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Pneumococcal Conjugate Vaccines Manufacturing By- Disha ,Kanika and Teena |
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Introduction Conjugate vaccines are produced on the principle that certain bacteria have polysaccharide outer coats that are poorly immunogenic especially in children below 2 years of age. By linking these outer coats to proteins (e.g. toxins), the immunogenicity of the polysaccharide is amplified.
E.g.; pneumococcal vaccines, Haemophilus influenzae type B vaccine. |
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Streptococcus pneumoniae Streptococcus pneumoniae, is a Gram-positive, alpha-hemolytic, aerotolerant anaerobic member of the genus Streptococcus.
This bacterium, S. pneumoniae was recognized as a major cause of pneumonia in the late 19th century, and is the subject of many humoral immunity studies.
Other pneumococcal diseases include acute sinusitis, otitis media, meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, and brain abscess. |
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Polysaccharide Streptococci have a polysaccharide capsule that acts as a virulence factor for the organism.
Resistance to phagocytosis is mediated by the polysaccharide capsule that forms a hydrophilic gel on the surface of the organism.
This gel shields the bacterium from antibodies and complement proteins. In addition, capsular sialic acid contributes to the anti phagocytic effect by inhibiting complement amplification and alternative pathway activation.
Intrinsic complement inactivation mechanisms, which degrade C3b bound to the bacterial surface and prevent further C3 deposition, are also facilitated by capsular sialic acid. |
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Of the 90 capsular types identified, only a few are common causes of pneumococcal disease. The seven most commonly isolated serotypes cover up to 85% of all pneumococcal strains causing invasive infections in children.
The available pneumococcal polysaccharide vaccines are composed of various pneumococcal serotypes, ranging from 7-23. They represent 85/90% of the serotypes that cause invasive infections in adults in industrialized countries.
Electron micrograph of Streptococcus pneumoniae and the associated pneumococcal capsular polysaccharide (labelled 6). The bacteria shows the typical diplococcus morphology of the pneumococcus.
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Carrier proteins A variety of proteins, including bacterial pilli, outer membrane proteins (OMPs), and excreted toxins of pathogenic bacteria, preferably in toxoid form, have been employed as carriers for carbohydrate antigens.
Most popular as carrier proteins are tetanus and diphtheria toxoids
Modified forms of bacterial toxoids have been developed, such as CRM197, a nontoxic analog of diphtheria toxoid.
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Protein-saccharide Linkage |
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Production: Overview |
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Toxoid bought from external source |
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Culturing of S. pneumoniae |
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Polysaccharide extraction |
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Polysaccharide extraction |
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Quality Control |
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Quantification of extracted polysaccharide |
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Toxoids Bacterial toxoids such as tetanus and diphtheria are mostly used as carrier proteins.
CRM 197 is also widely used and is a genetically toxoided variant.
CRM197 is isolated from cultures of Corynebacterium diphtheriae strain.
The carrier protein delivers a strong T cell involvement in immune response against the polysaccharide.
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Protein-saccharide conjugation Conjugation by reductive amination to the free Ne group of a lysine residue in an appropriate carrier protein. N acetylglucosamine (NAG)of polysaccharide from S. pneumoniae. Partial de – N –acetylation of NAG Treatment with nitrous acid leads to the formation of an anhydromannose residue with a free aldehyde group. Polysaccharides require activation before attachment
to the carrier protein. |
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Considerations for protein-saccharide ratio: The spacing and density of the saccharide on the protein are likely to have major impacts on the ability of the conjugate to induce an immune response.
If a conjugate contains appreciable amounts of free polysaccharide, dose calculations for animal experiments become unreliable.
Long-term storage of a conjugate can lead to partial depolymerization or decoupling of the saccharide antigen, which in turn will affect the saccharide-to-protein ratio.
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With high saccharide-to-protein ratios, essential carrier epitopes may be hidden from the immune system, preventing recognition of the conjugate as a T cell dependent antigen.
It is imperative that an optimal conjugation scheme be determined for each particular saccharide and protein combination.
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Stability studies and QC |
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Polysaccharide or
Oligosaccharide • Polysaccharide-protein ratio (NMR, HPAEC)
• Polysaccharide structure (NMR)
• Protein structure (circular dichroism)
• Carrier protein stability (CD, fluorescence)
• Stability of the glycan
(NMR, SEC-MALLS, free saccharide)
• Free saccharide (HPAEC)
• Location of the glycan chains on carrier
(HPLC-ESI-MS-MS) • Identity/structure (ESMS, PAGE, CD)
• Stability (ESMS, CD, fluorescence) Carrier
Protein • Identity/structure (NMR)
• Purity (NMR)
• Stability (NMR, SEC-MALLS)
• Size (SEC-MALLS, IEX, NMR) Polysaccharide-Protein
conjugate Coupling chemistry |
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Final bulk Polyvalent conjugate bulk Filling in containers Add preservatives
(ex : phenoxyethanol and formaldehyde) Add Stablizers
( ex : thimerosal) Sterility studies
( check bacterial and mycotic sterility) |
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Control tests on final product Identity Sterility Polysaccharide content Moisture content Endo-
toxin content Preserva- tives general safety tests pH Inspection of final containers |
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Final product The vaccine thus produced and formulated is ready for distribution, after proper labeling .
The label should contain appropriate information pertaining to the storage , stability and expiration of the vaccine.
The vaccine then proceeds to the processes of in vivo testing to ascertain safety, efficacy and immunogenicity.
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