The therapeutic monoclonal antibody (mAb) TGN1412 (anti-CD28 superagonist) caused near-fatal cytokine release syndrome (CRS) in all six volunteers during a phase-I clinical trial. Several cytokine release assays (CRAs) with reported predictivity for TGN1412-induced CRS have since been developed for the preclinical safety testing of new therapeutic mAbs. The whole blood (WB) CRA is the most widely used, but its sensitivity for TGN1412-like cytokine release was recently criticized. In a comparative study, using group size required for 90% power with 5% significance as a measure of sensitivity, we found that WB and 10% (v/v) WB CRAs were the least sensitive for TGN1412 as these required the largest group sizes (n = 52 and 79, respectively). In contrast, the peripheral blood mononuclear cell (PBMC) solid phase (SP) CRA was the most sensitive for TGN1412 as it required the smallest group size (n = 4). Similarly, the PBMC SP CRA was more sensitive than the WB CRA for muromonab-CD3 (anti-CD3) which stimulates TGN1412-like cytokine release (n = 4 and 4519, respectively). Conversely, the WB CRA was far more sensitive than the PBMC SP CRA for alemtuzumab (anti-CD52) which stimulates FcγRI-mediated cytokine release (n = 8 and 180, respectively). Investigation of potential factors contributing to the different sensitivities revealed that removal of red blood cells (RBCs) from WB permitted PBMC-like TGN1412 responses in a SP CRA, which in turn could be inhibited by the addition of the RBC membrane protein glycophorin A (GYPA); this observation likely underlies, at least in part, the poor sensitivity of WB CRA for TGN1412. The use of PBMC SP CRA for the detection of TGN1412-like cytokine release is recommended in conjunction with adequately powered group sizes for dependable preclinical safety testing of new therapeutic mAbs.

BackgroundThe 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.MethodsA 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.FindingsQuantitative 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.InterpretationThe 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.FundingFunding was from the UK DoH and the Wellcome Tust.

BackgroundAt 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.ResultsA 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.ConclusionAnalysis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1958-x) contains supplementary material, which is available to authorized users.

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.

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.