WO2020020364A1 - 一种制备重组人凝血因子ⅷ的方法 - Google Patents

一种制备重组人凝血因子ⅷ的方法 Download PDF

Info

Publication number
WO2020020364A1
WO2020020364A1 PCT/CN2019/097984 CN2019097984W WO2020020364A1 WO 2020020364 A1 WO2020020364 A1 WO 2020020364A1 CN 2019097984 W CN2019097984 W CN 2019097984W WO 2020020364 A1 WO2020020364 A1 WO 2020020364A1
Authority
WO
WIPO (PCT)
Prior art keywords
recombinant human
culture
factor viii
coagulation factor
cell
Prior art date
Application number
PCT/CN2019/097984
Other languages
English (en)
French (fr)
Inventor
胡伟伟
尚战强
杜敏
陆建胜
苏贤德
张哲文
谢一龙
费如翔
吴鼎
Original Assignee
正大天晴药业集团南京顺欣制药有限公司
正大天晴药业集团股份有限公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 正大天晴药业集团南京顺欣制药有限公司, 正大天晴药业集团股份有限公司 filed Critical 正大天晴药业集团南京顺欣制药有限公司
Priority to CN201980049311.8A priority Critical patent/CN112469821B/zh
Publication of WO2020020364A1 publication Critical patent/WO2020020364A1/zh
Priority to PH12021550195A priority patent/PH12021550195A1/en
Priority to ZA2021/00861A priority patent/ZA202100861B/en

Links

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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention belongs to the technical field of bioengineering, and relates to a method for preparing recombinant human coagulation factor VIII, and specifically relates to the use of screened human embryonic kidney cells, culturing cells expressing recombinant human coagulation factor ⁇ through a wave-type bioreactor, and a cell culture fluid Recombinant human coagulation factor VIII was isolated and purified.
  • Hemophilia is a genetic bleeding disorder in which congenital coagulation factor defects or mutations cause coagulation dysfunction. According to the corresponding coagulation factors, hemophilia is divided into hemophilia A, B, and C (ie, hemophilia A, B, C), of which hemophilia A accounts for 80% -85%.
  • alternative therapies such as infusion of plasma, factor VIII or IX are effective measures for the treatment of hemophilia.
  • plasma-derived coagulation products are likely to cause blood-borne virus contamination, genetically recombinant coagulation factors have become The main product for treating hemophilia.
  • Recombinant human coagulation factor VIII and natural coagulation factor VIII have similar biochemical, immune, and pharmacological properties, can effectively correct the bleeding tendency of hemophilia patients, and have a good therapeutic effect.
  • Recombinant human coagulation factors currently on the market include Recombinate (Baxter), Advate (Baxter), Kogenate FS (Bayer), ReFacto (pfizer), Xyntha (pfizer), Elocate (Biogen, B domain missing Factor VII and IgG1 Fc domain fusion protein) and Octanate (Octapharma).
  • the human embryonic kidney cell (HEK293) expression system has been used to prepare many therapeutic proteins containing recombinant coagulation factor VIII.
  • the expressed recombinant protein has complete post-translational modification and low immune response, but
  • the commonly used HEK293 cell culture technology has high barriers, severe cell agglomeration, and difficulty in maintaining the cell state.
  • the current large-scale cultivation of recombinant eight-factor cell lines mainly uses traditional stirred reactors, such as Chinese patent application CN103517919, which uses a stirred reactor to suspend by applying large shear stress (that is, a larger stirring rate).
  • WAVE wave bioreactor has high mixing efficiency, sufficient gas-liquid exchange, low foam and low shear force, which avoids the damage of the blade end and air bubbles of the stirred stainless steel reactor to the cells, so the cell state, cell viability and protein activity are all Higher than stirred stainless steel reactor.
  • CN107287265 discloses a method for preparing recombinant human coagulation factor VIII, culturing cells expressing recombinant human coagulation factor VIII through a wave type bioreactor, and separating and purifying the recombinant human coagulation factor VIII from a cell culture solution.
  • One aspect of the present invention provides a method for preparing recombinant human factor VIII, including:
  • a wave-type bioreactor is used to culture cells expressing recombinant human coagulation factor VIII, the cells are in the logarithmic growth phase and the cells are in good condition.
  • the temperature of the reactor culture is 36.5 ° C, the rotation speed is 16-18 rpm, and the angle is 6 -8 degrees, CO 2 concentration 6-10%, air 3-5lpm, 0-48 ⁇ 3 hours dissolved oxygen (DO) set to 40%, 48 ⁇ 3 to 66 ⁇ 3 hours DO set to 25%, 66 From 3 hours to the end of the culture, the DO is set to 40%, the culture time is 66-96 hours, and the culture method is fed-batch culture;
  • the recombinant human coagulation factor VIII is selected from a full-length recombinant human coagulation factor VIII, or a recombinant human coagulation factor ⁇ (BDDrFVIII) with a deleted B domain, and the sequence thereof can be found in SEQ ID ID NO: 1 of patent document CN107287265A. ).
  • the recombinant human factor VIII is selected from recombinant human factor VIII with a deleted B domain.
  • the cells of step (1) are selected human embryonic kidney cells 293 (HEK293) that express recombinant human factor VIII.
  • the cells of step (1) are suspension-adapted human embryonic kidney cells 293 (HEK293) expressing recombinant human factor VIII.
  • the cell of step (1) is human embryonic kidney cell 293 / N27-7, and the deposit number in China Type Culture Collection Center is CCTCC NO: C201828.
  • the above-mentioned human embryonic kidney cells 293 / N27-7 were selected through suspension and acclimation, and have been deposited in the China Type Culture Collection Center on December 28, 2017. The deposit address is Wuhan University, Wuhan, China, and the accession number is CCTCC.NO: C201828.
  • the speed of the wave type bioreactor in step (1) is 16-18 rpm. In some specific embodiments of the present invention, the speed of the wave type bioreactor is 17 rpm.
  • the angle of the wave-type bioreactor in step (1) is 6-8 degrees. In one embodiment of the present invention, the angle of the wave-type bioreactor is 7 degrees.
  • the CO 2 concentration in step (1) is 6-10%, and more preferably 10%.
  • the air in step (1) is set to 3-5 lpm, more preferably 3 lpm.
  • the culture time in step (1) is 66-96 hours, and more preferably 72-78 hours.
  • the medium used in step (1) is not particularly limited, as long as it is suitable for cell growth.
  • the medium used is 4 mmol / L glutamine, 0.02% antifoam C OptiCHO AGT medium with 1g / L Kolliphor P188.
  • the fed-batch culture is supplemented with 200 mmol / L glutamine to a concentration of 4 mmol / L within 24-72 hours of the culture period.
  • the wave type bioreactor of step (1) includes, but is not limited to, WAVE bioreactor (GE Healthcare), AppliFlex (Applikon), BIOSTAT RM (Sartorius Stedim Biotech), Celltainer (Celltainer Biotech), ReadyToProcess WAVE25 ( GE Healthcare), Xuri Cell Expansion System W25 (GE Healthcare), XRS20 (PallLife Sciences). More preferably, the wave type bioreactor of step (1) is selected from the WAVE bioreactor (GE Healthcare). In a specific embodiment of the present invention, the wave type bioreactor of step (1) is selected from a WAVE wave type reactor (GE Healthcare, model WAVE200).
  • the step (2) of harvesting the recombinant human factor VIII is performed in a solution containing Na + .
  • the method for preparing recombinant human factor VIII includes the following steps:
  • Preparation of primary seed liquid a frozen recombinant human coagulation factor VIII working cell bank (amount 1ml) from a liquid nitrogen tank, thawed in a 37 ° C water bath, and transferred to a CD OptiCHO containing about 19ml seed medium AGT (containing 4 mmol / L glutamine, 50 ⁇ g / ml zeocin) in a 125 ml cell culture shake flask was placed in a constant temperature incubator at 36.5 ° C and 10% CO 2 carbon dioxide at 130-170 rpm.
  • AGT containing 4 mmol / L glutamine, 50 ⁇ g / ml zeocin
  • the secondary seed solution was cultured in a WAVE wave reactor, the inoculation ratio was about 1/4, the inoculation density was about 0.75-1.2 ⁇ 10 6 cells / ml, and the culture medium was about CD OptiCHO AGT ( Contains about 4mmol / L glutamine, about 0.02% antifoam C, about 1g / L Kolliphor P188), secondary seed liquid culture parameter settings: culture temperature 36.5 °C, rotation speed 13-16rpm, angle 6 degrees, CO 2 concentration 6- 10%, air about 0.3-0.5 lpm, culture volume is about 8L.
  • the cell density of the secondary seed solution was about 3.0-4.8 ⁇ 10 6 cells / ml, the cells were subcultured and the cells were inoculated in a WAVE200 wave reactor.
  • WAVE200 wave reactor culture the inoculation ratio is about 1 / 4-1 / 3, the culture medium is CD OptiCHO AGT (containing about 4mmol / L glutamine, about 0.02% antifoam C, about 1g / L Kolliphor P188 ), Culture volume is about 100L, fed-batch culture, 200mmol / L glutamine is added to its concentration of 4mmol / L in the 24-72 hours of the culture cycle, harvested in the 72-78 hours of the culture cycle, WAVE wave reactor The main control parameters of cell culture are: culture temperature of 36.5 ° C, rotation speed of 16-18 rpm, angle of 6-8 degrees, CO 2 concentration of 6-10%, and air of 3-5 lpm.
  • the recombinant human coagulation factor ⁇ is harvested from the cell culture solution of step (4).
  • the harvest conditions are NaCl with a final concentration of about 0.5 mol / L, and the conductance is about 40-50 mS / cm at 2-8 ° C. After the solution is mixed Let stand for 45 minutes.
  • the preparation method of the present invention further includes a step of further purifying the recombinant human coagulation factor ⁇ harvested in step (5), wherein the purification step includes: deep filtration, unidirectional flow concentration, S / D virus inactivation, and affinity chromatography. , Anion exchange chromatography, hydrophobic chromatography, nanofiltration and ultrafiltration displacement buffer.
  • the present invention provides a human embryonic kidney cell for preparing recombinant human factor ⁇ , specifically, the human embryonic kidney cell is a human embryonic kidney cell 293 / N27-7, and the human embryonic kidney cell 293 / N27-7 described above. It was screened by suspension and acclimation and has been deposited in the China Type Culture Collection Center on December 28, 2017. The deposit address is Wuhan University, Wuhan, China, and the deposit registration number is CCTCC NO: C201828.
  • Another aspect of the present invention provides the application of human embryonic kidney cells in the preparation of recombinant human coagulation factor VIII.
  • the human embryonic kidney cells are human embryonic kidney cells 293 / N27-7, and the deposit registration number is CCTCC NO: C201828.
  • the application Methods including:
  • the human embryonic kidney cells 293 / N27-7 expressing recombinant human coagulation factor VIII are cultured in a wave type bioreactor, the cells are in the logarithmic growth phase and the cell condition is good, and the temperature of the reactor culture is about 36.5 °C, rotation speed 16-18rpm, angle 6-8 degrees, CO 2 concentration 6-10%, air 3-5lpm, 0-48 ⁇ 3 hours dissolved oxygen (DO) is set to about 40%, 48 ⁇ 3 to 66 ⁇ 3 hours DO is set to about 25%, 66 ⁇ 3 hours to the end of the culture. DO is set to about 40%, the culture time is 66-96 hours, and the culture mode is fed-batch culture;
  • Another aspect of the present invention provides a composition containing recombinant human factor VIII, wherein the recombinant human factor VIII is obtained by any one of the preparation methods of the present invention.
  • fed-batch culture refers to the continuous growth of cells and the continuous formation of products during the reactor culture process, and in the process, as nutrients are consumed, new nutrients are continuously added to the system to make cells grow and metabolize until The product was removed after the entire culture was completed.
  • the characteristic of fed-batch culture is that it can adjust the concentration of nutrients in the culture environment. On the one hand, it can avoid affecting the growth and metabolism of cells and the formation of products when the initial concentration of a certain nutritional component is too high; on the other hand, it can also Prevent certain limiting nutrients from being depleted during the culture process, affecting cell growth and product formation.
  • the WAVE wave bioreactor has high mixing efficiency, sufficient gas-liquid exchange, low foam and low shear force, which avoids the damage to the cells of the blade end and air bubbles of the stirred stainless steel reactor, so the cell state, cell viability, and Protein activity is higher than stirred stainless steel reactor;
  • Figure 1 Cell growth curves of different culture cycles of the WAVE wave bioreactor.
  • FIG. 1 Protein expression of WAVE wave bioreactor in different culture cycles.
  • Figure 3 Flow chart of purification of recombinant human factor VIII.
  • Cells Human embryonic kidney cells 293 / N27-7, deposited under CCTCC NO: C201828.
  • Preparation of first-stage seed liquid A frozen recombinant human coagulation factor ⁇ working cell bank cell (human embryonic kidney cell 293 / N27-7, self-made, 1ml volume) was taken from a liquid nitrogen tank, thawed in a 37 ° C water bath, and transferred Into a 125 ml cell culture shake flask containing 20 ml of seed medium CD OptiCHO AGT (containing 4 mmol / L glutamine (Life technologies), 50 ⁇ g / ml zeocin (Invotrogen), Life technologies), place at 37 ° C, 6 Incubate at 110-130 rpm in a -10% CO 2 carbon dioxide constant temperature incubator.
  • OptiCHO AGT containing 4 mmol / L glutamine (Life technologies), 50 ⁇ g / ml zeocin (Invotrogen), Life technologies
  • Preparation of secondary seed liquid WAVE wave reactor culture secondary seed liquid, the inoculation ratio is 1 / 4-1 / 3, the inoculation density is 0.6-1.0 ⁇ 10 6 cells / ml, and the culture medium is CD OptiCHO AGT (containing 4mmol / L glutamine (Life technologies), 0.02% antifoam C (SIGMA), 1g / L Kolliphor P188 (sigma), Life technologies), secondary seed liquid culture parameter settings: culture temperature 36.5 ° C, rotation speed 13 -16 rpm, angle 5-8 degrees, CO 2 concentration 6-10%, air 0.3-0.5 lpm.
  • the cells of the secondary seed solution are in the logarithmic growth phase and the cells are in good condition, they are inoculated into a WAVE wave reactor and a 30L stirred stainless steel reactor (Applikon company, model EZ-CONTROL).
  • inoculation ratio is 1 / 4-1 / 3
  • inoculation density is 0.6-1.0 ⁇ 10 6 cells / ml
  • culture medium is CD OptiCHO AGT (containing 4mmol / L glutamine (Life technologies Company), 0.02% antifoam C (SIGMA company), 1g / L Kolliphor P188 (sigma company, Life technologies company), fed-batch culture, 200mmol / L glutamine was added to it at 24-72 hours The concentration was 2-4 mmol / L, and a 200 g / L glucose (Sigma) solution was added to a glucose content of 2-4 g / L, and harvested at the 72nd hour of the culture cycle.
  • WAVE wave reactor cell culture main control parameter settings culture temperature 36.5 °C, rotation speed 16-18rpm, angle 6-8 degrees, CO 2 concentration 6-10% and air 3-5lpm.
  • the inoculation ratio is 1 / 4-1 / 3
  • the inoculation density is 0.6-1.0 ⁇ 10 6 cells / ml
  • the culture medium is CD OptiCHO AGT (containing 4mmol / L glutamine (Life technologies), 0.02% antifoam C (SIGMA), 1 g / L Kolliphor P188 (sigma), Life technologies), fed-batch culture, 200 mmol / L glutamine was added to the culture medium from 24-72 hours to Its concentration is 2-4 mmol / L, and a 200 g / L glucose (Sigma) solution is added to a glucose content of 2-4 g / L, and harvested at the 96th hour of the culture cycle.
  • samples were taken every 24 hours during the culture to measure cell viability (trypan blue method) and protein activity (one-stage method).
  • the main control parameters of the stirring stainless steel reactor are set as follows: culture temperature 37 ° C; stirring speed 100-130rpm; DO is automatically maintained at 40% by adding O 2 ; pH is adjusted by adding CO 2 and 0.5 mol / L sodium hydroxide solution.
  • the self-control was maintained at 7.00 ⁇ 0.20; the air was continuously ventilated with a ventilation volume of 500ml / min.
  • Table 1 shows the process of WAVE wave bioreactor and agitated stainless steel reactor and the culture effect.
  • WAVE wave bioreactor has high mixing efficiency, sufficient gas-liquid exchange, low foam and low shear force, which avoids the damage of the blade end and air bubbles of the stirred stainless steel reactor to the cells, so the cell state, cell viability and protein activity are all Higher than stirred stainless steel reactor.
  • WAVE wave bioreactor is used to culture cells at lower speeds (for example, 16 rpm to 18 rpm), cell viability and protein expression have also been unexpectedly improved.
  • Example 2 To the cell suspension (WAVE wave reactor culture) obtained in Example 1 was added a buffer solution containing sodium chloride and calcium chloride (10 mmol / L Hepes, 5 mmol / L calcium chloride dihydrate, 4 mol / L chloride Sodium, pH 7.2), so that the final concentration of sodium chloride is about 0.5 mol / L, the solution is mixed at 2-8 ° C and left for about 45 minutes, and the cells are removed by deep filtration and then filtered (0.22 ⁇ m) to remove any residual cell debris and particulate matter.
  • a buffer solution containing sodium chloride and calcium chloride (10 mmol / L Hepes, 5 mmol / L calcium chloride dihydrate, 4 mol / L chloride Sodium, pH 7.2), so that the final concentration of sodium chloride is about 0.5 mol / L, the solution is mixed at 2-8 ° C and left for about 45 minutes, and the cells are removed by deep filtration and then filtered (0.22 ⁇ m) to remove any residual cell
  • the cell clarified solution was concentrated in one direction, and concentrated about 3-4 times.
  • the concentrated solution was filtered through a 0.2 ⁇ m filter.
  • Virus inactivation was performed on the clarified cell harvest obtained in step (2) using 0.3% tributyl phosphate (TNBP) (v / v) and 1% Triton X-100. Virus inactivation was performed at 20-25 ° C for 45 minutes.
  • TNBP tributyl phosphate
  • the cell harvest solution obtained in step (3) was purified using a Quik Scale 140/550 chromatography column (column bed height 3.5 cm, diameter 14 cm, volume 540 ml, and the filler was VIIISelect gel (GE Healthcare)) for purification. It was used in the affinity layer.
  • the analysis buffers and steps are shown in Tables 2 and 3.
  • Q-Sepharose High Performance Packing (GE Healthcare) was packed into a Quik Scale 140/550 chromatography column so that the bed height was 3.5 cm, the diameter was 14 cm, and the volume was about 540 ml.
  • the eluate of step (4) is diluted about 10 times (2-8 ° C), so that the factor VIII can bind to the gel.
  • the buffers and chromatography steps used for anion chromatography are shown in Tables 4 and 5.
  • a Butyl Sepharose High Performance Packing (GE Healthcare) was packed into a chromatography column (SAC-Bio-100-500G-10) so that the bed height was 3.5 cm, the diameter was 10 cm, and the volume was about 110 ml.
  • Use the conductivity adjustment buffer to dilute the eluate in step (5) so that the conductivity value is 62 mS / cm (2-8 ° C). Buffers and chromatography steps for hydrophobic column chromatography are shown in Tables 6 and 7.
  • the filtrate obtained in step (7) was subjected to buffer replacement with a 10 kDa cellulose ultrafiltration membrane (Millipore).
  • the filtrate was concentrated to 0.1 to 0.25 mg / ml, and then the solution was changed.
  • the TMP was controlled to 0.6 bar during the exchange process, and the replacement buffer (3 mg / ml L-histidine, 0.7 mol / L sodium chloride, 5mmol / L calcium chloride, pH7.0) replace about 7 sample volumes, recover the sample, and finally add sucrose at a final concentration of 6mg / ml, control the final protein concentration to be not less than 0.1mg / ml, and sterilize with 0.2 ⁇ m
  • the original solution was filtered and stored at no higher than -70 ° C.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种制备重组人凝血因子Ⅷ的方法，包括利用波浪式生物反应器培养表达重组人凝血因子Ⅷ的人胚肾细胞293/N27-7以及从细胞培养液中分离并纯化重组人凝血Ⅷ因子。波浪生物反应器混合效率高，气液交换充分，泡沫少且剪切力低，避免了搅拌式不锈钢反应器浆叶端和气泡对细胞的伤害。

Description

一种制备重组人凝血因子Ⅷ的方法 技术领域
本发明属于生物工程技术领域，涉及一种制备重组人凝血因子Ⅷ的方法，具体涉及利用筛选的人胚肾细胞，通过波浪式生物反应器培养表达重组人凝血因子Ⅷ的细胞以及从细胞培养液中分离并纯化重组人凝血Ⅷ因子。
背景技术
血友病为一种先天性凝血因子基因缺陷或突变导致凝血功能障碍的遗传出血性疾病。根据相应的凝血因子将血友病分为甲型、乙型、丙型血友病(即血友病A、B、C)，其中血友病A占80％-85％。目前替代疗法如输注血浆、凝血因子Ⅷ或Ⅸ是治疗血友病有效措施，但由于血浆来源的凝血制品极易造成血源性病毒污染，基因重组类凝血因子因其安全性及有效性成为治疗血友病的主要产品。重组人凝血因子Ⅷ与天然凝血因子Ⅷ具有相似的生化、免疫及药理学特性，能有效纠正血友病患者的出血倾向，具有良好的治疗效果。目前上市的重组人凝血因子Ⅷ有Recombinate(Baxter公司)、Advate(Baxter公司)、Kogenate FS(Bayer公司)、ReFacto(pfizer公司)、Xyntha(pfizer公司)、Eloctate(Biogen公司，B结构域缺失的因子Ⅷ与IgG1Fc结构域的融合蛋白)以及Octanate(Octapharma公司)等。
作为哺乳动物表达细胞的一种，人胚肾细胞(HEK293)表达系统已经被用于制备许多包含重组凝血八因子的治疗蛋白，其表达的重组蛋白的蛋白翻译后修饰完全且免疫反应低，但常用的HEK293细胞培养工艺技术壁垒高，细胞结团严重，细胞状态难以维持。此外，目前重组八因子的细胞株的规模化培养主要采用的是传统搅拌式反应器，如中国专利申请CN103517919采用搅拌式反应器通过施加较大的剪切应力(即较大的搅拌速率)悬浮培养HEK293细胞，培养产生的重组人凝血八因子最高活性仅达到16IU/ml；中国专利申请CN102776260通过在培养过程中控制细胞培养液中血管性血友病 因子与人凝血八因子的活性比为1-10:1，获得的重组人凝血八因子的表达量最高也仅达到10-20国际单位/天/10 6细胞。因此，仍需要高效培养重组人凝血因子Ⅷ的方法。
WAVE波浪生物反应器混合效率高，气液交换充分，泡沫少且剪切力低，避免了搅拌式不锈钢反应器浆叶端和气泡对细胞的伤害，因此细胞状态、细胞活率及蛋白活性均高于搅拌式不锈钢反应器。CN107287265公开了制备重组人凝血因子Ⅷ的方法，通过波浪式生物反应器培养表达重组人凝血因子Ⅷ的细胞以及从细胞培养液中分离并纯化重组人凝血Ⅷ因子。
发明内容
本发明一方面提供一种制备重组人凝血因子Ⅷ的方法，包括：
(1)采用波浪式生物反应器培养表达重组人凝血因子Ⅷ的细胞，所述细胞处于对数生长期且细胞状态良好，所述反应器培养的温度为36.5℃，转速16-18rpm，角度6-8度，CO 2浓度6-10％，空气3-5lpm，0-48±3小时溶解氧(DO)设定为40％、48±3至66±3小时DO设定为25％、66±3小时至培养结束DO设定为40％，培养时间为66-96小时，培养方式为流加培养；
(2)从步骤(1)的细胞培养液中收获重组人凝血因子Ⅷ。
在一些方案中，所述重组人凝血因子Ⅷ选自全长的重组人凝血因子Ⅷ，或者B结构域缺失的重组人凝血因子Ⅷ(BDDrFⅧ，其序列可参见专利文献CN107287265A的SEQ ID NO:1)。在本发明的一个优选实施方案中，所述重组人凝血因子Ⅷ选自B结构域缺失的重组人凝血因子Ⅷ。
在一些方案中，步骤(1)的细胞为经筛选的表达重组人凝血因子Ⅷ的人胚肾细胞293(HEK293)。
在一些方案中，步骤(1)的细胞为经悬浮驯化的表达重组人凝血因子Ⅷ的人胚肾细胞293(HEK293)。
在一些方案中，步骤(1)的细胞为人胚肾细胞293/N27-7，在中国典型培养物保藏中心的保藏号为CCTCC NO:C201828。上述人胚肾细胞 293/N27-7经悬浮驯化筛选得来，已于2017年12月28日保藏于中国典型培养物保藏中心，保藏地址为中国武汉市武汉大学，保藏登记号为CCTCC NO:C201828。
在一些方案中，步骤(1)中波浪式生物反应器的转速为16-18rpm。在本发明的一些具体实施方案中，所述波浪式生物反应器的转速为17rpm。
在一些方案中，步骤(1)中波浪式生物反应器的角度为6-8度。在本发明的一个实施方案中，所述波浪式生物反应器的角度为7度。
在一些方案中，步骤(1)中CO 2浓度为6-10％，更优选为10％。
在一些方案中，步骤(1)中空气设置为3-5lpm，更优选为3lpm。
在一些方案中，其中步骤(1)中培养时间为66-96小时，更优选为72-78小时。
其中步骤(1)中所使用的培养基不特别限制，只要适合细胞生长即可，在本发明的一个优选实施方案中，所使用的培养基为含4mmol/L谷氨酰胺、0.02％antifoam C、1g/L Kolliphor P188的CD OptiCHO AGT培养基。
在一些方案中，其中步骤(1)中流加培养是在培养周期的24-72小时内补加200mmol/L谷氨酰胺至其浓度为4mmol/L。
在一些方案中，步骤(1)的波浪式生物反应器包括但不限于WAVE生物反应器(GE Healthcare)、AppliFlex(Applikon)、BIOSTAT RM(Sartorius Stedim Biotech)、Celltainer(Celltainer Biotech)、ReadyToProcess WAVE25(GE Healthcare)、Xuri细胞扩增系统W25(GE Healthcare)、XRS20(PallLife Sciences)。更优选的，步骤(1)的波浪式生物反应器选自WAVE生物反应器(GE Healthcare)。在本发明的一个具体实施方案中，步骤(1)的波浪式生物反应器选自WAVE波浪式反应器(GE Healthcare，型号WAVE200)。
在一些方案中，步骤(2)收获重组人凝血因子Ⅷ是在含有Na +的溶液中进行的。
在本发明的一个具体实施方案中，重组人凝血因子Ⅷ的制备方法包括以 下步骤：
(1)一级种子液制备：从液氮罐中取冻存的重组人凝血因子Ⅷ工作细胞库细胞一支(装量1ml)，37℃水浴解冻，转移至含约19ml种子培养基CD OptiCHO AGT(含4mmol/L谷氨酰胺、50μg/ml zeocin)的125ml细胞培养摇瓶中，放置于36.5℃、10％CO 2二氧化碳恒温培养箱中130-170rpm培养。细胞密度约3.0-4.8×10 6cells/ml时传代培养，传代密度约为0.8-1.2×10 6cells/ml，4-5次传代后将一级种子液接种于WAVE波浪式反应器(GE Healthcare，型号WAVE25)。
(2)二级种子液制备：WAVE波浪式反应器培养二级种子液，接种比例为约1/4，接种密度为约0.75-1.2×10 6cells/ml，培养基为约CD OptiCHO AGT(含约4mmol/L谷氨酰胺、约0.02％antifoam C、约1g/L Kolliphor P188)，二级种子液培养参数设置：培养温度36.5℃，转速13-16rpm，角度6度，CO 2浓度6-10％，空气约0.3-0.5lpm，培养体积为约8L。二级种子液的细胞密度处于约3.0-4.8×10 6cells/ml时传代培养且细胞状态良好时接种于WAVE200波浪式反应器。
(3)三级种子液制备：WAVE200波浪式反应器培养，接种比例为约1/4，接种密度为约0.75-1.2×10 6cells/ml，培养基为CD OptiCHO AGT(含约4mmol/L谷氨酰胺、约0.02％antifoam C、约1g/L Kolliphor P188)，三级种子液培养参数设置：培养温度约36.5℃，转速12-14rpm，角度4-6度，CO 2浓度6-10％，空气约1-2lpm，培养体积为30-35L，优选约32L。三级种子液的细胞密度处于约3.0-4.6×10 6cells/ml时传代培养且细胞状态良好时，于WAVE200波浪式反应器扩种进行终极培养。
(4)WAVE200波浪式反应器培养：接种比例为约1/4-1/3，培养基为CD OptiCHO AGT(含约4mmol/L谷氨酰胺、约0.02％antifoam C、约1g/L Kolliphor P188)，培养体积为约100L，流加培养，在培养周期的第24-72小时补加200mmol/L谷氨酰胺至其浓度为4mmol/L，培养周期第72-78小时收 获，WAVE波浪反应器细胞培养主控参数设定：培养温度36.5℃，转速16-18rpm，角度6-8度，CO 2浓度6-10％以及空气3-5lpm。
(5)从步骤(4)的细胞培养液中收获重组人凝血因子Ⅷ，收获条件为终浓度约0.5mol/L的NaCl，2-8℃下电导为约40-50mS/cm，溶液混合后静置45分钟。
本发明的制备方法进一步包括将步骤(5)收获的重组人凝血因子Ⅷ进一步纯化的步骤，其中所述纯化步骤包括：深层过滤、单向流浓缩、S/D病毒灭活、亲和层析、阴离子交换层析、疏水层析、纳滤和超滤置换缓冲液。
本发明再一方面提供一种用于制备重组人凝血因子Ⅷ的人胚肾细胞，具体的，人胚肾细胞是人胚肾细胞293/N27-7，上述人胚肾细胞293/N27-7经悬浮驯化筛选得来，已于2017年12月28日保藏于中国典型培养物保藏中心，保藏地址为中国武汉市武汉大学，保藏登记号为CCTCC NO：C201828。
本发明再一方面提供人胚肾细胞在制备重组人凝血因子Ⅷ中的应用，所述人胚肾细胞是人胚肾细胞293/N27-7，保藏登记号为CCTCC NO：C201828，所述应用方法，包括：
(1)采用波浪式生物反应器培养表达重组人凝血因子Ⅷ的人胚肾细胞293/N27-7，所述细胞处于对数生长期且细胞状态良好，所述反应器培养的温度为约36.5℃，转速16-18rpm，角度6-8度，CO 2浓度6-10％，空气3-5lpm，0-48±3小时溶解氧(DO)设定为约40％、48±3至66±3小时DO设定为约25％、66±3小时至培养结束DO设定为约40％，培养时间为66-96小时，培养方式为流加培养；
(2)从步骤(1)的细胞培养液中收获重组人凝血因子Ⅷ。
本发明再一方面提供一种含有重组人凝血因子Ⅷ的组合物，其中所述重组人凝血因子Ⅷ由本发明的任一项制备方法获得。
术语“流加培养”指反应器培养过程中细胞不断生长及产物不断形成，而在此过程中随着营养物质的消耗，不断地向系统中补充新的营养成分，使 细胞进一步生长代谢，直到整个培养结束后取出产物。流加培养的特点就是能够调节培养环境中营养物质的浓度，一方面，它可以避免在某种营养成分的初始浓度过高时影响细胞的生长代谢以及产物的形成；另一方面，它还能防止某些限制性营养成分在培养过程中被耗尽而影响细胞的生长和产物的形成。
本发明的制备方法具有如下有益效果：
(一)经悬浮驯化筛选的人胚肾细胞293/N27-7与常用人胚肾细胞293相比，降低了培养工艺技术壁垒，细胞悬浮性提高，降低了细胞结团及贴壁现象，细胞状态易于维持，利于在波浪式生物反应器中培养，相对CN107287265A记载的方法，本发明的细胞株在生产规模扩大至100L和/或培养周期缩短至72小时时仍保持较高蛋白表达量；
(二)WAVE波浪生物反应器混合效率高，气液交换充分，泡沫少且剪切力低，避免了搅拌式不锈钢反应器浆叶端和气泡对细胞的伤害，因此细胞状态、细胞活率及蛋白活性均高于搅拌式不锈钢反应器；
(三)WAVE波浪生物反应器采用较低的转速(例如16rpm-18rpm)培养细胞时，细胞活率和蛋白表达量也获得了出乎意料的提高。另外，进一步的纯化有效去除了宿主蛋白、DNA、亲和配基、聚合物、降解物以及细胞培养过程中带入的污染物，大大提高了重组人凝血因子Ⅷ的产量及蛋白活性。
附图说明
图1：WAVE波浪生物反应器不同培养周期的细胞生长曲线图。
图2：WAVE波浪生物反应器不同培养周期的蛋白表达量图。
图3：重组人凝血因子Ⅷ的纯化流程图。
具体实施方式
下面结合具体实施例对本发明进行进一步的描述，然而，本发明中的这些和其他实施例仅用于阐明而不限制本发明的范围。本领域技术人员应该理 解，对本发明技术特征所作的等同替换，或相应的改进，仍属于本发明的保护范围之内。
细胞：人胚肾细胞293/N27-7，保藏登记号为CCTCC NO：C201828。
细胞来源：正大天晴药业集团股份有限公司，自制。
实施例1考察WAVE波浪生物反应器与搅拌式不锈钢反应器细胞培养效果
一级种子液制备：从液氮罐中取冻存的重组人凝血因子Ⅷ工作细胞库细胞一支(人胚肾细胞293/N27-7，自制，装量1ml)，37℃水浴解冻，转移至含20ml种子培养基CD OptiCHO AGT(含4mmol/L谷氨酰胺(Life technologies公司)、50μg/ml zeocin(Invotrogen公司)，Life technologies公司)的125ml细胞培养摇瓶中，放置于37℃、6-10％CO 2二氧化碳恒温培养箱中110-130rpm培养。每天观察细胞状态，取样进行细胞计数和检测细胞活率(台盼蓝法)，细胞密度约3.0-4.0×10 6cells/ml时传代培养，传代密度约为0.6-1.0×10 6cells/ml，3-4次传代后将一级种子液接种于WAVE波浪式反应器(GE Healthcare，型号20/50EHT)。
二级种子液制备：WAVE波浪式反应器培养二级种子液，接种比例为1/4-1/3，接种密度为0.6-1.0×10 6cells/ml，培养基为CD OptiCHO AGT(含4mmol/L谷氨酰胺(Life technologies公司)、0.02％antifoam C(SIGMA公司)、1g/L Kolliphor P188(sigma公司)，Life technologies公司)，二级种子液培养参数设置：培养温度36.5℃，转速13-16rpm，角度5-8度，CO 2浓度6-10％，空气0.3-0.5lpm。二级种子液的细胞处于对数生长期且细胞状态良好时接种于WAVE波浪式反应器和30L搅拌式不锈钢反应器(Applikon公司，型号EZ-CONTROL)。
WAVE波浪式反应器细胞培养工艺：接种比例为1/4-1/3，接种密度为0.6-1.0×10 6cells/ml，培养基为CD OptiCHO AGT(含4mmol/L谷氨酰胺(Life technologies公司)、0.02％antifoam C(SIGMA公司)、1g/L Kolliphor P188(sigma公司)，Life technologies公司)，流加培养，在培养周期的第24-72 小时补加200mmol/L谷氨酰胺至其浓度为2-4mmol/L，补加200g/L葡萄糖(sigma公司)溶液至葡萄糖含量为2-4g/L，培养周期第72小时收获。培养过程中每24小时取样进行细胞计数检测细胞活率(台盼蓝法)，以及检测蛋白活性(一期法)。WAVE波浪反应器细胞培养主控参数设定：培养温度36.5℃，转速16-18rpm，角度6-8度，CO 2浓度6-10％以及空气3-5lpm。
WAVE波浪生物反应器不同培养周期的细胞生长曲线图如图1所示；WAVE波浪生物反应器不同培养周期的蛋白表达量如图2所示。
30L搅拌式不锈钢反应器细胞培养工艺：接种比例为1/4-1/3，接种密度为0.6-1.0×10 6cells/ml，培养基为CD OptiCHO AGT(含4mmol/L谷氨酰胺(Life technologies公司)、0.02％antifoam C(SIGMA公司)、1g/L Kolliphor P188(sigma公司)，Life technologies公司)，流加培养，在培养周期的第24-72小时补加200mmol/L谷氨酰胺至其浓度为2-4mmol/L，补加200g/L葡萄糖(sigma公司)溶液至葡萄糖含量为2-4g/L，培养周期第96小时收获。培养过程中每24小时取样进行细胞计数检测细胞活率(台盼蓝法)，以及检测蛋白活性(一期法)。搅拌式不锈钢反应器主控参数设定：培养温度37℃；搅拌转速100-130rpm；DO通过补加O 2自控维持在40％；pH值通过补加CO 2及0.5mol/L氢氧化钠溶液自控维持在7.00±0.20；空气持续通气，通气量为500ml/min。
WAVE波浪生物反应器与搅拌式不锈钢反应器工艺过程及培养效果对比如表1所示。
表1：WAVE波浪生物反应器与搅拌式不锈钢反应器工艺过程及培养效果对比
Figure PCTCN2019097984-appb-000001
WAVE波浪生物反应器混合效率高，气液交换充分，泡沫少且剪切力低，避免了搅拌式不锈钢反应器浆叶端和气泡对细胞的伤害，因此细胞状态、细胞活率及蛋白活性均高于搅拌式不锈钢反应器。此外，WAVE波浪生物反应器采用较低的转速(例如16rpm-18rpm)培养细胞时，细胞活率和蛋白表达量也获得了出乎意料的提高。
实施例2重组人凝血因子Ⅷ的纯化
(1)收获细胞培养液
向实施例1获得的细胞悬液(WAVE波浪式反应器培养)中加入含有氯化钠和氯化钙的缓冲液(10mmol/L Hepes，5mmol/L二水氯化钙，4mol/L氯 化钠，pH7.2)，使氯化钠的终浓度约为0.5mol/L，在2-8℃下，将溶液混合后静置约45分钟，通过深层过滤将细胞移除之后进行过滤(0.22μm)以除去任何残留的细胞碎片和颗粒物。
(2)单向流浓缩
将细胞澄清液进行单向流浓缩，浓缩约3-4倍，浓缩液经0.2μm过滤器过滤。
(3)S/D病毒灭活
使用0.3％的磷酸三丁脂(TNBP)(v/v)和1％的Triton X-100对步骤(2)获得的澄清细胞收获液进行病毒灭活。在20-25℃下进行病毒灭活45分钟。
(4)亲和层析
将步骤(3)获得的细胞收获液使用Quik Scale140/550层析柱(柱床高度3.5cm，直径14cm，体积约540ml，填料为ⅧSelect凝胶(GE Healthcare公司)进行纯化。用于亲和层析的缓冲液和步骤如表2和表3所示。
表2：用于亲和层析的缓冲液
Figure PCTCN2019097984-appb-000002
表3：亲和层析步骤
步骤 缓冲液 柱体积或时间
水洗 WFI 3CV
平衡 平衡缓冲液 5CV
上样 - -
上样后平衡 平衡缓冲液 6CV
淋洗 淋洗缓冲液 10CV
再平衡 平衡缓冲液 6CV
洗脱 洗脱缓冲液 4CV(出峰后开始收集)
平衡 平衡缓冲液 6CV
清洁1 清洁1缓冲液 6CV
清洁2 清洁2缓冲液 60分钟
水洗 去离子水 --
保存 20％乙醇 3CV
(5)阴离子层析
将Q-Sepharose High Performance填料(GE Healthcare公司)装填至Quik Scale 140/550层析柱中，使柱床高度为3.5cm，直径为14cm，体积约为540ml。
对步骤(4)的洗脱液进行约10倍稀释(2-8℃)，从而使凝血因子Ⅷ能与凝胶结合。用于阴离子层析的缓冲液和层析步骤如表4和表5所示。
表4：用于阴离子层析的缓冲液
Figure PCTCN2019097984-appb-000003
表5：阴离子柱层析步骤
步骤 缓冲液 柱体积或时间
CIP 0.5mol/LNaOH 30分钟
平衡 平衡缓冲液 6CV
上样 - -
平衡 平衡缓冲液 6CV
再平衡 清洗缓冲液 6CV
洗脱 洗脱缓冲液 整峰收集
清洗1 清洗1缓冲液 60分钟
平衡 平衡缓冲液 6CV
清洗2 清洗2缓冲液 60分钟
水洗 WFI --
保存 20％乙醇 3CV
(6)疏水柱层析
将Butyl Sepharose High Performance填料(GE Healthcare公司)装填至层析柱(SAC-Bio-100-500G-10)中，使柱床高度为3.5cm，直径为10cm，体积约为110ml。使用电导调节缓冲液稀释步骤(5)的洗脱液使其电导值为62mS/cm(2-8℃)。用于疏水柱层析的缓冲液和层析步骤如表6和表7所示。
表6：用于疏水层析的缓冲液
Figure PCTCN2019097984-appb-000004
表7：疏水层析步骤
步骤 缓冲液 柱体积或时间
CIP 0.5mol/LNaOH 30分钟
活化 再生缓冲液 4CV
平衡 平衡缓冲液 3CV
上样 - 流穿模式，整峰收集
平衡 平衡缓冲液 --
再生 再生缓冲液 3CV
水洗 WFI 3CV
清洁1 清洁1缓冲液 60分钟
水洗 WFI 6CV
清洁2 清洁2缓冲液 60
水洗 WFI --
保存 20％乙醇 3CV
(7)纳滤(除病毒过滤)
使用平衡缓冲液(10mmol/L Hepes，5mmol/L二水氯化钙，0.7mol/L氯化钠，pH7.2)平衡纳滤膜(PlanovaBioEx，Asahi-Kasei公司)，过滤步骤(6)获得的的疏水流穿液以除去无包膜病毒。
(8)超滤置换缓冲液
通过10kDa纤维素超滤膜(Millipore公司)对步骤(7)所得滤出液进行缓冲液置换。先对滤出液浓缩至0.1～0.25mg/ml，然后进行换液，换液过程控制TMP为0.6bar,用置换缓冲液(3mg/ml L-组氨酸，0.7mol/L氯化钠，5mmol/L氯化钙，pH7.0)置换约7个样品体积，对样品进行回收，最后加入终浓度为6mg/ml的蔗糖，控制最终蛋白浓度不小于0.1mg/ml,用0.2μm减菌过滤获到原液，并于不高于-70℃保存。

Claims (10)

  1. 一种制备重组人凝血因子Ⅷ的方法，包括：
    (1)采用波浪式生物反应器培养表达重组人凝血因子Ⅷ的细胞，所述细胞处于对数生长期，培养方式为流加培养；
    (2)从步骤(1)的细胞培养液中收获重组人凝血因子Ⅷ。
  2. 根据权利要求1的制备方法，其中步骤(1)所述细胞为经悬浮驯化的表达重组人凝血因子Ⅷ的人胚肾细胞293(HEK293)。
  3. 根据权利要求1-2任一项的制备方法，其中步骤(1)所述细胞为人胚肾细胞293/N27-7，保藏号为CCTCC NO:C201828。
  4. 根据权利要求1-3任一项的制备方法，其中所述重组人凝血因子Ⅷ选自全长的重组人凝血因子Ⅷ，或者B结构域缺失的重组人凝血因子Ⅷ。
  5. 根据权利要求1-4任一项的制备方法，其中步骤(1)中波浪式生物反应器的转速为16-18rpm，更优选为17rpm。
  6. 根据权利要求1-5任一项的制备方法，其中步骤(1)中空气设置为3-5lpm，更优选为3lpm。
  7. 根据权利要求1-6任一项的制备方法，其中步骤(1)中培养时间为66-96小时，优选为72-78小时。
  8. 一种含有重组人凝血因子Ⅷ的组合物，其中所述重组人凝血因子Ⅷ由权利要求1-7中任一项的制备方法获得。
  9. 一种用于制备重组人凝血因子Ⅷ的人胚肾细胞，所述人胚肾细胞是人胚肾细胞293/N27-7，保藏登记号为CCTCC NO:C201828。
  10. 权利要求9所述的人胚肾细胞在制备重组人凝血因子Ⅷ中的应用，所述应用方法包括：
    (1)采用波浪式生物反应器培养表达重组人凝血因子Ⅷ的人胚肾细胞293/N27-7，培养方式为流加培养；
    (2)从步骤(1)的细胞培养液中收获重组人凝血因子Ⅷ。
PCT/CN2019/097984 2018-07-26 2019-07-26 一种制备重组人凝血因子ⅷ的方法 WO2020020364A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN201980049311.8A CN112469821B (zh) 2018-07-26 2019-07-26 一种制备重组人凝血因子ⅷ的方法
PH12021550195A PH12021550195A1 (en) 2018-07-26 2021-01-26 Method for preparing recombinant human coagulation factor viii
ZA2021/00861A ZA202100861B (en) 2018-07-26 2021-02-08 Method for preparing recombinant human coagulation factor viii

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810859618 2018-07-26
CN201810859618.1 2018-07-26

Publications (1)

Publication Number Publication Date
WO2020020364A1 true WO2020020364A1 (zh) 2020-01-30

Family

ID=69181337

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/097984 WO2020020364A1 (zh) 2018-07-26 2019-07-26 一种制备重组人凝血因子ⅷ的方法

Country Status (4)

Country Link
CN (1) CN112469821B (zh)
PH (1) PH12021550195A1 (zh)
WO (1) WO2020020364A1 (zh)
ZA (1) ZA202100861B (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115819608A (zh) * 2022-10-08 2023-03-21 盛禾(中国)生物制药有限公司 一种纯化融合蛋白的方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070968A2 (en) * 2000-03-22 2001-09-27 Octagene Gmbh Production of recombinant blood clotting factors in human cell lines
CN102311495A (zh) * 2010-06-30 2012-01-11 上海同科生物科技有限公司 新型重组人凝血因子ⅷ及其生产方法
CN102321668A (zh) * 2011-07-06 2012-01-18 中国人民解放军军事医学科学院野战输血研究所 一种表达重组人凝血因子ⅶ的方法及其专用载体
CN103555759A (zh) * 2013-07-05 2014-02-05 兰诺生物技术无锡有限公司 利用家兔乳腺生物反应器制备第八凝血因子的方法
CN107287265A (zh) * 2016-03-31 2017-10-24 正大天晴药业集团南京顺欣制药有限公司 一种制备重组人凝血因子ⅷ的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2600886C2 (ru) * 2011-05-13 2016-10-27 Октафарма Аг Способ повышения продуктивности эукариотических клеток в продуцировании рекомбинантного fviii
CN103215308B (zh) * 2013-02-01 2015-12-23 中国科学院苏州生物医学工程技术研究所 表达重组人fviii的整合质粒、细胞株及其构建方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070968A2 (en) * 2000-03-22 2001-09-27 Octagene Gmbh Production of recombinant blood clotting factors in human cell lines
CN102311495A (zh) * 2010-06-30 2012-01-11 上海同科生物科技有限公司 新型重组人凝血因子ⅷ及其生产方法
CN102321668A (zh) * 2011-07-06 2012-01-18 中国人民解放军军事医学科学院野战输血研究所 一种表达重组人凝血因子ⅶ的方法及其专用载体
CN103555759A (zh) * 2013-07-05 2014-02-05 兰诺生物技术无锡有限公司 利用家兔乳腺生物反应器制备第八凝血因子的方法
CN107287265A (zh) * 2016-03-31 2017-10-24 正大天晴药业集团南京顺欣制药有限公司 一种制备重组人凝血因子ⅷ的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KOU, PENG ET AL: "Selection of Nanomembrane in Manufacturing Process of Recombinant Human Coagulation Factor VIH", CHINESE JOURNAL OF BIOLOGICALS, vol. 30, no. 2, 28 February 2017 (2017-02-28), pages 196 - 200 *
WANG, ZHUO ET AL.: "Expression of Recombinant Human Coagulation Factor VII in Flp-In-CHO Cells andIdentification of Expressed Product", CHINESE JOURNAL OF BIOLOGICALS, vol. 23, no. 3, 20 March 2010 (2010-03-20), pages 248 - 251 *
WINGE, STEFAN ET AL: "Development, upscaling and validation of the purification process forhuman-cl rhFVIII (Nuwiq), a new generation recombinant factor VIIIproduced in a human cell-line", PROTEIN EXPRESSION AND PURIFICATION, vol. 115, 30 November 2015 (2015-11-30), pages 165 - 175, XP055681863, ISSN: 1046-5928 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115819608A (zh) * 2022-10-08 2023-03-21 盛禾(中国)生物制药有限公司 一种纯化融合蛋白的方法

Also Published As

Publication number Publication date
ZA202100861B (en) 2022-05-25
CN112469821A (zh) 2021-03-09
CN112469821B (zh) 2021-12-14
PH12021550195A1 (en) 2022-02-21

Similar Documents

Publication Publication Date Title
CA2861270C (en) Perfusion bioreactor systems comprising a cell aggregate trap and methods of operating the same
KR20170076679A (ko) 파종 밀도 한계를 증가시키고 원하는 팽창 시간을 감소시키는 접착 세포 생물반응기에의 비접착세포의 파종
EP3487609A1 (en) Alternating tangential flow rapid harvesting
JP6282467B2 (ja) タンパク質を生産するための方法
CN111575244B (zh) 一种低Vero细胞残留DNA狂犬病疫苗原液的制备方法
DK2356247T3 (en) Process for the preparation of serum-free insulin-free factor vii.
WO2020020364A1 (zh) 一种制备重组人凝血因子ⅷ的方法
JP5096920B2 (ja) ウイルス物質の製造方法
CN107287265B (zh) 一种制备重组人凝血因子ⅷ的方法
CN109321519B (zh) Cho-k1悬浮驯化培养基及驯化方法
US20210268405A1 (en) Method for producing recombinant adenovirus
CN112094814B (zh) 一种通过灌流培养工艺制备腺病毒载体疫苗的方法
JP2001509675A (ja) 細胞培養およびウイルス増殖のための方法
CN111494615A (zh) 用于预防新型冠状病毒的重组腺病毒基因疫苗的生产方法
CN105579572B (zh) 用于净化高密度粗细胞培养收获物的方法
CN109479873B (zh) 一种脂肪组织保存液及其应用
CN108034634B (zh) 一种从经血中分离宫内膜间充质干细胞的方法
CN102776260A (zh) 一种高效表达重组人凝血八因子的方法
CN102268407B (zh) 一种rhIL-12工程细胞大规模无血清培养方法
CN112679601A (zh) 一种高比活性重组人促卵泡激素的制备方法
CN105950566A (zh) 一种利用生物反应器制备重组人5型腺病毒毒种库的方法
CN104212768A (zh) 用于培养微囊化重组中国仓鼠卵巢细胞的无蛋白培养基及其制备方法
Weiss Concentration of baboon endogenous virus in large‐scale production by use of hollow‐fiber ultrafiltration technology
CN109735588B (zh) 适用于片状载体袋的cho细胞生产分泌型蛋白的方法
CN117487747B (zh) 一种培养基及其在诱导干细胞分泌胰岛素中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19841492

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19841492

Country of ref document: EP

Kind code of ref document: A1