1

Discovery • Development • Production

2013 Pfenex, Inc.

Rapid, Cost Effective Microbial Strain Development Using BLI

Greg Cantin

2

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.

3

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

2013 Pfenex, Inc.

4

Pfēnex Expression Technology™

2013 Pfenex, Inc.

5

» 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™

2013 Pfenex, Inc.

6

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

2013 Pfenex, Inc.

7

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

2013 Pfenex, Inc.

8

‣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

2013 Pfenex, Inc.

9

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.

10

‣ 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

2013 Pfenex, Inc.

11

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

12

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.

13

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

2013 Pfenex, Inc.

14

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

15

Case Studies

2013 Pfenex, Inc.

16

Strain Engineering

2013 Pfenex, Inc.

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

17

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

18

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

2013 Pfenex, Inc.

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

20

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

21

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

22

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

23

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

2013 Pfenex, Inc.

24

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

25

Fully Integrated Process Development Capability

2013 Pfenex, Inc.

26

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

27

Example: Hexameric Tandem Repeat Protein, Strain Screening through Purification

2013 Pfenex, Inc.

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

28

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)

29

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

30

BLI Analysis of Purified Intact 4-4-20 mAb

2013 Pfenex, Inc.

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

31

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

2013 Pfenex, Inc.

32

Acknowledgements

Pfenex» Steve Maki» Jason Payne

» Nicole Glenn

» Luie Bentillo

» Michael Chtourou

» Jeff Allen

2013 Pfenex, Inc.

ForteBio (Pall)DARPA