Manufacturing vaccines for first-in-man clinical trials in an academic GMP

facility

Fred Porter PhDDuke Human Vaccine Institute

Envelope Manufacturing WorkshopRockville, MDJuly 20, 2017

Introduction

• Manufacturing has been a roadblock to testing new HIV vaccine candidates– Long development timelines and program delays– Costs limit the number of Phase I first-in-man studies

• To date, new vaccine candidates have been manufactured by CMOs or Industrial vaccine manufacturers with a commercialization focus

• DHVI has assembled vaccine development and GMP manufacturing team to streamline Phase I vaccine production

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Outline

• DHVI GMP organization• GMP Facilities• Development Approach: M5 gp120• Conclusions/Acknowledgements

3

Outline

• DHVI GMP organization

4

DHVI GMP OrganizationAn integrated and flexible team structure

Thomas DennyDHVI COO

Bart HaynesCHAVI-ID PI

DHVI Director

Geeta SwamyAssoc. Dean,

Regulatory Oversight & Research Inititavies

Munir AlamDirector of DHVI Protein

Purification and Antigenicity Characterization Amanda Parrish

Director, Quality /Regulatory Program

Daniel TonkinRegulatory Affairs

Scientist

James PeacockMgr, Upstream Mfg

Process Development

Ana Sanchez and Anna Fincher

DGP Program Management

Dan OzakiDuke GMP Facility Quality Manager

Duke University School of Medicine

Fred PorterSenior Director of

Product Development

Shauna Anderson & Open

QA Project Coordinators

Matt Tyson& Sravya KompalliGMP Downstream

Team

Kara AnastiAnalytics Team

Amy CaparoniGMP Operations

Manager

Michael Guerrero& George Barrett

GMP Upstream Team

Seth ThomasMgr, Downstream

Mfg Process Development

Diana MartikMgr, Mfg. Analytics

Team

David Milam James Baldwin

Jill WhitleyGMP Analytical

Team

Marcella Sarzotti-Kelsoe Director, QADVIP

Quality Leadership Team

Tony Moody, Chip Walter, and Geeta Swamy

Duke Clinical Vaccine Unit (DCVU)

HVTN Site

Kent WeinholdDirector, Duke Division

of Surgical Sciences

DCVU Clinical Staff6 Lead Coordinators

10 Clinical Research Staff3 Program Managers

Jae-Sung YuResearch Scientist

Cell Line Dev.

Maureen MaughanProduct

Development Leader.

Meghan SnareGMP Ops.Team

Technical Team20 team membersQuality/Regulatory Team

6 Team members

Clinical Unitand HVTN site

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Development Team

Manufacturing Campaign Team

Discovery

ScientistScientistScientist

Cell culture Development

ScientistEngineerEngineer

Purification Development

Scientist EngineerScientist

Analytical Development

ScientistAnalystAnalyst

GMP Operations

Ops LeadEngineerEngineer

Development and GMP Matrix TeamsEliminate knowledge gaps through end to end involvement

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Phase Appropriate Quality Systems• In compliance with FDA regulations• Designed with flexibility to suit phase I clinical

manufacturing needs

Quality Management Components of DGP QSU

ICH Q7 FDA Phase 1 CGMP

21 CFR Parts 210 and 211

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Outline

• DHVI GMP organization• GMP Facilities

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DHVI GMP Facilities• Dedicated labs for process development (10ml-50L scale)

– Cell Line Development– Cell Culture Development– Purification Development

• Analytical/ QC laboratory– Laboratory equipped for release testing and characterization of vaccine

products– UPLC, HPLC, ELISA, CE, SPR, BLI, Microfluidics (lab on-a-chip) and

molecular sizing instrumentation

• Clinical Manufacturing Suite– Fully commissioned facility with state of-the-art single use manufacturing

equipment– Flexible facility equipped for mammalian cell culture projects– 50L / 200L scale production capacity

• Support from research laboratories as needed9

DHVI GMP FacilitiesCell Culture Suite

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DHVI GMP FacilitiesPurification Suite

11

DHVI GMP FacilitiesFinal Bulk Fill Suite

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DHVI GMP FacilitiesManufacturing Support Area

13

DHVI GMP FacilitiesAnalytical Laboratory

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Coming soon: 2nd GMP Facility 4.9 M USD NIH grant awarded

• Increased capability and flexibility (proteins immunogens, mRNAs, others platforms)

• Completion date: 2019 15

Outline

• DHVI GMP organization• GMP Facilities• Development Approach: M5 gp120

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DHVI Development Approach • Focus on product

understanding and safety for Phase I

• Set manufacturing strategies to simplify clinical trial material delivery

• Accelerate development using standardized Envproduction platforms

• Leverage DHVI scientific expertise

Characterize vaccine products and processes. Limited focus on scale-up and

formulation

Define minimum targets to meet clinical objectives. Short GMP campaigns and stability programs. Limit use of CMO to

strategic partnerships

Build simple and efficient processes to reach project goals and shorten time to

clinic

Monitor critical immune epitopes from early development through product

release

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• Set “Minimum-go” targets for development

• Minimum quantity/quality to enable clinical testing

Performance Criteria

“go”Target

Upstream titer >40mg/L

purification yield > 25%

Drug Substance 2 g

Purity Target >95%

Vials 1,000

EnvSeq-2 vaccine immunogenM5 gp120

• Our first product is CH505 M5 gp120, the priming immunogen of the EnvSeq-2vaccine. (Bonsignori M, KorberB, et al. Cell 165: 449, 2016)

• CH505 M5 gp120 is the best Env of ~ 100 tested for binding to the UCA of the CH235

• Phase I dose : 300 µg with GLA-SE (TLR-4 agonist)

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Cell Line DevelopmentExpression Vector Design

• pOptiVEC expression vector− CMV promoter driven

bicistronic ORF− IRES driven expression of

DHFR

• Utilize CHO DG44 master cell bank produced by Charles River

• Evaluated 3 different leader sequences− M5 (wild-type envelope)− CD5− tPA

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Cell Line DevelopmentStable Pools for Product Expression

DNA Transfection and

HT- selection(~1 month)

Methotrexate amplification

(0.25- 1 uM MTX )(~1 month)

Methotrexate amplification(1-2 uM MTX)

(~1 month)

• Stable pools were derived through a three-round selection strategy

• 6 pools (3 each leader sequence, 2 selection strategies each)

• Stability was evaluated over 30 PDL based on: viability, doubling time and Env expression

• Selected pools had <20% change in Env expression

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Cell Line DevelopmentProductivity assessment

• An 8-day batch culture evaluation

• CHO pool tPA 1.0+ 2.0 MTX produced the highest titers of approx. 70 mg/L of M5 gp120 with good cell growth properties.

• CHP pool tPA + 1µm /2 µm MTX was selected for MCB production.

• MCB produced by BioReliance

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Cell Culture Process Development

• Cell culture process design focused on:– Simple and robust scale up to meet

productivity targets– Ease of implementation in the DHVI GMP

facility– Chemically defined, animal component free

process– Simplicity to facilitate future tech transfer

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Cell Culture Process

Chemically defined, animal

component free media with

single nutrient feed

Gibco CD OptiCHO media

Feed :Efficient Feed A+ (3x conc.)

Daily feed 1.5 % of initial working

volume, days 4 through 12

Glucose supplementation via feed

6 mM Glutamine bolus Day 0

Inoculation VCD:

0.5x106 viable cells/mL

Temperature: 37°C no temp shift

pH setpoint: 6.95 ± 0.1

Control at upper deadband with CO2

Control at lower deadband with 1M sodium carbonate

DO setpoint: 30%

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Aeration strategy: Control at setpoint (30%) with

Air and O2

Agitation Agitation setpoint

Determined by desired kLa range (10 – 15) P/V ≤ 100 W/m3

Use kLa & P/V data to scale

Cell Culture Process Control and Scale up

2 L

10 L

50 L

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Process Data – M5 Consistency Run

• Process consistency demonstration at small scale using the M5 cell pool MCB

• 2x 2L Sartorius single use bioreactor

• 1x 10L glass (5x scale up)

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Process Data – M5 Consistency Run

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Process Data – M5 Consistency Run

14 day peak titers from M5 CHO cell pools >400 mg/L

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Final M5 Cell Culture Process

Vial thaw (Passage 0)125 mL shake flask

25 mL working volume

Passage 11 mL shake flask

150 mL working volume

Passage 22 x 2 L shake flask

500 mL working volume per flask

Passage 3 (n-1)3 x 5 L shake flask

2500 mL working volume per flask

4 days

3 days

3 days

3 days

50 L BRX (n)40 L initial

working volume

14 days

Incubator Setpoints Temperature: 37°C CO2: 8 % Agitation: 120 rpm Humidity: 80%

Productivity: ~20g expected from a 50L GMP run

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Final M5 Cell Culture Process – Harvest and Depth Filtration

50 L BRX (n)

X0HC2 x 0.11 m2

D0HC1 x 0.55 m2

1° Downstream StepViral inactivation

Culture harvested on Production Day14

Depth filtration via Millistak+ PodsProcess flux 100 to 200 LMH

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Purification Process Development

• Purification process design focused on:– Step design to preserve activity and facilitate

efficient contaminant removal (HCP, DNA, viruses)

– Achieve yield target ( >25%)– Ease of implementation in the DHVI GMP

single use facility– Simplicity to facilitate future tech transfer

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M5 gp120 purification processInitial Observations

• An initial evaluation was conducted on M5 purified from the CHO RCB

• Low dimer content <5%• M5 instability at low pH (≤5.0) at room temperature• Informed process design

Intermediate Hold Stability (Room Temp)

M5

pH 5.0 Intermediate Hold Stability CH106 Binding: BLI

Time (sec)

nm

0 100 200 300 400 500 600 700 800 9000

0.1

0.2

24hr RT

72hr RT

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Capto DeVirS Capture

CHT Intermediate(pH 7.2)

Capto Adhere Polishing (pH 7.2)

Viresolve Pro

TangenX UFDF

0.22uM FiltrationBulk Drug Substance

Triton X-100 Viral Inactivation

M5 Process (7 steps)

Process Yield: >50%

pH 7.2

Load pH 6.0Elute pH 7.2

-Avoid low pH instability-Increase Yield

-Reduce complexity and buffer consumption

-Utilize orthogonal purification approaches

-Minimize number of steps-Avoid low pH instability

-Implement Single Use

-Increase Yield-Reduce Complexity

Final M5 purification process

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Purification process performance

Step Yield Dev Run 5 ConsistencyRun Assay

ViralInactivation /

Capture75% 56% RP-UPLC

Intermediate 116% 104% RP-UPLC

Polishing 93% 91% RP-UPLC

Nanofiltration 97% 95% A280

UFDF 95% 97% A280Overall Process

Yield56% 52% RPUPLC

A280

• Overall Process Productivity– 0.17g M5 DS / L Clarified

Harvest (Dev Run 5) – 0.18g M5 DS / L Clarified

Harvest (Consistency Run)

• ~8.5 g of bulk drug substance at 50L scale

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Development Lot Drug Substance SDS-PAGE comparison

250kD150kD100kD75kD

50kD

37kD

25kD20kD15kD

10kD

250kD150kD100kD75kD

50kD

37kD

25kD20kD15kD

10kD

1 2 3 4 5 6 7 1 2 3 4 5 6 7

Lane Upstream Source1 Marker2 Dev Run 2 – Shake

Flask3 Dev Run 3 – 10L4 Dev Run 4 – 2L5 Dev Run 5 – 2L 6 Consistency Run –

10L7 Marker

Gel: Novex NuPage BisTris 4-12%Marker: BioRad Precision Plus UnstainedLoading: 3ug M5 / well

Non-Reduced Reduced

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Characterization of M5 Drug Substance from Development LotsMethod Proposed

Acceptance Criteria/Spec.

R&D LotDevelopment #4 Development #5 Consistency Run :

10L

pH 6.3-6.7 6.53 6.54 6.48

RP-UPLC ≥95% product peak 97.5% 98.6% 97.9%

SEC-UPLC ≥95% monomer 97.5% 99.1% 98.0%

SDS-PAGE Reduced/NR

Comparable to reference standard

Comparable to reference standard

Comparable to reference standard

Comparable to reference standard

Absorption 280 nm 1.0 – 1.4 mg/mL 1.36 mg/mL 1.02 mg/mL 1.0 mg/mL

Antibody binding (SPR)1.CH235 UCA2. CH235 Intermediate I.4

Comparable to reference standard

CH235 UCA: KD = 1.24 µM

CH235 I.4: KD = 1.39 µM

CH235 UCA: KD = 1.67 µM

CH235 I.4: KD = 6.10 µM

Pending

Residual HCPELISA (Cygnus 1st

gen kit)

TBD: based on process

performance

1.8 µg/mg 2.9 µg/mg 11.6 µg/mg

HC DNA <10 ng/ dose < 2.76 pg/dose 2.31 pg/dose Pending

Analytical Characterization: Purity AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

Minutes2.60 2.65 2.70 2.75 2.80 2.85 2.90 2.95 3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60 3.65 3.70 3.75 3.80 3.85 3.90 3.95 4.00 4.05 4.10 4.15 4.20 4.25 4.30 4.35 4.40 4.45 4.50 4.55 4.60

Consistency RunDev Run #4Dev Run #5

RP-UPLC

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

Minutes0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00

Consistency RunDev Run #5

SEC-UPLC

Purity Range:97.5 – 98.6%

Purity Range:97.5 – 99.1%

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Next Steps• 50L demo run underway to confirm scale up

parameters• Preparations for engineering run in August• DAIDS Audit in September• GMP lot planned for October

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Outline

• DHVI GMP organization• GMP Facilities• Development Approach: M5 gp120• Conclusions/Acknowledgements

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DHVI Development Timeline Versus Industry Standard: Potential Time Savings to Phase I

2018Today

Sep Nov Jan Mar May Jul Sep Nov Jan Mar May Jul2016 2017 2018

Sep 1 - Dec 31 Cell Line DevelopmentJan 1 - Jun 30 Process Development

Jul 1 - Oct 1 Tech TransferOct 1 - Nov 30 GMP Drug Substance

Nov 30 - Jan 1 Release TestingJan 15 - Jan 30 GMP Drug Product

Jan 30 - Mar 15 Release TestingSep 1 - Mar 1 Cell Line Development

Mar 1 - Sep 1 Process DevelopmentSep 1 - Dec 1 Tech Transfer

Dec 1 - Mar 1 GMP Drug SubstanceMar 1 - May 15 Release Testing

May 15 - May 30 GMP Drug Product

May 30 - Aug 30 Release Testing

6 months difference

DHVI

Industry Standard

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Conclusions• DHVI has assembled a team focused on delivery of vaccine

products for Phase I

• We are have established state of the art equipment and facilities to develop vaccine candidates based on cell culture expression and other vaccine technologies

• We applied a phase-specific approach to development of M5 gp120

• Simplified approaches to cell line, upstream and downstream development were successfully applied with support of key analytical tools

• We are continuing to refine these approaches to shorten timelines for clinical trial material delivery

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Our Team, Partners and Support• Bart Haynes• Munir Alam• Scott Alderman• Richard Scearce• Jadrien Hill

• Kurt Last (Working Buildings)

• Shawn Geisel (Working Buildings)

• NIAID: VTRB group

• Carl Dieffenbach• Mary Marovich• Stuart Shapiro• Mike Pensiero

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AcknowledgementsSupported by:• National Institute of Allergy and Infectious Diseases (NIAID)• National Institutes of Health (NIH)• Division of AIDS (DAIDS)• U.S. Department of Health and Human Services (HHS)• Duke University School of Medicine

Duke Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID) #UM1-AI100645

HIV/AIDS Vaccine Facility (C06) #1C06OD023830

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