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Formulation Development Process of Multivalent Glycoconjugate 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

Outline
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Introduction
Rationale for glycoconjugate vaccines Process overview Prevnar® vaccine: 7 valent formulation Formulation development roles

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Liquid formulation and issues
Chemical and physical factors affecting stability Membrane filtration process

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Lyophilization and issues
Process steps Example of lyo cycle optimization

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Factors driving the development of glycoconjugate vaccines
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Capsular saccharides are important virulence components for several bacterial pathogens that cause serious diseases in infants. Bacterial saccharide capsules are major protective antigens (vaccine candidates) because antibodies to capsular epitopes promote killing of encapsulated bacteria.
Complement mediated lysis Complement mediated phagocytosis

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Factors driving the development of glycoconjugate vaccines
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Polysaccharides themselves are poor at stimulating an effective antibody response in the highest risk age groups (infants). Coupling T-cell independent saccharides to a Tcell 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 product profile
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HibTiter™ Haemophilus b Conjugate Vaccine (Diphtheria CRM197 protein conjugate), against Haemophilus influenzae type b approved and marketed worldwide (1990). 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). 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. Impact Major reduction in diseases caused by these bacterial pathogens.
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Preparation of saccharide-CRM197 conjugate vaccine
Periodate Oxidation

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H2N
CRM197

NH2 NH2

H2N Reductive Amination NaCNBH3 NaBH4
CRM197

OH

OH H2N
CRM197

CRM197

OH

+
OH OH

NH2 NH2

CRM197
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CRM197

H2N

Characterization and control of critical process steps in glycoconjugate vaccine production
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Polysaccharide Activation
Degree of activation (colorimetric assays) Molecular size (SEC-MALLS) Critical substituent groups, e.g. o-acetyl or pyruvyl (NMR, colorimetric assays)

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Saccharide-Protein Conjugate
Saccharide: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)
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6B

9V

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18C 19F

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Large scale fermentation and purification of saccharide

Each type of polysaccharide conjugated individually to the CRM197 protein carrier

The conjugates are mixed and formulated with AlPO4

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Formulation development’s roles
Develop a formulation that is safe, stable, robust, and cost effective
Define formulation conditions for DS and FP
- Using approved excipients
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Select appropriate container/closure
Compatibility with product Container closure integrity Convenience for shipping and storage

• Evaluate storage/stability
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Evaluate accelerated (and stress) and real-time data

Liquid formulation challenges for multivalent glycoconjugate vaccine
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Potential factors that could affect stability Chemical instability
Hydrolysis of saccharide antigens Fragmentation of protein carrier

Physical instability (process related)
Aggregation/precipitation Adsorption

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Aggregation of glycoconjugate vaccines
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Physical stresses
Pumping during bulk transfer Agitation during mixing Freezing and thawing cycles

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Adsorption onto hydrophobic surface
Liquid/solid and water/air interfacial interaction Local protein concentration can be 1000 X higher

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Strategies to manage protein adsorption
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Change container Change formulation:
Addition of excipients
- Surfactants - Stabilizers - Polymers - Amino acids

Formulate at higher dose and deliver with alternate dilution scheme

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Filtration considerations
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Particle removal in the 0.2 to 5.0 µm range
USP particulate standards for injectables “Sterilizing grade filter” -- sterile effluent produced when challenged with P. diminuta at 107 organisms per sq cm

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Regulatory requirements
Nondestructive integrity tests included in batch records - bubble point test - diffusion (pressure hold) test Information on extractables

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Filter capacity and scale-up
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Capacity -- process fluid volume fed to filter before exceeding the differential pressure drop limit (i.e., 20 psi) Trial runs on small area filter disc, then scaled-up to 10” cartridge
Flow decay Vmax trials (modified flow decay test)

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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 filtration
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:

2x10” Cartridges in series 4x10” Cartridges to fill Blend Tank
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Holding Tank

Lyophilization of glycoconjugate vaccines
Advantages
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Disadvantages
Economy of processing n Reconstitution steps required n Potential alteration of saccharide and protein conformation due to lyophilization-induced stress
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Reduced rates of chemical degradations Absence of physical stresses
agitation/shear forces pH changes moisture-induced aggregration

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Less dependence on cold chain storage
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Major process steps for lyophilization
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Freezing below Tg (i.e., -40 to -60°C) Primary drying
crystallized water removed by sublimation in vacuo

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Secondary drying
“bound” water is removed vacuum is released and vials are sealed in situ

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Considerations during freezing process
Freezing can induce stress:
Concentration of active product(s) and excipients occurs Ionic strength increases Excipients can crystallize or precipitate out pH can shift dramatically

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pH Shift during freezing of citric acid-disodium phosphate buffer system

freezing

thawing

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Considerations for primary drying
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Sublimation process
Product temperature needs to be below collapse temperature

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Vacuum conditions
High vacuum to be avoided Vacuum usually set at 10-30% of the vapor pressure of ice in the frozen product

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End of primary drying
Product attain the same temperature as the shelf temperature

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Considerations for secondary drying
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Shelf temp raised to the highest temperature possible consistent with product stability Vacuum level increased to “boil off” remaining water Endpoint determined by the level of moisture desired
General moisture range is 0.5 to 3% w/w Product stability should be evaluated at different residual moisture levels

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Poor cake quality observed for a multivalent pneumococcal glycoconjugate vaccine
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Freezing ramp, extremely fast

Amorphous 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
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Secondary drying longer than primary drying

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Thermal characterization of multivalent pneumococcal conjugate vaccine
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Collapse Temp. -36oC Freeze drying microscopy Ion Mobility -22 oC Electrical resistance Glass Transition Temp. Tg’ -38.3 oC Differential scanning calorimetry

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Key parameters in a lyo cycle development
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Sample Loading
Shelf temperature Time

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Primary Drying
Ramp rates Temperature -single or multiple Hold times Vacuum

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Freezing
Ramp rate Thermal treatment

- Annealing - Temperature - Hold time

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Secondary Drying
Ramp rates Temperature -single or multiple Hold times Vacuum

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Lyo cycle optimization Fractional factorial on 4 factors and 3 levels
Fractional Factorial Design
Expt. Number 1 2 3 4 5 6 7 8 9 10
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Primary Drying Temperature (oC) -35 -35 -35 -30 -30 -30 -25 -25 -25 -30

Primary Drying Time (Hours) 30 36 42 30 36 42 30 36 42 36

Ramp Rate (oC/minute) 0.2 0.4 0.6 0.4 0.6 0.2 0.6 0.2 0.4 0.4

Secondary Drying Time (Hours) 10 15 20 20 10 15 15 20 10 15

Results from lyo cycle optimization studies
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A rational design of experiments led to the development and optimization of an efficient lyo cycle. The length of the proposed lyo cycle was dependent on the primary drying temperature. The induction of thermal treatment and annealing appears to be a critical step, leading to elegant cake cosmetics. 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 cakes
Cake Properties
Shrinkage (Collapse) Meltback Cracks Puffing
Original Lyo Cycle

Texture Cake volume Pores (Holes) Froth (Foaming)

Crust or Glaze Color Bubbling

Optimized Lyo Cycle

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Nephelometry

Determination of serotype-specific antigenicity in multivalent conjugate vaccine formulation
Antigen and antibody are mixed in solution. Aggregates form and scatter light. The increase in scattered light is proportional to antigen concentration.

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% Recovery of serotype-specific antigenicity in multivalent conjugate vaccine formulation
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% Recovery

90 80 70 60 50 A B C D E F G H

Serotype
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N=3 lots Mean ± SD

Regulatory aspects of formulation development
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Every facet of Formulation Development is susceptible to regulation Formulation Process
“Guide to Inspections of Lyophilization of Parenterals”

Choice of Excipient
21CFR §210.3(b)8 21CFR §201.117 21CFR §210.3(b)(3)

Storage/Stability
ICH 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”

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