Rapid Manufacture and Release of a GMP Batch of...

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Rapid Manufacture and Release of a GMP Batch of Zaire Ebolavirus Glycoprotein Vaccine Made Using Recombinant

Baculovirus-Sf9 Insect Cell Culture Technology

Tim HahnSVP, Global Manufacturing Operations

Novavax, Inc.10 March 2015

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Presentation Overview

• Ebola Background

• Novavax Ebola/Mak Glyoprotein Vaccine

• Novavax saponin-based Matrix-M™ Adjuvant

• Rapid Manufacturing Timeline

• Pre-clinical Data

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Ebola Background

• A new virus was discovered in 1976‒ Caused hemorrhagic fever‒ Later named genus Ebolavirus ‒ Five Ebola virus species including Zaire Ebola virus (EBOV)

• From 1976 to 2013 ‒ Several outbreaks of Ebola Virus Disease (EBV)‒ Average EVD case fatality rate is about 50%‒ Most outbreaks in central and west Africa‒ Largest number of cases previously reported was 425 in 2000 outbreak‒ Estimate of total EVD deaths from 1976 to 2013 is 1590.

• On-going 2014 (to 2015) outbreak as of March 4, 2015‒ 23,969 cases‒ 9807 deaths

• 6-times the total from 1976 to 2013

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Span of Endemic

• Most cases in Guinea, Liberia, and Sierra Leone

• Infections also reported in:• Nigeria• Democratic Republic of Congo• Senegal• United States• Spain• Mali• United Kingdom

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“In memoriam: Tragically, five co-authors,who contributed greatly to public health and research efforts in Sierra Leone, contracted EVD and lost their battle with the disease before this manuscript could be published”

• In Sierra Leone May 2014 at funeral of a Guinea EVD case, attendees were infected with two distinct Ebola cluster 1,2 viruses leading to cluster 3 sustained human-to-human transmission.

• “Because many of the mutations alter protein sequences and other biologically meaningful targets, they should be monitored for impact on diagnostics, vaccines, and therapies critical to outbreak response.”

Guinea Ebola (EBOV) Gene Sequence for Cluster 3 VirusScience September 12, 2014

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Novavax EBOV/Mak GP recombinant nanoparticle vaccine

Recombinant EBOV/Makona Glycoprotein (GP)• GenBank #AIG96283• EBOV [H.sapiens -wt/SLE/2014/Makona-G3798]• Full length, unmodified GP gene• Synthetic, codon optimized• Cloned into a baculovirus vector rBV-GP

CLONINGBaculovirus vector

rBV-GP

EXPRESSION rBV-GP infected

Sf9 cells

PURIFICATIONEBOV GP

nanoparticlesEBOV GP 30-40nm

rBV-GPGP Sf9 cells

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Novavax EBOV/Makona Glycoprotein (GP) Vaccine

1Nonsynonymous AA: 2014 EBOV/Makona GP and 1976 EBOV/Mayinga GP

1Gire, et al, Science 12 Sept 2014

mAb 13F6

20 AA changes

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Anti-Ebola ELISA EC50 of mAb 13C6, 6D8 and KZ52

conccentration (ug/ml)

1e-5 1e-4 0.001 0.01 0.1 1 10 100 1000 100000

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4Ebola mAb ELISA

4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2c13c6 (Group01: Dilution vs Values) 0.0539 1.14 0.0623 3.46 0.998H13F6 (Group02: Dilution vs Values) 0.0485 1.26 1.18e+08 1.53e+05 0.999c6D8 (Group03: Dilution vs Values) 0.0243 1.02 0.0882 3.67 0.999KZ52 (Group04: Dilution vs Values) 0.0645 1.21 0.34 3.64 0.999

__________Weighting: Fixed

mAb EC50 (g/mL)13C6 0.062313F6 * -6D8 0.0882KZ52 0.34

2 aa change in 13F6 epitope on Ebola GP nanoparticle results in loss of recognition

KZ52, 13C6, 6D8

13F6

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Structure of Ebolavirus Glycoprotein

• The crystal structure of Ebolavirus GP reveals a three-

lobed chalice-like structure.

• The three GP1 subunits (colored blue and green),

mediate attachment to new host cells and are tethered

together by the three GP2 subunits (white).

• GP2 forms the protein machinery that drives fusion of

the viral membrane with the host cell.

• The human antibody KZ52 (yellow) binds the GP at the

base of the chalice, where it bridges GP1 to GP2,

before fusion of the membranes

Research conducted by J.E. Lee, M.L. Fusco, W.B. Oswald, A.J. Hessell, D.R. Burton, and E.O. Saphire (The Scripps Research Institute).Research Funding: U.S. National Institutes of Health, the Burroughs Wellcome Fund, and the Canadian Institutes of Health Research. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.Publication about this research: J.E. Lee, M.L. Fusco, W.B. Oswald, A.J. Hessell, D.R. Burton, and E.O. Saphire, "Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor," Nature 454, 177 (2008).

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Comparison of crystal structure and vaccine nanoparticle

Research conducted by J.E. Lee, M.L. Fusco, W.B. Oswald, A.J. Hessell, D.R. Burton, and E.O. Saphire (The Scripps Research Institute).Research Funding: U.S. National Institutes of Health, the Burroughs Wellcome Fund, and the Canadian Institutes of Health Research. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.Publication about this research: J.E. Lee, M.L. Fusco, W.B. Oswald, A.J. Hessell, D.R. Burton, and E.O. Saphire, "Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor," Nature 454, 177 (2008).

GP1

GP2

2D TEM – Manual coloration

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Matrix-M Adjuvant Background - Saponins

• Quillaja saponaria, the soap bark tree, is an evergreen tree native to Chile

• Saponins (glycosides of terpenes and steroids) are extracted and purified from the bark of the tree

General uses of saponins:

Food – foaming agent in beverages (produce a stable foam in beer)

Cosmetics – creams, facial cleansers, shampoos

Pharmaceuticals – Immune booster (adjuvant)

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Matrix-M Adjuvant Background - Nanoparticle

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Phase I: Manufacture 200L scale EBOV/Mak GP

Harvest ControlSf9 cells

Cel

l Cou

nt

Master rBV EBOV-GP

Sf9 WCB 200L Bioreactor

Working rBVEBOV-GP

Culture flasks 50L Wave

50L Wave

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Purification EBOV/Mak GP

Cell harvest

200L Bioreactor

Cell lysis: GP trimers

0.2 µm filtration

Centrifugation Filtration Anion exchange

Affinity Cation exchange EBOV GP30 – 40nm

Cell harvest

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EBOV/Mak GP Vaccine Manufacturing Timeline

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Trigger Event

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Project Start Date

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Passage 1 recombinant baculovirus

obtained

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Master Virus Seed (passage 2)

available

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Drug substance manufacturing

initiated to coordinate with MVS availability

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The purification process needed to be defined

prior to bioreactor harvest (and use available

buffers)

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Purification process defined in time to update SOPs and

batch records

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Drug product formulated and filled days after DS

was manufactured. Formulation was based

on protein assay

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A critical item was the development of a

“potency” assay for release and stability

studies. This was never on the primary critical

path

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MVS, DS, and DP release testing

occurred in parallel

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Batch and QC record review occurred in

parallel with processing and

testing

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A GMP batch for a Phase I clinical study was released 3 months from project start

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Matrix-M Manufacturing TimelineMatrix-M

Manufacturing was completed in 6 weeks and was never on the

critical path

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EBOV/Makona GP vaccine protected mice against lethal challenge with Ebola virus

Mice were challenged on day 42 with an intraperitoneal injection of 1,000 pfu mouse adapted EBOV/Mayinga 1976 strain

1One animal with an unrelated abdominal abscess and was dropped from the study.

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Vaccine Response Story to be continued…

Wednesday, March 11, 2:15-3:00

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Perspectives on Recombinant Baculovirus-Sf9 Platform Development and Manufacturing Process

H7N9 Influenza VLP Vaccine

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Comparable Upstream Processes for VLP and Nanoparticle Vaccines

Advantages:• One master cell bank• Rapid generation of recombinant baculovirus• Platform cell expansion process• Known purification options

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3-month Response Time from Gene Sequence to GMP Batch Release

Keys to success:• One master cell bank• Rapid generation of recombinant baculovirus• Platform cell expansion process• Known purification options• Rapid process confirmation at small scale• Rapid assay development• Forward processing of intermediates• Project management of timeline• Collaboration with suppliers and contract

service providers• Utilization of single-use technology

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Viral Threats

• Process & assay development• 1000L GMP production

• QC and QA for testing and release

• Vaccine candidate development• Preclinical study expertise

• Regulatory expertise

Response

Process Development & GMP Production

Recombinant Baculovirus-Sf9 Technology Enables a Rapid Response as a Countermeasure to Infectious Agent Threats

Clinical study expertise

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Ebola GP Vaccine - Author Recognition

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Ebola GP Vaccine – Employee Acknowledgements

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