WO2022157488A1 - Procédé de production d'un vecteur lentiviral recombiné - Google Patents

Procédé de production d'un vecteur lentiviral recombiné Download PDF

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WO2022157488A1
WO2022157488A1 PCT/GB2022/050137 GB2022050137W WO2022157488A1 WO 2022157488 A1 WO2022157488 A1 WO 2022157488A1 GB 2022050137 W GB2022050137 W GB 2022050137W WO 2022157488 A1 WO2022157488 A1 WO 2022157488A1
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cells
perfusion
lentiviral vector
tff
production
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PCT/GB2022/050137
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English (en)
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Vladimir Slepushkin
Kinga NOWICKA
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Autolus Limited
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    • 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
    • C12N2740/16011—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 a process for the production of recombinant lentiviral vectors.
  • the described method combines the use of a rocking motion bioreactor, pH adjustment and harvest by perfusion to provide advantageously high yields.
  • Accompanying methods of lentiviral vector particle purification are also described.
  • Recombinant viral vectors have become an important tool for cell therapy applications, such as adoptive cell therapies using genetically engineered T cells carrying a modified T cell receptor or chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • lentiviral vectors part of the retrovirus family are useful due to their ability to transduce and integrate into the genome of both dividing and non-dividing cells.
  • Vector particles may be produced by the transient transfection of human cells, such as human embryonic kidney (HEK) 293T cells, with plasmids encoding the viral genome components and the transgene of interest. The cells must then be cultured under suitable conditions for optimal production of the viral particles.
  • human cells such as human embryonic kidney (HEK) 293T cells
  • the resulting viral particles must then also be purified before being used to transduce immune cells. This purification process typically leads to loss of some viral particles and must be optimized to give acceptable yields.
  • the present inventors have developed a method for the production of recombinant lentiviral vectors using a combination of cell culture in a rocking-motion bioreactor and harvesting by perfusion. This method results in improved yield compared to prior art methods. Furthermore, the method may also include downstream purification steps adapted to the production method which further improve the final yield of purified lentiviral vectors.
  • the present invention provides a method for the production of a recombinant lentiviral vector, comprising:
  • the pore size of the perfusion membrane used for harvesting may be selected to optimise lentiviral vector recovery.
  • Preferred pore sizes include 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, and 2.5 pm.
  • a particularly preferred pore size is 1.8 pm.
  • the purification in step (g) includes at least one of
  • the filtration step may employ a filter having a pore size of from 0.1 to 0.8 pm. More preferably, the purification step (g) comprises:
  • the endonuclease treatment is carried out at 4°C for from 6 to 12 hours.
  • the cells may be transfected with a mixture of polyethyleneimine (PEI) and plasmids.
  • PEI polyethyleneimine
  • the induction agent may be sodium butyrate.
  • the final concentration of sodium butyrate is from 5 to 9 mM. More preferably the final concentration of sodium butyrate is 9 mM.
  • the pH adjustment in step (e) may be carried out with carbon dioxide or hydrochloric acid, or a combination of carbon dioxide and hydrochloric acid.
  • the rocking motion bioreactor may be set to either 25 rpm at a 9° rocking angle or 15 rpm at a 12° rocking angle.
  • the recombinant lentiviral vector may be harvested by perfusion at a perfusion rate of from lOL/day to 40L/day.
  • the perfusion membrane may have a pore size of from 0.5 pm to 2.5 pm, preferably from 1.0 pm to 2.0 pm, more preferably 1.5 pm to 2.0 pm, most preferably 1.8 pm.
  • the perfusion membrane may be located in a fixed position relative to the rocking motion bioreactor.
  • steps (a) to (f) are carried out at 37°C.
  • the cells are HEK293 cells or HEK293T cells. Where HEK293T cells are used, they may be adapted for growth in serum-free media.
  • FIG. 3 Cell viability during expansion and production phases in Flexsafe RM perfusion bag (Sartorius) Cell Culture Viability (%) in runs; 002,003,004,005,005,006 and 007 (A), Cell Culture Viability in runs with no pH shift; 004,005,007 and in run with pH shift 006 (B) Cell Culture Viability in runs with no perfusion post-transfection and post-induction; Sar003,004 and with perfusion post-transfection and post-induction; 005,006,007 (C)
  • FIG. 4 Functional titre Harvest I (48 PT) and Harvest II (&2 hrs PT) in Sartorius Flex perfusion bag Comparison of functional particles concertation (TU/ml) produced in bioreactor in runs; 002, 003, 004, 005, 006, and 007(A). Comparison of functional particles between the production bag and collection bags in in runs; 002, 003, 004, 005, 006, and 007 (B).
  • FIG. 1 FACS plots transfection efficiency cell culture transfected in bioreactor and non transfected cells.
  • the percentage of CAT 19- positive cells determined by flow cytometry represent transfection efficiency for samples harvested 24 hrs post induction in runs;
  • FIG. 7 MADLS results reporting presence of specific particle populations at each stage of the DSP process according to size (d.nm) and physical particle concentration (pp/mL): a) and b) “Small aggregates”, c) and d) “Vector”, e) and f) “Larger aggregates”.
  • Figure 8 Lentivrial recovery obtained by MADLS, p24 ELISA and infectivity assay. TFF- C R - TFF concentration retentate, TFF-C P - TFF concentration permeate, TFF-D R - TFF diafiltration retentate, TFF-D P - TFF diafiltration permeate, SF - sterile filtered final lentiviral vector product.
  • Figure 9 a) p24 concentration results obtained using ABL inc HIV-1 p24 Antigen Capture Assay, and b) recovery results after mass balancing.
  • H 24h - Harvest collected after 24h and treated with Denarase® H 48h - Harvest collected after 48h and treated with Denarase®, TFF-I - clarified Denarase® treated harvest/TFF input, TFF-C R - TFF concentration retentate, TFF-C P - TFF concentration permeate, TFF-D R - TFF diafiltration retentate, TFF-D P - TFF diafiltration permeate, SF - sterile filtered final lentiviral vector product.
  • Figure 10 a) Lentiviral titre (TU/mL) and b) recovery after mass balancing. Infectivity assay was performed only once and 50 000 cells per well were correctly seeded. H 24 - Harvest collected after 24h and treated with Denarase®, H 48 - Harvest collected after 48h and treated with Denarase®, TFF-I - clarified Denarase® treated harvest/TFF input, TFF- C R - TFF concentration retentate, TFF-C P - TFF concentration permeate, TFF-D R - TFF diafiltration retentate, TFF-D P - TFF diafiltration permeate, SF - sterile filtered final lentiviral vector product.
  • the present invention provides a method of producing recombinant lentiviral vectors.
  • the present inventors have found that a combination of the use of a rocking motion bioreactor, pH adjustment and harvest by perfusion provides advantageously high yields
  • the lentiviral particles are produced by cells which have been transfected with one or more plasmids carrying the required genes for the production of virus particles that include the sequence of the desired transgene.
  • the viral genes used may include gag (group specific antigen), pol (polymerase), and env (envelope).
  • gag sequence encodes the three main structural proteins: the matrix protein, nucleocapsid proteins, and capsid protein.
  • the pol sequence encodes the enzymes reverse transcriptase and integrase, the former catalyzing the reverse transcription of the viral genome from RNA to DNA during the infection process and the latter responsible for integrating the proviral DNA into the host cell genome.
  • the env sequence encodes for both SU and TM subunits of the envelope glycoprotein.
  • the rev gene may also be included for regulatory purposes.
  • the cells are transfected with a plasmid carrying the transgene of interest and a sequence named packaging signal (y) required for specific packaging of the viral RNA into newly forming virions.
  • This plasmid may also include two LTRs (long terminal repeats), which contain elements required to drive gene expression, reverse transcription and integration into the host cell chromosome. In this way, the transgene of interest is incorporated into the viral particles in a form ready for expression in target cells after transduction.
  • these genes may be divided onto separate plasmids in order to avoid the inadvertent production of RCLs (replication-competent lentiviruses) via recombination events.
  • the cells are transiently transfected with plasmids carrying all the required elements so that the cells transiently produce viral particles.
  • certain viral genes may be permanently incorporated into a cell to produce a packaging cell line.
  • Packaging cell lines need only be transfected with the plasmid carrying the transgene of interest and associated packaging signal in order to produce viral particles. Once a cell carries all the elements required for production of viral particles it may be termed a producer cell.
  • rocking motion bioreactor will be understood by those of skill in that art to relate to any device that achieves mixing of a fluid via a rocking or wave motion. Such devices are advantageous because they ensure gas exchange without the high levels of turbulence observed when other mixing methods, such as stirring or shaking, are employed. This is particularly important where the bioreactor is used to culture physically sensitive cells.
  • the wave motion in the fluid is usually achieved by placing the fluid container on a rocking plate.
  • a rocking motion bioreactor may also be referred to as a rocking plate bioreactor, rocking bioreactor or rocker bioreactor, wave bioreactor, or wave-mixed bioreactor.
  • the rate and angle of rocking can be adjusted to control the rate of gas exchange.
  • the present method typically makes use of serum-free media.
  • Serum-free media is advantageous because the resulting vectors are suitable for therapeutic use and more easily comply with Good Manufacturing Practice (GMP) requirements.
  • GMP Good Manufacturing Practice
  • the media Once the media has been inoculated with the desired cells, they must be expanded in order to reach a cell density that will provide the desired quantity of viral particles on an acceptable time scale. In most cases, this will take from 1 to 4 days, depending on the exact conditions and cell type.
  • the preferred cells are human embryonic kidney (HEK) 293 cells.
  • HEK293T cells which have been transformed with the SV40 T-antigen may also be used once adapted for growth in serum-free media. Methods for adapting cells for growth in serum-free media are available to the skilled person.
  • Transfection of the expanded cells is achieved by introduction of plasmids carrying the elements required for lentiviral particle production. Typically transfection is achieved using a mixture of polyethyleneimine (PEI) and the required plasmids. After transfection, viral gene expression is induced via treatment with an induction agent.
  • the induction agent may be sodium butyrate.
  • the final concentration of sodium butyrate may be from 5 to 13 mM. A preferred final concentration of sodium butyrate is 9 mM.
  • the present method also makes use of a pH adjustment during cell culture. More specifically, the pH of the media is adjusted such that the pH in the range of from 6.2 to 7.0. In particular, the pH of the media may be adjusted such that it is in the range of from 6.4 to 6.8. The preferred pH of the media after adjustment is 6.8.
  • the adjustment of pH may be carried out with carbon dioxide or hydrochloric acid, or a combination of carbon dioxide and hydrochloric acid
  • the cells are expanded for a period while lentiviral vector particles are produced. This may be from 2 to 8 days and is typically carried out at 37 °C while the rocking motion bioreactor operates to mix the media. Exemplary settings for the rocking motion bioreactor are 25 rpm at a 9° rocking angle or 15 rpm at a 12° rocking angle.
  • recombinant lentiviral vector particles are harvested from the culture medium by perfusion through a perfusion membrane.
  • the rate of perfusion must be selected in order to maximise the level of perfusate while maintaining the viability of the cells.
  • the present inventors have found a perfusion rate of from lOL/day to 40L/day to be optimal. Harvesting at 40L/day has been found to be particularly advantageous.
  • the pore size of the perfusion membrane must be selected to provide optimal results.
  • the present inventors have found pore sizes in the range of from 0.5 pm to 2.5 pm to be useful in the present invention. More specifically, pore sizes in the range of from 1.0 pm to 2.0 pm or from 1.5 pm to 2.0 pm are useful. Preferred pore sizes include 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, and 2.5 pm. The most preferred pore size is 1.8 pm.
  • the perfusion membrane may be located in a fixed position relative to the rocking motion bioreactor. For example, the membrane may be fixed to the bottom of the fluid container. Without wishing to be bound by any one theory, the inventors believe fixing the position of the perfusion membrane in this way reduces turbulence in the reactor and increases cell viability.
  • the present invention also provides a method of vector purification, for use in conjunction with the method of vector production.
  • the purification will include at least one of the following steps:
  • endonuclease for example Benzonase® or Denarase®
  • an endonuclease removes host cell DNA by digestion.
  • endonuclease treatment is carried out at from 2 to 8 °C, preferably at 4 °C.
  • the treatment may be carried out overnight or anywhere in the range of from 6 to 12 hours.
  • the amount of endonuclease used can be adjusted depending on the exact enzyme used. For Denarase a typical amount would be 2.5 U/mL.
  • Filtration of the perfusate removes cell debris and other material.
  • the present inventors have found the use of a 0.45 pm filter to be preferable.
  • an additional filtration step at the end of purification can be included.
  • a filter with a smaller filter may be used.
  • the present inventors have found a pore size of 0.22 pm to be useful at this stage.
  • Filtration with a 0.6/0.2 pm filter may also be used.
  • this filtration step may be the final step in the purification process.
  • Tangential flow filtration is a filtration technique in which the fluid to be filtered flows in parallel to a filter rather than perpendicular to it. This arrangement prevents clogging of the filters.
  • TFF is widely used in the bio-pharmaceutical industry, typically for concentration and/or diafiltration. In the present method, TFF is used to concentrate the lentiviral vectors in the media, forming the product. The use of a TFF filter with an appropriate molecular weight cut off also results in the removal of endonuclease and other impurities from the product.
  • a final buffer exchange step can be included so that the final product is in the desired buffer, which may for example be adapted for long term storage or for use in the transduction of cells.
  • the purification step comprises:
  • the below procedure provides details of the generation of CAT 19 Lenitviral Vector using suspension HEK293T-EX cells from RCB bank.
  • the cells were revived and expanded in Erlenmeyer Shake Flask (SF) and cultivated at 37 °C and 8% CO2 in a FreeStyleTM 293 Expression Medium.
  • SF Erlenmeyer Shake Flask
  • HEK293T cells grow to maximum cell density (typically to 4xlO 6 /mL) in several shake flasks.
  • Cells were harvested and used to inoculate approximately IL in a 10L Flexsafe RM perfusion bag to the final concentration of 5xl0 5 /mL.
  • the 10L bag was mounted on a Xuri Cell Expansion System W25 bioreactor system (Cytiva). The cells were further expanded to 5xlO 6 /mL for over 3 days. After inoculation, cell suspensions were cultured in the bioreactor using the following settings: 25 rocks/min, 9° rocking angle, an airflow of 0.2 L/min.
  • Cell expansion in Flexsafe® RM bag prior to transfection was optimized. In run 3 perfusion started 48 Hrs post inculcation at Vi volume per day and increased to 1 volume per day 24 hrs later. In all other runs perfusion started 24 hrs post-inoculation and increased to 1 volume per day 24 hrs later.
  • HEK293T cells adapted to serum-free media were transiently transfected with four plasmids required for third-generation lentivirus (LV) production.
  • Third generation selfinactivating LVs were produced using a four-plasmid system: pVSVf, pGAG-Pol, pRev, and LV transient gene. Plasmids were manufactured by Plasmid Factory or extracted with the EndoFree Plasmid kit (Qiagen) according to the manufacturer’s instructions. Cells were transfected typically on day 3. To facilitate entry of the DNA plasmid into the host cell Polyethyleneimine (PEI) and a total of 34 mg of plasmids were used.
  • PEI Polyethyleneimine
  • the transfection mix was prepared in a safety cabinet according to the protocol (small scale) DNA and PEI were separately mixed with serum-free culture medium, mixtures were combined and incubated at room temperature. The volume corresponding to the transfection mixture was removed from the bioreactor and the mixture was added into the bioreactor. The transfection mix was transferred to IL sterile bottle and welded on to the bioreactor bag. Two different methods of transfecting bioreactors were investigated. The direct assertion of IL transfection mix and pre-diluted transfection mix in an additional IL of FreeStyleTM 293 Expression Medium. Pre dilution of transfection mix in the medium was implemented to improve the mixing of transfection mix in the bioreactor. In runs 3 and 4 perfusion was stopped, and the culture was left overnight at the settings below.
  • the vector was collected using perfusion mode. The different settings were investigated to improve the vector particle recoveries:
  • the collection time differed depending on the setting used. 24 hrs for lOL/per day rate and 6 hrs for 40L/per day rate. In runs 3, 4, and 5 when collecting the vector at 40L/per day perfusion rate after 6 hrs perfusion mode was stopped and the settings were changed back to 25 rpm and 9° angle until the next day. In runs 6 and 7 perfusion continued at lOL/day rate for approximately 24hrs.
  • the collection bag was sampled, and the supernatant was centrifuged at 2000g for 5 mins and filtered through a 0.45 pm syringe filter. Samples were stored at -80 °C for further analysis.
  • Figure 1 shows the total cell count and cell culture viability during the expansion phase in Flexfase RM perfusion bag (Sartorius) and CellBag (GE) prior to transfection.
  • Cell expansion in the bioreactor allows decreased inoculation amount, thus decreasing the amount of material and working hours needed to expand the cells before inoculation. Therefore, cell expansion in Flexsafe RM perfusion bag and CellBag prior to Transfection was determined and optimized.
  • FIG. 3 shows cell viability during expansion and production phases in Flexfase RM perfusion bag (Sartorius). Each data point is derived from the average value of two measurements taken on the NC-250 Nucleocunter at that timepoint. Overall increase viability was observed in runs with perfusion posttransfection and post-induction (range; 56-59% no perfusion, 67-69% with perfusion at Harvest II time point) As can be seen from this data cultures with HCL addition compared to those with pH shift with CO2 only didn’t show a significant difference in cell viability at any point during the LV- production (range; 59-66.5% no pH shit and 67-69% with pH shift).
  • P24 protein concertation was analyzed to estimate the total particle concertation at Harvest I and Harvest II as well as in LV collection bags. The physical titre was then used to calculate P:I ratio.
  • Lentiviral material was prepared according to the method described in Example 1 and was kept refrigerated at 2-8 °C until arrangements were made to proceed with downstream processing (DSP).
  • the final filtration step was carried out using an ULTA Capsule HC 0.6/0.2pm 2” filter with 95% volume recovery rate.
  • In-house filter assemblies were created and sterile filtration was performed using Watson Marlow pump at 25 RPM.
  • TFF was carried out at a shear rate of 4000s' 1 on an AKTA Flux 6 using a UFP-500-C-6A hollowfibre, with a membrane area of 0.48 m 2 .
  • Diafiltration buffer in this experiment was 50 mM HEPES 70 mM NaCl 5% sucrose pH 7.
  • Multiangle dynamic light scattering was performed on the day of DSP execution with fresh samples. Five measurements per sample were taken and analysed according to manufacturer’s instructions (Malvern Panalytical). Samples for other analytical assays were collected and stored at -80°C until the assays could be performed. Statistical data analysis (t-test, One-way Anova) was performed using GraphPad Prism 8.4.2. Recovery calculations after mass balancing were based on the TFF input sample: 100

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Abstract

La présente invention concerne un procédé de production d'un vecteur lentiviral recombiné, comprenant les étapes suivantes : (a) inoculation de milieux sans sérum dans un bioréacteur à mouvement de bascule avec des cellules humaines ; (b) multiplication des cellules pendant 1 à 4 jours ; (c) transfection des cellules avec au moins un plasmide adapté à la production d'un vecteur lentiviral ; (d) induction des cellules avec un agent d'induction ; (e) ajustement du pH des milieux entre 6. 2 à 7,0, de préférence de 6,4 à 6,8, plus préférablement à 6,8 ; (f) multiplication des cellules pendant 2 à 8 jours tout en récoltant le vecteur lentiviral recombiné produit à partir du milieu de culture par perfusion à travers une membrane de perfusion ; et (g) éventuellement purification du vecteur lentiviral recombiné.
PCT/GB2022/050137 2021-01-19 2022-01-18 Procédé de production d'un vecteur lentiviral recombiné WO2022157488A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013076309A1 (fr) * 2011-11-24 2013-05-30 Genethon Système de production d'un vecteur lentiviral pouvant être mis à l'échelle compatible avec des applications pharmaceutiques industrielles
WO2015177501A1 (fr) * 2014-05-21 2015-11-26 Imperial Innovations Limited Vecteurs lentiviraux
WO2019175600A1 (fr) * 2018-03-16 2019-09-19 Oxford Biomedica (Uk) Limited Système de production de vecteurs viraux

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013076309A1 (fr) * 2011-11-24 2013-05-30 Genethon Système de production d'un vecteur lentiviral pouvant être mis à l'échelle compatible avec des applications pharmaceutiques industrielles
WO2015177501A1 (fr) * 2014-05-21 2015-11-26 Imperial Innovations Limited Vecteurs lentiviraux
WO2019175600A1 (fr) * 2018-03-16 2019-09-19 Oxford Biomedica (Uk) Limited Système de production de vecteurs viraux

Non-Patent Citations (1)

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Title
ANSORGE S ET AL: "Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures", THE JOURNAL OF GENE MEDICINE, JOHN WILEY & SONS, INC, US, vol. 11, no. 10, 1 October 2009 (2009-10-01), pages 868 - 876, XP002689780, ISSN: 1099-498X, [retrieved on 20090720], DOI: 10.1002/JGM.1370 *

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