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.     

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.     

Collaborative study for the calibration of a replacement International Standard for Tetanus Toxoid Adsorbed

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for Tetanus Toxoid Adsorbed. Two candidate preparations were included in the study, one of which was established as the 4th IS for Tetanus Toxoid Adsorbed at the WHO Expert Committee on Biological Standardization meeting in October 2010. This preparation was found to have a unitage of 490 IU/ampoule, based on calibration in guinea pig challenge assays. Results from mouse challenge assays suggest that the relative performance of two candidate preparations may differ significantly between guinea pigs and mice. The authors note that the number of laboratories that performed guinea pig challenge assays, which are used to calibrate and assign IU, is much lower than in previous collaborative studies and this may have implications for calibration of replacement standards in the future. The issue of assigning separate units to the IS for guinea pig and mouse assays is discussed. The study also assessed performance of the replacement standard in serological assays which are used as alternative procedures to challenge assays for tetanus potency testing. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays.     

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.     

Evaluation of Fourth International Standard for Whole Cell Pertussis Vaccine

Whole cell pertussis vaccine is still widely used in many countries. An International Standard is needed for its potency control. The Third International Standard for Pertussis Vaccine was prepared about 40 years ago and its replacement was recommended by the Expert Committee for Biological Standardisation (ECBS) of the WHO. Material in ampoules coded 94/532 was prepared as a candidate replacement and has been evaluated in international collaborative studies which consisted of two parts. The first part, to assess the suitability of the candidate standard by comparing it with the Second International Standard for Pertussis Vaccine (IS2) involved 14 laboratories in 11 countries. The second part to compare the candidate standard with the Third International Standard for Pertussis Vaccine (IS3) involved 16 laboratories in 14 countries. Since 1995 various other studies have included the international standards and the results of these are also considered in assessing likely continuity of the IU for potency of whole cell pertussis vaccine. The preparation in ampoules coded 94/532 was adopted by the WHO ECBS in October 2006 as the 4th International Standard for whole cell pertussis vaccine and assigned an activity of 40 IU per ampoule on the basis of the studies reported here.     

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.     

Collaborative study for the establishment of a European Phamacopoeia Biological reference preparation for Bordetella pertussis mouse antiserum for serological potency testing of acellular pertussis vaccines.

A collaborative study was organised by the European Directorate For the Quality of Medicines (EDQM) to assess the suitability of a candidate mouse antiserum as a European Pharmacopoeia Biological reference preparation (BRP) for acellular pertussis vaccine potency testing. The candidate antiserum was obtained by immunising mice with a five-component acellular pertussis vaccine: pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN) and Fimbrial 2/Fimbrial 3 (Fim 2&3).The study has been divided into two separate phases. Phase I was a pre-qualification study including three laboratories. This phase was aimed at pre-qualifying the candidate BRP (cBRP) and at documenting the impact of differences in the antibody detection methodology enzyme linked immunosorbent assay (ELISA) procedures on results of pertussis antisera calibration versus the currently used standard US standard pertussis antiserum (mouse) Lot 1 (SPAM-1) (United States Food and Drug Administration (USFDA) reference serum) and the cBRP. As no significant difference between the antibody titres determined by using the different ELISA methodologies was found, a large-scale study enrolling 13 laboratories (Phase II) was carried out, each participant performing its in-house methodology. Its aim was to calibrate the cBRP (in terms of the SPAM-1 reference) and to demonstrate its equivalence or superiority to internal references. The study showed that there was no difference in positive sera titres expressed relative to their corresponding internal reference (homologous situation) or the proposed standard (heterologous situation) reference. The cBRP can, therefore, reliably act as replacement for the in-house reference preparations. Further analysis of the outcome of this study enabled to assign to the cBRP a potency of 39, 138, 34 and 56 ELISA unit per millilitre, respectively, to its anti-PT, anti-FHA, anti-PRN and anti-Fim 2&3 antibody contents. The cBRP has been adopted by the European Pharmacopoeia Commission at its June 2000 session as Bordetella pertussis mouse anti-serum Ph Eur. BRP batch 1.     

WHO international reference reagents for human proinsulin and human insulin C-peptide

Candidate preparations for international reference reagents for immunoassays of human proinsulin and human insulin C-peptide were evaluated in an international collaborative study. With the authorization of the Expert Committee on Biological Standardization of WHO, the following preparations were established as international reference reagents: human proinsulin (84/611, nominal ampoule content 6 micrograms) and human insulin C-peptide (84/510, 10 micrograms). Each preparation is intended as a primary reference reagent for the calibration of immunoassays.     

Collaborative study for the validation of alternative in vitro potency assays for human tetanus immunoglobulins

An international collaborative study to validate 2 alternative in vitro methods for the potency testing of human tetanus immunoglobulin products was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). The study, run in the framework of the Biological Standardisation Programme (BSP) under the aegis of the European Commission and the Council of Europe, involved 21 official medicines control and industry laboratories from 15 countries.Both methods, an enzyme-linked immunoassay (EIA) and a toxoid inhibition assay (TIA), showed good reproducibility, repeatability and precision. EIA and TIA discriminated between low, medium and high potency samples. Potency estimates correlated well and both values were in close agreement with those obtained by in vivo methods. Moreover, these alternative methods allowed to resolve discrepant results between laboratories that were due to product potency loss and reporting errors.The study demonstrated that EIA and TIA are suitable quality control methods for tetanus immunoglobulin, which can be standardised in a control laboratory using a quality assurance system. Consequently, the Group of Experts on Human Blood and Blood Products of the European Pharmacopoeia revised the monograph on human tetanus immunoglobulins to include both the methods as compendial alternatives to the in vivo mouse challenge assay.     

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.     

WHO international reference reagents for bovine and porcine proinsulins

Candidate preparations for International Reference Reagents (IRR) for immunoassays of bovine and porcine proinsulin were evaluated in an international collaborative study. With the authorization of the Expert Committee on Biological Standardization of WHO, the following preparations were established as IRRs: bovine proinsulin (code 84/514, defined ampoule content 25 micrograms) and porcine proinsulin (84/528, 20 micrograms). The content of ampoules of these materials is defined in terms of mass rather than international units of activity, therefore they are IRR rather than International Standards. Both preparations are intended as primary reference reagents for the calibration of immunoassays.     

World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the “1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection” (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.     

Simplified activity evaluation of several tetanus vaccines.

The activity of several Tetanus Toxoids, Adsorbed, (commercial vaccines and references) were tested in mice in comparison with a standard, by a simple method, easier than the official challenge test (WHO and European Pharmacopoeia): the Tetanus Antitoxin level was titrated by agglutination of sensitized turkey red blood cells after immunization by the toxoids. Immuno-stimulation by the Pertussis component in associated vaccines was studied and the results with the conventional and the acellular Pertussis preparations were prepared. The method was also found to be suitable for Tetanus Toxoids, Non-Adsorbed, when a booster effect was used, except for the adjuvant-free polymerized antigen (POLAN) which did not require a booster, since it gave almost as good results as conventional adsorbed tetanus vaccines.     

Hepatitis A Immunoglobulin: An International Collaborative Study to Establish the Second International Standard

A collaborative study was carried out to assess the suitability of a candidate replacement material for the International Standard for hepatitis A immunoglobulin, which was found to be reactive for HCV RNA, and to calibrate it in International Units. The candidate standard, coded 97/646, was derived from a bulk of 16% immunoglobulin supplied by the Central Laboratory of the Netherlands Red Cross, Amsterdam, and diluted 1 in 2 in H2O resulting in a final immunoglobulin concentration of 8%. Sixteen laboratories from 11 countries participated in the study and contributed data from 64 assays performed using six commercial assay kits and four in-house methods. All assays were analysed as parallel line bioassays comparing assay response with log concentration. The overall mean potency of the candidate replacement immunoglobulin standard, 97/646, relative to the International Standard for hepatitis A immunoglobulin, was 98.6 IU/ml. A freeze-dried serum preparation, 97/648, was also calibrated in this study and had a potency of 22.64 IU/ml. The Second International Standard for hepatitis A immunoglobulin, human, was established by the World Health Organisation Expert Committee on Biological Standardisation in 1998 with a potency of 49 IU per ampoule when reconstituted in 0.5 ml.     

An international collaborative study on the First International Standard of bovine luteinizing hormone for immunoassay

An ampouled freeze-dried preparation of bovine pituitary luteinizing hormone (bLH), coded EHC-bLH-1, has been evaluated in an international collaborative study and shown to be suitable and sufficiently stable to serve as a standard for bLH. Eight laboratories provided immunoassay data, one laboratory provided receptor assay data, and bioassay data were obtained from 4 laboratories. The geometric mean potency estimate obtained by immunoassays, expressed as milliunits of the USDA bLH-B-5 preparation per ampoule, was 25.6, which is consistent with the result obtained by in-vivo bioassays. The geometric mean estimate obtained by receptor assays or by in-vitro bioassays was lower, i.e. 13.2 milliunits per ampoule. The reason for this discrepancy is currently under investigation. With the authorization of the Expert Committee on Biological Standardization of the World Health Organization this preparation was established in 1985 as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay with a unitage of 25 mi.u. per ampoule.     

European regulatory framework and practices for veterinary Leptospira vaccine potency testing

In Europe, the legal basis for requirements for medicinal products is described in the European Pharmacopoeia (Ph. Eur.) In the European Union, the Ph. Eur. is supplemented by several guidelines issued by the European Medicines Agency. Immunological veterinary products must comply with the Ph. Eur. monograph on veterinary vaccines and the accompanying texts, as well as specific monographs. The Ph. Eur. includes monographs on canine leptospirosis and bovine leptospirosis vaccines (inactivated). Both monographs require that an immunogenicity test be performed once in the target species during the life of a vaccine. The hamster challenge test is applied for batch potency testing of canine vaccines. Alternatively, serological tests or suitable validated in vitro tests to determine the content of one or more antigenic components indicative of protection may be performed. Vaccines for use in cattle are tested in a serological test in guinea pigs. The acceptance criteria in alternative tests are set with reference to a batch of vaccine that has given satisfactory results in the immunogenicity test. At a January 2012 European workshop, the suitability of the hamster potency test was questioned and unanimous agreement was reached that moving toward complete in vitro testing is possible and should be promoted.     

Comparison of requirements in the European Union and United States of America for pre-clinical viral safety testing of veterinary vaccines

Of paramount importance in ensuring the safety of live and inactivated veterinary vaccines is demonstration of freedom from extraneous agents in biological starting materials used in their production. Both the European Union (EU) and United States of America (US) provide regulations and guidelines on extraneous agent testing of veterinary vaccines including guidance from the Committee for Medicinal Products for Veterinary Use (CVMP), the European Pharmacopoeia (Ph. Eur.) and the USDA Code of Federal Regulations, Title 9 (9CFR). There are distinct requirements prescribed in EU and US regulations and guidelines. The differences in EU and US requirements for extraneous agent testing of starting materials are such that there may be occasions when no one test may satisfy both sets of regulations for a given scenario. For compliance with both, for global licensing purposes it may therefore be necessary to perform additional tests and/or to justify methods chosen from one set of regulations over another, based on a variety of factors.     

Evaluation of cell substrates for the production of biologicals: Revision of WHO recommendations: Report of the WHO Study Group on Cell Substrates for the Production of Biologicals, 22–23 April 2009, Bethesda,USA

Evaluating cell substrates for producing vaccines and other biologicals is one of the critical aspects in assuring quality and safety of these products. As part of its mission in setting standards for biological products, WHO provides recommendations for manufacturing and evaluating biologicals. Regular updates of the guidance documents are important to manufacturers and regulators worldwide. WHO Expert Committee on Biological Standardization (ECBS) identified a need for revising the requirements for cell substrates (WHO TRS 878, annex 1). In response, WHO established a Study Group (SG) in 2006 that prepared an updated set of recommendations for using cell substrates for the production of biologicals. A summary of the proposed changes that the SG made in 2007 is available at WHO web site (http://www.who.int/biologicals/publications/meetings/areas/vaccines/cells/en/index.html). Draft revised recommendations were circulated to regulators, manufacturers and other experts for comments in April 2009.The SG held its third meeting on 22–23 April 2009 to review progress in the revision and to propose further improvements. In addition, the experts discussed the need for reference preparations, reference cell banks, and standardization of testing methodologies. The SG proposed clarifications of the rationale for in vivo testing as well as the potential for applying new methods for in vitro testing for detecting microbial agents. In line with this, WHO should conduct review of the current manufacturers' practice in using tests for microbial agents and interpreting these results. Additionally, WHO should take a lead in developing an International Standard for nucleic acid amplification test (NAT) for detecting mycoplasma contamination in cell substrates. WHO Collaborating Centers will lead this initiative, involving other relevant institutions in this area. Finally, advice on the replacement of the WHO Vero reference cell bank 10–87 with respect to the source of cells and re-characterization of the bank was provided. The intended use of the replacement cell bank would be the same as for the current cell bank, which is to serve as a source of well-characterized cells for establishing master cell banks for the production of biologicals. The SG will report outcomes of its discussion to the ECBS at its next meeting in October 2009 for further considerations and advice regarding the proposed course of action.     

The Second International Standard for somatropin (recombinant DNA-derived human growth hormone): preparation and calibration in an international collaborative study.

A preparation of somatropin (recombinant DNA-derived human growth hormone) was prepared as lyophilised ampoules according to WHO procedures for international biological standards. The candidate preparation (98/574) was evaluated in an international collaborative study (16 laboratories, nine countries), with the following aims: (i) to determine the suitability of the preparation to serve as the International Standard for somatropin by studying its performance in the current range of physico-chemical and biological assay methods employed for somatropin; (ii) to assign a content in terms of the existing (first) International Standard for somatropin, using the currently recognised assay procedure (Size Exclusion High Performance Liquid Chromatography, SE HPLC); (iii) to confirm the specific biological activity of the candidate preparation; (iv) to confirm the stability of the candidate preparation. On the basis of the collaborative study WHO agreed that: the preparation in ampoules coded 98/574 is suitable to serve as the next WHO International Standard for somatropin; the preparation in ampoules coded 98/574 should be established as the second International Standard for somatropin, with a defined ampoule content of 1.95 mg total somatropin plus somatropin-related proteins per ampoule; the specific activity of the preparation should be defined as 3.0 IU/mg somatropin.     

Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human

The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.