High-throughput, high-titer CHO production of miniproteins with high yields and low endotoxin levels, surpassing traditional E. coli production methods.
Protein Sciences: Early-Stage R&D Insect Platform
WuXi Biologics provides protein production services utilizing insect cell culture due to its ability to produce proteins that are often difficult to express in prokaryotic or other eukaryotic systems. Insect cell platforms offer protein glycosylation and other post-translational modifications similar to mammalian cells with the added benefit of high productivity, fast turn-around times and cost-effective production.
Insect Cell Culture Platform Features
Protein Types Supported G protein-coupled receptors (GPCRs) Ion channel
Cell line screening: Sf9, Sf21, HighFive
Flashbac system to reduce timeline
Cell surface receptor Viral envelope protein Kinases Proteases and enzymes Protein complex Other intracellular proteins
Optimization of expression conditions at small scale (construct screening, MOI, harvest time… ) High-throughput purification system for purification of many variants
Scale up volumes up to 20 L
Routine Soluble Protein Production: 4-5 weeks
Gene Synthesis 1-2 weeks
Virus Generation and Pilot Expression 1.5 weeks
Scale up Expression, Purification (Affinity, IEX, and SEC) and QC 1.5 weeks
Production Platform Activity
Features
Timeline
QC and deliverable
Gene synthesis and Subcloning Baculovirus production
Free cloning into WuXi Biologics vector P0, P1, P2
2 weeks 1-2 weeks
4 μg DNA 10 8 PFU/mL virus titer
1 L – 20 L 4 x 40 mL Additional QC
1-step purification 2-3 step purification RP-HPLC, bioburden, DSF, DSC, SPR, LC/MS, SEC-MALS, peptide mapping, cIEF, residual DNA, ELISA, Western blot, freeze thaw study, micro-developability testing 2-4 weeks 2 weeks Purified protein, SDS-PAGE, LC/MS Purified protein or screening report, SDS-PAGE
Case Study: GPCR Production
Purified protein, SDS-PAGE, SEC-HPLC
Aim • Production of a high-quality GPCR protein in insect cells • Reconstitution of GPCR into nanodiscs Key Challenges • Expression level of GPCR was very low <0.2 mg/L • The protein was unstable and its conformation was very flexible • It was hard to reconstitute GPCR into nanodiscs Results • Expression constructs were optimized • ~1.5 mg/L was achieved after optimizing expression and purification protocol • Purity >90% • GPCR’s conformation was stabilized by suitable ligand • Successful assembly of GPCR-nanodiscs
Construct 1: Nt_BRIL
BRIL
ECL1
ECL2
ECL3
N-term
Extracellular
Nanodisc
W P P
Q V
Extracellular
S
Q
A Y N T I L S C O V C N
F
R
D M V Y A A Y C L L L I L I A F L N O V L V A I R E C W M L I P A
C
S I W V A L A Q L L V N L V A
F I F I O L A M M V A T L I N A C V S D
F V V Y M P A T L V
L N P
V L
Y O
P H
L N
Y V
S Y
C P
H I
F A
S Y
S F
S T F S A F V L M V L T Y A Y I V R Q L R I Q R V V
I T F P F A L C C I V F L G V V A M L T Q K K A M E F K K M S
A I F L S W V V M I L L R V R P S A
W Y L
Membrane Scaold Protein
V G L S N A N L
P L
4.5-5.6 nm
V P
Y I T F T N
F
Intracellular
Y N Y
~10.6 nm
T
L
R
A
L
K
M A V
M
T
K
F
A
Intracellular
A Q R A L
F
I
F
ICL2
ICL1
C-term
ICL3
BRIL
Construct 2: ICL3_BRIL
SEC-HPLC
150
M
Monomer
50 kd 75 kd 250 kd
100
GPCR
50
Aggregation
15 kd 20 kd
MSP
0
2
3
4
5
6
Time (min)
Case Study: Automated 2-step Purification of Kinases
Aim • Production of 13 of Kinase variants from Baculovirus pellets Key Challenges • Expression level is different for different variants • Urgent timeline Results • Successfully delivered >95% purity proteins in 1 week
NiNTA elution M T S SEC fraction
Flow-through of sample
mAU
2500
2000 1500 1000 500 0
SEC elution
mL
0
50
100
150
200
250
Automated 2-step purification
Automated 2-step purification results
Run time/sample Max. sample/week Manual operation during steps
4 h 16 No
Learn more: Protein Sciences: Early-Stage R&D Insect Platform
About WuXi Biologics
WuXi Biologics is a leading contract research, development and manufacturing organization (CRDMO) that provides end-to-end capabilities to healthcare organizations worldwide. With operations in China, the United States, Ireland, Germany, and Singapore, we enable our partners to effectively and efficiently bring biologics and vaccines to patients worldwide through our comprehensive and high-quality drug development model.
The world’s leading global single-source platform from concept to commercialization.
wuxibiologics.com | info@wuxibiologics.com
5-1-2024
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