2003 Seid Lyocycledevelopment

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 n

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 n

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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 n

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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 n

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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. 5

Preparation of saccharide-CRM197 conjugate vaccine H2N

+

Periodate Oxidation

NH2 CRM197

H2N

NH2

Reductive Amination NaCNBH3 NaBH4 CRM197

CRM197

CRM197 6

OH

CRM197

OH

OH

+ OH

OH

H2N

NH2 CRM197

H2N

NH2

Characterization and control of critical process steps in glycoconjugate vaccine production n

Polysaccharide Activation Degree of activation (colorimetric assays) Molecular size (SEC-MALLS) Critical substituent groups, e.g. o-acetyl or pyruvyl (NMR, colorimetric

assays) n

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) 7

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6B

9V

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18C 19F

23F

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 z

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

Evaluate accelerated (and stress) and real-time data 9

Liquid formulation challenges for multivalent glycoconjugate vaccine n

Potential factors that could affect stability Chemical instability ƒHydrolysis of saccharide antigens ƒFragmentation of protein carrier Physical instability (process related) ƒAggregation/precipitation ƒAdsorption

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Aggregation of glycoconjugate vaccines n

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 n n

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 n

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 n

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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 14

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 15

Holding Tank

Lyophilization of glycoconjugate vaccines Advantages n

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Reduced rates of chemical degradations Absence of physical stresses  agitation/shear forces  pH changes  moisture-induced

aggregration n

Less dependence on cold chain storage 16

Disadvantages Economy of processing n Reconstitution steps required n Potential alteration of saccharide and protein conformation due to lyophilization-induced stress n

Major process steps for lyophilization n

Freezing below Tg (i.e., -40 to -60°C)

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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 n

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 n

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

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Considerations for secondary drying n

Shelf temp raised to the highest temperature possible consistent with product stability

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Vacuum level increased to “boil off” remaining water

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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 n

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

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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 n

Collapse Temp. -36oC Freeze drying microscopy

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Ion Mobility -22 oC Electrical resistance

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Glass Transition Temp. Tg’ -38.3 oC Differential scanning calorimetry

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Key parameters in a lyo cycle development n

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Sample Loading

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Primary Drying

Shelf temperature

 Ramp rates

Time

 Temperature -single or

multiple

Freezing

 Hold times

Ramp rate Thermal treatment

- Annealing - Temperature - Hold time

 Vacuum n

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 25

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)

Secondary Drying Time (Hours)

0.2 0.4 0.6 0.4 0.6 0.2 0.6 0.2 0.4 0.4

10 15 20 20 10 15 15 20 10 15

Results from lyo cycle optimization studies n

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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

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Texture Cake volume Pores (Holes) Froth (Foaming)

Crust or Glaze Color Bubbling

Optimized Lyo Cycle

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

% Recovery

100 90 80 70 60 50 A

B

C

D

E

Serotype 29

F

G

H

N=3 lots Mean ± SD

Regulatory aspects of formulation development n

Every facet of Formulation Development is susceptible to regulation Formulation Process ƒ “Guide to Inspections of Lyophilization of Parenterals”

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

Storage/Stability ƒ ICH Guideline “Stability Testing of Biotechnological/Biological Products” ƒ FDA Guideline “Quality of Biotechnological Products: Stability Testing of

Biotechnological/Biological Products”

Container/Closure System ƒ “ FDA Guidance for Industry, Container Closure Systems for Packaging

Human Drugs and Biologics”

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