The development of a process and quality control methods for conjugate vaccine against streptococcus pneumoniae serotype 1

Includes abstract.Includes bibliographical references.Pneumonia is the leading cause of death in children worldwide and is estimated to kill 1.6 million children every year. Pneumonia affects children and families everywhere, but is most prevalent in sub–Saharan Africa and South–east Asia. Serotype 1 is responsible for up to 20 % of invasive pneumococcal diseases (IPD) in developing countries and has been the cause of several outbreaks in the African meningitis belt. Conjugate vaccines are effective in young children, induce immunological memory and reduce carriage. A conjugate vaccine against 7 serotypes (PCV7) was licensed in 2000 which resulted in a dramatic reduction of IPD. An increase in the number of cases due to non–vaccine serotypes (serotype replacement) led to the recent development and licensure of 10– and 13– valent conjugate vaccines that provide broader coverage. This thesis describes the development of purification and conjugation processes and associated analytical methods for the preparation of a Streptococcus pneumoniae serotype 1 polysaccharide (Pn1) conjugate vaccine. The Pn1 polysaccharide was purified following a two–step process utilising a differential filtration with ethanol. Analytical tests including size analysis, uronic acid composition, O–acetylation and purity (nucleic acids and protein) were optimized and performed on Pn 1 lots. The purified polysaccharide was found to meet World Health Organisation (WHO) specifications.The purified polysaccharide is viscous with a rigid structure that hampers full conjugation reactions and detailed characterisation. Size–reduction was performed and shown to have no impact on the structural integrity of the generated saccharide. The O–acetylated size–reduced polysaccharide was amenable to full nuclear magnetic resonance (NMR) characterisation to confirm the structural identity of Pn1 and determine the percentage of cell wall polysaccharide (CWPS) and the degree and position of O–acetylation present.Composition analysis was performed using published hydrolysis methods, however, they resulted in low recoveries and therefore alternative microwave assisted conditions were investigated followed by chromatographic separation and analysis. The size–reduced polysaccharide was conjugated to hydrazide–derivatized protein carriers via the polysaccharide carboxyl groups. The conjugates prepared using different activators were evaluated in mice and the immunogenicity data showed that they were non–inferior to two commercially available conjugate vaccines

Salts of S-(+)-Ibuprofen Formed via Its Reaction with the Antifibrinolytic Agents Aminocaproic Acid and Tranexamic Acid: Synthesis and Characterization

The paucity of multi-component compounds containing the non-steroidal anti-inflammatory drug (NSAID) S-(+)-ibuprofen (S-IBU) in combination with other drugs prompted the present study, which describes 1:1 salts of this active pharmaceutical ingredient (API) with the two most widely used antifibrinolytic APIs, namely 6-aminohexanoic acid (aminocaproic acid, ACA) and tranexamic acid (TXA), which are zwitterions in the solid state. Since NSAIDs are known to cause adverse side effects such as gastrointestinal ulceration, the presence of ACA and TXA in the salts with S-(+)-ibuprofen might counter these effects via their ability to prevent excessive bleeding. The salts were prepared by both the liquid-assisted grinding method and co-precipitation and were characterized by X-ray powder diffraction and single-crystal X-ray diffraction, thermal analysis, Fourier transform infrared spectroscopy, and solubility measurements. The X-ray analyses revealed a high degree of isostructurality, both at the level of their respective asymmetric units and in their extended crystal structures, with charge-assisted hydrogen bonds of the type N-H...O and O-H.. O featuring prominently. The thermal analysis indicated that both salts had significantly higher thermal stability than S-(+)-ibuprofen. Solubility measurements in a simulated biological medium showed insignificant changes in the solubility of S-(+)-ibuprofen when tested in the form of the salts (S-IBU) (TXA)

Genetic and structural elucidation of capsular polysaccharides from Streptococcus pneumoniae serotype 23A and 23B, and comparison to serotype 23F

Streptococcus pneumoniae is a globally important encapsulated human pathogen with approximately 100 different serotypes recognized. Serogroup 23 consists of serotype 23F, present in licensed vaccines, and emerging serotypes 23A and 23B. Here, we report the previously unknown structures of the pneumococcal capsular polysaccharides serotype 23A and 23B determined using genetic analysis, NMR spectroscopy, composition and linkage analysis and Smith degradation (of polysaccharide 23A). The structure of the serotype 23A capsular polysaccharide is: \u21924)-\u3b2-D-Glcp-(1\u21923)-[[\u3b1-L-Rhap-(1\u21922)]-[Gro-(2\u2192P\u21923)]-\u3b2-D-Galp-(1\u21924)]-\u3b2-L-Rhap-(1\u2192. This structure differs from polysaccharide 23F as it features a disaccharide backbone and the di-substituted \u3b2-Gal is linked to \u3b2-Rha as a side chain. This is due to the different polymerization position catalysed by the unusually divergent repeat unit polymerase Wzy in the 23A cps biosynthesis locus. Steric crowding in 23A, confirmed by molecular models, causes the NMR signal for H-1 of the di-substituted 2,3-\u3b2-Gal to resonate in the \u3b1-anomeric region. The structure of the serotype 23B capsular polysaccharide is the same as 23F, but without the terminal \u3b1-Rha: \u21924)-\u3b2-D-Glcp-(1\u21924)-[Gro-(2\u2192P\u21923)]-\u3b2-D-Galp-(1\u21924)-\u3b2-L-Rhap-(1\u2192. The immunodominant terminal \u3b1-Rha of 23F is more sterically crowded in 23A and absent in 23B. This may explain the reported typing cross reactions for serotype 23F: slight with 23A and none with 23B

Salts of S-(+)-Ibuprofen Formed via Its Reaction with the Antifibrinolytic Agents Aminocaproic Acid and Tranexamic Acid: Synthesis and Characterization

The paucity of multi-component compounds containing the non-steroidal anti-inflammatory drug (NSAID) S-(+)-ibuprofen (S-IBU) in combination with other drugs prompted the present study, which describes 1:1 salts of this active pharmaceutical ingredient (API) with the two most widely used antifibrinolytic APIs, namely 6-aminohexanoic acid (aminocaproic acid, ACA) and tranexamic acid (TXA), which are zwitterions in the solid state. Since NSAIDs are known to cause adverse side effects such as gastrointestinal ulceration, the presence of ACA and TXA in the salts with S-(+)-ibuprofen might counter these effects via their ability to prevent excessive bleeding. The salts were prepared by both the liquid-assisted grinding method and co-precipitation and were characterized by X-ray powder diffraction and single-crystal X-ray diffraction, thermal analysis, Fourier transform infrared spectroscopy, and solubility measurements. The X-ray analyses revealed a high degree of isostructurality, both at the level of their respective asymmetric units and in their extended crystal structures, with charge-assisted hydrogen bonds of the type N-H+⋅⋅⋅O− and O-H+⋅⋅⋅O− featuring prominently. The thermal analysis indicated that both salts had significantly higher thermal stability than S-(+)-ibuprofen. Solubility measurements in a simulated biological medium showed insignificant changes in the solubility of S-(+)-ibuprofen when tested in the form of the salts (S-IBU)−(ACA)+ and (S-IBU)−(TXA)+

Complexation between the Antioxidant Pterostilbene and Derivatized Cyclodextrins in the Solid State and in Aqueous Solution

Inadequate aqueous solubilities of bioactive compounds hinder their ability to be developed for medicinal applications. The potent antioxidant pterostilbene (PTB) is a case in point. The aim of this study was to use a series of modified water-soluble cyclodextrins (CDs), namely, hydroxypropyl β-CD (HPβCD), dimethylated β-CD (DIMEB), randomly methylated β-CD (RAMEB), and sulfobutyl ether β-CD sodium salt (SBECD) to prepare inclusion complexes of PTB via various solid, semi-solid, and solution-based treatments. Putative CD–PTB products generated by solid-state co-grinding, kneading, irradiation with microwaves, and the evaporative treatment of CD–PTB solutions were considered to have potential for future applications. Primary analytical methods for examining CD–PTB products included differential scanning calorimetry and Fourier transform infrared spectroscopy to detect the occurrence of binary complex formation. Phase solubility analysis was used to probe CD–PTB complexation in an aqueous solution. Complexation was evident in both the solid-state and in solution. Complex association constants (K1:1) in an aqueous solution spanned the approximate range of 15,000 to 55,000 M−1; the values increased with the CDs in the order HPβCD < DIMEB < RAMEB < SBECD. Significant PTB solubility enhancement factors were recorded at 100 mM CD concentrations, the most accurately determined values being in the range 700-fold to 1250-fold

Complexation between the Antioxidant Pterostilbene and Derivatized Cyclodextrins in the Solid State and in Aqueous Solution

Inadequate aqueous solubilities of bioactive compounds hinder their ability to be developed for medicinal applications. The potent antioxidant pterostilbene (PTB) is a case in point. The aim of this study was to use a series of modified water-soluble cyclodextrins (CDs), namely, hydroxypropyl β-CD (HPβCD), dimethylated β-CD (DIMEB), randomly methylated β-CD (RAMEB), and sulfobutyl ether β-CD sodium salt (SBECD) to prepare inclusion complexes of PTB via various solid, semi-solid, and solution-based treatments. Putative CD–PTB products generated by solid-state co-grinding, kneading, irradiation with microwaves, and the evaporative treatment of CD–PTB solutions were considered to have potential for future applications. Primary analytical methods for examining CD–PTB products included differential scanning calorimetry and Fourier transform infrared spectroscopy to detect the occurrence of binary complex formation. Phase solubility analysis was used to probe CD–PTB complexation in an aqueous solution. Complexation was evident in both the solid-state and in solution. Complex association constants (K1:1) in an aqueous solution spanned the approximate range of 15,000 to 55,000 M−1; the values increased with the CDs in the order HPβCD < DIMEB < RAMEB < SBECD. Significant PTB solubility enhancement factors were recorded at 100 mM CD concentrations, the most accurately determined values being in the range 700-fold to 1250-fold

Genetic and structural elucidation of capsular polysaccharides from Streptococcus pneumoniae serotype 23A and 23B, and comparison to serotype 23F.

Streptococcus pneumoniae is a globally important encapsulated human pathogen with approximately 100 different serotypes recognized. Serogroup 23 consists of serotype 23F, present in licensed vaccines, and emerging serotypes 23A and 23B. Here, we report the previously unknown structures of the pneumococcal capsular polysaccharides serotype 23A and 23B determined using genetic analysis, NMR spectroscopy, composition and linkage analysis and Smith degradation (of polysaccharide 23A). The structure of the serotype 23A capsular polysaccharide is: ->4)-b-D-Glcp-(1->3)-[[a-L-Rhap-(1->2)]-[Gro- (2->P->3)]-b-D-Galp-(1->4)]-b-L-Rhap-(1->. This structure differs from polysaccharide 23F as it features a disaccharide backbone and the di-substituted b-Gal is linked to b-Rha as a side chain. This is due to the different polymerization position catalysed by the unusually divergent repeat unit polymerase Wzy in the 23A cps biosynthesis locus. Steric crowding in 23A, confirmed by molecular models, causes the NMR signal for H-1 of the di-substituted 2,3-b-Gal to resonate in the a-anomeric region. The structure of the serotype 23B capsular polysaccharide is the same as 23F, but without the terminal a-Rha:->4)-b-D-Glcp-(1->4)-[Gro-(2->P->3)]-b-D-Galp-(1->4)-b-L-Rhap-(1->. The immunodominant terminal a-Rha of 23F is more sterically crowded in 23A and absent in 23B. This may explain the reported typing cross reactions for serotype 23F: slight with 23A and none with 23B