WO2019184996A1 - Gmp级无血清悬浮细胞大规模生产慢病毒的方法 - Google Patents

Gmp级无血清悬浮细胞大规模生产慢病毒的方法 Download PDF

Info

Publication number
WO2019184996A1
WO2019184996A1 PCT/CN2019/080215 CN2019080215W WO2019184996A1 WO 2019184996 A1 WO2019184996 A1 WO 2019184996A1 CN 2019080215 W CN2019080215 W CN 2019080215W WO 2019184996 A1 WO2019184996 A1 WO 2019184996A1
Authority
WO
WIPO (PCT)
Prior art keywords
culture
cells
liquid
transfection
time
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.)
Ceased
Application number
PCT/CN2019/080215
Other languages
English (en)
French (fr)
Inventor
洪谊
闫听
应降果
张豪杰
张露亿
张丽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuxi Cellular Biopharmaceutical Group Ltd
Shanghai Abelzeta Ltd
Original Assignee
Wuxi Cellular Biopharmaceutical Group Ltd
Shanghai Cellular Biopharmaceutical Group Ltd
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
Priority to SG11202009826PA priority Critical patent/SG11202009826PA/en
Priority to AU2019241301A priority patent/AU2019241301B2/en
Priority to JP2021501071A priority patent/JP2021519108A/ja
Priority to EP19775992.1A priority patent/EP3778880A4/en
Priority to KR1020207030733A priority patent/KR20200136463A/ko
Priority to US17/041,578 priority patent/US11845962B2/en
Application filed by Wuxi Cellular Biopharmaceutical Group Ltd, Shanghai Cellular Biopharmaceutical Group Ltd filed Critical Wuxi Cellular Biopharmaceutical Group Ltd
Publication of WO2019184996A1 publication Critical patent/WO2019184996A1/zh
Anticipated expiration legal-status Critical
Priority to US18/498,879 priority patent/US12180513B2/en
Priority to JP2024051544A priority patent/JP2024075766A/ja
Priority to US18/962,655 priority patent/US20250084386A1/en
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/12—Purification
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15051—Methods of production or purification of viral material
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for large-scale production of lentiviruses by GMP-grade serum-free suspension cells.
  • Gene therapy refers to the introduction of an exogenous therapeutic gene into a target cell to correct or compensate for a disease caused by a gene defect or abnormality, or a product of expression of a foreign gene acting on a disease target for therapeutic purposes.
  • recombinant chronic disease vector is a gene therapy vector based on HIV-1 (human immunodeficiency type I virus). Unlike general retroviral vectors, recombinant chronic disease vectors are capable of infecting both dividing cells and non-dividing cells. Recombinant chronic disease vectors have become the preferred transgenic vectors for CART cells and gene therapy because of their high biological titer and low immunogenicity in vitro and in vivo.
  • the mature HIV-1 virus is 100-120 nm in diameter and has a icosahedral symmetrical structure and a spherical shape. Under the electron microscope, a dense conical core with a viral RNA molecule and an enzyme, and the latter includes a reverse transcriptase, an integrase and a protease. (protease).
  • the outermost layer of HIV-1 is a lipoprotein envelope. There are two glycoproteins, surface protein (gp120) and intercalating protein (gp41), gp120 is a spike, and gp41 is a transmembrane protein.
  • the inner surface of the envelope is a matrix protein composed of P17, and the envelope is an envelope protein (P24)-encapsulated RNA.
  • the current recombinant lentiviral vector is a genetically engineered method that leaves only the packaging signal and the target gene transcription element in the lentiviral genome, but the structure of reverse transcriptase, envelope protein VSVG, gag/pol, rev, tat or The regulatory gene is dispersed on different vectors, and the disease-causing gene is deleted, thereby ensuring the safety of the recombinant lentiviral vector.
  • HEK293T is a cell line derived from human embryonic kidney epithelium. It is obtained by transfection of HEK 293 cell line by adenovirus E1A gene, and can express SV40 large T antigen, containing SV40 origin of replication and promoter region.
  • the eukaryotic expression vector containing the replication initiation site of the SV40 virus can efficiently replicate and transcribe in HEK 293T cells, thereby increasing the expression level of the foreign gene. Therefore, HEK293T cells are widely used in lentiviral packaging to obtain higher titers of lentiviral fluids.
  • HEK293T cells still have the following defects as lentivirus-producing cell lines: 1) There is a potential carcinogenic risk in the large T antigen. If the downstream process is not well removed, there is a certain risk in clinical treatment, and the large T antigen exists. Certain immunogenicity will increase the difficulty of clinical treatment; 2) HEK293T is an adherent cell, which is difficult to achieve industrialization by cell factory or rotary bottle production; 3) weak adherence, easy to use microcarrier technology Shedding, the toxicity of the drug is significantly reduced.
  • a method of producing a lentivirus by serum-free suspension cells comprising the steps of:
  • liquid exchange triggering condition includes:
  • the liquid changing operation comprises: discharging the cell-free clear liquid in the culture from the culture container, wherein the volume of the culture before the clear liquid is Vq1, and the culture is discharged after the clear liquid is discharged
  • the volume of the substance is Vh1, and the ratio of Vq1/Vh1 is 3-15 (preferably 4-7, more preferably 5-6); then, the culture medium is added to the culture vessel to form a packaging cell culture. ;
  • the transfection triggering conditions include:
  • the total amount of cells is 0.05 to 2 ⁇ 10 11 cells;
  • (t2) cell density is 0.5 to 5 ⁇ 10 6 cells / ml
  • the transfection operation comprises: mixing the production plasmid for producing the lentivirus with a transfection reagent, and then adding the culture vessel to introduce the packaging cells to form transfected packaging cells;
  • liquid exchange triggering condition includes:
  • the liquid exchange operation comprises: recovering the cell-free virus-containing material solution in the culture, wherein the volume of the culture before the liquid is recovered is Vq2, and the culture is recovered after recovering the clear liquid.
  • the volume is Vh2, and the ratio of Vq2/Vh2 is 3-15 (preferably 4-7, more preferably 5-6);
  • the culture liquid used in all the above steps is a serum-free cell culture liquid.
  • step (d) the volume of the packaging cell culture is Vb1, and the ratio of Vb1:Vq1 is (0.8-1.2):1.
  • step (j) the volume of the transfected cell culture is Vb2, and the ratio of Vb2:Vq2 is (0.8-1.2):1.
  • the culture in the step (c) and/or (h), is carried out under shaking conditions.
  • the shaking condition is a rocking speed of 10 to 30 rounds/min.
  • the amplitude of each swing is 1-20 cm, preferably 5-10 cm.
  • the culture fluids added in the different steps are the same or different culture fluids.
  • the pH is maintained between 7.0 and 7.35.
  • dissolved oxygen is maintained between 30% and 50% in steps (c) and/or (h).
  • the CO 2 concentration is maintained between 3-5%.
  • steps (d) and/or (i) the ratio of Vq1/Vh1 is 5-10.
  • the total time of steps (c), (d), and (e) is 72-216 hours. (before transfection)
  • the total time of steps (f), (g), (h), (i) and (j) is from 72 to 120 hours. (after transfection)
  • the multi-plasmid transfection employed comprises three plasmid transfections, four plasmid transfections.
  • the four plasmid transfection is transfected with plasmids CAR, gag/pol, rev, and VSVG.
  • the culture vessel is a disposable culture vessel.
  • the culture vessel has a volume of 20 to 120 L, preferably 30 to 100 L, more preferably 50 to 80 L.
  • the total number of viruses contained in the combined virus-containing serum is 1 x 10 12 Tu.
  • the combined virus-containing serum has a virus titer of 8 x 10 7 Tu/ml.
  • the packaging cell is a human embryonic kidney epithelial cell.
  • the packaging cell is human embryonic kidney epithelial cell HEK293F or a cell derived therefrom.
  • the serum-free medium was LV-MAX TM Production Medium (Gibco TM).
  • the purification comprises: ultrafiltration and chromatography.
  • the method of the present invention can be applied not only to large-scale production of 50 liters or more, but also to efficiently produce high-priced lentiviruses, and since the whole process uses serum-free culture conditions, animal origin introduced by using serum is avoided. The risk of proteins and other contaminants.
  • the fluctuations between the batches of lentiviruses produced by the method of the present invention are extremely small, thereby meeting the high requirements for GMP production for production quality.
  • the present invention has been completed on this basis.
  • packaging cell of the invention As used herein, the terms "packaging cell of the invention”, “packaging cell HEK293F”, “packaging cell HEK293F of the invention” and the like are used interchangeably and refer to the packaging and production of lentiviruses described in the first aspect of the invention. Cell.
  • the lentiviral packaging system which can be employed is not particularly limited, and preferably, a three-plasmid and four-plasmid system can be employed.
  • a particularly preferred packaging system is an HIV-1 derived lentiviral vector in which a four plasmid is used in place of the three plasmid system, the tat regulatory gene is knocked out, and the gag/pol and rev carrying plasmids are split into two , thereby reducing the possibility of generating a replicating virus and greatly increasing the security of the vector system.
  • the production of lentiviruses by using a four-plasmid system further ensures the safety and reliability of the lentiviral product.
  • the cells of the present invention are suspension cultured cells, and can be subjected to process amplification in conjunction with an automatic control system, thereby enabling large-scale production of recombinant lentiviral carrier material liquid, thereby making production cost controllable.
  • the cell culture process of the present invention adopts a one-time culture technique, using a serum-free, protein-free medium, eliminating the risk of contamination of the heterologous protein and mad cow disease virus in the final product, greatly improving the clinical application of the product.
  • the safety is thus used for the production of cellular or genetic drugs.
  • Medium serum-free, protein-free, chemically defined medium.
  • Culture conditions CO 2 is 3% to 5%; temperature is 30 to 37 degrees Celsius.
  • the present invention provides a method for large-scale production of lentiviruses by GMP-grade serum-free suspension cells.
  • the method of the invention comprises the steps of:
  • the liquid changing operation comprises: discharging the cell-free clear liquid in the culture from the culture container; and then adding a culture liquid to the culture container to form a packaging cell culture;
  • the transfection operation comprises: mixing the production plasmid for producing the lentivirus with a transfection reagent, and then adding the culture vessel to introduce the packaging cells to form transfected packaging cells;
  • liquid exchange operation comprises: recovering a cell-free virus-containing material solution located in the culture;
  • the culture liquid used in all the above steps is a serum-free cell culture liquid.
  • the liquid-changing triggering conditions are optimized to help reduce quality fluctuations in each production batch and to help obtain high-quality and high-yield medical grade lentiviruses.
  • the liquid exchange triggering conditions include:
  • the transfection trigger conditions are optimized to help reduce quality fluctuations in each production batch and to help obtain high quality and high yield medical grade lentiviruses.
  • the transfection triggering conditions include:
  • the total amount of cells is 0.05 to 2 ⁇ 10 11 cells;
  • (t2) cell density is 0.5 to 5 ⁇ 10 6 cells / ml
  • the method comprises the steps of:
  • Inoculation inoculated in 25 L culture medium, inoculation density 1-5 ⁇ 10 6 cells / ml;
  • the packaging cells are subcultured (during the pH and dissolved oxygen, the aeration ratio of the dissolved CO 2 , nitrogen, air, and the liquid exchange start mode are automatically adjusted), and the pH is controlled to be 6.9-7.4, preferably 7.0-7.3, more preferably 7.1. -7.2;
  • the first liquid change (when pH ⁇ 6.9, preferably ⁇ 7.0 can trigger a liquid change), for example 25L ⁇ 2-10L (preferably 3-7L);
  • Optional step after transfection, culture for 2-10 hours, preferably 4-6 hours, and then change the liquid again (first discharge a certain amount of the culture mixture, and then add the serum-free medium);
  • the culture is continued, and when the liquid exchange condition is triggered, the i-th change after the transfection is performed, wherein i is 2, 3, 4, and 5; each time the liquid is exchanged, the discharged liquid culture mixture is recovered.
  • Cell-free virus-containing serum recorded as the recovery liquid Ri, wherein the step is repeated 1, 2 or 3;
  • the combined virus-containing supernatant was subjected to purification treatment to obtain a purified lentiviral vector having a titer of 1 ⁇ 10 8 - 1 ⁇ 10 9 Tu/mL.
  • Example 1 was repeated except that the initial pH was 6.9-7.4, but during the passage, the real-time pH of the first culture was not monitored in real time, but was passaged at 24-96 hours.
  • Example 1 was repeated except that the CO 2 content was not adjusted.
  • the lentivirus biotitr is 20% of the preferred case.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Sustainable Development (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种GMP级无血清悬浮细胞大规模生产慢病毒的方法。该方法包括以下步骤：(a)提供包装细胞的种子液；(b)将所述种子液接种于第一培养液中；(c)进行包装细胞的传代培养；(d)当满足换液触发条件时，启动换液操作；(e)重复步骤(c)和(d) 1、2或3次；(f)当满足转染触发条件时，启动转染操作；(g)任选地进行转染后换液；(h)对经转染的包装细胞进行培养；(i)当满足换液触发条件时，启动收获与换液操作；(j)重复步骤(h)和(i) 1、2或3次；(k)将各次回收的回收液进行合并；和(l)纯化处理。其中，在各步骤中采用的培养液均为无血清细胞培养液。

Description

GMP级无血清悬浮细胞大规模生产慢病毒的方法 技术领域
本发明涉及生物技术领域，具体地涉及GMP级无血清悬浮细胞大规模生产慢病毒的方法。
背景技术
基因治疗(gene therapy)是指将外源治疗性基因导入靶细胞，以纠正或补偿因基因缺陷和异常引起的疾病，或通过外源基因表达的产物作用于疾病靶点，以达到治疗目的。
一类常用的重组慢病载体是以HIV-1(人类免疫缺陷I型病毒)为基础发展起来的基因治疗载体。与一般的逆转录病毒载体不同，重组慢病载体对分裂细胞和非分裂细胞均具有感染能力。重组慢病载体因其体内外生物滴度高和免疫原性低等优势，成为CART细胞和基因治疗的首选转基因载体。
成熟的HIV-1病毒直径100～120nm、呈20面体对称结构、球形，电镜下可见一致密圆锥状核心，内有病毒RNA分子和酶，后者包括逆转录酶、整合酶(integrase)和蛋白酶(protease)。HIV-1的最外层为脂蛋白包膜，膜上有表面蛋白(gp120)和相嵌蛋白(gp41)两种糖蛋白，gp120为刺突，gp41为跨膜蛋白。包膜内面为P17构成的基质蛋白(matrix)，包膜内为衣壳蛋白(P24)包裹的RNA。
目前的重组慢病毒载体是通过基因改造的方法，使慢病毒基因组中只留下包装信号和目的基因转录元件，而将逆转录酶、包膜蛋白VSVG、gag/pol、rev、tat等结构或调节基因分散在不同的载体上，同时删除致病基因，从而保证重组慢病毒载体的安全性。
HEK293T是一株来源于人胚胎肾上皮的细胞株，由HEK 293细胞系通过腺病毒E1A基因的转染而获得，能表达SV40的大T抗原、含有SV40复制起始点与启动子区。含有SV40病毒的复制起始位点的真核表达载体可以在HEK 293T细胞中实现高效的复制和转录，从而提高外源基因的表达水平。因此HEK293T细胞广泛应用于慢病毒包装，能获得较高滴度的慢病毒料液。
然而，HEK293T细胞作为慢病毒生产细胞株仍然存在以下缺陷：1)大T抗原存在潜在的致癌风险，下游工艺如果不能很好的对其去除，用于临床治疗存 在一定风险，且大T抗原存在一定的免疫原性，会增加临床治疗的难度；2)HEK293T是一株贴壁细胞，用细胞工厂或者转瓶生产较难真正实现工业化；3)贴壁能力较弱，使用微载体技术细胞容易脱落，产毒效能明显降低。
此外，虽然开发了一些基于悬浮细胞来生成慢病毒的技术，但是现有的生产方法仍存在一些不足，例如，不适合大规模生产，难以满足GMP生产的严格要求，生产出的病毒效价较低等。
因此，本领域迫切需要开发能够在无血清和悬浮培养培养下，通过包装细胞大规模高效生产慢病毒的方法。
发明内容
本发明的目的就是提供一种在无血清和悬浮培养培养下，通过包装细胞大规模高效生产慢病毒的方法。
在本发明的第一方面，提供了一种无血清悬浮细胞生产慢病毒的方法，包括步骤：
(a)提供一种用于生产慢病毒的包装细胞的种子液，所述的包装细胞是悬浮生长的包装细胞；
(b)将所述种子液接种于置于培养容器中的第一培养液中，获得第一培养物，其中，所述的第一培养液是无血清的细胞培养液，并且接种密度为1×10 6-5×10 6细胞/ml，并且第一培养液的体积为3-100升(较佳地5-50升)；
(c)对所述第一培养物进行包装细胞的传代培养，其中，传代培养的条件设定为在温度为30～38℃，溶氧为35～55％，CO 2浓度为2～10％和pH 6.9-7.4下进行；
(d)当满足换液触发条件时，启动换液操作，
其中，所述的换液触发条件包括：
(s1)实时pH≤6.9，较佳地≤7.0，较佳地≤7.05；
(s2)通过调控氧气、空气、氮气和/或CO 2浓度，实时pH值仍呈现下降趋势；和
(s3)传代培养时间≥72小时；
其中，所述换液操作包括：将位于所述培养物的无细胞清液从所述培养容器中排出，其中排出清液前所述培养物的体积为Vq1，而排出清液后所述培养 物的体积为Vh1，则Vq1/Vh1之比为3-15(较佳地4-7，更佳地5-6)；然后，向所述培养容器中补加培养液，形成包装细胞培养物；
(e)重复步骤(c)和(d)n次，n为1、2、或3次；
(f)当满足转染触发条件时，启动转染操作，
其中，所述的转染触发条件包括：
(t1)细胞总量为0.05～2×10 11个细胞；
(t2)细胞密度为0.5～5×10 6个细胞/ml；
(t3)细胞活率≥90％；和
(t4)总的培养时间≥72小时；
其中，所述的转染操作包括：将用于生产所述慢病毒的生产质粒与转染试剂混合后，加入所述培养容器，从而导入所述的包装细胞，形成经转染的包装细胞；
(g)任选地进行转染后换液；
(h)对经转染的包装细胞进行培养，其中，培养的条件设定为在温度为30～37℃，溶氧为35～55％，CO 2浓度为2～10％和pH 6.9-7.4下进行；
(i)当满足换液触发条件时，启动收获与换液操作，
其中，所述的换液触发条件包括：
(s1)实时pH≤6.9，较佳地≤7.0，较佳地≤7.05；
(s2)通过调控氧气、空气、氮气和/或CO 2浓度，实时pH值仍下降；和
(s3)传代培养时间≥72小时；
其中，所述换液操作包括：将位于所述培养物的无细胞的含病毒的料液进行回收，其中回收料液前所述培养物的体积为Vq2，而回收清液后所述培养物的体积为Vh2，则Vq2/Vh2之比为3-15(较佳地4-7，更佳地5-6)；
(j)重复步骤(h)和(i)m次，m为1、2、或3次，其中在重复之前，向所述培养容器中补加培养液，形成转染细胞培养物；
(k)将各次回收的回收液进行合并，获得经合并的含病毒的清液；和
(l)对所述的经合并的含病毒的清液进行纯化处理，从而得到经纯化的慢病毒载体；
其中，在上述所有步骤中采用的培养液均为无血清的细胞培养液。
在另一优选例中，在步骤(d)中，所述包装细胞培养物的体积Vb1，且Vb1:Vq1之比为(0.8-1.2)：1。
在另一优选例中，在步骤(j)中，所述转染细胞培养物的体积Vb2，且Vb2:Vq2之比为(0.8-1.2)：1。
在另一优选例中，所述的步骤(c)和/或(h)中，在摇动条件下进行培养。
在另一优选例中，所述的摇动条件是摇动速度为10～30次来回摆动(rocks)/min。
在另一优选例中，所述的每次摆动的幅度为1-20cm，较佳地5-10cm。
在另一优选例中，在不同步骤中添加的培养液是相同的或不同的培养液。
在另一优选例中，在步骤(c)和/或(h)中，将pH维持在7.0-7.35之间。
在另一优选例中，在步骤(c)和/或(h)中，将溶氧维持在30％－50％之间。
在另一优选例中，在步骤(c)和/或(h)中，将CO 2浓度维持在3－5％之间。
在另一优选例中，在步骤(d)和/或(i)中，将Vq1/Vh1之比为5－10。
在另一优选例中，步骤(c)、(d)、和(e)的总时间为72-216小时。(转染前)
在另一优选例中，步骤(f)、(g)、(h)、(i)和(j)的总时间为72-120小时。(转染后)
在另一优选例中，在步骤(f)中，采用的多质粒转染包括三质粒转染、四质粒转染。
在另一优选例中，所述的四质粒转染是用质粒CAR、gag/pol、rev、和VSVG进行转染。
在另一优选例中，所述的培养容器是一次性培养容器。
在另一优选例中，所述的培养容器的容积为20-120L，较佳地30-100L，更佳地为50-80L。
在另一优选例中，经合并的含病毒的清液中含有的病毒总数为1×10 12Tu。
在另一优选例中，经合并的含病毒的清液中病毒滴度为为8×10 7Tu/ml。
在另一优选例中，所述的包装细胞是人胚胎肾上皮细胞。
在另一优选例中，所述的包装细胞是人胚胎肾上皮细胞HEK293F或其衍生细胞。
在另一优选例中，所述的无血清培养基是LV-MAX TM Production Medium(Gibco TM)。
在另一优选例中，在步骤(l)中，所述的纯化包括：超滤和层析。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
具体实施方式
本发明人经过广泛而深入的研究，通过对生产工艺的探索和工艺参数的筛选，首次开发了一种GMP级大规模无血清悬浮细胞慢病毒生产的方法。本发明方法不仅可以适用于50升或更大体积的大规模生产，而且极其高效地制备高效价的慢病毒，而且由于全程采用无血清培养条件，因此避免了因使用血清而引入的动物源性蛋白和其它污染物的风险。此外，本发明方法生产的各批次慢病毒之间的波动性极小，从而可以满足GMP生产对于生产质量的高要求。在此基础上完成了本发明。
术语
如本文所用，术语“本发明的包装细胞”、“包装细胞HEK293F”、“本发明的包装细胞HEK293F”等可互换使用，指本发明第一方面中所述的用于包装和生产慢病毒的细胞。
包装细胞和包装体系
在本发明中，可采用的慢病毒包装体系没有特别限制，优选地，可以采用三质粒和四质粒系统。
一种特别优选的包装系统是采用HIV-1来源的慢病毒载体，其中采用四质粒代替三质粒系统，通过敲除tat调节基因，并把gag/pol和rev的携带质粒由一个拆成两个，从而减少了产生复制型病毒的可能性，大大增加载体系统的安全性。
在本发明的优选例中，通过使用四质粒系统进行慢病毒的生产，进一步确保慢病毒产品的安全可靠。
本发明的主要优点包括：
(a)本发明的细胞为悬浮培养细胞，配合自动控制系统，能进行工艺放大，从而能够大规模化生产重组慢病毒载体料液，使生产成本可控。
(b)本发明所述的细胞培养工艺采用一次性培养技术，使用无血清、无蛋白成分的培养基，免除了终产品存在异源蛋白和疯牛病病毒污染的风险，极大提高了产品临床应用的安全性，从而用于细胞或基因药品的生产。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
通用方法和材料
培养基：无血清、无蛋白且化学成分明确的培养基。
培养条件：CO 2为3％～5％；温度为30～37摄氏度。
(1)转染试剂的配置：
a)1×HBS(pH7.4)：将8.76g NaCl溶解于900ml超纯水，加入20ml1M的HEPES，调pH值到7.4，定容至1L，过滤(0.2μm滤膜)后储存于4℃备用。
b)称取125mg PEI粉末溶解于50ml 1×HBS(pH7.4)中，0.2μm滤膜过滤，储存于4℃备用。
实施例1
大规模生产慢病毒方法
本发明提供了一种GMP级无血清悬浮细胞大规模生产慢病毒的方法。
本发明的方法，包括步骤：
(a)提供一种用于生产慢病毒的包装细胞的种子液，所述的包装细胞是悬浮生长的包装细胞；
(b)将所述种子液接种于置于培养容器中的第一培养液中，获得第一培养物，其中，所述的第一培养液是无血清的细胞培养液，并且接种密度为1×10 6-5×10 6细胞/ml，并且第一培养液的体积为预定的大体积(如3-100升)；
(c)对所述第一培养物进行包装细胞的传代培养，其中，传代培养的条件设定为在温度为30～38℃，溶氧为35～55％，CO 2浓度为2～10％和pH 6.9-7.4下 进行；
(d)当满足换液触发条件时，启动换液操作，
其中，所述换液操作包括：将位于所述培养物的无细胞清液从所述培养容器中排出；然后，向所述培养容器中补加培养液，形成包装细胞培养物；
(e)重复步骤(c)和(d)n次，n为1、2、或3次；
(f)当满足转染触发条件时，启动转染操作，
其中，所述的转染操作包括：将用于生产所述慢病毒的生产质粒与转染试剂混合后，加入所述培养容器，从而导入所述的包装细胞，形成经转染的包装细胞；
(g)任选地进行转染后换液；
(h)对经转染的包装细胞进行培养，其中，培养的条件设定为在温度为30～37℃，溶氧为35～55％，CO 2浓度为2～10％和pH 6.9-7.4下进行；
(i)当满足换液触发条件时，启动收获与换液操作，
其中，所述换液操作包括：将位于所述培养物的无细胞的含病毒的料液进行回收；
(j)重复步骤(h)和(i)m次，m为1、2、或3次，其中在重复之前，向所述培养容器中补加培养液，形成转染细胞培养物；
(k)将各次回收的回收液进行合并，获得经合并的含病毒的清液；和
(l)对所述的经合并的含病毒的清液进行纯化处理，从而得到经纯化的慢病毒载体；
其中，在上述所有步骤中采用的培养液均为无血清的细胞培养液。
在本发明中，换液触发条件是经过优化的，有助于减少各生产批次的质量波动，并有助于获得高效价和高产量的医用级的慢病毒。典型地，所述的换液触发条件包括：
(s1)实时pH≤6.9，较佳地≤7.0，较佳地≤7.05；
(s2)通过调控氧气、空气、氮气和/或CO 2浓度，实时pH值仍呈现下降趋势；和
(s3)传代培养时间≥72小时；
在本发明中，转染触发条件是经过优化的，有助于减少各生产批次的质量波动，并有助于获得高效价和高产量的医用级的慢病毒。典型地，所述的转染触发条件包括：
(t1)细胞总量为0.05～2×10 11个细胞；
(t2)细胞密度为0.5～5×10 6个细胞/ml；
(t3)细胞活率≥90％；和
(t4)总的培养时间≥72小时；
典型地，在本发明的一个具体实施例中，所述方法包括步骤：
种子液的制备；
接种(接种于25L培养液)，接种密度1-5×10 6细胞/ml；
包装细胞传代培养(期间通过pH和溶氧，自动调控溶CO 2、氮气、空气的通气比例，以及换液启动模式)，控制pH在6.9-7.4，较佳地7.0-7.3，更佳地7.1-7.2；
第一次换液(当pH≤6.9，较佳地≤7.0可触发换液)，例如25L→2-10L(较佳地3-7L)；
补加无血清培养液，继续培养24-96小时，较佳地36-72小时，更佳地40-60小时；培养至300h时，细胞数为5×10 10；
加入质粒，对所述包装细胞进行多质粒转染；
任选的步骤：转染后，培养2-10小时，较佳地4-6小时，再次进行换液(先排放掉一定量的培养混合物，然后加入无血清培养液)；
通用步骤：在转染后，进行继续培养，当pH≤6.9，较佳地≤7.0可触发转染后的第一换液，并且在转染后的换液时，将排出的液态培养混合物(无细胞的含病毒的清液)进行回收，记为回收液R1，保存于2～8摄氏度；
换液后，继续培养，并且当触发换液条件时，进行转染后的第i次换液，其中i为2、3、4、和5；每次换液时，回收排出的液态培养混合物无细胞的含病毒的清液，记为回收液Ri，其中，该步骤重复1、2或3；
将各次回收的回收液进行合并，获得经合并的含病毒的清液；
对所述的经合并的含病毒的清液进行纯化处理，从而得到经纯化的慢病毒载体，滴度为1×10 8-1×10 9Tu/mL。
对比例1
重复实施例1，不同点在于：初始pH为6.9-7.4，但是传代过程中，不实时监控所述第一培养物的实时pH值，而是在24～96小时进行传代。
结果：培养至300h时，细胞数为4×10 9(少于优选例)。
对比例2
重复实施例1，不同点在于：不调节CO 2含量。
结果：慢病毒生物滴度为优选例的20％。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种无血清悬浮细胞生产慢病毒的方法，其特征在于，包括步骤：
    (a)提供一种用于生产慢病毒的包装细胞的种子液，所述的包装细胞是悬浮生长的包装细胞；
    (b)将所述种子液接种于置于培养容器中的第一培养液中，获得第一培养物，其中，所述的第一培养液是无血清的细胞培养液，并且接种密度为1×10 6-5×10 6细胞/ml，并且第一培养液的体积为3-100升；
    (c)对所述第一培养物进行包装细胞的传代培养，其中，传代培养的条件设定为在温度为30～38℃，溶氧为35～55％，CO 2浓度为2～10％和pH 6.9-7.4下进行，并且在所述传代培养过程中，监控所述第一培养物的实时pH值，并基于所述实时pH值对溶氧和/或CO 2浓度进行控制，从而维持pH在6.9-7.4之间；
    (d)当满足换液触发条件时，启动换液操作，
    其中，所述的换液触发条件包括：
    (s1)实时pH≤6.9，较佳地≤7.0，较佳地≤7.05；
    (s2)通过调控氧气、空气、氮气和/或CO 2浓度，实时pH值仍呈现下降趋势；和
    (s3)传代培养时间≥72小时；
    其中，所述换液操作包括：将位于所述培养物的无细胞清液从所述培养容器中排出，其中排出清液前所述培养物的体积为Vq1，而排出清液后所述培养物的体积为Vh1，则Vq1/Vh1之比为3-15；然后，向所述培养容器中补加培养液，形成包装细胞培养物；
    (e)重复步骤(c)和(d)n次，n为1、2、或3次；
    (f)当满足转染触发条件时，启动转染操作，
    其中，所述的转染触发条件包括：
    (t1)细胞总量为0.05～2×10 11个细胞；
    (t2)细胞密度为0.5～5×10 6个细胞/ml；
    (t3)细胞活率≥90％；和
    (t4)总的培养时间≥72小时；
    其中，所述的转染操作包括：将用于生产所述慢病毒的生产质粒与转染试剂混合后，加入所述培养容器，从而导入所述的包装细胞，形成经转染的包装 细胞；
    (g)任选地进行转染后换液；
    (h)对经转染的包装细胞进行培养，其中，培养的条件设定为在温度为30～37℃，溶氧为35～55％，CO 2浓度为2～10％和pH 6.9-7.4下进行，并且在所述传代培养过程中，监控所述第一培养物的实时pH值，并基于所述实时pH值对溶氧和/或CO 2浓度进行控制，从而维持pH在6.9-7.4之间；
    (i)当满足换液触发条件时，启动收获与换液操作，
    其中，所述的换液触发条件包括：
    (s1)实时pH≤6.9；
    (s2)通过调控氧气、空气、氮气和/或CO 2浓度，实时pH值仍下降；和
    (s3)传代培养时间≥72小时；
    其中，所述换液操作包括：将位于所述培养物的无细胞的含病毒的料液进行回收，其中回收料液前所述培养物的体积为Vq2，而回收清液后所述培养物的体积为Vh2，则Vq2/Vh2之比为3-15；
    (j)重复步骤(h)和(i)m次，m为1、2、或3次，其中在重复之前，向所述培养容器中补加培养液，形成转染细胞培养物；
    (k)将各次回收的回收液进行合并，获得经合并的含病毒的清液；和
    (l)对所述的经合并的含病毒的清液进行纯化处理，从而得到经纯化的慢病毒载体；
    其中，在上述所有步骤中采用的培养液均为无血清的细胞培养液。
  2. 如权利要求1所述的方法，其特征在于，所述的步骤(c)和/或(h)中，在摇动条件下进行培养。
  3. 如权利要求1所述的方法，其特征在于，在步骤(c)和/或(h)中，将pH维持在7.0-7.35之间。
  4. 如权利要求1所述的方法，其特征在于，在步骤(d)和/或(i)中，将Vq1/Vh1之比为5－10。
  5. 如权利要求1所述的方法，其特征在于，步骤(c)、(d)、和(e)的总时间为72-216小时。
  6. 如权利要求1所述的方法，其特征在于，在步骤(f)中，采用的多质粒转染包括三质粒转染、四质粒转染。
  7. 如权利要求1所述的方法，其特征在于，所述的培养容器是一次性培 养容器。
  8. 如权利要求1所述的方法，其特征在于，经合并的含病毒的清液中含有的病毒总数为1×10 12Tu。
  9. 如权利要求1所述的方法，其特征在于，所述的包装细胞是人胚胎肾上皮细胞。
  10. 如权利要求1所述的方法，其特征在于，在步骤(l)中，所述的纯化包括：超滤和层析。
PCT/CN2019/080215 2018-03-29 2019-03-28 Gmp级无血清悬浮细胞大规模生产慢病毒的方法 Ceased WO2019184996A1 (zh)

Priority Applications (9)

Application Number Priority Date Filing Date Title
AU2019241301A AU2019241301B2 (en) 2018-03-29 2019-03-28 Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells
JP2021501071A JP2021519108A (ja) 2018-03-29 2019-03-28 Ｇｍｐレベルの無血清懸濁液細胞を用いるレンチウイルスの大規模製造方法
EP19775992.1A EP3778880A4 (en) 2018-03-29 2019-03-28 Method for large-scale production of lentivirus by using gmp-level serum-free suspension cells
KR1020207030733A KR20200136463A (ko) 2018-03-29 2019-03-28 Gmp-수준 무혈청 현탁 세포 사용에 의한 렌티바이러스의 대규모 제조 방법
US17/041,578 US11845962B2 (en) 2018-03-29 2019-03-28 Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells
SG11202009826PA SG11202009826PA (en) 2018-03-29 2019-03-28 Method for large-scale production of lentivirus by using gmp-level serum-free suspension cells
US18/498,879 US12180513B2 (en) 2018-03-29 2023-10-31 Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells
JP2024051544A JP2024075766A (ja) 2018-03-29 2024-03-27 Ｇｍｐレベルの無血清懸濁液細胞を用いるレンチウイルスの大規模製造方法
US18/962,655 US20250084386A1 (en) 2018-03-29 2024-11-27 Method for large-scale production of lentivirus by using gmp-level serum-free suspension cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810273392.7A CN110317791A (zh) 2018-03-29 2018-03-29 Gmp级无血清悬浮细胞大规模生产慢病毒的方法
CN201810273392.7 2018-03-29

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US17/041,578 A-371-Of-International US11845962B2 (en) 2018-03-29 2019-03-28 Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells
US18/498,879 Continuation US12180513B2 (en) 2018-03-29 2023-10-31 Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells

Publications (1)

Publication Number Publication Date
WO2019184996A1 true WO2019184996A1 (zh) 2019-10-03

Family

ID=68058573

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/080215 Ceased WO2019184996A1 (zh) 2018-03-29 2019-03-28 Gmp级无血清悬浮细胞大规模生产慢病毒的方法

Country Status (8)

Country Link
US (3) US11845962B2 (zh)
EP (1) EP3778880A4 (zh)
JP (2) JP2021519108A (zh)
KR (1) KR20200136463A (zh)
CN (1) CN110317791A (zh)
AU (1) AU2019241301B2 (zh)
SG (1) SG11202009826PA (zh)
WO (1) WO2019184996A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113832112A (zh) * 2020-06-24 2021-12-24 重庆精准生物技术有限公司 一种制备慢病毒的混合方法及混合系统
JP2024515793A (ja) * 2021-04-27 2024-04-10 ノバルティス アーゲー ウイルスベクター生産システム

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110317791A (zh) * 2018-03-29 2019-10-11 西比曼生物科技（香港）有限公司 Gmp级无血清悬浮细胞大规模生产慢病毒的方法
CN110894494B (zh) * 2019-11-22 2022-09-27 广西梧州制药（集团）股份有限公司 一种大规模高密度悬浮培养293细胞高产腺病毒的方法
CN113265425A (zh) * 2021-05-08 2021-08-17 苏州博腾生物制药有限公司 基于悬浮细胞的car-cd19慢病毒制备工艺
CN114990077A (zh) * 2022-08-01 2022-09-02 深圳市先康达生命科学有限公司 一种慢病毒包装与纯化方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013076309A1 (en) * 2011-11-24 2013-05-30 Genethon Scalable lentiviral vector production system compatible with industrial pharmaceutical applications
CN107523555A (zh) * 2016-06-22 2017-12-29 武汉光谷人福生物医药有限公司 获得病毒的方法

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002348151A1 (en) 2001-11-05 2003-05-19 Genvec, Inc. Viral vector production methods and compositions
US20030175688A1 (en) 2002-03-15 2003-09-18 Rukmini Pennathur-Das Method for the purification and production of oncolytic adenoviruses
WO2007095336A2 (en) 2006-02-15 2007-08-23 The Immune Response Corporation Production of hiv
US7875446B2 (en) 2006-04-20 2011-01-25 Wyeth Llc Purification processes for isolating purified vesicular stomatitis virus from cell culture
RU2010124788A (ru) 2007-12-21 2012-01-27 Ваейт ЭлЭлСи (US) Генетически модифицированный ослабленный вирус везикулярного стоматита, композиции и способы их применения
US10041049B2 (en) 2008-11-03 2018-08-07 Janssen Vaccines & Prevention B.V. Method for the production of adenoviral vectors
EP2682168A1 (en) 2012-07-02 2014-01-08 Millipore Corporation Purification of biological molecules
CN103881984A (zh) * 2013-12-06 2014-06-25 深圳市赛百诺基因技术有限公司 无血清悬浮细胞生产重组腺病毒的方法及其药品制剂生产方法
FR3014901B1 (fr) 2013-12-17 2017-06-09 Genethon Procede de purification de virus ou vecteurs viraux enveloppes
CN104371982A (zh) 2014-10-21 2015-02-25 武汉维诺赛生物技术有限公司 一种慢病毒的纯化方法
EP3054007A1 (en) 2015-02-09 2016-08-10 Institut National De La Sante Et De La Recherche Medicale (Inserm) Recombinant adeno-associated virus particle purification comprising an immuno-affinity purification step
CN107043784A (zh) * 2016-02-05 2017-08-15 上海吉凯基因化学技术有限公司 一种慢病毒载体的制备方法
CN107841482B (zh) * 2016-09-21 2021-07-27 上海倍谙基生物科技有限公司 Mdck细胞无血清悬浮培养技术生产h9亚型流感疫苗
CN106434571A (zh) * 2016-10-19 2017-02-22 深圳源兴基因技术有限公司 一种wave无血清培养悬浮细胞制备重组腺病毒的方法
CN106474466B (zh) 2016-12-07 2018-04-13 申联生物医药（上海）股份有限公司 一种口蹄疫疫苗的制备方法
CN107384877B (zh) 2017-08-18 2020-09-08 深圳源兴基因技术有限公司 一种慢病毒的纯化方法
CN107630037A (zh) 2017-10-19 2018-01-26 和元生物技术（上海）股份有限公司 一种获得高纯度腺相关病毒载体的纯化工艺
CN110317832B (zh) 2018-03-28 2022-07-05 西比曼生物科技（香港）有限公司 Gmp级规模化制备重组慢病毒载体的纯化制剂的方法
CN110317791A (zh) * 2018-03-29 2019-10-11 西比曼生物科技（香港）有限公司 Gmp级无血清悬浮细胞大规模生产慢病毒的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013076309A1 (en) * 2011-11-24 2013-05-30 Genethon Scalable lentiviral vector production system compatible with industrial pharmaceutical applications
CN107523555A (zh) * 2016-06-22 2017-12-29 武汉光谷人福生物医药有限公司 获得病毒的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAI JINGJING; SONG XIAOPING; YAN JIGUI; WANG YAJIE: "Lentiviral Vector Packaging and Production Method", JOURNAL OF YANGTZE UNIVERSITY. NATURAL SCIENCE, vol. 11, no. 9, 25 March 2014 (2014-03-25), CN, pages 121 - 123, XP009523640, ISSN: 1673-1409, DOI: 10.16772/j.cnki.1673-1409.2014.09.054 *
SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
See also references of EP3778880A4
SVEN ANSORGE , OLIVIER HENRY , AMINE KAMEN : "Recent progress in lentiviral vector mass production", BIOCHEMICAL ENGINEERING JOURNAL , vol. 48, no. 3, 27 October 2009 (2009-10-27), pages 362 - 377, XP026886643, ISSN: 1369-703X, DOI: 10.1016/j.bej.2009.10.017 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113832112A (zh) * 2020-06-24 2021-12-24 重庆精准生物技术有限公司 一种制备慢病毒的混合方法及混合系统
JP2024515793A (ja) * 2021-04-27 2024-04-10 ノバルティス アーゲー ウイルスベクター生産システム

Also Published As

Publication number Publication date
US20250084386A1 (en) 2025-03-13
US20210363497A1 (en) 2021-11-25
AU2019241301A1 (en) 2020-10-22
AU2019241301B2 (en) 2025-04-17
CN110317791A (zh) 2019-10-11
EP3778880A1 (en) 2021-02-17
US20240060053A1 (en) 2024-02-22
KR20200136463A (ko) 2020-12-07
US11845962B2 (en) 2023-12-19
JP2021519108A (ja) 2021-08-10
EP3778880A4 (en) 2021-12-29
JP2024075766A (ja) 2024-06-04
SG11202009826PA (en) 2020-11-27
US12180513B2 (en) 2024-12-31

Similar Documents

Publication Publication Date Title
US12180513B2 (en) Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells
CN104136605B (zh) 与工业制药应用相容的可放大的慢病毒载体生产系统
KR102200278B1 (ko) 무혈청 현탁 세포 배양 시스템에서 재조합 렌티바이러스 벡터를 생성하기 위한 확대가능한 제작 방법
US9145564B2 (en) Persistently infective Sendai virus vector
CN113980916B (zh) 一种慢病毒的纯化方法
US20220056477A1 (en) Foamy viral vector compositions and methods for the manufacture of same
WO2023072178A1 (en) Methods for developing a cell line for producing virus in suspension cell culture
US12286639B2 (en) LDLR negative packaging cell line for the production of VSV-G pseudotyped retroviral vector particles or virus particles thereof
US20250084438A1 (en) Vectors for protein manufacture
US20250361491A1 (en) Methods of producing an enveloped virus
JP5778147B2 (ja) 遺伝子導入方法
AU2012352394B2 (en) Large commercial scale lentiviral vector production system and vectors produced thereby
HK40042109A (zh) Gmp级无血清悬浮细胞大规模生产慢病毒的方法
CN115916987A (zh) 填充床生物反应器中的慢病毒载体制造方法
WO2025219920A1 (en) Methods of producing an enveloped virus
WO2025219924A1 (en) Cell lines overexpressing vesicular stomatitis virus g
CA2858980C (en) Large commercial scale lentiviral vector production system and vectors produced thereby
HK1203360B (zh) 工业大规模慢病毒载体生产系统以及由此生产的载体

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: 19775992

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021501071

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019241301

Country of ref document: AU

Date of ref document: 20190328

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20207030733

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2019775992

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2019775992

Country of ref document: EP

Effective date: 20201029

WWR Wipo information: refused in national office

Ref document number: 1020207030733

Country of ref document: KR

WWR Wipo information: refused in national office

Ref document number: 1020207030733

Country of ref document: KR

WWC Wipo information: continuation of processing after refusal or withdrawal

Ref document number: 1020207030733

Country of ref document: KR