Expression of EGFP Reporter Protein with a Recombinant Vaccinia Virus - Comparison of Microcarrier and Cell Suspension Based Bioreactor Systems

1 Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bethesda, MD

2 Center for Biosystems Research, UMBI, College Park, MD 3 Department of Chemical Engineering, UMCP, College Park, MD

Nicole A. Bleckwenn1,2,3, William Bentley2,3, and Joseph Shiloach1

ABSTRACTA recombinant vaccinia virus expression system was studied as an alternative method to produce recombinant proteins in a scaleable bioreactor system. A recombinant vaccinia virus containing the gene for the reporter protein, enhanced green fluorescent protein (EGFP), was used to study the system parameters that affect final protein expression. Evaluations of a microcarrier based method for adherent cell growth and infection in a bioreactor were performed. Controllable parameters such as temperature and dissolved oxygen level during production were tested to observe the effect on the level of protein production. Additionally, a cell suspension system was compared to the microcarrier method to determine if the levels of cell growth, viral infection, and ultimately protein expression could be maintained with a simpler production scheme.

Objective - Develop a protein production process with recombinant vaccinia virus Vaccinia Virus

Transcription occurs in cytoplasm of infected cell Wide host range VOTE* inducible expression system for high, T7 promoter controlled expression Utilize previously constructed recombinant virus containing the gene for the reporter protein, enhanced green fluorescent protein (EGFP), to examine expression characteristics

High Cell Density HeLa and HeLa S3 cells – human cervical adenocarcinoma Compare attachment dependent growth on microcarriers to suspension growth

Develop bioreactor strategies for growth Study infection processes for these two types of systems

BACKGROUND

CONCLUSIONS

COMPARISON OF MICROCARRIER AND SUSPENSION SYSTEMS

Growth Comparison in Bioreactors

Higher dissolved oxygen and lower temperature during production increase expression levels Both microcarrier and suspension systems can produce proteins

MOI above 1 appears to be more detrimental to suspension production than microcarrier production Microcarrier system has a higher specific production Suspension system has a higher overall production due to a higher cell densitySpecial Thanks to Bernard Moss and Pat Earl (NIAID, NIH) for providing VOTE components and Refine Technology, Co. for providing ATF™ System

PARAMETER OPTIMIZATIONProduction with the Microcarrier System

DO Levels in Spinner Flasks(by changes in surface area to volume ratio of media in flasks)

Temperature in Spinner Flasks

Microcarrier Culture Suspension Culture

Time (hpi)

0 10 20 30 40 50 60 70E

GF

P ( g

/mil

lion

cel

ls a

t in

fect

ion

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

MOI 0 MOI 1 MOI 5 MOI 10

Time (hpi)

0 10 20 30 40 50 60 70

EG

FP

( g

/mil

lion

cel

ls a

t in

fect

ion

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

MOI 0 MOI 1 MOI 5 MOI 10

* Ward, G. A., Stover, C. K., Moss, B., and Fuerst, T. R. (1995). Stringent Chemical and Thermal Regulation of Recombinant Gene- Expression By Vaccinia Virus Vectors in Mammalian-Cells. Proceedings of the National Academy of Sciences of the United States of America 92, 6773-6777.

DO Levels in Bioreactor

Effect of MOI on Expression in Spinner Flasks

Protein Production Comparison in Bioreactors

Vacuum

LevelControl

FeedPump

WaterJacket

InletOutlet

D.O.

pH

Agit.

Temp.

FiltratePump

AirInlet

MeshScreenModule

Diaphragm

ATFController

Bioreactor Setup for Microcarrier Culture

Gas inlet into reactor headspace

LevelControl

FeedPump

WaterJacket

InletOutlet

D.O.

pH

Agit.

Temp.

Hollow Fiber Unit

CirculationPump

PermeatePump

Bioreactor Setup for Suspension CultureGas inlet into headspace or liquid

Time (hpi)

0 10 20 30 40 50 60 70

EG

FP

( g

/mil

lion

cel

ls a

t in

fect

ion

)

0.0

2.0

4.0

6.0

8.0

10.0

30% DO 50% DO

Time (h)

0 50 100 150 200 250

Via

ble

Cel

l Den

sity

(m

illi

on c

ells

/mL

)

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

Suspension Microcarrier

Time (hpi)

0 10 20 30 40 50 60 70

EG

FP

(m

g/L

)

0.0

2.0

4.0

6.0

8.0

10.0

12.0

Suspension Microcarrier

Specific Production Overall Production

Time (hpi)

0 10 20 30 40 50 60 70

EG

FP

( g

/mil

lion

cel

ls a

t in

fect

ion

)

0.0

2.0

4.0

6.0

8.0

10.0

SuspensionMicrocarrier

Time (hpi)

0 10 20 30 40 50 60 70

EG

FP

( g

/mil

lion

cel

ls a

t in

fect

ion)

0.0

2.0

4.0

6.0

8.0

10.0

12.034°C infected 37°C infected 34°C uninfected 37°C uninfected

Time (hpi)

0 10 20 30 40 50 60 70

EG

FP

( g

/mil

lion

cel

ls a

t in

fect

ion

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0SA/V=1.1 cm-1SA/V=1.4 cm-1SA/V=2.2 cm-1SA/V=4.3 cm-1