Poster Content as Presented at ISCT 2017

Development and Optimization of Large Scale Production of Adenoviral Vector and Autologous Insulin Producing Cells Using iCELLis® 500

and Xpansion® 200 Single-Use Bioreactors1Rachel Legmann, 2Andy Reniers, 1Nicholas Kohlstrom, 1Brian Gardell, 1Todd Sanderson, 1Lisa Bradbury, 1Jack Vicalvi,

1Heather Mallory, 2Fabien Moncaubeig, 3Vered Aviv, 3Keren Shternhall-Ron, 3Itai, Tzchori, 3Irit Meivar-Levy, 3Sarah Ferber 1Pall Life Sciences, 20 Walkup Dr, Westborough, MA, USA; 2Pall Life Sciences, Rue de Ransbeek 310, B-1120 Brussels, Belgium;

3Orgenesis Inc., 21 Sparrow Circle, White Plains, NY 10605, USA

INTRODUCTION

Cell and gene therapies have the potential of revolutionizing medicine in the 21st century. Bioprocessing of therapeutic agents for these therapies still faces many challenges during scale up. An autologous cell therapy approach that allows the diabetic patient to be a donor of their own therapeutic tissue has been developed by Orgenesis. To bring this cell therapeutic approach into pre-clinical and clinical phases, Pall optimized and scaled-up the current process of viral vectors production to an industrial process using the packed-bed iCELLis 500 single-use bioreactor that provides 3D controlled, system with low shear stress for adherent cells (Figure 1).

Figure 1

From current adenovirus production process to industrial process

The conventional flatware process that is currently used is tedious. Growing the cells in a 20 cell factory for large scale production is a major capital investment of approximately 800 cell stacks and 50 incubators as shown in Figure 1a.

The large industrial scale process as shown in Figure 1b has a small footprint of propagation. The Xpansion 200 bioreactor is being used as the N-1 seed train into the iCellis 500 production bioreactor.

The Xpansion 200 single-use bioreactor was used to amplify the human adult liver-derived cells from 10 million per biopsy to 1.8 billion cells required for curing a diabetic patient (Figure 2, Reniers et al, ESGCT 2015).

Figure 2

Industrial scale process for liver cell expansion and virus production

Pall Life Sciences provides process development intensification technologies and services for the biotech and biopharm industry. This includes single-use high-cell-density solutions. Pall has designed and developed the Xpansion bioreactor platform: a multi-plate bioreactor offering up to 122,400 cm2 of growth surface-that can be used for shear sensitive adherent cells such as stem cells (Reniers et al, ESGCT 2015).

A high yield of 1.0x1016 total infectious virus particles (ifu) was produced in an iCELLis 500 bioreactor using a bioreactor with 66 m2 of cell growth surface area by optimizing various key process parameters. The purified adenovirus were fully functional and successfully transduced the target liver cells. As a result, Orgenesis is moving forward with this process for pre-clinical and clinical testing of autologous insulin producing (AIP) cells for the treatment of diabetes.

MATERIALS AND METHODS

Materials

Biological materials: Human liver cells and hPDX-1,hNeuroD & hMafA adenoviruses (provided by Orgenesis), HEK293 cells (ATCC)

Reference adenoviruses amplified in flatware and purified by cesium chloride method (provided by O.D.260 Inc)

Growth media: DMEM (Thermo cat # 11965) supplemented with 10% FBS (Thermo cat# 26140)

Other reagents: DPBS (Thermo cat # 10010) and TrypLEu Select (Thermo cat# 12563)

HEK293 cells

a. Conventional virus production process b. Industrial virus production process

Thaw and recovery

N-1 �atware12 m2 growthSurface area

Flatware800 m2 growthsurface area

HEK293 cells

Thaw and recovery

Cellpropagation

Cellpropagation

Xpansion 200

N-1 Xpansionbioreactor asHEK293 cellsseed train to

iCELLis 500, 12 m2

iCELLis 500

3.0 x 1010 cells500 m2

growth andproduction

1000 LRecirculation

media

Virus particles yield in the �xed-bedbioreactor process production:

7.8 x 1016 VP /batch-500 m2

Virus particle yield in thecurrent process production: 8.1 x 1015 VP /batch-500 m2

iCELLis Nano, 4 m2

Starter virus

10-20 millionLiver cells Cell expansion

Trans-differentiation Adenoviral vectors

Ad5-PDX-1

Ad5-NeuroD1

Ad5-MafA

1.8 billion AIP Cells (β-like cells)

Cell culture support: CELLStacku 10 (Corning cat# 3320), Xpansion 50 plates bioreactor (Pall cat# 810122), Xpansion 200 plates bioreactor (Pall cat# 810155), iCELLis Nano 0.53 m2 bioreactor (Pall cat# 810039NS), iCELLis Nano 1.07 m2 bioreactor (Pall cat# 810061NS), iCELLis Nano 4 m2 bioreactor (Pall cat# 810042NS), iCELLis Nano 2.65 m2 bioreactor (Pall cat# 810206NS) iCELLis 500-66 m2 bioreactor (Pall cat# 810237), iCELLis 500, full starter kit, with CPC connectors (Pall cat # 810094), iCELLis Nano, full starter kit (Pall cat# 810210)

Methods

HEK293 host cells propagation performed in Xpansion bioreactor prior to seeding of iCELLis 500 bioreactor as shown in process flow chart (Figure 1)

Adenovirus production in iCELLis 500 bioreactor (Figure 1)

Liver cell propagation: process flow (Reniers et al, ESGCT 2015).

HEK293 cells were used in the iCELLis Nano and iCELLis 500 (66 m2) bioreactors at passage # 7&8

The iCELLis Nano process development and scalability strategy were performed as described in Figure 3

Flatware cultures for adenovirus production were performed in parallel to the iCELLis bioreactors as a production control

Bioreactor controller set points: ≤ pH-7.2 (no pH control below 7.2) and DO 50% air saturation

Target seeding density: 7000 cells/cm2

HEK293 culture duration for both growth and adenovirus production: 8 days

Analytics: Metabolites using Nova analyzer, pH meter for off-line analysis, Adeno X- infectivity titer (CloneTech), Death curve analysis (performed by Orgenesis on primary culture of liver derived cells) and Trans-differentiation efficiency by Real Time PCR analysis

Figure 3

Development and scalability strategy in iCELLis bioreactor

RESULTS

Figure 4

Optimized and scale-up Ad5 PDX-1 in iCELLis Nano bioreactors

The first step in the development strategy is to transfer the existing flatware process to bioreactor to demonstrate similarity. The second step is optimization and scalability within iCELLis Nano bioreactor working on various key process parameters to increase product yield (to drive reduce dose cost). When optimized process is locked two to three production runs are performed to validate consistency.

Figure 5

Scalability from development process in iCELLis Nano bioreactor to production process in iCELLis 500 (66 m2) bioreactor

Following the development of a robust and optimized process with the iCELLis Nano bioreactor, we then scaled up into the large scale production bioreactor, the iCELLis 500 bioreactor.

Tech transfer of existingprocess to iCELLis Nano

bioreactor (2 runs)

Optimization and scalabilityin iCELLis Nano bioreactor

Robustness

0.014 m2

iCELLis Nano(0.53-4 m2 scale)

iCELLis 500(66 m2 scale)

Scale-up to iCELLis500 (66 m2) bioreactor

1.00E+08

1.00E+09

1.00E+10

1.00E+11

Robust

Optimized process

Flatware iCELLis Nano0 0.5 1 1.5 2 2.5 3 Ti

ter y

ield

( IF

U P

DX-

1/cm

2 )

Vessel scale (growth surface area, m2)

Small Scale iCELLis Nano Bioreactor: Development Predictive Run

Large Scale iCELLis 500 Bioreactor: Manufacturing

Figure 6

Specific productivity: From bench to manufacturing scale

Linear adenovirus titer scalability from small iCELLis Nano scale bioreactor to iCELLis 500 bioreactor large scale. The optimized production process in each scale was consistent and the titer yield was eight fold higher in iCELLis bioreactor system than in the flatware.

CONCLUSIONS

iCELLis fixed-bed single use bioreactor was successfully used to scale-up adenovirus production generating 1.04x1016 ifu per batch using the iCELLis 500 (66 m2) scale bioreactor. Optimization of the culture parameters in the predictive iCELLis Nano bioreactor scale resulted in significant enhancement of the specific titer productivity by eight fold.

There was a good agreement in growth, metabolites and titer production between small scale iCELLis Nano bioreactor and large scale iCELLis 500 bioreactor. Simplification of the large scale process included reduced seeding cell density as well as using Xpansion multi-plates bioreactor as a seed train into the iCELLis 500 bioreactor. The iCELLis single-use fixed-bed bioreactors offer a solution for viral vector manufacturing in large quantities in an adherent environment. The results of the present study suggest that the adenoviruses amplified and purified by the industrial process is fully functional and comparable to the adenovirus that was produced by the current flatware process.

REFERENCES

1. Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells. Meivar-Levy et al, Journal of Transplantation, 2011:252387. doi: 10.1155/2011/252387

2. Industrialization of a Cell-Based Autologous Therapy Targeting Diabetes: Industrialization of a Liver Cell Proliferation Process from Petri Dish to the Xpansion Multiple Bioreactor. Reniers et al, ESGCT 2015, At Helsinki

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1.62E+09

1.36E+10

Total yield 3.6e14 ifu Total yield 1.04e16 ifu1.57E+10

1.00E+08

1.00E+09

1.00E+10

1.00E+11

Control iCELLis Nano (2.65) iCELLis 500 (66)

Tite

r ( if

u/cm

2 )

Specific Productivity Crude Harvest