Characterization of Glycoconjugate Vaccines from a Regulatory Perspective Willie F. Vann, Chief Laboratory of Bacterial Polysaccharides CBER/FDA Bethesda, Maryland USA
Many Pathogenic Bacteria are Encapsulated with Polysaccharides Capsule Haemophilus, E. coli, Neisseria Pneumococcus, Staphylococcus Serotype specific polysaccharides - Shields bacteria against phagocytosis by host Successful targets for vaccine development
Capsular Polysaccharides as Vaccine Targets Serotypes are chemically distinct and defined polysaccharide structures In most cases only a few serotypes are associated with most of the disease Most meningococcal disease associated with 5 of 13 serotypes A, B, C, Y, W135 Most of global pneumococcal disease is associated with less than 17% of the 90 serotypes Serotypes are stably expressed by pathogen
The Most Common Serotypes Causing Invasive Pneumococcal Disease (IPD) Globally
Why Glycoconjugate Vaccines? Polysaccharide vaccines introduced in the 1970s short-lived protection in adults not effective in children < 2 years old Conjugation of saccharides to proteins recruits CD4 + T cell help protective, high titer IgG anti-polysaccharide responses immunologic memory immunogenicity in infants
US Licensed Polysaccharide-Based Vaccines Polysaccharides Pneumococcal 23-valent polysaccharide Manufacturer Merck Meningococcal ACYW 135 polysaccharide Sanofi Vi Polysaccharide Sanofi Glycoconjugates Haemophilus type b Meningococcal ACYW 135 Pneumococcal 7-valent Pneumococcal 13-valent Meningococcal CY-Haemophilus type b Sanofi, Merck, GSK Sanofi, Novartis Pfizer Pfizer GSK
US Licensed Polysaccharide-Based Vaccines Polysaccharides Pneumococcal 23-valent polysaccharide Manufacturer Merck Meningococcal ACYW 135 polysaccharide Sanofi Vi Polysaccharide Sanofi Glycoconjugates Haemophilus type b Meningococcal ACYW 135 Pneumococcal 7-valent Pneumococcal 13-valent Meningococcal CY-Haemophilus type b Sanofi, Merck, GSK Sanofi, Novartis Pfizer Pfizer GSK
Goals of Characterization Ensure consistency of product potency and purity Ensure product safety Tools to assess stability of product
Key Features to Characterize Polysaccharide Vaccines Molecular Size Chemical Identity Chemical Structure Repeat structure Labile epitopes
Determination of Molecular Size Gel filtration on Sepharose 4B HPLC SEC Multiangle Laser Light Scattering (MALLS)
Gel filtration of Pn5 CPs on a Sepharose CL-4B 150 K D < X 100 V T 50 0-50 0 50 100 150 200 250 min
Composition and Identity Composition and identity by NMR Colorimetric and chromatographic assays Identity by serological assays
Fig. 1. Partial 500 MHz 1H NMR spectra of several CPSs used in vaccines, emphasizing differences in line width between different samples: (a) pneumococcal Type 4; (b) pneumococcal Type 23F CPS; (c) pneumococcal type 17F CPS. All spectra were collected at an indicated probe temperature of 70 deg. Jones, C. (2005) J. Pharm. Biomed. Anal. 38, 840-50
Key Features to Characterize Polysaccharide Vaccines Molecular Size Chemical Identity Chemical Structure Repeat structure Labile epitopes
Type 4 Type 9V Labile Epitopes Type 5
US Licensed Polysaccharide-Based Vaccines Polysaccharides Pneumococcal 23-valent polysaccharide Manufacturer Merck Meningococcal ACYW 135 polysaccharide Sanofi Vi Polysaccharide Sanofi Glycoconjugates Haemophilus type b Meningococcal ACYW 135 Pneumococcal 7-valent Pneumococcal 13-valent Meningococcal CY-Haemophilus type b Sanofi, Merck, GSK Sanofi, Novartis Pfizer Pfizer GSK
Glycoconjugate Vaccines Are More Complex than Polysaccharide Vaccines Starting material Polysaccharide or oligosaccharide Activated sugars Conjugate to protein carrier Conjugate
Relationship of Characterization to Structure Single point oligosaccharide Easiest Random Lattice Most Difficult
Some Characteristics of Licensed Polysaccharide Conjugate Vaccines Randomly coupled oligosaccharide or polysaccharides Protein carriers not always homogenous Structure of the active immunogen unknown Manufacturing consistency dependent entirely on manufacturing process consistency
Goals of Characterization Ensure consistency of product potency and purity Ensure product safety Tools to assess stability of product
Characterization of Individual Conjugates Properties of starting materials Polysaccharide size, structure and identity Carrier protein purity and size Degree of Activation Direct assessment Indirect assessment Proof of conjugation Measurement of conjugation Extent of coupling Free saccharide Characterization of reaction by-products De-activation of reactive intermediates Presence of potentially toxic materials Properties related to effectiveness in humans Molecular size
Characterization of Intermediates Polysaccharide Purity Chemical Identity Carrier Protein Molecular distribution Consistency
Carrier Proteins Used in Licensed Vaccines Tetanus toxoid Partially purified formaldehyde inactivated tetanus toxin CRM197 Purified point mutant of diphtheria toxin Neisseria outer membrane vesicles Diphtheria toxoid Partially purified formaldehyde inactivated diphtheria toxin
Characterization of Carrier Protein Purity HPLC SDS-PAGE Antigenic purity (Ex. Lf/mg N) Physical State SEC Chromatography Mass spectrometry Presence of Contaminating Saccharides
Modification of Tetanus Toxin Hc domain - MALDI-MS to determine number of incorporation sites -Trypsin digestion followed by liquid chromatography and mass spectrometery (LC-MS/MS) to determine positions of incorporation sites - Gold: peptide fragments, blue: modified residues - 9 sites of modification dispersed throughout protein
Measurement of conjugation Extent of coupling Saccharide/Protein Ratio Assay for unique conjugation moiety Free saccharide
Degree of Activation Direct assessment Reductive amination- aldehydes per saccharide Indirect assessment Hydroxyl activation Hydroxyl substitution per saccharide
Determination of Free Saccharide
Chromatographic Size exclusion Reverse phase Proof of conjugation SDS PAGE and/or Western Blot ELISA
Proof of Conjugation by PAGE 2. Unconjugated protein 4. and 5. Polysaccharide-protein conjugate SDS-PAGE Western Blot with polysaccharide specific antibody
Characterization of reaction by-products De-activation of intermediates Destruction activated moiety e.g. reduction Reaction excess small molecule Presence of potentially toxic materials Unreacted activation or coupling reagents Toxic by-products-e.g. cyanide, hydroxysuccinimide, DMSO, etc.
Characterization of Vaccine Identity of components Presence of important epitopes Amount of conjugate Free saccharide Stability
Type 4 Type 19F Type 5 Formulated Vaccine is a Multivalent Mixture Containing Conjugates with Diverse Chemistry
Characterization of Vaccine Identity of components - Specificity Antibody based assays often used to address requirements of specificity Presence of important epitopes Chemical assays for O-acetyl, pyruvate, etc.
Characterization of Vaccine Amount of conjugate Assay must distinguish free from conjugated saccharide Antibody based assay Chemical assays Free saccharide
Characterization as a Probe of Stability Stability indication assays Concentration of conjugate Free polysaccharide Preservation of labile epitopes Pyruvyl groups Ketoses O-acetyl Molecular size Size exclusion MALLS
Acknowledgements Laboratory of Bacterial Polysaccharides, DBPAP, OVRR, CBER