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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 Hahn
SVP, Global Manufacturing Operations
Novavax, Inc.
10 March 2015
1
Presentation Overview
22
•
Ebola Background
•
Novavax Ebola/Mak Glyoprotein Vaccine
•
Novavax saponin-based Matrix-M™ Adjuvant
•
Rapid Manufacturing Timeline
•
Pre-clinical Data
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
33
Span of Endemic
4
•
Most cases in Guinea, Liberia, and
Sierra Leone
•
Infections also reported in:
• Nigeria
• Democratic Republic of Congo
• Senegal
• United States
• Spain
• Mali
• United Kingdom
Presentation Overview
55
•
Ebola Background
•
Novavax Ebola/Mak Glyoprotein Vaccine
•
Novavax saponin-based Matrix-M™ Adjuvant
•
Rapid Manufacturing Timeline
•
Pre-clinical Data
Guinea Ebola (EBOV) Gene Sequence for Cluster 3 Virus
Science September 12, 2014
“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”
66
•
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.”
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
CLONING
Baculovirus vector
rBV-GP
EXPRESSION
rBV-GP infected
Sf9 cells
PURIFICATION
EBOV GP
nanoparticles
EBOV GP
30-40nm
GP
7
rBV-GP
Sf9 cells
Novavax EBOV/Makona Glycoprotein (GP) Vaccine
1Nonsynonymous
AA: 2014 EBOV/Makona GP and 1976 EBOV/Mayinga GP
20 AA changes
mAb 13F6
1Gire,
8
et al, Science 12 Sept 2014
Anti-Ebola ELISA EC50 of mAb 13C6, 6D8 and KZ52
Ebola mAb ELISA
4
KZ52, 13C6, 6D8
3
2
13F6
1
0
1e-5
1e-4
0.001
0.01
0.1
1
10
100
1000
10000
conccentration (ug/ml)
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D:
c13c6 (Group01: Dilution vs Values)
H13F6 (Group02: Dilution vs Values)
c6D8 (Group03: Dilution vs Values)
KZ52 (Group04: Dilution vs Values)
__________
Weighting: Fixed
mAb
13C6
13F6 *
A
0.0539
0.0485
0.0243
0.0645
B
1.14
1.26
1.02
1.21
C
D
R^2
EC
50 (g/mL)
0.0623
3.46
0.998
1.18e+08 1.53e+05
0.0882
3.67
0.34
3.64
0.0623
-
6D8
0.0882
KZ52
0.34
0.999
0.999
0.999
2 aa change in 13F6 epitope on Ebola GP nanoparticle results in loss of recognition
9
Structure of Ebolavirus Glycoprotein
•
The crystal structure of Ebolavirus GP reveals a threelobed 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).
10
Comparison of crystal structure and vaccine nanoparticle
GP1
GP2
2D TEM – Manual coloration
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).
11
Presentation Overview
1212
•
Ebola Background
•
Novavax Ebola/Mak Glyoprotein Vaccine
•
Novavax saponin-based Matrix-M™ Adjuvant
•
Rapid Manufacturing Timeline
•
Pre-clinical Data
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)
1313
Matrix-M Adjuvant Background - Nanoparticle
1414
Presentation Overview
1515
•
Ebola Background
•
Novavax Ebola/Mak Glyoprotein Vaccine
•
Novavax saponin-based Matrix-M™ Adjuvant
•
Rapid Manufacturing Timeline
•
Pre-clinical Data
Phase I: Manufacture 200L scale EBOV/Mak GP
Master rBV
EBOV-GP
Culture flasks
Cell Count
Sf9 WCB
50L Wave
16
Control
Sf9 cells
Working rBV
EBOV-GP
50L Wave
Harvest
200L Bioreactor
Purification EBOV/Mak GP
200L
Cell Bioreactor
harvest
Cell
harvest
Centrifugation
Affinity
17
Cell lysis: GP trimers
Filtration
Anion exchange
Cation exchange
0.2 µm filtration
EBOV GP
30 – 40nm
EBOV/Mak GP Vaccine Manufacturing Timeline
18
EBOV/Mak GP Vaccine Manufacturing Timeline
Trigger
Event
19
EBOV/Mak GP Vaccine Manufacturing Timeline
Project
Start Date
20
EBOV/Mak GP Vaccine Manufacturing Timeline
Passage 1
recombinant
baculovirus
obtained
21
EBOV/Mak GP Vaccine Manufacturing Timeline
Master Virus Seed
(passage 2)
available
22
EBOV/Mak GP Vaccine Manufacturing Timeline
Drug substance
manufacturing
initiated to coordinate
with MVS availability
23
EBOV/Mak GP Vaccine Manufacturing Timeline
The purification process
needed to be defined
prior to bioreactor harvest
(and use available
buffers)
24
EBOV/Mak GP Vaccine Manufacturing Timeline
Purification process
defined in time to
update SOPs and
batch records
25
EBOV/Mak GP Vaccine Manufacturing Timeline
Drug product formulated
and filled days after DS
was manufactured.
Formulation was based
on protein assay
26
EBOV/Mak GP Vaccine Manufacturing Timeline
A critical item was the
development of a
“potency” assay for
release and stability
studies. This was never
on the primary critical
path
27
EBOV/Mak GP Vaccine Manufacturing Timeline
MVS, DS, and DP
release testing
occurred in parallel
28
EBOV/Mak GP Vaccine Manufacturing Timeline
Batch and QC record
review occurred in
parallel with
processing and
testing
29
EBOV/Mak GP Vaccine Manufacturing Timeline
A GMP batch for a Phase I
clinical study was released
3 months from project start
30
Matrix-M Manufacturing Timeline
31
Matrix-M
Manufacturing was
completed in 6 weeks
and was never on the
critical path
Presentation Overview
3232
•
Ebola Background
•
Novavax Ebola/Mak Glyoprotein Vaccine
•
Novavax saponin-based Matrix-M™ Adjuvant
•
Rapid Manufacturing Timeline
•
Pre-clinical Data
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
33
animal with an unrelated abdominal abscess and was dropped from the study.
Vaccine Response Story to be continued…
Wednesday, March 11, 2:15-3:00
34
Perspectives on Recombinant Baculovirus-Sf9 Platform
Development and Manufacturing Process
H7N9 Influenza VLP Vaccine
3535
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
3636
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
3737
Recombinant Baculovirus-Sf9 Technology Enables a Rapid
Response as a Countermeasure to Infectious Agent Threats
Viral Threats
Response
Process Development & GMP Production
• Vaccine candidate development
• Preclinical study expertise
• Regulatory expertise
38
• Process & assay development
• 1000L GMP production
• QC and QA for testing and release
Clinical study expertise
Ebola GP Vaccine - Author Recognition
39
Ebola GP Vaccine – Employee Acknowledgements
40