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.

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.