Abhinav A. Shukla, Ph.D.Senior Vice President, Process Development & Manufacturing, KBI 

Biopharma

Prof. Barton Haynes, M.D.Director, Duke Human Vaccine Institute

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One of only three species ofthis birdPreys on flying fishLays a single egg directly on a cliff ledge

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

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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

• 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

PG9

PGT128

2F5

4E10

VRC01

Five HIV Env Regions Targeted By Broadly Neutralizing Antibodies

CD4 binding siteCD4 mimic‐ VRC01

HCDR3 binder‐ CH103N276‐dependent‐ HJ16

PGDM1400PG9, CH01VRC26

DH270, PGT128, PGT125, PGT135

35022, PGT151, ANC195

4E10, 2F5, 10E8, DH512

Marie Pancera, Peter Kwong

V1V2‐glycan

V3‐glycan

0.002

Co-Evolution of Transmitted/Founder Broad Neutralizing Antibodies From African Individual CH505

(CH505 bnAb‐ HIV‐1producer)

Antibody  (the CH103 HCDR3‐binder bnAb B Cell   lineage)

Transmitted/Founder Virus

41 weeks 

92 weeks 

14 weeks 

Week 4 Week 14Week 20Week 22Week 30Week 53Week 78Week 100Week 136Week 160

Onset breadth  Tier 2 

Virus Neutralization

Autologous Neutralization

Onset Broad Virus Neutralization

CH103‐CH106Isolated

CH

106

CH

103

CH

104

CH

105

H.X. Liao et al. Nature 496: 469; 2013

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: UCAT/F gp120 Kd = ~200 nM

Env:

CH103

CH505 wk53

CH505 wk78

CH505 wk100

CH103 lineage intermediateantibodies

CH505 TF

CH505 wk136

9H.X. Liao et al. Nature 496: 469; 2013

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: UCAT/F gp120 Kd = ~200 nM

Env:

CH103

CH505 wk53

CH505 wk78

CH505 wk100

CH103 lineage intermediateantibodies

CH505 TF

CH505 wk136

10H.X. Liao et al. Nature 496: 469; 2013

The key to patient access is creating processesthat can take these vaccine candidates intoclinical trials rapidly 

KBI Biopharma

Upstream Train I

Upstream Train II

ProA

VI

PolishVF

Bulk fill

KBI’s Cell Culture Manufacturing Facility

Purification Suite

2000L Prodn BRX200L Seed BRXWaveSF Harvest

2000L Prodn BRX200L Seed BRXWaveSF Harvest

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.

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

Virusfilter

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)

• 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

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• 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

• 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

• 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 envsworked 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

• 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

ScaleTemperature Set point 37.0 ± 0.5°C Temperature Shift 33.0 ± 0.5°C on Day 6DO Set point 30%pH Set point 6.90 ± 0.1

Agitation (1-impeller) 50 rpm → 55 rpm

Air overlay 1.6 SLPMAir Sparge 0.5 SLPM Max. Oxygen Sparge 5 SLPMMax. CO2 Sparge 5 SLPM

Medium CD OptiCHO + 8 mM Glutamine

Target VCD 0.50 x 106 cells/mLBase 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: CystineSupplement 3 addition: TyrosineSupplement 4 addition: Soy:Yeastolate Hydrolysate (2:3) 5g/L current wv each on Days 4 and 8

Supplement 5 addition: C1615Harvest Add 10g/L Hydrolysate on harvest

• 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.

• 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

• BIA Terminology

• BIA Technology (Surface Plasmon Resonance)

• Data (Sensogram)

Ligand ka (1/Ms) kd (1/s) KD (nM) Rmax(RU)

Chi² (RU²)

UCA 1.38E+04 7.51E-03 544 103.2 1.0IA2 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

• 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

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

Log

k'

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

Log

k'

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

Log

k'

Log [NaCl]

Lysozyme

Washes can be developed to disengage HCPs from the product rather than disrupt product-Protein A ligand interactions

96

116359243

34655

935491

05000

100001500020000250003000035000400004500050000

Null supernatant MAbSelecteluate (load =

nullsupernatant)

MAbSelecteluate (load =

null supernatant+ product)

Prosep A eluate(load = null

supernatant)

Prosep A eluate(load = null

supernatant +product)

Hos

t Cel

l Pro

tein

s (n

g/m

L) 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

CH

OP

(ppm

) nor

mal

ized

toco

ntro

l exp

erim

ent

Direction ofdesired trend

Protein A chromatography: Shukla, A. Hinckley, P. Host cell protein clearance during Protein Achromatography - development of an improved column wash step, Biotechnology Progress, 24, 1115-1121, 2008.

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 phasemodulators, Journal of Chromatography A, 1340, 151-156, 2014.

1

10

100

1000

10000

100000

Log HCP

 (pp

m)

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

w100GMP

w78Demo

w78 GMP

BDS 99.3% 98.9% 98.8% 98.9% 99.3% 99.2% 99.5% 99.6%

rHCP levelsCapto MMC Step Yield

SUB Biocompatibility

CH505w100

SPR activity concerns

RP-purity %pre-main peak levels

CH505w78CH505w53SPR activity

concerns

Molecule stability

SPR activity concerns

CH505TF

High %HMW by SEC-HPLC

• 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

CH505TF

CH505w100

CH505w78

CH505w53

B63521

Clone SelectionUpstream/Downstream Process Assessment &

confirmation of SPR activity

Verification of Compatibility with

Single Use Bioreactor

Pilot scaleRun

Engineering Run cGMP Run

• New ground has been broken with production of Envproteins 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

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 PDAnalytical development, Formulation Development, cGMP Manufacturing, QA/QC