Physico-chemical analysis

The main focus of the Physico-chemical Analysis Group is to evaluate the quality of saccharide-protein conjugate components and bacterial protein antigens. The following indicators of a vaccine’s quality are evaluated:

• consistency of production by HPLC Size-exclusion chromatography, and RP-HPLC (for lipids in outer membrane vesicles)
• integrity and potency (free/total saccharide) by HPAEC-PAD, colorimetric assays
• stability, through SEC/MALLS and optical spectroscopy methods (CD, fluorimetry).

Standards

Our laboratory characterises and evaluates the stability of polysaccharide standards established for the quantitative potency determination in Typhoid, Haemophilus influenzae type b and Meningococcal vaccines. Participation in WHO Collaborative Studies led by the NIBSC for assignment of unitage and to determine how the standard will perform in real situations is often of mutual benefit to the WHO-sponsored project and the individual participants.  

Control

We carry out formal batch release of licenced glyco-conjugate or protein based products for the U.K., European and rest of the world on Haemophilus influenze type b and serogroups A, C, W and Y meningococcal, pneumococcal and typhoid conjugate vaccines to the requirements of WHO and UK Independent Control Testing guidelines. Contract or collaborative evaluation of new products (covering GBS, meningococcal and typhoid vaccines), often for global markets, are also performed, for the purpose of quality comparisons through different approaches to explore the strengths and limitations of particular methods used for characterization or control.

Research 

Our laboratory evaluates new vaccines, protein antigens or carrier proteins by optical spectroscopic or chromatographic methods to look at conformation, size and integrity, and to study how these correlate with markers of immunogenicity. Circular dichroism and fluorescence spectroscopy are used to evaluate the 2° and 3° structure of proteins, while SEC-MALLS/Viscosity is utilized to more accurately gauge the quaternary structure, hydrodynamic radius, aggregation status and viscosity of vaccine materials in stability studies.

In addition to analyzing the protein components, we also evaluate saccharide and lipid components with HPAEC-PAD and RP-HPLC. Micro-flow imaging particle analysis (> 1 µm) adds a further dimension to formulation studies. Our long-term goal is to achieve a better understanding for the molecular basic of immunoprotection.

Key publications

Lockyer K, Gao F, Francis RJ, Eastwood D, Khatri B, Stebbings R, Derrick JP, Bolgiano B. (2020) Higher mass meningococcal group C-tetanus toxoid vaccines conjugated with carbodiimide correlate with greater immunogenicity. Vaccine 38, 2859-2869. https://doi.org/10.1016/j.vaccine.2020.02.012

Hitri K., Kuttel MM, De Benedetto G, Lockyer K, Gao F, Hansal P, Rudd TR, Beamish E, Rijpkema S, Ravenscroft N, Bolgiano B. (2019) O-acetylation of typhoid capsular polysaccharide confers rigidity and immunodominance by masking additional epitopes. Vaccine 37, 3866-3875.https://doi.org/10.1016/j.vaccine.2019.05.050

Beresford NJ, Martino A, Feavers IM, Corbel MJ, Bai X, Borrow R, Bolgiano B. Quality, immunogenicity and stability of meningococcal serogroups ACWY-CRM₁₉₇, DT and TT glycoconjugate vaccines. Vaccine 2017 Jun 10;35(28):3598-3606. http://dx.doi.org/10.1016/j.vaccine.2017.03.066

Otto RBD, Burkin K, Mulla SE, Crane DT, Bolgiano B. (2015) Patterns of binding of aluminum-containing adjvuants to Haemophilus influenza type b and meningococcal group C conjugate vaccines and components. Biologicals 43, 355-362. https://dx.doi.org/10.1016/j.biologicals.2015.06.008

Lockyer K, Gao F, Derrick JP, Bolgiano B.  (2015) Structural correlates of carrier protein recognition in tetanus toxoid conjugated bacterial protein vaccines. Vaccine 33, 1345-1352. http://dx.doi.org/10.1016/j.vaccine.2015.01.046

Martino A, Magagnoli C, De Conciliis G, D;Ascenzi S, Forster MJ, Allen L, Brookes C, Taylor S, Bai X, Findlow J, Feavers IM, Rodger A, Bolgiano B.  (2012) Structural characterization, stability and antibody recognition of chimeric NHBA-GNA1030: An investigational vaccine component against Neisseria meningitidis. Vaccine 30, 1330-1342.
https://doi.org/10.1016/j.vaccine.2011.12.066

Jumel K, Ho MM, Bolgiano B. (2002) Evaluation of Meningococcal C oligosaccharide conjugate vaccines by size exclusion chromatography/multi-angle laser light scattering. Biotechnology  Applied Biochemistry 36, 219-226. https://doi.org/10.1042/BA20020066

Qazi O, Bolgiano B, Crane D, Svergun DI, Konarev PV, Yao Z-P, Robinson CV, Brown KA & Fairweather N. (2007) The H_c fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disuphide bond.  J Mol Biol 365, 123-134.

https://doi.org/10.1016/j.jmb.2006.09.050

Ho MM, Bolgiano B, Corbel MJ. (2000) Assessment of the stability and immunogenicity of meningococcal oligosaccharide C-CRM₁₉₇ conjugate vaccines. Vaccine, 19, 716-725. https://doi.org/10.1016/S0264-410X(00)00261-9 

Crane DT, Bolgiano B, Jones C. (1997) Comparison of the diphtheria mutant toxin, CRM197, with a Haemophilus influenzae  type-b polysaccharide-CRM197 conjugate by optical spectroscopy. European Journal of Biochemistry, 246, 320-327. https://doi.org/10.1111/j.1432-1033.1997.00320.x