Evaluation of candidate International Standards for meningococcal capsular polysaccharide groups W and Y.

Two candidate International Standards for meningococcal capsular group W and Y (MenW and MenY, respectively) polysaccharides were assessed for their suitability as quantitative standards in various physicochemical assays. The study was designed to evaluate the intended purpose of these standards, namely, to standardize the quantification of the respective polysaccharide content in meningococcal polysaccharide and conjugate vaccines and their intermediate components. Twelve laboratories from eleven different countries participated in the collaborative study of candidate preparations for International Standards for MenW and MenY polysaccharide (coded 16/152 and 16/206, respectively). Unitage was assigned using the Resorcinol assay. Our proposals, on the basis of data from the Resorcinol assay were: 1) candidate standard for MenW polysaccharide (16/152) to be assigned a content of 1.015 ± 0.071 mg MenW polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.13, corresponding to a 95 % level of confidence) and 2) candidate standard for MenY polysaccharide (16/206) be assigned a content of 0.958 ± 0.076 mg MenY polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.26, corresponding to a 95 % level of confidence). The amount of polysaccharide per ampoule remained consistent under all stability conditions over a 36-month period.

International collaborative study to evaluate and calibrate two recombinant L chain Ferritin preparations for use as a WHO International Standard.

OBJECTIVES: To evaluate and calibrate two candidate preparations for the 4th International Standard for Ferritin (Human, Recombinant) (codes: 19/118 and 19/162) against the 3rd International Standard for Ferritin (Human, Recombinant) (code: 94/572), and three serum commutability samples in an international collaborative study involving 12 laboratories in nine countries. METHODS: Eleven of the 12 participating laboratories performed Ferritin quantitation using automated assay platforms and one laboratory used a manual ELISA kit. RESULTS: There was better overall agreement between all laboratories and between assay methods for the potency of preparation 19/118 than for preparation 19/162. The overall geometric mean potency (from all methods) of the candidate 4th International Standard, 19/118, was 10.5 µg/ampoule, with inter-laboratory variability, expressed as % geometric coefficient of variation (GCV), of 4.7%. Accelerated stability studies have predicted both 19/118 and 19/162 to be very stable for long term storage at -20 °C. CONCLUSIONS: The candidate 4th International Standard for Ferritin (Human, Recombinant) (19/118) has been shown to be immunologically similar to the 3rd International Standard for Ferritin (Human, Recombinant) (94/572). It was recommended to and accepted by the WHO Expert Committee on Biological Standardization that 19/118 be established as the 4th International Standard for Ferritin (Human, Recombinant) with an assigned potency of 10.5 µg/ampoule and expanded uncertainty limits 10.2-10.8 µg/ampoule (95% confidence; k=2.23).

Chemical probes targeting the kinase CK2: a journey outside the catalytic box.

CK2 is a protein kinase that plays important roles in many physio-pathological cellular processes. As such, the development of chemical probes for CK2 has received increasing attention in the past decade with more than 40 lead compounds developed. In this review, we aim to provide the reader with a comprehensive overview of the chemical probes acting outside the highly-conserved ATP-site developed to date. Such probes belong to different classes of molecules spanning from small molecules to peptides, act with a range of mechanisms of action and some of them present themselves as promising tools to investigate the biology of CK2 and therefore develop therapeutics for many disease areas including cancer and COVID-19.

Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study.

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase's biology, with wide-reaching implications for drug development.

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

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.

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

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.

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

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.

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

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-IgGR1, 2011. 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-IgGR1, 2011 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-IgGR1, 2011.

Development of small cyclic peptides targeting the CK2α/ß interface.

In this work, an iterative cycle of enzymatic assays, X-ray crystallography, molecular modelling and cellular assays were used to develop a functionalisable chemical probe for the CK2α/ß PPI. The lead peptide, P8C9, successfully binds to CK2α at the PPI site, is easily synthesisable and functionalisable, highly stable in serum and small enough to accommodate further optimisation.

A fragment-based approach leading to the discovery of inhibitors of CK2α with a novel mechanism of action.

CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is through allosteric inhibition. The recently identified αD site represents a promising binding site for allosteric inhibition of CK2α. The work presented herein describes the development of a series of CK2α allosteric inhibitors through iterative cycles of X-ray crystallography and enzymatic assays, in addition to both fragment growing and fragment merging design strategies. The lead fragment developed, fragment 8, exhibits a high ligand efficiency, displays no drop off in activity between enzymatic and cellular assays, and successfully engages CK2α in cells. Furthermore, X-ray crystallographic analysis provided indications towards a novel mechanism of allosteric inhibition through αD site binding. Fragments described in this paper therefore represent promising starting points for the development of highly selective allosteric CK2 inhibitors.

The First WHO International Standard for Harmonizing the Biological Activity of Bevacizumab.

Several Bevacizumab products are approved for clinical use, with many others in late-stage clinical development worldwide. To aid the harmonization of potency assessment across different Bevacizumab products, the first World Health Organization (WHO) International Standard (IS) for Bevacizumab has been developed. Two preparations of a Bevacizumab candidate and comparator were assessed for their ability to neutralize and bind vascular endothelial growth factor (VEGF) using different bioassays and binding assays in an international collaborative study. Relative potency estimates were similar across different assays for the comparator or the duplicate-coded candidate sample. Variability in relative potency estimates was reduced when the candidate standard was used for calculation compared with various in-house reference standards, enabling harmonization in bioactivity evaluations. The results demonstrated that the candidate standard is suitable to serve as an IS for Bevacizumab, with assigned unitages for VEGF neutralization and VEGF binding activity. This standard coded 18/210 was established by the WHO Expert Committee on Biological Standardization, which is intended to support the calibration of secondary standards for product development and lifecycle management. The availability of IS 18/210 will help facilitate the global harmonization of potency evaluation to ensure patient access to Bevacizumab products with consistent safety, quality and efficacy.

The First WHO International Standard for Adalimumab: Dual Role in Bioactivity and Therapeutic Drug Monitoring.

The expanded availability of adalimumab products continues to widen patient access and reduce costs with substantial benefit to healthcare systems. However, the long-term success of these medicines is highly dependent on maintaining consistency in quality, safety and efficacy while minimizing any risk of divergence during life-cycle management. In recognition of this need and demand from global manufacturers, the World Health Organization (WHO) Expert Committee on Biological standardization established the WHO 1st International standard (IS) for Adalimumab (coded 17/236) in October 2019 with a defined unitage ascribed to each of the individual bioactivities evaluated in the study namely, TNF-α binding, TNF-α neutralization, complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity. For development of the IS, two candidate standards were manufactured as per WHO recommendations. Analysis of extensive datasets generated by testing of a common set of samples including the candidate standards by multiple stakeholders including regulatory agencies using their own qualified assays in a large international collaborative study showed comparable biological activity for the tested candidates for the different activities. Use of a common standard significantly decreased the variability of bioassays and improved agreement in potency estimates. Data from this study clearly supports the utility of the IS as an important tool for assuring analytical assay performance, for bioassay calibration and validation, for identifying and controlling changes in bioactivity during life-cycle management and for global harmonization of adalimumab products. In addition, in a separate multi-center study which included involvement of hospital and clinical diagnostic laboratories, the suitability of the adalimumab IS for therapeutic drug monitoring assays was examined by analysis of data from testing of a common blind coded panel of adalimumab spiked serum samples representative of the clinical scenario along with the IS and in-house standards in diverse immunoassays/platforms. Both commercially available and in-house assays that are routinely used for assessing adalimumab trough levels were included. Excellent agreement in estimates for adalimumab content in the spiked samples was observed regardless of the standard or the method with inter-laboratory variability also similar regardless of the standard employed. This data, for the first time, provides support for the extended applicability of the IS in assays in use for therapeutic drug monitoring based on the mass content of the IS. The adalimumab IS, in fulfilling clinical demand, can help toward standardizing and harmonizing clinical monitoring assays for informed clinical decisions and/or personalized treatment strategies for better patient outcomes. Collectively, a significant role for the adalimumab IS in assuring the quality, safety and efficacy of adalimumab products globally is envisaged.

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.

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.

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

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 (n = 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.

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

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.

Efficient development of stable and highly functionalised peptides targeting the CK2α/CK2ß protein-protein interaction.

The discovery of new Protein-Protein Interaction (PPI) modulators is currently limited by the difficulties associated with the design and synthesis of selective small molecule inhibitors. Peptides are a potential solution for disrupting PPIs; however, they typically suffer from poor stability in vivo and limited tissue penetration hampering their wide spread use as new chemical biology tools and potential therapeutics. In this work, a combination of CuAAC chemistry, molecular modelling, X-ray crystallography, and biological validation allowed us to develop highly functionalised peptide PPI inhibitors of the protein CK2. The lead peptide, CAM7117, prevents the formation of the holoenzyme assembly in vitro, slows down proliferation, induces apoptosis in cancer cells and is stable in human serum. CAM7117 could aid the development of novel CK2 inhibitors acting at the interface and help to fully understand the intracellular pathways involving CK2. Importantly, the approach adopted herein could be applied to many PPI targets and has the potential to ease the study of PPIs by efficiently providing access to functionalised peptides.

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

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.