A Vaccine Approach against HIV-1, Manufacturing Env proteins: from Bench to Bedside Abhinav A.Shukla, Ph.D. Senior Vice President, Process Development & Manufacturing, KBI Biopharma Prof.Barton Haynes,M.D.Director,Duke Human Vaccine Institute

1. Abhinav A. Shukla, Ph.D.
Senior Vice President, Process Development & Manufacturing, KBI
Biopharma
Prof. Barton Haynes, M.D.
Director, Duke Human Vaccine Institute

2. 2
One of only three species of
this bird
Preys on flying fish
Lays a single egg directly on a
cliff ledge

3. • 36.9 million people living with HIV/AIDS
• Still > 1 million deaths per year
3

4. The HIV-1 Arms Race--Mapping the Virus and
Antibody From the Time of Transmission
The initial neutralizing
antibody response to HIV
“autologous nAb”
15%- Broadly
neutralizing
antibody
The transmitted-
founder virus
Escape virus
HIV-1 Antibody
-ID

5. • Multiple previous vaccine approaches have failed
• 1987: First clinical trial with a gp160 subunit vaccine
• 1999: Vaxgen Phase III clinical trials started in Thailand
• 2000: NIAID (National Institute of Allergy and Infectious
Diseases) forms HVTN (HIV Vaccine Trials Network)
• 2003: RV144 US/Thai Phase III clinical trial started
• 2004: Both Vaxgen vaccine candidates failed to create
immunity
• 2009: RV144 trial showed marginal protective effect
• Multiple immunogens may be needed
5

6. PG9
PGT128
2F5
4E10
VRC01

7. Five HIV Env Regions Targeted By
Broadly Neutralizing Antibodies
CD4 binding site
CD4 mimic‐ VRC01
HCDR3 binder‐ CH103
N276‐dependent‐ HJ16
PGDM1400
PG9, CH01
VRC26
DH270, PGT128,
PGT125, PGT135
35022, PGT151,
ANC195
4E10, 2F5, 10E8,
DH512
Marie Pancera, Peter Kwong
V1V2‐glycan
V3‐glycan

8. 0.002
Co-Evolution of Transmitted/Founder Broad
Neutralizing Antibodies From African Individual CH505
(CH505 bnAb‐ HIV‐1
producer)
Antibody (the CH103 HCDR3‐
binder bnAb B Cell
lineage)
Transmitted/
Founder Virus
41 weeks
92 weeks
14 weeks
Week 4
Week 14
Week 20
Week 22
Week 30
Week 53
Week 78
Week 100
Week 136
Week 160
Onset breadth
Tier 2
Virus Neutralization
Autologous
Neutralization
Onset Broad
Virus Neutralization
CH103‐CH106
Isolated
CH106
CH103
CH104
CH105
H.X. Liao et al. Nature 496: 469; 2013

9. CH505 Envelopes selected as
vaccine immunogens
CH505 transmitted-founder (TF) and Env variants
generated during viral evolution drove affinity maturation
of CH103 bnAb lineage
Antibody: UCA
T/F gp120 Kd = ~200 nM
Env:
CH103
CH505
wk53
CH505
wk78
CH505
wk100
CH103 lineage intermediate
antibodies
CH505
TF
CH505
wk136
9
H.X. Liao et al. Nature 496: 469; 2013

10. CH505 Envelopes selected as
vaccine immunogens
CH505 transmitted-founder (TF) and Env variants
generated during viral evolution drove affinity maturation
of CH103 bnAb lineage
Antibody: UCA
T/F gp120 Kd = ~200 nM
Env:
CH103
CH505
wk53
CH505
wk78
CH505
wk100
CH103 lineage intermediate
antibodies
CH505
TF
CH505
wk136
10
H.X. Liao et al. Nature 496: 469; 2013
The key to patient access is creating processes
that can take these vaccine candidates into
clinical trials rapidly

11. KBI Biopharma
Upstream Train I
Upstream Train II
ProA
VI
Polish
VF
Bulk fill
KBI’s Cell Culture Manufacturing Facility
Purification Suite
2000L Prodn BRX200L Seed BRXWaveSF Harvest
2000L Prodn BRX200L Seed BRXWaveSF Harvest

12. Programs in mammalian clinical manufacturing at KBI
~ 16 IND filings per year supported via development & manufacturing
efforts
Shukla, A., Mostafa, S., Wilson, M., Lange, D. Vertical Integration of Disposables in Biopharmaceutical
Drug Substance Manufacturing, Bioprocess International, 10(6), 34-47, 2012.
Gottschalk, U., Shukla, A. Single-use disposable technologies for biopharmaceutical manufacturing,
Trends in Biotechnology, 31(3), 147-154, 2013.

13. a customer and science-focused
contract development & manufacturing organization
Media and Feed preparation utilizing disposable
mixing, filtration and storage systems
Disposable shake flasks or
disposable spinner flasks
MCB or
WCB vial
Disposable expansion
reactor
Disposable
seed bioreactor
Disposable production
bioreactor
Disposable fluid path
centrifuge
Disposable depth
filtration system
0,2 µm
filter
Hold vessels
(Bags)
Hold vessel
(bag)
Disposable fluid path
purification system
Disposable
mixing tank
0,2 µm
filter
Retentate
Permeate
PD
Disposable fluid path
purification system
Disposable
mixing tank
0,2 µm
filter
BPC
Virus
filter
BPC
0,2 µm
filter
BPC
BPC
Sterile bulk fill and
sampling bags
Buffer preparation utilizing disposable mixing,
filtration and storage systems
0,2 µm
filter
Disposable fluid path
UF/DF system
Aseptic
connection
Hold vessel
(bag)
Hold vessel
(bag)
Hold vessel
(bag)
Hold vessel
(bag)
Hold vessel
(bag)

14. • HIV‐1 Env consists of a trimer of homodimers of gp120 and the gp141
transmembrane protein and has 25 N‐linked glycosylation sites
• gp120 binds target CD4 receptors
• gp120 is heavily glycosylated with glycans comprising 50% of its mass
• Several neutralizing antibodies bind to the glycan structures which are also
responsible for correct folding of these proteins (Go et al, Journal of Virology,
2015, 89(16), 8245‐8257)
• High mannose glycans are the predominant glycoform
14

15. • Need for a Platform process for Env proteins
• Creating a process platform can help save time in
development and enable human trials to proceed
• Clinical trials in this space are proof‐of‐concept studies and
likely even more antigens will be needed
• Key aspects of the platform
• CHO DG44 cell line, cell culture process development,
downstream process development, analytical methods
development
• Use of antigenic binding to human antibodies as a key tool for
confirming process decisions
• Process issues encountered & solved for multiple antigens

16. • The mAb platform (left) employs
the same unit operations in the
same order; however, variables
within the unit operations are
optimized for the target protein
Shukla, A., Thommes, J. Advances in large-scale production of monoclonal antibodies &
related proteins, Trends in Biotechnology, 28(5), 253-261, 2010.
• For the ProA unit operation
(right) the parameters in gray
are templated across
molecules; whereas, the
factors in yellow require
molecule specific definition

17. • Parameters shaded in
gray are defined across
molecules. Parameters
shaded in yellow require
molecule specific
optimization
• The MTX amplification
concentration varied
across the development
of the 5 gp120 envs
worked on to date
• A 2nd round of limited
dilution cloning was
employed for 2 of the 5
gp120 envs worked on to
date
Expression Plasmid
Generation
Stable Pool
Selection
Clone Scale-Up and
Productivity Screening
Limiting Dilution
Cloning
Shake Flask Fed-
Batch Evaluation
Expression Stability
Evaluation
3L Bioreactor
Evaluation

18. • Parameters shaded in gray are
defined across molecules.
Parameters shaded in yellow
require molecule specific
optimization
• For all Env molecules the
operating parameters, basal
medium, feed type and some of
the supplement additions have
defined
• The need for additional
supplements is molecule specific
• Reasons for supplement
addition:
» Biocompatability in SU
bioreactors
» Increase in productivity
Scale
Temperature Set point 37.0 ± 0.5°C
Temperature Shift 33.0 ± 0.5°C on Day 6
DO Set point 30%
pH Set point 6.90 ± 0.1
Agitation (1-impeller) 50 rpm → 55 rpm
Air overlay 1.6 SLPM
Air Sparge 0.5 SLPM
Max. Oxygen Sparge 5 SLPM
Max. CO2 Sparge 5 SLPM
Medium CD OptiCHO + 8 mM Glutamine
Target VCD 0.50 x 106
cells/mL
Base 1M Sodium carbonate
Feed Type: LTI Feed A+B (1:1)
15% on Day 0, 10% current wv each
on Days 3, 6, and 9
Supplement 1 addition: HT Supplement 1X current wv each on Days 0 and 4
Supplement 2 addition: Cystine
Supplement 3 addition: Tyrosine
Supplement 4 addition: Soy:Yeastolate
Hydrolysate (2:3)
5g/L current wv each on Days 4 and 8
Supplement 5 addition: C1615
Harvest Add 10g/L Hydrolysate on harvest

19. • Accelerated development approaches critical for non‐
platform molecules
• Rapid experimentation made possible by integrated use of
high through cell culture and high throughput analytics
Rameez, S.; Mostafa, S. S.; Miller, C.; Shukla, A. A.
High-throughput miniaturized bioreactors for cell
culture
process development: Reproducibility, scalability
and control.
Biotechnology Progress 2014, (30): 718-727.

20. • Challenge:
• Susceptibility of CH505TF to proteolytic activity
was a concern from project initiation
• Solution:
• A mixture of soy and yeast hydrolysate is
added to the clarified harvest after depth
filtration to serve as an alternative substrate
for proteases
• Harvest/clarification and the capture
chromatography step occur on the same day

21. • BIA Terminology
• BIA Technology (Surface Plasmon Resonance)
• Data (Sensogram)

22. Ligand ka (1/Ms) kd (1/s) KD (nM)
Rmax
(RU)
Chi²
(RU²)
UCA 1.38E+04 7.51E-03 544 103.2 1.0
IA2 7.79E+03 1.08E-04 13.9 95.63 0.1
IA3.2 1.88E+04 2.67E-04 14.2 240.6 9.2
UCA IA2
IA3.2

23. • Parameters shaded in gray are
defined across molecules.
Parameters shaded in yellow
require molecule specific
optimization
• Load and elution conditions for
three of the unit operations
require molecule specific
definition given the heterogeneity
of this class of molecules
• Env antigens structurally sensitive
to hydrophobic surfaces, hence
HIC not employed

24. log k’ = A – Blog(csalt) + C(csalt)
where csalt is the mobile phase salt concentration in molar units and A, B and C are constants
Melander, W.; El Rassi, Z.; Horvath, Cs. Journal of Chromatography, 469, 3-27, 1989.
• Key step for capture, HCP removal and selection of
“SPR active” species
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
2.10 2.30 2.50 2.70
Logk'
Log [NaCl]
mAb1
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.50 2.00 2.50
Logk'
Log [NaCl]
RNase
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
2.60 3.10 3.60
Logk'
Log [NaCl]
Lysozyme

25. Washes can be developed to disengage HCPs from the product
rather than disrupt product-Protein A ligand interactions
96
11635
9243
34655
935491
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Null supernatant MAbSelect
eluate (load =
null
supernatant)
MAbSelect
eluate (load =
null supernatant
+ product)
Prosep A eluate
(load = null
supernatant)
Prosep A eluate
(load = null
supernatant +
product)
HostCellProteins(ng/mL)
Normalized Yield vs. normalized CHOP for a
variety of washes on MAbSelect Protein A
0%
20%
40%
60%
80%
100%
120%
140%
0% 20% 40% 60% 80% 100% 120%
Yield normalized to control experiment
CHOP(ppm)normalizedto
controlexperiment
Direction of
desired
trend
Protein A chromatography: Shukla, A. Hinckley, P. Host cell protein clearance during Protein A
chromatography - development of an improved column wash step, Biotechnology Progress,
24, 1115-1121, 2008.

26. 0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0%
Normalized HCP
Recovery
HCP vs. Recovery after Intermediate Wash for Capto MMC Polishing
baseline
Wolfe, L., Barringer, C., Mostafa, S., Shukla, A. Multimodal chromatography:
characterization of protein binding and selectivity enhancement through mobile phase
modulators, Journal of Chromatography A, 1340, 151-156, 2014.

27. 1
10
100
1000
10000
100000
Log HCP (ppm)
Downstream Process
Platform HCP Clearance
TF Demo
TF ENG
TF GMP
w100 Demo
w100 ENG
w100 GMP
w78 Demo
w78 GMP
SEC‐HPLC % Main Peak
Sample ID
TF
Demo
TF
ENG
TF
GMP
w100
Demo
w100
ENG
w100
GMP
w78
Demo
w78
GMP
BDS 99.3% 98.9% 98.8% 98.9% 99.3% 99.2% 99.5% 99.6%

28. rHCP levels
Capto MMC
Step Yield
SUB
Biocompatibility
CH505w100
SPR activity
concerns
RP-purity
%pre-main
peak levels
CH505w78CH505w53
SPR activity
concerns
Molecule
stability
SPR activity
concerns
CH505TF
High %HMW
by SEC-
HPLC

29. • Increasing selectivity of capture step is essential since a
particular range of glycoforms is selected
• Lectin affinity chromatography:
• Lectins from plant sources are heterogenous
• cGMP sources of resin are not existent
• Can recombinant lectins be expressed by E. coli
fermentation
• Immunoaffinity based approach
• Use bnAbs as immunoaffinity ligands
• Advantage: can be eluted by sugars

30. CH505TF
CH505w100
CH505w78
CH505w53
B63521
Clone Selection
Upstream/Downstream
Process Assessment &
confirmation of SPR activity
Verification of
Compatibility with
Single Use Bioreactor
Pilot scale
Run
Engineering
Run
cGMP Run

31. • New ground has been broken with production of Env
proteins using stable CHO cell lines
• Several issues encountered and resolved – both technical
and strategic
• Complexity of this series of molecules is significant but not
insurmountable
• Close coordination of efforts between teams has enabled
this progression e.g. SPR activity assay at DHVI and Process
Development efforts at KBI Biopharma
• Process platforms can significantly help progression of non‐
mAbs to the clinic, particularly when a series of molecules
are being developed

32. Duke Human Vaccine
Institute:
Dr. Barton Haynes,
Prof. Thomas Denny, Dr.
Munir Alam and team
Dr. Gerry Kovacs and team
Dr. Michael Pensiero and team
Cell line development, Upstream & Downstream PD
Analytical development, Formulation Development,
cGMP Manufacturing, QA/QC