Kamal Rashid, Ph.D.

Director & Research Professor

Biomanufacturing Education & Training Center

Worcester Polytechnic Institute

Massachusetts, USA

Cphl Worldwide October 24th, 2017 Messe, Frankfort

Germany

Innovative Bioreactor Systems for Enhancing Vero Cell growth

Cell Culture Products

Viral Vaccine

Biopharmaceutical Proteins

(Biologics)

Monoclonal Antibody

Production

Why Vero Cells ?

• Vero cells are known for stability

• Vero cell lines are well documented for performance and quality of the yield

• Received US FDA and other regulatory agencies approval.

• Acceptable Worldwide as a well established cell line most attractive for cell-based vaccine production

• Other cell lines:

*MDCK *Per C6 *SF9

Rational

• Viral diseases are a challenge to the World biomedical communities

• Influenza is a major cause of concern

• Possibility of a pandemic & stock piles

• Cell-based vaccines takes shorter lead time

• Bioreactor systems are available for scale-up

• Problems with egg-based vaccines:– Low titer

– Problems with availability of high quality, pathogen free eggs

• Provide detailed studies on the utility of a packed-bed bioreactor systems for the production of vaccines utilizing Vero cells as a model for anchorage dependent cell lines.

• Compare the single-use bioreactor system-Eppendorfs BioBLUR- to the traditional re-usable bioreactors.

• Demonstrate the advantages of the packed-bed system in a semi-continuous mode of operation for long-term growth of Vero Cells.

Specific Objectives

Media

Selection

Vero Cell Process Development

Cell Line

Selection

Bioreactor

Selection

DMEM+ 5% FBS Packed-Bed

Vero

Experimental Approach

DMEM ( GlutaMAXTM)Media was used for high density growth of Vero cells.

• Contains 4.1 g/L Glucose

• Supplemented with:

– 5% FBS from Gibco- Reduced %

– Pre-supplemented with stable form of L-Glutamine that prevent ammonia build-up

Medium

• Vero Cells. African Green Monkey Kidney cells (Vero 76

ATCC 158) were utilized in this study.

• Vero cells are anchorage dependent cells.

• Typically grown on T-Flasks and Roller Bottles.

• For scale up, microcarriera are used in a pitched-blade

bioreactor.

• There are no published records of growing Vero cells in

the Packed-Bed bioreactor system.

Cells

• Vero cells have been used extensively in vaccine production since the 1960.

• They are also utilized in the detection of verotoxins that are the cause of hemorhagic syndrome in humans.

• Vero cells are susceptible to a wide variety of viruses including:

Polioviruses, Simian Viruses, Rubellaviruse Adenoviruses, Influenza viruses

Vero Cells and Vaccine Production

Packed-Bed Basket Technology

Stirred Tank Technology

Cell Immobilization

Technology

BasketTechnology

Combines the Best of

Both Technologies

Packed-Bed Bioreactors

Magnetically coupled bearing housing

Harvest tube

Medium lift impeller

Packed bed - basket with Fibra-Cel disks

Ring sparger

Aeration cage (special design)

Draft tube

Foam elimination cage (special design)

Medium inlet tube

Gas in

The Basket Design

Fibra-Cel Disks

• Surface area of Fibra-Cel disks 1,200 cm2/g

• Packe-Bed bioreactor 1.7L working volume provides 102,000 cm2 which is:

• Equal to 120 roller bottles (850 cm2)

• Equal to 453 T-flasks (225cm2)

• Equal to 4080 T-flasks (25cm2)

o

o Source: Eppendorf data

Seed Train Development

Packed-Bed Bioreactor System

MIXING & MASS TRANSFER

The impeller rotation creates negative pressure in the

hollow impeller tube causing medium to circulate

uniformly over the entire basket

Advantages:

- Reduced shear force

- High mass transfer of nutrients

- Can use with suspension cells as well as

anchorage dependent cells

Experimental Approach

• Vero cells were seeded directly into the Bioreactor in Medium

• Seeding Cell Density 1 x 105 cells/mL

• Perfusion started three days after seeding the Packed-Bed Bioreactor

• 30 minutes were allowed for complete entrapment

• Impeller speed initially set to 40 rpm

• The experiment continued for 21 days with periodic glucose addition or media exchanges.

The Comparative Study: Blue vs Glass

Growth Parameters

• Temperature set at 370C• Dissolved Oxygen (DO) set at 40% saturation• pH set at 7.1• Agitation set at 80 rpm• Air Flow: 0.004-1.0 SLPM

Growth:

• Daily glucose up-take: calculate total glucose consumed in 24 hours

• Daily Lactate production: calculate total lactate produced in 24 hours

• Cedex Bio from Roche Custom

Biotech was utilized in

Glucose and Lactate

measurement

• Growth and viability in T-Flasks during seed train

development were measured utilizing Cedex Cell Analyzer

Measured Variables

Results

Glucose Up-Take

HOURS IN CULTURE

GLUCOSE CONSUMPTION FEEDING SCHEDULE

0 4.01

21 3.77

45 3.57

67 3.29

92.5 2.86 Glucose feed shot

92.6 3.95

116.5 3.58 Media Exchange

141.8 2.97

142 3.91 Glucose feed shot

164.5 3.42

190.5 2.73

190.6 4.9 Media Exchange

214.5 4.35

238.5 3.71 Glucose feed shot

259.1 3.27

259.3 4.04 Media Exchange

284.5 3.29

308.25 2.49

308.35 4.81 Glucose feed shot

332.5 4.06

356.5 3.45

381.5 2.99

381.6 3.78 Media Exchange

406.6 3.12

430.6 2.44

430.8 5.02 Glucose feed shot

454.8 4.53

480.8 4.04

503.1 3.7

Glucose and Lactate Concentrations

Total glucose consumption (Gluctotal) in the glass

and the single-use bioreactor-21 days

Grams of glucose consumed hourly

(Gluctotal/h)

Glucose consumption per measurement

interval (Gluci) plotted against time

Glucose Consumption Rate to Cell Density

0

5

10

15

20

25

30

35

40

45

0 5 10 15 20

Ca

lcu

late

d c

ell

de

nsity (

X1

06

ce

lls/m

L)

Time (day)

Calculated Vero cell growth curve

From Han and Sha Application Note 2017

• Bench-top packed-bed bioreactors are ideal for small batch sized

productions especially with Vero cell or for vaccine production.

• Statistically they are similar to the pitched-blade bioreactors in terms

of cell growth.

• Production can be enhanced significantly in perfusion mode with

packed-bed basket technology:

– No reseeding with new batches of cells required as in Pitched

blade,

saves time and effort

– Reduced chance of contamination

– Reduced chance of loss of viability

– Virus production can become continuous with the amount of

space available for new cells to attach to

Concluding Remarks

Packed-Bed Process for Vero Cells

• High cell density- Increased surface area.

• Disks remain stationary

• Cells grow in a 3-D environment

• Little Shear effects

• Medium exchange without cell loss

• Packing density: 50% of vessel working volume

• Bottom line: Vero Cell can be efficiently expanded in packed-bed bioreactors with Fibra-Cel disks as growth support even with lower seeding densities

CHO Cells in the Packed Bead Bioreactor

Media

Optimization

Cell Culture Process Development

Expression

System

Bioreactor

Selection

- Invitrogen- CD-CHO Medium

-Pitched-Blade

-Packed-Bed

rCHO Cell Line

Packed-Bed vs Pitch Blade

Pitched-Blade BioreactorPacked-Bed Bioreactor

Project Timeline

Day 0

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7-15

Glu/Lac &Cell Count

Seed5x105

Cells/ml

InducePre & Post Glu/Lac& Cell Count

Seed5x105

Cells/ml

Glucose & Lactate Levels

Perfusion ½LPre & Post Glu/Lac

Perfusion 1LPre & Post Glu/Lac

Perfusion 2LInducePre & Post Glu/Lac

Perfusion 2L E.O.D.Pre & Post Glu/Lac

Glu/Lac &Cell Count

Glu& Lac

Growth and Production Patterns

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100 120 140 160

time

Ce

lls,s

ub

str

ate

,pro

du

cts

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100 120 140 160 180 200

time

Ce

lls,s

ub

str

ate

,pro

du

cts

Batch Periodic Exchange

Glucose up-take: Pitched-Blade/ Packed-Bed

(A) single-use [CelliGen BLU]; and (B) reusable [glass]. From: Taylor, Barnett & Rashid, Bioprocessing J. 2013

ALKP Production rCHO Cells

ALKP production in pitched-blade (Batch) or packed-bed

bioreactor systems (perfusion) mode of operation. Values are

average of two trials independent trials for each experiment

From: Taylor, Barnett & Rashid, Bioprocess J. 2012

Acknowledgments

• Joseph Duffy; Chris Bellerive; Dan Mardirosian; Worcester Polytechnic Institute, Worcester, MA

• Shannon Guertin; AbbVie, Inc. Worcester, MA

• Ma Sha; Eppendorf, Inc. Enfield, CT

Thank You

Questions ??