Scaleable Protein Production in Anchorage Scaleable Protein Production in Anchorage Dependent Mammalian CellsDependent Mammalian Cells

1 Biotechnology Unit, NIDDK,National Institutes of Health, Bethesda, MD 2 Center for Biosystems Research, UMBI, College Park, MD 3 Department of Chemical Engineering, UMCP, College Park, MD

Nicole A. Bleckwenn123, William Bentley23, and Joseph Shiloach1ABSTRACT

Recombinant protein production is a common method for providing clinical and commercial quantities of human therapeutic agents. Methods of production include recombinant bacterial fermentations, transfected or transformed cell culture, and virally infected cell culture. A potential protein production method with anchorage dependent mammalian cells has been evaluated. The system utilizes a recombinant vaccinia virus with the VOTE expression system that can potentially be used to produce large, highly glycosylated recombinant proteins, which may be difficult to produce by other means. A reporter protein, enhanced green fluorescent protein (EGFP), has been used to study the characteristics of this protein production method. Growth, infection and production parameters such as multiplicity of infection, culture volume during infection, infection duration, inducer concentration and timing of inducer addition were studied in monolayer culture. These results were then used to establish conditions for studies in microcarrier spinner flask and 1.5 L bioreactor cultures. Production processes utilizing the selected conditions will be described, together with an evaluation of the expression system.

Objective - Develop an alternative protein production process with recombinant vaccinia virus Necessary components

– Vaccinia Virus Transcription occurs in cytoplasm of infected cell Wide host range VOTE expression system for high, T7 promoter controlled expression

– High cell density HeLa cells – human cervical adenocarcinoma Attachment dependent – growth on microcarriers

Develop bioreactor strategy for growth Study infection process and parameters affecting expression

Project ScopeBACKGROUND

VOTE* Expression System

* 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.

Recombinant Vaccinia Virus Construction

vT7lacOI

HeLa Cells

Transfect

Infect

vEGFP-His6

PlaquePurify

Insert EGFP-His6

pVOTE.2-EGFP-His6

EGFP-His6

pEGFP-N1

EGFP

pSecTag2A-EGFP

EGFP-His6

InsertEGFP

Infected and Induced

Infected only

Uninfected

Vacuum

LevelControl

FeedPump

WaterJacket

InletOutlet

D.O.

pH

Agit.

Temp.

FiltratePump

AirInlet

MeshScreenModule

Diaphragm

ATFController

Time (hpi)

0 50 100 150 200 250

Via

ble

Ce

ll D

en

sit

y(m

illio

n c

ells

/mL

)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

ATF Feeding Run 1ATF Feeding Run 2Batch Run

Parameters Tested Result

Multiplicity of infection (MOI, pfu/cell) 1.0

Volume during infection Lowest Possible (0.5 mL)

Serum concentration during infection None

Infection duration 1 hr

Inducer concentration 1.0 mM IPTG

Inducer addition timing At time of infection

Time (hpi)

0 10 20 30 40 50 60 70

Intr

acel

lula

r E

GF

P(

g/m

illio

n c

ells

at

infe

ctio

n)

0.0

1.0

2.0

3.0

4.00.10.5 1.0 5.0 10.0 uninfected

Time (hpi)

0 10 20 30 40 50 60 70

Ext

race

llula

r E

GF

P(

g/m

illio

n c

ells

at

infe

ctio

n)

0.0

1.0

2.0

3.0

4.00.10.51.05.010.0uninfected

Time (hpi)

10 20 30 40 50 60 70

EG

FP

(g

/mill

ion

ce

lls a

t in

fec

tio

n)

0.0

2.0

4.0

6.0

8.0

IntracellularExtracellular

Infected Uninfected

Fluo

resc

ence

Ligh

t

FUTURE WORK

MOI Evaluation in Spinner Flasks

RESULTS – Cell Growth

Bioreactor Setup for Microcarrier Culture

ATF Unit Mechanism of ActionA view through the sight glass as the diaphragm moves through an exhaust and pressure cycle

Batch versus ATF Feeding Strategy

6-Well Plate Infection Experiments

RESULTS – Protein Expression

Infection in 1.5 L Bioreactor

lac O EMC SLO

Induced

InactiveRepressor

Target GeneExpression

T7 gene I gpt PL lac IPE/L TT PT7 PE/LTarget

Active Repressor

T7 gene I

lac O

gpt PL lac IPE/L TT

EMC SLO

PT7 PE/LTarget

Uninduced

Cytodex 3 MicrocarriersWith Attached HeLa Cells

More reactor studies–Dissolved oxygen, temperature, feeding strategy

Test production in other cell lines–MRC-5, Vero, HEK 293, etc.

Try other proteins (gp120, hGC-1) Verify post-translational processing

Special Thanks to Bernard Moss and Pat Earl (NIAID, NIH) for providing VOTE componentsRefine Technologies for providing ATF System

Picture of Bioreactor