Download Advances in the Drosophila Expression System

Advances in the Drosophila Expression System
W.A. de Jongh, CSO
New Cells, New Vaccines VII
18 March, 2013
Overview
• About ExpreS2ion Biotechnologies
• Drosophila S2 and The ExpreS2 system
• Two vaccine development case studies
EXPRES2ION Biotechnologies
• Founded in 2010 in Copenhagen, Denmark
• A CRO spin-off from Affitech A/S
• Proprietary technology platform for production of proteins
based on Drosophila S2 insect cells
• > 10 years experience with S2 cells from research to GMP
production for human clinical trials
Drosophila S2 insect cells
• Derived from the late stages of Drosophila melanogaster (fruit fly)
embryos (Schneider, 1972)
• Doubling times of 18-24 hrs (both serum-containing and serum-free
medium)
• Cell size: 9-10 µm in diameter
• Grow well between 5 and 350E6 cells/ml in suspension
• Grow at 23ᵒC to 27ᵒC
• Very robust
The ExpreS2 System
An integrated and proprietary protein expression platform
for the biopharmaceutical industry
Know-How
Expression
vectors
Transfection
reagent
Expression
Host
Culture
medium
•
•
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•
•
From construct to protein in 6 weeks
GLP protein production
Vector and cell line development
Upstream process development
Scale-up and transfer to cGMP
Advantages of the ExpreS2 System
• High success rate for complex proteins and VLPs
• High yields for secreted proteins
• Very fast to protein and stable process
•
•
•
•
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Very high cell densities without aggregation
Straightforward scalability, from lab to cGMP
Robust system
Homogeneous product
Regulatory acceptance & reduced risk of human viral
contaminants
Saves time in
R&D
Flexibility &
Compliance
in Production
ExpreS2 Improves Yields
Medium development
Vector development
300
100
100
pExpreS2-1
90
RANKL
Mg/L
RANKL (mg/L)
Mg/L RANKL
pExpreS2-2
80
70
60
50
40
30
20
10
0
Hybrid_RNA+
Hybrid
pAc5.1/V5
pMT/V5.His.A
Control
Med.1
Med.2
Med.3
Med.4
Med.5
Med.6
ExpreS2ion
*Adaptation to individual media were performed
Quick to Protein and Stable Process
Transfection
Stable polyclonal cell line
- High stability
Week 1
Week 3
Week 6
Purified protein
candidates from
bioreactor
1st screening of protein
candidates
High density
frozen cell
lines
3-4 Weeks
What makes the ExpreS2 system different
Compared to
Baculovirus insect
cell systems
• Non-viral, Non-lytic
• More homogeneous product
• High reproducibility and consistency between
batches
• High yield for secreted proteins
• Long-term stability of frozen cell stocks
Compared to
other S2 systems
•
•
•
•
Increased yield
Protocols for large-scale production
Including Perfusion processes
Support for process development and cGMP
production
More Homogeneous Product
• Consistent Glycosylation
BEVS produced Hemagglutinin A from H5N1 bird flu material in:
Sf21
Sf9
Compared to:
Stable S2 cell line
3xHA2
HA1
HA2
Effect of different MOI’s and harvest time points
BEVS: Baculovirus Expression Vector System
MOI: Multiplicity of infection
Consecutive days of harvest
from a bioreactor running perfusion
rProteins Made in Insect Cells
are Clinically Validated
Baculovirus-based Products on the market
gsk
Dendreon
Protein Sciences
HPV vaccine –Cervarix®
Prostate cancer – Provenge®
Flublok
Market
Market
FDA approved
Drosophila S2 products in clinical development
Pharmexa
TxCells
Merck Inc.
Hawaii Biotech
Pharmexa
Copenhagen University
HER-2+ breast cancer vaccine
Crohn’s disease
Tetravalent Dengue fever vaccine
West Nile virus vaccine
Bone metastatic cancer vaccine
Placental malaria vaccine
Phase 2
Phase 2
Phase 1
Phase 1
Late PC
Late PC
Role of ExpreS2 in Malaria Vaccine Development
Copenhagen University
Placental Malaria
The Jenner Institute,
Oxford University
Blood stage malaria
Vaccine development case study 1:
Placental Malaria Vaccine Development
Assc. Prof. Ali Salanti, Prof. Thor Theander
Placental Malaria Vaccine
VAR2CSA from Plasmodium falciparum
was identified by CU as a possible vaccine
target (2003)
CU Malaria case study
Antigen Selection and Expression
VAR2CSA is a complex 350 KDa protein,
with 7 domains.
Using Biosensor technology, the core CSA
binding site within the DBL2X and minimal
binding region (ID1-DBL2Xb; 62 Kda) were
identified (CU)
CU Malaria case study
Screening Antigen Variants with ExpreS2
Variant
construction
Shake-flask
production
(500ml)
1L-15L
production
CU Malaria case study
• Screening capability: ~40 variants have
been tested
• Expression ability and level
• Initial immune response
• Immune response, cross-reactivity
• Scalability
Relative expression level of 34
VAR2CSA truncation variants
Immunogenicity and Cross-Reactivity
AB induction (rats)
VAR2CSA fragments
-ABs raised in rats can
block binding
Cross-strain reactivity
VAR2CSA fragments induce
cross-reactive ABs to
parasite subtypes
Cross reactivity of 2 variants shown .
Y axe - Proportion of binding
CU Malaria case study
Application of Single-Use Technology
• Flexiblity
• Reduced cost of goods
Current Status and Future Work
 Minimal VAR2CSA regions can be expressed as functional proteins
in ExpreS2
 The proteins induce functional antibodies in various animal
species
 Variant selection is completed
• Upstream & downstream development is ongoing
• Scale-up and transfer to CMO for GMP production
• Phase Ia and Ib within 3 years
Vaccine development case study 2:
Development of a Broadly-Neutralising Vaccine
against Blood-Stage P. falciparum Malaria
Dr. Simon Draper
Next-Generation Anti-Merozoite Vaccines
1. Are there CONSERVED and
HIGHLY SUSCEPTIBLE target
antigens in the Mz?
2. Is it possible to develop a
strain-transcending anti-Mz
vaccine
Rh5 Identified as Vaccine target
MEROZOITE
Erythrocyte Invasion by
P. falciparum Merozoite
RH5
RH5 is essential in culture – KOs failed
RH5 has an essential interaction with BSG
Basigin
Red blood cell
Parasitophorous
Vacuole Formation
and Invasion
Inward Motion
Driven by
Actinomyosin
Motor
Highly conserved
(Potentially highly cross-reactive)
RBC
Merozoite
Attachment
Protein in adjuvant vaccine100% protection in primate model
Resealing of RBC
and Parasitophorous
Vacoule Membrane
The Problem?
• Rh5 is extremely hard to produce
– Failure:
• E.coli
• Yeast
• Baculovirus
– Partial success in transient Hek293
• Low yields ~1mg/L
• A fusion protein, not native Rh5
ExpreS2 system expresses Rh5 at high levels
PfRh5 S2
supernatant
70
Rh5
55
TFF/buffer
exchange/filtration
S2 produced Rh5 binds Basigin
Affinity
Chromatography
Kd = 0.9µM
1
Size Exclusion
Chromatography &
concentration
Kathryn Hjerrild, Matt Higgins, Kate Wright
1 2
2
Initial Process Optimization
Application of single use and perfusion technology
Batch
Fed Batch
ATF-Perfusion
The ATF (Alternating Tangential Flow) method concentrates cells and protein
Production using Fed-batch and
High-Density Perfusion Culture
Fed-Batch
ATF-Perfusion
Process Dependent Rh5 Yields
• Hek293 yield = 1mg/L
• S2 yield ...
Concentrated Perfusion/
Concentrated Fed-batch
Take Home Messages
• Successful expression of difficult proteins
• Well suited to Vaccine manufacture (clinical track record)
• High yields for secreted proteins (multiple processing options)
• Fast (6 weeks)
• Great for R&D (speed, ease of use, high success rate)
Acknowledgments
Assc. Prof Ali Salanti
Prof. Thor Theander
Assc. Prof. Morten Agertoug Nielsen
Mafalda Dos Santos Marques Resende
Besim Berisha
Simon Draper
Sandy Douglas
Joe Illingworth
Kathryn Hjerrild
Charlotte Dyring
Anette Strøbæk
Stine Clemmensen
Carsten Leisted
Lars Poulsen
Sancha Salgueiro
Selected Customers and Collaboration Partners
Questions?
GLYCOSYLATION
Insect (S2)
CHO
Human
N-glycosylations found in RANKL based on MALDI-TOF analysis
0 N-glycosylation
+1 N-glycosylation
+2 N-Glycosylation
Native
Glycosylated RANKL1.12
- 5 different Mw’s
N-deglycosylated
RANKL1.12
34
C C+F
C
C
C+F
C C+F C+F
The parasite expresses proteins on the surface of infected red
blood cells, which allows it to bind different tissues in the body
IRBC
Infected erythrocyte
VAR2CSA
CSA
Placental tissue