IBC’s Recombinant Protein & Complex Biologic Development and Purification, March 5, 2010

1. Virulence Factor-Based Drug Discovery
A Novel Approach for Identifying Drug Targets for
Autoimmune and Inflammatory Diseases
David Bienvenue, Ph.D.
VLST Corporation

2. Viral Logic Systems Technology
VLST
Office view: Office view:
July 8th, 2009, 3:43 PM The other 364 days of 2009
• Privately-held company founded in 2004
• ~35 Employees
• Focused on exploiting viral evolution to develop novel
biotherapeutics
• Based in Seattle, WA

3. Virulence Factors as a
Novel Route to Therapeutics
• Some viral proteins modulate/suppress host immune system
• Facilitate viral infection and influence severity of disease
• Can be homologous or unrelated to host genes
• Targets of viral proteins validated as treatment methods for
autoimmune/inflammatory illness
Drug Development Strategy
Identify Identify Define biologic Develop therapeutics
virulence cellular consequences mimicking
factors targets of interaction virulence factors

4. Discovery of Soluble Viral TNF Receptor
Key Step in Development of Enbrel®
TNFR2
Enbrel® (p75) Shope fibroma
(Entanercept) virus-T2
38 % sequence
identity
Fc
Smith et al (1990) Science 248: 1019, Smith et al (1991) BBRC 176: 335

5. Cytokines, Chemokines and Their
Receptors Encoded by Herpes Viruses
Alcami (2003)
Nat Rev
Immunol 3: 36

6. Genomic Scale Search
for Viral Virulence Factors
Analyzed >200 viral genomes
•Pox
>18,000 viral proteins
•Herpes
in VLST database •Adeno
•Asfar
Bioinformatic Expert System
>6,000 viral protein clusters of similar Selection Criteria
proteins
•Topology – anchor, secreted,
transmembrane
•Homology to human proteins
•Species infected by virus
•Pfam motifs
>600 putative virulence factors •Non-essential for viral replication
identified and queued for screening

7. Identification of Host Targets of
Virulence Factors
Bioinformatic mining for Transiently express affinity- Target
virulence factors tagged viral proteins identification
by LC-LTQ MS
Bind target(s) from
supe and lysates from
VF immune-related cell
lines, using tandem
VF affinity tag
Synthesize
viral genes VF

8. Viral Protein Expression
Target Discovery is a Numbers Game
Proteins expressed
Proteins screened
Targets identified
• Maximize number of viral factors going into screen
• Minimize effort, reagents spent on non-expressors
• Evaluate different vectors and cell lines with Freestyle
transfection reagent
• Scale up via technology, not FTE’s!

9. Effect of Cell line, Vector in Freestyle
Comparison of Day 4 Titers
Cells:
60 56.3
51.9 50.5 293-EBNA
50
40.9 43.1 43.6 40.6 40.0
45.2 293-F
40 36.8
CHO-F
ug/mL
30.7
30 26.7
20
CHO-EBNA
10
0 Vectors
F- GS
F
GS
9
S
F
409
EF
In-house
- 409
- GS
09
HEF
F- 40
CHE
- CHE
EB- G
EB- 4
F- CH
-EB
-EB-
293F
293F
EB- C GS
CHO
CHO
-EB
293F
293-
CHO
293-
CHO
CHO
CHEF
293-
CHO
•Same gene in different vectors and cell lines
•Similar expression levels with all combinations tried

10. N- And C-Tagged Virulence Factors
To Increase # Identified Targets
Affinity Resin HAC
Tag
• “HAC”- tandem affinity tag on one end of virulence factor
• Position of HAC may block binding, affect expression
• Gene synthesize both N- and C-term. tagged vectors
• >90% express at least one version, ~20% increase over
expressing C-tag alone
• Increases probability of identifying targets, in some cases,
only one version binds target

11. 24-well Shaker Plate Prescreen
• Reagent, time, effort wasted on large-
scale transfections if viral protein
doesn’t express
• Developed expression pre-screen of
CHO-EBNA transients Western blot
expression using 24-well shaker
plates
• Can be used to make relative
comparisons between different
expression constructs, media, etc.

12. Platform Highlights
•Identified numerous immunologically relevant
targets
•Validated targets of 4 approved, 11
investigational drugs
•Partnership with Novo Nordisk in 2008 to provide
research targets
•Therapeutic programs based on viral targets
entering the clinic in 2010/2011

13. Therapeutic Proteins Based on
Virulence Factor Platform
Possible approaches:
•Recombinant viral proteins to treat autoimmune diseases
•Focus on acute indications to avoid immunogenicity from
long-term dosing
•Not currently being pursued
•If virus makes a homologue to a human protein (eg. soluble
viral TNFαR), use recombinant human protein
•Develop antibodies to bind to/block the targets of viral proteins

14. Challenges of Non-mAb Therapeutics
• mAb-like expression levels may not be achievable
• mAb-like purification process steps may not be
feasible
• Platform processes may not be available
• More complex glycosylation patterns
• Greater stability issues with non-native
structures/fusion proteins
• Despite potential challenges, unique therapeutic
approaches makes the effort worthwhile

15. Case study: VLST-007
Human Viral
•Viral protein interrupts signaling by forming
heterodimer with human transmembrane protein
VLST’s approach
•Make soluble Fc-fusion protein of bind to and block
signaling through binding partner

16. Case study: VLST-007
• Extracellular domain fused to IgG1 Fc
• 7 putative N-linked glycosylation sites (14 in dimer)
– Process may impact glycoforms, potency
• Use “mAb-like” process, but customize as necessary
– Anion exchange flow through step not possible
• Initial process development performed at VLST, tech transfer
of process and reagents to CMO
• Collaborative effort to trouble shoot, assess impact of
changes on product quality/activity

17. VLST-007 Development Challenges
CEX Ligand-Dependent Aggregation
Fractogel Fractogel
Mabselect SP XL Eluate
SO3(S) Eluate SO3 (M)
Eluate Eluate
•Strong CEX ligands induced product
aggregation, as observed on SEC-HPLC
•Recommended that CMO switch resin (after
tech transfer had already begun)
•CMO accommodated change with no
impact to time-line
GE CM FF TOYO CM
Eluate 650M

18. VLST and CMO Develop mAb-Like,
Scaleable cGMP Process
CHO Fed-batch Depth filter Low pH viral Nanofiltration
>1 g/L inactivation
clarification
ProA C
H+ H+
H+
H+H+
E
X
HIC
BDS TFF
•10L PD process yield: 75%
•400L Engineering run process yield: 78%
Confidential

19. Leveraging Technology to Boost Titer
CHO XD® Comparison
2L, 50L XD® and Fed-Batch
10
•DSM performed evaluation, utilized
9 same media and feeds as VLST’s
GMP process
8
•No extensive custom media
Product concentration (g/L)
7 optimization necessary
6 •Titer Boost:
From 1.2 to 9.2 g/L
5
in 12 days (7x)
4
50L XD
3 2L XD
Fed-batch
2
1
0
0 2 4 6 8 10 12 14 16 18
Time (days)

20. Case Study: VLST-018
• Mechanism validated by viral biology- multiple
viral homologues mimic a membrane-bound
human protein
• Generate soluble Fc-fusion of extracellular
domain
• Multiple N-linked glycosylation sites
• Generate production cell lines, perform PD
and tech transfer to CMO

21. Generation of Production CHO Cell Lines
•Codon-optimize gene, and clone into
two different proprietary expression
vectors
•Screen transfectants via Clonepix
•Use to weed out low-expressing clones
•Permitted thousands of colonies to be
pre-screened
•Titer results significantly better (40%)
with top GS clones, will utilize for future
cell line campaigns

22. Purification Development
High-throughput Screening
96-well plate Chromatography
•Screen multiple conditions
quickly and easily
•Facilitates use of DOE
•Rapidly identified lead CEX
resin based on capacity, using
little protein

23. Lessons Learned/Conclusions
•Leverage new technology, rather than adding
FTE’s, to do PD better, faster
•24-well prescreen
•Alternatively-tagged constructs
•Clonepix
•Alternative cell culture methods, XD, perfusion
to increase titer
•Utilize vendor’s expertise and help if possible
•Plate-based chromatography development

24. Acknowledgements
Protein Sciences Group
• Jeff Bartron
• Chris Tompkins
• Laura Hajny
• Patrick Mosher
• Ryan Kelly
• Ryan Merrill
Bioinformatics and Proteomics
• Stefan Ponko, Ph.D.
• Ajamete Kaykas, Ph.D.