Evaluation of candidate International Standards for meningococcal capsular polysaccharide groups W and Y.

Two candidate International Standards for meningococcal capsular group W and Y (MenW and MenY, respectively) polysaccharides were assessed for their suitability as quantitative standards in various physicochemical assays. The study was designed to evaluate the intended purpose of these standards, namely, to standardize the quantification of the respective polysaccharide content in meningococcal polysaccharide and conjugate vaccines and their intermediate components. Twelve laboratories from eleven different countries participated in the collaborative study of candidate preparations for International Standards for MenW and MenY polysaccharide (coded 16/152 and 16/206, respectively). Unitage was assigned using the Resorcinol assay. Our proposals, on the basis of data from the Resorcinol assay were: 1) candidate standard for MenW polysaccharide (16/152) to be assigned a content of 1.015 ± 0.071 mg MenW polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.13, corresponding to a 95 % level of confidence) and 2) candidate standard for MenY polysaccharide (16/206) be assigned a content of 0.958 ± 0.076 mg MenY polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.26, corresponding to a 95 % level of confidence). The amount of polysaccharide per ampoule remained consistent under all stability conditions over a 36-month period.

Saccharide dosage content of meningococcal polysaccharide conjugate vaccines determined using WHO International Standards for serogroup A, C, W, Y and X polysaccharides.

Potency of meningococcal polysaccharide-protein conjugate vaccines relies on the polysaccharide content to prevent meningitis. NIBSC, as the official national control laboratory in UK, analysed ten different mono- and multi-meningococcal conjugate vaccines, using established International Standards for meningococcal serogroups A, C, W, Y and X, by resorcinol or HPAEC-PAD assay. Most saccharide contents were within ±20% of their claimed content for licensure with taking different O-acetylation levels into consideration, with only MenC content in two vaccines below (by 60% and 54%) the labelled value, however, previous study showed different dosage was not necessarily correlated to the immunogenicity of those vaccines. This study demonstrated the use of International Standards to quantify saccharide content in polysaccharide-based vaccines with different percentage of O-acetylation. These International Standards are suitable to serve as either quantitative standard or calibrator of in-house standards, with supplied stability data.

International collaborative study for establishment of the 2nd WHO International Standard for Haemophilus influenzae type b polysaccharide.

In this report we present the results of a collaborative study for the preparation and calibration of a replacement International Standard (IS) for Haemophilus influenzae type b polysaccharide (polyribosyl ribitol phosphate; 5-d-ribitol-(1 â†’ 1)-ß-d-ribose-3-phosphate; PRP). Two candidate preparations were evaluated. Thirteen laboratories from 9 different countries participated in the collaborative study to assess the suitability and determine the PRP content of two candidate standards. On the basis of the results from this study, Candidate 2 (NIBSC code 12/306) has been established as the 2nd WHO IS for PRP by the Expert Committee of Biological Standards of the World Health Organisation with a content of 4.904 ± 0.185mg/ampoule, as determined by the ribose assays carried out by 11 of the participating laboratories.

Patterns of binding of aluminum-containing adjuvants to Haemophilus influenzae type b and meningococcal group C conjugate vaccines and components.

The basis of Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroup C (MenC) glycoconjugates binding to aluminum-containing adjuvants was studied. By measuring the amount of polysaccharide and protein in the non-adsorbed supernatant, the adjuvant, aluminum phosphate, AlPO4, was found to be less efficient than aluminum hydroxide, Al(OH)3 at binding to the conjugates, at concentrations relevant to licensed vaccine formulations and when equimolar. At neutral pH, binding of TT conjugates to AlPO4 was facilitated through the carrier protein, with only weak binding of AlPO4 to CRM197 being observed. There was slightly higher binding of either adjuvant to tetanus toxoid conjugates, than to CRM197 conjugates. This was verified in AlPO4 formulations containing DTwP-Hib, where the adsorption of TT-conjugated Hib was higher than CRM197-conjugated Hib. At neutral pH, the anionic Hib and MenC polysaccharides did not appreciably bind to AlPO4, but did bind to Al(OH)3, due to electrostatic interactions. Phosphate ions reduced the binding of the conjugates to the adjuvants. These patterns of adjuvant adsorption can form the basis for future formulation studies with individual and combination vaccines containing saccharide-protein conjugates.

A physico-chemical assessment of the thermal stability of pneumococcal conjugate vaccine components.

Physico-chemical analysis of pneumococcal polysaccharide (PS)-protein conjugate vaccine components used for two commercially licensed vaccines was performed to compare the serotype- and carrier protein-specific stabilities of these vaccines. Nineteen different monovalent pneumococcal conjugates from commercial vaccines utilizing CRM197, diphtheria toxoid (DT), Protein D (PD) or tetanus toxoid (TT) as carrier proteins were incubated at temperatures up to 56°C for up to eight weeks or were subjected to freeze-thawing (F/T). Structural stability was evaluated by monitoring their size, integrity and carrier protein conformation. The molecular size of the vaccine components was well maintained for Protein D, TT and DT conjugates at -20°C, 4°C and F/T, and for CRM197 conjugates at 4°C and F/T. It was observed that four of the eight serotypes of Protein D conjugates tended to form high molecular weight complexes at 37°C or above. The other conjugated carrier proteins also appeared to form oligomers or 'aggregates' at elevated temperatures, but rarely when frozen and thawed. There was evidence of degradation in some of the conjugates as evidenced by the formation of lower molecular weight materials which correlated with measured free saccharide. In conclusion, pneumococcal-Protein D/TT/DT and most CRM197 bulk conjugate vaccines were stable when stored at 2-8°C, the recommended temperature. In common between the conjugates produced by the two manufacturers, serotypes 1, 5, and 19F were relatively less stable and 6B was the most stable, with types 7F and 23F also showing good stability.

Evaluation of a candidate International Standard for Meningococcal Group C polysaccharide.

Meningococcal group C (MenC) plain polysaccharide (PS) and conjugate vaccines are primarily evaluated by physicochemical methods to ensure that batches are consistently manufactured. As different assays are employed to quantify the MenC PS content of final formulations and bulk intermediaries, there is a need for an International MenC PS Standard to calibrate internal references used in the different laboratories. Twelve laboratories from nine different countries participated in a collaborative study to determine the MenC PS content of a candidate International Standard MenC PS preparation (08/214) and to assess its suitability. On the basis of the results from this study the candidate standard 08/214 was established as an International Standard for the quantification of MenC PS content in vaccines and components. It has a content of 1.192 ± 0.192 mg MenC PS/ampoule (expanded uncertainty with coverage factor of k = 2.365 corresponding to a 95% level of confidence), as determined by the resorcinol assays carried out by eight of the participating laboratories. The standard is available from The National Institute of Biological Standards and Control who act as guardians and distributors of the material under the auspices of WHO.

Immunogenicity and thermal stability of a combined vaccine against Haemophilus influenzae type b and Neisseria meningitidis serogroup C diseases.

The immunogenicity, structure and stability of a combined conjugate vaccine against Haemophilus influenzae type b and meningococcal serogroup C (Hib/MenC) were investigated. A rat model for immunogenicity showed that antibody responses to Hib and MenC in the combined vaccine were similar to or higher than those of individual conjugates given alone, or concomitantly at separate sites. At elevated temperatures, the combination vaccine was slightly more stable than a monovalent Hib-TT vaccine, with respect to molecular size, which could be attributed to differences in the formulations. Following 5 weeks incubation at 56 degrees C, there was some dissociation of high molecular weight conjugate without significant loss of saccharide integrity; however, this did not significantly affect the vaccine immunogenicity, demonstrating the stability of this lyophilized vaccine.

A retrospective study on the quality of Haemophilus influenzae type b vaccines used in the UK between 1996 and 2004.

Following the reduction in efficacy of Hib-TT vaccines in the primary immunization schedule observed in the UK between 1999 and 2003, batches of vaccine manufactured by two different companies were retrospectively examined by the National Institute for Biological Standards and Control. The study evaluated 41 batches of the Hib-TT vaccines manufactured between 1994 and 2003, assaying potency (total PRP saccharide content), integrity (% free saccharide), consistency (molecular sizing), and immunogenicity, as well as reviewing data previously obtained at the time of release. The study indicated the stability of the lyophilized final fill vaccines to extend well past their assigned shelf-lives, and found no trends in the endotoxin content, total saccharide or % free saccharide content. A trend towards slightly larger conjugates was observed over time in Hib-TT A, evidenced in both the manufacturer's data obtained at the time that samples were submitted for testing and in data obtained from the retrospective analysis. The study confirmed that that there had been no significant change in the quality of the Hib vaccines that could possibly account for the change reported in their protective efficacy in the UK. The study also demonstrated the value of independent testing of vaccines from the time of licensure and in the ongoing monitoring and re-examination of selected batches, as necessary, to assure their continuing quality, safety and consistency.