Frasch, Carl

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Engineering Conferences International

Vaccine Technology IISession IV:  Conjugate VaccinesAlbufeira, Portugal      June 1-6, 2008Analytical and Manufacturing Challenges:Preparation of BacterialPolysaccharide ConjugatesCarl E. Frasch, Ph.D.Biologics ConsultantConjugate Vaccines:  Manufacturing and quality controlTopics to be discussed:• Characteristics of a conjugate vaccine• Considerations  in production of a conjugate-- Polysaccharide activation-- Conjugation• Quality control and lot release• Improving conjugation efficiency (yields)Properties of a good polysaccharide-protein conjugate vaccine• Has defined chemical composition and structure• Can be manufactured with consistent physical and chemical characteristics• Is safe  - has no inherent toxicity• Induces high avidity bactericidal or opsonic antibodies• Induces boostable IgG antibody in infants• Primes for response upon natural exposure  to the polysaccharideSome currently licensed bacterial polysaccharideprotein conjugate vaccinesOrganism        Vaccine              First          Saccharide   Protein       Vaccinemanufacturer    Licensed                             carrier          dose (μg)Haemophilusinfluenzae bWyethMerckSanofiGSK1988198919931996Hib oligoHib Sz redHib PSHib PSCRM197OMPCTet ToxTet Tox107.51010Meningococcal WyethBaxterNovartisSanofi1999200020002005C Sz redC De OAcC oligoA,C,Y,W Sz redCRM197Tet ToxCRM197Diph Tox1010104 eachPneumococcal      Wyeth            2000             4, 6B, 9V,        CRM197 214, 18C, 19F                   (4μg 6B)23FMeningo/Hib          GSK                 2005             Hib, MenC      Tet Tox       5 eachMajor physical and chemical variablesin production of glycoconjugate vaccines1. Size of the polysaccharide or oligosaccharide2. Chemistry for activation of the polysaccharide3. Choice of carrier protein4. Saccharide - protein conjugation chemistry5. Saccharide loading onto protein carrier, ie,Saccharide to protein ratioGeneral process for manufacture of a conjugate vaccine Polysaccharide FermentationCarrier protein (toxoid) FermentationPurificationPurification (detoxification)Polysaccharide activationConjugation then purificationConjugate vaccineMaking a conjugateFor a polysaccharide to be chemically linkedto a protein, the polysaccharide must beactivated, that is, chemically modified.Methods include:• Reductive amination• Cyanylation• CarbodiimideActivation ChemistryImportant difference between methods used toactivate the polysaccharide for conjugationPeriodate activation for reductive amination:Activates by cutting carbon-carbon bonds between adjacent carbons having -OH groups to create activealdehyde groupsCyanylationActivtes by randomly changing –OH groupsto active –CN groupsCarbodiimideActivates at carboxyl groups by replacing –OH withcarbodiimidePeriodate oxidation of polysaccharidesImportant lot release tests forpolysaccharide-protein conjugate vaccines• Purity of polysaccharide and carrier protein • Saccharide of known molecular size distribution• Degree of saccharide activation• Molecular size of conjugate (a stability measure)• Vaccine Potency:¾ Polysaccharide to protein ratio in conjugate¾ Percent non-conjugated (free) saccharide present in monovalent conjugate bulksNewer physical methods to analyze polysaccharide component of a conjugate Identity NMR Structure NMR Purity NMR Quantitation              NMR Stability SEC-HPLC, NMR Size SEC-HPLC, MALLS600-MHz proton spectrum of pneumococcaltype 17F polysaccharideAnomeric region Ring region Acetyls MethylsStability indicating quality control tests for monovalent bulks and final vaccine• Change in the molecular size of conjugate during storage• Increase in free (unbound) saccharide over time• Change in conjugate solubility during storage• Change in pH during storage• Change in degree of adsorption, if vaccine adjuvant adsorbedConjugation efficiency (yields)• It is very difficult to discern manufacturing yields• Some manufacturers report high yields based uponrecovery of the carrier protein (not relevant)• Some do not account for losses during activation• Yields can be much higher when both the polysaccharideand protein are activated before conjugationYields:¾ Utilize native amino groups – about 10 to 30%¾ Utilize activated protein       -- about 50 %New Aldehyde Conjugation Chemistry using activated proteins to improve yields1.  Hydrazone formationAldehyde/hydrazide/reduction Benzaldehyde/hydrazideActivates through the carboxyl groups, not aminoHydrazones are the condensation of hydrazide or hydrazine with aldehyde2.  Oxime formationCreates a highly reactive aminooxy groupSchematics for Preparation of Polysaccharide-Tetanus Toxoid Conjugate usingHydrazide ChemistryTetanus toxoid + Hydrazine  → TT-HEDCPolysaccharide + NaIO4 → Activated PS→ ConjugateConjugates made with hydrazide activated tetanus toxoid(TT-H) versus unmodified tetanus toxoid (TT)TTTT-HConjConjSE-HPLCConclusions9 There are several methodologies for manufacture of saccharide-protein conjugate vaccines9 The polysaccharide or oligosaccharide must be chemicallyactivated for conjugation to occur9 It is important to carefully consider the chemistry used forsaccharide acvtivation9 Activation of both the protein and the polysaccharide willimprove conjugation yields9 Quality control and lot release testing are described in WHO Technical Report series publications for Hib, meningococcal and pneumococcal conjugate vaccines

Analytical and Manufacturing Challenges in Preparation of Bacterial Polysaccharide Conjugates

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Analytical and Manufacturing Challenges in Preparation of Bacterial Polysaccharide Conjugates

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