Collaborative study for the calibration of a replacement international standard for diphtheria toxoid adsorbed

We present the results of a collaborative study for the characterization of a preparation of diphtheria toxoid adsorbed, and its calibration in terms of the 3rd International Standard (IS) for Diphtheria Toxoid Adsorbed. Calibration was performed using established World Health Organization (WHO) and European Pharmacopoeia (Ph. Eur.) protection models. Two candidate toxoid preparations were included in the study, one of which was adopted as a replacement Ph. Eur. Biological Reference Preparation (BRP, batch 4) in February 2009. The second candidate preparation was found to have a unitage of 213 IU/ampoule based on the calibration by in vivo bioassay in 19 laboratories in 16 countries, and was established as the 4th IS for Diphtheria Toxoid Adsorbed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2009.The study also assessed performance of the replacement standard in mouse and guinea pig serological assays which are used as alternative procedures for diphtheria potency testing. Participants tested both candidate preparations and potency was expressed in relative terms only. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays and that the Vero cell assay may be suitable for calibration of future replacement standards.     

Calibration of replacement international standard and European Pharmacopoeia Biological Reference Preparation for Diphtheria Toxoid, Adsorbed.

We report here the characterisation of a preparation of diphtheria toxoid, adsorbed, and its calibration by twenty laboratories in fourteen countries in terms of the Second International Standard (I.S.) for Diphtheria Toxoid, Adsorbed, coded sample A (DIXA) using the established World Health Organisation (WHO)/European Pharmacopoeia (Ph Eur) challenge methods. The replacement standard preparation was found to have a unitage of 160 IU/ampoule on the basis of its calibration by in vivo bioassay. Stability was assessed within the collaborative study, and as part of candidate characterisation. Results suggest that the replacement standard will have satisfactory stability. This study also provided an opportunity to investigate serology as alternative to in vivo bioassay for potency testing of diphtheria vaccines. Six laboratories participated by performing serology according to in-house protocol. The calibration of the replacement standard in a mouse Vero cell assay gave a significantly higher results than in the established WHO/Ph Eur methods. Based on the results of this study and with the agreement of participants, the candidate standard was established as the Third International Standard for Diphtheria Toxoid, Adsorbed (coded 98/560) by the WHO Expert Committee of Biological Standardization in October 1999. The same preparation was also established as the second Ph Eur Biological Reference Preparation (Ph Eur BRP, batch no. 3) by the Steering Committee of the Biological Standardisation Programme of the European Directorate for the Quality of Medicines and approved by the European Pharmacopoeia Commission.     

Optimization of the formulation for a candidate lyophilised tetanus toxoid reference preparation

Tetanus toxoid is a vital primary reference material used for standardization of assays required to establish the antigenic purity of tetanus toxoid for vaccine production. Several formulations were assessed and ampouled fills of each formulation lyophilised. The relative Lf content determined by Ramon flocculation, SRD, and ELISA assays was measured. The stability of the tetanus toxoid activity in each formulation was assessed by accelerated degradation studies. Formulations containing glycine were not suitable in flocculation tests but both sorbitol and trehalose formulations were. The trehalose/sodium chloride formulation had a good appearance, showed good activity in all assays and maintained its activity best under stress conditions. This formulation has been applied to a large scale batch of ampoules prepared as a WHO candidate replacement standard, evaluated in a collaborative study and accepted as a replacement WHO IS for use in flocculation test (WHO ECBS, October 2007, ref no BS/07.2061). The stability of this formulation was also excellent for the large scale batch. The benefits of using thermal analysis and freeze drying microscopy coupled with small scale lyophilisation trials in order to screen formulations for the preparation of batches of biological reference materials are demonstrated.     

Calibration of replacement international standards of diphtheria and tetanus toxoids for use in flocculation test

The 1st International Reference Reagents (IRR) of Diphtheria and Tetanus Toxoids for Flocculation Test (DIFT and TEFT) were established by the WHO in 1988. These reagents are essential for the standardization of assays used to calculate Lf units of toxoids. Candidate replacement materials were provided by several European vaccine manufacturers and were formulated and freeze-dried at NIBSC. This paper provides a summary of the results of an international collaborative study including 18 laboratories from 16 countries, which examined the candidate replacement materials in a variety of methods. Materials 02/176 and 04/150 were proposed and adopted by the Expert Committee on Biological Standardization of WHO in October 2007 as 2nd WHO International Standards of Diphtheria and Tetanus Toxoid for use in Flocculation Test. The replacement standards were assigned the value of 1100 and 690Lf/ampoule, respectively, based on results of flocculation tests carried out using provided reagents. Material coded 02/176 fully complied with the WHO specifications for stability, residual moisture content, precision of fill and sterility. Stability of material coded 04/150 was slightly lower than expected but predictions were based only on 2-year data and were to be further monitored, post-adoption.     

Quantitative estimation of diphtheria and tetanus toxoids. 4. Toxoids as international reference materials defining Lf-units for diphtheria and tetanus toxoids

The Lf-unit, which is used in the control of diphtheria and tetanus toxoid production and in some countries also to follow immunization of horses for production of antitoxins, has hitherto been defined by means of antitoxin preparations. A diphtheria toxoid and a tetanus toxoid preparation, both freeze-dried, were examined in an international collaborative study for their suitability to serve as reference reagents in the flocculation tests and for defining the Lf-units. It was shown that flocculation tests using the reference toxoids are very reproducible and reliable and the WHO Expert Committee on Biological Standardization established: the toxoid called DIFT as the International Reference Reagent of Diphtheria Toxoid for Flocculation Test with a defined content of 900 Lf-units of diphtheria toxoid per ampoule; and the toxoid called TEFT as the International Reference Reagent of Tetanus Toxoid for Flocculation Test with a defined content of 1000 Lf-units of diphtheria toxoid per ampoule.     

精制破伤风类毒素产毒及精制工艺的改进

用超滤、硫酸铵二段盐析法取代等电点沉淀法后,精制破伤风类毒素（精破类）的纯度由８０７Ｌｆ／ｍｇＰＮ提高到１８８３Ｌｆ／ｍｇＰＮ,纯度提高一倍以上。使用双胨培养基取代酪素培养基后,产毒水平由４７Ｌｆ／ｍｌ提高到８８Ｌｆ／ｍｌ（ｔ＝６．４６,ｐ＜０．００１）;用新法精制后,精破类纯度分别为１９４９Ｌｆ／ｍｇＰＮ及１７８５Ｌｆ／ｍｇＰＮ（ｔ＝０．３３４,ｐ＞０．０５）,引用双胨培养基后可提高产毒水平,但不影响精破类的纯度。     

Comparative analysis of antigenic strength and in vivo serum antibodies concentration of tetanus toxoid vaccine adsorbed in Pakistan

Clostridium tetani produce tetanospasmin, a potent exotoxin; that causes tetanus or lockjaw disease. Scientists developed an anti-tetanus toxoid to protect the body from the spasm's neurotoxic effect. In Pakistan recently, 478 cases of neonatal tetanus were reported. The study was carried out at The National Control Laboratory for Biologicals Islamabad, aiming to decipher the effectiveness of the most frequently used tetanus toxoid vaccine adsorbed in Pakistan in comparison to standard reference vaccine having earlier known consistent values. The vaccines included domestic public sector, domestic private sector, imported private sector I, and imported private sector II. The triplicate experiments on purebred Swiss albino mice were performed by immunizing with Tetanus toxoid and then tested parallel with standard reference vaccine. Various analytical tests were performed on the test organism that included flocculation test/identity test, antibody quantification using enzyme-linked immunosorbent assay (ELISA), potency test, abnormal toxicity test, osmolality, pH test, liquid sub-visible particle test, and sterility test. Results of all the vaccines were compared in comparison with the standard reference vaccine. Absorbances of test vaccines were recorded at the lowest dilution by ELISA. The domestic private sector, imported private sector I, imported private sector II and standard reference vaccine were flocculated at mean dilution (Mean: 0.24, 95% CI: 0.1903–0.2897), and the domestic public sector was flocculated at mean dilution (Mean: 0.23, 95% CI: 0.2052–0.2548). All the products were found within the normal ranges where it was concluded that the maximum average titer of 2.81 was observed at dilution 10^?1.6, indicating that these vaccines were adequate/suitable for the prevention of tetanus.