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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

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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

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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

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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, kenneth.thompson@thermofisher.com

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