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Analytical Characterization of
Vaccines
Virus-Like Particle Vaccines
Challenge
Steven Pincus, PhD
Head of Analytical and Quality Operations
Novavax, Inc. , Rockville MD
Historical Vaccines
Live attenuated virues or bacteria
MMR
Smallpox
YFV
BCG
Inactivated vaccines
Polio
JEV
Rabies
Subunit vaccines
Polysaccharides
Enveloped
Seasonal and Pandemic Influenza
- Novavax HA-NA-M1 VLPs
HA and NA
Spikes
Lipid bilayer
M1 helical
matrix
120nm
Identity
Potency
Dose confirmation
Purity
Secondary structural characteristics
Stability
Dose Confirmation
Potency
Antigen modifications
Secondary structural characteristics
Comparability
CBER/NIBSC
Novavax
Reference antiserum
Sheep anti-HA
Sheep anti-rHA
Reference antigen
HA cloned
Purified
3 wks
SRID reference
antiserum
rHA
Sheep
Hyperimmunized
6 9 weeks
Purity
Baculovirus
gp64 envelope
p39 capsid
ubiquitin
minor structural proteins
HA0
gp64 BV
NA
M1 dimer/tubulin
p39 BV capsid
M1
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%
VLP Aggregation
Wyatt technology
Field Flow Fractionation online static and dynamic LS detection
No aggregation
pH 7.2
pH 4
178
1435
160
268
180
284
Comparability
Biochemical Characterization
Carbohydrate
Fatty acid
Lipid
Oligosaccharides
gA Map
Possible Oligosaccharide Assignment
Hex3HexNAc2
Hex3HexNAc2DeoxyHex1
Hex3HexNAc3
Hex5HexNAc2
Hex6HexNAc2
Hex7HexNAc2
Hex8HexNAc2
Hex9HexNAc2
Hex5HexNAc2
Hex3HexNAc4DeoxyHex1
Hex9
Hex7HexNAc2
Hex8HexNAc2
Hex9HexNAc2
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
Phospholipid
60
50
BV A/NC Lipids
SF9 Lipids
Weight, %
40
30
20
10
0
Cholesterol
PC
SM
PE
Outline
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
TLR receptors
Cytokine profiles
MHC class I vs II
Recombinant DNA-generated antigens
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 (Europe)
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
Formulation
Excipient compatibility, stability,
biological activity
Manufacturability
Characterization
Complementary physicochemical
analytics
Particle size
Zeta potential
-5
-10
-15
*
G
LA
-
SE
-20
SE
Immunomodulator
HPLC-CAD
Visual appearance
Peak Area
15000
10000
5000
LOD: 227 ng
Ave RSD: 5.6%
0
0
50
100
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
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
squalene
O/W emulsion
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
Q&A
Your Moderator
Tamlyn Oliver
Managing Editor
Genetic Engineering & Biotechnology News