Determining trace amounts and the origin of formaldehyde impurity in Neisseria meningitidis A/C/Y/W-135-DT conjugate vaccine formulated in isotonic aqueous 1× PBS by improved C18-UPLC method.

The ability to accurately measure and report trace amounts of residual formaldehyde impurity in a vaccine product is not only critical in the product release but also a regulatory requirement. In many bacterial or viral vaccine manufacturing procedures, formaldehyde is used either at a live culture inactivation step or at a protein de-toxification step or at both. Reported here is a validated and improved C18-UPLC method (developed based on previously published C-8 HPLC method) to determine the traces of formaldehyde process impurity in a liquid form Neisseria meningitidis A/C/Y/W-135-DT conjugate vaccine formulated in isotonic aqueous 1× PBS. UPLC C-18 column and the conditions described distinctly resolved the 2,4-DNPH-HCHO adduct from the un-reacted 2,4-DNPH as detected by TUV detector at 360 nm. This method was shown to be compatible with PBS formulation and extremely sensitive (with a quantitation limit of 0.05 ppm) and aided to determine formaldehyde contamination sources by evaluating the in-process materials as a track-down analysis. Final nanogram levels of formaldehyde in the formulated single dose vialed vaccine mainly originated from the diphtheria toxoid carrier protein used in the production of the conjugate vaccine, whereas relative contribution from polysaccharide API was minimal.

Determination of free formaldehyde in vaccines and biological samples using solid-phase microextraction coupled to GC-MS.

A headspace solid-phase microextraction method coupled to GC-MS was successfully developed for the trace determination of formaldehyde in veterinary bacterial and human vaccines, and diphtheria-tetanus antigen. The formaldehyde was derivatized by means of the Hantzsch reaction prior to extraction and subsequent determination. Three different types of solid-phase microextraction fibers, polar, and nonpolar poly(dimethylsiloxane) and polyethylene glycol were prepared by using a sol-gel technique. The effects of different parameters such as type of fiber coating, extraction time and temperature, desorption conditions, agitation rate, and salt effect were investigated. Under the optimized conditions, the detection limit of the method was 979 ng/L using the selected ion-monitoring mode. The interday and intraday precisions of the developed method under the optimized conditions were below 13%, and the method shows linearity in the range of 1.75-800 µg/L with a correlation coefficient of 0.9963. The optimized method was applied to the determination of formaldehyde from some biological products. The results were satisfactory compared to the standard method.

Development and evaluation of chitosan microspheres for tetanus, diphtheria and divalent vaccines: a comparative study of subcutaneous and intranasal administration in mice.

CONTEXT: There is a need to use the new technologies to induce immunity with minimum number of vaccination sessions to ensure compliance with reducing cost. OBJECTIVES: To develop single shot vaccines of tetanus, diphtheria and divalent toxoids microsphere's formulations and to induce their immune response after intranasal and subcutaneous administration in mice. MATERIALS AND METHODS: The microspheres were prepared using different concentrations of chitosan. Microsphere's morphology, particle size analysis, encapsulation efficiency and antigen integrity were performed and the best formulations were selected for in vitro and in vivo testing in mice. RESULTS: The developed microspheres have a yield percent of 70.3-91.5%. In vitro release of antigens indicated that tetanus release was increased up to 75 and 81% post T5 and TD5 formulations respectively, whereas diphtheria cumulative release increased up to 74 and 69% post D3 and TD5, respectively. DISCUSSION: Antibody levels produced were lower than that obtained from alum adsorbed vaccine but higher than the minimum level required to induce immunogenicity (>0.01 IU/mL). The subcutaneous route of administration was superior over the intranasal route in producing higher antibody levels. CONCLUSION: Chitosan microspheres were developed successfully and prove that chitosan represents a good candidate for vaccines delivery.

Evaluation of a diphtheria and tetanus PLGA microencapsulated vaccine formulation without stabilizers.

Polymeric microspheres containing diphtheria and tetanus toxoids were prepared without protein stabilizers. A vaccine containing 2 Lf(tetanus) and 0.4 Lf(diphtheria) was injected either in BALB/c mice or in guinea-pigs. As control, a group received the alum-adsorbed unencapsulated toxoids. In mice, on day 44 one group and control received a booster and at day 111 the other group received the same booster dose. Before de booster, all groups had very low neutralizing antibodies, as determined by Toxin binding inhibition assay. One week after booster all groups had high antibody titers, especially those immunized with microencapsulated vaccine, which were at least 5 times higher than those immunized with alum vaccine for both antigens. Besides, guinea pigs receiving lower dose had antibodies titers as high as 60 UI/mL, and 30 times higher than those immunized with alum vaccine. Therefore by using an encapsulated vaccine without any kind of protein stabilizer we were able to induce in vivo protective responses irrespective of observed in vitro protein degradation by HPLC. Manipulating the vaccination schedule at the same time to the toxoids encapsulation does not only increase the antibody titers but also their specificity.

Trace determination of free formaldehyde in DTP and DT vaccines and diphtheria-tetanus antigen by single drop microextraction and gas chromatography-mass spectrometry.

An immersed single drop microextraction (SDME) method was successfully developed for the trace enrichment of formaldehyde from DTP and DT vaccines and diphtheria-tetanus antigen. The formaldehyde was derivatized by means of the Hantzsch reaction. The dehydropyridine derivative was extracted into a microdrop of chloroform that suspended in a 4 ml sample solution for a preset time. The microdrop was then retracted into the microsyringe and injected directly into a gas chromatography-mass spectrometry (GC-MS) injection port. Effects of different parameters such as the type of solvent, extraction time, stirring rate, and temperature were studied and optimized. The limit of detection was 0.22 ng/l and relative standard deviation (RSD) value was 6.2% (n=5). The regression coefficient was satisfactory (r(2)=0.992) and linear range was obtained from 1 to 500 ng/l.

Subcutaneous nodule after vaccination with an aluminum-containing vaccine.

Persistent subcutaneous nodules may arise after vaccination or allergen desensitization. The swelling might appear as a result of a specific histiocytic reaction to aluminum, which is used in many preparations to hasten immune response. A wide range of such vaccines are used in national childhood vaccination programs. Such nodules are frequently itchy or painful with local skin alterations. The condition tends to resolve spontaneously, although long-term observation is recommended. We describe the clinical history of a 10-year-old girl who presented with an itchy subcutaneous nodule that appeared five months after her second DiTe revaccination.

Aluminium assay and evaluation of the local reaction at several time points after intramuscular administration of aluminium containing vaccines in the Cynomolgus monkey.

Aluminium hydroxide and aluminium phosphate have been widely used as vaccine adjuvants with a good safety record for several decades. The recent observation in human deltoid muscle of macrophage aggregates containing aluminium hydroxide spicules and termed Macrophagic Myofasciitis (MMF) has encouraged research on aluminium salts. This study was conducted in order to further investigate the clearance of aluminium at the vaccine injection site and the features of induced histopathological lesions. Two groups of 12 monkeys were immunised in the quadriceps muscle with Diphtheria-Tetanus vaccines, which were adjuvanted with either aluminium hydroxide or aluminium phosphate. Three, six or twelve months after vaccination, four monkeys from each group were sacrificed and histopathological examination and aluminium assays were performed on quadriceps muscle sections. Histopathological lesions, similar to the MMF described in humans, were observed and were still present 3 months after aluminium phosphate and 12 months after aluminium hydroxide adjuvanted vaccine administration. An increase in aluminium concentration, more marked in the area of the lesions, was also observed at the 3- and 6-month time points. These findings were localised at the injection site and no similar changes were observed in the distal or proximal muscle fragments. We conclude from this study that aluminium adjuvanted vaccines administered by the intramuscular route trigger histopathological changes restricted to the area around the injection site which persist for several months but are not associated with abnormal clinical signs.

Spontaneously reported adverse reactions after diphtheria-tetanus revaccination at 4-6 years of age--a comparison of two vaccines with different amounts of diphtheria toxoid.

On 1 January 1996, diphtheria-tetanus revaccination at the age of 5 years was implemented in the Danish Childhood Vaccination Programme. Initially, a combined DT vaccine containing 25 Lf diphtheria toxoid was used. Due to a high frequency of spontaneously reported adverse reactions, however, concerns were raised that the diphtheria dose was too high, and it was reduced to 6.25 Lf. This survey presents the rates of spontaneously reported adverse reactions following diphtheria-tetanus revaccinations of 4-6-year-old from 1996 to 2002. The change to the lower dose of diphtheria toxoid resulted in a remarkable reduction in the yearly rates of injection site reactions: in 1996, the rate was 180 injection site reactions per 100,000 vaccinations; from 1998 to 2002, this changed to between 12 and 24 reactions per 100,000 vaccinations. Furthermore, the rates of systemic reactions such as fever were reduced.

Water accessibility, aggregation, and motional features of polysaccharide-protein conjugate vaccines.

A relaxometric investigation of a nontoxic mutant of diphtheria toxin and of its conjugates with capsular polysaccharides of different groups of Neisseria meningitidis was performed. The insertion of polysaccharides chains alters dramatically the hydrodynamic properties of the protein. The model-free analysis of the (1)H nuclear magnetic relaxation dispersion profiles of their water solutions shows: i), a reduced protein hydration with respect to the carrier protein alone; ii), a much larger flexibility of the conjugates with respect to a compact macromolecule of the same molecular weight; and iii), a strong tendency to aggregate. The above findings are largely independent on the nature of the polysaccharide and thus provide a fairly general picture of the dynamic properties of glycoconjugate proteins.

An experimental divalent vaccine based on biodegradable microspheres induces protective immunity against tetanus and diphtheria.

In an endeavor towards development of multivalent vaccines based on biodegradable microspheres, we tested the immunologic performance of several divalent microsphere formulations against tetanus and diphtheria. Microspheres were made by separate microencapsulation of tetanus and diphtheria toxoid in poly(lactide-co-glycolide) by either spray-drying or coacervation. Guinea pigs were subcutaneously immunized by a single injection of the divalent vaccines or, for control, an equivalent dose of a licensed vaccine containing both antigens adsorbed on aluminium hydroxide. All microsphere formulations were strongly immunogenic, irrespective of particle size and hydrophobicity. End point titers of ELISA antibodies, mainly of the IgG1 subtype, were comparable to those obtained after immunization with the licensed vaccine. The microspheres provided increasing levels of antibodies, during the 16 weeks of testing, and the antibodies were weakly polarized towards tetanus. The induced antibodies were also toxin neutralizing, as determined for both diphtheria (1-4 IU/mL) and tetanus (5-9 IU/mL) 8 weeks after immunization. These neutralization levels were several orders of magnitude above the level considered minimum for protection (0.01 IU/mL). When the animals were challenged with tetanus or diphtheria toxins 6 weeks after immunization, microsphere vaccines produced protective immunity that was comparable to or better than that induced by the licensed divalent vaccine. In conclusion, this study showed that a single administration of biodegradable microsphere vaccines provided protective immunity against diphtheria and tetanus, and that this immunization approach might be feasible for multivalent vaccines.