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.

Complement mediated lysisComplement mediated phagocytosis

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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.

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Wyeth glycoconjuate vaccine Wyeth glycoconjuate vaccine product profileproduct profilen HibTiter™ Haemophilus b Conjugate Vaccine (Diphtheria CRM197

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

Physical instability (process related)Aggregation/precipitationAdsorption

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Aggregation of glycoconjugate vaccinesAggregation of glycoconjugate vaccines

nPhysical stressesPumping during bulk transfer Agitation during mixingFreezing and thawing cycles

nAdsorption onto hydrophobic surfaceLiquid/solid and water/air interfacial interactionLocal protein concentration can be 1000 X higher

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Strategies to manage protein adsorptionStrategies to manage protein adsorption

n Change containern Change formulation:

Addition of excipients- Surfactants- Stabilizers- Polymers- Amino acidsFormulate at higher dose and deliver with alternate dilution scheme

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Filtration considerationsFiltration considerations

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

Advantagesn Reduced rates of

chemical degradations n Absence of physical

stressesagitation/shear forcespH changesmoisture-induced aggregration

n Less dependence on cold chain storage

Disadvantagesn Economy of

processingn Reconstitution steps

requiredn Potential alteration of

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

Fractional Factorial Design

Expt. Number

Primary Drying

Temperature (oC)

Primary Drying Time

(Hours)

Ramp Rate (oC/minute)

Secondary Drying Time

(Hours)

1 -35 30 0.2 10 2 -35 36 0.4 15 3 -35 42 0.6 20 4 -30 30 0.4 20 5 -30 36 0.6 10 6 -30 42 0.2 15 7 -25 30 0.6 15 8 -25 36 0.2 20 9 -25 42 0.4 10

10 -30 36 0.4 15

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Results from lyo cycle optimization studiesResults from lyo cycle optimization studies

n A rational design of experiments led to the development and optimization of an efficient lyo cycle.

n The length of the proposed lyo cycle was dependent on the primary drying temperature.

n The induction of thermal treatment and annealing appears to be a critical step, leading to elegant cake cosmetics.

n Scale-up lyo runs gave good cake appearance, low moisture level, as well as acceptable stability at 2-8°and RT.

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A comparison of lyophilized cakesA comparison of lyophilized cakes

Optimized Lyo CycleOriginal Lyo Cycle

Cake PropertiesShrinkage (Collapse) Texture Crust or GlazeMeltback Cake volume ColorCracks Pores (Holes) BubblingPuffing Froth (Foaming)

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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”