Physicochemical studies of a novel adjuvant and conjugate vaccines

Mensah, Priscilla Darling Naa-Ahimah

Physicochemical studies of a novel adjuvant and conjugate vaccines

Doctoral Thesis

2010

Publisher

University of Cape Town

Department

Department of Chemistry

Faculty

Faculty of Science

Abstract

South African children currently receive vaccines against Diphtheria, Tetanus, Pertussis (administered as DTP), Hepatitis B (HBV) and Haemophilus influenzae type b (Hib) at 6, 10 and 14 weeks. The use of combination vaccines provides a means of avoiding the logistical problems and costs associated with multiple injections of these different vaccines. A local vaccine manufacturer is in the process of developing a combined tetravalent DTP-HBV as well as a liquid pentavalent DTP-HBV-Hib vaccine. The Hib vaccine is a glycoconjugate in which Haemophilus influenzae type b capsular polysaccharide (polyribosylribitolphosphate or PRP) is conjugated to a carrier protein. The conjugate is immunogenic in infants who have a higher risk of infection while the polysaccharide vaccine is not. The compatibility of the Hib antigenic component in the presence of the other antigens and adjuvant presents a challenge. Aluminium containing adjuvants have been the most widely used adjuvants in human vaccines. Aluminium hydroxide has been found to catalyse the hydrolysis of PRP when added to Hib conjugate vaccines. Although this can be circumvented by the use of aluminium phosphate, there is a need for new adjuvants that elicit broader immune responses. This thesis presents a study of the size, structure and composition of a locally developed experimental adjuvant called Pheroid™ by use of NMR spectroscopy and Coulter Counter. NMR analysis of Pheroid™ formulations provided a structural fingerprint for the formulations and indicated the relative proportions of the major components present, whereas their particle size distribution was profiled using a Coulter Counter. The average size distribution was similar in the formulations tested including those that had been activated using nitrous oxide. The primary focus of this thesis was the application of appropriate physicochemical procedures for evaluation of the locally manufactured Hib vaccine alone and when in combination with DTP-HBV-Hib. Investigating the stability and integrity of Hib conjugate vaccines requires determination of the total saccharide and unbound or free saccharide which is expressed as the percentage of free saccharide present. In the absence of a suitable Hib conjugate, model compounds such as human serum albumin (HSA) , meningococcal group A polysaccharide (PsA) and the derived conjugate (Mn A-IT) were used to investigate three methods of free saccharide separation: solid phase extraction (SPE), acid precipitation using deoxycholate (DOC/HCI) and ultrafiltration (UF). At physiological pH, the binding capacity was low and so the SPE method was not investigated further. For Mn A-IT the DOC/HCI method generally gave lower free saccharide values than the UF method; this was attributed to entrapment and coprecipitation of free saccharide by DOC/HCI. In contrast, washing steps in the UF method ensured good free saccharide recovery. A colorimetric assay for phosphorus and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) were investigated for saccharide quantification in Mn A-TT vaccines. The HPAEC-PAD method for the monomer (mannosamine-6-phosphate from acid hydrolysis) permitted higher specificity and sensitivity for the free saccharide analysis compared to the phosphate assay. The DOC/HCI and UF methods were compared by their application to Mn A-TT samples subjected to an accelerated stability study.

Description

Includes abstract.

Includes bibliographical references.

Keywords

Chemistry

Reference:

Collections

PhD / Doctoral

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