A PROCESS FOR THE PRODUCTION OF ADENOVIRUS

Abstract

A Process for the Production of Adenovirus The present disclosure relates to a continuous process for the manufacture of an adenovirus wherein the process comprises the steps: A) continuously culturing, in a vessel, mammalian cells infected with the adenovirus in the presence of media suitable for supporting the cells such that the virus replicates, wherein the cells are capable of supporting viral replication, and B) isolating from the media the virus produced from step a) wherein the isolation of virus is not subsequent to a cell lysis step, wherein viable cells for virus infection and production are maintained in the vessel at a level suitable for replicating the virus for the period of continuous manufacture, wherein the process comprises at least one media change or addition and at least one cell change or addition. The disclosure also extends to viruses populations obtained or obtainable from the method.

Claims

1. A continuous process for the manufacture of an adenovirus wherein the process comprises the steps: A) continuously culturing, in a vessel, mammalian cells infected with the adenovirus in the presence of media suitable for supporting the cells such that the virus replicates, wherein the cells are capable of supporting viral replication, and B) isolating from the media the virus produced from step A) wherein the isolation of virus is not subsequent to a cell lysis step, wherein viable cells for virus infection and production are maintained in the vessel at a level suitable for replicating the virus for the period of continuous manufacture, wherein the process comprises at least one media change or addition and at at least one time point post infection at least some of the cells are changed or cells are added.

2. A process according to claim 1, wherein the virus has a hexon and fibre from a group B adenovirus.

3. A process according to claim 1, wherein the virus is replication competent.

4. A process according to claim 1, wherein the continuous manufacturing period comprises at least two virus replication cycles.

5. A process according to claim 1, wherein each virus replication cycle is in the range of from 30 to 300 hours.

6. A process according to claim 1, wherein the process produces at least 50,000 virus particles per cell at one or more time points post infection.

7. A process according to claim 1, wherein viable cells for virus infection and production are maintained in the vessel at a level suitable for replicating the virus by the addition of cells to the culture.

8. A process according to claim 1, wherein cells are removed from the cultures at one or more time points post infection.

9. A process according to claim 1, wherein the mammalian cells are selected from the group comprising HEK, CHO, Hela, Vero, A549, PerC6 and GMK, in particular HEK293.

10. A process according to claim 1, wherein the multiplicity of infection is 5 to 50 vp/cell.

11. A process according to claim 1, wherein the cells are infected with a starting concentration of virus of 1-4×10.sup.6 vp/ml.

12. A process according to claim 1, wherein a perfusion culture is employed.

13. A process according to claim 1, wherein a suspension culture is employed.

14. A process according to claim 1, wherein an adhesion culture is employed.

15. A process according to claim 1, wherein the process further comprises a purification step, selected from a CsCl gradient, chromatography step such as ion-exchange chromatography in particular anion-exchange chromatography, and a combination thereof.

16. A process according to claim 1 which comprises at least one media change or addition.

17. A process according to claim 1, which further comprises formulating the virus in a buffer suitable for storage.

18. A process for the manufacture of an adenovirus comprises the steps: a. culturing, in a vessel, mammalian cells infected with the adenovirus in the presence of media suitable for supporting the cells such that the virus replicates, wherein the cells are capable of supporting viral replication, wherein the starting seed density of the virus is in the range 1 to 2×10.sup.6 vp/ml (such as 1×10.sup.6 vp/ml) and the multiplicity of infection is in the range 5 to 20; and b. performing a lysis step in the period 24 to 75 hours post virus infection to harvest the virus from the cells.

19. A process according to claim 18, wherein the virus is a group B virus.

20. A process according to claim 18, wherein the process comprises at least one media change or addition.

21. A process according to claim 18, wherein at at least one time point post infection at least some of the cells are changed or cells are added.

22. A virus or formulation obtained or obtainable from this process described in claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0530] FIG. 1 shows a schematic representation of adenovirus production process

[0531] FIG. 2 shows a cell culturing arrangement for continuous virus manufacture

[0532] FIG. 3 shows a plot of seed density vs yield

[0533] FIG. 4 shows a plot of virus produced per cell at various time points post infection for shake flasks A & B with a Multiplicity of Infection of 12.5 and seed cell density of 1×10.sup.6 and media change

[0534] FIG. 5 shows a plot of virus produced per cell at various time points post infection for shake flasks C & D with a Multiplicity of Infection of 12.5 and seed cell density of 4×10.sup.6 and NO media change

[0535] FIG. 6 shows that media changes increase virus yield

[0536] FIG. 7 shows percentage viabilty of cells vs duration of infection

[0537] FIG. 8 shows percentage of cell viability at various times post infection for DOE (??) Fflask A & B with a multiplicity of infection of 12.5 and seed density of 1×10.sup.6 with media change

[0538] FIG. 9 shows percentage of virus in the supernatant for various time points post infection for DOE flasks A & B with a multiplicity of infection of 12.5 and a seed density of 1×10.sup.6 with media change

[0539] FIG. 10 shows percentage of virus in the supernatant for various time points post infection for DOE flasks C & D with a multiplicity of infection of 12.5 and a seed density of 4×10.sup.6 with no media change

[0540] FIG. 11 shows the virus product distribution of a 5 L bioreactor process with cells infected with EnAd at an MOI of 12.5 ppc and a cell density of 1×10.sup.6 cell/ml.

[0541] FIG. 12 shows the total virus particles produced per cell over time for a process employing a seed density of 1.9×10.sup.6 cells/ml and the culture was infected with EnAd at 50 ppc

[0542] FIG. 13 shows cell viability over time for a process employing 12.5 ppc and a seed density of 1×10.sup.6 vp/ml

[0543] FIG. 14 shows cell viability over time for a process employing 50 ppc and a seed density of 1.9×10.sup.6 vp/ml

[0544] FIG. 15 shows bioreactor data for two process one employing a seed density of 1×10.sup.6 vp/ml and 12.5 ppc and a second process employing 1.9×10.sup.6 vp/ml and 50 ppc.

[0545] FIG. 16 shows cell viability over time for a process employing a seed density of 2.2×10.sup.6 viable cells/mL and 50 ppc infection

[0546] FIG. 17 shows the cell distribution for a process employing a seed density of 2.2×10.sup.6 viable cells/mL and 50 ppc infection

[0547] FIG. 18 shows viability of control cultures employing a seed density of 2.2×10.sup.6 viable cells/mL and infected with 50 ppc but no cell suspension removal or addition of fresh cells and medium.

[0548] FIG. 19 shows virus distribution for control cultures employing a seed density of 2.2×10.sup.6 viable cells/mL and infected with 50 ppc but no cell suspension removal or addition of fresh cells and medium.

[0549] FIG. 20 shows cumulative virus distribution for a process employing a seed density of 2.2×10.sup.6 viable cells/mL and 50 ppc infection and a control process omitting no cell suspension removal and no addition of fresh cells and medium.

[0550] FIG. 21 shows cell viability over time for a process employing 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection in a shake flask experiment.

[0551] FIG. 22 shows virus distribution over time for a process employing 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection in a shake flask experiment.

[0552] FIG. 23 shows cell viability over time for a control process employing 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection in a shake flask experiment where omitted was cell suspension removal and no addition of fresh cells and medium.

[0553] FIG. 24 shows virus distribution over time for a control process employing 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection in a shake flask experiment where omitted was cell suspension removal and no addition of fresh cells and medium.

[0554] FIG. 25 shows cumulative total virus yields for a process employing 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection in a shake flask experiment and a control process where omitted was cell suspension removal and no addition of fresh cells and medium.

[0555] FIG. 26 shows viability of cells in a process with 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection wherein 95% suspension was removed at various time points & replaced with 47.5 mL uninfected cells in fresh medium cell density of 1.0×10.sup.6 viable cells/ml.

[0556] FIG. 27 shows virus distribution in a process with 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection wherein 95% suspension was removed at various time points & replaced with 47.5 mL uninfected cells in fresh medium cell density of 1.0×10.sup.6 viable cells/ml.

[0557] FIG. 28 shows cumulative total virus in a process with 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection wherein 95% suspension was removed at various time points & replaced with 47.5 mL uninfected cells in fresh medium cell density of 1.0×10.sup.6 viable cells/ml and a control process.

EXAMPLES

Example 1

[0558] Suspension culture and serum-free media adapted HEK293 cells are grown and expanded to a cell density of 1×10.sup.6 viable cells/mL at >90% viability in 30 mL EX-CELL serum-free medium in multiple 125 mL shake-flasks at 100 rpm. Some of the flasks are then infected with EnAd virus using a multiplicity of infection (MOI) of 50 virus particles per cell (ppc), while the remainder are left uninfected and are maintained at a cell density between 0.5 and 2×10.sup.6 viable cells/mL and viability >75% by passaging and used for adding back to the infected cell cultures as indicated below. At various time points post infection, 10 mL of the culture is removed from one of the shake flasks and replaced with 10 mL of uninfected HEK293 cells in fresh EX-CELL medium at 1×10.sup.6 viable cells/mL and >90% viability. The 10 ml of infected cell culture suspension is centrifuged, the supernatant is stored for analysis and the cell pellet is lysed in 1 mL fresh medium (3× freeze-thaw) which is then clarified by centrifugation prior to storing for analysis. The infected shake flask cultures are then re-cultured until the cell viability decreased to <75% and then cultures are terminated, centrifuging and processed to generate supernatant and cell lysate fractions (as above) which are stored for analysis. Throughout the experiment, small aliquots of suspension (50 μl) are removed from each flask for cell and viability counts using a Burker cell hemacytometer and trypan blue staining, as well as pH measurements.

[0559] Total and infectious EnAd particles in cell lysate and supernatant samples are determined by HPLC and immunostaining infection assays, respectively. The amount of host cell DNA in supernatant samples is determined by real time qPCR and the amount of host cell protein in supernatants is determined using a HEK293 Host Cell Protein ELISA kit using affinity purified goat anti-HEK antiserum. Total EnAd virus yields and levels of host cell DNA and protein were compared for the different processing time points.

Example 2

[0560] An experimental protocol is employed as described in Example 1 but with 15 mL of the 30 mL suspension being removed at the various time points and replaced with 15 mL uninfected cells in fresh medium instead of 10 mL volumes.

Example 3

[0561] An experimental protocol is employed as described in Example 1 but in this experiment the fresh uninfected cells added back to the infected culture flasks are resuspended in the virus-containing supernatant removed from that same flask (keeping back 2×500 μl aliquots for analysis) and the volume adjusted to 10 mL prior to adding back to the original flask.

Example 4

[0562] An experimental protocol is employed as described in Example 3 but with 15 mL of the 30 mL suspension being removed at the different time points and replaced with uninfected cells resuspended in 15 mL of the virus-containing supernatant instead of 10 mL volumes.

Example 5 to 8

[0563] A set of four further experiments employing the protocols detailed in examples 1-4, but rather than terminating the experiment after one round of suspension removal and replacement with fresh cells, the cultures are maintained on a repeating suspension removal and fresh cell replacement protocol which is continued until maintenance of sufficient viable cells could not be sustained (e.g. due to build-up of cell debris or other cellular products or lack of sufficient nutrients). The frequency of suspension removals and replacements were guided by daily cell counting and viability information together with visual observations.

Example 9

[0564] A single vial of suspension HEK293 cells are thawed and expanded in shake flasks prior to expansion to a 3 L working volume in a 5 L stirred-tank (glass vessel) bioreactor. The bioreactor controller is set to parameters of 37° C., a pH setpoint of 7.4, dissolved oxygen (DO) of 50, an airflow rate of 100 mL/min, and the agitation at 100 rpm. After the bioreactor system is equilibrated, an initial volume of 1.5 L EX-CELL culture medium is seeded at a viable cell density of 5×10.sup.5 cells/mL and then expanded to a working volume of 3 L maintaining >90% viability and targeting a density of 1.8 to 2.2×10.sup.6 cells/mL for infection with EnAd virus. Perfusion and media exchange is initiated at the 3 L stage via hollow fibre tangential flow filtration (TFF). The TFF cartridge had a 0.2 um pore size, 0.5 mm lumen diameter, and a surface area of 790 cm.sup.2. The TFF assembly was pre-sterilized by autoclaving, and then attached to the bioreactor through the use of a sterile tubing welder. Using a peristaltic pump, the cell culture is recirculated through the TFF system. No backpressure was placed onto the retentate line, while a second peristaltic pump is placed onto the permeate line to provide a measured flow rate out of the system. The perfusion flow rate was set to 1 vessel volume per day (i.e. 3 L/24 h). Once the target cell density was reached, the culture was infected with EnAd at an MOI of 50 ppc.

[0565] In parallel with the bioreactor culture, shake flask cultures of HEK293 cells are established and maintained at a cell density of 0.5-2×10.sup.6 cells/mL and >90% viability by regular passaging and these cells are used as source of uninfected cells to add into the bioreactor as described.

[0566] Collection of the perfusion permeate was started 48 hours post infection and perfusion permeate samples are taken at regular time points, with non-infected cells from the second suspension flasks added back to the infected cells in the bioreactor, maintaining the cell density at 2×10.sup.6 cells/mL and cell viability >75% viability. Total and infectious EnAd particles in cell permeate samples were determined by HPLC and immunostaining infection assays, respectively. The amount of host cell DNA in supernatant samples was determined by real time qPCR and the amount of host cell protein in was determined using a HEK293 Host Cell Protein ELISA kit using affinity purified goat anti-HEK antiserum. Total EnAd virus yields and levels of host cell DNA and protein at various time points are compared. The bioreactor culture is actively maintained by the perfusion and fresh uninfected cell replacement procedure until the cell viability could not be maintained above 50%.

[0567] Collected virus-containing permeate samples are pooled and virus purified by a process previously established for GMP manufacture of EnAd virus (outlined below) such that analytical assay data could be compared to appropriate standard virus preparations that had been manufactured without the elements of continuous manufacturing described herein.

EnAd Virus Purification

[0568] Virus is purified from the cell-free permeate samples collected from the bioreactor at different time points. These permeate samples are first treated with Benzonase® to digest host cell DNA and then concentrated and buffer exchanged by tangential flow filtration (TFF) in preparation for the first of a two-step purification process in the downstream which involves the selective capture and elution of EnAd using two different anion exchange chromatography resins. The first primary capture step (e.g. Sartobind resin) is followed by a second “polishing” purification step (e.g. CIM-Q) to reduce host cell residuals further. The purified virus is then buffer exchanged into the final formulation buffer using a second TFF step prior to the material being stored frozen at −80° C. prior to analyses.

Example 10

[0569] An experimental protocol as described in example 9 is employed with an MOI of 100 used for the EnAd infection of the HEK293 cells.

Example 11

[0570] For this experiment, a Design of Experiment (DOE) approach was followed and customised using JMP software in order to evaluate effects of different culture parameters on virus yields and distribution into supernatant or cells. The different variables, range of each variable and responses used for the design of this study are shown in Table 1.

TABLE-US-00002 TABLE 1 Variables and responses for design of experiment Factors/Variables Range Responses Multiplicity of 12.5-50 ppc Yield: Virus particles/cell Infection (MOI) Seeding Density 1 × 10.sup.6-4 × 10.sup.6 cells/ml Virus distribution: % SN, % CVL Media change Yes/No % Viability Duration of 40-96 hrs Host cell proteins Infection (DOI)

[0571] One vial of HEK293 cells from a working cell bank (WCB) was thawed at 37° C. and expanded in 75 cm.sup.2 cell culture flasks using Ex-Cell 293 medium supplemented with 6 mM L-glutamine (growth medium). After 4 days of incubation the cells were further passaged (Passage 1) and once a viable cell count of 1.2×10.sup.7 cells at a density of >1.0×10.sup.6 viable cells/mL was achieved (approximately 3-5 days after passage 1), the cells were transferred into sterile shaker flasks (Passage 2). The cultures were monitored, and when the cell number had doubled to ≧1.2×10.sup.6 viable cells/mL, the cells were further passaged approximately every 3 or 4 days. Cell numbers were monitored throughout the cell expansion phase by counting to ensure cell density was maintained at a minimum cell density of 0.5-0.6×10.sup.6 viable cells/mL.

[0572] Cell counting was performed using automatic cell counter (Invitrogen) and Trypan Blue staining (Invitrogen). Cell numbers were expanded further in 1 L sterile shaker flasks until the required cells for the experiment were generated.

[0573] The cell suspension was centrifuged at 300 g for 5 minutes and the cell pellet resuspended in fresh growth medium and the cell suspension transferred to shaker flasks at a working volume of 40 ml in each flask with the seeding cell density adjusted according to Table-2. Each shaker flask was labelled appropriately.

TABLE-US-00003 TABLE 2 Experiment design set up Duration of Shaker Infection flask MOI Seed Cell Total Media [DOI] ID (ppc) Density Volume Cells Change (hrs) A 12.5 1.00E+06 40 4.00E+07 Yes 40, 48, 60, 72, 96 B 12.5 1.00E+06 40 4.00E+07 Yes 40, 48, 60, 72, 96 C 12.5 4.00E+06 40 1.60E+08 No 40, 48, 60, 72, 96 D 12.5 4.00E+06 40 1.60E+08 No 40, 48, 60, 72, 96 E 31.25 2.50E+06 40 1.00E+08 No 40, 48, 60 72, 96 F 31.25 2.50E+06 40 1.00E+08 No 40, 48, 60, 72, 96 G 31.25 2.50E+06 40 1.00E+08 Yes 40, 48, 60, 72, 96 H 50 1.00E+06 40 4.00E+07 No 40, 48, 60, 72, 96 I 50 4.00E+06 40 1.60E+08 Yes 40, 48, 60, 72, 96 Negative 0 1.00E+06 40 4.00E+07 NA NA control Note: 1.00E+06, 1e.sup.6 and 1 × 10.sup.6; 4.00E+07, 4e.sup.7 and 4 × 10.sup.7 etc are equivalent cell number descriptors

[0574] One vial of EnAd working virus seed stock (WVSS) was removed from −70° C. storage and thawed at room temperature. Infection of the shaker flasks A to I was performed using a multiplicity of infection (MOI or ppc) according to Table-2. A negative control flask was not infected but maintained throughout the duration of infection. All shaker flasks were placed in a shaking incubator at +37° C., 5% CO.sub.2 and 120 rpm. Media change was performed on shake flasks A, B, G and I at 24 hrs post infection by removing the supernatant after centrifugation at 300 g for 5 min and resuspending the cell pellet in 40 ml fresh growth medium in each flask.

TABLE-US-00004 TABLE 3 Virus calculations for infections Volume Shaker AEX titer Total vp of Virus flask ID Cells/ml Volume Total cells ppc of virus needed added (μl) A 1.00E+06 40 4.00E+07 12.5 1.35E+11 5E+08 3.7 B 1.00E+06 40 4.00E+07 12.5 1.35E+11 5E+08 3.7 C 4.00E+06 40 1.60E+08 12.5 1.35E+11 2E+09 14.8 D 4.00E+06 40 1.60E+08 12.5 1.35E+11 2E+09 14.8 E 2.50E+06 40 1.00E+08 31.25 1.35E+11 3.125E+09    23.1 F 2.50E+06 40 1.00E+08 31.25 1.35E+11 3.125E+09    23.1 G 2.50E+06 40 1.00E+08 31.25 1.35E+11 3.125E+09    23.1 H 1.00E+06 40 4.00E+07 50 1.35E+11 2E+09 14.8 I 4.00E+06 40 1.60E+08 50 1.35E+11 8E+09 59.2

[0575] At 40, 48, 60 and 72 hours post infection, 4.1 ml samples were taken from each flask for analyses and at 96 hours post-infection all the remaining cell suspensions were harvested. 2×500 μl of the samples at each time point were used for cell count and viability analysis. The remaining 4.0 ml was used for analysis of virus distribution between the supernatant and cell pellet. This was determined by centrifugation of the infected cell culture suspension and the supernatant stored for analysis and the cell pellet lysed in fresh medium (3× freeze-thaw) which was then clarified by centrifugation prior to storing for analysis.

[0576] Total viral particle concentrations (vp) in the Crude Viral Lysate (CVL) and supernatant (SN) samples were measured by AEX-HPLC assay. During AEX-HPLC analysis, it was known that host cell proteins (HCP) elute at the beginning of the elution run and thus HCP content can also be determined by analysis of the chromatogram and the size of the HCP peak area.

[0577] Cell percentage viability and percentage trypan blue stained cells (which represent both dead cells and “leaky” cells that are not yet functionally dead but have their membrane integrity compromised such that the trypan blue dye can enter the cell) is represented in Table 4. Total number of virus particles per shaker flask culture and the percentage of viral particles in the SN and CVL for each sample time point are represented in Table 5.

[0578] Results from this DOE experiment were fitted using the ‘least square fit model’ using JMP software analysis to assess the relationship between different variables and the effects of the variable on responses in relation to viral yield (vp/cell). Three interaction relationships were observed as significant in the yield per cell model, which were 1) seeding density to DOI; 2) MOI to DOI and 3) media change to DOI.

[0579] Seeding density had the largest statistical effect on total virus production. Higher yields per cell were observed at lower cell seeding density (FIG. 3). Highest yield of 198,143 vp/cell (FIGS. 3 & 4) was observed at the lowest cell density of 1e.sup.6 cells/ml (infected with 12.5 ppc) compared to 4e.sup.6 cells/ml (infected with 12.5 ppc) where the viral yield per cell was significantly less at 5922 vp/cell (FIGS. 3 & 5). Total virus yields per flask were highest at the higher cell density and MOI conditions (flask I). Media change also had a significant statistical effect on yield (FIG. 6). Shaker flasks which had media changes post 24 hrs infection (Shaker flask A, B, G and I) had higher virus yield compared to the shaker flask which had no media change. Results are shown in FIGS. 4, 5 and 6.

[0580] At higher duration of infection (DOI) cell viability decreased and the percentage of leaky and dead cells increased. Viability and % leaky/dead data is represented in Table 4 and shown in FIGS. 7 and 8. Leaky cells are defined as cells which appear to have an intact cell membrane but the membrane is permeable to trypan blue stain. Dead cells are trypan blue stained but the cell membrane is no longer intact.

[0581] At 1e.sup.6 cells/ml seeding density infected with 12.5 ppc with a media change, more than 93% of virus was observed in supernatant at 96 hrs post infection (FIG. 9). At a higher cell density of 4e.sup.6 cells/ml, infected with the same 12.5 ppc with no media change, there was no virus observed in supernatant (FIG. 10).

TABLE-US-00005 TABLE 4 Viability data of shaker flasks time point Avg viable Avg leaky Average dead (h) Flask ID cells/ml cells/ml cells/ml Total Viability % Leaky % 40 Neg 9.6E+05 5.0E+04 0.0E+00 1.0E+06 95 5 control A 4.9E+05 7.0E+04 1.7E+05 7.3E+05 68 10 B 3.2E+05 1.9E+05 1.3E+05 6.4E+05 50 30 C 4.3E+06 1.1E+06 8.4E+05 6.3E+06 68 18 D 5.2E+06 4.1E+05 5.0E+05 6.1E+06 85 7 E 5.6E+05 2.4E+06 7.4E+05 3.7E+06 15 64 F 2.7E+06 5.8E+05 3.4E+05 3.6E+06 75 16 G 3.7E+06 7.8E+05 4.6E+05 4.9E+06 75 16 H 4.3E+05 2.0E+05 1.2E+05 7.5E+05 58 26 I 2.4E+06 5.3E+05 3.0E+05 3.2E+06 74 16 48 A 3.0E+05 7.0E+04 5.0E+04 4.2E+05 71 17 B 1.2E+06 2.7E+05 1.6E+05 1.6E+06 74 17 C 5.2E+06 7.4E+05 3.1E+05 6.2E+06 83 12 D 5.2E+06 8.9E+05 4.0E+05 6.5E+06 80 14 E 2.8E+06 9.8E+05 3.6E+05 4.1E+06 68 24 F 2.3E+06 9.1E+05 3.0E+05 3.5E+06 65 26 G 2.9E+06 2.3E+06 4.5E+05 5.6E+06 51 41 H 7.6E+05 6.2E+05 1.9E+05 1.6E+06 49 39 I 1.7E+06 1.1E+06 4.3E+05 3.3E+06 53 34 65 FA 4.5E+05 7.7E+05 1.5E+05 1.4E+06 33 56 B 3.4E+05 8.5E+05 9.5E+04 1.3E+06 27 66 C 4.0E+06 1.9E+06 8.8E+04 6.0E+06 67 31 D 4.6E+06 1.9E+06 1.0E+05 6.6E+06 69 29 E 1.9E+06 1.6E+06 1.5E+05 3.6E+06 52 44 F 1.9E+06 1.6E+06 7.5E+04 3.5E+06 53 44 G 2.3E+06 3.8E+06 2.6E+05 6.3E+06 36 60 H 3.8E+05 1.0E+06 5.5E+04 1.5E+06 26 70 I 1.1E+06 1.8E+06 2.4E+05 3.1E+06 34 58 72 A 3.9E+05 1.1E+06 1.0E+05 1.6E+06 25 69 B 2.8E+05 9.3E+05 1.1E+05 1.3E+06 21 70 C 4.7E+06 2.1E+06 1.8E+05 7.0E+06 67 30 D 4.5E+06 2.3E+06 1.9E+05 7.0E+06 64 33 E 4.9E+05 4.0E+05 2.5E+04 9.1E+05 53 44 F 2.0E+06 2.2E+06 3.8E+04 4.2E+06 48 51 G 1.5E+06 3.6E+06 2.5E+05 5.4E+06 28 68 H 3.2E+05 9.3E+05 6.5E+04 1.3E+06 24 71 I 9.1E+05 2.5E+06 1.5E+05 3.5E+06 26 70 96 A 8.8E+04 9.1E+05 5.8E+04 1.1E+06 8 86 B 1.2E+05 1.1E+06 4.0E+04 1.3E+06 9 87 C 6.1E+05 5.4E+06 1.1E+05 6.1E+06 10 88 D 6.5E+05 5.1E+06 1.0E+05 5.8E+06 11 87 E 3.8E+05 2.6E+06 5.0E+04 3.0E+06 13 86 F 4.6E+05 3.0E+06 6.3E+04 3.5E+06 13 85 G 3.3E+05 4.4E+06 3.5E+05 5.1E+06 6 87 H 1.5E+05 9.5E+05 4.0E+04 1.1E+06 13 83 I 2.3E+05 2.4E+06 1.3E+05 2.8E+06 8 87 Neg control 3.1E+06 1.7E+05 0.0E+00 3.3E+06 95 5

TABLE-US-00006 TABLE 5 AEX-HPLC assay results of ColoAd1 in Supernatant (SN) and CVL (intracellular) Produced Total vp Produced Produced Infection Sample Total vp vp/cell (% in vp/cell vp/cell Total vp time (h) Detail (% in SN) (SN) CVL) (CVL) (SN + CVL) yield/flask 40 Flask A 0 0 100 41222 41222 1.65 × 10.sup.12 48 Flask A 20 15509 80 61724 77232 3.09 × 10.sup.12 65 Flask A 69 127823 31 56114 183937 7.36 × 10.sup.12 72 Flask A 98 163705 2 3274 166980 6.68 × 10.sup.12 96 Flask A 93 185298 7 13893 199192 7.97 × 10.sup.12 40 Flask B 0 0 100 36293 36293 1.45 × 10.sup.12 48 Flask B 18 19867 82 89433 109301 4.37 × 10.sup.12 65 Flask B 71 126002 29 52553 178555 7.14 × 10.sup.12 72 Flask B 98 147558 2 2951 150510 6.02 × 10.sup.12 96 Flask B 93 184024 7 13070 197094 7.88 × 10.sup.12 40 Flask C 0 0 100 5843 5843 9.35 × 10.sup.11 48 Flask C 0 0 100 5706 5706 9.13 × 10.sup.11 65 Flask C 0 0 100 3767 3767 6.03 × 10.sup.11 72 Flask C 0 0 100 3631 3631 5.81 × 10.sup.11 96 Flask C 0 0 0 0 0 0 40 Flask D 0 0 100 6001 6001 9.60 × 10.sup.11 48 Flask D 0 0 100 6772 6772 1.08 × 10.sup.12 65 Flask D 0 0 100 3488 3488 5.58 × 10.sup.11 72 Flask D 0 0 100 4178 4178 6.68 × 10.sup.11 96 Flask D 0 0 0 0 0 0 40 Flask E 0 0 100 20149 20149 2.01 × 10.sup.12 48 Flask E 30 7142 70 16276 23418 2.34 × 10.sup.12 65 Flask E 56 11305 44 8813 20117 2.01 × 10.sup.12 72 Flask E 64 14559 36 8302 22861 2.29 × 10.sup.12 96 Flask E 85 17386 15 3172 20558 2.06 × 10.sup.12 40 Flask F 0 0 100 25088 25088 2.51 × 10.sup.12 48 Flask F 26 7574 74 21086 28660 2.87 × 10.sup.12 65 Flask F 57 12324 43 9413 21737 2.17 × 10.sup.12 72 Flask F 69 18240 31 8220 26460 2.65 × 10.sup.12 96 Flask F 88 20295 12 2725 23020 2.30 × 10.sup.12 40 Flask G 11 7003 89 57008 64010 6.40 × 10.sup.12 48 Flask G 38 30548 62 48833 79382 7.94 × 10.sup.12 65 Flask G 59 46073 41 31699 77772 7.78 × 10.sup.12 72 Flask G 66 57663 34 29199 86862 8.69 × 10.sup.12 96 Flask G 81 65738 19 15773 81510 8.15 × 10.sup.12 40 Flask H 28 19075 72 49192 68267 2.73 × 10.sup.12 48 Flask H 53 55309 47 48091 103400 4.14 × 10.sup.12 65 Flask H 79 98126 21 25714 123841 4.95 × 10.sup.12 72 Flask H 81 107729 19 25036 132765 5.31 × 10.sup.12 96 Flask H 92 105893 8 8775 114668 4.59 × 10.sup.12 40 Flask I 19 6169 81 26235 32404 5.18 × 10.sup.12 48 Flask I 39 20457 61 31430 51887 8.30 × 10.sup.12 65 Flask I 71 38093 29 15738 53831 8.61 × 10.sup.12 72 Flask I 72 44409 28 17336 61746 9.88 × 10.sup.12 96 Flask I 81 47634 19 11180 58814 9.41 × 10.sup.12

Example 12

[0582] Key DOE parameters indicated from the experiment described in Example 11 in shake flasks were assessed in a 5 L bioreactor. HEK293 cell expansion and bioreactor preparation was performed as described in Example 9. Cell counting was performed using automatic cell counter and trypan blue staining. Once viable cells totaling 7.5e.sup.8 cells were attained in shake flasks, the cells were used to inoculate the 5 L bioreactor. The bioreactor was infected with EnAd at an MOI of 12.5 ppc when the target cell density of 1e.sup.6 cell/mL was achieved.

[0583] At 24, 40, 48, 65 and 70 hrs post infection samples (20 ml) were taken for cell count, viability and total virus concentration. The supernatant was separated from the cells by centrifugation and stored at −80° C. until viral particle concentrations analysis by AEX-HPLC was performed. The cell pellet samples were prepared as outlined in Example 11 and stored at −80° C. until HPLC analysis. The results are shown in Tables 6.

[0584] Under these conditions, the majority of the virus was present in the CVL for all time points (FIG. 11) with no significant virus present in the supernatant until 71 h post infection. At 71 hrs post-infection, 96% of EnAd virus was observed in the cell pellet (Table 6 and FIG. 11) with only 4% present in supernatant (Table 6). A total of 428,868 virus particles were extracted from the cells by chemical lysis and cell debris removed by clarification (refer to post lysis numbers in table 6).

[0585] Cell viability at 71 hour post infection with 12.5 ppc was 58% compared to 85% at TO (FIG. 13). At 71 hour post infection with 50 ppc (see Example 13), the cell viability was typically below 30%.

TABLE-US-00007 TABLE 6 AEX-HPLC assay results of EnAd Infec- tion Total vp time total vp % total vp % vp/cell vp/cell yield from point SN SN CVL CVL (SN) (CVL) bioreactor 24 0.00E+00 0 2.37E+13 100 0 7914 2.40E+13 40 0.00E+00 0 4.74E+13 100 0 15796 4.70E+13 48 0.00E+00 0 6.18E+13 100 0 20605 6.20E+13 65 0.00E+00 0 3.79E+14 100 0 126367 3.80E+14 71 2.81E+13 4 6.06E+14 96 9383 202069 6.30E+14 Post 1.28E+15 99 8.67E+12 1 425979 2889 1.30E+15 lysis

Example 13

[0586] An experimental protocol as described in Example 12 was employed with a target cell density at infection of 1.9e.sup.6 cells/ml and the culture was infected with EnAd at an MOI of 50 ppc. Samples were taken at 24, 48, 60 and 70 hrs post-infection. The viral particle concentrations of the samples were analysed with AEX-HPLC and the results are shown in Tables 7.

[0587] At 71 hrs post-infection, 59% (89, 485 vp) of EnAd virus was observed in the supernatant (Table 8) with 41% (63, 081 vp) present in the cell viral lysate (Table 7 and FIG. 12). A total of 213, 981 virus particles were extracted from the cells by chemical lysis and cell debris removed by clarification (Refer to post lysis numbers in Table 7).

[0588] In this experiment, the bioreactor culture parameters of 1.9e.sup.6 cell/mL infected with 50 ppc produced half the yield (213981 vp/cell) compared to the parameters outlined in Example 12 where 428,868 vp/cell were produced at 1e6 cell/mL infected with 12.5 ppc.

[0589] Cell viability at 71 hour post infection with 50 ppc was 26% compared to 96% at TO (FIG. 14). Cell viability at 71 hour post infection with 12.5 ppc (in Example 12) was 58% compared to 85% at TO (FIG. 13). The low MOI (12.5 ppc) at infection may account for the higher (post infection) cell viability (58%) at 71 h which potentially contributed to the 2 fold higher viral production post lysis in Example 12 compared to this study (FIG. 15).

TABLE-US-00008 TABLE 7 AEX-HPLC assay results of EnAd Infection Total vp time total vp total vp vp/cell vp/cell yield form point SN % SN CVL % CVL (SN) (CVL) bioreactor 24 0.00E+00 0 1.57E+14 100 0 27550 1.60E+14 48 1.95E+14 23 6.49E+14 77 34141 113941 8.40E+14 60 3.59E+14 43 4.80E+14 57 62966 84245 8.40E+14 71 5.10E+14 59 3.60E+14 41 89485 63081 8.70E+14 Post lysis 1.11E+15 100 0.00E+00 0 213981 0 1.20E+15

Example 14

[0590] An experimental protocol was employed as described in Example 1 but with a shaker flask working volume of 25 ml, cell density of 2.2×10.sup.6 viable cells/mL and 50 ppc infection. To explore principles of a continuous manufacturing approach, at various time points 20 mL of the 25 mL cell suspension (80%) was removed and replaced with 20 mL non-infected cells at the same cell density as at the start (2.2×10.sup.6 viable cells/mL) in fresh medium. The experiment was continued for 7 days and on each day (Day 1, Day 2, Day 3, Day 4, Day 5, Day 6 and Day 7) the 20 mL post infection samples were taken for cell count, viability and total virus concentrations in supernatant and CVL. Post infection cell counting was performed using Hemocytometer and Trypan Blue stain (Invitrogen). The supernatant was separated from the cells by centrifugation and stored at −80° C. for viral particle concentration analysis by AEX-HPLC. The cell pellet samples were prepared as outlined in Example 11 and stored at −80° C. until HPLC analysis. The viability results are shown in Table 8 and the HPLC results for supernatant and CVL are shown in Table 9.

[0591] Control shaker flasks as the experimental control for this experiment were set up with a working volume of 25 ml, cell density of 2.2×10.sup.6 viable cells/mL and infected with 50 ppc. No cell suspension removal or addition of fresh cells and medium were undertaken with these control flasks post infection. These controls were terminated at Day 3 post infection. Cell count and viability were assessed daily and day 3 post-infection samples were taken for cell count, viability and total virus concentrations. Samples were processed as described in Example 11 for supernatant and CVL analysis by HPLC. The results are shown in Table 9.

[0592] The cell viability on Day 1 was 94% which decreased overtime with the lowest viability at 5% on Day 7. The percentage of leaky cells increased over time with the highest amount present on Day 7(86%) (Refer to Table 8 and FIG. 16). The control cell viability at Day 1 was also 94% which decreased with the lowest viability on Day 3 (34%). The percentage leaky cells increased over time with the highest on Day 3 (64%) (Table 8 and FIG. 18).

TABLE-US-00009 TABLE 8 Viability data in shaker flasks Avg Avg Avg viable leaky dead Avg Avg Avg Timepoint cells 8 cells 8 cells 8 viable (leaky) dead (h) squares squares squares cells cells cells Total Viability % Leaky % Day 1 55 0 3.5 2.20E+06 0.00E+00 0.00E+00 2.00E+06 94 6 Day 2 47.75 10.5 0 1.91E+06 4.20E+05 0.00E+00 2.33E+06 72 18 Day 3 32.5 22.5 2.25 6.50E+05 4.95E+05 4.50E+04 1.19E+06 55 42 Day 4 12 85.5 7.25 2.40E+05 1.86E+06 1.45E+05 2.24E+06 11 83 Day 5 7 75.25 6.5 1.40E+05 1.64E+06 1.30E+05 1.91E+06 7 86 Day 6 21 69.75 8 4.20E+05 1.56E+06 1.60E+05 2.14E+06 20 73 Day 7 5.75 83.25 8.75 1.15E+05 1.84E+06 1.75E+05 2.13E+06 5 86 Control 55 0 3.5 2.20E+06 0.00E+00 0.00E+00 2.00E+06 94 6 Day 1 Control 20 6.5 0 8.00E+05 2.60E+05 0.00E+00 1.06E+06 75 25 Day 2 Control 64.75 120 4 1.30E+06 2.40E+06 8.00E+04 3.78E+06 34 64 Day 3

[0593] The percentage of virus in the supernatant varied from day 3 to day 7 with maximum on day 5 and 6 (46 & 47%, respectively). By day 7 only 17% was present in the supernatant (Table 9 and FIG. 17). The control at day 3 had 74% of the virus present in the supernatant (FIG. 19). For the control CVL, at day 3 only 26% virus remained in the cell, where as in the continuous manufacturing test flasks 53% remained in the cell from day 1 to 7 with 83% present in the cell at day 7 (Table 9, FIG. 19) For this study, the total cumulative viral particles generated by the daily addition of fresh non-infected cell to the infected cell cultures over 7 days was 1.36e.sup.13 vp which was 7-fold higher than the amount generated in the control flasks (2.0e.sup.12 vp). The total amount in the supernatant over 7 days was 3.7e.sup.12 vp which represented 27% of the total vp (1.36e.sup.13 vp), with 73% present in the CVL during the 7 days. In comparison, the viral distribution of the control was 74% (1.5e.sup.12 vp) present in the supernatant with 26% (5.2e.sup.11 vp) present in the CVL (Table 9 and 10, FIG. 20).

TABLE-US-00010 TABLE 9 AEX-HPLC assay results of EnAd Days post Total vp Total vp vp/cell vp/cell vp/cell infection SN % SN CVL % CVL (SN) (CVL) (SN + CVL) Total vp Day1 0.0E+00 0% 4.4E+11 100% 0 10104 10104 4.45E+11 Day2 3.3E+11 23% 1.1E+12 77% 7560 24973 32533 1.43E+12 Day3 6.8E+11 26% 1.9E+12 74% 15367 43736 59103 2.60E+12 Day4 7.5E+11 24% 2.4E+12 76% 17055 54515 71570 3.15E+12 Day5 9.6E+11 46% 1.1E+12 54% 21859 25821 47680 2.10E+12 Day6 5.3E+11 47% 6.0E+11 53% 12002 13677 25679 1.13E+12 Day7 4.6E+11 17% 2.3E+12 83% 10497 52621 63118 2.78E+12 Control 1.5E+12 74% 5.2E+11 26% 27435 9522 36957 2.0E+12

TABLE-US-00011 TABLE 10 Total virus particles Experiment Total VP SN Total VP CVL Total VP Cumulative Total 3.71E+12 (27%) 9.84E+12 (73%) 1.36E+13 VP (Day 1-7) Control  1.5E+12 (74%)  5.2E+11 (26%)  2.0E+12

Example 15

[0594] An experimental protocol was employed as described in Example 14 but with a shaker flask working volume of 50 ml, cell density of 1.0×10.sup.6 viable cells/mL and 12.5 ppc infection. At various time points 40 mL of the 50 mL suspension (80%) was removed and replaced with 40 mL uninfected cells at 1.0×10.sup.6 viable cells/mL in fresh medium. The experiment was continued for 5 days and on each day (Day 1, Day 2, Day 3, Day 4 and Day 5) post infection samples were taken for cell count, viability and total virus concentration. Post infection cell counting was performed using Hemocytometer and Trypan Blue stain (Invitrogen). The supernatant was separated from the cells by centrifugation and stored at −80° C. until viral particle concentrations analysis by AEX-HPLC was performed. The cell pellet samples was prepared as outlined in Example 11 and stored at −80° C. until HPLC analysis. The viability results are shown in Tables 11. The HPLC results for supernatant and CVL are in Table 12.

[0595] The cell viability on Day 1 was 78% which decreased overtime with the lowest viability at Day 7(16%). The percentage leaky cells increased over time with highest amount present on Day 3 (82%) (Table 11 and FIG. 21). The control cell viability at Day 1 was 73% which decreased with the lowest viability recorded on Day 3(23%). The percentage leaky cells increased over time and highest on Day 3(70%) (FIG. 23).

TABLE-US-00012 TABLE 11 Viability data of shaker flasks Avg Avg Avg viable leaky dead Avg Timepoint cells 8 cells 8 cells 8 viable Avg leaky Avg dead (h) squares squares squares cells cells cells Total Viability % Leaky % Day 1 50.3 12.8 1.3 1.01E+06 2.55E+05 2.50E+04 1.29E+06 10.5 20 Day 2 53.3 22.5 2.3 1.07E+06 4.50E+05 4.50E+04 1.56E+06 56 41 Day 3 3.8 27.5 6.3 7.50E+04 5.50E+05 1.25E+05 7.50E+05 30 82 Day 4 6.8 28.5 6.0 1.35E+05 5.70E+05 1.20E+05 8.25E+05 24 65 Day 5 8.0 34.3 2.3 1.60E+05 6.85E+05 4.50E+04 8.90E+05 17 76 Control 18 4 3 7.35E+05 1.68E+05 1.03E+05 1.01E+06 73 17 Day-1 Control 20 40 6 3.93E+05 8.06E+05 1.20E+05 1.32E+06 30 61 Day-2 Control 17 50 5 3.34E+05 1.00E+06 1.06E+05 1.44E+06 23 70 Day-3

[0596] The percentage of virus in the supernatant varied from day 1 to day 5 with maximum on day 4 (28%, respectively). By day 5 only 18% was present in the supernatant (Table 12 and FIG. 22). The control (flasks which did not have removal and replacement of cells and medium) at day 3 had 81% of the virus present in the supernatant and 19% in CVL.

[0597] For this control CVL, at day 3 only 19% virus remained in the cell, where as in the flasks having daily cell and media replacements ≧72% remained cell associated from day 1 to 5 with 82% present in the cell pellets at day 5 (Table 12).

[0598] In this study, the total viral particles generated by the addition of fresh non-infected cells to infected cells over 5 days was 2.33e.sup.13 vp which was 3-fold higher than the amount generated in the control (7.6e.sup.12). The total amount in the supernatant over 5 days was 4.4e.sup.12 vp which represented 19% of the total vp (2.33e.sup.13), with 81% present in the CVL during the 5 days. In comparison, the viral distribution of the control cultures was 81% (6.10e.sup.12) present in the supernatant with 19% (1.48e.sup.12) present in the CVL (Table 12 and 13, FIG. 25).

TABLE-US-00013 TABLE 12 AEX-HPLC assay results of EnAd Total VP Total VP vp/cell vp/cell vp/cell Sample ID SN % SN CVL % CVL (SN) (CVL) (SN + CVL) Total VP Day 1 0.0E+00 0% 6.1E+11 100% 0 15357 15357 6.1E+11 Day 2 0.0E+00 0% 2.6E+12 100% 0 63820 63820 2.6E+12 Day 3 1.2E+12 19% 5.1E+12 81% 29834 128393 158227 6.3E+12 Day 4 2.0E+12 28% 5.2E+12 72% 50298 129956 180254 7.2E+12 Day 5 1.2E+12 18% 5.4E+12 82% 30008 134004 164012 6.6E+12 control 6.1E+12 81% 1.48E+12  19% 153565 36964 190529 7.6E+12

TABLE-US-00014 TABLE 13 Total virus particles Experiment Total VP SN Total VP CVL Total VP Cumulative Total 4.40E+12 1.89E+13 2.33E+13 VP (Day 1-5) control 6.10E+12 1.48E+12 7.60E+12

Example 16

[0599] An experimental protocol was employed as described in Example 15 but at various time points 47.5 mL of the 50 mL suspension (95%) was removed and replaced with 47.5 mL uninfected cells in fresh medium at the same cell density of 1.0×10.sup.6 viable cells/mL. This study was run in parallel with that in Example 15 and used the same control flasks. The viability results are shown in Tables 14. The HPLC results for supernatant and CVL are shown in Table 15.

[0600] The cell viability on Day 1 was 78% which decreased overtime with the lowest viability at Day 5 (16%). The percentage leaky cells increased over time with highest amount present on Day 5 (77%) (Table 14 and FIG. 26). The control cell viability at Day 1 was 73% which decreased with the lowest viability recorded on Day 3 (23%). The percentage leaky cells increased over time and highest on Day 3 (70%) (FIG. 23).

TABLE-US-00015 TABLE 14 Viability data of shaker flasks Avg viable Avg leaky Avg dead Timepoint (h) cells cells cells Total Viability % Leaky % Day 1 1.01E+06 2.55E+05 2.50E+04 1.29E+06 78 20 Day 2 1.07E+06 4.50E+05 4.50E+04 1.56E+06 68 29 Day 3 7.50E+04 5.50E+05 1.25E+05 7.50E+05 30 73 Day 4 1.35E+05 5.70E+05 1.20E+05 8.25E+05 16 69 Day 5 1.60E+05 6.85E+05 4.50E+04 8.90E+05 18 77 Control Day-1 7.35E+05 1.68E+05 1.03E+05 1.01E+06 73 17 Control Day-2 3.93E+05 8.06E+05 1.20E+05 1.32E+06 30 61 Control Day-3 3.34E+05 1.00E+06 1.06E+05 1.44E+06 23 70

[0601] From Day 1 to 5 no virus present in the supernatant, all viruses remained in CVL (Table 15 and FIG. 27).

[0602] For this experiment, the total viral particles generated by the daily removal of suspension and addition of fresh non-infected cell to infected cells over 5 days was 1.96e.sup.13 vp which was 3 fold higher than the amount generated in the control (7.6e.sup.12). The total amount of virus in the CVL over 5 days was 1.96e.sup.13 vp which represented 100% of the total vp (1.96e.sup.13). In comparison, the viral distribution of the control was 81% (6.10e.sup.12) present in the supernatant with 19% (1.48e.sup.12) present in the CVL (Table 16 and FIG. 28).

TABLE-US-00016 TABLE 15 AEX-HPLC assay results of ColoAd1 Total VP Total VP vp/cell vp/cell vp/cell Sample ID SN % SN CVL % CVL (SN) (CVL) (SN + CVL) Total VP Day 1 0.00E+00 0% 9.7E+11 100% 0 20513 20513 9.7E+11 Day 2 0.00E+00 0% 1.5E+12 100% 0 31209 31209 1.5E+12 Day 3 0.00E+00 0% 5.0E+12 100% 0 105575 105575 5.0E+12 Day 4 0.00E+00 0% 5.8E+12 100% 0 122263 122263 5.8E+12 Day 5 0.00E+00 0% 6.3E+12 100% 0 133257 133257 6.3E+12 control  6.1E+12 81% 1.48E+12  19% 153565 36964 190529 7.6E+12

TABLE-US-00017 TABLE 16 Total virus particles Experiment Total VP SN Total VP CVL Total VP Cumulative Total 0.00E+00 1.96E+13 1.96E+13 VP (Day 1-5) control 6.10E+12 1.48E+12 7.60E+12