Method for large-scale preparation of purified recombinant lentivir vectors in GMP grade

Abstract

A method for large-scale preparation of a purified GMP-grade recombinant lentiviral vector preparation is provided, comprising the following steps: (a) providing a raw material, a feed solution having a volume Va, containing the recombinant viral vector to be purified; (b) subjecting the feed solution to a microfiltration process to obtain a microfiltrate having a volume Vb, containing the recombinant viral vector; (c) optionally concentrating the filtrate to obtain a concentrated filtrate having a volume Vc; (d) purifying the filtrate obtained in the previous step by chromatography to obtain a crude product containing the recombinant viral vector; and (e) liquid-exchanging and finely purifying the crude product obtained in the previous step to obtain a purified recombinant viral vector.

Description

Technical Field

[0001] The present invention relates to the technical field of biology, and particularly to a method for the large-scale preparation of a purified preparation of a GMP-grade recombinant lentiviral vector.

Background Art

[0002] Gene therapy refers to introducing an exogenous therapeutic gene into target cells to correct or compensate for diseases caused by gene defects or abnormalities, or to achieve the purpose of treatment by acting on disease targets through products expressed by exogenous genes.

[0003] The exogenous gene can be transduced or delivered by a viral vector or a non-viral vector. Common non-viral vectors include liposomes, dendrimers, unnatural cationic polymers, natural polysaccharides, etc. Non-viral gene delivery vectors are relatively safe and stable, but their transfection efficiencies are usually low. The viral vector packages the exogenous gene in the outer shell of a natural virus and uses the ability of the virus to infect host cells to introduce the exogenous gene into the cell. Common viral vectors include recombinant retrovirus (rRV), recombinant lentivirus (rLV), recombinant adenovirus (rAd), recombinant adeno-associated virus (rAAV), etc. The viral vector has a much higher transduction efficiency than the non-viral vector and is particularly suitable for infecting target cells that are difficult to infect, such as lymphocytes.

[0004] Recombinant lentiviral vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus-1). Unlike conventional retroviral vectors, recombinant lentiviral vectors have the ability to infect both dividing and non-dividing cells. Due to their high biological titer in vivo and in vitro, low immunogenicity, and other advantages, recombinant lentiviral vectors have become the preferred transgenic vectors for CART cells and gene therapy.

[0005] Current recombinant lentiviral vectors employ genetic modification methods to leave only the original transcription of the packaging signal and target gene in the lentiviral genome, while distributing the reverse transcriptase, envelope protein VSVG, gag-pol, and other structural genes across two to three vectors, and simultaneously deleting the pathogenic gene. Mature lentiviral particles are produced by simultaneously transfecting 293T cells with multiple vectors, then packaged within the cells, and secreted from the 293T cells into the culture supernatant, which can be obtained by ultracentrifugation or chromatographic purification.

[0006] The conventional laboratory method for obtaining lentiviruses is ultracentrifugation. While this method is simple, it cannot be scaled up industrially, and the prepared lentiviral vectors may contain high levels of endotoxins, BSA, HCP, or nucleic acids and other residues, making them unsuitable for direct use in the human body.

[0007] Furthermore, existing chromatographic purification methods also suffer from drawbacks such as complex processes, low yields, and insufficient purity, making them largely unsuitable for large-scale industrial production and GMP-grade production.

[0008] Therefore, there is an urgent need in this field to develop novel and efficient methods for preparing purified lentiviral vectors that are suitable for large-scale production and meet the requirements of GMP-grade production. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The objective of the present invention is to provide an efficient method for preparing purified lentiviral vectors that are suitable for large-scale production and meet the requirements of GMP-grade production.

[0010] Another object of the present invention is to provide recombinant lentiviruses purified by this method, as well as purified preparations containing recombinant lentiviruses, and applications thereof. [Means for solving the problem]

[0011] In a first embodiment of the present invention, a method for large-scale purification of recombinant viral vector preparations is provided. This method includes the following steps: (a) A step of providing a raw material which is a feed solution of volume Va containing a recombinant viral vector to be purified; (b) A step of performing microfiltration on the supply liquid to obtain a microfiltered filtrate of volume Vb containing the recombinant viral vector; (c) If necessary, a step of concentrating the filtrate to obtain a concentrated filtrate of volume Vc; (d) A step of purifying the filtrate obtained in the preceding step by chromatography to obtain a crude product containing a recombinant viral vector; (e) A step in which the crude product obtained in the preceding step is liquid-exchanged and refined to obtain a purified recombinant viral vector.

[0012] Here, the chromatography is selected from anionic chromatography, molecular exclusion chromatography, multimode composite resin chromatography, or a combination thereof. [Modes for carrying out the invention]

[0013] In another preferred embodiment, Va ≥ 100 L (or 100 to 500 L).

[0014] In another preferred embodiment, the method further includes the following steps after step (e): (f) A step of replacing the purified recombinant viral vector with a virus freezing medium containing the recombinant viral vector by liquid exchange of the purified recombinant viral vector; (g) After the liquid exchange described above, the virus is filtered and sterilized to obtain a sterile recombinant viral vector.

[0015] In another preferred embodiment, the virus includes a lentivirus.

[0016] In another preferred embodiment, this method complies with GMP conditions.

[0017] In another preferred embodiment, in step (d), molecular exclusion chromatography and anion chromatography are performed sequentially, sequentially, or simultaneously.

[0018] In another preferred embodiment, the anionic resin is selected from Capto Q, Capto ImpRes, and Capto DEAE.

[0019] In another preferred embodiment, a multimodal composite chromatography resin such as "Capto adhere ImpRes" or "Capto core 700" is used.

[0020] In another preferred embodiment, the chromatography purification may involve first performing primary purification by anion chromatography, followed by more refined purification by multimode complex chromatography.

[0021] In another preferred embodiment, the purification by chromatography can be first carried out by primary purification using multimode composite chromatography, and then by fine purification using anion chromatography.

[0022] In another preferred embodiment, the purification by chromatography can be carried out by connecting two multimode chromatography resins in series and then simultaneously adsorbing and removing impurities and capturing viruses.

[0023] In another preferred embodiment, after concentration, the time for the purification treatment of the feed solution by chromatography is 10 L / 30 min.

[0024] In another preferred embodiment, the processing speed of the purification by chromatography is such that the filtrate processed by chromatography is 20 L / 60 min.

[0025] In another preferred embodiment, the weight - volume ratio of the chromatography medium to the filtrate subjected to chromatography is 500 mL:10 L filtrate.

[0026] In another preferred embodiment, the pore size of the bacterial filter is 0.2 μM.

[0027] In another preferred embodiment, the chromatography medium is selected from Capto Q ImpRes.

[0028] In another preferred embodiment, in step (d), the purified recombinant lentiviral vector has one or more of the following characteristics: (p1) The biological titer of the recombinant lentiviral vector is 1.06x10

[0029] , , , , 9 , Tu / mL; (p2) BSA residue < 50 ng / mL; (p3) Endotoxin < 1 EU / mL.

[0029] In another preferred embodiment, the filtrate (including concentrated or unconcentrated filtrate) is subjected to ribozyme treatment before chromatographic purification.

[0030] In another preferred embodiment, the ribozyme treatment includes adding 10 U / ml of ribozyme and incubating it at 37°C for 30 minutes.

[0031] In another preferred embodiment, in step (b), a hollow fiber column for microfiltration is used for microfiltration (microfiltration).

[0032] In another preferred embodiment, the hollow fiber column for microfiltration is a microfiltration membrane having a cutoff value of 0.4 to 1.0 μm (preferably 0.45 to 0.8 μm).

[0033] In another preferred embodiment, in step (c), the ratio of Vb to Vc (Vb / Vc) is 5 to 50, preferably 10 to 30, and more preferably 15 to 25.

[0034] In another preferred embodiment, in step (c), concentration is carried out by ultrafiltration.

[0035] In another preferred embodiment, the ultrafiltration employs an ultrafiltration membrane having a cutoff value of 100 to 800 K.

[0036] In another preferred embodiment, the cutoff value of the hollow fiber column for ultrafiltration is 200 to 1000 K, preferably 300 to 500 K.

[0037] In another preferred embodiment, ultrafiltration employs a hollow fiber column and an ultrafiltration system for ultrafiltration.

[0038] In another preferred embodiment, the ultrafiltration system is selected from AKTA Flux 6 and AKTA Ready Flux.

[0039] In a second aspect of the present invention, a purified recombinant lentivirus prepared by the present method is provided.

[0040] In a third aspect of the present invention, a preparation comprising a purified recombinant lentivirus is provided.

[0041] In another preferred embodiment, the preparation is a pharmaceutical composition.

[0042] In another preferred embodiment, the pharmaceutical composition contains a pharmaceutically acceptable carrier.

[0043] A fourth aspect of the present invention provides a purification apparatus used in the above method. The purification apparatus is (S1) Any first container for holding the raw material for the recombinant lentivirus to be purified; (S2) A microfiltration unit used to perform microfiltration of the recombinant lentivirus to be purified and to obtain the microfiltered filtrate; (S3) Any concentration unit used to concentrate the filtrate in order to obtain a concentrated filtrate; (S4) A chromatography purification unit used to purify the filtrate from the microfiltration unit or the concentration unit by chromatography to obtain a purified recombinant lentiviral vector; (S5) A collection unit used to collect the purified recombinant lentiviral vector; Includes.

[0044] In another preferred embodiment, the first vessel, the microfiltration unit, the concentration unit, the chromatography purification unit, and the collection unit are in fluid communication.

[0045] In another preferred embodiment, the chromatography purification unit includes a molecular exclusion chromatography unit and an anion chromatography unit.

[0046] In another preferred embodiment, the molecular exclusion chromatography unit and the anion chromatography unit are independent of each other.

[0047] In another preferred embodiment, the molecular exclusion chromatography unit and the anion chromatography unit are integrated.

[0048] In another preferred embodiment, the purification apparatus further includes: (S6) A ribozyme processing unit equipped with an additive device for adding ribozymes.

[0049] In another preferred embodiment, the ribozyme processing unit further includes an incubation device for incubating the filtrate to which the ribozyme has been added.

[0050] It should be understood that, within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., embodiments) can be combined to form new or preferred technical solutions. Due to space limitations, these will not be described here.

[0051] Detailed explanation Following extensive and detailed research, and through large-scale screening and exploration of purification conditions, the inventors unexpectedly developed a rapid and simple method for the large-scale GMP-grade purification of recombinant lentiviruses with superior purification efficacy. In the method provided by the present invention, by using specific purification media and specific purification steps and conditions, manufacturing raw materials containing recombinant lentiviruses can be purified in a highly efficient, rapid, and large-scale manner, resulting in a highly purified recombinant lentivirus preparation with fewer impurities and no endotoxins. This completes the present invention.

[0052] term As used herein, the term “composite filler resin” refers to Capto Q, Capto ImpRes, or Capto DEAE.

[0053] As used herein, "composite filler resin" means chromatography using a composite filler resin.

[0054] As used herein, the terms “recombinant lentivirus” and “lentiviral vector” refer to lentiviral vectors that can be used in an interchangeable manner and are produced by introducing a specific plasmid into a specific packaging cell. Typically, these lentiviral vectors may be used in subsequent reactions to transfect a given cell (including human and non-human mammalian cells) for therapeutic or non-therapeutic purposes.

[0055] By using the GE AKTA device and a combination (not limited to) of the new generation Capto Core700 and Capto adhere ImpRes resins, high-purity lentiviral preparations can be rapidly obtained by the methods illustrated in the present invention.

[0056] Purified recombinant lentiviral vector preparations prepared by the method provided by the present invention can be used for the production of cells or gene therapies.

[0057] The present invention has the following main advantages: By purifying lentiviruses using "Capto Core 700" in combination with "Capto adhere ImpRes," high-purity lentivirus preparations can be rapidly obtained. [Examples]

[0058] The present invention will be further described below in relation to specific embodiments. It should be understood that these embodiments are intended to illustrate the present invention and not to limit its scope. Experimental methods in the following embodiments, without specific conditions, generally follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts refer to weight percentages and parts by weight.

[0059] Example 1: (1) Collection of feed solution: Collect the lentivirus feed solution.

[0060] (2) Microfiltration and clarification: a) Connect a 0.45-0.8 μM microfiltration hollow fiber column to an AKTA Flux 6 system and test its integrity; b) Sterilize the connected AKTA Flux 6 system with 1M NaOH; c) Wash the AKTA Flux 6 system with sterile water for injection; d) Wash the AKTA Flux 6 system with sterile 1XPBS; e) Pour 20L of recombinant lentivirus supply solution into the supply solution bucket in two batches, perform microfiltration, and collect the filtrate.

[0061] (3) Ultrafiltration concentrate: a) Connect a 300-800K ultrafiltration hollow fiber column to an AKTA Flux 6 system and test its integrity; b) Sterilize the connected AKTA Flux 6 system with 1M NaOH; c) Wash the AKTA Flux 6 system with sterile water for injection; d) Wash the AKTA Flux 6 system with sterile 1XPBS; e) Ultrafiltration concentration of the microfiltration lentivirus supply solution is performed using a 300-800K ultrafiltration column and an AKTA Flux 6 system, and the filtrate is discarded; f) Concentrate the lentivirus feed solution from 20L to 1-2L.

[0062] (4) Nuclease treatment: a) Add nuclease at a rate of 10-1000 U / mL to 1-2 L of lentivirus supply solution and mix well; b) Incubate overnight at 2-8°C.

[0063] (5) Operation to remove impurities and capture viruses by using Capto Core700 and Capto adhere ImpRes in conjunction: a) Connect Capto Core700 500mL and Capto adhere ImpRes 500mL in series and install them in an AKTA pure150 chromatography system; b) Sterilize the connected AKTA Flux 150 system with 1M NaOH; c) Wash the AKTA Flux 150 system with sterile water for injection; d) Wash the AKTA pure150 system with sterile lentivirus frozen medium; e) remainder; f) Load 1-2 L of supply solution, and use 20-50 mM Tris-Cl / 1-1.5 M NaCl for elution after loading, and collect the elution peaks.

[0064] (6) Ultrafiltration and liquid exchange: a) Connect a 300-800K ultrafiltration hollow fiber column to an AKTA Flux 6 system to verify the integrity of the test; b) Sterilize the connected AKTA Flux 6 system with 1M NaOH; c) Wash the AKTA Flux 6 system with sterile water for injection; d) Wash the AKTA pure6 system with sterile lentivirus frozen medium; e) Ultrafiltration and fluid exchange of the microfiltration lentivirus feed solution are performed using a 300-800K ultrafiltration hollow fiber column and an AKTA Flux 6 system, and the filtrate is discarded; f) Collect 100-300 mL of recombinant lentiviral vector.

[0065] (7) Filtration sterilization, subpackaging, and cryopreservation: a) Use a 0.2 μM filter to filter the purified lentivirus supply; b) Package the finished product in 1 mL / tube; c) Lentivirus preparations should be stored in an ultra-low temperature refrigerator (≤-70°C).

[0066] I. Results: (1) Final lentivirus product concentration 2-4 × 10 9 / mL (2) BSA <50 ng / mL; (3) HCP <1 ng / mL; (4) Nucleic acid residues <5pg / mL; (5) RCL negative.

[0067] II. Conclusion By using a 0.45-0.8 μM microfiltration hollow fiber column, a 300-800 K hollow fiber column, and a "Capto Core700 + Capto adhere Imp Res" composite packing material, high-purity lentivirus preparations could be rapidly obtained by performing clarification filtration, concentration, liquid exchange, and impurity removal in a stepwise manner.

[0068] All documents referenced in this invention are cited in this application by reference as each document is cited individually. Furthermore, based on the above teachings of this invention, those skilled in the art can understand that various changes or modifications can be made to the invention, and these equivalent forms also fall within the scope defined by the claims appended to this application.

Claims

1. A method for the large-scale purification of recombinant viral vectors, comprising the following steps: (a) A step of providing a raw material which is a feed solution containing a recombinant viral vector to be purified, wherein the volume of the feed solution is 20 L or more; (b) A step of performing microfiltration on the supply liquid to obtain a microfiltered filtrate containing the recombinant viral vector, wherein the microfiltration is performed using a hollow fiber column for microfiltration, and the hollow fiber column for microfiltration includes a microfiltration membrane having a cutoff value of 0.4 to 1.0 μm; (c) A step of concentrating the filtrate to obtain a concentrated filtrate, wherein the concentration is performed using a hollow fiber column for ultrafiltration having a cutoff value of 200 to 1000 K; (d) A step of purifying the filtrate by chromatography to obtain a crude product containing a recombinant viral vector; (e) A step of obtaining a purified recombinant viral vector by liquid exchange and purification of the crude product; Includes, Here, the chromatography in step (d) is anion exchange chromatography, and the eluate contains 1 to 1.5 M NaCl. A method wherein the recombinant viral vector is a recombinant lentiviral vector.

2. The method according to claim 1, wherein in step (d), the purified recombinant lentiviral vector has one or more of the following characteristics: (p1) The biological titer of the recombinant lentiviral vector is approximately 1.06 x 10⁻⁶ ９ It is Tu / mL; (p2) BSA<50ng / mL; (p3) Endotoxin < 1 EU / mL.

3. The method according to claim 1, wherein in step (c), concentration is performed by ultrafiltration.