Publications by authors named "Peter Rigsby"

69 Publications

An international collaborative study to establish the WHO 3rd International Standard for Thrombin: Communication from the ISTH SSC subcommittee on factor XIII and fibrinogen.

J Thromb Haemost 2021 Mar;19(3):852-858

Biostatistics Section, National Institute for Biological Standards and Control, South Mimms, UK.

The calibration of thrombin products relies on the World Health Organization (WHO) 2nd International Standard (IS) for Thrombin (01/580) which defines the international unit (IU) for thrombin potency. With stocks of the 2nd IS (01/580) running low, an international collaborative study was organized to calibrate a replacement. Twenty laboratories from 13 countries took part in the study and measured the potency of two candidate replacement standards (coded 01/578 and 19/188) relative to the 2nd IS. In total, 111 valid assays were returned, which were a combination of plasma/fibrinogen clotting assays and chromogenic assays. Variation between and within laboratories was low, with inter- and intra-laboratory geometric coefficient of variation (GCV) generally <5% for all assay methods and substrates. For 01/578, potency estimates by clotting assays (101.1 IU/ampoule) were significantly lower than estimates by chromogenic assays (111.5 IU/ampoule). Mean potency estimates for 19/188 were 90.4 IU/ampoule by clotting assay and 88.1 IU/ampoule by chromogenic assay, which was not a statistically significant difference. The close ratio between clotting and chromogenic assay potency estimates for 19/188 suggests it has a higher α-thrombin content than 01/578 and is equivalent to the current IS (01/580). Accelerated degradation studies predicted excellent long-term stability profiles for preparations 01/580, 01/578, and 19/188. Based on the results of this study, the WHO Expert Committee on Biological Standardization established 19/188 as the 3rd IS for Thrombin with a potency of 90 IU/ampoule in August 2020.
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http://dx.doi.org/10.1111/jth.15207DOI Listing
March 2021

Saccharide dosage content of meningococcal polysaccharide conjugate vaccines determined using WHO International Standards for serogroup A, C, W, Y and X polysaccharides.

Biologicals 2021 Jan 28. Epub 2021 Jan 28.

Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, Hertfordshire, EN6 3QG, UK.

Potency of meningococcal polysaccharide-protein conjugate vaccines relies on the polysaccharide content to prevent meningitis. NIBSC, as the official national control laboratory in UK, analysed ten different mono- and multi-meningococcal conjugate vaccines, using established International Standards for meningococcal serogroups A, C, W, Y and X, by resorcinol or HPAEC-PAD assay. Most saccharide contents were within ±20% of their claimed content for licensure with taking different O-acetylation levels into consideration, with only MenC content in two vaccines below (by 60% and 54%) the labelled value, however, previous study showed different dosage was not necessarily correlated to the immunogenicity of those vaccines. This study demonstrated the use of International Standards to quantify saccharide content in polysaccharide-based vaccines with different percentage of O-acetylation. These International Standards are suitable to serve as either quantitative standard or calibrator of in-house standards, with supplied stability data.
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http://dx.doi.org/10.1016/j.biologicals.2021.01.001DOI Listing
January 2021

Development of the first reference antibody panel for qualification and validation of cytokine release assay platforms - Report of an international collaborative study.

Cytokine X 2020 Dec;2(4):100042

Novartis Institutes for BioMedical Research, Klybeckstrasse 141, Basel CH-4002, Switzerland.

Immunomodulatory therapeutics such as monoclonal antibodies (mAb) carry an inherent risk of undesired immune reactions. One such risk is cytokine release syndrome (CRS), a rapid systemic inflammatory response characterized by the secretion of pro-inflammatory cytokines from immune cells. It is crucial for patient safety to correctly identify potential risk of CRS prior to first-in-human dose administration. For this purpose, a variety of in vitro cytokine release assays (CRA) are routinely used as part of the preclinical safety assessment of novel therapeutic mAbs. One of the challenges for the development and comparison of CRA performance is the lack of availability of standard positive and negative control mAbs for use in assay qualification. To address this issue, the National Institute for Biological Standards and Control (NIBSC) developed a reference panel of lyophilised mAbs known to induce CRS in the clinic: human anti-CD52, mouse anti-CD3 and human superagonistic (SA) anti-CD28 mAb manufactured according to the respective published sequences of Campath-1H® (alemtuzumab, IgG1) , Orthoclone OKT-3® (muromonab, IgG2a) and TGN1412 (theralizumab, IgG4), as well as three isotype matched negative controls (human IgG1, mouse IgG2a and human IgG4, respectively). The relative capacity of these control mAbs to stimulate the release of IFN-γ, IL-2, TNF-α and IL-6 in vitro was evaluated in eleven laboratories in an international collaborative study mediated through the HESI Immuno-safety Technical Committee Cytokine Release Assay Working Group. Participants tested the NIBSC mAbs in a variety of CRA platforms established at each institution. This paper presents the results from the centralised cytokine quantification on all the plasma/supernatants corresponding to the stimulation of immune cells in the different CRA platforms by a single concentration of each mAb. Each positive control mAb induced significant cytokine release in most of the tested CRA platforms. There was a high inter-laboratory variability in the levels of cytokines produced, but similar patterns of response were observed across laboratories that replicated the cytokine release patterns previously published for the respective clinical therapeutic mAbs. Therefore, the positive and negative mAbs are suitable as a reference panel for the qualification and validation of CRAs, comparison of different CRA platforms (e.g. solid vs aqueous phase), and intra- and inter-laboratory comparison of CRA performance. Thus, the use of this panel of positive and negative control mAbs will increase the confidence in the robustness of a CRA platform to identify a potential CRS risk for novel immunomodulatory therapeutic candidates.
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http://dx.doi.org/10.1016/j.cytox.2020.100042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789043PMC
December 2020

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies.

Vaccines (Basel) 2020 Nov 9;8(4). Epub 2020 Nov 9.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA.

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals ( = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A "pooled serum" (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1.
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http://dx.doi.org/10.3390/vaccines8040666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712758PMC
November 2020

Establishment of a WHO Reference Reagent for anti-Mullerian hormone.

Reprod Biol Endocrinol 2020 Aug 15;18(1):86. Epub 2020 Aug 15.

Biotherapeutics Division, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, UK.

Background: There is a need for a reference material to support the development and ensure the quality of immunoassays for human AMH. A batch of ampoules, coded 16/190, containing lyophilised recombinant AMH was evaluated in a WHO Collaborative Study. The aims of the study were to determine the AMH content in terms of the calibration of each immunoassay method, to predict long-term stability and to assess the suitability of the preparation to calibrate AMH immunoassays.

Methods: Study participants were asked to report the AMH content of specific dilutions of coded ampoules of 16/190 and a comparator preparation containing approximately half the AMH content. In each assay, participants also reported the AMH content of 22 patient samples to assess commutability. A robust all-laboratory geometric mean of the content estimates was determined using the laboratory geometric mean estimates. Commutability was assessed using a difference in bias approach. Stability was predicted by the measurement of thermally accelerated degradation samples.

Results: Seven laboratories performed twenty-one immunoassay method-platform combinations, sixteen of which provided data which met the validity criteria, giving a consensus geometric mean estimate of AMH content of 511 ng/ampoule (95% CI, 426-612, n = 16, GCV 42%) and a robust geometric mean of 489 ng/ampoule. By contrast, the GCV% for the all-laboratory geometric mean of the relative content estimates for the comparator sample to 16/190 was 12%. Commutability was assessed using 20 of the 22 representative patient samples. Of the valid assays, 16/190 was within the limits of acceptable commutability for 6 methods, partially commutable for a further 3 methods and non-commutable when measured by 7 methods. The preparation was predicted to be highly stable when stored at - 20 °C.

Conclusion: The majority of methods met the validity criteria. Content estimates showed a high between-method variability, yet assays exhibited a similar proportionality of response as demonstrated using the comparator sample. 16/190 was commutable in some but not all methods. On the basis of these results, it was agreed by the WHO Expert Committee on Biological Standardization to establish 16/190 as a WHO Reference Reagent for AMH with a content defined by consensus immunoassay of 489 ng/ampoule.
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http://dx.doi.org/10.1186/s12958-020-00641-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429692PMC
August 2020

Evaluation of a standardised Vi poly-l-lysine ELISA for serology of Vi capsular polysaccharide antibodies.

Biologicals 2020 Jul 20;66:21-29. Epub 2020 Jun 20.

Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG, UK. Electronic address:

Typhoid vaccines based on protein-conjugated capsular Vi polysaccharide (TCVs) prevent typhoid in infants and young children. Analysis of the serum anti-Vi IgG response following immunisation against typhoid confirms the immunogenicity of TCVs and forms an important part of the pathway to licensing. Comparative studies could expedite the licencing process, and the availability of a standardised ELISA method alongside the 1st International Standard (IS) 16/138 for anti-typhoid capsular Vi polysaccharide IgG (human) will facilitate this process. To this end, a non-commercial ELISA based on a coat of Vi and poly-l-lysine (Vi-PLL ELISA) was evaluated by 10 laboratories. Eight serum samples, including IS 16/138, were tested in the standardised Vi-PLL ELISA (n = 10), a commercial Vi ELISA (n = 3) and a biotinylated Vi ELISA (n = 1). Valid estimates of potencies relative to IS 16/138 were obtained for all samples in the Vi-PLL ELISA and the commercial ELISA, with good repeatability and reproducibility evident from the study results and concordant estimates obtained by the two ELISA methods. The study demonstrates that the Vi-PLL ELISA can be used in clinical trial studies to determine the immunogenicity of TCVs.
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http://dx.doi.org/10.1016/j.biologicals.2020.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391004PMC
July 2020

The Use of Next-Generation Sequencing for the Quality Control of Live-Attenuated Polio Vaccines.

J Infect Dis 2020 Nov;222(11):1920-1927

Division of Virology, National Institute for Biological Standards and Control, Potters Bar, United Kingdom.

Background: Next-generation sequencing (NGS) analysis was compared to the current MAPREC (mutational analysis by polymerase chain reaction and restriction enzyme cleavage) assay for quality control of live-attenuated oral polio vaccine (OPV).

Methods: MAPREC measures reversion of the main OPV attenuating mutations such as uracil (U) to cytosine (C) at nucleotide 472 in the 5' noncoding region of type 3 OPV. Eleven type 3 OPV samples were analyzed by 8 laboratories using their in-house NGS method.

Results: Intraassay, intralaboratory, and interlaboratory variability of NGS 472-C estimates across samples and laboratories were very low, leading to excellent agreement between laboratories. A high degree of correlation between %472-C results by MAPREC and NGS was observed in all laboratories (Pearson correlation coefficient r = 0.996). NGS estimates of sequences at nucleotide 2493 with known polymorphism among type 3 OPV lots also produced low assay variability and excellent between-laboratory agreement.

Conclusions: The high consistency of NGS data demonstrates that NGS analysis can be used as high-resolution test alternative to MAPREC, producing whole-genome profiles to evaluate OPV production consistency, possibly eliminating the need for tests in animals. This would be very beneficial for the quality assessment of next-generation polio vaccines and, eventually, for other live-attenuated viral vaccines.
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http://dx.doi.org/10.1093/infdis/jiaa299DOI Listing
November 2020

An international collaborative study to establish the WHO 4th International Standard for Streptokinase: Communication from the SSC of the ISTH.

J Thromb Haemost 2020 06;18(6):1501-1505

Haemostasis Section, Biotherapeutics Division, National Institute for Biological Standards and Control, South Mimms, Herts, UK.

Streptokinase is used worldwide as a cost-effective treatment for acute myocardial infarction. Manufacturers use the World Health Organization (WHO) International Standard (IS) for Streptokinase to potency label their products, ensuring consistent, safe, and effective dosing. Stocks of the third IS for streptokinase (coded 00/464) are running low, and an international collaborative study was organized to calibrate a replacement. A total of 15 laboratories from nine countries took part, using chromogenic and/or fibrin clot lysis methods to determine the potency of two candidate preparations, coded 16/356 (sample B) and 16/358 (sample C), relative to the third IS (00/464). A third sample (88/824, sample A), which was used in the collaborative studies to establish the second and third IS, was also included. There was good agreement in potency estimates from different assay methods and low variability both within and between laboratories. Long-term stability modeling indicated the candidates are very stable. Comparison of potency estimates for 88/824 (sample A) with potencies calculated in previous studies revealed a variability of only 1.9% over the course of three collaborative studies spanning 30 years and more than 50 years of streptokinase standardization. This indicates excellent continuity of the International Unit (IU) and assay methods. Following agreement by study participants and Scientific and Standardization Committee experts of the International Society on Thrombosis and Haemostasis, the WHO Expert Committee on Biological Standardization established 16/358 (sample C) as the fourth IS for Streptokinase with a potency of 1013 IU per ampoule in October 2019.
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http://dx.doi.org/10.1111/jth.14787DOI Listing
June 2020

The first World Health Organization International Standard for in vitro biological activity of darbepoetin.

Biologicals 2020 Jan 18;63:33-38. Epub 2019 Dec 18.

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Hertfordshire, EN6 3QG, UK.

The expiry of patents protecting the manufacture and sale of therapeutic darbepoetin products is expected to lead to the emergence of biosimilar products. In response to this, the first World Health Organization (WHO) International Standard (IS) for darbepoetin has been developed. A lyophilized preparation of darbepoetin, coded 17/204, was evaluated in an international collaborative study, the results of which suggest that the candidate preparation is suitable to serve as an IS. This material defines the International Unit (IU) of in vitro biological activity of darbepoetin and should be used to calibrate of in vitro potency assays of darbepoetin preparations. It is envisaged that widespread use of the IS will promote the consistency and harmonization of darbepoetin in vitro bioassay measurements in laboratories worldwide. Each ampoule contains 100,000 IU of darbepoetin activity. The IU is not intended to revise product labelling or dosing requirements, decisions regarding which lie solely with the regulatory authority. Additionally, the IS is not intended to define the specific activity of darbepoetin, as this may differ between products in the future. Finally, the IS is not intended to serve any regulatory role in defining biosimilarity (i.e. as a reference medicinal product).
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http://dx.doi.org/10.1016/j.biologicals.2019.12.004DOI Listing
January 2020

Expansion of the 1st WHO international standard for antiserum to respiratory syncytial virus to include neutralisation titres against RSV subtype B: An international collaborative study.

Vaccine 2020 01 8;38(4):800-807. Epub 2019 Nov 8.

Division of Virology, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar, Herts EN6 3QG, UK. Electronic address:

An International Standard to harmonise results from RSV subtype A neutralisation assays was generated and established by the World Health Organization in 2018. Here we report on a study to expand the use of that standard to include neutralisation assays using human sera against RSV subtype B and to test its ability to harmonise neutralisation titres from neutralisation assays including complement. The study included 11 laboratories from 6 countries. All participants used their own in-house virus neutralisation assay and their own virus stocks. The study samples comprised the current International Standard (16/284) and its potential replacement (16/322), individual sera from naturally infected humans, a monoclonal antibody to RSV (palivizumab) and samples from the BEI Resources panel of human antiserum and immune globulin to RSV. Of the 11 laboratories that took part in the study, 5 returned data from neutralisation assays with and without the inclusion of serum complement. The study showed that inter-laboratory variability in neutralisation titres was significantly reduced when values were expressed relative to 16/284 or 16/322. Complement did not affect the ability of the International Standard to decrease inter-laboratory variability as the standard was able to reduce the differences between titres from assays with and without complement. Based on these results, we will recommend to the WHO Expert Committee on Biological Standardisation (ECBS) that 16/284 and 16/322 be expanded in their use to include neutralisation assays against RSV/B.
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http://dx.doi.org/10.1016/j.vaccine.2019.10.095DOI Listing
January 2020

Harmonization of Zika neutralization assays by using the WHO International Standard for anti-Zika virus antibody.

NPJ Vaccines 2019 14;4:42. Epub 2019 Oct 14.

1Division of Virology, National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG UK.

During outbreaks of emerging viruses, such as the Zika outbreak in 2015-2016, speed and accuracy in detection of infection are critical factors to control the spread of the disease; often serological and diagnostic methods for emerging viruses are not well developed and validated. Thus, vaccines and treatments are difficult to evaluate due to the lack of comparable methods. In this study, we show how the 1st WHO International Standard for anti-Zika antibody was able to harmonize the neutralization titres of a panel of serological Zika-positive samples from laboratories worldwide. Expression of the titres in International Unit per millilitre reduced the inter-laboratory variance, allowing for greater comparability between laboratories. We advocate the use of the International Standard for anti-Zika virus antibodies for the calibration of neutralization assays to create a common language, which will permit a clear evaluation of the results of different clinical trials and expedite the vaccine/treatment development.
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http://dx.doi.org/10.1038/s41541-019-0135-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791859PMC
October 2019

A WHO Reference Reagent for lupus (anti-dsDNA) antibodies: international collaborative study to evaluate a candidate preparation.

Ann Rheum Dis 2019 12 5;78(12):1677-1680. Epub 2019 Sep 5.

Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Introduction: Antibodies against double-stranded DNA (anti-dsDNA) are a specific biomarker for systemic lupus erythematosus (SLE). The first WHO International Standard (IS) for anti-dsDNA (established in 1985), which was used to assign units to diagnostic tests, was exhausted over a decade ago.

Methods: Plasma from a patient with SLE was first evaluated in 42 European laboratories. The plasma was thereafter used by the National Institute for Biological Standards and Control to prepare a candidate WHO reference preparation for lupus (anti-dsDNA) antibodies. That preparation, coded 15/174, was subjected to an international collaborative study, including 36 laboratories from 17 countries.

Results: The plasma mainly contained anti-dsDNA, other anti-chromatin antibodies and anti-Ku. The international collaborative study showed that the field would benefit from 15/174 as a common reference reagent improving differences in performance between different assays. However, no statistically meaningful overall potency or assay parallelism and commutability could be shown.

Conclusion: 15/174 cannot be considered equivalent to the first IS for anti-dsDNA (Wo/80) and was established as a WHO Reference Reagent for lupus (oligo-specific) anti-dsDNA antibodies with a nominal value of 100 units/ampoule. This preparation is intended to be used to align test methods quantifying levels of anti-dsDNA antibodies.
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http://dx.doi.org/10.1136/annrheumdis-2019-215845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900249PMC
December 2019

Evaluation of a capture antigen ELISA for the characterisation of tetanus vaccines for veterinary use.

Biologicals 2019 Sep 27;61:8-14. Epub 2019 Aug 27.

National Institute for Biological Standards and Control, Division of Bacteriology, South Mimms, Potters Bar, EN6 3QG, United Kingdom. Electronic address:

We previously developed an ELISA assay for detection of tetanus toxoid antigen in tetanus vaccines for human use. Tetanus vaccines for veterinary use are qualitatively different to those used in humans, often containing a larger number and variety of non-tetanus antigens in the multi-valent products, and adjuvants that are not found in human vaccines. We assessed performance of the capture ELISA with a range of veterinary tetanus vaccines as a first step towards development of an immunoassay as a potential in vivo potency substitute. Nine tetanus vaccines were tested and all produced a good dose response in the ELISA. The shape of the dose response curve for the whole vaccine compared to a matched non-adjuvanted tetanus toxoid antigen was more comparable for vaccines containing a non-aluminium adjuvant than products containing aluminium adjuvants. Elution of the antigen from aluminium adjuvant did not improve the comparability of the dose response curve but did increase the total amount of tetanus antigen available for detection. The ELISA was highly specific for tetanus with no signal obtained for a large number of non-tetanus antigens. These results suggest that a capture ELISA assay can be applied to a control strategy for veterinary tetanus vaccines.
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http://dx.doi.org/10.1016/j.biologicals.2019.08.003DOI Listing
September 2019

A new WHO reference reagent for activated blood coagulation factor X (FXa), human (15/102).

J Thromb Haemost 2020 01;18(1):255-257

Biostatistics Group, National Institute for Biological Standards and Control, South Mimms, UK.

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http://dx.doi.org/10.1111/jth.14623DOI Listing
January 2020

Comparison of Serologic Assays for Middle East Respiratory Syndrome Coronavirus.

Emerg Infect Dis 2019 10 17;25(10):1878-1883. Epub 2019 Oct 17.

Middle East respiratory syndrome coronavirus (MERS-CoV) was detected in humans in 2012. Since then, sporadic outbreaks with primary transmission through dromedary camels to humans and outbreaks in healthcare settings have shown that MERS-CoV continues to pose a threat to human health. Several serologic assays for MERS-CoV have been developed globally. We describe a collaborative study to investigate the comparability of serologic assays for MERS-CoV and assess any benefit associated with the introduction of a standard reference reagent for MERS-CoV serology. Our study findings indicate that, when possible, laboratories should use a testing algorithm including >2 tests to ensure correct diagnosis of MERS-CoV. We also demonstrate that the use of a reference reagent greatly improves the agreement between assays, enabling more consistent and therefore more meaningful comparisons between results.
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http://dx.doi.org/10.3201/eid2510.190497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759245PMC
October 2019

The third international standard for anti-D immunoglobulin: international collaborative study to evaluate candidate preparations.

Vox Sang 2019 Oct 19;114(7):740-748. Epub 2019 Jul 19.

Biotherapeutics Group, National Institute for Biological Standards and Control, Hertfordshire, UK.

Background And Objectives: The purpose of the study was to evaluate a lyophilized anti-D immunoglobulin preparation to serve as a replacement WHO International Standard for the calibration of potency assays of anti-D immunoglobulin products. Such products are used to prevent haemolytic disease of the foetus and newborn due to maternal alloanti-D.

Materials And Methods: The candidate 3rd International Standard for anti-D immunoglobulin (16/332) was evaluated and calibrated against the 2nd International Standard for anti-D immunoglobulin (01/572), along with a coded duplicate, a second candidate preparation (16/278) and a comparability sample (16/272) in an international collaborative study. Twenty of 21 laboratories in 15 countries performed one or more of the three European Pharmacopoeia reference methods.

Results: The overall geometric mean potency (from all methods) of the candidate 3rd International Standard, 16/332, was 296·6 IU/ampoule, with inter-laboratory variability, expressed as % GCV, of 4·7%. SE-HPLC of the immunoglobulin preparations demonstrated combined monomeric and dimeric IgG peak areas of >95% for all samples. Accelerated stability studies have shown both 16/332 and 16/278 to be very stable for long-term storage at -20°C.

Conclusions: Preparation 16/332 was established by the World Health Organisation Expert Committee on Biological Standardization as the 3rd International Standard for anti-D immunoglobulin with an assigned potency of 297 IU/ampoule.
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http://dx.doi.org/10.1111/vox.12822DOI Listing
October 2019

Continued provision of WHO International Standards for total and free PSA: Content and commutability of replacement preparations.

Clin Biochem 2019 Sep 8;71:58-66. Epub 2019 Jul 8.

Biotherapeutics Group, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom. Electronic address:

Objectives: Replacements are required for the WHO International Standards (IS) for free PSA, coded 96/668 and total PSA (90:10), coded 96/670, which were established in 1999 to support efforts to harmonise PSA assays and address non-equimolarity. An important consideration is that the introduction of the replacements should have minimal impact on PSA measurements.

Design And Methods: We report the development of a replacement strategy, informed by field assessment of preparations through an external quality assessment scheme and the subsequent evaluation of the candidate ISs in worldwide collaborative studies.

Results: By immunoassay, data from participants confirmed the value assigned to the current standards. Robust geometric mean estimates of the free PSA content of the candidate replacement for 96/668 coded 17/102 was 0.533 μg/ampoule (n = 21). The ratio of the content estimates of 17/102:96/668 was 0.516 (GCV 12.5%, n = 21). Robust geometric mean estimates of the total PSA content of the candidate replacement for 96/670, coded 17/100, was 0.505 μg/ampoule (n = 22). The ratio of the content estimates of 17/100:96/670 was 0.490 (GCV 5.3%, n = 22). Through concomitant measurement of a panel of 15 representative patient samples, the candidate ISs were shown to exhibit commutability with patient samples that was comparable with that of the current ISs.

Conclusion: On the basis of these results, the preparations coded 17/102 and 17/100 were established by the WHO Expert Committee on Biological Standardization as the 2nd ISs for free and total PSA (PSA-ACT+free PSA) respectively, with assigned contents of 0.53 μg/ampoule and 0.50 μg/ampoule.
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http://dx.doi.org/10.1016/j.clinbiochem.2019.07.007DOI Listing
September 2019

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

Cytometry B Clin Cytom 2019 11 20;96(6):508-513. Epub 2019 Feb 20.

Biotherapeutics group, National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK.

Background: Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations.

Method: In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated.

Results: The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method.

Conclusions: Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899615PMC
November 2019

Differences in Antigenic Structure of Inactivated Polio Vaccines Made From Sabin Live-Attenuated and Wild-Type Poliovirus Strains: Impact on Vaccine Potency Assays.

J Infect Dis 2020 02;221(4):544-552

Division of Virology, National Institute for Biological Standards and Control, Herts, United Kingdom.

Background: Following the declaration of wild-type 2 poliovirus eradication in 2015, the type 2 component was removed from the live-attenuated oral polio vaccine (OPV). This change implies a need to improve global coverage through routine immunization with inactivated polio vaccine (IPV), to ensure type 2 immunity. Several manufacturers use Sabin OPV strains for IPV production (sIPV), rather than the usual wild-type strains used for conventional IPV (cIPV). However, in contrast to cIPV, potency assays for sIPV have not been standardized, no international references exist, and no antigen units have been defined for a sIPV human dose. Thus, sIPV products from different manufacturers cannot be compared, and the relationship between antigenicity and immunogenicity of sIPV is not well understood.

Methods: A collaborative study was conducted in which laboratories used different methods to measure the antigen content of a set of sIPV and cIPV samples with an aim to identify a suitable reference for sIPV products.

Results: The study revealed differences in the reactivity of antibody reagents to cIPV and sIPV products.

Conclusions: Homologous references are required to measure the antigen content of IPV products consistently. The first World Health Organization international standard for sIPV was established, with new, specific Sabin D-antigen units assigned.
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http://dx.doi.org/10.1093/infdis/jiz076DOI Listing
February 2020

Establishment of the WHO 2nd International Standard Factor V, plasma (16/374): communication from the SSC of the ISTH.

J Thromb Haemost 2019 Apr 28;17(4):695-697. Epub 2019 Feb 28.

National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.

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http://dx.doi.org/10.1111/jth.14403DOI Listing
April 2019

Evaluation of two WHO First International Standards for Vi polysaccharide from Citrobacter freundii and Salmonella enterica subspecies enterica serovar Typhi.

Biologicals 2019 Jan 27;57:34-45. Epub 2018 Nov 27.

Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, Hertfordshire, EN6 3QG, UK.

Numerous Vi capsular polysaccharide (Vi PS) conjugate vaccines to protect young children and infants from Typhoid are either licensed or under development. These vaccines are evaluated by laboratory methods to ensure their potency and that quality requirement are met. International Standard (IS) preparations of Vi PS are needed to calibrate and harmonise these assays. Twenty laboratories from 12 countries participated in a collaborative study to evaluate two candidate ISs: Citrobacter freundii Vi PS (NIBSC code 12/244) and Salmonella enterica serovar Typhi Vi PS (16/126). On the basis of returned results and stability profiles, these standards were established by the WHO Expert Committee on Biological Standardization in Oct 2017 as the First WHO IS for C. freundii Vi PS with a content of 1.94 ± 0.12 mg Vi PS per ampoule (expanded uncertainty with coverage factor of k = 2.11 corresponding to a 95% level of confidence) and the First WHO IS for S. Typhi Vi PS with a content of 2.03 ± 0.10 mg Vi PS per ampoule (expanded uncertainty with coverage factor of k = 2.11), as determined by quantitative NMR. The study also showed the ISs are suitable for physicochemical and immuno assays used for the quantitation of the Vi PS component in Vi PS and conjugate vaccines.
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http://dx.doi.org/10.1016/j.biologicals.2018.11.004DOI Listing
January 2019

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

MAbs 2019 01 5;11(1):13-25. Epub 2018 Nov 5.

a Division of Biotherapeutics , National Institute for Biological Standards and Control (NIBSC) , South Mimms , Potters Bar, Hertfordshire , UK.

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.
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http://dx.doi.org/10.1080/19420862.2018.1532766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343779PMC
January 2019

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

Vaccine 2018 11 30;36(50):7641-7649. Epub 2018 Oct 30.

Division of Virology, National Institute for Biological Standards and Control (NIBSC), South Mimms, Potters Bar, Herts EN6 3QG, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.vaccine.2018.10.087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838659PMC
November 2018

Establishment of the first International Standard for human anti-typhoid capsular Vi polysaccharide IgG.

Biologicals 2018 Nov 7;56:29-38. Epub 2018 Sep 7.

Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom.

Vi capsular polysaccharide (Vi) conjugate vaccines, which can prevent typhoid in infants and young children, are being developed. Comparative immunogenicity studies are facilitated by an International Standard (IS) for human anti-Vi IgG. 16/138, a pool of sera from volunteers which received either Vi conjugate vaccine or plain Vi vaccine, was assessed as an IS alongside U.S. reference reagent Vi-IgG. Samples were tested in a commercial ELISA (n = 7), a standardised ELISA based on biotinylated Vi (n = 7) and in-house ELISAs (n = 7). Valid estimates were obtained for the potency of all samples in the commercial ELISA, and the commutability of 16/138 and Vi-IgG was evident for the commercial ELISA and in-house ELISAs based on a coating of Vi and protein. The WHO Expert Committee on Biological Standardization established 16/138 as the first IS for anti-Vi IgG with 100 IU per ampoule and assigned 163 IU per vial of Vi-IgG.
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http://dx.doi.org/10.1016/j.biologicals.2018.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238147PMC
November 2018

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

Biologicals 2018 Nov 24;56:63-66. Epub 2018 Aug 24.

Haemostasis Section, Biotherapeutics Group, National Institute of Biological Standards and Control (NIBSC), South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.

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.
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http://dx.doi.org/10.1016/j.biologicals.2018.08.007DOI Listing
November 2018

WITHDRAWN: Establishment of the first International Standard for human anti-typhoid capsular Vi polysaccharide IgG.

Biologicals 2018 Jun 23. Epub 2018 Jun 23.

Center for Vaccine Development, University of Maryland Baltimore, 685 West Baltimore Street, Room 480, Baltimore, MD, USA.

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http://dx.doi.org/10.1016/j.biologicals.2018.05.007DOI Listing
June 2018

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

Biologicals 2018 May 20;53:39-50. Epub 2018 Mar 20.

Division of Virology, Beijing 100050, China. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biologicals.2018.03.001DOI Listing
May 2018

Establishment of the World Health Organization First International Standard for Factor XII, Plasma, Human.

Front Med (Lausanne) 2018 28;5:46. Epub 2018 Feb 28.

Haemostasis Section, National Institute for Biological Standards and Control, Potters Bar, United Kingdom.

Until recently, the role of factor XII (FXII) in hemostasis was not considered to be important since patients with FXII deficiency do not present with bleeding. The activation of FXII by agents including mast cells and platelet polyphosphates suggests that it may have a role in thrombogenesis. The inhibition of FXII therefore presents an option for antithrombotic therapy, and antibodies and inhibitors are already in development. Assays for FXII will be required to support these technologies, and an international standard (IS) for FXII would be useful for the development of these methods and for the clinical monitoring of patients. The purpose of this study was to develop an IS for FXII, with values for functional activity (FXII:C) and antigen (FXII:Ag). Double-spun normal plasma was pooled, filled into siliconized glass ampoules, and freeze-dried to prepare the candidate material. Data from 20 laboratories using the one-stage clotting assay were used to assign the functional activity value in units (u). The antigen value was calculated using data from eight laboratories that carried out antigen assays. Each laboratory was requested to collect two local normal plasma pools. Units of activity and antigen were calculated relative to these pools, as is usual for new coagulation factor analytes. The amount of activity or antigen in 1 ml of normal plasma from each pool was taken to be 1 unit. A total of 566 donors were used across the pools for the FXII:C study and 216 donors for the FXII:Ag study. The overall geometric mean per ampoule for FXII:C was 0.86 u and for FXII:Ag was 0.80 u. The inter-laboratory variation was 10 and 11%, respectively (expressed as the geometric coefficient of variation). Based on these data, the candidate was deemed suitable for use as an IS for FXII. In 2017, the candidate was established by the World Health Organization (WHO) Expert Committee on Biological Standardization as the WHO first IS for blood coagulation FXII, Plasma (National Institute for Biological Standards and Control code 15/180). The values assigned were 0.86 international units (IU) of functional activity (FXII:C) per ampoule and 0.80 IU/ampoule of antigen (FXII:Ag).
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http://dx.doi.org/10.3389/fmed.2018.00046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835526PMC
February 2018

Recommendations of the VAC2VAC workshop on the design of multi-centre validation studies.

Biologicals 2018 Mar 1;52:78-82. Epub 2018 Feb 1.

Zoetis, UK. Electronic address:

Within the Innovative Medicines Initiative 2 (IMI 2) project VAC2VAC (Vaccine batch to vaccine batch comparison by consistency testing), a workshop has been organised to discuss ways of improving the design of multi-centre validation studies and use the data generated for product-specific validation purposes. Moreover, aspects of validation within the consistency approach context were addressed. This report summarises the discussions and outlines the conclusions and recommendations agreed on by the workshop participants.
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http://dx.doi.org/10.1016/j.biologicals.2018.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278876PMC
March 2018

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

MAbs 2018 01 3;10(1):129-142. Epub 2017 Nov 3.

b Technology Development and Infrastructure Division, National Institute for Biological Standards and Control, South Mimms , Potters Bar , Hertfordshire , United Kingdom.

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.
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http://dx.doi.org/10.1080/19420862.2017.1386824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836816PMC
January 2018