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I. ExpiSf™ Chemically Defined Medium
Figure 2. Consistent growth and protein production with ExpiSf medium. (A) ExpiSf CD
Medium (blue line) shows more consistent doubling time over 14 passages compared to
yeastolate containing media. (B) ExpiSf CD Medium (Blue line) have higher peak cell densities
(~20x106 cells/ml) compared to yeastolate Medium (Red line). (C,D) Consistent growth and
protein expression in the ExpiSf media across four lots of media..
INTRODUCTIONThe Baculovirus Expression Vector System (BEVS) is a versatile platform for
expression of individual recombinant proteins as well as multimeric protein
complexes, virus-like particles, and membrane proteins. Recently, BEVS has
demonstrated particular utility in the commercial-scale manufacture of vaccines
and gene therapy vectors. Unlike mammalian expression systems that have
long since transitioned to serum-free, chemically defined culture media,
relatively little innovation has taken place in insect expression systems, with
insect cells continuing to rely on undefined, yeastolate-containing culture media
that can exhibit significant cell growth and protein expression variability from lot-
to-lot. Here, we describe the ExpiSf™ Expression System, the first chemically
defined insect expression system that enables high-yield production of proteins
and viral particles with consistent performance run after run using a fast,
streamlined workflow. The system also enables faster baculovirus generation
with an improved transfection reagent and a chemically defined enhancer to
boost protein expression. We demonstrate scalability of the ExpiSf system from
small to large scale (WAVE™ and Bioreactor) for the production and purification
of recombinant proteins, virus-like particles (VLPs), and recombinant adeno-
associated viruses (AAV). Together, the ExpiSf Expression System enables
consistent production of diverse classes of recombinant macromolecules with
significant improvements in titers compared to alternative BEVS platforms.
Kenneth Thompson1, Maya Yovcheva1, Sara Barnes1, Melissa Cross1, Katy Irvin1, Natasha Lucki2, Henry Chiou2, and Jonathan Zmuda1
1Thermo Fisher Scientific, Inc., 7335 Executive Way, Frederick, MD 217042Thermo Fisher Scientific, Inc., 5781 Van Allen Way, Carlsbad, CA 92008
ExpiSf™ Expression system: A Chemically Defined Baculovirus-Based
System for Enhanced Protein and Virus Production in Sf9 Cells
Figure 1. Systems based approach to optimize Baculovirus-based protein expression.
For Research Use Only. Not for use in diagnostic procedures.
© 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
ExpiSf™ CD Medium Attributes:
• Yeastolate free and Chemically-defined (CD)
• Animal origin-free (AOF), serum-free and protein-free
• No supplementation required
• One media for virus generation and protein expression
• Manufactured under cGMP
• Consistent cell growth and protein expression over multiple media lots
• Consistent Performance for over 12 months
• Available in AGT™ dry powder format for large scale applications
Figure 3. Characterization of ExpiSf9 cells adapted to ExpiSf CD medium. (A) ExpiSf9
cells. (B) Consistent growth over passages. Lines represents the growth of ExpiSf9 cells in
ExpiSf CD Medium at passage 4 (Blue line) and passage 17 (Red Line) (C) Consistent
protein expression over passages.
II. ExpiSf9™ Cells adapted in ExpiSf™ CD Medium
ExpiSf9™ attributes:• Adapted for high-density culture in Chemically Defined Medium
• ~24 hour doubling time
• Optimized for high-density infections
• Stable growth and expression profiles over 25+ passages
Figure 9. Production and purification of recombinant Adeno-Associated Virus (rAAV).
(A) Higher rAAV2 genome titers in ExpiSf System compared to Sf900II traditional workflow.
(B) Scalable rAAV2 production in ExpiSf system. Shows comparable rAAV2 titers between 125
mL shake flask and Finesse® 3L stir-tank bioreactor. (C) AKTA chromatogram showing strong
elution peak of rAAV6 purified on POROS™ CaptureSelect™ AAVX Affinity Resin. (D)
Coomassie gel on fractions from rAAV6 purification run in (C). Total rAAV6 yield of 1.1 x 1014
vector genomes per liter of culture.
VI. Protein Characterization in ExpiSf™ System
VII. rAAV production in ExpiSf™ System
Figure 7. Expression and Purification of Secreted Alkaline Phosphatase (SEAP). (A) SEAP
activity measured by chemiluminescence assay for protein expressed in Sf-900 II Medium and
ExpiSf System (B) Size Exclusion Chromatograph of SEAP Purified from Sf-900 II Medium and
ExpiSf System (C) SDS-PAGE of SEAP purified from Sf-900 II Medium and ExpiSf System (D)
Glycan profiles of SEAP expressed in Sf-900 II Medium and ExpiSf System
Secreted Proteins
G-protein coupled receptors
A B
C D
Sample %HMW %Purity %LMW
1.SEAP Purified from Sf900-II Medium 0.3 94.8 4.9
2.SEAP Purified from ExpiSf System 2.1 95.4 0.5
S f-9 0 0 I I M e d iu m E x p iS f S y s te m
0
1
2
3
4
5
Re
lati
ve
Lu
min
ec
en
ce
Un
its
(x
10
5)
III. ExpiFectamine™ Sf Transfection Reagent
Figure 4. Characterization of ExpiFectamine™ Sf Transfection Reagent. (A) Baculovirus titers
obtained at Day 3 and Day 4 following Adherent and Suspension protocols. (B) The ExpiSf system
P0 virus produces equivalent protein titers compared to P1 or P2 virus obtained following the
classical adherent workflow.
Faster time to protein with Expifectamine Sf transfection reagent
ExpiFectamine™ Sf Transfection
Reagent Attributes:
• Optimized suspension protocol reduces
time to protein by half
• No time consuming viral amplification
• Large scale P0 virus production in as
little as 3 days post-transfection to be
used directly in protein expression runs
Figure 5. Characterization of ExpiSf™ Enhancer. (A) ExpiSf Enhancer, used in conjunction with
ExpiSf CD Medium and ExpiSf9 cells, generated 3-fold higher GFP titers than a traditional Sf9
workflow; ExpiSf Enhancer nearly doubled protein titers compared to the ExpiSf System without
enhancer addition. (B) Addition of ExpiSf Enhancer 16-24hr prior to infection gives the highest protein
titer improvement.
IV. ExpiSf™ Enhancer
ExpiSf™ Enhancer Attributes:
• Essential for obtaining high protein titers
• Needs to be added18-24hr before infection
• Optimized for ExpiSf CD Medium
V. Superior Growth and Expression in ExpiSf™ system
Figure 6. Superior growth and protein expression compared to yeastolate containing medium.
(A) Cell growth of ExpiSf9 cells in ExpiSf CD Medium (Blue Line) and four different yeastolate
containing media. (B-D) Comparison of the ExpiSf System to traditional Sf9 workflows using various
yeastolate-containing media (1-5). Expression levels of an Fc fusion protein, green fluorescent
protein (GFP), and tumor necrosis factor-alpha (TNF-a) were higher in the ExpiSf expression system
compared to those obtained using a traditional Sf9 workflow.
1 2 3 4 5 E x p i S f
S y s t e m
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
GF
PT
ite
r (
mg
/L)
T r a d i t i o n a l S f 9 w o r k f l o w
1 2 3 4 5 E x p i S f
S y s t e m
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
Fc
fu
sio
n t
ite
r (
mg
/L)
T r a d i t i o n a l S f 9 w o r k f l o w
1 2 3 4 5 E x p i S f
S y s t e m
0
2 0
4 0
6 0
8 0
TN
Fa
tit
er
s (
mg
/L)
T r a d i t i o n a l S f 9 w o r k f l o w
A
B
C
D
0 1 2 3 4 5 6 7 8
0
5
1 0
1 5
2 0
2 5
D a y s i n c u l t u r e
VC
D (
x1
06
ce
lls
/mL
)
Y C M e d i u m 1
Y C M e d i u m 3
Y C M e d i u m 2
Y C M e d i u m 4
E x p i S f C D M e d i u m
Y C M e d i u m 5
Figure 8. Expression of Cannabinoid receptor type 2 in the ExpiSf system. (A) Time course
study determined that 48 hours post-infection is the optimal collection time point. (B) Viability of
ExpiSf9 cells during the time course study in (A). (C) Total CB2 molecules measured by flow
cytometry assay. Shows comparison of total CB2 produced in ExpiSf system versus in Sf900II
media following a traditional Sf9 workflow.
A B C
T r a d i t i o n a l S F 9
W o r k f l o w
E x p i S f S y s t e m
0
2 1 01 1
4 1 01 1
6 1 01 1
8 1 01 1
1 1 01 2
To
ta
l C
B2
2 4 3 2 4 0 4 8 5 6
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
H o u r s p o s t - i n f e c t i o n
CB
2/c
ell
2 4 3 2 4 0 4 8 5 6
0
2 0
4 0
6 0
8 0
1 0 0
H o u r s p o s t - i n f e c t i o n
Via
bil
ity
(%
)
Figure 10. Superior expression of an enveloped and non-enveloped VLPs in ExpiSf
System. (A) Chikungunya virus like particles expressed in Sf900II and ExpiSf System. (B)
Human papilloma virus like particles expressed in Sf900II and ExpiSf System
VIII. Expression of VLPs in ExpiSf system
A B
Corresponding author: Kenneth Thompson, [email protected]
A B
C D
S h a k e F l a s k F i n e s s e B i o r e a c t o r
1 09
1 01 0
1 01 1
1 01 2
Ge
no
me
tite
r (v
g/m
L)
1 2 5 m L 3 L
1 2 3
0
2 1 01 0
4 1 01 0
6 1 01 0
8 1 01 0
1 1 01 1
D a y s p o s t - i n f e c t i o n
Ge
no
me
tit
er
(v
g/m
L) S f 9 0 0 I I
E x p iS f
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