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Discovery • Development • Production
2013 Pfenex, Inc.
Rapid, Cost Effective Microbial Strain Development Using BLI
Greg Cantin
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Corporate Overview
» Located in San Diego, California USA
» 35 FTEs primarily in SoCal, with satellite offices in St. Louis and Boston
» 23,000 square feet of space in Sorrento Valley housing capabilities in biologics research, development and commercialization
» Founded as a platform technology company; now a product development company with four business units
» Global partnerships in the US, EU and Asia
2013 Pfenex, Inc.
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What We MakeBiosimilars» Protein Development» Partners: Biosimilar Manufacturers
Vaccines» Anthrax, Malaria, Pandemic and Seasonal Flu» Partners: Government, Large Pharma
Strain Engineering- Lead Proteins, Components & Antigens» Macromolecules, Vaccine Antigens, Antibody Fragments,
Vaccine Components, Cytokines» Partners: Large Pharma, Biotech, Academia
Reagent Proteins» Vaccine Components, Vaccine Antigens, “Custom Proteins”» Customers: Academia, Industry, Distributors
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Pfēnex Expression Technology™
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» P. fluorescens: Gram negative, non-pathogenic, metabolically versatile organism
» Develop effective high throughput growth and assay methods» 96 well growth and assay methods for production of soluble, active protein
» Rapid, effective, scaled down fermentation development
» Comprehensive DSP development capability
» Accelerates pre-clinical and clinical development
Discards the traditional, linear and iterative approach, adopts parallel, HTP method for microbial strain
development
CELL BIOLOGY EXPRESSION STRAIN
Pfēnex Expression Technology™
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Strain Libraries and Vectors to Enhance Protein Expression
Sequence the genome of P. fluorescens strain MB101 » 6.5 mega base genome, > 6300 genes, 1.4 X E. coli » Use bioinformatics to annotate strain
Identify targets for engineering » Gene annotation» Transcription array, proteome analysis – apply to cultures undergoing
target protein expression
Create strains » Targeted gene deletion/insertion, precise molecular biology tools» Mapped transposon insertions» Overexpression of helper proteins on separate plasmid, multiple
inducible promoters
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Thousands of unique components can be seamlessly integrated to enable strain engineering for optimal protein production
The State of the Art for Bacterial Strain Engineering
The Pfenex Toolbox
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‣High throughput parallel processing‣ Speed, Quality and Yield create significant
advantages in real and opportunity costs
STRAIN CONSTRUCTION SEED HTP EXPRESSION
HARVEST& ANALYSIS
plasmids host strains electroporate andgrow transformantson selective media
inoculate seedculture into
HTP mediumreplicate cultures
will be grown for 24h
induce andincubate for 24h
Thousands of strains evaluated in parallel in <5 weeks
Robotically Enabled High Throughput Growth and Expression
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Challenge: To accurately analyze target in complex mixture of solids, protein, DNA, etc.
QUANTITY ADDRESSED QUALITY ADDRESSED
1st TierAnalysis
2nd TierAnalysis
SAMPLES:1000s SAMPLES:10s to 100s
BLI allows both quantity and quality measurements in crude cell extracts
Measure Fragments, Aggregates, etc.
HTP Expression30-50 OD achieved
Measure target mass yield
High Throughput Analytical Capability
2013 Pfenex, Inc.
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‣ Showing the optimal plasmid expression strategy‣ Showing the optimal host strain phenotypes
Pfenex explores a much larger experimental design space, defined by interactions between expression strategy and host phenotype
“Expression Space”: Plasmid & Host Strain Effects
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Impact of Host Strain Phenotype on Target Gene expression
2013 Pfenex, Inc.
» In most cases the parallel processing, toolbox approach yield numerous strains expressing at least some target
» In the illustrated case the only strain giving significant expression was over-expressing one particular protein folding modulator
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Fermentation scouting » 5 strains» Up to 9 variable induction conditions each» Milligrams of protein
Text
Fermentation confirmation » 2-4 strains» 2-4 variable induction conditions each» Grams of protein
Enables rapid (<4 weeks) production of protein to speed product progression through development value chain
24-unit 4 mL 8-unit 1.0L
Scaled Down Fermentation Assessment Accelerates Development
2013 Pfenex, Inc.
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Production Strain Producing High Titers of High Quality Protein in ~ 8 WeeksProduction Strain Producing High Titers of High Quality Protein in ~ 8 Weeks
Hundreds of Expression Strains Cultured in ParallelHundreds of Expression Strains Cultured in Parallel
High-Throughput Analytical: Quality, Titer & FunctionHigh-Throughput Analytical: Quality, Titer & Function
Strain Down-SelectionStrain Down-Selection
High-Throughput Analytical: Quality, Titer & Function
High-Throughput Analytical: Quality, Titer & Function
DNA + Pfēnex Expression Technology™ Toolbox
High-Throughput Analytical:Quality, Titer & Function
High-Throughput Analytical:Quality, Titer & Function
16 Parallel 1L Fermentations16 Parallel 1L Fermentations
48 Parallel μ-Scale Fermentations48 Parallel μ-Scale Fermentations
8 w
eeks
Next Generation Expression Technology
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BLI Methods Developed at Pfenex
2013 Pfenex, Inc.
Protein Type Expressed in
Pfenex Platform
Biosensor Configuration
Antibody derivative (multiple types)
Immobilized Protein L or antigen
Fab (multiple) Immobilized antigen
Fab’ Immobilized antigen
Mab Immobilized small molecule
Vaccine #1 Immobilized kinasedomain
Vaccine #2Immobilized
antibody to vaccine tag
Cytokine Immobilized receptor
Growth factor (multiple)
Immobilized growth factor receptor
Dozens of BLI methods developed Dozens of BLI methods developed
Thousands of assays performed on different protein product types
Thousands of assays performed on different protein product types
Variety of immobilization strategies utilized with Dip and Read SensorsVariety of immobilization strategies
utilized with Dip and Read Sensors
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Case Studies
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Strain Engineering
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Protein Type Alternative Host Pfenex Results
Fab Yeast: quality issues, low yield10-20X yield improvement high quality at 1L scale
Microbial Outer Membrane Protein
E. coli: completely insoluble expression; poor quality
Soluble active expression 20 g/L
Growth Factor Yeast: low yield, degradation, glycosylation
20X yield improvement at HTP scale, high quality, active
Therapeutic Enzyme E. coli: undesirable isoforms, quality issues
10X yield improvement; no isoform issues
Human Cytokine E. coli: inclusion bodies; no soluble expression
Soluble active expression; elimination of refold step
Multimeric Antibody Derivative CHO: low expression(<10 mg/L)
20X yield improvement; soluble active protein; scaled to 1L in 8 weeks
Vaccine Antigen Native organism; cost issues 10X yield improvement
» >85% Success Rate in programs failing in another host
Exa
mpl
es o
f Sta
lled
Dev
elop
men
t
…Creating Success from Failure
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Example: Granulocyte Colony Stimulating Factor (G-CSF) binding to G-CSF Receptor
2013 Pfenex, Inc.
Real-time binding curves of varying amounts of G-CSF in null lysate
Streptavidin -coated biosensor
BiotinylatedG-CSF receptor
G-CSF
Biosensor configuration for G-CSF analysis
» Develop low-cost production of G-CSF (filgrastim) as a biosimilar in the therapeutic protein market; G-CSF currently produced in E.coli as an inclusion body
» Use BLI method to screen Pfenex strains for soluble and functional G-CSF in lysate» Good sensitivity and range for G-CSF, and low interference from null lysate
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G-CSF Expression Analysis: 1st Tier Screen Using BLI
2013 Pfenex, Inc.
240 strains (three replicates each)
A C D E F G H I J K LB
Rel
ativ
e B
LI A
ctiv
ity
Evaluate G-CSF expression in 240 strains (12 plasmids x 20 host strains)
Approximately 30 strains were selected based on specific expression levels and diversity of host and secretion leaders for further analysis
Plasmid:
G-CSF Expression Analysis: 2nd Tier Screen Using SDS-CGE
SDS-CGE analysis shows soluble band co-migrating with standard
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null
null
MW
MW
STD
STD
1 2 3 4 5 6 7 8 9 10 11 12 13
14 15 16 17 18 19 20 21 22 23 24 25 26 27
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G-CSF Fermentation Evaluation Using BLI
2013 Pfenex, Inc.
Unit
0
100
200
300
400
500
600
700
800
0 5 10 15 20 25Post-induction Time (hr)
u1u2 u3u4u5 u6 u7 u8
G-CSF expression reproduced at 1-L scale using scouting
conditions from DOE at mini-bioreactor scale
G-CSF expression reproduced at 1-L scale using scouting
conditions from DOE at mini-bioreactor scale
+ + - -
Induction condition factorsA B C D
Rel
ativ
e B
LI a
ctiv
ity
Rel
ativ
e B
LI a
ctiv
ity
DOE at MBR scale (4 mL) shows induction condition factors A and
B support higher expression
DOE at MBR scale (4 mL) shows induction condition factors A and
B support higher expression
Fermentation Scouting Fermentation Confirmation
A and Bconditions
Further optimization experiments can be designed based on A and
B factors
Further optimization experiments can be designed based on A and
B factors
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Small Disulfide Bonded Protein Strain Screening
2013 Pfenex, Inc.
» 100s of strains screened in HTP expression study» Soluble, secreted target shown to be active in multiple binding assays» Both secretion leader (plasmid) and host strain appear to influence expression » Five strains advanced to fermentation evaluation
275ng010 013 016 018 042 056 057 060 089 096 097 100 129 132 133 136 138 140 230 236 237 238 261 268 269 276 280 null Std Std+ null
* * *
* co- expressed folding modulator
0
100
200
Plasmid: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
EACH RED BAR ILLUSTRATES RANGE OF VOLUMETRIC TITERS FOR 3 REPLICATES (BLACK DOTS) OF EACH STRAIN
Binding activity
SDS-CGE
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Small Disulfide Bonded Protein Fermentation Evaluation
post-induction samples except for * pre-induction samples
* *
REDUCED NON- REDUCED
SDS-CGE
LC/MS
2013 Pfenex, Inc.
» Increased yields at fermentation scale >5X following fermentation evaluation and confirmation at 1 L scale
» Leader properly processed» Strain selected for RCB
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Example: Antibody Fragment Rapid Transition from Discovery to Development
WEEK
0 1 2 3 4 5 6 7 8 9 10
Receive target sequence from discovery; gene
synthesis
Deliver >50mg/
99% pure
Deliver 500mg/
99% pure
Produce Interim
Standard
See Boehringer Ingelheimcase study (Pfenex.com)
Antibody Fragment: Gene 0.5 g; 99% Pure Protein in < 10 Weeks
STRAIN SCREEN
FERMENTATION SCOUTING
PURIFICATION
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Antibody Fragment (Fab)
0
100
200
300
400
500
Volu
met
ric A
ctiv
ity (u
g/m
L)
Leader AHeavy Chain Secretion Leader:
Leader F
Leader B Leader C Leader D Leader E Leader A Leader B Leader C Leader D Leader E
Leader ALight Chain Secretion Leader:
DC432Strains (three replicates each)
0
100
200
300
400
500
Volu
met
ric A
ctiv
ity (u
g/m
L)
Leader AHeavy Chain Secretion Leader:
Leader F
Leader B Leader C Leader D Leader E Leader A Leader B Leader C Leader D Leader E
Leader ALight Chain Secretion Leader:
DC432Strains (three replicates each)
Problem: Low Yield on High Dose Requirement ProductSolution: Choice of Secretion Leaders Influences Expression
and Activity
BLI
2013 Pfenex, Inc.
» Soluble expressed Fab protein bound to target antigen in BLI (activity) assay» Yields of soluble expressed Fab in hundreds of expression strains ranged
from ~90 to 490 µg/mL at 96 well scale» 4 fold further improvement at 1 L fermentation scale
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Fully Integrated Process Development Capability
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Rapid Strain Construction and
Screening
Fermentation Scouting and Optimization
Primary Recovery
*Purification
*Characterization and Analysis
Strain EngineeringThousands of strains- rapid cloning, periplasmicexpression, 96-well screening
Process AnalyticalRobotic sample processing, microchip SDS-CGE analysis and biolayer interferometry binding assays
Fermentation DevelopmentMultiple strains each evaluated in multiple scalable fermentation processes
Protein PurificationPrimary recovery and chromatography options evaluated in parallel microtiter plate format
Product Quality AnalysisDetailed characterization (MALDI, QTOF MS, RP-HPLC, SEC, fluorescence); impurity analysis (HCP, DNA, LPS)
Aut
omat
ion-
enab
led
proc
ess
anal
ytic
al
Sol
uble
, act
ive
prot
ein
Pfenex Parallel Development Approach
2013 Pfenex, Inc.
* BLI also used in these steps
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Example: Hexameric Tandem Repeat Protein, Strain Screening through Purification
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mg/
L (B
LI)
SDS-CGE
STAGE 1: 1000 STRAINS TESTED BY BINDING AFFINITY, UP TO 1 g/L STAGE 2: 5 STRAINS ASSESSED IN MINI-
BIOREACTORS, UP TO 2 g/L
0
2
4
6
8
10
12
14
0 5 10 15 20 25
g/L So
l Tar
get (B
LI )
Post-induction time (hr)
CS594-0222 (2)CS594-0222 (7)CS594-0233 (4)CS594-0233 (6)CS594-0236 (1)CS594-0236 (4)CS594-0385 (4)CS594-0385 (8)
STAGE 2: 4 STRAINS ASSESSED IN 1 L FERMENTATIONS, UP TO 10 g/L
PURIFICATION 30 g LOT
PURIFIED SAMPLE ACTIVITY TESTED USING BLI (STANDARD CURVE, QUANTITATIVE MODE)
ACTIVITY AS EXPECTED
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Example: Analysis of Purified Fab #2
2013 Pfenex, Inc.
BLI Binding Traces of Purified Sample and Standard at the Same Concentration
bind
ing
dept
h (n
m)
time (sec)
Reference Standard (blue)
Pfenex-produced Fab (red)Streptavidin -coated
biosensor
Biotinylatedantigen
Fab
Biosensor configuration for Fab analysis
» No difference in calculated dissociation constant, Kd (data not shown)» Pfenex-produced Fab activity also comparable in vitro by cell-based
assay (data not shown)
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Purified Nef Protein BLI Analysis
2013 Pfenex, Inc.
association (10 min) dissociation (15 min)
[Nef]
696 nM
348 nM
174 nM
87 nM
44 nM
22 nM
BLI kinetic traces for binding to Hck SH3 domain Purified Nef
Calculated KD for purified Nef = 186 ± 20 nM
Biotinlyated Hck SH3 domain NefNefStreptavidin
SensorStreptavidin
Sensor
BLI sensor configuration for Nef detection
PxxP domain of Nef specifically interacts with SH3 domain of Src family kinases1,2
1Geyer et al. EMBO reports 2: 580-585, 20012Karkkainen et al. EMBO reports 7: 186-191, 2006
Heating Nef at 70ºC/4M urea for 30 min shows no binding (not shown)
Nef – Hck SH3 domain affinity:Reported in literature: KD = 250 nM3
3Lee et al. Cell 85: 931-942, 1996
overlays of 4 reps at 6 concentrations
Binding activity observed by BLI analysis comparable to literature values
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BLI Analysis of Purified Intact 4-4-20 mAb
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association (15 min) dissociation (15 min) [mAb]
64 nM32 nM16 nM
8 nM
4 nM
2 nM
BLI kinetic traces for binding to fluoresceinPurified 4-4-20 mAb
association (15 min) dissociation (15 min) [mAb]
64 nM
Calculated KD for purified 4-4-20 = 122 pM
16 nM
8 nM
4 nM
2 nM
Calculated KD for commercial anti-fluorescein mAb = 619 pM
Reported KD for 4-4-20 mAb = 59 pM1
1JBC 271: 5338-5346, 1996
Anti-fluorescein mAb standard
overlay of 4 reps at 6 concentrations
Analysis of chromatography fractions by BLI allows faster assessment of elution profile
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Speed of Development – Opportunity Cost Avoidance » Extensive expression toolbox: expression strategies and host strains » Robust high throughput methods developed to identify best strains quickly» Rapid fermentation optimization and reliable scale-up to thousands of liters» More leads through development, allowing more and faster “go, no go” decisions» Success rates upwards of 82%, after previous failure in other expression systems
Product Quality» Correct disulfide bonding» Secretion leader processing fidelity» Very low product heterogeneity » No glycosylation » Scalability of production- small scale to commercial quantities
Cost of Goods » High specific and volumetric expression of target protein» High cell density, defined mineral salts medium» Two day fermentation turnaround» Simplified and scalable downstream processing methods, periplasmic release; no unique
processing equipment required
Pfēnex Expression Technology™ Value
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Acknowledgements
Pfenex» Steve Maki» Jason Payne
» Nicole Glenn
» Luie Bentillo
» Michael Chtourou
» Jeff Allen
2013 Pfenex, Inc.
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