WO2019184995A1 - Gmp级规模化制备重组慢病毒载体的纯化制剂的方法 - Google Patents
Gmp级规模化制备重组慢病毒载体的纯化制剂的方法 Download PDFInfo
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Definitions
- the present invention relates to the field of biotechnology, and in particular to a method for preparing a purified preparation of a recombinant lentiviral vector on a GMP scale.
- 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.
- the foreign gene can be transduced or delivered by a viral vector or a non-viral vector.
- the commonly used non-viral vectors are liposomes, dendrimers, non-natural cationic polymers, natural polysaccharides and the like. Non-viral gene delivery vectors are safer and more stable, but their transfection efficiency is usually lower.
- the viral vector is a foreign gene packaged into the outer shell of a natural virus, and the foreign gene is introduced into the cell by utilizing the infectivity of the virus to the host cell.
- Common viral vectors include retrovirus (rRV), recombinant lentivirus (rLV), recombinant adenovirus (rAd), and recombinant adeno-associated virus (rAAV).
- the transduction efficiency of viral vectors is much higher than that of non-viral vectors, and is particularly suitable for infecting target cells, such as lymphocytes, which are difficult to infect.
- the recombinant chronic disease vector is a gene therapy vector developed on the basis of HIV-1 (human immunodeficiency type I virus). It distinguishes between a general retroviral vector, which is 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 current recombinant lentiviral vector is a genetically engineered method that leaves only the packaging signal and the target gene transcript in the lentiviral genome, and disperses the structural genes such as reverse transcriptase, envelope protein VSVG, and gag-pol in 2 On ⁇ 3 vectors, the disease-causing gene was deleted at the same time.
- the mature lentiviral particles are obtained by co-transfecting 293T cells into a cell and then secreting them into the culture supernatant by 293T cells, which can be obtained by ultracentrifugation or chromatographic purification.
- the method used by conventional laboratories to obtain lentiviruses is ultracentrifugation. Although this method is simple but cannot be industrially amplified, the prepared lentiviral vector may have high endotoxin, BSA, HCP or nucleic acid residues, and cannot be directly used. In the human body.
- the existing chromatographic purification methods also have the disadvantages of cumbersome steps, low yield and high purity, and are difficult to meet the requirements of industrial large-scale production and GMP-level production.
- Another object of the present invention is to provide a recombinant lentivirus purified by the method, and a purified preparation containing the recombinant lentivirus and use thereof.
- a method of scale-up purification of a recombinant viral vector preparation comprising the steps of:
- the liquid is subjected to microfiltration treatment to obtain a microfiltration-treated filtrate, wherein the filtrate contains the recombinant viral vector, the volume of the filtrate is Vb;
- the chromatography is selected from the group consisting of anion chromatography, size exclusion chromatography, multi-mode composite resin chromatography, or a combination thereof.
- Va 100 liters (or 100-500 liters);
- the method further comprises after step (e):
- the virus comprises a lentivirus
- the method is GMP compliant.
- step (d) size exclusion chromatography and anion chromatography are carried out sequentially, sequentially or simultaneously.
- the anionic resin is selected from the group consisting of Capto Q, Capto ImpRes, Capto DEAE.
- a multimodal composite chromatography resin Capto adhere ImpRes, Capto core 700 is employed.
- the chromatographic purification treatment is performed by anion chromatography crude purification followed by multi-mode complex chromatography purification.
- the chromatographic purification treatment is performed by multi-mode conformation chromatography and then subjected to anion chromatography purification.
- the chromatographic purification treatment is to carry out impurity adsorption removal and virus capture while the two multi-modes are matched in series with the chromatography resin.
- the chromatographic purification treatment time of the concentrated liquid is 10 L/30 min.
- the chromatographic purification treatment is carried out at a rate of 20 L of filtrate to be chromated/60 minutes.
- the weight ratio of the chromatographic medium to the filtrate to be chromatographed is 500 mL: 10 L of filtrate,
- the sterilizing filter has a pore size of 0.2 ⁇ M.
- the chromatographic medium is selected from the group consisting of Capto Q ImpRes.
- the purified recombinant lentiviral vector has one or more characteristics selected from the group consisting of:
- the filtrate (including the concentrated or unconcentrated filtrate) is subjected to ribozyme treatment prior to performing the chromatographic purification treatment
- the ribozyme treatment comprises: adding 10 U/ml of ribozyme and incubating at 37 ° C for 30 min.
- step (b) a microfiltration hollow fiber column is used for microfiltration
- the microfiltration hollow fiber column is a microfiltration membrane having a cut-off of 0.4-1.0 micrometers (preferably 0.45-0.8 micrometers).
- the ratio of Vb to Vc is 5 to 50, preferably 10 to 30, more preferably 15 to 25.
- step (c) concentration is carried out by ultrafiltration.
- the ultrafiltration uses an ultrafiltration membrane having a cut-off of 100 to 800K.
- the ultrafiltration hollow fiber column has a cutoff value of 200-1000K, preferably 300-500K.
- the ultrafiltration is carried out using an ultrafiltration hollow fiber column and an ultrafiltration system.
- the ultrafiltration system is selected from the group consisting of AKTA flux 6, AKTA readyflux.
- a purified recombinant lentivirus prepared by the method is provided.
- a formulation comprising the purified recombinant lentivirus.
- the formulation is a pharmaceutical composition
- the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
- a purification apparatus for the method comprising:
- (S1) an optional first container for holding a raw material of the recombinant lentivirus to be purified
- (S2) a microfiltration unit, wherein the microfiltration unit is used for microfiltration treatment of the recombinant lentivirus to be purified, thereby obtaining a microfiltration-treated filtrate;
- (S5) a collection unit for collecting the purified recombinant lentiviral vector.
- the first container, the microfiltration unit, the concentration unit, the chromatographic purification unit, and the collection unit are in fluid communication.
- the chromatographic purification unit comprises: a molecular exclusion chromatography unit and an anion chromatography unit.
- the molecular exclusion chromatography unit and the anion chromatography unit are each independent;
- the size exclusion chromatography unit and the anion chromatography unit are integrated.
- the purification device further comprises:
- (S6) A ribozyme treatment unit comprising an addition device for adding a ribozyme.
- the ribozyme processing unit further includes an incubation device for incubating the filtrate to which the ribozyme is added.
- the inventors have extensively and intensively studied, and through the extensive screening and exploration of purification conditions, for the first time, a rapid, simple and excellent purification method of GMP-scale large-scale purification of recombinant lentivirus has been unexpectedly developed.
- the purification raw material containing recombinant lentivirus can be purified extremely efficiently, rapidly and on a large scale by using a specific purification medium and specific purification steps and conditions, thereby obtaining high purification, low impurity and no internal content.
- Recombinant lentiviral preparation of toxin The present invention has been completed on this basis.
- composite filler resin refers to Capto Q, Capto ImpRes, Capto DEAE.
- composite filler resin refers to chromatography using a composite filler resin.
- lentiviral vector As used herein, the terms "recombinant lentivirus", “lentiviral vector” and the like are used interchangeably and refer to a lentiviral vector produced by introducing a particular plasmid into a particular packaging cell. Typically, these lentiviral vectors are useful for subsequent therapeutic or non-therapeutic purposes of transfecting predetermined cells, including cells of humans and non-human mammals.
- the method set forth in the present invention rapidly obtains a high purity lentiviral formulation by using a GE AKTA device using a combination of, but not limited to, a new generation of Capto Core 700 and Capto adhere ImpRes resin.
- the recombinant lentiviral vector purified preparation prepared by the method of the invention can be used for the production of cells or gene drugs.
- High purity lentiviral formulations can be obtained quickly by combining the combination of Capto Core700 and Capto adhere ImpRes for lentiviral purification.
- AKTA Flux 6 system is cleaned by sterile 1X PBS;
- AKTA Flux 6 system is cleaned by sterile 1X PBS;
- microfiltered lentiviral solution is subjected to ultrafiltration through a 300-800K ultrafiltration column and an AKTA Flux 6 system, and the filtrate is discarded;
- nuclease a concentration of 10 to 1000 U / mL into a 1 ⁇ 2L lentiviral solution and mixing;
- AKTA Flux 6 system is cleaned by sterile lentiviral cryopreservation
- the microfiltered lentiviral solution is subjected to ultrafiltration through a 300-800K ultrafiltration column and an AKTA Flux 6 system, and the filtrate is discarded;
- the lentiviral preparation is stored in an ultra-low temperature freezer ( ⁇ -70 ° C).
- High-purity lentivirus can be obtained quickly and efficiently by using 0.45-0.8 ⁇ M microfiltration hollow fiber column, 300-800K hollow fiber column and Capto Core700+Capto adhere ImpRes composite packing for stepwise clarification filtration, concentration, exchange and impurity removal. preparation.
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Abstract
Description
Claims (10)
- 一种规模化纯化重组病毒载体制剂的方法,其特征在于,所述方法包括以下步骤:(a)提供一含待纯化的重组病毒载体的原料,所述原料是料液,所述料液的体积为Va;(b)将所述料液进行微滤处理,从而获得经微滤处理的滤液,其中所述滤液含有所述重组病毒载体,所述滤液的体积为Vb;(c)任选地对所述滤液进行浓缩处理,从而获得经浓缩的滤液,所述经浓缩的滤液的体积为Vc;(d)对所述的上一步骤获得的滤液进行层析纯化处理,从而获得含重组病毒载体的粗纯产物;(e)对所述的上一步骤获得的粗纯产物,进行换液和精纯,获得纯化的重组病毒载体;其中,所述的层析选自下组:阴离子层析、分子排阻层析、多模式复合树脂层析、或其组合。
- 如权利要求1所述的方法,其特征在于,所述的阴离子树脂选自下组:Capto Q,Capto ImpRes,Capto DEAE。
- 如权利要求1所述的方法,其特征在于,所述的层析纯化处理是先进行阴离子层析粗纯,然后进行多模式复合层析精纯。
- 如权利要求1所述的方法,其特征在于,在步骤(d)中,所述的经纯化的重组慢病毒载体具有选自下组的一个或多个特征:(p1)重组慢病毒载体生物滴度1.06x10 9Tu/mL;(p2)BSA残留<50ng/mL;(p3)内毒素<1EU/mL。
- 如权利要求1所述的方法,其特征在于,在步骤(c)中,通过超滤进行浓缩。
- 一种用权利要求1所述方法制备的纯化的重组慢病毒。
- 一种制剂,其特征在于,所述制剂含有权利要求5所述的纯化的重组慢病毒。
- 一种用于权利要求1所述方法的纯化装置,其特征在于,包括:(S1)任选的用于盛放待纯化的重组慢病毒的原料的第一容器;(S2)微滤单元,所述的微滤单元用于对待纯化的重组慢病毒进行微滤处理,从而获得经微滤处理的滤液;(S3)任选的浓缩单元,所述的浓缩单元用于对所述滤液进行浓缩处理,从而获得经浓缩的滤液;(S4)层析纯化单元,所述的层析纯化单元用于对来自微滤单元或来自浓缩单元的滤液进行层析纯化处理,从而获得经纯化的重组慢病毒载体;和(S5)收集单元,所述收集单元用于收集所述经纯化的重组慢病毒载体。
- 如权利要求8所述的纯化装置,其特征在于,所述的层析纯化单元包括:分子排阻层析单元和阴离子层析单元。
- 如权利要求8所述的纯化装置,其特征在于,所述的纯化装置还包括:(S6)核酶处理单元,所述核酶处理单元包括用于添加核酶的添加装置。
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Application Number | Priority Date | Filing Date | Title |
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KR1020207030556A KR20200136448A (ko) | 2018-03-28 | 2019-03-28 | Gmp 등급의 재조합 렌티바이러스 벡터의 정제된 제제의 대규모 제조 방법 |
SG11202009824WA SG11202009824WA (en) | 2018-03-28 | 2019-03-28 | Method for large-scale preparation of purified preparation of recombinant lentiviral vector at gmp grade |
JP2020552824A JP2021518757A (ja) | 2018-03-28 | 2019-03-28 | Gmpグレードでの組換えレンチウイルベクターの精製調製物の大規模調製のための方法 |
EP19778302.0A EP3778905A4 (en) | 2018-03-28 | 2019-03-28 | PROCESS FOR THE LARGE-SCALE PRODUCTION OF CLEANED RECOMBINANT LENTIVIRAL VECTORS IN GMP QUALITY |
US17/042,125 US20210009966A1 (en) | 2018-03-28 | 2019-03-28 | Method for large-scale preparation of purified preparation of recombinant lentiviral vector at gmp grade |
AU2019241300A AU2019241300A1 (en) | 2018-03-28 | 2019-03-28 | Method for large-scale preparation of purified preparation of recombinant lentiviral vector at GMP grade |
US17/034,168 US20210147826A1 (en) | 2018-03-28 | 2020-09-28 | Method for large-scale preparation of purified preparation of recombinant lentiviral vector at gmp grade |
JP2023210248A JP2024041758A (ja) | 2018-03-28 | 2023-12-13 | Gmpグレードでの組換えレンチウイルベクターの精製調製物の大規模調製のための方法 |
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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 |
CN110714029A (zh) * | 2019-11-06 | 2020-01-21 | 无锡生基医药科技有限公司 | 一种慢病毒载体全封闭生产的方法及系统 |
CN111876393A (zh) * | 2020-06-30 | 2020-11-03 | 恒瑞源正(上海)生物科技有限公司 | 一种大规模快速生产高纯度高活性慢病毒载体的方法 |
CN111876392A (zh) * | 2020-06-30 | 2020-11-03 | 恒瑞源正(上海)生物科技有限公司 | 一种大规模快速生产病毒载体的方法 |
CN116590346B (zh) * | 2020-09-15 | 2024-05-14 | 上海药明巨诺生物医药研发有限公司 | 一种慢病毒载体纯化方法 |
CN113373120B (zh) * | 2021-06-18 | 2022-04-26 | 浙江康佰裕生物科技有限公司 | Gmp级逆转录病毒载体的纯化方法与应用 |
CN116769736B (zh) * | 2023-08-17 | 2023-11-14 | 赛奥斯博生物科技(北京)有限公司 | 一种快速纯化慢病毒的生产工艺 |
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CN110317832B (zh) | 2022-07-05 |
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US20210147826A1 (en) | 2021-05-20 |
EP3778905A1 (en) | 2021-02-17 |
EP3778905A4 (en) | 2021-12-22 |
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