Utility of reference materials for Zika Virus nucleic acid testing.

The emergence of Zika virus (ZIKV) in the Americas has resulted in increased nucleic acid amplification testing (NAT) of clinical samples and blood donations. New molecular diagnostic assays have been developed resulting in a corollary requirement for ZIKV reference material. To address this we have produced and calibrated two African lineage ZIKV reference materials: a highly concentrated secondary standard (NIBSC: 16/110) and a lower concentration external quality control (QC) reagent (NIBSC: 16/124) and compared their performance in three ZIKV NAT assays in relation with the First International Standard (IS) for Zika Virus NAT assays (PEI: 11468/16). In summary the African lineage ZIKV reference materials were detected by all three assays. The ZIKV lineage did not affect the performance of the secondary standard. The external QC reagent (16/124) was detected by all three assays highlighting its suitability for use as a low positive control to monitor assay performance on a regular basis. The relative potency of 16/110 to the IS was 5.49E+06IU/mL (95% CI: 1.46E+06-2.06E+07) and 16/124 to 16/110 was 8.36E+03 (95% CI: 7.83E+03-8.92E+03). The global availability of African lineage ZIKV reference materials will facilitate standardization of ZIKV molecular diagnostic assays between and within laboratories whilst preserving the IS.

The first World Health Organization International Standard for infliximab products: A step towards maintaining harmonized biological activity.

Due to the increase in the number of infliximab products, the need for global harmonization of the bioactivity of this monoclonal antibody was recognized by the World Health Organization (WHO). In response, the National Institute for Biological Standards and Control (NIBSC) developed the first international standard (IS) for infliximab, which targets tumour necrosis factor (TNF). Each ampoule is assigned values of 500 IU of TNF neutralizing activity and 500 IU of binding activity. Two preparations of infliximab were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as an IS for the in vitro biological activity of infliximab. The study involved participants using in vitro cell-based bioassays (TNF neutralization, antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity) and binding assays. The results of this study showed that the candidate preparation, coded 16/170, is suitable as an IS for infliximab bioactivity. This infliximab IS from NIBSC, is intended to support in vitro bioassay calibration and validation by defining international units of bioactivity. The proposed unitages, however, are not intended to revise product labelling or dosing requirements, as any decisions regarding this relies solely with the regulatory authorities. Furthermore, the infliximab IS is not intended for determining the specific activity of products, nor to serve any regulatory role in defining biosimilarity. We briefly discuss the future use of WHO international standards in supporting the global harmonisation of biosimilar infliximab products.

Establishment of the first WHO International Standard for antiserum to Respiratory Syncytial Virus: Report of an international collaborative study.

Respiratory Syncytial Virus (RSV), a leading cause of lower respiratory tract illness, has been a focus of vaccine development efforts in recent years. RSV neutralisation assays are particularly useful in the evaluation of immunogenicity of RSV vaccine candidates. Here we report a collaborative study that was conducted with the aim to establish the 1st International Standard for antiserum to RSV, to enable the standardisation of results across multiple assay formats. Two candidate standards were produced from serum samples donated by healthy adult individuals. 25 laboratories from 12 countries, including university laboratories, manufacturers/developers of RSV vaccines and public health laboratories, participated in the study. The study samples comprised the two candidate standards, NIBSC codes 16/284 and 16/322, naturally infected adult sera, age stratified naturally infected paediatric sera, sera from RSV vaccine clinical trials in maternal and elderly subjects, a monoclonal antibody to RSV (palivizumab), two cotton rat serum samples and samples from the BEI Resources panel of human antiserum and immune globulin to RSV. The collaborative study showed that between-laboratory variability in neutralisation titres was substantially reduced when values were expressed relative to those of either of the two candidate international standards. Stability of 16/284 and 16/322 maintained for 6 months at different temperatures showed no significant loss of activity (relative to that at -20 °C storage temperature) at temperatures of up to +20 °C. Based on these results, 16/284 was established as the 1st International Standard for antiserum to RSV, with an assigned unitage of 1000 International Units (IU) of anti-RSV neutralising antibodies per vial, by the WHO Expert Committee on Biological Standardisation, with 16/322 suitable as a possible replacement standard for 16/284.

Calibration of the 7th British Working Standard for factors II, IX and X, concentrate.

Seven laboratories from 5 different countries participated in the calibration of the 7th British Working Standard (BWS) for blood coagulation factors II, IX and X. The candidate, 15/182, was assayed for Factors II and X potencies against the 4th International Standard (IS) for Factors II and X, Concentrate (11/126) and for Factor IX potency against the 5th IS for Factor IX, Concentrate (14/148). Intra-laboratory GCVs for all 3 factors were less than 10%, with the majority less than 5%. Inter-laboratory GCVs were 3.4%, 3.2% and 2.3% for FII, IX and X respectively. All participants agreed with the value assigned and preparation 15/182 was established by NIBSC in October 2017 as the 7th BWS for FII, IX, X Concentrate with potencies of 6.0 IU/ampoule, 6.7 IU/ampoule and 4.9 IU/ampoule for FII, IX and X respectively.

Establishment of the 1st WHO International Standard for anti-EV71 serum (Human).

Enterovirus A71 (EV71) is the major causative agent of severe and fatal hand, foot and mouth disease. There is plenty of evidence that EV71 has circulated widely in the Western Pacific Region for the last twenty years. Vaccines against EV71 are already available or under development. A collaborative study to establish the 1st WHO International Standard for anti-EV71 serum (Human) was conducted to ensure that methods used to measure the serum neutralizing activity or antibody levels against EV71 are accurate, sensitive and reproducible. Two candidate samples as well as a third candidate reference containing low anti-EV71 antibody titre were produced from plasma samples donated by healthy individuals. All three serum samples exhibited good levels of neutralizing antibodies against a wide range of EV71 strains of various genotypes. The study showed that between laboratory variations in neutralization titres were significantly reduced when values were expressed relative to those of either of the two candidate sera. Sample 14/140 was established as the WHO 1st International Standard for anti-EV71 serum (human), 14/138 as its potential replacement and 13/238 as a WHO Reference Reagent, with assigned unitage of 1,000, 1090 and 300 International Units (IU) of anti-EV71 neutralizing antibodies per ampoule, respectively.

International standards for monoclonal antibodies to support pre- and post-marketing product consistency: Evaluation of a candidate international standard for the bioactivities of rituximab.

The intrinsic complexity and heterogeneity of therapeutic monoclonal antibodies is built into the biosimilarity paradigm where critical quality attributes are controlled in exhaustive comparability studies with the reference medicinal product. The long-term success of biosimilars will depend on reassuring healthcare professionals and patients of consistent product quality, safety and efficacy. With this aim, the World Health Organization has endorsed the need for public bioactivity standards for therapeutic monoclonal antibodies in support of current controls. We have developed a candidate international potency standard for rituximab that was evaluated in a multi-center collaborative study using participants' own qualified Fc-effector function and cell-based binding bioassays. Dose-response curve model parameters were shown to reflect similar behavior amongst rituximab preparations, albeit with some differences in potency. In the absence of a common reference standard, potency estimates were in poor agreement amongst laboratories, but the use of the candidate preparation significantly reduced this variability. Our results suggest that the candidate rituximab standard can support bioassay performance and improve data harmonization, which when implemented will promote consistency of rituximab products over their life-cycles. This data provides the first scientific evidence that a classical standardization exercise allowing traceability of bioassay data to an international standard is also applicable to rituximab. However, we submit that this new type of international standard needs to be used appropriately and its role not to be mistaken with that of the reference medicinal product.

The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum.

BACKGROUND: At a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study. RESULTS: A pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-119, MSP-142, MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule. CONCLUSION: Analysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross-comparisons of results of vaccine trials performed in different centres/with different products; (2) to facilitate standardization and harmonization of immunological assays used in epidemiology research; and (3) to allow optimization and validation of immunological assays used in malaria vaccine development.

Preparation, calibration and evaluation of the First International Standard for human C-peptide.

BACKGROUND: Measurement of C-peptide by immunoassay contributes to the diagnosis of a number of disorders related to ß cell function. Stocks of the current international reference reagent (IRR) for C-peptide, used to calibrate these immunoassays, are exhausted, and this report summarises the international study to establish a replacement World Health Organization (WHO) international standard (IS) to maintain the availability of a globally available reference material and support efforts to standardise C-peptide assays. METHODS: The study was conducted in three phases; phase I involved the assignment of a value to a primary calibrant in mass units by amino acid analysis and phase II applied this value to the calibration of a candidate standard, 13/146, by reversed phase high-performance liquid chromatography (RP-HPLC) assay. In phase III, the candidate standard was compared to the first IRR by current immunoassays to assess its suitability to serve as an IS. RESULTS: Calibration of the candidate standard by RP-HPLC gave a final estimated content of 8.64 µg/ampoule with expanded uncertainty of 8.21-9.07 µg/ampoule (95% confidence; k=2.45). The candidate standard also appears sufficiently stable to serve as an IS, based on HPLC analysis of accelerated thermal degradation samples of 13/146, and was also shown to have appropriate immunological activity. A difference in bias approach was used to assess the commutability of 13/146 with human serum and urine samples. With the exception of two laboratories, the candidate standard demonstrated commutability with respect to the serum and urine samples included in this study. CONCLUSIONS: The candidate standard, 13/146, is suitable to serve as the First International Standard for human C-peptide, and it has been formally adopted by the Expert Committee on Biological Standardisation of the WHO.

Evaluation of candidate international standards for meningococcal serogroups A and X polysaccharide.

Polysaccharide (PS) based meningococcal vaccines are primarily evaluated by physicochemical methods to ensure batches are consistently manufactured. As PS content is determined by different methods across numerous laboratories, there is a need for International Standards (IS) to calibrate the assays. Following the successful introduction of the WHO Meningococcal group C (MenC) IS in 2011, NIBSC initiated projects to prepare similar standards for groups A, W, Y and X (MenA/W/Y/X) to standardise all meningococcal- PS based vaccines. On the basis of results from a collaborative study to evaluate preparations of MenA and MenX PS, both were established by the WHO Expert Committee on Biological Standardization in Oct 2015 as; the First WHO International Standard for the Meningococcal Group A polysaccharide with a content of 0.845 ± 0.043 mg MenA PS per ampoule (expanded uncertainty with coverage factor of k=2.45 corresponding to a 95% level of confidence); the First WHO International Standard for the Meningococcal Group X polysaccharide with a content of 0.776 ± 0.089 mg MenX PS per ampoule (expanded uncertainty with coverage factor of k=2.45), as determined by quantitative NMR. The standards are available from NIBSC, who act as guardians and distributors of the material under the auspices of WHO.

Comparison of platform technologies for assaying antibody to Ebola virus.

BACKGROUND: The recent Ebola outbreak in West Africa led to the use of a variety of different platform technologies for assaying antibodies because of the difficulties of handling the live virus. The same types of method could be applied rapidly to other infections when they emerge. There is a need to compare quantitative results of different assays, which means that the assays must measure similar parameters and give comparable results. METHODS: A collaborative study was carried out to establish an International Reference Reagent through WHO. Nine samples were sent to 16 laboratories and the results from 22 different assays compared to those obtained by neutralisation assays using the wild type virus. FINDINGS: Quantitative correlation with the wild type neutralisation assays was very variable but generally poor, with only five of the twenty-two assays giving a correlation coefficient of 0.7 or greater; the five best assays included methods based on wild type and VSV pseudotype neutralisation and ELISA. They could be applicable to other rapidly emerging diseases. The remaining assays including neutralisation of lentiviral pseudotypes need further development. INTERPRETATION: The assay platform should be chosen with care to ensure that it is fit for purpose. Many of the assays were not suitable for quantitation of antibody levels, a finding that is not surprising given the urgency with which they had to be implemented but some may be of generic value. Antibody titres in samples from a vaccine trial were comparable to those from convalescent patients or lower. FUNDING: Funding was from the UK DoH and the Wellcome Tust.

International collaborative study on the 3rd WHO International Standard for hepatitis B surface antigen.

BACKGROUND: The WHO International Standard (IS) for hepatitis B surface antigen (HBsAg) is used to standardize HBsAg assays. Stocks of the 2nd IS for HBsAg are depleted. The proposal to establish its replacement was endorsed by WHO in 2012. OBJECTIVE: Preparation of a freeze-dried candidate 3rd IS (NIBSC 12/226); evaluation of its suitability in a WHO international collaborative study; calibration of its potency in International Units (IU). STUDY DESIGN: The 3rd IS is based on plasma-derived, purified, inactivated HBsAg from Vietnam. Qualitative and quantitative HBsAg assays were used to evaluate 12/226 alongside the 2nd IS and 1st IS. Blinded study samples included a duplicate of 12/226, a negative control and two diluted plasma samples representing hepatitis B virus (HBV) genotypes A and B. RESULTS: Twelve laboratories from 9 countries returned 22 data sets from 15 methods. The overall geometric mean potency of 12/226 is 47.3IU/mL (±13% CV) when compared to the 2nd IS with HBV subgenotype A2. The 3rd IS has HBV subgenotype B4 with a heterogeneous HBsAg subtype population of ayw1 and adw2. Some genotype-dependent effects on the inter-laboratory variability were observed but overall mean potencies were virtually identical irrespective of the IS used for calibration. Stability studies indicate that the candidate is stable for long-term use. CONCLUSIONS: 12/226 was established in October 2014 by the WHO Expert Committee on Biological Standardization as the 3rd IS for HBsAg with a potency of 47.3IU per ampoule maintaining the continuity in the standardization of HBsAg assays.

Establishment of the first WHO Erythropoietin antibody reference panel: Report of an international collaborative study.

A panel of 9 fully human monoclonal antibodies against human erythropoietin (EPO) with defined characteristics (non-neutralizing, neutralizing, various isotypes, affinities) representative of those evident in antibody-mediated pure red cell aplasia (PRCA) and non-PRCA patients were formulated and lyophilized. The panel was evaluated in a multi-centre international collaborative study comprising eighteen different laboratories using different assay platforms including those in routine use. These included binding assays, some based on use of novel technologies and neutralization assays predominantly employing EPO responsive cell-lines. Results showed that detection and titre varied depending on antibody characteristics and the method used. Only selective assay platforms were capable of detecting the diverse repertoire of EPO antibodies in the panel indicating that some clinically relevant antibodies are likely to be missed in some assays. Importantly, the clinical samples from PRCA patients were distinguished as antibody-positive and the healthy donor serum as antibody negative across all different platforms tested. For neutralization, data was generally consistent across the assays for the different samples regardless of the cell-line and the assay conditions. The heterogeneity in data from the study clearly indicated the need for reference standards for consistency in detecting and measuring EPO antibodies across different assay platforms for monitoring the safety and efficacy of erythropoiesis stimulating agents. Therefore, the WHO ECBS at its meeting in October'15 established the EPO antibody panel, available from NIBSC, to facilitate decision-making on assay selection for testing antibodies against human EPO, for evaluating assay performance of antibody assays for clinical use, for assay validation and for standardization.

International collaborative study for establishment of the 2nd WHO International Standard for Haemophilus influenzae type b polysaccharide.

In this report we present the results of a collaborative study for the preparation and calibration of a replacement International Standard (IS) for Haemophilus influenzae type b polysaccharide (polyribosyl ribitol phosphate; 5-d-ribitol-(1 â†’ 1)-ß-d-ribose-3-phosphate; PRP). Two candidate preparations were evaluated. Thirteen laboratories from 9 different countries participated in the collaborative study to assess the suitability and determine the PRP content of two candidate standards. On the basis of the results from this study, Candidate 2 (NIBSC code 12/306) has been established as the 2nd WHO IS for PRP by the Expert Committee of Biological Standards of the World Health Organisation with a content of 4.904 ± 0.185mg/ampoule, as determined by the ribose assays carried out by 11 of the participating laboratories.

Establishment of the first international standard for PEGylated granulocyte colony stimulating factor (PEG-G-CSF): report of an international collaborative study.

We assessed the feasibility of developing a suitable international reference standard for determination of in vitro biological activity of human sequence recombinant PEG-G-CSF products with a 20kD linear PEG linked to the N-terminal methionyl residue of G-CSF (INN Filgrastim), produced using a conjugation process and coupling chemistry similar to that employed for the lead PEGfilgrastim product. Based on initial data which showed that the current WHO 2nd international standard, IS for G-CSF (09/136) or alternatively, a PEG-G-CSF standard with a unitage traceable to the G-CSF IS may potentially serve as the IS for PEG-G-CSF products, two candidate preparations of PEG-G-CSF were formulated and lyophilized at NIBSC. These preparations were tested by 23 laboratories using in vitro bioassays in a multi-centre collaborative study. Results indicated that on the basis of parallelism, the current WHO 2nd IS for G-CSF or any of the PEG-G-CSF samples could be used as the international standard for PEG-G-CSF preparations. However, because of the variability in potency estimates seen when PEG-G-CSF preparations were compared with the current WHO 2nd IS for G-CSF, a candidate PEG-G-CSF was suitable as the WHO IS. The preparation 12/188 was judged suitable to serve as the WHO IS based on in vitro biological activity data. Therefore, the preparation coded 12/188 was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2013 as the WHO 1st IS for human PEGylated G-CSF with an assigned in vitro bioactivity of 10,000IU per ampoule.