Originally presented at the HIV Vaccine Manufacturing Workshop –July 19th& 20th, 2017 by Abhinav A. Shukla, Ph.D.Senior Vice PresidentDevelopment & ManufacturingKBI Biopharma, Durham NC

1. HIV Vaccines Process
Development &
Manufacturing – pitfalls
& possibilities
Abhinav A. Shukla, Ph.D.
Senior Vice President
Development & Manufacturing
KBI Biopharma, Durham NC
HIV Vaccine Manufacturing Workshop – July 19th & 20th, 2017

2. 2
High throughput
process development
(HTPD)
Modulating glycosylation
a) Cell line b) Cell culture media
Platforms for mAbs
Platforms for
non-mAbsReducing cycle time
in production brx
a) Frozen inoculum
b) High density seed
Selective washes for
HCP removal
Rapid PC/PV
leveraging
HTPD tools
Proprietary cell
lines
a) DG44
b) Horizon GS-CHO
Continuous processing
a) Perfusion
b) Downstream
capture
A3 implementa-
-tion
ELP Protein A
Multimodal
chrom.

3. 3
Durham, North Carolina
- Cell Line Development
- Cell Culture cGMP Manufacturing
- Analytical QC, Formulation, Stability
- Mass Spec Core Facility
RTP, North Carolina
- Cell Culture Process Development
- Analytical development
Boulder, Colorado
- Cell Line Development
- Microbial Process Development
- Microbial cGMP Manufacturing
- Analytical, QC, Formulation, Stability
- Particle Characterization Core Facility
The Woodlands, Texas
- Cell Therapy
- Bioassays
San Diego, California
- Analytical Technologies
United States

4. 4
Durham, North Carolina
- Cell Line Development
- Cell Culture cGMP Manufacturing
- Analytical QC, Formulation, Stability
- Mass Spec Core Facility
RTP, North Carolina
- Cell Culture Process Development
- Analytical development
Selexis
Geneva, Switzerland
- Cell Line
Development
Leuven, Belgium
- Analytical/QC
Services
Europe

5. Programs in mammalian clinical manufacturing at KBI
• Primary emphasis 2011 – 2016 on clinical entry stage programs
• Strong emphasis on science &
• ~ 10-14 IND filings per year supported via development &
manufacturing efforts
• Also supported several stand-alone programs for late-stage process
characterization studies
• Manufacturing capacity ~ 30 batches per year
New areas for KBI
• Acquisition of Selexis to integrate leading high yielding cell line
technology into KBI
• Expansion of train II in manufacturing to boost manufacturing
capacity to > 50 batches per year

6. Selexis
• Leading independent cell line in biotech industry
• > 5 g/L titers for mAbs
• Significant track record with hard to express proteins
• Selexis Genetic Elements enable enhanced transcription via high
expression vectors

7. SUREtechnology Platform™ Delivers Short Timelines and
Flexibility to Translate in a Range of Workflows
7
RCB STABILITY DATA
8 WEEKS
RCBSTABILITYDATAREPORT
EXPANSION
RCB CULTURING
EVALUATION
REPORT GENERATION
EXPANSION
MODULE S
RCB Stability
MCB BARCODING
4 WEEKS
MCBCLONALITYREPORT
MAPPING
INTEGRATION SITES
INTEGRITY AT JUNCTIONS
TRANSGENE INTEGRITY
COPY NUMBER
REPORT GENERATION
MAPPING
MODULE W
Whole Genome Sequencing
MANUFACTURING AND SAFETY INTELLIGENCE
 ICH COMPLIANT
 TRACEABILITY
CMO
DNAORDERING
DNAEXPRESSIONVECTORS
 CONTRACT SIGNATURE
 DNA ORDERING
 READY TO TRANSFECT
 PREDICTABLE TITER AND PRODUCTIVITY
 HIGH PRODUCING CLONAL CELL LINE
3 WEEKS
3 WEEKS
5 WEEKS
RCBCOMPLETED
7 WEEKS 2 WEEKS
14 WEEKS
TRANSFECTION
MODULE 1
DNA Construction
MODULE 2
Research Cell Bank Generation
CODON
OPTIMIZATION
LEADER PEPTIDE
ANALYSIS
DNA CLONING
TRANSFECTION
SELECTION OF STABLE POOLS
CSA
CLONEPIX
ENRICHED CLONE POOL
BANKING
SCCO TRANSFECTION
SELECTION
CSA
CLONEPIX
CSA
CYTENA
RCB BANKING
EXPANSION
FED BATCH
CULTURE
RESEARCH CELL BANK (RCB)
DEVELOPMENT
RCB DELIVERYSCC 1 + SCC 2
VECTOR
CONSTRUCTION
ENRICHED POOLS
 PROOF OF CONCEPT
 TRANSFER TO CMO
©Selexis 2017 CONFIDENTIAL - NOT FOR DISTRIBUTION

8. Upstream Train I
Upstream Train II
BPC BPC BPC
ProA
VI
Polish
VF
Bulk fill
KBI’s Cell Culture Manufacturing Facility
Purification Suite #1
2000L Prodn BRX200L Seed BRXWaveSF Harvest
2000L Prodn BRX #1
200L Seed BRX
Wave
SF
Harvest
50L Seed BRX
2000L Prodn BRX #2
Purification Suite #2
BPC BPC BPC
ProA
VI
Polish
VF
Bulk fill
UF
UF

9. 9
Biologics Life Cycle
Pre-Clinical Phase I Phase II Phase III
Process Development
Process
Characterization
Process
Validation
Process Monitoring
& Improvement
FIH Process
• Deliver clinical process
quickly
• Platform process
• Clinical Supply
Submission &
Approval
Lifecycle
management
BLA Prep &
PAI
Commercial Process
• Deliver manufacturing process for
registrational trials and market
• Design keeping large-scale manufacturing in
mind
• Improve productivity, efficiency, robustness,
manufacturability, COGs
• Analytical characterization and method
development
Process Characterization and Validation
• Develop IPC strategy through understanding of process inputs and
outputs (design space)
• Scale-down characterization and validation studies
• Large-scale process validation to demonstrate process consistency
• BLA preparation
• Supporting documents for licensure inspections
• Post-commercial process improvements (CI)
• Post-commercial process monitoring
FIH process Commercial process

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

11. • 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

12. • 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

13. 1
10
100
1000
10000
100000
LogHCP(ppm)
Downstream Process
PlatformHCP 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%

14. 14
Fractogel SO3
CHT Type I
CM Sepharose HP
UF/DF #2
Viral Filtration
SP Sepharose HP
UF/DF #1
Harvest
Viral Inactivation
BDS
Product capture
Product polishing. rHCP, rDNA clearance
Product polishing. rHCP clearance
Product polishing. rHCP clearance
1% Triton X-100 for 30-60min, dedicated viral inactivation step
Dedicated virus removal step
Step Purpose
Buffer Exchange
Buffer exchange into formulation buffer

15. UV 1_280 UV 2_254 Cond Conc B pH Fraction Injection Run Log
950900850800750700650600550500450400
120
110
100
90
80
70
60
50
40
30
20
10
55
50
45
40
35
30
25
20
15
10
5
4.A.2
^^^^^
6.A.4
^
6.A.2
^
5.C.5
^
5.C.3
^
5.C.1
^
5.B.4
^
5.B.2
^
5.A.5
^
5.A.3
^
5.A.1
^
3.C.4
^
3.C.2
^
3.B.5
^
3.B.3
^
3.B.1
^
3.A.4
^
3.A.2
^
2.C.5
^
2.C.3
^
2.C.1
^
2.B.4
^
2.B.2
^
2.A.5
^
2.A.3
^
2.A.1
^
1.C.4
^
1.C.2
^
1.B.5
^
1.B.3
^
1.B.1
^
1.A.4
^
1.A.2
^^^^
4.A.1
^^
HolduntilConditionOccurredAirse...>
Datacollectionsettingsadjustedfo...>
Datacollectionsettingsadjustedfo...>
End_Block(Issued)(Processing)(...>
End_Block(Issued)(Processing)(...>
0 1 2 3 4 5 6 7 8 9
0-600mM
NaCl gradient
Fraction #
CH106 Octet Binding
Kon Koff KD (nm) X^2 R^2
0 Not tested due to absence of CH505w53 as confirmed by RP-titer
1 9.48E+03 1.40E-04 14.7 0.0287 0.9827
2 7.21E+03 1.47E-04 20.4 0.0183 0.9935
3 6.92E+03 1.41E-04 20.3 0.0729 0.9977
4 1.29E+04 1.11E-04 8.6 0.0434 0.9971
5 1.60E+04 7.34E-05 4.6 0.0665 0.9973
6
2.40E+04 1.52E-04 6.3 0.0424 0.9981
7 4.09E+04 3.03E-04 7.4 0.0766 0.9881
8 3.70E+04 2.96E-04 8.0 0.0917 0.9891
9 1.82E+04 1.83E-04 10.1 0.107 0.9964
The first three fractions have a weaker affinity for CH106 than the
later eluting fractions
15

16. • w53 requiring more NaCl for elution (later fractions) has better dose response than earlier
eluting fractions
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 100 200 300 400 500 600 700 800 900
EndPointBindingSignal
Concentration (nM)
w53 Dose Response Binding
Fraction 1 (2B4-2C2)
Fraction 2 (2C3-2C5)
Fraction 3 (3A1-3A3)
Fraction 4 (3A4-3B1)
Fraction 5 (3B2-3B4)
Fraction 6 (3B5-3C2)
Fraction 7 (3C3-3C5)
Fraction 8 (5A1-5A4)
Fraction 9 (5A5-5C2)
16

17. • Dose response curves for VRC01 and CH103_IA_3.1 are
comparable to DHVI control for CHT Eluate and BDS (July 2016)
17
DHVI Control
CHT Eluate
BDS
VRC01 Dose Response CH103_IA_3.1 Dose Response

18. Cell Line Development
Jun2013
Aug2016
Oct2014
18
Jun2016
Mayw017
Scale Up
Assessment
(Demo Run)
May2015
Process Development
Proposed Timing
Actual Timing
• Additional development needed for downstream process
required to address product activity concerns
• Subsequent activities stage-gated resulted in further delays
Tech Transfer and Manufacturing
Dec2016

19. 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
CH505TF
(transient)*
*Collaboration between ABL and KBI. ABL performed upstream activities and KBI performed downstream and release activities
Successful IND

20. • Project delayed due to lack of adequate antigenicity
• Change of capture step from Capto MMC to Fractogel SO3
enabled capture of more appropriately glycosylated
species
• ForteBio Octet (BLI) binding used as a rapid
troubleshooting tool for guiding downstream process
alterations
• Significantly higher throughput than SPR (Surface Plasmon
Resonance)

21. • HIV vaccine envelope is trimeric (3 gp120 + 3 gp41 subunits)
• HIV ENV is weakly immunogenic, trimeric structure could boost
immunogenicity
• Soluble form with gp120 linked to gp41 by a disulfide bond (Prof.
John Moore, Cornell – J. Virology, 2002)
• SOSIP trimer of gp140 with favorable antigenic properties
developed (BG505 SOSIP.664) (Sanders and Moore,
Immunological Reviews, 2017)

22. • Molecule exists as a non-covalent trimer of covalently
associated heterodimer, gp120 and gp41, approaching 700
kDa in size – exceeding the size traditionally encountered
in bioprocessing
• Process utilized several unit operations typically not
considered scalable (custom affinity chromatography and
size exclusion chromatography), but were considered key
to success of the program
• Due to the custom nature and long lead time for many of
the manufacturing materials and equipment, process
delays were encountered
• Devising a robust viral clearance strategy

23. 23
• Consistent cell culture growth profiles from Development to
cGMP manufacturing
Clarified Harvest Titer
(g/L)
Run Name 2G12 BLI1 PGT145
BLI1
Demonstrati
on Run (200
L)
0.103 0.114
cGMP Run
(200 L)
0.085 N/A2
1 PGT145 antibody binds gp140 trimer only; 2G12
antibody measures total gp140 protein
2 PGT145 BLI assay was not employed in cGMP
campaign due to demo run observation of matrix
interference with cell culture supernatant

24. 24
Master Cell Bank – MCB
4 Shake Flask Passages
12 days in Shake Flasks
Inoculum volume is increased
from 35 mL (Vial Thaw) to 4.0
L in Shake Flasks (Passage 4)
XDR-200 – Production Run
14-15 Days in XDR-200
(Final volume: ~190-200 L)
Passage 5 – 50L Wave
3 Days in 50 L Wave
(Final volume: ~20.5 L)
Total time from Vial Thaw to Harvest of the XDR-200 should be 29-30 days
Harvestandtransfertodownstreamprocessing

25. Harvest
• Depth/sterile filtration for cell removal and clarification
Harvest UF
• Due to slow binding kinetics over capture column long residence times are required for capture. To
reduce 2G12 volume and cycle time requirements harvest is concentrated to 1/5th the harvest volume.
Capture by 2G12
• Utilizes 2G12 antibody to bind Aggregate/Trimer/Dimer/Monomer from concentrated harvest and
reduce host cell contaminants. Product elution is achieved with 3M MgCl2
Post Capture UF/DF
• Product is concentrated and buffer exchanged due to product’s reduced stability in 2G12 elution buffer.
Inactivation/Adsorption
• Detergent inactivation implemented to meet guidelines established by ICH Guideline Q5A: Viral Safety
Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin
• Adsorption step implemented to remove detergent from product stream quickly and efficiently to
mitigate risk to product degradation
Harvest
Harvest UF
Capture 2G12
UF/DF
Inactivation/Ads
ProA Flow Through
CaptoAdhere
Viral Filtration
Conc UF
SEC
UF/DF
Bulk Fill
Unit operations likely to require little to no optimization and provide a platform approach for future molecules

26. ProA Flow Through
• Due to high amount of leached 2G12 antibody from capture, ProA affinity capture of residual antibody is
required. Other process steps do not effectively clear residual 2G12 Antibody.
CaptoAdhere
• Step designed to remove residual host cell contaminants and achieve additional orthogonal viral
clearance safety.
Viral Filtration
• Planova 20N viral filtration implemented to meet guidelines established by ICH Guideline Q5A: Viral
Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin
Concentration UF
• Product is concentrated to control loading over subsequent size exclusion chromatography step.
Size Exclusion Chromatography
• Designed to separate product related variants (Monomer/Dimer/Aggregate) and also provides
additional removal of host related impurities
Final UF/DF
• Final concentration and buffer exchange into formulation buffer.
Harvest
Harvest UF
Capture 2G12
UF/DF
Inactivation/Ads
ProA Flow Through
CaptoAdhere
Viral Filtration
Conc UF
SEC
UF/DF
Bulk Fill

27. • Utilizes bnAb 2G12, which exclusively recognizes oligomannose-type glycans
on gp120 residues on the surface of HIV-1 – provides highly specific binding
reducing host cell contaminants and ensures glycosylated product
• Use of highly specific affinity ligand could provide robust platform approach to
minimize downstream process development requirements
• Ligand currently is not stable over multiple cycles
– limiting resin lifetime
• Sanitization procedure is difficult and current
method significantly decreases binding capacity –
ligand is highly susceptible to commonly used
affinity bioprocess resin cleaning protocols
Rapid capacity decline
in 8-12 min of exposure

28. • Viral clearance – will it be adequate?
• Triton X-100 removal by adsorption – uptake kinetics
• Cycling & best way to sanitize 2G12 affinity resin
Step LRV XMuLV LRV MMV
2G12 ≥ 5.08 1.07
Triton
Inactivation/
Adsorption
5.96 N/A
CaptoAdhere 3.35 ≥ 6.26
Viral
Filtration
≥ 5.23 ≥ 5.00
Total ≥ 19.62 ≥ 12.33
Purity SEC-HPLC rHCP Purity SEC-HPLC rHCP
Sample
PGT145/
Conc (A280)
%Trimer ng/mg
PGT145/
Conc (A280)
%Trimer ng/mg
Post 2G12 UF/DF 40% 82% 39,287 96% 88% 63,773
Capto Adhere Product 73% 96% 1,053 93% 91% 1,095
Size Exclusion Chromatography 96% 100% 10 108% 100% 7
Small Scale Large Scale
3 L Bioreactor 200 L Bioreactor
Small Scale Large Scale
Test Assay 3 L Bioreactor 200 L Bioreactor
Quantity A280 0
Potency BLI PGT145 108% 121%
SEC-HPLC %Trimer 100% 96%
RP-UPLC 95% 89%
rHCP (ng/mg) 5 6
rProA (ng/mg) <LOQ (0.2 ng/mg) <LOQ (0.2 ng/mg)
rDNA (pg/mg) <LOQ (15 pg/mg) <LOQ (10 pg/mg)
r2G12 (ng/mg) <LOQ (8 ng/mg) <LOQ (8 ng/mg)
rTriton (%w/v) <LOQ (0.002%) <LOQ (0.002%)
Other SDS-PAGE (Cleavage) Comparable to Ref. Comparable to Ref.
Purity
Safety

29. Sep2015
200L Scale Up Assessment
(Demo Run)
Tech Transfer and Manufacturing
Sep2016
Jul2016
29
Nov2016
Apr2017
• Initial SOW did not include GMP manufacturing
• Program required customized equipment and materials resulting
in long lead time for many of the manufacturing materials and
equipment, program delays were encountered between
development and scale up activities Proposed Timing
Actual Timing
Process Transfer and Development
Apr2016

30. • New solutions & technologies do take some investment in
time & money to create a smoother path
Full
Development
New vaccine
candidates
cGMP
manufacturing
Translational
Science
&
Focused
investigations

31. 31
Conclusions
• Process, analytical and formulation development for a
novel type of proteins cannot be taken for granted
• Requires significant scientific ability and focus
• Unique match with KBI’s scientific and technical
capabilities
• KBI increasing depth in cell line development & boosting
manufacturing capacity
• Timelines, budgets and expectations need to reflect the
development hurdles that can arise with a novel set of
complex biologics
• True partnership & collaboration needed