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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
n Introduction
Rationale for glycoconjugate vaccines
Process overview
Prevnar® vaccine: 7 valent formulation
Formulation development roles
n Liquid formulation and issues
Chemical and physical factors affecting stability
Membrane filtration process
n Lyophilization and issues
Process steps
Example of lyo cycle optimization

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Factors driving the development of
glycoconjugate vaccines

n Capsular saccharides are important virulence


components for several bacterial pathogens that
cause serious diseases in infants.
n 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 Polysaccharides themselves are poor at


stimulating an effective antibody response in
the highest risk age groups (infants).
n Coupling 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
product profile
n 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.

Impact
Major reduction in diseases caused by these bacterial
pathogens.
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Preparation of saccharide-CRM197
conjugate vaccine
H2N NH2
Periodate Oxidation + CRM197

H2N NH2

Reductive Amination
NaCNBH3
NaBH4

CRM197
OH
OH
CRM197 OH +
H2N NH2
OH
CRM197
OH CRM197
CRM197
H2N NH2
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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)
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4 6B 9V 14 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
n 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
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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
n 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 Change container
n 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
n 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 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 decay
Vmax 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
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 Holding Tank


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

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Major 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 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
n 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
n Vacuum level increased to “boil off” remaining water
n 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
n Primary drying above collapse temperature
 –100C versus –340C
n Secondary drying longer than primary drying

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Thermal characterization of multivalent
pneumococcal conjugate vaccine

n Collapse Temp. -36oC


Freeze drying microscopy
n Ion Mobility -22 oC
Electrical resistance
n Glass Transition Temp. Tg’ -38.3 oC
Differential scanning calorimetry

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Key parameters in a lyo cycle development

n Sample Loading n Primary Drying


Shelf temperature  Ramp rates
Time  Temperature -single or
multiple
n Freezing  Hold times
Ramp rate  Vacuum
Thermal treatment
- Annealing
n Secondary Drying
- Temperature  Ramp rates
- Hold time  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. Primary Primary Ramp Rate Secondary
Number Drying Drying (oC/minute) Drying Time
Temperature Time (Hours)
(oC) (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 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 cakes

Cake Properties
Shrinkage (Collapse) Texture Crust or Glaze
Meltback Cake volume Color
Cracks Pores (Holes) Bubbling
Puffing Froth (Foaming)

Original Lyo Cycle 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

100
% Recovery

90

80

70

60

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A B C D E F G H

Serotype N=3 lots


Mean ± SD

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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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