Characterization of Glycoconjugate Vaccines from a Regulatory Perspective

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1 Characterization of Glycoconjugate Vaccines from a Regulatory Perspective Willie F. Vann, Chief Laboratory of Bacterial Polysaccharides CBER/FDA Bethesda, Maryland USA

2 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

3 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

4 The Most Common Serotypes Causing Invasive Pneumococcal Disease (IPD) Globally

5 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

6 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

7 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

8 Goals of Characterization Ensure consistency of product potency and purity Ensure product safety Tools to assess stability of product

9 Key Features to Characterize Polysaccharide Vaccines Molecular Size Chemical Identity Chemical Structure Repeat structure Labile epitopes

10 Determination of Molecular Size Gel filtration on Sepharose 4B HPLC SEC Multiangle Laser Light Scattering (MALLS)

11 Gel filtration of Pn5 CPs on a Sepharose CL-4B 150 K D < X 100 V T min

12 Composition and Identity Composition and identity by NMR Colorimetric and chromatographic assays Identity by serological assays

13 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,

14 Key Features to Characterize Polysaccharide Vaccines Molecular Size Chemical Identity Chemical Structure Repeat structure Labile epitopes

15 Type 4 Type 9V Labile Epitopes Type 5

16 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

17 Glycoconjugate Vaccines Are More Complex than Polysaccharide Vaccines Starting material Polysaccharide or oligosaccharide Activated sugars Conjugate to protein carrier Conjugate

18 Relationship of Characterization to Structure Single point oligosaccharide Easiest Random Lattice Most Difficult

19 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

20 Goals of Characterization Ensure consistency of product potency and purity Ensure product safety Tools to assess stability of product

21 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

22 Characterization of Intermediates Polysaccharide Purity Chemical Identity Carrier Protein Molecular distribution Consistency

23 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

24 Characterization of Carrier Protein Purity HPLC SDS-PAGE Antigenic purity (Ex. Lf/mg N) Physical State SEC Chromatography Mass spectrometry Presence of Contaminating Saccharides

25 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

26 Measurement of conjugation Extent of coupling Saccharide/Protein Ratio Assay for unique conjugation moiety Free saccharide

27 Degree of Activation Direct assessment Reductive amination- aldehydes per saccharide Indirect assessment Hydroxyl activation Hydroxyl substitution per saccharide

28 Determination of Free Saccharide

29 Chromatographic Size exclusion Reverse phase Proof of conjugation SDS PAGE and/or Western Blot ELISA

30 Proof of Conjugation by PAGE 2. Unconjugated protein 4. and 5. Polysaccharide-protein conjugate SDS-PAGE Western Blot with polysaccharide specific antibody

31 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.

32 Characterization of Vaccine Identity of components Presence of important epitopes Amount of conjugate Free saccharide Stability

33 Type 4 Type 19F Type 5 Formulated Vaccine is a Multivalent Mixture Containing Conjugates with Diverse Chemistry

34 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.

35 Characterization of Vaccine Amount of conjugate Assay must distinguish free from conjugated saccharide Antibody based assay Chemical assays Free saccharide

36 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

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38 Acknowledgements Laboratory of Bacterial Polysaccharides, DBPAP, OVRR, CBER