Analytical Control Strategies for Aluminum Adjuvants

Chris Farrell Vaccine Analytical Development Vaccine Bioprocess R&D Merck Research Labs, Merck & Co., Inc.

Impact of Vaccines

Leon Farrant, CDC, Matthew Herper, Forbes, 19Feb2013.

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More Than Half of the Top 15 Vaccines of 2012 Contain Aluminum Adjuvants (in bold)

1. Prevnar 13®/Prevenar 13, Pfizer, $3.7B USD

2. Gardasil®, Merck, $1.9B USD

3. PENTAct-HIB, Sanofi Pasteur, $1.5B USD

4. Infanrix/Pediarix, GlaxoSmithKline, $1.2B USD

5. Fluzone® (High-Dose/Intradermal/Vaxigrip/Mutagrip), Sanofi Pasteur, $1.2B USD

6. Hepatitis franchise (Havrix/Engerix-B/Twinrix), GlaxoSmithKline, $986M USD

7. Varivax, Merck, $846M USD

8. Menactra, Sanofi Pasteur, $735M USD

9. Zostavax, Merck, $651M USD

10. RotaTeq®, Merck, $648M USD

11. Synflorix®, GlaxoSmithKline, $587M USD

12. Pneumovax®23, Merck, $580M USD

13. Rotarix, GlaxoSmithKline, $549M USD

14. Adacel, Sanofi Pasteur, $469M USD

15. Prevnar/Prevenar 7, Pfizer, $399M USD

http://www.genengnews.com/insight-and-intelligenceand153/top-15-vaccines-of-2012/77899844/?page=1.

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Aluminum Adjuvants Have a Strong Safety Profile

• Adjuvants used in prophylactic vaccines need to have pristine safety records since they are given to normal, healthy populations

• Primary limitations for use of novel adjuvants: safety issues

• Demonstrated safety profile over seven decades

• Local reactions – Granulomas – Subcutaneous nodules – Erythema – Contact hypersensitivity

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Lindblad E. et al. Safety Evaluation of Vaccine Adjuvants, in Vaccine Adjuvants and Delivery Systems,

Edited by Manmohan Singh, © 2007 John Wiley & Sons, Inc.

A Brief History of Aluminum Adjuvants

Early work/discovery 1920s-1940s

• Development of diphtheria subunit toxoid, weak immunogenicity (Smith, von Behring, 1913)

• Search for agents to potentiate immune response added to tetanus and diphtheria toxoid vaccines (Ramon, 1925)

• First demonstration of the antigenic value of toxoid before and after precipitation with potassium alum (Glenny, 1926)

Broadening use 1940s-1970s

• Clinical studies examining effectiveness (1930s, 40s, and 50s)

Ott, G et al. Development of Vaccine Adjuvants: A Historical Perspective in Vaccine Adjuvants

and Delivery Systems, Edited by Manmohan Singh, © 2007 John Wiley & Sons, Inc.

Material

Dose

injected

Lf units

injected

Number of days after

injection before pigs

became Schick negative

Toxoid “A”

original

1.3 cc 8 units 15, 15, 15, 15

Alum filtrate 4.0 cc 8 units 13, 15, 16, 18

Alum precipitate

emulsion

2.0 cc

1.0 cc

0.1 cc

80 units

40 units

4 units

11, 11, 13, 14

12, 12, 12, 12, 12, 12

12, 14, 14, 14

Toxoid “B”

original

1.0 cc 6 units 18, 27, 27, 40

Alum precipitate

emulsion

1.0 cc

0.1 cc

60 units

6 units

10, 11, 12, 12, 13, 13, 18, 21

14, 14, 17, 17

Table 1.

Antigenic Value of Toxoid Before and After

Precipitation With Potassium Alum

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A Brief History of Aluminum Adjuvants

Physical/chemical characterization 1990s-2000s

• Physical properties (Hem)

• Effects on antigen structure (Middaugh, Carpenter)

New insights into mechanisms of action 2000s

• NLRP3 inflammasome stimulation (Eisenbarth, 2008)

• NLRP3-independent signaling

– PGE2 (Kuroda, 2011)

– Uric acid, cell damage, release of innate immune signals (Kool, 2011)

– Direct membrane binding (Flach, 2011)

Ott, G et al. Development of Vaccine Adjuvants: A Historical Perspective in Vaccine Adjuvants

and Delivery Systems, Edited by Manmohan Singh, © 2007 John Wiley & Sons, Inc.

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What are Aluminum Adjuvants?

Aluminum hydroxide (Al(OH)3)

• Crystalline aluminum oxyhydroxide

• Primary particle: fibers, 4.5 x 2.2 x 10 nm

• Form irregular agglomerates 1-20 mm in diameter

• Positively charged at phys. pH, PZC=11

• e.g., Alhydrogel

Aluminum phosphate (AlPO4)

• Amorphous aluminum hydroxyphosphate

• Primary particles: Plate-like, 50 nm

• Form irregular agglomerates 1-20 mm in diameter

• Negatively charged at phys. pH PZC ~5-7

• e.g., Adju-Phos

Alum (KAl(SO4)·12H20)

• Component of alum-precipitated vaccines

• Aluminum hydroxide that contains some sulfate anions as well as some anions that are used in the buffer (e.g., phosphate)

• PZC depends on precipitation process, usually low (0.3-0.6)

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Baylor NM, Egan W, Richman P. Aluminum salts in vaccines-US perspective. Vaccine. 2002, S18-S23.

Al Al Al O O

OH OH OH

P:Al ~0

Al Al Al

PO4 OH PO4

OH OH

P:Al ~1

Aluminum hydroxide

(Al(OH)3)

Aluminum phosphate

(AlPO4)

Aluminum Adjuvant Analytical Assays

An important part of the vaccine manufacturing and development process

Compendial assays

• Raw material and excipient release

Release assays

• Disposition decisions

Characterization assays

• Inform comparability studies and investigations

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Compendial Testing: Identity, Strength, Quality, Purity

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Compendial Testing: Identity, Strength, Quality, Purity

Raw materials and excipients

• Monographs

– e.g., sodium chloride, aluminum hydroxide

Drug substance or drug product

• General chapters (where applicable)

– e.g., microbial tests

• Microbial enumeration tests USP <61>

– e.g., chemical tests

• pH USP <791>

Ph Eur > USP > JP

• Appropriateness of compendial assay to use as per ICH guidelines (ICH Q4B)

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1. ep7.6, 31Mar2013; 2. USP 36 NF 31 01Dec2013; 3. JP XVI 24Mar2011.

Ph Eur1 USP2 JP3

Aluminum

Hydroxide

Identity: Acid dissolve, base ppt Identity: IR <197K>, Aluminum

<191> (acid dissolve, base ppt)

Identity: Qualitative tests

<1.09> for aluminum salt

Assay: Complexometric titration (2.5.11) Assay: Complexometric titration Assay: Complexometric

titration

Testing: pH (2.2.3), Adsorption power, sedimentation, chlorides (2.4.4),

nitrates, sulfates (2.4.13), ammonium (2.4.1), arsenic (2.4.2), iron

(2.4.9), heavy metals (2.4.8), bacterial endotoxins (2.6.14)

Testing: Acid-neutralizing

capacity <301>, pH <791>,

chloride <221>, sulfate <221>,

arsenic <211>, heavy metals

<231>

Testing: Acidity or alkalinity,

chloride <1.03>, sulfate

<1.14>, nitrate, heavy metals

<1.07>, arsenic <1.11>,

acid-consuming capacity

Aluminum

Phosphate

Identity: Aluminum (2.3.1), phosphates (2.3.1) Identity: Aluminum <191> (acid

dissolve, base ppt)

N/A, no monograph

Assay: Complexometric titration of aluminum, potentiometric end-point

determination (2.2.20)

Assay: phenolphtalein TS-KNO3-

NaOH titration

Testing: pH (2.2.3), Peroxides, chlorides (2.4.4), soluble phosphates,

sulfates (2.4.13), soluble aluminum (2.5.11), arsenic (2.4.2), heavy

metals (2.4.8), acid-neutralizing capacity, residue on ignition, microbial

contamination (2.6.12, 2.6.13)

Testing: pH <791>, Soluble

phosphate, sulfate <221>, arsenic

<211>, heavy metals <231>,

chloride

Alum

Identity: Aluminum (2.3.1), potassium (2.3.1), sulfates (2.3.1) Identity: Aluminum <191>, sulfate

<191>

Identity: Qualitative tests

<1.09> for aluminum salt,

potassium salt, and sulfate

Assay: Complexometric titration of aluminum (2.5.11) Assay: Complexometric titration Assay: Complexometric

titration

Testing: Solution S (appearance is clear (2.2.1) and colorless (2.2.2)),

pH (2.2.3), ammonium (2.4.1), iron (2.4.9), heavy metals (2.4.8)

Testing: Heavy metals <231>,

iron, loss on drying <731>

Testing: Heavy metals

<1.07>, iron <1.10>, arsenic

<1.11>

Aluminum Adjuvant Release Assays

Identity

• P:Al ratio

– ICP-AES

Strength

• Aluminum content

– ICP-AES

Quality

• pH

• Appearance

Purity

• Sterility

– Filtration sterility

• Endotoxin

– LAL test

• Sodium borate

– ICP-AES

• Sodium chloride

– Titration

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Aluminum Adjuvant Characterization Assays

• Particle size by laser diffraction

– Case study: Design of experiments (DoE) around manufacturing parameters

• Surface charge by point of zero charge (PZC)

– Case study: pH excursion during manufacturing

• Adsorption capacity (AdCap)

• Hydrodynamic properties by sedimentation

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Characterization Assay: Particle Size by Laser Diffraction

Instrument

• Malvern© Mastersizer 2000™

– Size limits: 20 nm-2000 mm

• Advantages

– Large size range

– Easy to use

• Disadvantages

– Low throughput

– Moderate sample volume requirements

– No morphology information

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Characterization Assay: Particle Size by Laser Diffraction

How does it work?

• Sample preparation

– Sample is dispersed to the correct concentration and delivered to optical bench

• Measurement

– Measurement of scattering pattern (angle, intensity) of samples that pass through analyzer beam

• Data analysis

– Raw data (angle, intensity) is fit to scattering model (Mie theory) and converted to particle size distribution

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Characterization Assay: Particle Size by Laser Diffraction

Critical parameters

• Sample preparation – Dilution effects – Sample handling – Degassing of diluent

• Measurement – Sample mixing – measurement parameters

• Standardization of testing – Gentle, robust – Assess operator differences

• Data analysis – Number-based vs volume-based

distributions

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Characterization Assay: Particle Size by Laser Diffraction Case Study (DoE)

• DoE around manufacturing parameters (eg, tank size, pH titration addition rate, pH titration hold time, mix speed)

• Assessed comparability with historical data

Analytical

• Noted operator differences

– One analyst tested historical samples to set comparability alert limits

– Two analysts tested DoE samples, noted differences in particle size

• Sample introduction, sample handling

• Led to method standardization

Process

• After method standardization, differences still present for one parameter (tank size)

– Hypothesis

• Tank size affects efficiency of mixing, particle size

– Path forward

• Used particle sizing data to help determine manufacturing parameters going into comparability

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Characterization Assay: Surface Charge by PZC

PZC

• Defines the pH at which a substance is electrically neutral

• Analogous to isoelectric point (pI) of a protein

Instrument

• Malvern© Zetasizer Nano ZS®

Zetasizer Nano User Manual, 4.0, May2008.

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Characterization Assay: Surface Charge by PZC

How does it work?

• Determine electrophoretic mobility by laser Doppler Velocimetry

• Calculate zeta potential by applying the Henry equation

• Determine zeta potential at different pH

• PZC is pH at which zeta potential is 0

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Zetasizer Nano User Manual, 4.0, May2008

Characterization Assay: Surface Charge by PZC

• Surface properties (eg, PZC) determined by chemical composition

– P:Al ratio

• PZC can be used for adjuvant selection

– Electrostatic attraction

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RinellaJV, WhiteJL, HemSL. (1998) Effect of pH on the Elution

of Model Antigens from Aluminum-containing Adjuvants.

J Col & Interface Sci, 205-161-165.

Al Al Al O O

OH OH OH

Al Al Al

PO4 OH PO4

OH OH

P:Al ~0

P:Al ~1

Aluminum hydroxide (Al(OH)3)

Aluminum phosphate (AlPO4)

Characterization Assay: Surface Charge by PZC Case Study (pH)

Analytical

• Drifting pH of buffers

– Inaccurate PZC values

– Failure of comparability

• Assay change, measure pH of buffers day of measurement

Process

• Manufacturing DoE study

• One sample had a pH excursion during manufacturing (high pH)

• Sample impact confirmed by PZC assay

– Historical PZC range: 6.9-7.4

– Sample PZC out of historical range: 5.5

• Result: exclusion of sample from DoE study

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Characterization Assay: Adsorption Capacity

Adsorption capacity

• Mass of protein that can adsorb per unit mass of adjuvant

• µg BSA/µg Al

Related to

• Surface area

• Surface charge

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BSA on AlOH

Seeber.Hem. Predicting the adsorption of proteins by aluminum

containing adjuvants. Vaccine9:201. Mar1991.

Characterization Assay: Adsorption Capacity

How does it work?

• Mix protein and aluminum adjuvant

– Binding a series of BSA dilutions to a fixed concentration of aluminum adjuvant, incubate

• Measure unbound protein

– Spin down, collect supt (unbound protein), measure by BCA assay

• Langmuir Isotherm

• Linear Langmuir Isotherm

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BSA on AlOH

Characterization Assay: Hydrodynamic Properties by Sedimentation

Settling velocity

• Rate at which suspended particles settle in still fluid

• Measure change in bed height over a set period of time (24 hr)

• Factors affecting settling velocity highlighted by Stokes’ law

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Loaded sample (10 cm mark)

T=0 hours Wintrobe tube

Settled height of adjuvant sample.

T=24 hr

Measuring Change (Δ) in Bed Height

w = settling velocity

= density, with subscripts (p) for particle and (f) for fluid

g = acceleration due to gravity

r = radius of particle

m = dynamic viscosity of the fluid

Emerging Orthogonal Characterization Assays

LUMiSizer

• Size/hydrodynamic properties

Micro flow imaging (MFI)

• Size/imaging

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LUMiSizer: Size and Hydrodynamic Properties

• Analytical centrifugation with light transmittance readout

– Measure up to 12 samples simultaneously

– Particle size 20 nm-100 µm

• Dispersion fingerprints

– Sedimentation velocity

– Particle size distribution

• Potential uses/applications

– Rapid screening tool

• Damage due to freezing or agitation

• Product consistency

– Similar trends seen with particle size by laser diffraction

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http://www.lumamericas.com.

Bottom

Sedimentation

0

T = 260 s Top

130

Tra

nsm

issio

n [%

]

50

Position [mm]

Image

Analysis

MFI: Size and Visualization

Instrument:

• ProteinSimple® DPA 4100 Particle Analyzer

Technique:

• Imaging and analysis of particles as liquid passes through a flow cell

Measures particles by

• Sample is drawn through a precision flow cell

• Images are acquired (multiple frames/second)

• All particles in frame are detected and measured

Size range

• 2 to 400 µm

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http://www.proteinsimple.com/mfi_overview.html.

Summary

• Aluminum adjuvants are critical components of life-saving vaccines

• Aluminum adjuvants have a demonstrated track record of safety and efficacy

• Analytical assays are an important part of the vaccine manufacturing and development process – Compendial assays

• Raw material and excipient release – Release assays

• Disposition decisions – Characterization assays

• Inform comparability studies and investigations

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Acknowledgements

• Merck Research Laboratories – Vaccine Analytical Development

• Amy Gallagher, Bettiann Waldner, Chris Hamm, Van Hoang, John MacNair, Liman Wang

– Vaccine Drug Product Development • David Thiriot, Bill Smith, Pat Ahl, Chris Mensch,

Harrison Davis – Raw Material Release

• Carol Thomas

• Merck Manufacturing Division – Leo Xu, Korie Jones, Garry Takle

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