Report of a collaborative study to calibrate the Second International Standard for parvovirus B19 antibody

A collaborative study was undertaken to assess the suitability of a replacement for the First International Standard for parvovirus B19 IgG, human serum and to calibrate it in IU. The proposed standard, which is a pool of sera from 16 US blood donors, was assayed along with the First International Standard, a coded duplicate of the proposed standard and a plasma sample from a single blood donor. Nine laboratories from eight countries participated in the studies and five different assay kits were used. Two kits contained VP1+VP2, one kit contained VP1 only and two kits, one of which was used by five participants contained VP2 only. Differences in detection of the proposed standard and the individual plasma were observed with assay kits containing different antigens, VP1, VP2 or VP1+VP2. However, since VP1 is a minor capsid protein and on its own does not assemble into virus like particles and the dominant response in individuals appears to be against VP2, it was considered reasonable to utilize only the data from kits containing VP2 antigen for the calibration of the proposed standard. The results of this study demonstrated that the proposed standard coded 01/602 was suitable to serve as the replacement International Standard for parvovirus B19, serum IgG, and this preparation was established as the Second International Standard for parvovirus B19 antibody, plasma human, with an assigned unitage of 77 IU per ampoule by the Expert Committee on Biological Standardisation of the World Health Organisation in February 2003.     

A collaborative study to establish the International Reference Reagent for hepatitis B vaccine containing plasma-derived hepatitis B surface antigen

A collaborative study was conducted to establish a suitable international reference reagent for hepatitis B vaccine for use in immunogenicity assays. The limiting dilution required to induce antibodies in 50% of the test animals was determined for the proposed international reference reagent and three other plasma-derived hepatitis B vaccines. The minimum antigenic dose of these preparations varied widely (100-fold range) between laboratories. However, the expression of potencies of vaccines relative to the proposed International Reference Reagent reduced the variation between laboratories to within a 10-fold range. The reference reagent is intended for use in assays of hepatitis B vaccines in mouse (or guinea-pig) immunogenicity studies. For products made by different procedures, clinical trials in humans are necessary to establish a correlation between the immunogenic potency in animals and man.     

Standardization of therapeutic,urinary gonadotrophins: An update on the use and availability of International Standards for Menotrophin

The potencies of therapeutic preparations of gonadotrophins of human, urinary origin, which comprise a heterogenous mix of isoforms with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) bioactivities, are standardized by WHO International Standards (IS). We report here, the evaluation, through an international collaborative study, of a candidate preparation, coded 10/286, to replace the 4th IS, 98/704, for human, urinary FSH and LH (Menotrophin) which has been used for many years for the potency assignment of therapeutic preparations using bioassays. The mean FSH and LH bioactivities of 10/286, determined by in vivo bioassays in terms of 98/704, were 183 IU per ampoule (95% confidence limits 165–202) and 177 IU per ampoule (95% confidence limits 159–197), respectively.     

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.     

Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies

A collaborative study was designed to asses two freeze-dried human plasma preparations containing anti-Treponema pallidum antibodies, 05/132 and 05/122, for their suitability as international reference reagents for syphilis serology. Both preparations are intended as replacements of the first international standard (IS) for syphilitic serum antibodies (HS). Samples were tested by eight laboratories using the T. pallidum passive particle agglutination assay (TPPA), the venereal disease research laboratory test (VDRL) and the rapid plasma reagin test (RPR). In addition a range of immunoassays was also used. The outcome of the collaborative study revealed that candidate standard 05/132 contains T. pallidum-specific IgG and IgM and is reactive in VDRL or RPR, and that 05/122 contains T. pallidum-specific IgG but is not reactive in either the VDRL or RPR test. Both 05/132 and 05/122 are reactive in the TPPA. On the basis of these results the Expert Committee on Biological Standardization of the World Health Organization designated 05/132 as the 1st IS for human syphilitic plasma IgG and IgM with a unitage of 3 IU per ampoule relative to HS and 05/122 as the 1st IS for human syphilitic plasma IgG with a unitage of 300 mIU per ampoule relative to 05/132.     

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.     

Calibration of replacement international standard and European pharmacopoeia biological reference preparation for tetanus toxoid, adsorbed.

Here we report the characterisation of a preparation of tetanus toxoid, adsorbed, and its calibration by 27 laboratories in 19 countries in a joint international collaborative study co-sponsored by World Health Organization (WHO) Expert Committee of Biological Standardization (ECBS) and the European Biological Standardisation Programme of European Directorate for the Quality of Medicines (EDQM), Council of Europe. Calibration was in terms of the Second International Standard (I.S.) for Tetanus Toxoid, Adsorbed, by the established WHO/European Pharmacopoeia (Ph Eur) challenge methods. The replacement standard preparation was found to have a unitage of 469 IU/ampoule on the basis of its calibration in guinea-pigs and 496 IU/ampoule on the basis of its calibration in mice. Assessment, both within the collaborative study and as part of candidate characterisation, indicated satisfactory stability of the candidate preparation. This study also provided some information on the effect of mouse strain on potency testing of tetanus vaccines. A limited assessment of the impact of the replacement standard on testing of current production batches of vaccines was also carried out by four manufacturers. This study did not directly address the serological approaches to potency testing. However, one laboratory offered data from mouse serology assay, which gave comparable estimates to in vivo mouse bioassay.Based on the results of this study and with the agreement of participants, the candidate standard was established as the Third International Standard for Tetanus Toxoid, Adsorbed (coded 98/552) by the WHO Expert Committee of Biological Standardization (ECBS) in November 2000. The same preparation was also established as the second Ph Eur Biological Reference Preparation (Ph Eur BRP, batch no. 2) by the Steering Committee of the Biological Standardisation Programme of the EDQM and approved by the European Pharmacopoeia Commission.     

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.     

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.     

Preparation of a respiratory syncytial virus human reference serum for use in the quantitation of neutralization antibody.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, young children and the elderly. Yet, the development of a vaccine to protect against RSV infection still remains an unmet need. At present, immune responses to experimental vaccines under investigation are usually evaluated by ELISA and/or by neutralization assays against RSV. However, both types of assays are generally performed somewhat differently at different laboratories. An important step towards standardization of serology is the use of a standard human reference serum enabling normalization of results generated within and between laboratories. To fill this need, we prepared and characterized a human reference serum against the A2 strain of respiratory syncytial virus. The serum represents a pool of more than 400 individual human sera obtained from commercial sources. The sera were screened and selected on the basis of individual RSV neutralization titers. A final neutralization titer of 973 (95% C.I., 884-1072) was assigned to the final reference serum pool after it was tested three times in the presence of 10% guinea pig complement and a titer of 286 (95% C.I., 243-337) was assigned to the serum when it was tested in the absence of an exogenous complement source. Sterilely reconstituted lyophilized aliquots of the serum exhibited a stable neutralization titer for at least 1 month at room temperature and at 4 degrees C, as well as after 5 weekly freeze-and-thaw cycles at -20 degrees C. In the lyophilized state, the neutralization titer of the lyophilized reagent was stable for at least 6 months, the last time point tested. Two additional smaller pools of serum with high and medium neutralization titers of 2692 and 575, respectively, were also produced in parallel for use as positive controls and were designated as control sera. The reference serum can be used to normalize neutralization and/or other RSV-specific assay results from different laboratories and the control sera can be used for quality control purposes or as part of a panel to test operator proficiency. Individual lyophilized aliquots of the reference and control sera may be obtained from the US National Institute of Allergy and Infectious Diseases (NIAID) Reference Reagent Repository.     

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.     

Evaluation of a candidate International Standard for Meningococcal Group C polysaccharide

Meningococcal group C (MenC) plain polysaccharide (PS) and conjugate vaccines are primarily evaluated by physicochemical methods to ensure that batches are consistently manufactured. As different assays are employed to quantify the MenC PS content of final formulations and bulk intermediaries, there is a need for an International MenC PS Standard to calibrate internal references used in the different laboratories. Twelve laboratories from nine different countries participated in a collaborative study to determine the MenC PS content of a candidate International Standard MenC PS preparation (08/214) and to assess its suitability. On the basis of the results from this study the candidate standard 08/214 was established as an International Standard for the quantification of MenC PS content in vaccines and components. It has a content of 1.192 ± 0.192 mg MenC PS/ampoule (expanded uncertainty with coverage factor of k = 2.365 corresponding to a 95% level of confidence), as determined by the resorcinol assays carried out by eight of the participating laboratories. The standard is available from The National Institute of Biological Standards and Control who act as guardians and distributors of the material under the auspices of WHO.     

Collaborative study to establish a proficiency panel of hepatitis B surface antigens for evaluating in vitro diagnostics

Hepatitis B virus (HBV) infection is a critical health issue. In hospitals, in vitro diagnostic devices (IVDs) are used to detect HBV infections. The Taiwan Food and Drug Administration has prepared a candidate hepatitis B surface antigen (HBsAg) proficiency panel as an analytical standard to evaluate the sensitivity of the postmarketing surveillance of HBV IVDs. The sensitivity and stability of the proficiency panel, which comprises 5 HBsAg positive sera (SN-A to E) and 1 negative serum, was determined by a collaborative study involving 8 collaboratories. The potency of the candidate proficiency panel was consistent among all collaboratories. The potencies of sera SN-A to E in semiquantitative and quantitative analyses were 2.823–0.011 and 2.229–0.009 IU/mL, respectively. The mean coefficients of variation (CV) of intra and interday precision among the collaboratories were <7%. Accelerated degradation and long-term storage tests were used as stability assays. The candidate HBsAg proficiency panel exhibited good stability under different storage conditions, while all CVs were <6%. In conclusion, this candidate HBsAg proficiency panel is credible and reliable. Therefore, it can be used for postmarketing surveillance of HBV IVDs and quality assessment by clinical researchers and IVD manufacturers.     

The International Standard for Kanamycin

The International Reference Preparation of Kanamycin has been replaced by the International Standard for Kanamycin. The potency of the standard is based upon a collaborative study in ten laboratories in ten countries. Each ampoule of the International Standard of Kanamycin is defined as containing the activity of 10345 International Units of Kanamycin.     

Analytical Bias in the Measurement of Serum 25-Hydroxyvitamin D Concentrations Impairs Assessment of Vitamin D Status in Clinical and Research Settings

Measured serum 25-hydroxyvitamin D concentrations vary depending on the type of assay used and the specific laboratory undertaking the analysis, impairing the accurate assessment of vitamin D status. We investigated differences in serum 25-hydroxyvitamin D concentrations measured at three laboratories (laboratories A and B using an assay based on liquid chromatography-tandem mass spectrometry and laboratory C using a DiaSorin Liaison assay), against a laboratory using an assay based on liquid chromatography-tandem mass spectrometry that is certified to the standard reference method developed by the National Institute of Standards and Technology and Ghent University (referred to as the ‘certified laboratory’). Separate aliquots from the same original serum sample for a subset of 50 participants from the Ausimmune Study were analysed at the four laboratories. Bland-Altman plots were used to visually check agreement between each laboratory against the certified laboratory. Compared with the certified laboratory, serum 25-hydroxyvitamin D concentrations were on average 12.4 nmol/L higher at laboratory A (95% limits of agreement: -17.8,42.6); 12.8 nmol/L higher at laboratory B (95% limits of agreement: 0.8,24.8); and 10.6 nmol/L lower at laboratory C (95% limits of agreement: -48.4,27.1). The prevalence of vitamin D deficiency (defined here as 25-hydroxyvitamin D <50 nmol/L) was 24%, 16%, 12% and 41% at the certified laboratory, and laboratories A, B, and C, respectively. Our results demonstrate considerable differences in the measurement of 25-hydroxyvitamin D concentrations compared with a certified laboratory, even between laboratories using assays based on liquid chromatography-tandem mass spectrometry, which is often considered the gold-standard assay. To ensure accurate and reliable measurement of serum 25-hydroxyvitamin D concentrations, all laboratories should use an accuracy-based quality assurance system and, ideally, comply with international standardisation efforts.     

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.     

A collaborative assay of the proposed third British Reference Preparation for Pertussis Vaccine and of the relative potencies of the second International Standard and the second British Reference Preparation for Pertussis Vaccine

A collaborative assay has been carried out to estimate the mouse protective potency of a freeze-dried preparation of Bordetella pertussis (88/522) intended to serve as the third British Reference Preparation for Pertussis Vaccine (third BRP). The opportunity was also taken of reassessing the relationship between the second International Standard for Pertussis Vaccine and the second British Reference Preparation for Pertussis Vaccine (second BRP). Workers in nine laboratories took part in the study and together completed 19 assays which were considered to be statistically valid. Based on the results of the study it is proposed that ampouled preparation code number 88/522 be established as the third BRP with an assigned potency of 50 IU per ampoule. The evidence of this study also suggests that the relationship between the second International Standard for Pertussis Vaccine and the second BRP has not changed significantly since they were originally established.     

Development and calibration of a standard for the protein content of granulocyte colony-stimulating factor products

This collaborative study characterizes a homogeneous standard for the protein content determination of granulocyte colony-stimulating factor (G-CSF) products with traceability of the measurement. The Kjeldahl method was used to determine the average protein content of G-CSF bulk as 2.505 mg/ml (95% C.I: 2.467-2.543 mg/ml, GCV 4.0%). Using G-CSF bulk as a traceability benchmark, the protein content of the final freeze-dried standard using reverse phase HPLC (RP-HPLC) was 215.4 μg protein per ampoule (95% C.I: 212.407-218.486 μg/ampoule, GCV 3.4%). A comparative study showed that there was no difference between using Filgrastim CRS (European Pharmacopeia G-CSF reference standard) and freeze-dried homogeneous standard when quantifying G-CSF protein content by RP-HPLC (P > 0.05). However, there were significant differences in the G-CSF protein content obtained using a serum albumin standard by Lowry assay and a G-CSF standard with RP-HPLC. Therefore, use of RP-HPLC with a freeze-dried homogeneous standard would eliminate the systematic errors introduced when using a serum albumin standard because of the differences in protein composition between the standard and the sample. It would also be helpful to use this method to compare the quality of G-CSF biosimilar products in situations where the protein content has been calibrated using various standards.     

International collaborative study: evaluation of proposed International Reference Reagent of pertussis antiserum (mouse) 97/642.

A freeze dried preparation of mouse serum in vials coded 97/642 containing antibodies to five pertussis antigens [pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbriae type 2 and 3 (Fim 2 and 3)] has been assessed for its suitability as an international reference reagent in an international collaborative study by thirteen laboratories in nine countries. This serum has been compared with U.S. Standard Pertussis Antiserum (mouse) Lot No. 1 (US Lot 1), which has been in use since 1995, for antibodies for each antigen. Calibration of the proposed International Reference Reagent of Pertussis Antiserum (pIRR) in terms of US Lot 1 gives results which are broadly consistent between laboratories for antibodies to each antigen, although the between-laboratory differences are larger than those seen for comparison of identical sera. Calibration of two positive control sera in terms of the pIRR gave similar between laboratory variability of estimates to that obtained when the same sera were calibrated in terms of US Lot 1. Overall continuity of estimates is maintained if units are assigned to the pIRR based on its calibration in terms of US Lot 1 in this study. Data presently available indicate that the pIRR is sufficiently stable to serve as a reference reagent. It was therefore recommended, with the agreement of all participants, that the preparation in vials coded 97/642 be established as the First International Reference Reagent for Pertussis Antiserum, mouse, with assigned unitages 16 units of anti-PT per vial, 143 units of anti-FHA per vial and 30 units of anti-PRN per vial based on its calibration in terms of US Lot 1. These unitages are also consistent with calibration of 97/642 in terms of the Japanese preparations JNIH-11 for anti-FHA and of JNIH-12 for anti-PT. Purified antigens for Fim 2 and Fim 3 are not readily available and an arbitrary value of 32 units per vial is suggested for anti-Fim 2 and 3 mixture. These recommendations were agreed by the Expert Committee on Biological Standardization of the World Health Organization.     

Standardization: a Progress Report

The introduction of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products requires the availability of well-characterized reference reagents. Working reagents for hepatitis C virus RNA, hepatitis B virus DNA, HIV-1 RNA and human parvovirus B19 DNA have been established at NIBSC and at many other laboratories (both official medicinal control laboratories and commercial laboratories). However, as these reagents have been characterised independently, it is difficult to compare results from assays using different working reagents. Recently, a WHO International Standard was established for HCV RNA NAT assays. This standard has been calibrated in International Units (IU) and provides a common standard against which all working reagents can be calibrated. Collaborative studies to characterise two further candidate International Standards for HBV DNA and HIV-1 RNA NAT assays have been completed.