METHOD FOR PRODUCING VIRUS

Abstract

The invention relates to a method for preparing an adenovirus comprising a) providing a host cell in a medium capable of supporting growth of said host cell b) contacting said host cell with an adenovirus c) incubating to allow infection of said cell by said adenovirus d) incubating to allow production of adenovirus by said host cell wherein said host cell is, or is derived from, a HEK293 cell, and wherein the medium comprises BalanCD HEK293. The invention also relates to adenovirus produced, and to compositions comprising said adenovirus.

Claims

1. A method for preparing an adenovirus comprising a) providing a host cell in a medium capable of supporting growth of said host cell b) contacting said host cell with an adenovirus c) incubating to allow infection of said cell by said adenovirus d) incubating to allow production of adenovirus by said host cell wherein said host cell is, or is derived from, a HEK293 cell, and wherein the medium comprises BalanCD HEK293

2. A method according to claim 1 wherein feed is added to said medium, and wherein the feed comprises BalanCD HEK293 Feed.

3. A method according to any preceding claim wherein said host cell comprises a HEK293-T-REx cell, an Expi293F cell, or an Expi293F inducible cell.

4. A method according to any preceding claim wherein infection of said cell by said adenovirus is carried out at a cell density of at least 2e6 cells/mL, optionally wherein infection of said cell by said adenovirus is carried out at a cell density of between 4e6 cells/mL to 7e6 cells/mL, preferably about 6e6 cells/mL.

5. A method according to any preceding claim wherein feed is added to said medium in an amount of 5% of starting medium volume every 24-48 hours.

6. A method according to any preceding claim wherein feed is added to said medium in the period 48 hrs before infection to 48 hrs after infection.

7. A method according to any preceding claim wherein said adenovirus is, or is derived from, a simian adenovirus.

8. A method according to any preceding claim wherein said adenovirus is, or is derived from, a species E simian adenovirus.

9. A method according to any preceding claim wherein the adenovirus is ChAdOx1 or ChAdOx2.

10. A method according to claim 9 wherein the adenovirus is ChAdOx1.

11. A method according to claim 10 wherein the adenovirus is ChAdOx1 nCoV-19.

12. A method according to any preceding claim wherein said adenovirus comprises a nucleotide sequence capable of directing expression of Ad5 E4orf6 in said host cell.

13. A method according to claim 12 wherein said Ad5 E4orf6 comprises the sequence of Uniprot Accession Number: Q6VGT3.

14. A method according to any preceding claim wherein said adenovirus comprises a heterologous nucleotide sequence capable of directing expression of an antigen of interest.

15. A method according to any preceding claim wherein said antigen of interest comprises, or consists of, SARS-CoV2 spike protein.

16. A method according to any preceding claim wherein said heterologous nucleotide sequence is under the control of the Tet Repressor.

17. A method according to any preceding claim wherein the host cell expresses the Tet Repressor.

18. A method according to any preceding claim wherein said host cell is contacted with said adenovirus at a multiplicity of infection (MOI) of 3 to 10.

19. A method according to any preceding claim wherein said adenovirus is produced at a yield of at least 210.sup.14 vp/litre of medium.

20. A method according to any preceding claim wherein said method comprises use of a fed batch culture.

21. A method for preparing an adenovirus wherein said method comprises use of a fed batch culture.

22. An adenovirus prepared by a method according to any preceding claim.

23. A composition comprising an adenovirus according to claim 22.

24. A composition according to claim 23 which is a pharmaceutical composition.

25. A composition according to claim 24 which is a vaccine composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0284] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

[0285] FIG. 1 shows graphs, bar charts and a table.

[0286] FIG. 2 shows graphs, diagrams and tables.

[0287] FIG. 3 shows graphs, tables and a diagram.

[0288] FIGS. 4A and 4B show graphs of medium adaptation.

[0289] FIGS. 5A and 5B show graphs of growth and feeding.

[0290] FIG. 6A and 6B show a bar chart and a table of yield in shake flasks (ChAdOx1 luciferase)(Experiment CJ7).

[0291] FIG. 7 shows a bar chart of yield in shake flasks (ChAdOx2 GFP) (Experiment CJ9)

[0292] FIG. 8 shows a plot of yield in shake flasks (ChAdOx1 Lassa) (Experiment CJ9)

[0293] FIG. 9 shows a bar chart of volumetric productivity in different culture media.

[0294] FIG. 10 shows graphs of volumetric productivity and cell-specific productivity at different cell densities at infection.

EXAMPLES

[0295] All results in Examples 1-10 were obtained with BalanCD293 medium/feed combination & HEK293 T-REx cells.

[0296] ChAdOx1 nCoV-19 manufacturing scale up: 50 L and 200 L runs were performed at Pall Biotech, Portsmouth, UK.

Example 1: Growth Of ChAdOx1 Luciferase in Shake Flasks

[0297] We refer to FIG. 6. We refer FIG. 1A.

[0298] D indicates use of the medium/feed strategy stated in Methods, including 2-fold dilution immediately prior to infection. Lack of D indicates the strategy was modified by the omission of this 2-fold dilution.

Example 2: Growth of ChAdOx2 GFP in Shake Flasks

[0299] We refer to FIG. 7. We refer to FIG. 1B.

Example 3: Growth of ChAdOx1 LassaSpike in Shake Flasks

[0300] We refer to FIG. 8. We refer to FIG. 1B.

Example 4: Growth of ChAdOx1 Luciferase in 3L Stirred Tank Bioreactor

[0301] A 3L stirred tank reactor (STR) was infected and harvested as previously described (Fedosyuk et a1 2019 Vaccine vol 37 pages 6951-6961), with the exception of the use of the BalanCD293 medium/feed strategy, with a starting cell density of 4e6 cells/mL (diluted from 8e6 cells/mL) and an MOI of 10. Yield is shown in the Table below:

TABLE-US-00008 TABLE ChAdOx1 luciferase upstream process yield from 3L STR (experiment CJ10) VP/mL IU/mL P:I BalanCD luciferase bioreactor 1 5.8E+11 1.4E+10 41

Example 5: 50 L Stirred Tank Bioreactor Production & Purification of ChAdOx1 nCoV-19

[0302] A 40 L culture was infected and harvested in a 50 L STR, as previously described (Fedosyuk et al 2019 Vaccine vol 37 pages 6951-6961 (doi: 10.1016)), with the exception of the use of the BalanCD293 medium/feed strategy, with a starting cell density of 2e6 cells/mL (diluted from 4e6 cells/mL) and an MOI of 10.

[0303] The product was subsequently purified using a strategy as previously described (Fedosyuk et a1 2019 Vaccine vol 37 pages 6951-6961 (doi: 10.1016)). The final purified product yield was 1.810.sup.11 VP per mL of the upstream process (FIG. 1E-G).

Example 6: Comparative Data

[0304] Here we illustrate the improvement (e.g. in yield) compared to known methods. Comparative data is presented.

[0305] The inventor asserts that the benchmark in the art is yields <1e11 VP/mL for non-perfusion processes.

[0306] Firstly, specific to simian Adenoviruses, we refer to FIG. 1B.

[0307] This shows side-by-side comparisons for 2 ChAdOx1 & 1 ChAdOx2 viruses.

[0308] This clearly shows the prior art process performs at a much lower productivity, and the methods of the invention deliver a clear technical benefit compared to the prior art process.

[0309] In addition, we refer to Table 1 in Fedosyuk et al 2019. This shows productivity of the known process across 8 runs with 3 viruses. The highest is 1.4e11 VP/mL and the mean is <1e11 VP/mL (=1e14 VP/L).

Example 7: Production/Upstream Process

[0310] We initially investigated medium/feed combinations for a fed-batch USP. BalanCD293 medium and feed (Fujifilm) was found to support growth of the producer cells (HEK293 T-rex, Thermo) to 1.210.sup.7 cells/mL with high viability (FIG. 1A). Using this combination in small-scale production of adenovirus vectors of two serotypes and carrying three transgenes, we attained productivity exceeding 510.sup.11 virus particles (VP) per mL, around 5-fold greater than typically obtained in our previous USP (FIG. 1B). To our knowledge such productivity has not previously been reported from a non-perfusion USP: a fall in cell-specific productivity to <110.sup.5 VP/cell is commonly observed at cell densities exceeding 110.sup.6 cells/mL (Kamen, A. and O. Henry, Development and optimization of an adenovirus production process. J Gene Med, 2004. 6 Suppl 1: p. S184-92).

[0311] Using ChAdOx1 nCoV-19 starting material, we initially investigated the optimal multiplicity of infection (MOI), cell density and time of harvest (FIG. 1C-D). An MOI of 10, cell density of 2-310.sup.6/mL and time of harvest 42-48 hours post infection were selected for further work. This USP was subsequently scaled up, with USP productivities in 10 L, 50 L and 200 L stirred tank reactors (STR) in the range 2-410.sup.11 VP/mL, and acceptable cell growth and metabolite profiles (FIGS. 1E-F).

[0312] Early STR batches made use of a DSP very similar to that we had previously described, which again achieved recovery of 50-60% and quality characteristics compliant with a regulator-accepted specification for product for clinical use (FIG. 1G) (Fedosyuk et al 2019 Vaccine vol 37 pages 6951-6961).

[0313] We refer to FIG. 1 which shows development of fed batch process.

[0314] Panel A shows cell counts (solid lines, filled symbols) and viability (dashed lines, open symbols) attained during growth of HEK293 T-rex cells in BalanCD medium with (triangles) or without (squares) feed, as compared to the CD293 medium (circles, Thermo) used in our previous process. In the case of the BalanCD medium +feed condition, feed (5% v/v) was added at 36 and 108 hours.

[0315] Panel B shows small-scale USP productivity of ChAdOx1-luciferase, ChAdOx1-LassaGP, and ChAdOx2-GFP with BalanCD medium/feed (infected at 410.sup.6 cells/mL, MOI=10) as compared to our previously established conditions in CD293 medium (infected at 110.sup.6 cells/mL, MOI=3) (Fedosyuk et al 2019 Vaccine vol 37 pages 6951-6961).

[0316] ChAdOx1-luciferase infections were performed in a 3L bioreactor; the other two viruses were produced in 30 mL volume in shake flasks.

[0317] Panels C-D show small-scale USP productivity of ChAdOx1 nCoV-19 in shake flasks at 30 mL working volume at MOI=3 (C) and MOI=10 (D) respectively. Legend indicates cell density represented by each line, in million cells/mL at point of infection.

[0318] Infectious unit (IU) titers broadly paralleled VP titers; results are representative of two replicate experiments.

[0319] For panels A-D, points indicate median and error bars show range of results for 2-3 replicate flasks.

[0320] Panels E-F: Examples of 50 L and 200 L USP runs (carried out by Pall Biotech). (E) shows cell growth (solid lines) and viability (dashed lines). (F) shows glucose (solid lines) and lactate (dashed lines) concentrations.

[0321] Panel G: Examples of quality of drug substance from 50 L and 200 L runs (carried out by Cobra).

Example 8: Purification/Downstream Process

[0322] The inventors proceeded to scale the process to 1000-2000 L. Handling of the large volumes of lysate, diafiltration buffer and waste during the initial TFF step was identified as a process bottleneck. We therefore developed a simplified DSP by loading the clarified lysate directly on an AEX membrane, followed by a single TFF diafiltration polish/formulation step. As well as removing the initial TFF step, this process change provides the option of execution of clarification and AEX as a single unit operation (FIG. 2A). Small-scale experiments were used to demonstrate feasibility and optimise conditions of the AEX step (FIG. 3A-D), prior to execution of the complete revised DSP at 10 L scale (FIG. 2B). Binding capacity of the AEX membrane was reduced (to c. 310.sup.13 VP per mL of membrane, as compared to >510.sup.13 VP with loading of a diafiltered feed), but it was typically possible to recover c. 90% of loaded virus in the eluate. After a final TFF step, overall recovery was typically c. 70% and host cell protein and DNA were reduced to acceptable levels (FIG. 2C). Efficient recovery of adenovirus from such a direct load AEX has not, to the inventors' knowledge, previously been disclosed. The ability to achieve this enabled the simplification of the DSP from four steps to two or three.

[0323] The simplicity and high productivity of the method of the invention are beneficial from an economic perspective. Process economic modelling of drug substance manufacturing suggests a cost of goods of <USD 2 per dose (FIG. 2D). Final prices may be somewhat higher due to additional costs, for example fill and finish.

Example 9: Distributed Manufacturing

[0324] COVID-19 poses a unique challenge for vaccine manufacturing, both due to the volume and speed required, and due to concerns about equitable vaccine distribution and so-called vaccine nationalism. Distributed manufacturing (manufacturing the same vaccine across multiple facilities in different countries) is a potential solution to these issues, but requires a readily transferable process (Gomez, P. L. and J. M. Robinson, Vaccine Manufacturing. Plotkin's Vaccines, 2018: p. 51-60.e1). Our process is well-suited to this strategy as it uses unit operations which are standard across the bioprocess industry, single-use product-contact materials throughout, and a viral vector of good biosafety (BSL1-2, dependent upon jurisdiction). Many contract manufacturing organisations (CMOs) are already equipped to execute such processes with little or no capital expenditure. We therefore pursued a distributed manufacturing strategy from an early stage of the pandemic. ChAdOx1 nCoV-19/AZD1222 drug substance is now being manufactured at 1000 L scale in facilities in multiple countries (FIG. 2E).

[0325] We refer to FIG. 2 which shows Improved process, enabling cost-effective large-scale distributed manufacture

[0326] Panel A shows schematics of previous and revised DSPs. The dashed box indicates the feasibility of execution of depth filter clarification and AEX as a single unit operation. Panel B shows chromatogram obtained when running in-line depth filter clarification and AEX at 10 L scale. Absorbance at 280 nm is shown (line showing 4-5 arbitrary units between 50 and 100 minutes), and conductivity in also shown (other line). For schematic of process skid, see FIG. 3E. A 150 mL Sartobind Q capsule was loaded at 3.810.sup.13 VP per mL of membrane. Numerals indicate stages: 1=loading, 2=wash, 3=elution, 4=1M sodium hydroxide sanitisation.

[0327] Panel C shows product recovery and quality from the 10 L process shown in Panel B and after final formulation by TFF.

[0328] Panel D tabulates modelled costs of drug substance production at 200 L scale. This includes capital for purchase of all equipment needed for the process; many facilities do not require this.

[0329] Panel E shows, in dark grey, countries in which ChAdOx1 nCoV-19/AZD1222 is currently being manufactured.

[0330] We refer to FIG. 3 which shows small-scale optimisation of anion exchange with direct loading of clarified lysate.

[0331] Panel A shows gradient elution of ChAdOx1-luciferase from Sartobind Q anion exchange membrane. Filtered lysate containing 910.sup.13 VP of ChAdOx1-luciferase was loaded onto a 3 mL Sartobind nano Q capsule, followed by elution with a gradient of increasing salt concentration. Two peaks were observed (chromatogram) and analysed. Coomassie-stained SDS-PAGE, with comparison to virus purified by caesium chloride gradient ultracentrifugation (+C), shows that peak 1 contains impurities (notably free hexon protein) while Peak 2 contains virus. This was corroborated by infectivity assay. n.d. indicates not detected.

[0332] Panel B shows initial estimation of binding capacity and product recovery by step elution. Clarified lysate containing 210.sup.14 VP of ChAdOx1 nCoV-19 was loaded onto a 3 mL Sartobind nano Q capsule. Binding capacity and quantity of product bound was determined by collection of serial fractions of flow-through during loading (step 1). After washing with equilibration buffer (step 2), product was eluted with a step to 39-40 mS/cm (step 3); steps 4 and 5 indicate regeneration with 1M NaCl and 1M NaOH respectively. Flowthrough and elution fractions were analysed by qPCR to calculate binding capacity and recovery: 10% breakthrough occurred at a load of 3.410.sup.13 VP per mL of bed volume; 90% of bound product was recovered.

[0333] Panel C shows optimisation of salt concentration/conductivity during loading and washing. Three runs were performed, in each of which filtered lysate containing 210.sup.14 VP of ChAdOx1 nCoV-19 was loaded onto a 3 mL Sartobind nano Q capsule, after adjustment of conductivity to the indicated values by addition of salt. Each run used wash buffer with conductivity matching the load. The chromatogram overlays results from the three runs. Eluates were analysed by Coomassie-stained SDS-PAGE, qPCR and HCP ELISA. Recovery and HCP are tabulated: the basal condition (24-25 mS/cm) achieved nearly 2-log.sub.10 reduction in HCP; increasing the conductivity of the load and wash achieved modest improvement in HCP clearance, with substantially reduced recovery with loading at 30-31 mS/cm. As virus in the flowthrough was not quantified, recovery is shown as % of loaded product (rather than bound product, as in panel B); the low recovery for the basal condition here thus reflects the overloading of the column.

[0334] Panel D shows initial results obtained using a 150 mL Sartobind Q capsule with relatively low load challenge. Clarified lysate containing 7.410.sup.14 VP of ChAdOx1 nCoV-19 was loaded, using conditions as indicated, based upon the results of the experiment shown in Panel C. The eluate was analysed by qPCR, HCP ELISA and infectivity assay. Recovery as % of loaded product, HCP and P:I ratio are tabulated.

[0335] Panel E illustrates system used for in-line clarification and anion exchange (P: pump; DF: depth filter; F: 0.2 m filter), results of which are shown in FIG. 2B-C.

Viruses

[0336] The ChAdOx1 nCoV-19, ChAdOx1 Lassa-GP, ChAdOx1 luciferase and ChAdOx2 GFP vectors used here have previously been described.sup.1-4. Virus used as seed to infect shake flask cultures and as standards in quality control assays was produced by caesium chloride density-gradient ultracentrifugation by the Jenner Institute Viral Vector Core Facility. Virus used as seed to infect 50 L and 200 L cultures was prepared using our previously described process in 3 L shake flasks or bioreactors, up to the point of the first tangential flow filtration (TFF) step.sup.5. After this the concentrated and diafiltered lysate was aliquoted and frozen at 80 C.

Cells and Upstream Process

[0337] HEK293 T-rex cells (ThermoFisher) were banked and adapted to low-serum suspension culture in CD293 medium (ThermoFisher) as previously described.sup.5. Cells were then adapted to increasing proportions of BalanCD293 medium (Fujifilm-Irvine Scientific), supplemented with 4 mM GlutaMAX (ThermoFisher), over one week.

[0338] For upstream process experiments, seed culture at 2 the specified final density was diluted by addition of 1 volume of fresh medium to reach the cell density specified for each experiment at the point of infection. A multiplicity of infection of 10 was used unless otherwise stated.

[0339] Each feed was with 0.05 volumes of BalanCD293 feed (Fujifilm-Irvine Scientific). Pre-infection, cultures were fed on the day cell density exceeded 110.sup.6 cells/mL. Cultures for which the intended cell density at the point of infection was 310.sup.6 cells/mL received a second pre-infection feed when cell density exceeded 410.sup.6 cells/mL. Post-infection, all cultures were fed at 0.5 h and 22 h after infection.

[0340] Shake flask experiments were performed in Erlenmeyer flasks (Corning), with a working volume of 25-35 mL in a 125 mL flask unless otherwise stated. BioBlu 3c and 14c (Eppendorf) single-use bioreactor vessels were used in accordance with the manufacturers' instructions. A GX bioreactor controller unit and C-BIO software (both from Global Process Control) were used to control both vessel types. Dissolved oxygen (DO) was regulated at a setpoint of 55% air saturation by addition of medical air via macrosparger. pH was regulated in the range 7.2-7.3 as previously described.sup.5.

[0341] 50 L and 200 L upstream processes were performed using Pall Allegro stirred tank reactors (STRs).

[0342] Unless otherwise stated, bioreactors were seeded at 0.4-0.610.sup.6 cells/mL in c. 35% of the maximum working volume. Antifoam C emulsion (SigmaAldrich) was used in 50 L and 200 L STRs. 0.05 culture volumes of BalanCD feed was added when the density reached to 1.010.sup.6 culture cells/mL. At a cell density of 4.010.sup.6/mL (range 3.0-6.010.sup.6), cells were diluted with 1 volume of medium and infected, using an MOI of 10 unless otherwise stated. 0.05 volumes of BalanCD feed were added 30 minutes after infection, and again after 22 hours.

Lysis, Nucleic Acid Digestion and Clarification

[0343] Lysis was performed as previously described.sup.5, in the culture vessel, with the exception that the concentration of Benzonase (MerckMillipore) was reduced to 15 units/mL. Lysis was initiated at 42-48 h after infection, with the exception of the productivity kinetic experiments shown in FIGS. 1C-D. Two hours after addition of lysis buffer, clarification was initiated, using Millistak+ HC Pro CoSP depth filters as in our previous work.sup.5. During 200 L runs, an Advanced MVP skid (Pall Biotech) was used for filtration steps.

Tangential Flow And Bioburden Reduction Filtration

[0344] Tangential flow filtration was performed essentially as we have previously described.sup.5, scaled appropriately and with the following modifications. Where TFF was performed before anion exchange (AEX), i.e. for the 200 L run producing product as reported in FIG. 1G, only 2-fold concentration was performed, prior to 6 diavolumes of diafiltration. For TFF after AEX, Omega T-series 300 kDa cut-off flat sheet filters (Pall Biotech) were used. For TFF during 200 L runs, an Allegro CS 4500 single-use TFF skid was used (Sartorius). A Supor EKV 0.2 m filter was used for bioburden reduction filtration after the final TFF.

Anion Exchange Chromatography

[0345] Where preceded by TFF (run reported in FIG. 1G), AEX was performed as previously reported.sup.5, with scaling of the chromatography capsule and buffer volumes based upon anticipated binding capacity of 710.sup.13 VP per mL of membrane volume.

[0346] For direct-load AEX (loading clarified lysate), small-scale studies (Supplementary FIG. 1) were performed using an Akta Pure (GE) and 3 mL bed volume/8 mm bed height SartobindQ Nano capsules (Sartorius). Equilibration buffer comprised 20 mM Tris-HCL pH8.0, 1 mM MgCl.sub.2, 0.1% v/v polysorbate 20, 5% w/v sucrose; this was also used as a base for wash buffers. Elution buffer comprised 20 mM Tris-HCL pH8.0, 1 mM MgCl.sub.2, 0.1% v/v polysorbate 20, 5% w/v sucrose, 600 mM NaCl, except where salt concentration was varied, as stated. Adjustment of the conductivity of the sample and wash buffers, to target values as stated in the descriptions of individual experiments, was performed using 5M NaCl (Sigma).

[0347] Column equilibration was in accordance with the manufacturer's instructions. After loading, capsule was washed with 10 MV of equilibration buffer before elution step (both at 5 MV/min).

[0348] For the direct-load AEX purification from a 10 L bioreactor (FIG. 2B) a peristaltic pump-driven rig was constructed, as shown in Supplementary FIG. 1E, incorporating a CoSP depth filter (as above), Millipak-20 0.2 m filter, and 150 mL/8 mm bed height Sartobind Q capsule (Sartorius), plus single-use UV absorbance, conductivity and pressure sensors (Pendotech). Buffers, column equilibration, sample loading, washing and elution were as described above, with the exception that a flow rate of 0.7 membrane volumes/minute was used for sample loading, washing and elution.

Product Quantification and Assessment Of Product Quality

[0349] Product quantification was as previously reported, using qPCR and UV spectrophotometry assays for viral particles in impure and pure samples respectively, and an immunostaining-based infectivity assay.sup.5.

[0350] Residual host-cell protein (HCP) was quantified using the HEK293 HCP ELISA kit (Cygnus Technologies), according to the manufacturer's instructions. Residual host cell DNA was quantified using a previously reported quantitative PCR method targeting a 94 base pair amplicon within the Alu repeats.sup.6. The lower limit of quantification was 100 pg/mL for intact HEK293 DNA.

References to Examples 7, 8 and 9

[0351] 1 van Doremalen, N. et al. ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. Nature, doi: 10.1038/s41586-020-2608-y (2020).

[0352] 2 Purushotham, J., Lambe, T. & Gilbert, S. C. Vaccine platforms for the prevention of Lassa fever. Immunol Lett 215, 1-11, doi: 10.1016/j.imlet.2019.03.008 (2019).

[0353] 3 Dicks, M. D. et al. Differential immunogenicity between HAdV-5 and chimpanzee adenovirus vector ChAdOx1 is independent of fiber and penton RGD loop sequences in mice. Sci Rep 5, 16756, doi: 10.1038/srep16756 (2015).

[0354] 4 Morris, S. J. S., Sarah; Spencer, Alexandra J.; Gilbert, Sarah C. Simian adenoviruses as vaccine vectors. Future Virology 11, 649-659, doi: 10.2217/fvl-2016-0070 (2016).

[0355] 5 Fedosyuk, S. et al. Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components. Vaccine, doi: 10.1016/j.vaccine.2019.04.056 (2019).

[0356] 6 Zhang, W. et al. Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples. J Pharm Biomed Anal 100, 145-149, doi: 10.1016/j.jpba.2014.07.037 (2014).

Example 10: Antigen Repression

[0357] Here we show data which illustrate that the antigen repression, such as provided by the most suitable HEK293 T-REx cells, delivers the advantage of high productivity (enhanced productivity compared to non-repression of antigen).

[0358] The conditions used were the BalanCD293 fed batch production process (upstream process) as described above.

[0359] The data show that for the most suitable specific product (ChAdOx1 nCoV-19) there is a strong enhancement of productivity with repression compared to without repression.

[0360] The data also show that this is not an essential feature of the invention (i.e. repression is not always necessary for production of all adenoviruses), but is rather an advantageous embodiment.

[0361] Thus it is shown that the benefit of the method of the invention (fed batch upstream process) is indeed generalisable to other cell lines (i.e. to cells other than the most suitable HEK293 T-REx cells).

[0362] Suitably when the adenovirus comprises nucleic acid encoding an antigen capable of being expressed which is inhibitory to the adenovirus production, expression of said antigen is repressed.

Results

[0363] 1. Chadox1-nCov19 in T-REx

[0364] a. with tetracycline (which removes the repression, allowing antigen expression):

[0365] 4.3E+09 VP/mL

[0366] b. without tetracycline (i.e. with the antigen repressed): 2.4E+11 VP/mL

[0367] 2. In Expi293 cells (i.e. no tet repression)

[0368] a. Chadox1-ncov19 (antigen expressed): 1.1E+10 VP/mL

[0369] b. Chadox1-luciferase (antigen expressed but the luciferase protein is relatively non-interfering for adeno production): 1.9E+11 VP/mL

Example 11: Production in EXPI293 Inducible Cells

[0370] Here we show data that illustrate that the use of BalanCD media with the HEK293-derived cells, Expi293F inducible cells (available from Thermo Fisher (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA 02451, USA) Catalogue number: A39241) achieves a more than 1.5 fold increase in volumetric productivity compared with the same cells grown in the Expi293 medium. This increased volumetric productivity is demonstrated for both culture in a shaker, and culture in a stirred tank reactor.

Methods

[0371] For both the experiments with Expi293F-inducible cells in BalanCD medium (in shaker and stirred tank reactor), frozen cells were revived in 30 mL volume of Expi293 medium. 48 hours later cells were split 1:2 in the same volume with 15 ml of fresh medium. 24 hours later, cell density was adjusted to 0.5e6 cells/ml final viable cell density (VCD) with 75% volume (22.5 mL) Expi293 medium and 25% (7.5 mL) BalanCD medium. 24 hours later further adapted the cell by adjusting the density to 1 e6 cells/ml final VCD with 50% volume (15 mL) Expi293 medium and 50% (15 mL) BalanCD medium. 48 hours later, adjust density to 0.5e6 cells/ml final VCD with BalanCD medium. Thereafter cells were diluted with 100% BalanCD medium.

[0372] For the experiments in the shaker:

[0373] Expi293F inducible cells were cultured in 125 mL shake flasks in duplicates. 0.05 culture volumes (i.e. 5% of the starting medium volume) of BalanCD feed was added when the density reached 1e6 culture cells/mL. At a cell density of 4.4e6 cells/ml, cells were diluted with 1 volume of medium (final volume of 30 mL) and infected, using an MOI of 5. Subsequently, 0.05 volumes of BalanCD feed were added 30 minutes after infection, and again after 22 hours. The cultures were then harvested 47 hour after infection. At harvest 1/9 volume of 10 Lysis buffer and 100 IU/ml benzonase was added into each flask and incubated for 2 hours before they were diluted 1:200 in A438 formulation buffer for qPCR or non-diluted for infectivity analysis. All samples were frozen at 80 C. before analysis.

[0374] For the experiments in the reactor:

[0375] 3 L upstream processes were performed using 3cBioBlu Eppendorf stirred tank reactors (STRs). The process was run as above, with the exception that dissolved oxygen was maintained at 55% and pH was adjusted with 7.5% sodium bicarbonate. The cultures were then harvested 47 hour after infection, as above.

Results

[0376] FIG. 9, left and middle columns show the comparison of volumetric productivity of ChAdOx1-nCoV19 in a shaker using Expi293F inducible cells in Expi293 medium (left column) and BalanCD medium (middle column). This shows a more than 1.5 fold increase in volumetric productivity when using BalanCD medium compared to Expi293 medium.

[0377] FIG. 9, right column shows the volumetric productivity of ChAdOx1-nCoV19 in a reactor (3c Bioblu) using Expi293F inducible cells in a 3L scale using BalanCD medium. This demonstrates that the increased volumetric productivity resulting from the use of BalanCD medium is maintained when using a different batch fed systems.

Conclusion

[0378] These data clearly demonstrate that the use of BalanCD media in a fed batch system results in an increased volumetric yield across different HEK293-derived cell types, and that this increase is consistent across different fed batch systems.

Example 12

[0379] This Example shows that the use of BalanCD medium and feed with Expi293 inducible cells allows at least maintenance of cell-specific productivity at cell densities exceeding 210.sup.6 cells/mL at point of infection. BalanCD medium therefore permits higher volumetric productivities than have previously been reported for adenovirus production using fed batch processes.

Methods

[0380] Two similar experiments were performed to assess volumetric productivity and cell-specific productivity at different cell densities at the point of infection.

[0381] In the first experiment (corresponding to top two panels of FIG. 10) cells were cultured in 125 mL shake flasks in duplicates for each of the cell densities, 0.05 culture volumes of BalanCD feed was added when the density reached 1.0 and 4.010.sup.6 culture cells/mL. The cells were then infected at different densities (2.0, 3.0, 4.0, 6.010.sup.6 cells/ml), using an MOI of 5. Subsequently, 0.05 volumes of BalanCD feed were added 30 minutes after infection, and again after 22 hours. The cultures were then harvested 47 hour after infection. At harvest 1/9 volume of 10 Lysis buffer and 100 IU/ml benzonase was added into each flask and incubated for 2 hours before they were diluted 1:200 in A438 formulation buffer for qPCR. All samples were frozen at 80 C. before analysis.

[0382] In the second experiment (corresponding to bottom two panels of FIG. 10) cells were cultured in 125 mL shake flasks in duplicates for each of the cell densities, 0.05 culture volumes of BalanCD feed was added when the density reached 1.0 and 5.010.sup.6 cells/mL. The cells were then infected at different densities (2.0, 4.0, 6.0, 9.010.sup.6 cells/ml), using an MOI of 5. Subsequently, 0.05 volumes of BalanCD feed were added 30 minutes after infection, and again after 22 hours. The cultures were then harvested and samples prepared for qPCR as above.

[0383] Both experiments were performed with ChAdOx1-nCoV19.

Results

[0384] Both experiments demonstrate that the cell-specific productivity can be maintained at cell densities exceeding 210.sup.6 cells/mL at point of infection. This therefore allows a higher volumetric productivity than methods which involve use of a cell density of equal to or less than 210.sup.6 cells/mL at point of infection. Both of these experiments demonstrated that the highest volumetric productivity is achieved with a cell density at infection of 6.010.sup.6 cells/ml

[0385] Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to these precise embodiments and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.