Collaborative study for the calibration of a replacement International Standard for diphtheria toxoid for use in flocculation test.

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for diphtheria toxoid for use in flocculation test and its calibration in Lf units. Calibration was performed using Ramon flocculation method, standardized using the 2nd IS. The candidate standard was assigned a unitage of 1870 Lf/ampoule based on results from 25 laboratories in 15 different countries and was established as the 3rd IS for diphtheria toxoid for use in flocculation test by the WHO Expert Committee on Biological Standardization (ECBS) in October 2015. The study also assessed the use of alternative methods for measuring Lf. Participants were asked to determine the Lf value of the candidate standard using an Enzyme Linked Immunosorbent Assay (ELISA) established at NIBSC, or other suitable in-house method. 10 laboratories performed ELISA according to the NIBSC protocol, 1 laboratory performed flocculation using laser-light scattering according to an in-house protocol, and 1 laboratory performed another in-house ELISA. Results suggest these methods may provide suitable alternatives to the Ramon flocculation test, subject to validation, and that the new standard could act as a suitable reference preparation in these methods.

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 European pharmacopoeia biological reference preparation for diphtheria vaccine (adsorbed) batch 4.

A collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the National Institute for Biological Standards and Control (NIBSC) to establish replacement batches of the current World Health Organization (WHO) International Standard (IS) and European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for Diphtheria Vaccine (Adsorbed). Two candidates were assayed against the current 3rd IS/BRP batch 3 for Diphtheria Vaccine (Adsorbed) with an assigned potency of 160 IU/ampoule using established WHO/Ph. Eur. challenge methods in guinea pigs as described in the Ph. Eur. general chapter 2.7.6. Assay of diphtheria vaccine (adsorbed). Twenty-one laboratories (regulatory organisations and manufacturers) from 17 countries participated in the study. Two freeze-dried, stabilised diphtheria vaccine (adsorbed) preparations were included in the study: Preparation A (07/218) and Preparation B (07/216). As stocks of the 3rd IS were very low, the Diphtheria vaccine (adsorbed) BRP batch 3, which is identical to the 3rd IS but which was kept at the EDQM, was used for the calibration (coded Preparation C). The majority of participants performed 2 independent challenge tests. Five laboratories performed the intradermal challenge test, 16 laboratories performed the systemic challenge test. For Preparation A, the unweighted geometric mean potency estimate (with 95 % confidence limits) for all laboratories that provided valid results (n = 17) was 97.2 (89.5-105.6) IU/ampoule. For systemic challenge assays (n = 14) the unweighted geometric mean potency was 97.0 (88.1-106.7) IU/ampoule. The between-laboratory GCV was 17.4 % for all assays and 18.0 % for systemic challenge assays. There was no significant difference in estimates for intradermal or systemic challenge (p = 0.45). For Preparation B the unweighted geometric mean potency estimate (with 95 % confidence limits) for all laboratories that provided valid results (n = 19) was 213.4 (185.7-245.4) IU/ampoule. For systemic challenge assays (n = 14) the unweighted geometric mean potency was 202.0 (170.4-239.5) IU/ampoule. The between-laboratory GCV was 33.5 % for all assays and 34.3 % for systemic challenge assays. For both preparations there is a good agreement between results obtained from systemic and intradermal challenge methods. Greater between-laboratory variability was observed for systemic assays than for intradermal challenge assays, although the small number of intradermal assays performed in the study makes comparison difficult. The GCV for all assays was 17.4 % for potency estimates of Preparation A and 33.5 % for Preparation B. This compares favourably with the calibration of the current standard where the between-laboratory GCV for all assays was 28.3 %. From the collaborative study both Preparation A and Preparation B appeared suitable to replace the current Diphtheria vaccine (adsorbed) BRP batch 3. Due to the similarity of Preparation A with the current BRP batch 3, the Ph. Eur. Commission adopted Preparation A as Diphtheria vaccine (adsorbed) BRP batch 4 with an assigned potency of 97 IU/ampoule.

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.

Investigation of immunity level against diphtheria and reinforcement of immunity by booster vaccination for infection control staff in Okayama prefecture.

The prevalence of immunity against diphtheria among Okayama local government staff members involved in diphtheria infection control was measured. Diphtheria booster vaccination was administered to staff members with low antitoxin levels (<0.1 IU/ml) in order to reinforce of immunity. Ninety-one (36.7%) of 248 staff members, 20-69 years of age, had fully protective antitoxin levels (> or =0.1 IU/ml), and the remaining 157 (63.3%) showed levels of <0.1 IU/ml. The rate of full protection was higher in females (44.9%) than in males (22.8%) and was also higher in the diphtheria-pertussis mixed vaccine (born in 1958-1967) and diphtheria-pertussis-tetanus mixed vaccine (born in 1968-) (58.3-61.0%) groups than in diphtheria vaccine (born in 1948-1957) and non-vaccinated (born until 1947) (7.4-18.9%) groups. Though antitoxin levels of 13 (68.4%) out of 19 staff members given booster vaccinations increased to 0.1 IU/ml, 50% of these individuals then showed levels of <0.1 IU/ml after 3 years. Most of the staff members with antitoxin levels of > or =0.1 IU/ml in the non-booster vaccination group maintained their immunity levels for 2-4 years, independent of their history of vaccination. To ensure that staff members of the local government have fully protective antitoxin levels against diphtheria, periodical confirmation of antitoxin levels and booster vaccination should both be systematically carried out.

Quality control of routine, experimental and real-time aged diphtheria toxoids by in vitro analytical techniques.

Physicochemical and immunochemical techniques can be used to assess the quality of diphtheria toxoid vaccines. In a previous paper [Metz B, Jiskoot W, Hennink WE, Crommelin DJA, Kersten GFA. Physicochemical and immunochemical techniques predict the quality of diphtheria toxoid vaccines. Vaccine 2003;22(2):156-67], techniques were introduced which indicated toxoid quality with respect to safety and potency: SDS-PAGE, primary amino group determination, fluorescence/denaturation, circular dichroism and biosensor analyses. These analyses were performed with experimental toxoids from one toxin batch. In the present study, the quality of regular vaccine batches of different manufacturers, the properties of real-time aged products and a number of experimental toxoids were investigated, using the above-mentioned analytical techniques. We had the unique opportunity to analyse toxoids that were up to 40 years old. The real-time aged diphtheria toxoids showed hardly any structural differences as compared to the recently prepared products in both the analytical chemical techniques and the conventional potency and safety tests. The analytical assays discriminated between regular diphtheria toxoids and experimental toxoids prepared by methylation, acetylation or glutaraldehyde treatment. The analytical data showed a clear correlation with potency and safety of these toxoids. Based on the results, we refined the described physicochemical and immunochemical criteria that a standard diphtheria toxoid has to meet. We recommend further validation of these techniques for quality control of diphtheria toxoid vaccine because of their high precision and easy performance as compared to conventional in vivo procedures.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccine (part 2).

The study is a contribution to the EDQM's efforts to meet some of the expectations of the 3 Rs: Replacement, Reduction and Refinement of animal assays as proposed by Russell and Burch in 1959 and adopted by the European Union in 1986, and specifically to validate alternative assays to replace, for batch-release purposes, the European Pharmacopoeia (Ph. Eur.) in vivo direct challenge procedures for the potency determination of diphtheria toxoid vaccines. The study results may be used in support of the replacement of the multi-dilution direct challenge procedures in different animal models by a single dilution serology test, where appropriate, and to use sera from the same animals for potency testing of several components in combined vaccines. With regard to the latter, the present study explores the possibility of testing both diphtheria and tetanus toxoid potencies using serum from the same animals.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines-part 1.

A collaborative study on the evaluation of an alternative functional assay, the Vero cell method, to the Ph. Eur. in vivo challenge procedures for potency determination of diphtheria toxoid in 6 different combined vaccines was initiated in January 2001. The study was an extension of a previous study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. To allow interim evaluation of test results and to monitor study progress, the project was divided into three consecutive phases. The results of Phase I and II studies are presented in this report. Pre-validation (Phase I) study, performed in two laboratories, indicated that comparable diphtheria potency estimates were obtained in the Ph. Eur. direct intradermal challenge assay in guinea pigs, in Vero cell assay and in indirect ELISA for five vaccines of different potencies (range of estimates: ca. 20-200 IU/ml). The correlation coefficients between the challenge assay and the Vero cell assay corresponded to those between the challenge assay and ELISA, confirming that the antibodies play an important role in protection and that predominantly protective/neutralising antibodies are present in guinea pigs, at the time point investigated. It was observed, for Vero cell assays, that about 16-35 (9-28 in Phase II study) fold lower titre of individual serum samples were obtained when using equine, rather than guinea pig reference serum. The study also provided preliminary information that sera from the same guinea pigs may be used for potency determination of both diphtheria and tetanus toxoid components of vaccines. In Phase II, another five laboratories analysed a subset of the vaccines included in Phase I study plus an additional vaccine. Four laboratories performed the lethal challenge assay and one laboratory carried out the intradermal challenge assay. All laboratories also performed the Vero cell assay and both ELISA for diphtheria antitoxin and ELISA for tetanus antitoxin. One laboratory also performed the tetanus ToBI assay. The correlation coefficient (r) between Vero cell assay and ELISA for diphtheria antitoxin ranged from 0.76 to 0.91 in the different laboratories. The correlation between diphtheria serological assays and challenge assays were confirmed satisfactory as ca. 90 per cent of serum-estimates lead to correct prediction of mortality. All laboratories had identical rankings of the vaccines in all serological assays and in the valid challenge assays. The ranking order was identical to assumed/provided potency for the highest and the lowest vaccine. Two of the vaccines had an inversion in some assays and laboratories. As these two vaccines have almost identical potencies in all assays, these inversions are not significant. As the vaccine doses were optimised for the diphtheria component, serum anti-tetanus toxoid/toxin activities varied widely between the vaccines, making it questionable to apply a parallel line model to calculate exact potencies. However, the dose levels used showed a clear regression and good linearity in general. DTaP vaccines containing the IPV component did not always meet the present Ph. Eur. requirements in the serological assays. It should be further investigated in the Phase III study if this is a general feature of such combined vaccines. Preliminary investigations on samples from two laboratories indicate that the neutralising activity of type 1, 2 and 3 polioviruses can also be detected, in a dose-dependent way. Further studies are in progress with serum samples from other laboratories. In the light of results obtained in the first two phases, it is recommended to proceed with Phase III study to investigate reliability of the in vitro assays. In Phase III it will also be further investigated whether the serological assays for D and T components are suitable for the control of the multi-component vaccines currently marketed in Europe.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines - extended study: correlation of serology with in vivo toxin neutralisation.

Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.

Consistency testing of diphtheria and tetanus to replace potency testing for lot release.

Diphtheria and tetanus vaccines are among the most effective and safe vaccines in the EPI programme. Their mechanism of toxicity and clinical protection is well documented and toxin neutralising antibodies induced by the vaccines are generally accepted as correlates of protection. Despite these positive aspects there are still no generally accepted methods to estimate their potency for routine lot release. Some of these tests use large numbers of animals and rely on lethal challenge tests. Consequently there are ethical and financial barriers to perform these tests. Test results expressed in IU, depend on the animal species or strain used and there is limited information about their predictive value for clinical protection. WHO, supported by the ECBS, has made a proposal to harmonise the current methods into simplified consistency tests, after clinical safety and efficacy, as well as consistency in manufacturing has been established to the satisfaction of the National Regulatory Authority. In principle these tests aim for a proof of consistency in biochemical and immunological characteristics in comparison with the lots shown to be clinically safe and effective. Given that many manufacturers have recently made, or are planning to make clinical trials with new combination vaccines in the near future, recent clinical data are already available, or will be soon, on the clinical safety and efficacy of the D and T components present in these combination vaccines. This will create a unique opportunity to compare the biochemical and immunological characteristics of routinely produced vaccine lots with the clinical lots and to use the consistency approach as suggested by WHO for lot release purpose. Background information and an update will be given about the proposed consistency approach of WHO for routine lot release of the D and T components in vaccines.

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.

An in vitro approach in quality control of toxoid vaccines.

For routine immunogenicity testing of traditionally produced vaccines, animal tests are required by regulatory authorities, with potency estimated in International Units. A new concept focuses on assuring immunogenicity by monitoring batch-to-batch consistency in production. This concept is used for well-defined biologicals such as hormones. Through the use of immunochemical and bio- and physiochemical techniques the traditional products can be characterised as completely as possible. Developments in in vitro methodologies offer opportunities for immunogenicity testing in vitro. This study describes the possibilities for applying the consistency concept to the traditional products, tetanus and diphtheria toxoids. The sources of variation in these products were studied by flocculation time, SDS-PAGE, biosensor analysis, gel permeation chromatography and in vitro cytokine production studies. Batch-to-batch variation was shown using these in vitro techniques. Results indicate that it is possible to apply the consistency concept in the quality control of traditional vaccines like tetanus and diphtheria toxoids.

Statistical evaluation of numbers of animals to be used in vaccine potency testing: a practical approach.

Some of the guidelines for potency testing of vaccines issued by regulatory bodies such as the European Pharmacopoeia (EP) and WHO are detailed and stringent (e.g. EP monograph for Newcastle Disease (ND) Vaccine (inactivated)), whereas others only stipulate that the number of animals used should be sufficient to meet the required accuracy (e.g. EP monograph for Hepatitis A vaccine (inactivated)). Simulation studies in our laboratory using historical ND potency test data indicated that the number of animals specified in the monograph is too high; a considerable reduction from 10 to seven animals per group does not substantially influence the precision of the results. Multipoint models (e.g. EP monograph for Tetanus Vaccine (adsorbed)) require at least three dilutions per vaccine for testing for response linearity. However, when historical data clearly show that in the range used the response curves are linear, it is superfluous to verify this in every test. Furthermore, linearity has little priority for a valid parallel line assay calculation. A simulation study using historical Diphtheria potency test data showed that calculations using two dilutions per vaccine in relatively small groups of animals produced results comparable to those obtained from the full assay. This procedure still enables calculation of the relative potency, in contrast to the 1 + 1 method, which gives only a pass or fail result, while the number of animals required is only slightly higher. This method could be applied in cases where the 1 + 1 method fails. In conclusion, by providing guidelines on methods in which proven consistency in production and testing may be taken into account, manufacturers are more stimulated to look for other (cheaper) ways to test the potency of a vaccine using less animals.

Standardization of acellular pertussis vaccine by assay of serum neutralizing antibodies to pertussis toxin (antitoxin): analogy with diphtheria toxoid.

Immunity conferred by vaccination with cellular pertussis vaccines and pertussis wanes so that adults are now the main reservoir of B. pertussis. Similar to diphtheria toxoid, acellular pertussis vaccines are suitable for vaccination of adults and it is probable that addition of pertussis toxoid to DT will be recommended for this age group. If this can be achieved, it is easy to predict that pertussis will be eliminated and the possibility of eradicating B. pertussis will become feasible. We propose that, despite the relative inaccuracy of correlating levels of antitoxin with individual immunity, a standardized assay and reference antiserum for pertussis toxoid will control acellular pertussis vaccines as was achieved with diphtheria toxoid.

An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

Experimental study on estimating the immunogenic potency of diphtheria and/or tetanus toxoids in monovaccines and associated vaccines.

The results obtained using the "classical" active immunoprotection tests on guinea pigs, biometrically interpreted by the regression analysis in "PROBIT" systems revealed that the biopreparations of the Cantacuzino Institute meet the validity conditions related to the parallelism of the "slopes" enabling the calculation of ED50 and of the relative potencies against the reference preparations. The results also showed that the relative potencies of the biopreparations tested by the two methodologies were very close. The geometrical means and the geometrical standard deviations of the titrers obtained in mice pointed to the similarity of the values following inoculation of the Cantacuzino Institute preparations on the one hand and of the international reference preparation on the other.

Use of in vitro Vero cell assay and ELISA in the United States potency test of vaccines containing adsorbed diphtheria and tetanus toxoids.

Current United States (US) regulations for potency testing of vaccines containing adsorbed diphtheria and tetanus toxoids require in vivo toxin neutralization (TN) tests on the pooled sera of immunized guinea pigs. To reduce the number of animals required for testing, two in vitro tests have been evaluated, the Vero cell assay for diphtheria antitoxin and ELISA for IgG antibody to tetanus toxin; these have been correlated with in vivo TN tests. In the Vero cell method, diphtheria antitoxin titres of the guinea pig sera, obtained four weeks after immunization as per US potency requirements, were markedly dependent on the toxin dose level used in the assay. A toxin dose level termed the Lcd/1 dose (limit of cytopathic dose at a IU/ml) for Vero cells gave comparable estimates of antitoxin activity to the in vivo TN test performed at L+/1 dose of toxin. When lower dose levels of toxin were used in the Vero cells (Lcd/10 to Lcd/1000), diphtheria antitoxin levels in four weeks guinea pig sera were two to 11.7 times lower than with the Lcd/1 dose level. The most likely reason for these differences is that guinea pig sera a 4 weeks are of lower avidity than the equine antitoxin standard. Antibodies of low avidity bind antigen less well at low reactant concentrations. Therefore, to obtain similar estimates of diphtheria antitoxin in the Vero cell method and in vivo TN test, the use of toxin dose for the Vero cell method similar to that for the in vivo TN test is suggested. Another alternative, in which any dose of toxin may be used for the Vero cell method, is the use of a reference guinea pig serum (calibrated in IU/ml by the in vivo TN test at L+/1 level of toxin) that has similar avidity or similar immunization status as the test sera (i.e. 4 week serum). IgG antibodies to tetanus toxin in guinea pig sera were found early in the course of immunization when tetanus antitoxin could not be detected by TN test. Tetanus toxin IgG antibody levels of guinea pig sera calculated in IU/ml against an ELISA guinea pig reference serum (calibrated in IU/ml by TN test) depended upon the immunization status of the animals. To obtain similar estimates of tetanus antibodies in IU/ml by TN and ELISA, the ELISA reference guinea pig serum should have similar immunization status (and presumably similar avidity) as the test serum (i.e. six week serum). We propose that the Vero cell method and ELISA deserve further evaluation to determine whether they can replace in vivo TN tests for titration of diphtheria and tetanus antitoxins in the US potency test.