Increasing Recombinant Protein Expression in Insect Cells

expressionsystems.com

Increasing Recombinant Protein Expression in Insect Cells

Increasing Recombinant

Protein Expression in Insect

Cells


• An Insect and Mammalian Cell Culture

Media Company

• Contract Manufacturing


Platform to Produce Recombinant Protein

Cells

Media

Vector

Culture Vessel


Cell Type

• Cell line choice is influenced by a number of

factors

– What is the goal?

• ie virus production is typically done in Spodoptera cells

– Is there a licensing fee?

– How easy is it to grow?

– Does it actually express my protein of interest?

– Does the produced protein have the characteristics I

want?


Trichoplusia vs Spodoptera

80 kD Protein 15 kD Protein

60

120

50

100

40

80

mg/L

30

20

PRO

Sf9

60

40

PRO

Sf9

10

20

0

Day 3 Day 4

0

Day 3 Day 4

Expression of secreted proteins. Both cell lines were infected at an

MOI of 1.


Do the Glycosylation Patterns Seen

Suit My Protein?

• Eukaryotic

processing but

not mammalian

type

• This may or may

not influence

application

• Absence of

terminal sialic

acids is often

unacceptable for

therapeutics

Watanabe, S. et al. J. Biol. Chem.

2002;277:5090-5093


Altering Glycosylation Patterns

• Transgenic insect cell

lines were made by

transforming with

mammalian genes

• SfSWT-1 cells were

transformed with 5

mammalian

glycosyltransferases

Expression of rTSH is

enhanced compared to

parental Sf9 cells

ng/ml

2500

2000

1500

1000

500

0

Sf9

SfSWT-1


Vector Choice

• Shuttle vectors allow for a variety of

modifications

– Targeting signals, purification tags, promoter

choice, enhancers

• Vectors for stable cell line

• Basis of lytic vectors is AcMNPV with

modifications of the backbone

– Deletions, manipulations for ease of use


Promoter Usage

Expression of a

secreted protein in

PRO cells

• Secretion enhanced

with honeybee mellitin

signal

Cells were infected at

an MOI of 1 and

harvested day 4

mg/L

90

80

70

60

50

40

30

20

10

0

p10

pH

*In progress*

p6.9


Media Manipulations

• Serum supplemented media

• Serum-free and protein-free formulations

• Additives

• Future Directions-

– Chemically defined

– Optimized for stable cells versus shorter term

lytic production runs


Supplementation over course of

production

Secreted Protein

mg/L

100

90

80

70

60

50

40

30

20

10

0

Day 3 Day 4

PRO

PRO PBA

Cells were infected at an MOI of 1. Production Boost Additive was

delivered as 5% of the total culture volume 18 hours post infection.


Timing of Viral Infection

Experiment:

Take Sf9 cells from seed stock and

inoculate flask at 10 6 /ml

Let grow overnight so they are in

log phase

40

35

30

25

mg/L 20

15

10

5

Mid Conc

High Conc

Infect at MOI of 1

0

7

1 2 3 4 5

9

6

8

7

5

6

5

4

3

2

U/ml

4

3

2

Mid Conc

High Conc

1

0

0 1 2 3 4 5

1

0

1 2 3 4 5


Low MOI Experiment

• Hypothesis: Infecting with a low MOI may

increase overall recombinant protein yield

• Sf9 cells were infected rBV expressing

human transferrin as a model of a secreted

protein

• Samples taken over timecourse to determine

cell count, viability, spread of bv infection,

and protein production


gp64 Staining Over Range of

MOI

% Positive

100

90

80

70

60

50

40

30

20

10

0

1 2 3 4 5

Day of Infection

1.E+00

1.E-01

1.E-02

1.E-03

1.E-04

1.E-05

• gp64 expression demonstrates spread of

virus


Total Number of Infected Cells

Increases with Lower MOI

6

# of Infected Cells per ml

5

4

3

2

1

0

1 2 3 4 5

Days of Infection

1.E+00

1.E-01

1.E-02

1.E-03

1.E-04

1.E-05

• Uninfected cells do not undergo growth arrest

leading to more targets for viral spread


Viral Burst Size

Cells grown in suspension in ESF 921

• Viral infection determined by gp64 staining

• Titers determined by flow cytometric method

• Assumes generation time of 24 hours

• Estimation based on low MOI expt with Sf9 cells:

131 viruses per generation


What is the predicted amount of

MOI

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

virus produced and resulting MOI?

Virus

Produced

3.12 x 10 8

1.01 x 10 8

1.64 x 10 7

# of

targets

3.15 x 10 6

4.84 x 10 6

4.48 x 10 6

Effective

MOI

x10 6 /ml

6

5

4

3

2

1

0

# of Infected Cells

1

2

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)


What is the predicted amount of

MOI

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

virus produced and resulting MOI?

Virus

Produced

3.12 x 10 8

1.01 x 10 8

1.64 x 10 7

# of

targets

3.15 x 10 6

4.84 x 10 6

4.48 x 10 6

Effective

MOI

99

x10 6 /ml

6

5

4

3

2

1

0

# of Infected Cells

1

2

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)


What is the predicted amount of

MOI

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

virus produced and resulting MOI?

Virus

Produced

3.12 x 10 8

1.01 x 10 8

1.64 x 10 7

# of

targets

3.15 x 10 6

4.84 x 10 6

4.48 x 10 6

Effective

MOI

99

21

x10 6 /ml

6

5

4

3

2

1

0

# of Infected Cells

1

2

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)


What is the predicted amount of

MOI

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

virus produced and resulting MOI?

Virus

Produced

3.12 x 10 8

1.01 x 10 8

1.64 x 10 7

# of

targets

3.15 x 10 6

4.84 x 10 6

4.48 x 10 6

Effective

MOI

99

21

3.7

x10 6 /ml

6

5

4

3

2

1

0

# of Infected Cells

1

2

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)


Does the estimated MOI meet our

predictions?

MOI

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

Virus

Produced

3.12 x 10 8

1.01 x 10 8

1.64 x 10 7

# of

targets

3.15 x 10 6

4.84 x 10 6

4.48 x 10 6

Effective

MOI

99

21

3.7

Percentage of Infected Cells

80

70

60

50

40

30

20

10

0

1

2

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)


Taking it a step further-

Predicting spread in the 10 -3 culture

MOI

1

10(-1)

10(-2)

10(-3)

10(-4)

Virus

Produced

3.12 x 10 8

1.01 x 10 8

1.64 x 10 7

Assume

1.64 x 10 6

# of

targets

3.15 x 10 6

4.84 x 10 6

4.48 x 10 6

Effective

MOI

99

21

3.7

x10 6 /ml

6

5

4

3

2

1

# of Infected Cells

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

10(-5)

0

1

2


Taking it a step further-

Predicting spread in the 10 -3 culture

MOI

1

10(-1)

10(-2)

10(-3)

10(-4)

Virus

Produced

3.12 x 10 8

1.01 x 10 8

1.64 x 10 7

Assume

1.64 x 10 6

# of

targets

3.15 x 10 6

4.84 x 10 6

4.48 x 10 6

5.12 x 10 6

Effective

MOI

99

21

3.7

x10 6 /ml

6

5

4

3

2

1

# of Infected Cells

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

10(-5)

0

1

2


Taking it a step further-

Predicting spread in the 10 -3 culture

MOI

1

10(-1)

10(-2)

10(-3)

10(-4)

Virus

Produced

3.12 x 10 8

1.01 x 10 8

1.64 x 10 7

Assume

1.64 x 10 6

# of

targets

3.15 x 10 6

4.84 x 10 6

4.48 x 10 6

5.12 x 10 6

Effective

MOI

0.32

99

21

3.7

x10 6 /ml

6

5

4

3

2

1

# of Infected Cells

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

10(-5)

0

1

2


Taking it a step further-

Predicting spread in the 10 -3 culture

• MOI of 0.32 times 5.12

x 10 6 targets = 1.64 x 10 6

infected cells/ml

• # of infected cells

determined by cell

counts and staining:

• 1.83 x 10 6 0

x10 6 /ml

6

5

4

3

2

1

# of Infected Cells

1

10(-1)

10(-2)

10(-3)

10(-4)

10(-5)

1

2


Production of human Transferrin

35

30

mg/L

25

20

15

10

5

MOI = 1

MOI = 10(-1)

MOI = 10(-2)

MOI = 10(-3)

MOI = 10(-4)

0

1 2 3 4 5 6 7


Low MOI with PBA

• Hypothesis: The high cell density that

occurs when using a low MOI may benefit

from a nutritive boost to maintain cell

health

• Experiment: Infect Sf9 cells at an MOI of

10 -2 . Add PBA after the bulk of the cells are

infected with baculovirus.


Production of human Transferrin

45

40

mg/L

35

30

25

20

15

10

5

1 % PBA

5% PBA

10% PBA

No PBA

Control

0

Day 3 Day 4 Day 5 Day 6

• PBA was added 36 hours after the addition of virus

• Cell count at the time of PBA addition was between 10-

12x10 6 cells/ml


Low MOI Expression in PRO cells

• PRO cells are derived

from T.ni embryos

Cells were infected at

an MOI of 1 and 0.1

• 72 hour harvest shows

higher MOI to be

superior

25

20

15

10

5

MOI=0.1

MOI=1

0

1 2 3


Low MOI Expression in PRO cells

• PRO cells are derived

from T.ni embryos

Cells were infected at an

MOI of 1 and 0.1

• 72 hour harvest shows

higher MOI to be superior

• Waiting an extra day can

have dramatic benefits

90

80

70

60

50

40

30

20

10

0

1 2 3 4

MOI=0.1

MOI=1


Platform-Build Your Own?

Cells:

Sf9, Sf21,

Tni, Ea4, S2,

Bm4, Dpn1,

etc etc

Culture Vessels:

Fermentor

WAVE

Fernbach

Plates

Scale:

mls to Ls

Vectors:

Lytic-Bacmid Bacpak, etc

Stable

Transient

Promoter choices

Media:

Serum-free

Protein-free

Animal-free

Additives


High Throughput Screening

• Rapid screening to speed time to target

• Two cell lines to maximize possibility of

detecting product

• Plaque purification of virus can wait until

candidates are selected

• Small scale expressions (


Production of Protein of Interest

• Pick cell line that creates product with most

desirable characteristics

• Pick vectors that help streamline purification

downstream

– Purification tags, enhancement of solubility

• Pick stable vector, plaque purify, maintain low

passage virus

• Maximize cell density during production

• Fine tune media formulation specific to protein


Acknowledgments

Brooks Hayes

Aaron McKinney

Ron Makishima

Allen Rodrigo-

University of Auckland

Don Jarvis-

University of Wyoming

Loy Volkman-

UC Berkeley

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