Publications by authors named "Meenu Wadhwa"

66 Publications

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

Biomolecules 2021 Oct 30;11(11). Epub 2021 Oct 30.

Division of Biotherapeutics, National Institute for Biological Standards and Control, Hertfordshire EN6 3QG, UK.

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.
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http://dx.doi.org/10.3390/biom11111610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8615914PMC
October 2021

Therapeutic use of specific tumour necrosis factor inhibitors in inflammatory diseases including COVID-19.

Biomed Pharmacother 2021 Aug 28;140:111785. Epub 2021 May 28.

NIBSC, MHRA, Blanche Lane, South Mimms, Hertfordshire EN6 3QG, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biopha.2021.111785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162906PMC
August 2021

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

Front Immunol 2021 15;12:636420. Epub 2021 Apr 15.

Analytical and Biological Sciences Division, National Institute for Biological Standards and Control, Medicines and Healthcare Products Regulatory Agency, Potters Bar, United Kingdom.

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 1 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.
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http://dx.doi.org/10.3389/fimmu.2021.636420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082443PMC
September 2021

Maintaining 'standards' for biosimilar monoclonal antibodies.

Nat Biotechnol 2021 03;39(3):276-280

National Institute for Biological Standards and Control (NIBSC), South Mimms, United Kingdom.

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http://dx.doi.org/10.1038/s41587-021-00848-0DOI Listing
March 2021

Regulatory challenges with biosimilars: an update from 20 countries.

Ann N Y Acad Sci 2021 05 21;1491(1):42-59. Epub 2020 Nov 21.

Pharmaceuticals and Medical Devices Agency, Tokyo, Japan.

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.
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http://dx.doi.org/10.1111/nyas.14522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247359PMC
May 2021

WHO implementation workshop on guidelines on procedures and data requirements for changes to approved biotherapeutic products, Seoul, Republic of Korea, 25-26 June 2019.

Biologicals 2020 May 17;65:50-59. Epub 2020 Jan 17.

World Health Organization, Switzerland.

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.
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http://dx.doi.org/10.1016/j.biologicals.2019.12.008DOI Listing
May 2020

Recommendations for the Development and Validation of Immunogenicity Assays in Support of Biosimilar Programs.

AAPS J 2019 12 2;22(1). Epub 2019 Dec 2.

BioAgilytix Labs, Durham, North Carolina, USA.

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.
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http://dx.doi.org/10.1208/s12248-019-0386-yDOI Listing
December 2019

The impact of thioredoxin reduction of allosteric disulfide bonds on the therapeutic potential of monoclonal antibodies.

J Biol Chem 2019 12 14;294(51):19616-19634. Epub 2019 Nov 14.

Division of Biotherapeutics, The National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom

Therapeutic 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 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 interchain 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 tumor necrosis factor (TNF) neutralization by two anti-TNF mAbs. Conversely, Trx reduction decreased the antiproliferative activity of an anti-tyrosine kinase-type cell-surface receptor HER2 mAb. In all of the mAbs, Fc receptor binding was abrogated by Trx activity, with significant loss in both complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) activity of the mAbs tested. We also confirmed that without alkylation, Trx-reduced interchain 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 .
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http://dx.doi.org/10.1074/jbc.RA119.010637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926469PMC
December 2019

Bioanalytical strategies in determining immunogenicity.

Bioanalysis 2019 Sep;11(17):1535-1537

Welwyn, Hertfordshire, UK.

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http://dx.doi.org/10.4155/bio-2019-0233DOI Listing
September 2019

Harmonization and standardization of immunogenicity assessment of biotherapeutic products.

Bioanalysis 2019 Sep;11(17):1593-1604

Welwyn, Hertfordshire, UK.

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.
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http://dx.doi.org/10.4155/bio-2019-0202DOI Listing
September 2019

WHO International Standards and reference preparations for cytokines and growth factors.

Cytokine 2019 11 8;123:154797. Epub 2019 Aug 8.

Cytokine & Growth Factors Section, Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), Medicines and Healthcare products Regulatory Agency, Hertfordshire, UK. Electronic address:

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http://dx.doi.org/10.1016/j.cyto.2019.154797DOI Listing
November 2019

WHO International Standards and Reference Preparations for Cytokines and Growth Factors.

J Interferon Cytokine Res 2019 07;39(7):438-439

2 ICIS Standards Committee Member, Cytokine and Growth Factors Section, Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), Medicines and Healthcare Products Regulatory Agency (MHRA), Hertfordshire, United Kingdom.

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http://dx.doi.org/10.1089/jir.2019.0078DOI Listing
July 2019

WHO International Standards and Reference Preparations for Cytokines and Growth Factors.

J Leukoc Biol 2020 01 29;107(1):159-160. Epub 2019 Apr 29.

ICIS Standards Committee, Cytokine & Growth Factors Section, Biotherapeutics Group, National Institute for Biological Standards and Control (NIBSC), Medicines and Healthcare Products Regulatory Agency, Hertfordshire, United Kingdom.

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http://dx.doi.org/10.1002/JLB.3LT0419-120DOI Listing
January 2020

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

Bioanalysis 2018 Dec 29;10(23):1897-1917. Epub 2018 Nov 29.

US FDA, Silver Spring, MD, USA.

The 2018 12 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 , issues 22 and 24 (2018), respectively.
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http://dx.doi.org/10.4155/bio-2018-0285DOI Listing
December 2018

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

Bioanalysis 2018 Dec 29;10(24):1973-2001. Epub 2018 Nov 29.

Genentech, South San Francisco, CA, USA.

The 2018 12 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 full immersion in 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 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of , issues 22 and 23 (2018), respectively.
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http://dx.doi.org/10.4155/bio-2018-0287DOI Listing
December 2018

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

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

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

Due to the increase in the number of infliximab products, the need for global harmonization of the bioactivity of this monoclonal antibody was recognized by the World Health Organization (WHO). In response, the National Institute for Biological Standards and Control (NIBSC) developed the first international standard (IS) for infliximab, which targets tumour necrosis factor (TNF). Each ampoule is assigned values of 500 IU of TNF neutralizing activity and 500 IU of binding activity. Two preparations of infliximab were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as an IS for the in vitro biological activity of infliximab. The study involved participants using in vitro cell-based bioassays (TNF neutralization, antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity) and binding assays. The results of this study showed that the candidate preparation, coded 16/170, is suitable as an IS for infliximab bioactivity. This infliximab IS from NIBSC, is intended to support in vitro bioassay calibration and validation by defining international units of bioactivity. The proposed unitages, however, are not intended to revise product labelling or dosing requirements, as any decisions regarding this relies solely with the regulatory authorities. Furthermore, the infliximab IS is not intended for determining the specific activity of products, nor to serve any regulatory role in defining biosimilarity. We briefly discuss the future use of WHO international standards in supporting the global harmonisation of biosimilar infliximab products.
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http://dx.doi.org/10.1080/19420862.2018.1532766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343779PMC
January 2019

Endothelial cell functions impaired by interferon in vitro: Insights into the molecular mechanism of thrombotic microangiopathy associated with interferon therapy.

Thromb Res 2018 03 6;163:105-116. Epub 2018 Feb 6.

Section of Cytokines and Growth Factors, Division of Biotherapeutics, National Institute for Biological Standards and Control, United Kingdom.

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.
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http://dx.doi.org/10.1016/j.thromres.2018.01.039DOI Listing
March 2018

WHO informal consultation on development of guidelines on procedures and data requirements for changes to approved biotherapeutic products, Seoul, Republic of Korea, 27-28 April 2017.

Biologicals 2018 Mar 27;52:83-91. Epub 2017 Dec 27.

Department of Medical Sciences, Ministry of Public Health, Tivanon Road, Nonthaburi 11000, Thailand. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biologicals.2017.12.002DOI Listing
March 2018

Reprint of "Anti-therapeutic antibodies and their clinical impact in patients treated with the TNF antagonist adalimumab".

Cytokine 2018 01 23;101:70-77. Epub 2017 Nov 23.

Biotherapeutics Group, Cytokines and Growth Factor Section, National Institute for Biological Standards and Control, Medicines and Healthcare products Regulatory Agency, Blanche Lane, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.

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.
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http://dx.doi.org/10.1016/j.cyto.2017.11.007DOI Listing
January 2018

Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic monoclonal antibodies.

J Pharm Biomed Anal 2017 Sep 9;143:188-198. Epub 2017 Jun 9.

Cytokines and Growth Factors Section, Biotherapeutics Group, NIBSC, Blanche Ln, South Mimms, Potters Bar EN6 3QG, UK.

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.
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http://dx.doi.org/10.1016/j.jpba.2017.06.004DOI Listing
September 2017

Establishment of the first WHO International Standard for etanercept, a TNF receptor II Fc fusion protein: Report of an international collaborative study.

J Immunol Methods 2017 08 10;447:14-22. Epub 2017 Mar 10.

European Directorate for the Quality of Medicines & HealthCare, 7 allée Kastner, CS 30026, F-67081 Strasbourg, France.

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.
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http://dx.doi.org/10.1016/j.jim.2017.03.007DOI Listing
August 2017

Anti-therapeutic antibodies and their clinical impact in patients treated with the TNF antagonist adalimumab.

Cytokine 2017 08 6;96:16-23. Epub 2017 Mar 6.

Biotherapeutics Group, Cytokines and Growth Factor Section, National Institute for Biological Standards and Control, Medicines and Healthcare products Regulatory Agency, Blanche Lane, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.

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.
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http://dx.doi.org/10.1016/j.cyto.2017.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484178PMC
August 2017

Influence of Escherichia coli chaperone DnaK on protein immunogenicity.

Immunology 2017 03 7;150(3):343-355. Epub 2016 Dec 7.

Faculty of Biology, Medicine and Health, School of Biological Sciences, The University of Manchester, Manchester, UK.

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.
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http://dx.doi.org/10.1111/imm.12689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290234PMC
March 2017

Editor's Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response.

Toxicol Sci 2016 10 30;153(2):258-70. Epub 2016 Jun 30.

*Faculty of Life Sciences, The University of Manchester, C1256, Michael Smith Building, Dover St, Manchester M13 9PT, UK;

Protein aggregation is associated with enhanced immunogenicity of biotherapeutics. As a result, regulatory guidelines recommend screening for aggregation during bioprocessing. However, the mechanisms underlying the enhanced immunogenicity of aggregates are poorly understood. In the investigations described herein, the immunogenicity in mice of a humanized single chain variable antibody fragment (scFv) purified after expression in Escherichia coli has been examined. Reproducible scFv aggregates were obtained within the subvisible particle size range (mean diameter 2 µm) using thermal and mechanical stresses. Intraperitoneal immunization of BALB/c strain mice with 1 mg/ml of aggregated or monomeric scFv induced similar IgG and IgG1 antibody responses. In contrast, aggregate preparations stimulated significantly higher levels of anti-scFv IgG2a antibody than did the monomer. In comparative studies, aggregates of ovalbumin (OVA) within the subvisible particle size range were prepared by stir stress, and their immunogenicity compared with that of monomeric OVA in mice. Aggregated and monomeric OVA induced similar anti-OVA IgG and IgG1 antibody responses, whereas IgG2a antibody levels were significantly higher in aggregate-immunized mice. Furthermore, cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were consistent with aggregated preparations inducing a T helper (Th) 1-type response. Aggregated proteins within the subvisible range were therefore shown to induce a preferential Th1 type response, whereas monomeric proteins elicited a selective Th2 response. These data indicate that protein aggregation can impact on both the vigor and quality of immune responses.
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http://dx.doi.org/10.1093/toxsci/kfw121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036615PMC
October 2016

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

J Immunol Methods 2016 08 10;435:32-42. Epub 2016 May 10.

Biotherapeutics Group, NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.

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.
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http://dx.doi.org/10.1016/j.jim.2016.05.005DOI Listing
August 2016

IL-27 Promotes Proliferation of Human Leukemic Cell Lines Through the MAPK/ERK Signaling Pathway and Suppresses Sensitivity to Chemotherapeutic Drugs.

J Interferon Cytokine Res 2016 05 27;36(5):302-16. Epub 2016 Jan 27.

Cytokines and Growth Factors Section, Biotherapeutics Group, National Institute for Biological Standards and Control , Potters Bar, United Kingdom .

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.
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http://dx.doi.org/10.1089/jir.2015.0091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854210PMC
May 2016

Quality and Batch-to-Batch Consistency of Original and Biosimilar Epoetin Products.

J Pharm Sci 2016 Feb 9;105(2):542-550. Epub 2016 Jan 9.

Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, 3584 CG Utrecht, the Netherlands. Electronic address:

Comprehensive physicochemical characterization and biological assays are essential parts in assessing quality attributes of biologicals. Here, we compared the quality of different marketed recombinant human erythropoietin (epoetin) products: originators, Eprex and NeoRecormon as well as 2 biosimilars, Retacrit and Binocrit. In addition, assessment of batch-to-batch variability was included by collecting 2 or more batches of each product. Common assays which included sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance size-exclusion chromatography, asymmetrical flow field-flow fractionation, capillary zone electrophoresis, and potency testing were used. Of the tested products and among batches of single products, variations in epoetin content, isoform profiles, and potency were found. Ultimately, this study demonstrated the high quality of epoetin products with some degree of variation among products and batches, confirming the "similar but not identical" paradigm of biologicals.
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http://dx.doi.org/10.1016/j.xphs.2015.10.019DOI Listing
February 2016

Immunogenicity assessment of biotherapeutic products: An overview of assays and their utility.

Biologicals 2015 Sep 3;43(5):298-306. Epub 2015 Jul 3.

8 Old Forge Close, Welwyn AL6 0SR, United Kingdom.

Biotherapeutic products (BTPs) are the fastest growing medicines in the pharmaceutical market. Despite their clinical success, the immunogenicity of BTPs continues to be a major concern. Assessment of immunogenicity as well as appropriate interpretation of immunogenicity data is therefore, of critical importance for defining safety profile of these products for the purpose of their licensure and use. In the past decade, much progress has been made towards how immunogenicity should be studied. This article reflects the content of the brief presentation on principles of methods used for immunogenicity assessment and their merits and limitations given at the first World Health Organization (WHO) implementation workshop on rDNA derived biotherapeutic products held in the Republic of Korea in May 2014 to support the case studies on immunogenicity presented and discussed during the workshop. The purpose of this article is to provide an overview of the methods used for assessing immunogenicity of biotherapeutic products (BTPs) and the most important considerations in interpreting results in the context of regulatory overview of these products.
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http://dx.doi.org/10.1016/j.biologicals.2015.06.004DOI Listing
September 2015

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

J Immunol Methods 2015 Jan 25;416:17-28. Epub 2014 Oct 25.

Biotherapeutics Group, NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.

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.
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http://dx.doi.org/10.1016/j.jim.2014.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334095PMC
January 2015

Standardization of human IL-29 (IFN-λ1): establishment of a World Health Organization international reference reagent for IL-29 (IFN-λ1).

J Interferon Cytokine Res 2014 Nov 23;34(11):876-84. Epub 2014 Jun 23.

1 Cytokine and Growth Factors Section, Biotherapeutics Group, NIBSC, Medicines and Healthcare Products Regulatory Agency , Hertfordshire, United Kingdom .

Human interleukin-29 (IL-29), a helical cytokine with interferon-like activities, is currently being developed as a clinical biotherapeutic to treat chronic hepatitis C infection and some cancers. As such, the World Health Organization (WHO) has recognized a need for biological standardization of IL-29 and the establishment of an internationally available reference reagent of IL-29. In order to accomplish this, an international collaborative study that evaluates WHO candidate reference reagents of IL-29 was instigated by the National Institute for Biological Standards and Control (NIBSC) in 2010 and was carried out in the succeeding year. Two preparations of human sequence recombinant IL-29, one expressed in murine NS0 cells and the other in Escherichia coli, were formulated and lyophilized at NIBSC before evaluation in the collaborative study for their suitability to serve as a reference reagent. The preparations were tested by 6 laboratories from 4 countries using in vitro bioassays and also evaluated for thermal stability within the NIBSC laboratory. On the basis of the results of the collaborative study, both preparations, 07/212 (NS0-derived) and 10/176 (E. coli-derived) were judged sufficiently active and stable to serve as a reference reagent. However, since IL-29 produced in E. coli is in development for clinical applications, it was recommended that the preparation coded 10/176 be established as the WHO international reference reagent for human IL-29. This recommendation was accepted, and the IL-29 preparation coded 10/176 was formally established by the WHO ECBS at its meeting in October 2012 as the WHO international reference reagent for IL-29 with an assigned unitage of 5,000 reference units per ampoule.
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http://dx.doi.org/10.1089/jir.2014.0015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216994PMC
November 2014
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