CN113337562B - 使用CHO细胞高效发酵生产rhCG的方法 - Google Patents
使用CHO细胞高效发酵生产rhCG的方法 Download PDFInfo
- Publication number
- CN113337562B CN113337562B CN202110562698.6A CN202110562698A CN113337562B CN 113337562 B CN113337562 B CN 113337562B CN 202110562698 A CN202110562698 A CN 202110562698A CN 113337562 B CN113337562 B CN 113337562B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- cho
- cells
- rhcg
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 31
- 230000004151 fermentation Effects 0.000 title claims abstract description 31
- 101100468640 Danio rerio rhcgl2 gene Proteins 0.000 title claims abstract description 16
- 101150053759 rhcg gene Proteins 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title description 9
- 241000699802 Cricetulus griseus Species 0.000 title description 2
- 210000001672 ovary Anatomy 0.000 title description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims abstract description 30
- 239000001963 growth medium Substances 0.000 claims abstract description 24
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 claims abstract description 15
- 229940048086 sodium pyrophosphate Drugs 0.000 claims abstract description 15
- 235000019818 tetrasodium diphosphate Nutrition 0.000 claims abstract description 15
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 13
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims description 37
- 239000012526 feed medium Substances 0.000 claims description 5
- 229960000890 hydrocortisone Drugs 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 229950000785 hydrocortisone phosphate Drugs 0.000 abstract description 11
- 229910021380 Manganese Chloride Inorganic materials 0.000 abstract description 8
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 abstract description 8
- 239000011565 manganese chloride Substances 0.000 abstract description 8
- 235000002867 manganese chloride Nutrition 0.000 abstract description 7
- 229940099607 manganese chloride Drugs 0.000 abstract description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 4
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 4
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000012879 subculture medium Substances 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229910001437 manganese ion Inorganic materials 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 2
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 229930064664 L-arginine Natural products 0.000 description 2
- 235000014852 L-arginine Nutrition 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229960005261 aspartic acid Drugs 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960000304 folic acid Drugs 0.000 description 2
- 235000019152 folic acid Nutrition 0.000 description 2
- 239000011724 folic acid Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 102000005396 glutamine synthetase Human genes 0.000 description 2
- 108020002326 glutamine synthetase Proteins 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 230000008844 regulatory mechanism Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- BBMHARZCALWXSL-UHFFFAOYSA-M sodium dihydrogenphosphate monohydrate Chemical compound O.[Na+].OP(O)([O-])=O BBMHARZCALWXSL-UHFFFAOYSA-M 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010013883 Dwarfism Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 208000007984 Female Infertility Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010021928 Infertility female Diseases 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 201000001880 Sexual dysfunction Diseases 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940061607 dibasic sodium phosphate Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000012803 optimization experiment Methods 0.000 description 1
- TWHXWYVOWJCXSI-UHFFFAOYSA-N phosphoric acid;hydrate Chemical compound O.OP(O)(O)=O TWHXWYVOWJCXSI-UHFFFAOYSA-N 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 231100000872 sexual dysfunction Toxicity 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 210000002993 trophoblast Anatomy 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Endocrinology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Reproductive Health (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本申请提供了一种使用CHO细胞表达重组rhCG的补料培养发酵方法，包括在发酵混合培养基中加入氯化锰，或者在补料培养基中加入氢化可的松和焦磷酸钠。本申请还提供了所述方法用的相应培养基。
Description
技术领域
本申请属于蛋白领域和细胞培养领域，具体地，本申请提供了使用CHO细胞高效发酵生产rhCG的方法及其相应培养基。
背景技术
人绒毛膜***(hCG)由胎盘滋养细胞合成的激素，由α亚基、β亚基组成，目前广泛用于治疗女性***、性机能障碍、流产、侏儒症、皮肤瘙痒、皮痒、肿瘤等疾病。
hCG最早以孕妇尿液为原料，通过沉淀、离子交换层析等方法制备，含较多杂质、来源不确定、有传染疾病风险以及批间差异、易发生过敏反应等，在临床使用中存在风险。之后研究者把研发重点放在了利用重组技术制备和生产，并在临床上逐渐取代尿源HCG。
虽然有昆虫细胞、神经细胞等宿主的尝试，但目前rhCG生产中最常用的细胞仍然为为CHO细胞。但CHO细胞中蛋白质的表达调控机理、蛋白质翻译后修饰调控***、细胞生长代谢调控机制等环节仍然缺少深入认识，所以在外源基因的整合策略、高表达菌株的筛选、高效培养基配置、培养工艺调控等方面的理论指导水平都还比较低，相关研究都更多地依赖大量的优化筛选和优化实验来获得较优的操作条件。不仅研究和生产效率低、耗时长、成本高，而且受偶然性因素影响较大。虽然国外先进企业已经通过开发谷氨酰胺合成酶(GS)***、定制无血清个性化培养基、优化高密度微载体培养工艺和补料策略、应用低成本和低剪切力生物反应器等手段，rhCG的高水平表达仍然需要较长的培养时间(＞20天)和较高的生产成本。
因此，本领域中仍然有进一步改进CHO细胞生产rhCG技术的需求。
发明内容
前期对发酵参数和细胞等因素的优化后，我们尝试了对发酵混合培养基和补料培养基的改进，研究了多种特殊添加剂对CHO表达rhCG的影响，发现在培养基中加入锰离子、氢化可的松和焦磷酸钠可以有效提高rhCG表达水平。
一方面，本申请提供了一种使用CHO细胞表达重组rhCG的补料培养发酵方法，包括在发酵混合培养基中加入氯化锰，或者在补料培养基中加入氢化可的松和焦磷酸钠。
进一步地，加入的氯化锰浓度为3mg/ml，加入的氢化可的松和焦磷酸钠浓度为0.05mg/ml和3mg/ml。
进一步地，发酵时间为12日。
进一步地，将密度1.5*106cells/ml以上的细胞接种20L到提前一夜已经预培养的50L发酵混合培养基里，接种完的当天，定为第一天，在培养第3、6、9天时分别加入10L补料培养基。
进一步地，培养参数为：36.5±1℃、pH控制在6.8-7.2、溶氧控制在20-70％，转速控制在60±5转/分。
另一方面，本申请提供了一种CHO细胞表达重组rhCG用培养基，包括发酵混合培养基和补料培养基，其中发酵混合培养基中加入了氯化锰，或者补料培养基中加入了氢化可的松和焦磷酸钠。
进一步地，加入的氯化锰浓度为3mg/ml，加入的氢化可的松和焦磷酸钠浓度为0.05mg/ml和3mg/ml。
另一方面，本申请提供了氯化锰、氢化可的松和/或焦磷酸钠在制备CHO细胞表达重组rhCG用培养基中的应用。
进一步地，培养基中氯化锰浓度为3mg/ml，氢化可的松和焦磷酸钠浓度为0.05mg/ml和3mg/ml。
附图说明
图1为CHO细胞发酵生产rhCG的基本流程。
图2为发酵培养第6日时hCG各亚基的相对丰度。
具体实施方式
实施例1发酵所用的细胞、培养基及其他试剂
发酵所用CHO细胞为申请人自制，使用LipofectamineTM2000脂质体转染试剂将携带rhCGα、β亚基的载体转染入CHO细胞K1株中制成，该细胞随后被无血清驯化并已经在申请人rhCG的试验性生产中运用超过18个月。
申请人为发酵过程配制的培养基：
传代培养基：
CD CHO AGT 24.3mg/ml、一水磷酸二氢2.69mg/ml、磷酸氢二钠4.33mg/ml、L-精氨酸550mg/ml、L-天冬酰胺1300mg/ml、L-天冬氨酸400mg/ml；
发酵混合培养基：
CD CHO AGT 24.3mg/ml、CD OPTICHO AGT 9.66mg/ml、一水磷酸二氢钠2.69mg/ml、磷酸氢二钠4.33mg/ml、生物素0.15mg/ml、叶酸11.5mg/ml、肌醇120mg/ml、氯化钾300mg/ml、葡萄糖5500mg/ml、氯化钠2100mg/ml、大豆水解蛋白30mg/ml；
补料培养基：
BalanCD CHO Feee3 66mg/ml、一水磷酸二氢钠2.69mg/ml、磷酸氢二钠4.33mg/ml、L-精氨酸2000mg/L、L-天门冬氨酸4500mg/L、维生素C15mg/L、维生素H 5mg/L叶酸30mg/L、肌醇400mg/L、维生素B12 5mg/L、氯化钾0.0015mg/L、F-68 950mg/L、葡萄糖18000mg/L；
HCG ELISA试剂盒：日本东曹；
MnCl2：济南凯创化工有限公司；
CD CHO AGT：gibco
CD OPTICHO AGT：gibco
BalanCD CHO Feee3：irvine scientific
焦磷酸钠：食品级，山东冠特生物工程有限公司；
氢化可的松：上海麦克林生化科技有限公司；
其他包括PCR试剂和仪器在内的试剂和仪器均为常规国产。
实施例2基本发酵生产过程
细胞复苏：
1)取细胞冻存管，置于37℃水浴锅内直至冻存的细胞悬液融化。
2)在吸取冻存细胞转至装有6～7ml CHO传代培养基的离心管内，离心1000rpm，5分钟，去除上清。
3)用分次吸取10mlCHO传代培养基轻轻吹打离心管内底部的成团细胞。
4)接种至装有10mlCHO传代培养基的125ml摇瓶内，使最终体积约为20ml，接种细胞密度为0.4-1.0*106个细胞/ml。将摇瓶置于二氧化碳培养箱中36.5±1℃、5±3％CO2、125±10rpm进行培养。
传代：
1)摇瓶培养：将细胞摇瓶放置于二氧化碳培养箱中36.5±1℃、5±3％CO2、125±10rpm进行培养。每天进行细胞计数，当细胞密度达到1.5*106细胞/ml以上时，进行传代，加入CHO传代培养基，调整细胞密度为0.4-1.0*106个细胞/ml。
2)wave培养：当细胞密度达1.5*106细胞/ml以上时，在把摇瓶内的细胞液收集到无菌瓶中，通过无菌接管机接种到细胞培养袋子中，调整细胞密度为0.4-1.0*106个细胞/ml，将细胞培养袋置于36.5±1℃、20±5rpm摇摆式仪上进行培养。当细胞密度达到1.5*106细胞/ml以上时，进行传代，加入CHO传代培养基，调整细胞密度为0.4-1.0*106个细胞/ml。
发酵：(100L规模)：
当20L体积的细胞培养袋内细胞密度1.5*106细胞/ml以上时，接种20L到提前一夜已经预培养的50L发酵混合培养基里，接种完的当天，定为第一天，在培养第3、6、9天时分别加入10L补料培养基。培养至12天发酵结束(培养参数为：36.5±1℃、pH控制在6.8-7.2、溶氧控制在40-60％，转速控制在60±5转/分)。
发酵结束后和发酵过程中使用实施例1所述的试剂盒检测HCG水平，使用现有文献中的引物PCR检测α亚基、β亚基转录水平(α亚基：上游CTGGTCACATTGTGCTATCTTTC、下游TGAGGTGACGTTCTTTTGGAC；β亚基：上游ATGTTCCAGGGGCTGCTGCTGTT、下游CGCGGTAGTTATTGCACCACACCACCTGA)
实施例3发酵过程的优化
经过前期对发酵参数和细胞等因素的优化，我们的方法在12天时间内实现了180mg/L左右的重组蛋白的表达水平。为了进一步提高产量/在类似产量下降低成本，我们自行对定制培养基进行了进一步改进。参考现有技术，尝试向培养基中加入MnCl2(发酵混合培养基中加入3mg/ml)，氢化可的松，焦磷酸钠，结果如下：
表1优化细胞株和发酵混合培养基中添加锰离子对表达水平的影响(三罐样本平均值)
进一步研究转录水平结果如图2所示：我们发现优化后的细胞株中β亚基与α亚基比率明显超过了初始细胞株，这意味着在优化后的细胞株中α亚基转录表达可能是限制产量的因素，而在发酵混合培养基中加入MnCl2可以有效提高α亚基转录水平进而提高产量；
表2补料培养基中加入氢化可的松和焦磷酸钠对表达水平的影响(优化后的细胞株，三罐样本平均值)
在补料培养基中单独加入氢化可的松或焦磷酸钠并无明显增产效果，而氢化可的松和焦磷酸钠的组合则可以实现明显提高重组蛋白表达量的效果(初步分子是补料中两者配合可以抑制细胞生长，停滞细胞周期以累积产物)。
组合锰离子、氢化可的松和焦磷酸钠后，我们在12日培养时间中实现了280.22mg/L的高表达水平。
Claims (3)
1.一种使用CHO细胞表达重组rhCG的补料培养发酵方法，其特征在于，将密度1.5*106cells/ml以上的细胞接种到发酵混合培养基里，接种完的当天，定为第一天，在培养第3、6、9天时分别加入补料培养基；补料培养基中包括浓度为0.05 mg/ml的氢化可的松和3mg/ml的焦磷酸钠。
2.根 据权利要求1所述的方法，其中发酵时间为12日。
3.根据权利要求1所述的方法，其中发酵过程中的参数为：36.5±1℃、pH控制在6.8-7.2、溶氧控制在20-70%，转速控制在60±5转/分。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110562698.6A CN113337562B (zh) | 2021-05-24 | 2021-05-24 | 使用CHO细胞高效发酵生产rhCG的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110562698.6A CN113337562B (zh) | 2021-05-24 | 2021-05-24 | 使用CHO细胞高效发酵生产rhCG的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113337562A CN113337562A (zh) | 2021-09-03 |
| CN113337562B true CN113337562B (zh) | 2022-06-03 |
Family
ID=77470997
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110562698.6A Active CN113337562B (zh) | 2021-05-24 | 2021-05-24 | 使用CHO细胞高效发酵生产rhCG的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113337562B (zh) |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6924124B1 (en) * | 2002-08-23 | 2005-08-02 | Immunex Corporation | Feeding strategies for cell culture |
| US20110137012A1 (en) * | 2007-04-26 | 2011-06-09 | Chugai Seiyaku Kabushiki Kaisha | Cell culture method using amino acid-enriched medium |
| CN101633894B (zh) * | 2009-08-20 | 2010-12-01 | 北京芳能科技有限公司 | 一种纤细裸藻的培养基及其开放式高密度养殖方法 |
| US20110052591A1 (en) * | 2009-08-28 | 2011-03-03 | Board Of Regents Of The University Of Texas System | Variants of Thyroid Stimulating Hormone Beta |
| NO2760138T3 (zh) * | 2012-10-01 | 2018-08-04 | ||
| WO2016170113A1 (en) * | 2015-04-24 | 2016-10-27 | Ferring B.V. | Method of production of gonadotrophin |
| US10119117B2 (en) * | 2016-01-28 | 2018-11-06 | Nanogen Pharmaceutical Biotechnology Co., Ltd | Universal, glycosylation enhancer, completely chemically defined medium formulation |
| CN111454877B (zh) * | 2019-01-22 | 2023-03-21 | 鲁南制药集团股份有限公司 | 一种cho细胞培养方法 |
| CN110042137B (zh) * | 2019-05-14 | 2023-02-28 | 上海赛迈生物科技有限公司 | 高密度灌注培养重组cho细胞生产人促卵泡激素的方法、培养基及其应用 |
| WO2021254296A1 (zh) * | 2020-06-14 | 2021-12-23 | 浙江大学 | 一种生物活性物质组合物、包含所述组合物的无血清培养基及其用途 |
-
2021
- 2021-05-24 CN CN202110562698.6A patent/CN113337562B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113337562A (zh) | 2021-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110607268B (zh) | 一株高产l-缬氨酸的基因工程菌及发酵生产l-缬氨酸方法 | |
| EP3473725B1 (en) | Fermentation technique with pichia yeast expressing recombinant protein | |
| JP6479974B2 (ja) | 大規模エクリズマブ産生細胞培養を再現する方法 | |
| CN116121161A (zh) | 一种生产麦角硫因的基因工程菌及其构建方法与应用 | |
| CN113337562B (zh) | 使用CHO细胞高效发酵生产rhCG的方法 | |
| KR102473375B1 (ko) | 재조합 미생물, 그 제조방법 및 보효소 q10의 생산에 있어서 그의 사용 | |
| CN1191369C (zh) | 一种用代谢工程菌生产腺苷甲硫氨酸的方法 | |
| CN114854659B (zh) | 一种麦角硫因生产工艺及其应用 | |
| CN115449499A (zh) | 一种脱氮生丝微菌及其发酵制备吡咯喹啉醌的方法 | |
| CN114480359B (zh) | 一种高密度发酵生产阿洛酮糖3-差向异构酶的方法 | |
| CN114574377B (zh) | 一株产腺苷蛋氨酸的酿酒酵母工程菌及其应用 | |
| Nie et al. | A novel strategy on the high-cell-density cultivation of Candida utilis for the enhanced production of glutathione | |
| WO2013058516A1 (en) | Recombinant vector, recombinant yeast containing the vector, and mass-producing method of glucose oxidase using the yeast | |
| CN113667650A (zh) | 一种11α-羟化酶突变体及其应用 | |
| CN114634902B (zh) | 用于生产阿洛酮糖差向异构酶的培养基和培养方法及其应用 | |
| CN114990138B (zh) | 基因、表达载体、菌株及在提高头孢菌素c产量中的应用 | |
| CN116200360B (zh) | 一种futCB突变体及生物合成2’-岩藻糖基乳糖的方法 | |
| CN113493745B (zh) | 生产头孢菌素c的基因工程菌及其构建方法 | |
| CN116875475A (zh) | 一株高产***素合成前体的酵母菌株及其构建方法和应用 | |
| CN116970504A (zh) | 一种高产菌体蛋白的毕赤酵母菌及其生产含nmn的单细胞蛋白的方法 | |
| CN118272328A (zh) | 咖啡酰辅酶a连接酶突变型蛋白、其编码基因、重组载体、重组菌和应用 | |
| CN115820443A (zh) | 一种高产s-腺苷-l-蛋氨酸的酿酒酵母工程菌及其应用 | |
| CN118127093A (zh) | 一种利用大肠杆菌细胞工厂发酵合成麦角硫因的方法 | |
| CN118048285A (zh) | 一种稳定生产l-甲硫氨酸的基因工程菌及其构建方法与应用 | |
| KR0143760B1 (ko) | 효모추출물을 사용하여 재조합 단백질의 생산성을 향상시키는 발효 방법 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |