Formulation Development Process Formulation Development Process of Multivalent Glycoconjugate of Multivalent Glycoconjugate Vaccines Vaccines Robert Seid Senior Director, Formulation Development Wyeth Vaccines Research North Carolina Biotechnology Conference “Technical and Regulatory Issues for the Qualification/Validation of Processes for Biological Product Development” October 2, 2003
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Formulation Development Process Formulation Development Process of Multivalent Glycoconjugate of Multivalent Glycoconjugate Vaccines Vaccines
Robert SeidSenior Director, Formulation DevelopmentWyeth Vaccines Research
North Carolina Biotechnology Conference“Technical and Regulatory Issues for the Qualification/Validation of Processes for Biological Product Development”October 2, 2003
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OutlineOutlinen Introduction
Rationale for glycoconjugate vaccinesProcess overviewPrevnar® vaccine: 7 valent formulationFormulation development roles
n Liquid formulation and issuesChemical and physical factors affecting stabilityMembrane filtration process
n Lyophilization and issuesProcess stepsExample of lyo cycle optimization
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Factors driving the development of Factors driving the development of glycoconjugate vaccinesglycoconjugate vaccines
nCapsular saccharides are important virulence components for several bacterial pathogens that cause serious diseases in infants.
nBacterial saccharide capsules are major protective antigens (vaccine candidates) because antibodies to capsular epitopes promote killing of encapsulated bacteria.
Factors driving the development of Factors driving the development of glycoconjugate vaccinesglycoconjugate vaccines
nPolysaccharides themselves are poor at stimulating an effective antibody response in the highest risk age groups (infants).
nCoupling T-cell independent saccharides to a T-cell dependent protein allows the infant immune system to provide T-cell help to B-cells to produce a boostable IgG antibody of high affinity to the saccharide antigen.
protein conjugate), against Haemophilus influenzae type b approved and marketed worldwide (1990).
n MeningitecTM, Meningococcal group C conjugate vaccine(Diphtheria CRM197 protein conjugate) is a novel Meningococcal C conjugate vaccine that is approved and marketed in Europe (1999).
n Prevnar®, Pneumococcal 7-valent Conjugate Vaccine(Diphtheria CRM197 protein conjugate) is a novel 7-valent pneumococcal conjugate vaccine approved and marketed worldwide (2000), sold as Prevenar® outside of US.
ImpactMajor reduction in diseases caused by these bacterial pathogens.
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Preparation of saccharidePreparation of saccharide--CRMCRM197197conjugate vaccineconjugate vaccine
CRM197Periodate Oxidation
Reductive AminationNaCNBH3NaBH4
+H2N NH2
H2N NH2
OH
CRM197
CRM197
CRM197CRM197
OH
OH
OHOH
+
CRM197
H2N NH2
H2N NH2
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Characterization and control of critical Characterization and control of critical process steps in glycoconjugate vaccine process steps in glycoconjugate vaccine productionproductionnPolysaccharide Activation
Degree of activation (colorimetric assays)Molecular size (SEC-MALLS)Critical substituent groups, e.g. o-acetyl or pyruvyl (NMR, colorimetric assays)
nSaccharide-Protein ConjugateSaccharide:protein ratio (colorimetric assays)Free sugar (physical separation and colorimetric assays)Free protein (SEC-HPLC)Molecular size distribution (size exclusion chromatography)Freedom from conjugate chemicals (colorimetric assays)Protein modification (amino acid analysis)
44 6B6B 9V9V 1414 18C18C 19F19F 23F23F
77--VV
The conjugates are mixed and formulated with AlPO4
Each type of polysaccharide conjugated individually to the CRM197 protein carrier
Large scale fermentation and purification of saccharide
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Formulation development’s rolesFormulation development’s rolesDevelop a formulation that is safe, stable, robust, and cost effective
Define formulation conditions for DS and FP- Using approved excipients
n Select appropriate container/closureCompatibility with productContainer closure integrity Convenience for shipping and storage
• Evaluate storage/stabilityEvaluate accelerated (and stress) and real-time data
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Liquid formulation challenges for multivalent Liquid formulation challenges for multivalent glycoconjugate vaccine glycoconjugate vaccine
nPotential factors that could affect stabilityChemical instability
Hydrolysis of saccharide antigensFragmentation of protein carrier
nParticle removal in the 0.2 to 5.0 µm rangeUSP particulate standards for injectables“Sterilizing grade filter” -- sterile effluent produced when challenged with P. diminuta at 107 organisms per sq cm
nRegulatory requirementsNondestructive integrity tests included in batch records- bubble point test- diffusion (pressure hold) test
Information on extractables
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Filter capacity and scaleFilter capacity and scale--upup
n Capacity -- process fluid volume fed to filter before exceeding the differential pressure drop limit (i.e., 20 psi)
n Trial runs on small area filter disc, then scaled-up to 10” cartridge
Flow decayVmax trials (modified flow decay test)
n Scale up equation:Volume cartridge will process = [Cartridge filter area / Disc filter area] X Volume fed to Disc
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From small scale to large scale From small scale to large scale filtrationfiltration
Results from small scale studies on 47mm filter disc: • Consistent volume output capacity• Consistent protein recovery• Low filter extractables• 100% integrity tested
Formulation process for manufacture:
Blend Tank Holding Tank
4x10” Cartridges
2x10” Cartridgesin series
to fill
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Lyophilization of glycoconjugate vaccinesLyophilization of glycoconjugate vaccines
saccharide and protein conformation due to lyophilization-induced stress
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Major process steps for lyophilizationMajor process steps for lyophilization
n Freezing below Tg (i.e., -40 to -60°C)n Primary drying
crystallized water removed by sublimation in vacuo
n Secondary drying“bound” water is removedvacuum is released and vials are sealed in situ
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Considerations during freezing process Considerations during freezing process
Freezing can induce stress:Concentration of active product(s) and
excipients occursIonic strength increasesExcipients can crystallize or precipitate out pH can shift dramatically
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pH Shift during freezing of citric acidpH Shift during freezing of citric acid--disodiumdisodiumphosphate buffer systemphosphate buffer system
thawing
freezing
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Considerations for primary dryingConsiderations for primary drying
n Sublimation process Product temperature needs to be below collapse temperature
n Vacuum conditionsHigh vacuum to be avoided Vacuum usually set at 10-30% of the vapor pressure of ice in the frozen product
n End of primary dryingProduct attain the same temperature as the shelf temperature
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Considerations for secondary dryingConsiderations for secondary drying
n Shelf temp raised to the highest temperature possible consistent with product stability
n Vacuum level increased to “boil off” remaining watern Endpoint determined by the level of moisture desired
General moisture range is 0.5 to 3% w/wProduct stability should be evaluated at different residual moisture levels
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Poor cake quality observed for a multivalent Poor cake quality observed for a multivalent pneumococcal glycoconjugate vaccinepneumococcal glycoconjugate vaccine
n Freezing ramp, extremely fastAmorphous phase with high water concentration- Decrease in collapse temperature
Formation of very small ice crystals- Slowing of sublimation in the primary drying phase
n Primary drying above collapse temperature–100C versus –340C
n Secondary drying longer than primary drying
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Thermal characterization of multivalent Thermal characterization of multivalent pneumococcal conjugate vaccinepneumococcal conjugate vaccine
n Collapse Temp. -36oCFreeze drying microscopy
n Ion Mobility -22 oCElectrical resistance
n Glass Transition Temp. Tg’ -38.3 oCDifferential scanning calorimetry
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Key parameters in a lyo cycle developmentKey parameters in a lyo cycle development
nSample LoadingShelf temperatureTime
nFreezingRamp rateThermal treatment
- Annealing- Temperature- Hold time
n Primary DryingRamp ratesTemperature -single or multipleHold timesVacuum
n Secondary Drying Ramp ratesTemperature -single or multipleHold timesVacuum
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Lyo cycle optimizationLyo cycle optimizationFractional factorial on 4 factors and 3 levelsFractional factorial on 4 factors and 3 levels
Nephelometry Nephelometry Determination of serotypeDetermination of serotype--specific antigenicity in multivalent conjugate specific antigenicity in multivalent conjugate vaccine formulationvaccine formulation
Antigen and antibody are mixed in solution. Aggregates form andAntigen and antibody are mixed in solution. Aggregates form andscatter light. The increase in scattered light is proportional scatter light. The increase in scattered light is proportional to to antigen concentration.antigen concentration.
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% Recovery of serotype% Recovery of serotype--specific antigenicity specific antigenicity in multivalent conjugate vaccine formulationin multivalent conjugate vaccine formulation
50
60
70
80
90
100
A B C D E F G H
Serotype
% R
ecov
ery
N=3 lotsMean ± SD
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Regulatory aspects of formulation Regulatory aspects of formulation developmentdevelopment
n Every facet of Formulation Development is susceptible to regulation
Formulation Process “Guide to Inspections of Lyophilization of Parenterals”
Choice of Excipient21CFR §210.3(b)821CFR §201.11721CFR §210.3(b)(3)
Storage/StabilityICH Guideline “Stability Testing of Biotechnological/Biological Products”FDA Guideline “Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products”
Container/Closure System“ FDA Guidance for Industry, Container Closure Systems for Packaging Human Drugs and Biologics”