Professional Documents
Culture Documents
of Multivalent Glycoconjugate
Vaccines
Robert Seid
Senior Director, Formulation Development
Wyeth Vaccines Research
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Factors driving the development of
glycoconjugate vaccines
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Factors driving the development of
glycoconjugate vaccines
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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
7-V
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
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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
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Major process steps for lyophilization
n Secondary drying
“bound” water is removed
vacuum is released and vials are sealed in situ
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Considerations during freezing process
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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
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Poor cake quality observed for a multivalent
pneumococcal glycoconjugate vaccine
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Thermal characterization of multivalent
pneumococcal conjugate vaccine
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Key parameters in a lyo cycle development
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Results from lyo cycle optimization studies
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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)
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Nephelometry
Determination of serotype-specific antigenicity in multivalent conjugate
vaccine formulation
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% Recovery of serotype-specific antigenicity
in multivalent conjugate vaccine formulation
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% Recovery
90
80
70
60
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A B C D E F G H
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Regulatory aspects of formulation
development
n 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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