gen vaccine webinar.pdf

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 OliverManaging Editor

Genetic Engineering & Biotechnology News



Steven Pincus, Ph.D.Head of Analytical and Quality Operations

Novavax

Analytical Characterization of Vaccines

Virus-Like Particle Vaccines Challenge

Steven Pincus, PhDHead 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

rHAHA cloned

Purified

3 wks

SRID reference

antiserum

Sheep

Hyperimmunized

6 – 9 weeks

HPLC Alternative to SRID

Purity

LC/Mass Spec Analysis of Proteins

In B/Florida/4/06 VLPs

HA0

gp64 BV

NA

M1 dimer/tubulin

p39 BV capsid

M1

Influenza

• target HA, NA

• M1

Baculovirus

• gp64 envelope

• p39 capsid

• ubiquitin

• minor structural proteins

Sf9 host proteins

• alpha-actin and tubulin

• HSP 70 (chaperon)

• several housekeeping proteins

Performed under contract by John Hopkins University

1. No αTubulin was detected in Baculovirus

2. No αTubulin 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.

α-Tubulin is present within VLP

0

2

4

6

8

10BV Ref 266.3.3

Sf9 Lysate Ref 1-26-10

VLP H5N1 #2

VLP H5N1 #3

VLP H5N1 #4

0%

1,28%

4.59% 5.00%

1.69%

%

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 VLP’s, Lot#: 11508-1 (Stage B)

180µg HA/mL178 1435

Seasonal Trivalent VLP’s, Lot#:

75508008-2A (05/06 strains) 30µg

HA/mL/ strain

160 268

Seasonal Trivalent VLP’s, Lot#:

75508013-1 (08/09 strains) 120µg

HA/mL/ strain

180 284

Comparability

Biochemical Characterization

• Carbohydrate

• Fatty acid

• Lipid

Carbohydrate Analysis 2008-2009 Trivalent

VLP Vaccine

gF Map gA Map

Oligosaccharides

Consistent with the presence of truncated complex

type and/or high Mannose structures expected from

insect cells

Possible Oligosaccharide Assignment

Hex5

Hex3HexNAc2

Hex6

Hex3HexNAc2DeoxyHex1

Hex3HexNAc3

Hex7

Hex5HexNAc2


Hex8

Hex5HexNAc2


Hex9

Hex7HexNAc2

Hex8HexNAc2

Hex9HexNAc2

Possible Oligosaccharide Assignment

Hex3HexNAc2


Hex3HexNAc3

Hex5HexNAc2

Hex6HexNAc2

Hex7HexNAc2

Hex8HexNAc2

Hex9HexNAc2

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

0

5

10

15

20

25

30

35

40

C14:0 M

YRISTATE

C16:0 P

ALMIT

ATE

C16:1 P

ALMIT

OLEATE

C18:0 S

TEARATE

C18:1n9 O

LEATE

C18:1n7 V

ACCENATE

C20:0 E

ICOSANOATE

C20:1 1

1-EIC

OSENOATE

C22:0 B

EHENATE

Per

cen

tag

e

VLP

SF9 Cells

BV

0

10

20

30

40

50

60

70

saturated monounsaturated

Per

cen

t, %

VLP

BV

There are detectable

differences in the fatty acid

content of VLP, Sf9 cells

and BV.

Cholesterol and Zwitterionic Lipid Composition

0

10

20

30

40

50

60

Cholesterol PC SM PE

Weig

ht,

%

H1N1 A/NC

BV A/NC Lipids

SF9 Lipids

Lipid composition of VLP differs from BV and host cells

Phospholipid

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



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 Reedas a non-profit biotech for globalhealth

•~90 employees, ~35 withadvanced 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

H5N1+AS03

H5N1

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 inweakened immunesystem (e.g. geriatric)

– Response broadening

• For subunit/recombinantvaccines critical enablingcomponent

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 ofcytokines, MHC, co-stimulatory molecules

• APC migration to T-cellarea of lymph nodes

• Modification of intra-cellular 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. 1-31Emulsion 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 byimmunology 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

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

oiloil

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)

Imiquimod (TLR7) Poly(I:C) (TLR3)R848 (TLR7/8)

QS21

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 pressurehomogenization, sonication, sterile filtration

oiloil

Adjuvant Product Considerations

• Components

– Source, purity, biocompatibility, affordability, stability

• Formulation

– Excipient compatibility, stability, biological activity

– Manufacturability

• Characterization

– Complementary physicochemical analytics

Immunomodulator

Adjuvant Physicochemical CharacterizationEmulsion Zeta Potential

SE

GLA

-SE

-20

-15

-10

-5

0

*

Zeta

po

ten

tial

(mV

)

Zeta potential

HPLC-CAD

Visual appearance

Particle size

How CAD Works

• 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

A mass sensitive detector

for the determination of any non-volatile

and many semi-volatile chemical species

TLR4 Agonist Quantitation

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,


Linear gradient: 0-5 min: 50% A

15-20 min: 10% A

25-30 min: 50% A

0 50 1000

5000

10000

15000

Adjuvant Conc. (mg/ml)

Peak A

rea

LOD: 227 ng

Ave RSD: 5.6%

Raw Material Purity: Emulsion Oil

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

Waters Atlantis C18 column

A: 75:15:10 (v/v/v) MeOH:CHCl3:H2O,


B: 50:50 (v/v) MeOH:CHCl3,


Linear gradient: 0 min: 100% A

45-50 min: 10% A

55-60 min: 100% A

Sample: 4 mg/ml, 50 ml injection

Effect of Oil Purity on Emulsion Stability

0

50

100

150

200

250

DM 1 wk 2 wk 1 mon 3 mon

Time

Z-a

vg (

nm

)

stable

metastable

unstable

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

Oil Emulsifier Emulsion Stability at 3 months

shark squalene Soy PC Stable




olive squalene (N85) Soy PC Unstable

olive squalene (WT97) Soy PC Unstable

olive squalene (WT97) Soy PC Metastable

olive squalene (WT97) Soy PC Stable

olive squalene (WT97) Soy PC Stable

olive squalene (N92) Soy PC Stable

shark squalene DOPC Stable

olive squalene (WT97) DOPC Metastable

Multi-Component Formulation Analysis

O/W emulsion

Liposome

mV

mV

squalene

egg phosphatidylcholine emulsifier

TLR4 agonist

DPPC

cholesterol

DPPG

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 non-chromophore immunomodulators, excipient raw materials, and complete adjuvant formulations

• Complemented by other analytical technqiues, HPLC-CAD has proven to be an important tool for IDRI’s 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 bygrant #42387 from the Bill andMelinda Gates Foundation



Modern Vaccine and Adjuvant Production and

Characterization

Q&A



Your Moderator

Tamlyn OliverManaging Editor

Genetic Engineering & Biotechnology News



Steven Pincus, Ph.D.Head of Analytical and Quality Operations

Novavax



Chris Fox, Ph.D.Scientist I/Lead Formulations Engineer

Infectious Disease Research Institute



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

gen vaccine webinar.pdf

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