Search

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “Context of Use – COU”); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “context of use” [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term “Context of Use – COU”); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

The protein engineering and formulation of therapeutic proteins for prolonged shelf-life remain a major challenge in the biopharmaceutical industry. Understanding the influence of mutations and formulations on the protein structure and dynamics could lead to more predictive approaches to their improvement. Previous intrinsic fluorescence analysis of the chemically denatured granulocyte colony-stimulating factor (G-CSF) suggested that loop AB could subtly reorganize to form an aggregation-prone intermediate state. Hydrogen deuterium exchange mass spectrometry (HDX-MS) has also revealed that excipient binding increased the thermal unfolding transition midpoint (

The World Health Organization has provided specific guidance for biosimilar products to assist regulators, manufacturers and other professionals involved in the development and evaluation of these products. The development and approval of biosimilars are important for health care, as they allow the marketing of safe, efficacious and affordable biological products. Since the first biosimilars were approved in the EU in 2006, a series of biosimilars have been approved in many countries/geographical regions. This manuscript provides the figures on the status of approved biosimilars in 16 countries based on the information from regulatory experts and from publicly available data. It is clear that increasing numbers of biosimilars are now available in many countries and provide more options for treatments. It is expected that adoption of biosimilars will allow affordable health care and greater patient access to important medicinal products. It will also contribute to the overall WHO goal recognized by the World Health Assembly in 2014 by adopting a resolution on access to biotherapeutic products including biosimilars and on ensuring their quality, safety and efficacy.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9CAR-T Immunogenicity; PCRVaccine Assay Performance; ADA Assay ComparabilityCut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, GeneCell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/OralMultispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/OralMultispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, GeneCell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9CAR-T Immunogenicity; PCRVaccine Assay Performance; ADA Assay ComparabilityCut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, GeneCell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/OralMultispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9CAR-T Immunogenicity; PCRVaccine Assay Performance; ADA Assay Comparabil ityCut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

The World Health Organization (WHO) issued guidelines for the regulatory evaluation of biosimilars in 2009 and has provided considerable effort toward helping member states implement the evaluation principles in the guidelines into their regulatory practices. Despite this effort, a recent WHO survey (conducted in 2019–2020) has revealed four main remaining challenges: unavailable/insufficient reference products in the country; lack of resources; problems with the quality of some biosimilars (and even more with noninnovator products); and difficulties with the practice of interchangeability and naming of biosimilars. The following have been identified as opportunities/solutions for regulatory authorities to deal with the existing challenges: (1) exchange of information on products with other regulatory authorities and accepting foreign licensed and sourced reference products, hence avoiding conducting unnecessary (duplicate) bridging studies; (2) use of a “reliance” concept and/or joint review for the assessment and approval of biosimilars; (3) review and reassessment of the products already approved before the establishment of a regulatory framework for biosimilar approval; and (4) setting appropriate regulatory oversight for good pharmacovigilance, which is essential for the identification of problems with products and establishing the safety and efficacy of interchangeability of biosimilars.

Publisher Copyright: © 2022 The WHO informal consultation was held to promote the revision of WHO guidelines on evaluation of similar biotherapeutic products (SBPs) adopted by the Expert Committee on Biological Standardization (ECBS) in 2009. It was agreed in the past consultations that the evaluation principles in the guidelines are still valid, but a review was recommended to provide more clarity and case-by-case flexibility. The opportunity was therefore taken to review the experience and identify areas where the current guidance could be more permissive without compromising its basic principles, and where additional explanation could be provided regarding the possibility of reducing the amount of data needed for regulatory approval. The meeting participants applauded the leading role taken by the WHO in providing a much-needed streamlined approach for development and evaluation of SBPs which will provide efficient and cost-effective product development and increase patient access to treatments. It was recognized that the principles as currently described in the draft WHO guidelines are based on sound science and experience gained over the last fifteen years of biosimilar approvals. However, since these guidelines when finalised will constitute the global standard for biosimilar evaluation and assist national regulatory authorities in establishing revised guidance and regulatory practice in this complex area, it was felt that further revision and clarity on certain perspectives in specific areas was necessary to dispel uncertainties arising in the current revised version. This report describes the principles in the draft guidelines, including topics discussed and consensus reached.

Therapeutic monoclonal antibodies (mAbs) are used to manage a wide range of cancers and autoimmune disorders. However, mAb-based treatments are not always successful, highlighting the need for a better understanding of the factors influencing mAb efficacy. Increased levels of oxidative stress associated with several diseases are counteracted by the activities of various oxidoreductase enzymes, such as thioredoxin (Trx), which also reduces allosteric disulfide bonds in proteins, including mAbs. Here, using an array of in vitro assays, we explored the functional effects of Trx-mediated reduction on the mechanisms of action of six therapeutic mAbs. We found that Trx reduces the inter-chain disulfide bonds of the mAbs, after which they remain intact but have altered function. In general, this reduction increased antigen-binding capacity, resulting in, for example, enhanced tumour necrosis factor (TNF) neutralization by two anti-TNF mAbs. Conversely, Trx reduction decreased the anti-proliferative activity of an anti–tyrosine kinase-type cell-surface receptor HER2 (HER2) mAb. In all the mAbs, Fc receptor binding was abrogated by Trx activity, with significant loss in both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) activity of the mAbs tested. We also confirmed that without alkylation, Trx-reduced inter-chain disulfide bonds reoxidize, and ADCC activity is restored. In summary, Trx-mediated reduction has a substantial impact on the functional effects of an mAb, including variable effects on antigen binding and Fc function, with the potential to significantly impact mAb efficacy in vivo.

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.

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.

Coronavirus disease 2019 (COVID-19) has caused significant devastation globally. Despite the development of several vaccines, with uncertainty around global uptake and vaccine efficacy, the need for effective therapeutic agents remains. Increased levels of cytokines including tumour necrosis factor are significant in the pathogenesis of COVID-19 and associated with poor outcomes including ventilator requirement and mortality. Repurposing tumour necrosis factor blocker therapy used in conditions such as rheumatoid arthritis and inflammatory bowel disease seems promising, with early feasibility data showing a reduction in circulation of pro-inflammatory cytokines and encouraging the evaluation of such interventions in preventing disease progression and clinical deterioration in patients with COVID-19. Here, we examine the biological activities of tumour necrosis factor inhibitors indicative of their potential in COVID-19 and briefly outline the randomised control trials assessing their benefit-risk profile in COVID-19 therapy., Graphical abstract

Etanercept, a recombinant human tumor necrosis factor (TNF) receptor Fc fusion protein is an effective treatment option in adults with rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis or plaque psoriasis and paediatrics with juvenile idiotypic arthritis and plaque psoriasis. Patent expiration in Europe and intense development of various etanercept products worldwide triggered a need for an international reference standard to facilitate determination of biological activity. Therefore, three candidate preparations of etanercept were lyophilized and evaluated in a multi-centre collaborative study comprising twenty eight laboratories from 15 countries for their suitability to serve as an international standard for the bioactivity of TNF receptor II Fc fusion proteins (international nonproprietary name, Etanercept). The preparations were tested for neutralization activity against the third TNF-α international standard (IS) in different in vitro cell-based assays, e.g., cytotoxicity, apoptosis and reporter gene methods. Regardless of the assay and the amount of TNF-α IS used, potency estimates for the different preparations were very similar. An indication of the inhibitory activity of etanercept in terms of the biological activity of the TNF-α IS based on ED50 data derived from a limited number of laboratories using a cytotoxicity assay was also derived. Results indicated that the candidate preparation coded 13/204 was stable and suitable to serve as an international standard for the biological activity of etanercept. Therefore, the preparation coded 13/204 was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2015 as the WHO first International Standard for TNF receptor II Fc fusion protein (INN, etanercept) with an assigned in vitro bioactivity of 10,000IU per ampoule. It should be noted that this first-in-class international standard for a Fc fusion protein, available from the National Institute for Biological Standards and Control and also as a biological reference preparation (BRP) from the European Directorate for the Quality of Medicines and Healthcare, is intended for controlling the performance of biological assays for etanercept and to support the establishment of in-house bioassay standards. This standard is not intended for describing the labelling or dosage of etanercept therapeutic products or for use as a comparator (reference product) for biosimilarity determination.

The first global workshop on implementation of the WHO guidelines on procedures and data requirements for changes to approved biotherapeutic products adopted by the WHO Expert Committee in 2018 was held in June 2019. The workshop participants recognized that the principles based on sound science and the potential for risk, as described in the WHO Guidelines on post-approval changes, which constitute the global standard for product life-cycle management are providing clarity and helping national regulatory authorities in establishing guidance while improving time-lines for an efficient regulation of products. Consequently, the regulatory situation for post-approval changes and guideline implementation is changing but there is a disparity between different countries. While the guidelines are gradually being implemented in some countries and also being considered in other countries, the need for regional workshops and further training on post-approval changes was a common theme reiterated by many participants. Given the complexities relating to post-approval changes in different regions/countries, there was a clear understanding among all participants that an efficient approach for product life-cycle management at a national level is needed to ensure faster availability of high standard, safe and efficacious medicines to patients as per the World Health Assembly Resolution 67.21.

Understanding of the determinants of immunogenicity, the testing paradigm, the impact of antibody attributes on clinical outcomes and regulatory guidance is leading to harmonized practices for immunogenicity assessment of biotherapeutics. However, generation of robust immunogenicity data for inclusion in product labels to support clinical practice continues to be a challenge. Assays, protocols and antibody positive controls/standards need to be developed in sufficient time to allow assessment of clinical immunogenicity using validated methods and optimized protocols. Standardization and harmonization play a significant role in achieving acceptable results. Harmonization in the postapproval setting is crucial for a valid interpretation of the product's immunogenicity and its clinical effects. Efforts are ongoing to standardize assays where possible for antibody measurement and for measuring product/drug levels by producing reference standards. Provision of such standards will help toward personalized treatment strategies with better patient outcomes.

For biosimilar drug development programs, it is essential to demonstrate that there are no clinically significant differences between the proposed biosimilar therapeutic (biosimilar) and its reference product (originator). Based on a stepwise comprehensive comparability exercise, the biosimilar must demonstrate similarity to the originator in physicochemical characteristics, biological activity, pharmacokinetics, efficacy, and safety, including immunogenicity. The goal of the immunogenicity assessment is to evaluate potential differences between the proposed biosimilar product and the originator product in the incidence and severity of human immune responses. Establishing that there are no clinically meaningful differences in the immune response between the products is a key element in the demonstration of biosimilarity. An issue of practical, regulatory, and financial importance is to establish whether a two-assay (based on the biosimilar and originator respectively) or a one-assay approach (based on the biosimilar) is optimal for the comparative immunogenicity assessment. This paper recommends the use of a single, biosimilar-based assay for assessing immunogenic similarity in support of biosimilar drug development. The development and validation of an ADA assay used for a biosimilar program should include all the assessments recommended for an innovator program (10-16, 29). In addition, specific parameters also need to be evaluated, to gain confidence that the assay can detect antibodies against both the biosimilar and the originator. Specifically, the biosimilar and the originator should be compared in antigenic equivalence, to assess the ability of the biosimilar and the originator to bind in a similar manner to the positive control(s), as well as in the confirmatory assay and drug tolerance experiments. Practical guidance for the development and validation of anti-drug antibody (ADA) assays to assess immunogenicity of a biosimilar in comparison to the originator, using the one-assay approach, are described herein.

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.

IL-27 is a pleiotropic cytokine of the IL-6/IL-12 family with diverse biological functions. Previous in vivo studies have suggested the antitumor activities of IL-27 in animal models, whereas clinical observations indicate the link of IL-27 in tumor progression. IL-27 has recently been shown to cause inhibition of proliferation on primary leukemic cells from pediatric patients, but information on its role in human leukemic cell lines is limited. In the present study, we investigated the ability of IL-27 to regulate cell growth and survival of various human leukemic cell lines. Our results showed that in human leukemic cell lines coexpressing both IL-27R chains, IL-27Rα and gp130, IL-27 did not inhibit cell growth, but caused dose-dependent proliferation of the acute myeloid leukemic cell line, OCI-AML5, and the erythroleukemic cell lines, TF-1, UT-7, and UT-7/EPO. Consistent with this, IL-27 promoted cell survival and reduced TNF-α-induced apoptosis of the leukemic cell lines. IL-27 also decreased the responsiveness of the leukemic cells to chemotherapeutic drugs, cytarabine and daunorubicin. We observed that IL-27 induced the activation of STAT1/3 and ERK1/2 in the leukemic cells. Growth stimulation by IL-27 was suppressed by the specific MEK inhibitor, U0126, indicating that IL-27-induced cell proliferation is mainly mediated through the activation of the MAPK/ERK signaling pathway. The present study is the first demonstration of the proliferative and antichemotherapeutic properties of IL-27 in human leukemic cell lines, suggesting that IL-27 can play an unfavorable role in tumor growth and can be an important determinant in the chemoresponsiveness of certain subtypes of human leukemia.

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9–13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event – a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies’ input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis, issues 22 and 24 (2018), respectively.

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.

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.

In April 2017, WHO convened an informal consultation to develop WHO guidelines on procedures and data requirements for changes to approved biotherapeutic products. The objective of the meeting was to review the draft of WHO guidelines and the comments received from the public consultation. The guidelines were recognized by the participants as a tool for regulatory convergence and harmonization. Regulation of changes to approved biotherapeutic products is a key in ensuring that products of consistent quality, safety and efficacy are distributed after they receive authorization or licensure. Participants agreed that the guidelines would contribute to assuring the continued quality, safety and efficacy throughout the life-cycle of biotherapeutics as well as continuity in supply and access. In the meeting, participants further requested WHO should assist national regulatory authorities in improving technical expertise in the evaluation of biotherapeutics and their post-approval changes by organizing implementation workshops and developing case studies and e-training modules on various technical topics. At its meeting in October 2017, the WHO Expert Committee on Biological Standardization formally adopted the WHO guidelines on procedures and data requirements for changes to approved biotherapeutic products.

Introduction Interferon (IFN)-α and IFN-β approved for treatment of chronic hepatitis C viral infection and multiple sclerosis respectively have been linked to thrombotic microangiopathy (TMA) affecting renal function. Since the molecular mechanisms underlying this severe complication remain largely unclear, we aimed to investigate whether IFN affects directly in vitro endothelial cell functions associated with angiogenesis and blood haemostasis, as well as endothelial cell-derived vasodilators of nitric oxide (NO) and prostacyclin. Methods Proliferation and survival of human umbilical vein endothelial cells (HUVECs) were measured by BrdU incorporation and alamarBlue assays. Angiogenesis was evaluated in co-cultures of HUVECs and human dermal fibroblasts. Fibrinolysis molecules were measured with ELISA. NO and prostacyclin were measured using a fluorescent NO-specific probe and a competitive enzyme immunoassay, respectively. Results HUVEC proliferation was dose-dependently inhibited by IFN-β1a and IFN-β1b, but not by IFN-α2a and IFN-α2b. Consistently, IFN-β1a and IFN-β1b also reduced survival of HUVECs, but this again was not observed with IFN-α. However, both IFN subtypes inhibited VEGF-induced development of capillary-like structures, but the effect of IFN-α was less potent than IFN-β. In addition, both IFN subtypes upregulated interferon inducible protein 10 production from treated co-cultures while suppressing angiogenesis. Furthermore, intracellular NO generation was reduced by IFN-α2a and IFN-β1a, whereas prostacyclin release from HUVECs was not affected by IFN. Importantly, both IFN-β1a- and IFN-β1b-treated HUVECs showed a marked reduction in urokinase-type plasminogen activator release and a much greater secretion of plasminogen activator inhibitor-1 than tissue-type plasminogen activator compared with untreated cells, suggesting decreased fibrinolytic activity. IFN-α, however was less effective in modulating the fibrinolysis system. Conclusions We demonstrate the detrimental effects of IFN on endothelial cell functions mediated with angiogenesis and fibrinolysis, which could potentially cause the loss of physiological endothelium thromboresistance and facilitate the development of vascular complications in a clinical setting. Mechanistically, our findings have implications for understanding how IFN therapy can foster the development of TMA.

Traditional antibody dependent cellular cytotoxicity (ADCC) assays use donor derived natural killer (NK) or peripheral blood mononuclear cells, but donor genetic variability and the technically challenging nature of the assay means that alternative in vitro assay formats are required. We explored the utility of two reporter gene cell lines, the J2 and J9, as surrogate effector cells for ADCC assays. Both express the ADCC relevant Fcγ receptor CD16, crosslinking of which leads to firefly luciferase expression. For anti-CD20 rituximab and anti-HER2 trastuzumab (both IgG1 monoclonal antibodies, mAbs) a dose dependent firefly luciferase response was observed exclusively in the presence of their respective targets, representing the molecular interaction which potentiates ADCC activity. Importantly, both surrogate effector and NK cell based assays gave statistically similar values for rituximab ADCC activity. Increased engagement with target cell bound mAbs was determined to be cytotoxic for the J2 and J9 cell lines at the assay end point (at which luciferase expression is measured). However, use of the J9 cells containing the constitutively expressed renilla luciferase gene enabled data normalisation and corrected for fluctuations in both cell number and viability providing an advantage over currently available surrogate effector cell-lines. Abrogated ADCC activity with IgG4 mAbs, but enhanced activity with an IgG1 non-fucosylated mAb, was seen with the J9 cell line, as expected. Additionally, two rituximab products (biosimilars in development) with similar binding by flow cytometry, N-glycan profiles using HPLC and CD16 binding by surface plasmon resonance showed comparable ADCC activity to Mabthera. The ADCC activity of another anti-CD20 mAb, ofatumumab, reported only with primary cell based assays to date was also measured. This is the first report of a dual reporter gene based ADCC assay.

Patients treated with the TNF antagonist adalimumab develop anti-therapeutic antibodies (ATA), the prevalence of which varies depending on the assay used. Most assays are compromised due to the presence of adalimumab in the clinical samples. Our objective was to develop an antibody assay, applicable for clinical testing, which overcomes the limitation of therapeutic interference and to further determine the relationship between ATA development, adalimumab levels and disease activity in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) or ankylosing spondylitis (AS). Use of an electrochemiluminescence platform permitted development of fit-for-purpose immunoassays. Serum samples from patients, taken prior to and at 12 and 24weeks of treatment, were retrospectively analysed for levels of adalimumab and ATA. Overall, the antibody prevalence was 43.6% at 12weeks and 41% at 24weeks of treatment. Disruption of immune complexes by acid dissociation, a strategy often adopted for this purpose, only marginally increased the antibody prevalence to 48.7% and 46% at 12 and 24weeks respectively. We found that antibody formation was associated with decreasing levels of circulating adalimumab, but no direct effect on disease activity was evident as assessed using DAS28 for RA patients and BASDAI for PsA and AS patients. However, a negative correlation of free adalimumab trough levels with disease activity scores was observed. Data showed that adalimumab levels can serve as an indicator of ATA development which can then be confirmed by ATA testing. Monitoring of both therapeutic and antibodies should be considered during adalimumab therapy to allow clinicians to personalise treatments for maximal therapeutic outcomes.

Cytokines regulate many aspects of cell growth and differentiation and play pivotal roles in the orchestration of immune defence against invading pathogens. Though 'self' proteins, they are potentially immunogenic and can give rise to anti-cytokine autoantibodies (aCA). The main foci of the article are a critical summary of the various methodologies applied for detecting and measuring aCA and a broad review of studies of the occurrence, characterization and clinical relevance of aCA in normal healthy individuals, patients with autoimmune diseases or microbial infections and aCA in patients whose disease is treated with recombinant cytokine products. The need for technical and methodological improvement of assays, including validation and standardization, together with approaches to harmonize calculation and reporting of results is also discussed.

Two candidate preparations of human sequence recombinant Interleukin-2 (IL-2) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement international standard. The preparations were tested by eight laboratories using in vitro bioassays and immunoassays. The candidate preparation 86/500 was judged suitable to serve as a replacement international standard based on the data obtained for activity and stability. On the basis of the results reported here, the preparation coded 86/500 was established by the WHO Expert Committee on Biological Standardisation (ECBS) in 2012 as the WHO 2nd IS for human IL-2 with an assigned value for IL-2 activity of 210IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies exclusively on the estimates calculated relative to the WHO 1st IS for continuity of the IU.

A subset of patients with relapsing-remitting multiple sclerosis (RRMS) on therapy with interferon beta (IFNβ) develop neutralising anti-drug antibodies (ADA) resulting in reduced, or loss of, therapeutic efficacy. The aims were to characterise the relative contributions of anti-IFNβ antibody isotypes to drug neutralising activity, ability of these antibodies to cross-react with endogenous IFNβ, to form immune complexes and activate complement. IFNβ-specific ADA were measured in plasma from RRMS patients treated with IFNβ1a (Rebif®). Neutralisation of endogenous and therapeutic IFNβ by ADA was determined by IFNβ bioassay. IFNβ-ADA profile was predominantly comprised of IgG1 and IgG4 antibody isotypes. The contribution of IgG4-ADA towards neutralising activity was found to be minimal. Neutralising IFNβ-ADA blocks endogenous IFNβ activity. ADA interaction with therapeutic IFNβ results in immune complex formation and complement activation. In summary, IgG1 and IgG4 IFNβ-ADA have the ability to neutralise therapeutic and endogenous protein and to activate complement., Highlights • IgG4 and IgG1 contributes to IFNβ-ADA profile • Neutralising IFNβ-ADA cross reacts and blocks endogenous IFNβ activity. • ADA-IFNβ results in IC formation and complement activation

Aim To determine if cytokine release with a solid phase assay is predictive of adverse responses for a range of therapeutic mAbs. Methods Cytokine ELISAs and a multi-array system were used to compare responses generated by different therapeutic mAbs using a solid phase assay. Flow cytometry was employed to determine the cellular source of those cytokines. Results Only TGN1412 and muromonab-CD3 stimulated CD4+ T-cell mediated cytokine release characterized by significant (all P < 0.0001) IFNγ, TNFα, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and IL-22 release, comparable with T-cell mitogen. Significantly greater (P < 0.0001) IL-2 release with TGN1412 (2894–6051 pg ml−1) compared with muromonab-CD3 (62–262 pg ml−1) differentiated otherwise comparable cytokine responses. Likewise, TGN1412 stimulated significantly more (P = 0.0001) IL-2 producing CD4+ T-cells than muromonab-CD3 and induced Th1, Th2, Th17 and Th22 subsets that co-release this cytokine. Significant TNFα release was observed with bevacizumab (P = 0.0001), trastuzumab (P = 0.0031) and alemtuzumab (P = 0.0177), but no significant IL-2 release. TGN1412 and muromonab-CD3 caused pro-inflammatory cytokine release despite significantly (both P < 0.0001) increasing numbers of T-cells with a regulatory phenotype. Conclusions The severity of the adverse response to TGN1412 compared with muromonab-CD3 and other therapeutic mAbs correlates with the level of IL-2 release.

Recombinant erythropoietin (rHuEPO) is used extensively to treat anaemia associated with chronic kidney disease. However, the development of neutralizing antibodies (NAbs) to rHuEPO can result in the development of antibody-mediated pure red cell aplasia (PRCA). The detection of NAb in patient sera by in vitro bioassay relies on the inhibition of a cellular response to rHuEPO. Current bioassays for rHuEPO measure proliferation in responsive cell lines such as the erythroleukaemic cell lines, UT-7 and UT-7/EPO, the latter sensitized to EPO. Using these cell lines, we show the dose-responsive induction of both PIM1 and EGR1 gene expression in UT-7 cells and of EGR1 in UT-7/EPO cells. The expression of EGR1 in UT-7/EPO cells in response to rHuEPO was comparable to the proliferative response measured by (3)H-thymidine incorporation and could be inhibited by serum from a patient with NAb-mediated PRCA in a dilution-dependent manner. Bioassays based on the induction of endogenous gene expression are comparable to current bioassays but are considerably quicker given that incubation time is decreased from 2-3 days to 50 min. Measurement of EGR1 gene expression in response to rHuEPO in UT-7/EPO cells offers a rapid, non-radioactive and automatable alternative to current assays for the detection of rHuEPO NAbs.

Highlights • ECL-based assays for measurement of adalimumab and adalimumab antibodies. • Performance of ECL antibody assay not significantly improved by acid dissociation. • Negative correlation between levels of antibody and free adalimumab. • Negative correlation between adalimumab level and disease activity scores., Patients treated with the TNF antagonist adalimumab develop anti-therapeutic antibodies (ATA), the prevalence of which varies depending on the assay used. Most assays are compromised due to the presence of adalimumab in the clinical samples. Our objective was to develop an antibody assay, applicable for clinical testing, which overcomes the limitation of therapeutic interference and to further determine the relationship between ATA development, adalimumab levels and disease activity in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) or ankylosing spondylitis (AS). Use of an electrochemiluminescence platform permitted development of fit-for-purpose immunoassays. Serum samples from patients, taken prior to and at 12 and 24 weeks of treatment, were retrospectively analysed for levels of adalimumab and ATA. Overall, the antibody prevalence was 43.6% at 12 weeks and 41% at 24 weeks of treatment. Disruption of immune complexes by acid dissociation, a strategy often adopted for this purpose, only marginally increased the antibody prevalence to 48.7% and 46% at 12 and 24 weeks respectively. We found that antibody formation was associated with decreasing levels of circulating adalimumab, but no direct effect on disease activity was evident as assessed using DAS28 for RA patients and BASDAI for PsA and AS patients. However, a negative correlation of free adalimumab trough levels with disease activity scores was observed. Data showed that adalimumab levels can serve as an indicator of ATA development which can then be confirmed by ATA testing. Monitoring of both therapeutic and antibodies should be considered during adalimumab therapy to allow clinicians to personalise treatments for maximal therapeutic outcomes.

Summary The production of anti‐drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics. It is known that various factors, including aggregation and the presence of process‐related impurities, can modify and augment the immunogenic potential of proteins. The purpose of the investigations reported here was to characterize in mice the influence of aggregation and host cell protein impurities on the immunogenicity of a humanized single‐chain antibody variable fragment (scFv), and mouse albumin. Host cell protein impurities within an scFv preparation purified from Escherichia coli displayed adjuvant‐like activity for responses to the scFv in BALB/c strain mice. The 70 000 MW E. coli chaperone protein DnaK was identified as a key contaminant of scFv by mass spectrometric analysis. Preparations of scFv lacking detectable DnaK were spiked with recombinant E. coli DnaK to mimic the process‐related impurity. Mice were immunized with monomeric and aggregated preparations, with and without 0·1% DnaK by mass. Aggregation alone enhanced IgM and IgG2a antibody responses, but had no significant effect on total IgG or IgG1 responses. The addition of DnaK further enhanced IgG and IgG2a antibody responses, but only in the presence of aggregated protein. DnaK was shown to be associated with the aggregated scFv by Western blot analysis. Experiments with mouse albumin showed an overall increase in immunogenicity with protein aggregation alone, and the presence of DnaK increased the vigour of the IgG2a antibody response further. Collectively these data reveal that DnaK has the potential to modify and enhance immunogenicity when associated with aggregated protein.

B-cell activating factor (BAFF) is a type II transmembrane glycoprotein belonging to the tumour necrosis factor ligand superfamily. Active soluble forms of BAFF are generated either by cleavage of the extracellular domain or by recombinant DNA technology. The current bioassay for measuring the activity of soluble BAFF involves stimulation of the proliferation of mouse splenic B-cells in the presence of goat anti-mouse IgMmicro chain which is rather cumbersome and lengthy and yields variable results. We have therefore developed an alternative functional assay which relies on the ability of BAFF to induce an apoptotic response in human rhabdomyosarcoma cells. For this, we constructed a chimeric receptor containing the ectodomain of the MuBAFF-R--the major cell receptor for BAFF--and the endodomain of the HuTRAIL-R2--one of the two functional receptors for TRAIL--which is known to contain a death domain and trigger apoptosis. When the chimeric receptor was expressed in the TRAIL-sensitive human rhabdomyosarcoma cell line KD4 clone 21, recombinant BAFF of either human or mouse sequence stimulated apoptosis, similar to TRAIL, in a dose-dependent manner. The transfected cell population, called FL17, expressing the MuBAFF-R/ HuTRAIL-R2 thus provided the basis of a novel functional bioassay for BAFF that is simple and relatively fast to perform. The construction of the chimeric receptor, development of the transfected cells expressing this receptor and the development of sensitive and reproducible bioassays for BAFF and anti-BAFF neutralising antibodies are described.

Biosimilar medicinal products (biosimilars) have become a reality in the European Union and will soon be available in the United States. Despite an established legal pathway for biosimilars in the European Union since 2005 and increasing and detailed regulatory guidance on data requirements for their development and licensing, many clinicians, particularly oncologists, are reluctant to consider biosimilars as a treatment option for their patients. Major concerns voiced about biosimilars relate to their pharmaceutical quality, safety (especially immunogenicity), efficacy (particularly in extrapolated indications), and interchangeability with the originator product. In this article, the members and experts of the Working Party on Similar Biologic Medicinal Products of the European Medicines Agency (EMA) address these issues. A clear understanding of the scientific principles of the biosimilar concept and access to unbiased information on licensed biosimilars are important for physicians to make informed and appropriate treatment choices for their patients. This will become even more important with the advent of biosimilar monoclonal antibodies. The issues also highlight the need for improved communication between physicians, learned societies, and regulators.

One candidate preparation of human sequence recombinant transforming growth factor-β3 (TGF-β3) was formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for its suitability to serve as an international standard. The preparation was tested by 8 laboratories using in vitro bioassays and immunoassays. The candidate preparation 09/234 was judged suitable to serve as an international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/234 was established by the WHO Expert Committee on Biological Standardisation (ECBS) as the WHO 1st IS for human TGF-β3 with an assigned value for TGF-β3 activity of 19,000 IU/ampoule.

In August 2010, the World Health Organization and the Korea Food & Drug Administration jointly organized the first implementation workshop of WHO guidelines on evaluating similar biotherapeutic products (SBPs) at the global level. The objective of the Workshop was to facilitate implementation of the newly adopted WHO Guidelines into the practice of national regulatory authorities (NRAs). WHO Guidelines were recognized by the workshop participants as a tool for harmonizing regulatory requirements worldwide. By reviewing and practicing several case studies, better understanding and consensus on the principles of clinical trial designs were reached. However, variations in terms of the national requirements for quality, safety and efficacy of these products revealed diversity in the regulatory expectations in different countries and regions. In addition, lack of terminology for the products developed as copy products (so called "me too" products) with a partial comparability to an RBP, led to a great diversity in evaluating as well as naming these products. The workshop participants proposed the following actions: a) NRAs should make efforts to build their capacities for regulation of SBPs; b) WHO should revise WHO Guidelines for assuring the quality of products prepared by recombinant DNA technology (WHO TRS 814) and continue monitoring progress with the implementation of the Guidelines on evaluating SBPs. Publication of the outcome of the Workshop was recognized as another action that WHO should coordinate.