Virus Production From Bench Scale To Industrial Scale: New Technologies That Intensify, Simplify Virus Manufacturing

And Increase Overall Process EfficiencyA. Onraedt , P. Lefebvre , M. Kremer (Pall Life Sciences)

Contributing to the complexities of developing viral vaccines are the availability of purpose built process technologies to manufacture virus. Current upstream and downstream technologies either are scaled-out laboratory tools, including 2D growthsurfaces for adherent cells, or are derived from other biotech processes, where the size of the molecule or its shear sensitivity are not as critical as in virus manufacturing. The streamlined viral manufacturing platform that will be discussed is basedon technologies that are specifically developed for large-scale manufacture of virus for vaccine or gene therapy applications. The result is a process that simplifies virus manufacturing and reduces manufacturing footprint. It also increases the processefficiency and reduces cost-of-goods versus traditional approaches.

For the adherent culture of the host cell line, the iCELLis® fixed-bed bioreactor from Pall Life Sciences is designed to achieve high cell densities and good virus productivity by combining controlled growth conditions and a large growth surface areain a small reactor volume. This has been demonstrated on a wide range of viruses including MVA, Bovine Herpes, Paramyxovirus, Influenza, Adenovirus, rAAV and more in various production cell lines, including VERO, HEK293 and MDBK. Scale-up of the cell culture and virus production from benchtop iCELLis bioreactors to up to 500 m² has been realized. Specific virus productivity in the iCELLis bioreactors at benchtop and industrial scale was similar or better than that achieved instatic culture or roller bottles. In combination with Pall’s virus downstream processing solution, the iCELLis bioreactor offers a versatile platform for industrial scale virus production.

SUMMARY

The single-use iCELLis bioreactor containes a compact fixed-bed with carriers made of medical grade polyester. Thismatrix provides a large growth surface area in a relatively small volume: the iCELLis design provides up to 500 m2 in only25 liters of fixed-bed. Commercially available iCELLis bioreactors range in size from 0.53 to 500 m2 with two compactiondensities of carriers (Table 1).

Table 1Commercially available iCELLis bioreactor size specifications

iCELLis Nano Bioreactor iCELLis 500 BioreactorFixed-Bed (FB) (Process Development) (Large-Scale Production)Compaction Density FB Height (cm) FB Volume (L) FB Surface Area (m2) FB Volume (L) FB Surface Area (m2)

96 g/L 2 0.04 0.53 5 66

(C 1) 4 0.08 1.06 10 133

13 m2/L 10 0.2 2.67 25 333

144 g/L 2 0.04 0.8 5 100

(C 1.5) 4 0.08 1.6 10 200

19.5 m2/L 10 0.2 4.0 25 500

SUMMARY

PALL VIRUS MANUFACTURING PLATFORM

Virus analyticswith BLI (Octet®, Blitz®) Cell debris removal

with V100P depth �lter

Final sterilizing �ltrationFurther formulation & �lling

Puri�cation with Mustang®

AEX and Cadence™ SU TFFVirus production in iCELLis 500

with BLI (OctetVirus analytics

)®, Blitz®with BLI (OctetVirus analytics

with V100P depth �lterCell debris r

with V100P depth �lteremoval Cell debris r

with V100P depth �lterwith V100P depth �lterwith V100P depth �lterwith V100P depth �lter

Final sterilizing �ltrationFinal sterilizing �ltrationFinal sterilizing �ltrationFurther formulation & �lling

Final sterilizing �ltrationFurther formulation & �lling

Final sterilizing �ltrationFurther formulation & �lling

Final sterilizing �ltration

oduction in iCELLis 500Virus production in iCELLis 500oduction in iCELLis 500 AEX and Cadence™ SU TFFAEX and Cadence™ SU TFFPuri�cation with Mustang

AEX and Cadence™ SU TFF®Puri�cation with Mustang

AEX and Cadence™ SU TFFVirus pr

ICELLIS BIOREACTOR

TRANSFER AND SCALE-UP OF A HEK293 CELL CULTUREPROCESS FOR PRODUCTION OF ADENOVIRUS

RESULTS

Table 2Results of HEK293 cell culture four days after inoculation in small- and large-scale iCELLis bioreactors.

Average AverageFixed Bed Fixed Bed Cell Density Total

Bioreactor Surface Height Fixed Bed Reached Cells perScale Area (m2) (cm) Volume (L) (cells/cm2) Bioreactor

iCELLis nano 1.06 4 0.08 2.8 to 3.8 x 105 3.5 x 109

iCELLis 500 133 4 10 2.7 to 3.4 x 105 4.1 x 1011

Analysis of glucose and lactate (Figure 3) at both scales in comparison to a 5-tray Cell Factory control indicated that cellmetabolism was comparable between small and large scale iCELLis bioreactors and the standard 2-D process.

Figure 3Comparability of Glucose and Lactate Profiles of HEK293 culture in the iCELLis 133 m2 bioreactor (Blue), iCELLisnano bioreactor 1.06 m2 (Green) and 5-tray Cell Factory (Red).

Figure 2The iCELLis nano bioreactor system(from 0.53 to 4 m2)

Figure 1The iCELLis 500 control system and bioreactor

Evenly distributed medium circulation is powered by a built-in magnetic mixer, ensuring low shear stress. The cell culturemedium flows through the fixed-bed from the bottom to the top and returns to the bottom of the bioreactor as a waterfall,resulting in a high mass transfer co-efficient (kLa).In the iCELLis bioreactors, the following parameters can be measured and controlled: pH, DO, biomass (using a biomassprobe), temperature, gas flow rate, agitation, pressure and medium recirculation/perfusion rate.The unique waterfall oxygenation together with gentle agitation, biomass immobilization and process control provides afavored growth environment that can increase the cell specific productivity of the desired protein or virus.

EXPERIMENTAL APPROACH

HardwareiCELLis nano bioreactor (from 0.53 to 4 m2 )

iCELLis 500 bioreactor (133 m2)

Metabolites were analyzed with a Bioprofile-100 bio-analyzer (Nova Biomedical, MA, USA)

Biological MaterialHEK293 cells were grown in serum-supplementedmedium

Cell Culture ParametersAn existing process using HEK293 cells for the productionof adenovirus was first transferred from multi-tray systemsto an iCELLis nano bioreactor (0.53 m2, 40 mL of fixed-bed)by keeping equivalent cell culture parameters:

Temperature, pH and DO (% saturation with air)

Multiplicity of infection (pfu/cell)

Time of infection

Cell seeding density (cells/cm2 and cells/mL)

Culture duration

Additional experiments were performed with lower cell densities at inoculation in order to reduce the number ofpreculture steps at large scale. The following parameters werealso optimized for cell growth and virus productivity:

Compaction of carriers inside the fixed-bed (96 g/L or 144 g/L)

Linear velocity of medium through the fixed-bed (cm/s)

Fixed-bed height (2, 4 or 10 cm)

0

1

2

3

4

5

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

5CS

nano (1,06 m2)

large (133 m2)

Time (days)

L

Glu

cose

(g/L

)

5

5

4

3

2

1 Glu

cose

(g/L

)

5CS

nano (1,06 m

large (133 m

5CS

)2nano (1,06 m

)2large (133 m

0.5 0

1

0

Glu

cose

(g/L

)

2 1.5 1 Time (days)

3.5 3 2.5 Time (days)

4.5 4

Table 3Results for different viruses from various cell lines at different bioreactor scales.

Surface Productivity Total Virus Scale Up of ControliCELLis SA. (Virus Titer per cm2) Extrapolated to Process Required

Cells Virus Tested (m2) iCELLis Control Units 500 m2 iCELLis to Match iCELLis 500CEF MVA 0.07* 3.5E+06 1.0E+07 pfu/cm2 1.7E+13 pfu 780 CF-10MDBK Bovine 4 2.2E+07 1.3E+07 pfu/cm2 1.1E+14 pfu 310 L MC (6g/L)

Herpes 20* 1.7E+07 8.5E+13 pfu 240 L MC (6g/L)Virus 66 3.3E+07 1.6E+14 pfu 460 L MC (6g/L)

A549 rAAV 0.53 5.0E+08 3.0E+08 vg/cm2 2.6E+15 vg 1300 CF-10Adenovirus 2.67 9.8E+09 9.6E+09 TCID50/cm2 4.9E+16 TCID50 810 CF-10

Vero Influenza 4 3.8E+06 1.3E+06 TCID50/cm2 1.9E+13 TCID50 540 L MC (6g/L)20* 2.5E+06 1.3E+13 TCID50 360 L MC (6g/L)

Paramyxovirus 2.67 6.4E+05 5.7E+04 TCID50/cm2 3.2E+12 TCID50 8900 CF-102.9E+08 5.3E+07 Vp/cm2 1.5E+15 vp 4300 CF-10

Undisclosed 40* Confidential 794 CF-10 Lytic Virus 133 (serum-free)

660* 1238 CF-10 (+serum)

*Not a standard bioreactor size. Please refer to table 1 where all commercially available bioreactor sizes are specified

VIRUS PRODUCTION RESULTS IN ICELLIS

© 2015, Pall Corporation. Pall, , Blitz, Cadence, iCELLis, Mustang, and Octet are trademarks are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 3/15, GN15.6201Contact: 800.717.7255 (USA and Canada) • 1.516.484.5400 (Outside USA and Canada) • www.pall.com/bioreactors • www.pall.com/vaccines • E-mail: vaccines@pall.com

This summary of experiments demonstrates that the fixed-bed design of the iCELLis bioreactor enables high cell densities to be achieved and maintained in both small and large bioreactor volumes. Different processes have been easily scaled upby keeping cell culture conditions and process parameters identical to the standard 2-D cell culture process. The iCELLis bioreactor can be inoculated at a very low cell density, leading to a dramatic simplification of seed train operations and asignificant reduction of development timelines. In conclusion, large biomass amplification and excellent virus productivities, combined with the advantages of a fully closed single-use system with low shear stress, make the iCELLis fixed-bedbioreactor a simple and straightforward solution for industrial production of viruses. In combination with Pall's virus downstream processing solution, it brings an industrial virus production platform with a reduced footprint that is fully automatablefor better control and that brings overall process economics.

Outer shell, top sectionwith probes and ports

Fixed-bed retaining ring

Fixed-bed filled with 500 m2 macrocarriers

Fixed-bed support

Pump housing

Impeller

Pump housing ring

Outer shell, bottom section

Double-jacket

0 5

10 15 20 25 30

0 0.5 1 1.5 2 2.5

Time (days)

T

Lact

ate

(mM

)

3 3.5 4 4.5

5CS

nano (1,06 m2)

large (133 m2)

30

Lact

ate

(mM

)

30 25 20 15 10 5

5CS

nano (1,06 m

large (133 m

5CS

)2nano (1,06 m

)2large (133 m

0.5 0

5 0

Time (days)

2 1.5 1

3.5 3

Time (days)

2.5

4.5 4

CONCLUSION: VIRUS PRODUCTION INTENSIFICATION AND SIMPLIFICATION

Sequence of automated, single-use technologies optimized for high virus yield and quality, process robustness and optimized process economics: