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Modern Vaccine and Adjuvant

Production and Characterization

Modern Vaccine and Adjuvant Production and


Characterization
Broadcast Date: Wednesday, April 27, 2011

Time: 11:00 am EDT, 8:00 am PDT


Sponsored by

Modern Vaccine and Adjuvant


Production and Characterization

Your Moderator

Tamlyn Oliver
Managing Editor
Genetic Engineering & Biotechnology News

Modern Vaccine and Adjuvant


Production and Characterization

Steven Pincus, Ph.D.


Head of Analytical and Quality Operations
Novavax

Analytical Characterization of
Vaccines
Virus-Like Particle Vaccines
Challenge
Steven Pincus, PhD
Head of Analytical and Quality Operations
Novavax, Inc. , Rockville MD

April 27, 2010

Historical Vaccines
Live attenuated virues or bacteria
MMR
Smallpox
YFV
BCG

Inactivated vaccines
Polio
JEV
Rabies
Subunit vaccines
Polysaccharides

Analytical Characterization of Historical


Vaccines
Antigenic Dose/Potency
Infectious units
SRID
Potency in animal model
Stability
Maintain infectious titer or antigen dose
Maintain potency
Identity
Western blot
Serum neutralization
immunofluoresence

Recombinant Virus-Like Particle (VLP)


Vaccines
Non-enveloped
Hepatitis B vaccines (recombinant)
- Recombivax HB (Merck)
- Engerix B (GSK)

Human Papilloma Virus Vaccines (recombinant)


- Gardasil (Merck)
- Cervarix (GSK)
- Made in insect (Lepidoptera) cells
- Recently licensed in the U.S.

Enveloped
Seasonal and Pandemic Influenza
- Novavax HA-NA-M1 VLPs

Recombinant Influenza VLPs:


Pleomorphic Spherical Particles

HA and NA
Spikes
Lipid bilayer
M1 helical
matrix

120nm

Analytical Characterization Protein VLPs


Release

Identity
Potency
Dose confirmation
Purity
Secondary structural characteristics

Stability

Dose Confirmation
Potency
Antigen modifications
Secondary structural characteristics

Comparability

Identity and Potency

CBER/Novavax SRID HA Reference Reagents


are Interchangeable
Reagent

CBER/NIBSC

Novavax

Antigen used for


immunization

Purified bromelian cleaved


HA from flu virus grown in
eggs

Purified recombinant HA (rHA)


produced in insect cells

Reference antiserum

Sheep anti-HA

Sheep anti-rHA

Reference antigen

Purified whole inactivated


influenza produced in eggs

Influenza VLPs produced in


insect cells

HA cloned
Purified
3 wks

SRID reference
antiserum

rHA

Sheep
Hyperimmunized
6 9 weeks

HPLC Alternative to SRID

Purity

LC/Mass Spec Analysis of Proteins


In B/Florida/4/06 VLPs
Influenza
target HA, NA
M1

Baculovirus

gp64 envelope
p39 capsid
ubiquitin
minor structural proteins

HA0
gp64 BV
NA
M1 dimer/tubulin

Sf9 host proteins


alpha-actin and tubulin
HSP 70 (chaperon)
several housekeeping
proteins

Performed under contract by John Hopkins University

p39 BV capsid
M1

-Tubulin is present within VLP


10

BV Ref 266.3.3
Sf9 Lysate Ref 1-26-10
VLP H5N1 #2

4.59%

5.00%

VLP H5N1 #3

VLP H5N1 #4
4
2

1.69%
1,28%
0%

1. No Tubulin was detected in Baculovirus


2. No Tubulin was detected in non-Zwittergent treated VLP samples
3. Concentration of Tubulin in VLP samples treated with 1% Zwittergent
was 1.3-3.9 times higher than in SF9 lysate.

VLP Aggregation
Wyatt technology
Field Flow Fractionation online static and dynamic LS detection
No aggregation

Different trivalent seasonal or monovalent VLPs similar size and


distribution
RMS Radius (root mean square)/Rh (radius of hydration) Radius
ratio ~ 1:1
Spherical shells with open center

Particle sizing Malvern Zetasizer


No evidence of aggregation in VLP samples
After acid treatment can detect aggregates
Sample

Particle Size (nm)

pH 7.2

pH 4

H5N1 VLPs, Lot#: 11508-1 (Stage B)


180g HA/mL

178

1435

Seasonal Trivalent VLPs, Lot#:


75508008-2A (05/06 strains) 30g
HA/mL/ strain

160

268

Seasonal Trivalent VLPs, Lot#:


75508013-1 (08/09 strains) 120g
HA/mL/ strain

180

284

Comparability

Biochemical Characterization
Carbohydrate
Fatty acid
Lipid

Carbohydrate Analysis 2008-2009 Trivalent


VLP Vaccine
gF Map

Oligosaccharides

Possible Oligosaccharide Assignment


Hex5
Hex3HexNAc2
Hex6
Hex3HexNAc2DeoxyHex1
Hex3HexNAc3
Hex7
Hex5HexNAc2
Hex3HexNAc3DeoxyHex1
Hex8

gA Map
Possible Oligosaccharide Assignment
Hex3HexNAc2
Hex3HexNAc2DeoxyHex1
Hex3HexNAc3
Hex5HexNAc2
Hex6HexNAc2
Hex7HexNAc2
Hex8HexNAc2

Hex9HexNAc2

Hex5HexNAc2

Hex3HexNAc4DeoxyHex1
Hex9
Hex7HexNAc2
Hex8HexNAc2
Hex9HexNAc2

Consistent with the presence of truncated complex


type and/or high Mannose structures expected from
insect cells

Potential Insect Cell Glycoallergens


Alpha 1 3 fucose
Plants and some insects
glycoproteins
Very low or absent in Sf9
(S. frugiperda) cells
Galactose-alpha-1,3-galactose
Food allergen
Significant levels High5 (T. ni) cells
Very low level in Sf9 cells

No Evidence of potential glycoallergens alpha 1,3


fucose and alpha 1,3 galactose linkages in H5N1,
2005-2006 or 2008-2009 VLPs

Fatty Acid Analysis


80% of fatty acids belong to 4 classes
C16.0 Palmitate, C16.1 Palmitoleate, C18.0 Stearate, C18.1 n9
Oleate

Seasonal and H5N1 pandemic VLPs differ in their total


percentage of saturated vs unsaturated fatty acids
Seasonal longer chains
Higher saturated/unsaturated ratio in seasonal suggesting greater
lateral segregation

VLP fatty acid lower content of saturated fatty acids than


baculovirus and may bud from different membrane
regions

Fatty Acid Composition


40

Percentage

35
30

VLP
SF9 Cells

25
20

BV

15
10

C
14
:0

Y
R
C
IS
16
TA
:0
C
TE
P
16
A
LM
:1
P
IT
A
A
LM
TE
IT
O
C
18
LE
:0
A
TE
S
TE
C
AR
18
AT
:1
C
n
E
18
9
O
:1
LE
n7
A
V
C
TE
A
20
C
C
:0
E
E
N
C
IC
A
20
TE
O
:1
S
A
11
N
-E
O
A
IC
TE
O
S
EN
C
22
O
:0
A
TE
B
E
H
E
N
A
TE

5
0

70
60

VLP
BV

Percent, %

50
40
30
20
10
0
saturated

monounsaturated

There are detectable


differences in the fatty acid
content of VLP, Sf9 cells
and BV.

Phospholipid
60

Cholesterol and Zwitterionic Lipid Composition


H1N1 A/NC

50

BV A/NC Lipids
SF9 Lipids

Weight, %

40

30

20

10

0
Cholesterol

PC

SM

PE

Lipid composition of VLP differs from BV and host cells

Conclusions and Implications


New Vaccines can be Analytically Characterized using
methods developed for biological and monoclonal
antibodies
Virus-like particles can be characterized and may allow
designation as well-characterized biological

Modern Vaccine and Adjuvant


Production and Characterization

Chris Fox, Ph.D.


Scientist I/Lead Formulations Engineer
Infectious Disease Research Institute

Characterizing Vaccine Adjuvant


Formulations by HPLC-CAD
Christopher Fox
GEN-Dionex Webinar
April 27, 2011

Infectious Disease Research Institute (IDRI)


Founded in 1993 by Steve Reed
as a non-profit biotech for global
health
~90 employees, ~35 with
advanced degrees
Diseases: leishmaniasis,
tuberculosis, malaria, influenza,
leprosy, chagas
Capabilities: vaccinology (antigen discovery, adjuvants,
formulations), drug discovery (medicinal chemistry), process
sciences, cGMP manufacturing, clinical/regulatory, biz develop/legal
Funded by BARDA, NIH, BMGF, DARPA, PATH, WHO, Eli Lilly,
Murdock Charitable Trust, American Leprosy Missions, and several
public-private partnerships (2010 budget ~$24 million)

Outline

Introduction to vaccine adjuvants


History of vaccine adjuvant development
Modern vaccine adjuvant considerations
HPLC-CAD Analysis
Quantification of TLR4 agonist
Raw material purity
Nanoparticle formulation analysis

Conclusions and Recommendations

Adjuvants (Adjuvare = to help)


Added to a vaccine to improve the immune response
Increase antibody titers
Induce cell-mediated immunity
Reduce antigen dose,
number of doses
Enable immunization in
weakened immune
system (e.g. geriatric)
Response broadening

H5N1+AS03
H5N1

For subunit/recombinant
vaccines critical enabling
component
Carter et al. BioDrugs 2008, 22:279

Classification of Adjuvants
Immunomodulatory molecules
Directly stimulate immune cells
Ex: TLR agonists, saponins, bacterial
exotoxins

Delivery systems
Present antigen to immune system
Ex: Mineral salts, emulsions, liposomes

Combinations
Antigens or immunomodulators associated
with delivery systems
Ex: AS04, AS01, MPL-SE

Mechanisms of Action
Promote antigen uptake by APCs
Stimulation of APCs
Upregulation of
cytokines, MHC, costimulatory molecules

APC migration to T-cell


area of lymph nodes
Modification of intracellular trafficking

Seubert et al. in J Immunol 2008, 180:5402

History of Vaccine Adjuvant Development


1920s to 1970s: Alum and oil-based adjuvant
development
Agar, tapioca, bread crumbs, metallic salts, etc.
Alum-antigen combos most successful
DTP (containing alum) licensed in 1948

Water-in-oil emulsions (CFA, IFA)


IFA in influenza and polio vaccines

1970s to 1990s: Small molecules and particulate


vehicles

Bacterial cell wall derivatives (LPS, MDP, TDM)


dsRNA: Poly(I:C) and Poly(A:U)
Saponins (Quil A)
Liposomes, polymeric spheres
Ott et al. in Vaccine Adjuvants and Delivery Systems, Wiley-Interscience, Hoboken, NJ, 2007, p. 1-31
Emulsion image from Freund et al. J Immunol 1944, 48:325

History of Vaccine Adjuvant Development


1990s to present: Rational design of adjuvants and
delivery systems
Adjuvant development aided by
immunology progress

TLR receptors
Cytokine profiles
MHC class I vs II
Recombinant DNA-generated antigens

New adjuvant molecules and delivery vehicles

MPL and analogues


QS21
Imidazoquinolines
CpG
Oil-in-water emulsions

oil

Ott et al. in Vaccine Adjuvants and Delivery Systems, Wiley-Interscience, Hoboken, NJ, 2007, p. 1-31
TLR3-dsRNA image from Liu et al. Science 2008, 320:379

Approved Adjuvants or in Clinical Trials


Approved (US)
Alum (grandfathered 80+ years, contained in many
vaccines)
MPL-alum (2009 in Cervarix)
Conditional approval in the event of pandemic influenza:
MF59, AS03

Approved (Europe)

MF59 (seasonal and pandemic flu vaccines)


AS03 (pandemic flu)
MPL-alum (Cervarix, Fendrix)
Virosomes (seasonal flu)

Clinical trials
AS01, AS02, MPL-SE, CpG, Montanide, R848
Image from healthbeautynews.com

Structures of Immunomodulators

GLA
(TLR4)
QS21

R848 (TLR7/8)

Imiquimod (TLR7)

Poly(I:C) (TLR3)

Adjuvant Formulations
Aqueous
Soluble molecules or suspensions

Alum
Aluminum hydroxide or aluminum phosphate
1-10 mm aggregate particles

Oil-in-water emulsions
~100 nm emulsified oil droplets

Lipid vesicles
Liposomes, niosomes, virosomes
~100 nm lipid or surfactant vesicles

Manufacturing techniques
High speed mixing, high pressure
homogenization, sonication, sterile filtration

oil

Adjuvant Product Considerations


Components
Source, purity, biocompatibility,
affordability, stability

Formulation
Excipient compatibility, stability,
biological activity
Manufacturability

Characterization
Complementary physicochemical
analytics

Adjuvant Physicochemical Characterization


Emulsion Zeta Potential

Particle size

Zeta potential

-5
-10
-15

*
G

LA
-

SE

-20
SE

Zeta potential (mV)

Immunomodulator

HPLC-CAD

Visual appearance

How CAD Works


A mass sensitive detector
for the determination of any non-volatile
and many semi-volatile chemical species

The eluent from the column is


nebulized to form droplets
The droplets are dried to form
neutral particles
These particles are charged
The charge is then measured

The size of the particle is related to


the total charge measured and the
concentration in the peak

TLR4 Agonist Quantitation

Peak Area

15000

10000

5000

LOD: 227 ng
Ave RSD: 5.6%
0
0

50

Adjuvant Conc. (mg/ml)

100

Waters Atlantis C18 column


A: 75:15:10 (v/v/v) MeOH:CHCl3:H2O,
20 mM ammonium acetate, 1% acetic acid
B: 50:50 (v/v) MeOH:CHCl3,
20 mM ammonium acetate, 1% acetic acid
Linear gradient: 0-5 min: 50% A
15-20 min: 10% A
25-30 min: 50% A

Raw Material Purity: Emulsion Oil

Waters Atlantis C18 column


A: 75:15:10 (v/v/v) MeOH:CHCl3:H2O,
20 mM ammonium acetate, 1% acetic acid
B: 50:50 (v/v) MeOH:CHCl3,
20 mM ammonium acetate, 1% acetic acid
Linear gradient: 0 min: 100% A
45-50 min: 10% A
55-60 min: 100% A
Sample: 4 mg/ml, 50 ml injection

Fox et al. Coll Surf B: Biointerfaces 2008, 65:98

Effect of Oil Purity on Emulsion Stability

250

Z-avg (nm)

200

stable
metastable
unstable

Oil
shark squalene
shark squalene
shark squalene
shark squalene

Emulsifier
Soy PC
Soy PC
Soy PC
Soy PC

Emulsion Stability at 3 months


Stable
Stable
Stable
Stable

olive squalene (N85)


olive squalene (WT97)
olive squalene (WT97)
olive squalene (WT97)
olive squalene (WT97)
olive squalene (N92)

Soy PC
Soy PC
Soy PC
Soy PC
Soy PC
Soy PC

Unstable
Unstable
Metastable
Stable
Stable
Stable

shark squalene
olive squalene (WT97)

DOPC
DOPC

Stable
Metastable

150

100

50

0
DM

1 wk

2 wk

1 mon

3 mon

Time

Fox et al. Coll Surf B: Biointerfaces 2008, 65:98

Multi-Component Formulation Analysis


mV

squalene

O/W emulsion

egg phosphatidylcholine emulsifier

TLR4 agonist

mV

DPPG

Liposome

DPPC

cholesterol
TLR4 agonist

Conclusions
Adjuvants critical component of next-generation
vaccines
Vaccine adjuvants include wide range of
immunomodulatory molecules and formulations
Empirical approach in adjuvant development is being
replaced by rational design and thorough
physicochemical characterization
HPLC-CAD facilitates sensitive detection of nonchromophore immunomodulators, excipient raw
materials, and complete adjuvant formulations
Complemented by other analytical technqiues, HPLCCAD has proven to be an important tool for IDRIs
vaccine adjuvant analytical laboratories

Acknowledgments
IDRI

Steve Reed
Darrick Carter
Tom Vedvick
Tim Dutill
Susan Lin
Sandra Sivananthan
Ryan Anderson

WHO
Martin Friede
This research was supported by
grant #42387 from the Bill and
Melinda Gates Foundation

Modern Vaccine and Adjuvant


Production and Characterization

Modern Vaccine and Adjuvant Production and


Characterization

Q&A

Modern Vaccine and Adjuvant


Production and Characterization

Your Moderator

Tamlyn Oliver
Managing Editor
Genetic Engineering & Biotechnology News

Modern Vaccine and Adjuvant


Production and Characterization

Steven Pincus, Ph.D.


Head of Analytical and Quality Operations
Novavax

Modern Vaccine and Adjuvant


Production and Characterization

Chris Fox, Ph.D.


Scientist I/Lead Formulations Engineer
Infectious Disease Research Institute

Modern Vaccine and Adjuvant


Production and Characterization

Thank You For Attending


Modern Vaccine and Adjuvant Production and Characterization
Broadcast Date: Wednesday, April 27, 2011
Time: 11:00 am EDT, 8:00 am PDT
Sponsored by