Upload
duonglien
View
318
Download
10
Embed Size (px)
Citation preview
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.
2
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.
3
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
4
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
5
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.
6
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)
7
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
8
Compendial Testing: Identity, Strength, Quality, Purity
9
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)
10
11
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
12
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
13
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
14
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
15
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
16
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
17
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.
18
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
19
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
20
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
21
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
22
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
23
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
24
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
25
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
26
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
27
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
28
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
29