A Chemically-Defined Baculovirus-Based Expression System for Enhanced

Protein Production in Sf9 Cells

Maya Yovcheva1, Sara Barnes1, Kenneth Thompson1, Melissa Cross1, Katy Irvin1, Mintu Desai1, Natasha Lucki2, Henry Chiou2,
Jonathan Zmuda1

1Thermo Fisher Scientific, Inc., 7335 Executive Way, Frederick, MD 21704

2Thermo Fisher Scientific, Inc., 5781 Van Allen Way, Carlsbad, CA 92008

INTRODUCTION AND ABSTRACT III. Characterization of ExpiFectamine™ Sf Transfection VII. ExpiSf System Scalability
Here, we present data on the performace of a novel Sf9-based Baculovirus Reagent
expression system based upon a yeastolate-free, animal origin-free, 1200

chemically-defined, high-density culture medium that allows for Sf9 cells to ExpiFectamine™ Sf Transfection Reagent 25 100 125 m L 1000

P r o t e in T it e r ( G F P ) m g / L
250 m L

V C D ( x 1 0 c e ll/m L )
reach densities nearly twice as high as those attained in traditional yeastolate- • Convenient protocol 20
500 m L 800

% V ia b ility
1L
containing media. Additionally, Sf9 cells adapted to grow to high densities in • Scalable virus production
15
600

6
50

the yeastolate-free media were generated and a new, high-efficiency Bacmid 10

400
• Adherent- 6-well plate to T-25 flask format
transfection reagent was developed to allow for the generation of high titer 5
200

baculovirus stocks. Together, with the addition of protein expression enhancer • Suspension - 4mL to 100mL or greater 0
2 4 6 8
0
0
D a y s in c u ltu r e F la s k s iz e 125 m L 250 m L 500 m L 1L 2L 3L
these improvements allow for the optimization of a new expression protocol
A B C u ltu r e v o lu m e 25 m L 50m L 100m L 200m L 400m L 1000m L

that takes advantage of the high cell densities achievable with the new
chemically-defined medium and adapted Sf9 cells, as well as high multiplicity 1 1 0
D a y 3 H a rv e s t
in fe c t io u s v ir u s p a r tic le s /m L

in fe c t io u s v ir u s p a r tic le s /m L
10
D a y 4 H a rv e s t 10
1400
of infection (MOI), to significantly improve protein titers and enable lot -to- lot 15
100
1200

V ia b le c e lls /m L (x 1 0 )
consistency of both cell growth and protein expression in a defined media

6
80
1 1 0
1000

G F P ( m g /L )
9
9
10
formulation.

% V ia b ility
10 60 800

600
40

1 1 0 8 5 400
Media 10 8
20
200
In f e c t io n
0 0 0
-4 -3 -2 -1 0 1 2 3 4 5 3 4 5
1 1 0 7
10 7 D a y in C u lt u r e
D a y s P o s t In fe c tio n
A d h e re n t S u s p e n s io n Kept at 4oC F r o z e n in L N 2 a n d t h a w e d W a v e s y s te m 1 2 5 m L F la s k
Optimized W a v e s y s te m F la s k

Baculovirus Figure 8. ExpiSf System can be scaled up or scaled down

Expression ExpiSf System is directly scalable from 125mL to 1L flask size. Comparable cell growth (A) and protein
System expression (B) were achieved at 125 rpm shake speed. The ExpiSf System also can be scaled down to 24
deep well plate for cell growth (C) and protein expression (D).

VIII. Protein Expression in ExpiSf System vs Traditional

Systems Figure 9. Superior
growth and protein
Figure 1. System based approach to optimize Baculovirus-based protein expression E x p iS f C D M e d iu m
expression compared
S u p p lie r 1
system D S u p p lie r 2 to yeastolate
A S u p p lie r 3 B
containing medium
I. Consistency of ExpiSf™ CD Medium in Sf9 Cell
S u p p lie r 4
1500 25 80
Protein Titer (GFP) mg/L

S u p p lie r 5
(A) Cell growth of

T N F a t it e r s ( m g /L )
V C D ( x 1 0 c e ll/m L )
Culture
20
1000
60
ExpiSf9 cells in ExpiSf
15
40
>5x CD Medium (Blue Line)

6
and four different
ExpiSf™ CD Medium Attributes:
500
10
20
yeastolate containing
5

• Yeastolate free and Chemically-defined (CD) 0

P1 P2 P0 0
medium
Classical Adherent ExpiFectamine
TM
Sf 0 1 2 3 4 5 E x p iS f
S ys te m (B) Expression of Protein
• Animal origin-free (AOF), serum-free and protein-free Workflow Suspension Workflow 0 2 4 6 8
S u p p lie r
D a y s in c u ltu r e A in five yeastolate
C D containing medium and
• No supplementation required
Figure 4. Characterization of ExpiFectamine™ Sf Transfection Reagent and 1000
140
ExpiSf System (Last Bar)

F c f u s io n t it e r ( m g /L )
120
• One media for virus generation and protein expression baculovirus generation 800
(C) Expression of Protein
G F P T it e r ( m g /L )
>3x 100
>4x B in five yeastolate
• Manufactured under cGMP (A) Baculovirus Titers obtained at Day 3 and Day 4 from Adherent and Suspension Protocol 600 80

60 containing medium and

(B) Baculoviruses can be frozen at -80 or LN2 for longer storage. Slight reduction in titer is typically 400
40 ExpiSf System (Last Bar)
• Consistent cell growth and protein expression over multiple media lots observed, but when accounted for it does not affect protein expression 200
20 (D) Expression of Protein
(C) Optimized suspension protocol allows for reduction of the time to protein in half
• Consistent Performance for over 12 months C in five yeastolate
0 0
1 2 3 4 5 E x p iS f
(D) Equivalent protein titers can be obtain by using P0 from ExpiFectamine Sf compared to P1 or P2 1 2 3 4 5 E x p iS f
S ys te m S ys te m

S u p p lie r containing medium and

• Formulated for high density Sf9 cell growth from classical adherent workflow S u p p lie r
ExpiSf System (Last Bar)
IV. Characterization of ExpiSf™ Enhancer
A B ExpiSf™ Enhancer Attributes:
IX. Protein Characterization in ExpiSf System
E x p iS f C D M e d iu m Y e a s to la te M e d iu m
E x p iS f C D M e d iu m

80
Y e a s to la te m e d iu m 1 25 100
• Essential for obtaining high protein titers Secreted Proteins Figure 10. Expression
Y e a s to la te m e d iu m 2 and Purification of
V C D ( x 1 0 c e ll/m L )
D o u b lin g tim e (h o u r s )

Y e a s to la te m e d iu m 3
20 80
• Needs to be added18-24hr before infection A B
Secreted Alkaline
% V ia b ility

R e la tiv e L u m in e c e n c e U n its (x 1 0 )

60
5

15 60
• Optimized for ExpiSf CD Medium 5
Phosphatase (SEAP)
6

40 10 40
A B
4
(A) SEAP activity
3 measured by
5 20
20
2 chemiluminesence assay
500
0 0 1200
1
Sample %HMW %Purity %LMW for protein expressed in
0 0 2 4 6 8 10 1.SEAP Purified from Sf900-II Medium 0.3 94.8 4.9
Sf-900 II Medium and
P r o t e in T it e r ( G F P ) m g / L

1000 400
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 D a y s in c u ltu r e 0 2.SEAP Purified from ExpiSf System 2.1 95.4 0.5
ExpiSf System (B) Size
P r o t e in T it e r ( m g /L )

S f - 9 0 0 II M e d iu m E x p iS f S y s t e m
Passages
800
300
Exclusion Chromatograph
600
of SEAP Purified from Sf-
C 25 100 D 1200 D a y 3 H a rv e s t D a y 4 H a rv e s t
900 II Medium and ExpiSf
200
C D
1000
400 System (C) SDS-PAGE of
20 80
SEAP purified from Sf-900
V C D ( x 1 0 c e lls /m L )

G F P T it e r ( m g /L )

100
800 200
II Medium and ExpiSf
% V ia b ility

15 60
600 0 0
System (D) Glycan
6

r
r

T r a d itio n a l S f9 E x p iS f S y s te m E x p iS f S y s te m
profiles of SEAP
h

h
e

2
c

10 Lot 1 40
1

3
n

400 W o r k flo w (N o e n h a n c e r) ( P lu s e n h a n c e r )
a

expressed in Sf-900 II
h

Lot 2
n
E

T im in g o f a d d itio n
Figure 5. Characterization of ExpiSf™ Enhancer
)

Medium and ExpiSf

(-

5 Lot 3 20 200
Lot 4
0 (A)ExpiSf Enhancer, used in conjunction with ExpiSf CD Medium and ExpiSf9 cells, generated 3-fold System
0 0
0 1 2 3 4 5 6 7 8
T r a d itio n a l S F 9 Lot Lot 2 Lot Lot
higher GFP titers than a traditional Sf9 workflow; ExpiSf Enhancer nearly doubled protein titers
D a y s in c u ltu r e
compared to the ExpiSf System without enhancer addition.
W o r k f lo w 1 3

E x p iS f C D M e d iu m
4

(B) Addition of ExpiSf Enhancer 18-24hr prior to infection gives the highest protein titer improvement. G-protein coupled receptors
A B Figure 11. Expression
Figure 2. Characteristics of ExpiSf9 CD Medium V. Protein Expression Workflow 1 .0  1 0 1 2
and Purification of
8 .0  1 0 1 1
(A) ExpiSf CD Medium (blue line) shows more consistent doubling time over 14 passages compared to Cannabinoid receptor
T o ta l C B 2

three other yeastolate containing media. (B) ExpiSf CD Medium (Blue line) have higher peak cell 6 .0  1 0 1 1
type 2
densities (~20x106 cells/ml) compared to yeastolate Medium (Red line). 4 .0  1 0 1 1 (A) Total CB2 Molecules
(C) Consistent growth in the ExpiSf Media across 4 different media lots. measured by flow
2 .0  1 0 1 1
(D) Consistent protein expression in the ExpiSf Media for over 4 ExpiSf CD Medium lots cytometry assay for
0 protein expressed in Sf-
II. Growth of ExpiSf9™ Cells adapted in ExpiSf™ CD
S f 9 0 0 II M e d iu m E x p iS f S y s t e m
900 II Medium and
ExpiSf System
Medium (B) Schematic
representation of the
ExpiSf9™ Cells cell line attributes: C
25000
D
100
CB2 Structure (C) Time
Figure 6. Protein Expression Workflow course study determined
• Adapted for high-density culture in Chemically Defined Medium 20000 80
that 48hpi is the optimal
• ~24 hour doubling time VI. Comparison of protein expression between ExpiSf collection point in the
C B 2 /c e ll

% v ia b le

15000 60

ExpiSf System (D)

• Optimized for high-density infections System and High Five cells 10000 40
Viability of the ExpiSf9
5000 20
cells post infection
• Stable growth and expression profiles over 25+ passages
0 0 during the time course
24 32 40 48 56 24 32 40 48 56
study
A B C H o u r s p o s t -in f e c t io n H o u r s p o s t -in f e c t io n

25 100 500

CONCLUSIONS
V C D ( x 1 0 c e ll/m L )

P r o t e in T ite r m g /L

20 80 400

We describe a system-based approach for enhancing levels of protein

% V ia b ility

15 60 300
6

10 40 200 production in insect cells using Bac-to-Bac generated viruses that allows for
5 Passage 4
Passage 17
20 100 production of proteins at levels exceeding those of the most of the popular
0
0 5 10
0 0 systems used at the moment. This performance enhancement was made
Passage 8 Passage 23
D a y s in c u ltu r e possible through the incorporation of multiple novel reagents, including: (1)
Figure 3. Characterization of Sf9 cells adapted to ExpiSf Chemically-Defined medium Figure 7. Protein expression in ExpiSf System and High Five cells Chemically Defined Culture Medium that allows for high density cell growth
(A) ExpiSf9 cells morphology (A) SEAP activity measured by chemiluminesence assay for protein expressed High Five cells in and infection, (2) Sf9 Cells Adapted to grow optimally in the CD Medium, (3)
(B) Consistent growth over passages. Lines represents the growth of ExpiSf9 cells in ExpiSf CD Medium Expressive medium and in the ExpiSf System
(B)GFP protein titers measured by fluorescent assay for protein expressed High Five cells in
an optimized transfection reagent for baculovirus generation, (4) a novel pre-
at passage 4 (Blue line) and passage 17 (Red Line) (C) Consistent Protein Expression over passages.
Protein titers at passage 8 (Blue Bar) and passage 23 (Red Bar) Expressive medium and in the ExpiSf System infection expression enhancer solution, and (6) a simple-to-perform workflow.
For Research Use Only. Not for use in diagnostic procedures.
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