocythaemic sera. Acta Haematol 1958;20:350-5
2． De Sauvage FJ, Hass PE, Spencer SD, et al. Stimulation of megakaryocytopoiesis by the c-Mpl ligand. Nature 1994;369:583-8
3． Lok S, Kaushansky K, Holly RD, et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994;369:565-8
4． Bartly TD, Bogenberger J, Hunt P, et al. Identification and cloning of a megacaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 1994;77:1117-24
5． Sohma Y, Akahori H, Seki N, et al. Molecular cloning and chromosomal localization of the human thrombopoietin gene. FEBS Lett 1994;353:57-61
6． David J. Kuter, Pamela Hunt, William Sheridan, et al. Thrombopoiesis and thrombopoietins: Molecular, Cellular, Preclinical, and Clinical Biology. 1997; Humana Press US.
7． Takashi Kato, Atsushi Matsumoto, Kinya Ogami, et al. Native thrombopoietin: structure and function. Stem Cells 1998; 16:322-8
8． Wendling F. Thrombopoietin: its role from early hematopoiesis to platelet production. 1999; 84(2) : 158-66
9． Alexandra J, Valerie F, Marielle A, et al. Human thrombopoietin structure-function relationships: identification of functionally important residues. Biochem. J.1998;333: 729-34
10． K Kaushansky. Thrombopoietin. N Engl J Med. 1998;339(11) : 746-54
11． Frederic J. de Sauvage. Philip E.Hass, et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994;369:533-8
12． 刘丽，王颖，田生礼等。血小板生成素糖基化与其体内生物学活性关系的研究，免 疫学杂志 1999；15(2) ：103-5
13． Thomas GR. Thibodeaux H. Errett CJ. et al. In vivo biological effects of various forms of thrombopoietin in a murine model of transient pancytopenia. Stem Cells 1996; 14 Suppl l(l):246-55
14． Kaushansky K. Broudy VC. Lin N. et al. Thrombopoietin, the Mpl ligand, is essential for
full megakaryocyte development. Proc Natl Acad Sci U S A 1995 Apr 11;92(8) :3234-8
15． Ulich TR. del Castillo J. Yin S. et al. Megakaryocyte growth and development factor ameliorates carboplatin-induced thrombocytopenia in mice. Blood 1995 Aug 1;86(3) :971-6
16． Hokom MM. Lacey D. Kinstler OB. et al. Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice. Blood 1995 Dec 15;86(12) :4486-92
17． Molineux G. Hartley C. McElroy P. et al. Megakaryocyte growth and development factor accelerates platelet recovery in peripheral blood progenitor cell transplant recipients. Blood 1996 Jul 1;88(1) :366-76
18． Marker LA. Marzec UM. Hunt P. et al. Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates. Blood 1996 Jul 15;88(2) :511-21
19． Farese AM. Hunt P. Boone T. et al. Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates. Blood 1995 Jul1;86(1) :54-9
20． Fanucchi M. Glaspy J. Crawford J. et al. Effects of polyethylene glycol-conjugated recombinant human megakaryocyte growth and development factor on platelet counts after chemotherapy for lung cancer. N Engl J Med. 1997; 336:404-9
21． Basser RL. Rasko JEJ. Clarke K. et al. Randomized blinded, placebo-controlled phase I trial of pegylated recombinant human megakaryocyte growth and development factor with filgrastim after dose-intensive chemotherapy in patients with advanced cancer. Blood 1997; 89: 3118-28
22． Vadhan-Raj S. Murry LJ. Bueso-Ramos C, et al. Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer. Ann Int Med. 1997; 126: 673-81
23． Vadhan-Raj S. Verchraegen C. McGarry L. et al. Recombinant human thrombopoietin(rhTPO) attenuates high-dose carboplatin (?)-induced thrombocytopenia in patients with gynecologic malignancy. Blood 1997(suppl 1) (abstr)
24． Vadhan-Raj S. Patel S. Broxmeyer HE. et al. Phase I-II investigation of recombinant human
25．金冬雁，黎孟枫等译。《分子克隆实验指南》(第二版)，1992，科学出版社
26．《中国生物制品规程》1995年版P20，《人二倍体细胞建株、检定及制备疫苗规程》
27．《中国生物制品规程》1995年版P244，人凝血因子Ⅷ效价测定
28. Mosmann, T. J. Immun. Meth. 1983; 65:55-63
29. Ronald Hill, Jack Levin. Partial purification of thrombopoietin using lectin chromatography. Exp. Hematol. 1986;14:752-9
30. Sialic acid in polysaccharide vaccines. EP 1997:69
31. Subramani S. Mulligan R. Berg P. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. Mol. Cell. Biol. 1981; 1:854-64
32. Kaufman R. J. Sharp P.A. Amplification and expression sequence cotransfected with a modular dihydrofolate reductase complementary DNA gene. J.Mol. Biol. 1982; 159:601-21
33. Kaufman R. J. Sharp P.A. Construction of a modular dihydrofolate reductase cDNA gene: Analysis of signals utilized for efficient expression. Mol. Cell. Biol. 1982; 2:1304-19
34．鄂征主编，《组织培养和分子生物学实验技术》，1995年版，北京出版社
35. W. Craig Cullen, T. P. Mcdonald. Comparision of stereologic techniques for the quantitation of megakaryocyte size and number. Exp. Hematol. 1986; 14:782-8
36. Barbara W. Grant, William L. et al. Quantitation of human in vitro megakaryocytopoiesis by radioimmunoassay. Blood 1987; 69(5): 1334-9
2． De Sauvage FJ, Hass PE, Spencer SD, et al. Stimulation of megakaryocytopoiesis by the c-Mpl ligand. Nature 1994;369:583-8
3． Lok S, Kaushansky K, Holly RD, et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994;369:565-8
4． Bartly TD, Bogenberger J, Hunt P, et al. Identification and cloning of a megacaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 1994;77:1117-24
5． Sohma Y, Akahori H, Seki N, et al. Molecular cloning and chromosomal localization of the human thrombopoietin gene. FEBS Lett 1994;353:57-61
6． David J. Kuter, Pamela Hunt, William Sheridan, et al. Thrombopoiesis and thrombopoietins: Molecular, Cellular, Preclinical, and Clinical Biology. 1997; Humana Press US.
7． Takashi Kato, Atsushi Matsumoto, Kinya Ogami, et al. Native thrombopoietin: structure and function. Stem Cells 1998; 16:322-8
8． Wendling F. Thrombopoietin: its role from early hematopoiesis to platelet production. 1999; 84(2) : 158-66
9． Alexandra J, Valerie F, Marielle A, et al. Human thrombopoietin structure-function relationships: identification of functionally important residues. Biochem. J.1998;333: 729-34
10． K Kaushansky. Thrombopoietin. N Engl J Med. 1998;339(11) : 746-54
11． Frederic J. de Sauvage. Philip E.Hass, et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994;369:533-8
12． 刘丽，王颖，田生礼等。血小板生成素糖基化与其体内生物学活性关系的研究，免 疫学杂志 1999；15(2) ：103-5
13． Thomas GR. Thibodeaux H. Errett CJ. et al. In vivo biological effects of various forms of thrombopoietin in a murine model of transient pancytopenia. Stem Cells 1996; 14 Suppl l(l):246-55
14． Kaushansky K. Broudy VC. Lin N. et al. Thrombopoietin, the Mpl ligand, is essential for
full megakaryocyte development. Proc Natl Acad Sci U S A 1995 Apr 11;92(8) :3234-8
15． Ulich TR. del Castillo J. Yin S. et al. Megakaryocyte growth and development factor ameliorates carboplatin-induced thrombocytopenia in mice. Blood 1995 Aug 1;86(3) :971-6
16． Hokom MM. Lacey D. Kinstler OB. et al. Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice. Blood 1995 Dec 15;86(12) :4486-92
17． Molineux G. Hartley C. McElroy P. et al. Megakaryocyte growth and development factor accelerates platelet recovery in peripheral blood progenitor cell transplant recipients. Blood 1996 Jul 1;88(1) :366-76
18． Marker LA. Marzec UM. Hunt P. et al. Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates. Blood 1996 Jul 15;88(2) :511-21
19． Farese AM. Hunt P. Boone T. et al. Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates. Blood 1995 Jul1;86(1) :54-9
20． Fanucchi M. Glaspy J. Crawford J. et al. Effects of polyethylene glycol-conjugated recombinant human megakaryocyte growth and development factor on platelet counts after chemotherapy for lung cancer. N Engl J Med. 1997; 336:404-9
21． Basser RL. Rasko JEJ. Clarke K. et al. Randomized blinded, placebo-controlled phase I trial of pegylated recombinant human megakaryocyte growth and development factor with filgrastim after dose-intensive chemotherapy in patients with advanced cancer. Blood 1997; 89: 3118-28
22． Vadhan-Raj S. Murry LJ. Bueso-Ramos C, et al. Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer. Ann Int Med. 1997; 126: 673-81
23． Vadhan-Raj S. Verchraegen C. McGarry L. et al. Recombinant human thrombopoietin(rhTPO) attenuates high-dose carboplatin (?)-induced thrombocytopenia in patients with gynecologic malignancy. Blood 1997(suppl 1) (abstr)
24． Vadhan-Raj S. Patel S. Broxmeyer HE. et al. Phase I-II investigation of recombinant human
25．金冬雁，黎孟枫等译。《分子克隆实验指南》(第二版)，1992，科学出版社
26．《中国生物制品规程》1995年版P20，《人二倍体细胞建株、检定及制备疫苗规程》
27．《中国生物制品规程》1995年版P244，人凝血因子Ⅷ效价测定
28. Mosmann, T. J. Immun. Meth. 1983; 65:55-63
29. Ronald Hill, Jack Levin. Partial purification of thrombopoietin using lectin chromatography. Exp. Hematol. 1986;14:752-9
30. Sialic acid in polysaccharide vaccines. EP 1997:69
31. Subramani S. Mulligan R. Berg P. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. Mol. Cell. Biol. 1981; 1:854-64
32. Kaufman R. J. Sharp P.A. Amplification and expression sequence cotransfected with a modular dihydrofolate reductase complementary DNA gene. J.Mol. Biol. 1982; 159:601-21
33. Kaufman R. J. Sharp P.A. Construction of a modular dihydrofolate reductase cDNA gene: Analysis of signals utilized for efficient expression. Mol. Cell. Biol. 1982; 2:1304-19
34．鄂征主编，《组织培养和分子生物学实验技术》，1995年版，北京出版社
35. W. Craig Cullen, T. P. Mcdonald. Comparision of stereologic techniques for the quantitation of megakaryocyte size and number. Exp. Hematol. 1986; 14:782-8
36. Barbara W. Grant, William L. et al. Quantitation of human in vitro megakaryocytopoiesis by radioimmunoassay. Blood 1987; 69(5): 1334-9