Publications by authors named "Lakshmi Amaravadi"

35 Publications

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback ( - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

Bioanalysis 2021 Mar 29;13(5):295-361. Epub 2021 Jan 29.

Health Canada, Ottawa, ON, Canada.

The 14 edition of the Workshop on Recent Issues in Bioanalysis (14 WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14 WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global 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 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.
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http://dx.doi.org/10.4155/bio-2021-0005DOI Listing
March 2021

Protein Biomarker Quantification by Immunoaffinity Liquid Chromatography-Tandem Mass Spectrometry: Current State and Future Vision.

Clin Chem 2020 02;66(2):282-301

Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, IN.

Immunoaffinity-mass spectrometry (IA-MS) is an emerging analytical genre with several advantages for profiling and determination of protein biomarkers. Because IA-MS combines affinity capture, analogous to ligand binding assays (LBAs), with mass spectrometry (MS) detection, this platform is often described using the term hybrid methods. The purpose of this report is to provide an overview of the principles of IA-MS and to demonstrate, through application, the unique power and potential of this technology. By combining target immunoaffinity enrichment with the use of stable isotope-labeled internal standards and MS detection, IA-MS achieves high sensitivity while providing unparalleled specificity for the quantification of protein biomarkers in fluids and tissues. In recent years, significant uptake of IA-MS has occurred in the pharmaceutical industry, particularly in the early stages of clinical development, enabling biomarker measurement previously considered unattainable. By comparison, IA-MS adoption by CLIA laboratories has occurred more slowly. Current barriers to IA-MS use and opportunities for expanded adoption are discussed. The path forward involves identifying applications for which IA-MS is the best option compared with LBA or MS technologies alone. IA-MS will continue to benefit from advances in reagent generation, more sensitive and higher throughput MS technologies, and continued growth in use by the broader analytical community. Collectively, the pursuit of these opportunities will secure expanded long-term use of IA-MS for clinical applications.
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http://dx.doi.org/10.1093/clinchem/hvz022DOI Listing
February 2020

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

Bioanalysis 2019 Dec 10;11(24):2207-2244. Epub 2019 Dec 10.

Janssen R&D, Spring House, PA, USA.

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 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, immunogenicity and gene therapy. 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, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 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 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of , issues 22 and 23 (2019), respectively.
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http://dx.doi.org/10.4155/bio-2019-0271DOI Listing
December 2019

A sensitive antibody-free 2D-LC-MS/MS assay for the quantitation of myostatin in the serum of different species.

Bioanalysis 2019 May;11(10):957-970

Bioanalytical & Biomarker Development, Takeda Pharmaceuticals International Co., 125 Binney Street, Cambridge, MA 02142, USA.

Myostatin (MSTN) is an attractive therapeutic target for the treatment of muscle degeneration-related diseases and is being evaluated as a target engagement biomarker. A sensitive 2D-LC-MS/MS assay was developed to quantify MSTN in different animal species. Sample preparation involved SDS denaturation of serum proteins followed by tryptic digestion and peptide enrichment by SPE. The assay was validated with LLOQ of 2.5 ng/ml in rat and monkey serum. The precision was within 13.7%, and the bias was within ±12.6% for all quality control samples in authentic matrices. This new assay was successfully applied to measure MSTN in mouse, rat, monkey and human serum. The total MSTN in rat and monkey serum was elevated following administration of an MSTN inhibitor.
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http://dx.doi.org/10.4155/bio-2018-0311DOI Listing
May 2019

An LC-MS/MS approach to assess total and free protein target in the serum of cynomolgus monkey.

Bioanalysis 2019 Mar 15;11(5):393-406. Epub 2019 Mar 15.

Bioanalytical & Biomarker Development, Takeda Pharmaceuticals International Co., 125 Binney Street, Cambridge, MA 02142, USA.

Aim: Develop LC-MS/MS-based assays to measure total and free complement C5 in cynomolgus monkey serum as a target engagement biomarker for pharmacokinetic/pharmacodynamic correlation study. Materials & methods/results: The C5-specific signature peptide derived from pellet digestion of serum proteins with and without prior immunodepletion of the drug-bound C5 by protein A beads was quantified to assess free and total C5 levels, respectively. Conditions for immunodepletion by protein A were optimized to ensure complete depletion of IgGs (and drug-bound C5). The effect of sample dilution on drug-target dissociation and thus free C5 measurement was evaluated by applying a mathematical simulation.

Conclusion: The procedure described here allows for the assessment of protein target engagement, aiding in pharmacokinetic/pharmacodynamic correlation analysis and human dose projection.
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http://dx.doi.org/10.4155/bio-2018-0294DOI Listing
March 2019

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.

Amador Bioscience, Pleasanton, CA, USA (formerly of OncoMed, Redwood City, 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

A novel LC-MS/MS assay to quantify dermatan sulfate in cerebrospinal fluid as a biomarker for mucopolysaccharidosis II.

Bioanalysis 2018 Jun 4;10(11):825-838. Epub 2018 Jun 4.

Shire, 300 Shire Way, Lexington, MA 02421, USA.

Aim: The study aimed to develop an LC-MS/MS assay to measure dermatan sulfate (DS) in human cerebrospinal fluid (CSF).

Methods & Results: DS was quantified by ion pairing LC-MS/MS analysis of the major disaccharides derived from chondroitinase B digestion. Artificial CSF was utilized as a surrogate for calibration curve preparation. The assay was fully validated, with a linear range of 20.0-4000 ng/ml, accuracy within ±20%, and precision of ≤20%. CSF samples from mucopolysaccharidoses (MPS) II patients showed an average of 11-fold increase in DS levels compared with controls.

Conclusion: The described assay is capable of differentiating DS levels in the CSF of MPS II patients from controls and can be used to monitor disease progression and therapeutic responses.
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http://dx.doi.org/10.4155/bio-2018-0025DOI Listing
June 2018

2017 White Paper on recent issues in bioanalysis: a global perspective on immunogenicity guidelines & biomarker assay performance (Part 3 - LBA: immunogenicity, biomarkers and PK assays).

Bioanalysis 2017 Dec 5;9(24):1967-1996. Epub 2017 Dec 5.

BioMarin Pharmaceutical, San Rafael, CA, USA.

The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals 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 analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 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 2017 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. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.
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http://dx.doi.org/10.4155/bio-2017-4974DOI Listing
December 2017

Recommendations for the Assessment and Management of Pre-existing Drug-Reactive Antibodies During Biotherapeutic Development.

AAPS J 2017 11 6;19(6):1576-1586. Epub 2017 Nov 6.

Clinical Laboratory Sciences, DSAR, Sanofi R&D, Framingham, Massachusetts, USA.

Anti-drug antibodies (ADA) pose a potential risk to patient safety and efficacy and are routinely monitored during clinical trials. Pre-existing drug-reactive antibodies are present in patients without prior drug exposure and are defined by their ability to bind to a component of the drug. These pre-existing drug-reactive antibodies are frequently observed and could represent an adaptive immune response of an individual who has been previously exposed to antigens with structural similarities to the biotherapeutic. Clinical consequences of these antibodies can vary from no impact to adverse effects on patient safety, exposure, and efficacy, and are highly dependent on biotherapeutic modality, disease indications, and patient demographics. This paper describes how the immunogenicity risk assessment of a biotherapeutic integrates the existence of pre-existing drug-reactive antibodies, and provides recommendations for risk-based strategies to evaluate treatment-emergent ADA responses.
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http://dx.doi.org/10.1208/s12248-017-0153-xDOI Listing
November 2017

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material.

AAPS J 2017 11 2;19(6):1550-1563. Epub 2017 Oct 2.

Genentech/Roche, 11 Genentech/Roche, 1 DNA Way, South San Francisco, CA, 94080, USA.

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.
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http://dx.doi.org/10.1208/s12248-017-0146-9DOI Listing
November 2017

Recommendations for clinical biomarker specimen preservation and stability assessments.

Bioanalysis 2017 Apr 15;9(8):643-653. Epub 2017 May 15.

Alliance Pharma, 17 Lee Boulevard, Malvern, PA, USA.

With the wide use of biomarkers to enable critical drug-development decisions, there is a growing concern from scientific community on the need for a 'standardized process' for ensuring biomarker specimen stability and hence, a strong desire to share best practices on preserving the integrity of biomarker specimens in clinical trials and the design of studies to evaluate analyte stability. By leveraging representative industry experience, we have attempted to provide an overview of critical aspects of biomarker specimen stability commonly encountered during clinical development, including: planning of clinical sample collection procedures, clinical site training, selection of sample preservation buffers, shipping logistics, fit-for-purpose stability assessments in the analytical laboratory and presentation of case studies covering widely utilized biomarker specimen types.
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http://dx.doi.org/10.4155/bio-2017-0009DOI Listing
April 2017

Evidence of activation of the Nrf2 pathway in multiple sclerosis patients treated with delayed-release dimethyl fumarate in the Phase 3 DEFINE and CONFIRM studies.

Mult Scler 2017 Dec 3;23(14):1875-1883. Epub 2017 Feb 3.

Biogen, Inc., Cambridge, MA, USA.

Background: Delayed-release dimethyl fumarate (DMF) is an approved oral treatment for relapsing forms of multiple sclerosis (MS). Preclinical studies demonstrated that DMF activated the nuclear factor E2-related factor 2 (Nrf2) pathway. DMF and its primary metabolite monomethyl fumarate (MMF) were also shown to promote cytoprotection of cultured central nervous system (CNS) cells via the Nrf2 pathway.

Objective: To investigate the activation of Nrf2 pathway following ex vivo stimulation of human peripheral blood mononuclear cells (PBMCs) with DMF or MMF, and in DMF-treated patients from two Phase 3 relapsing MS studies DEFINE and CONFIRM.

Methods: Transcription of Nrf2 target genes NADPH:quinone oxidoreductase-1 (NQO1) and heme-oxygenase-1 (HO1) was measured using Taqman® assays. RNA samples were isolated from ex vivo-stimulated PBMCs and from whole blood samples of 200 patients each from placebo, twice daily (BID) and three times daily (TID) treatments.

Results: DMF and MMF induced NQO1 and HO1 gene expression in ex vivo-stimulated PBMCs, DMF being the more potent inducer. Induction of NQO1 occurred at lower DMF concentrations compared to that of HO1. In DMF-treated patients, a statistically significant induction of NQO1 was observed relative to baseline and compared to placebo. No statistical significance was reached for HO1 induction.

Conclusion: These data provide the first evidence of Nrf2 pathway activation from two large pivotal Phase 3 studies of DMF-treated MS patients.
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http://dx.doi.org/10.1177/1352458517690617DOI Listing
December 2017

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 3 - LBA, biomarkers and immunogenicity).

Bioanalysis 2016 Dec;8(23):2475-2496

OncoMed Pharmaceuticals, Redwood City, CA, USA.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 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. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
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http://dx.doi.org/10.4155/bio-2016-4989DOI Listing
December 2016

Biomarker measurements: how far have we come and where are we heading?

Bioanalysis 2016 Dec;8(23):2383-2386

Translational Medicine & Early Development, Sanofi-Genzyme, Framingham, MA, USA.

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http://dx.doi.org/10.4155/bio-2016-4987DOI Listing
December 2016

Population PK-PD analyses of CD25 occupancy, CD56 NK cell expansion, and regulatory T cell reduction by daclizumab HYP in subjects with multiple sclerosis.

Br J Clin Pharmacol 2016 11 3;82(5):1333-1342. Epub 2016 Aug 3.

Receptos, a wholly owned subsidiary of Celgene, San Diego, CA, 92121, USA.

Aim: Daclizumab high yield process (HYP) is a humanized IgG1 monoclonal antibody that binds to the α-subunit of the interleukin-2 receptor and is being developed for treatment of multiple sclerosis (MS). This manuscript characterized the pharmacokinetic-pharmacodynamic (PK-PD) relationships of daclizumab HYP in subjects with MS.

Methods: Approximately 1400 subjects and 7000 PD measurements for each of three biomarkers [CD25 occupancy, CD56 natural killer (NK) cell count, regulatory T cell (Treg) count] from four clinical trials were analyzed using non-linear mixed effects modelling. Evaluated regimens included 150 or 300 mg subcutaneous (s.c.) every 4 weeks.

Results: CD25 occupancy was characterized using a sigmoidal maximum response (E ) model. Upon daclizumab HYP treatment, CD25 saturation was rapid with complete saturation occurring after approximately 7 h and maintained when daclizumab HYP serum concentration was ≥5 mg l . After the last 150 mg s.c. dose, unoccupied CD25 returned to baseline levels in approximately 24 weeks, with daclizumab HYP serum concentration approximately ≤1 mgl 1L. CD56 NK cell expansion was characterized using an indirect response model. Following daclizumab HYP 150 mg s.c. every 4 weeks, expansion plateaus approximately at week 36, at which the average maximum expansion ratio is 5.2. After the last dose, CD56 NK cells gradually declined to baseline levels within 24 weeks. Treg reduction was characterized by a sigmoidal E model. Average maximum reduction of 60% occurred approximately 4 days post 150 mg s.c. dose. After the last dose, Tregs were projected to return to baseline levels in approximately 20 weeks.

Conclusions: Robust PK-PD models of CD25 occupancy, CD56 NK cell expansion and Treg reduction by daclizumab HYP were developed to characterize its key pharmacodynamic effects in the target patient population.
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http://dx.doi.org/10.1111/bcp.13051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061794PMC
November 2016

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 3--LBA, biomarkers and immunogenicity).

Bioanalysis 2015 Dec 4;7(24):3107-24. Epub 2015 Dec 4.

Sanofi, Framingham, MA, USA.

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 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 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.
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http://dx.doi.org/10.4155/bio.15.226DOI Listing
December 2015

Workshop report: Crystal City V--quantitative bioanalytical method validation and implementation: the 2013 revised FDA guidance.

AAPS J 2015 Mar 31;17(2):277-88. Epub 2014 Dec 31.

U.S. Food and Drug Administration, Silver Spring, MD, USA.

In September 2013, the FDA released a draft revision of the Bioanalytical Method Validation (BMV) Guidance, which included a number of changes to the expectations for bioanalysis, most notably the inclusion of biomarker assays and data. To provide a forum for an open, inclusive discussion of the revised draft BMV Guidance, the AAPS and FDA once again collaborated to convene a two-and-a-half day workshop during early December 2013 in Baltimore, MD, USA. The resulting format embodied extensive open discussion and each thematic session included only brief, concise descriptions by Agency and industry representatives prior to opening the floor discussion. The Workshop was built around four thematic sessions (Common Topics, Chromatographic, Ligand-Binding Assays, and Biomarkers) and a final session with international regulators, concluding with a review of the outcomes and recommendations from the thematic sessions. This Workshop report summarizes the outcomes and includes topics of agreement, those where the FDA will consider the Industry's perspective, and those where the workshop provided a first open dialogue. This article will be available to the bioanalytical community at http://www.aaps.org/BMV13 .
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http://dx.doi.org/10.1208/s12248-014-9696-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365089PMC
March 2015

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 3 - LBA and immunogenicity).

Bioanalysis 2014 ;6(24):3355-68

Biogen Idec Inc., Cambridge, MA, USA.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for Large molecules bioanalysis using LBA and Immunogenicity. Part 1 (Small molecules bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input) were published in the Bioanalysis issues 6(22) and 6(23), respectively.
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http://dx.doi.org/10.4155/bio.14.283DOI Listing
August 2015

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 2 - hybrid LBA/LCMS, ELN & regulatory agencies' input).

Bioanalysis 2014 ;6(23):3237-49

Pfizer, Andover, MA, USA.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input. Part 1 (Small molecules bioanalysis using LCMS) was published in the Bioanalysis issue 6(22) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the Bioanalysis issue 6(24).
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http://dx.doi.org/10.4155/bio.14.279DOI Listing
August 2015

In vivo maintenance of human regulatory T cells during CD25 blockade.

J Immunol 2015 Jan;194(1):84-92

Biogen Idec, Cambridge, MA 02142

Regulatory T cells (Tregs) mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction, and instability are implicated in the pathogenesis of numerous autoimmune diseases. There is considerable interest in therapeutic modulation of the IL-2 pathway to treat autoimmunity, facilitate transplantation tolerance, or potentiate tumor immunotherapy. Daclizumab is a humanized mAb that binds the IL-2 receptor a subunit (IL-2R a or CD25) and prevents IL-2 binding. In this study, we investigated the effect of daclizumab-mediated CD25 blockade on Treg homeostasis in patients with relapsing-remitting multiple sclerosis. We report that daclizumab therapy caused an ~50% decrease in Tregs over a 52-wk period. Remaining FOXP3+ cells retained a demethylated Treg-specific demethylated region in the FOXP3 promoter, maintained active cell cycling, and had minimal production of IL-2, IFN- g, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2R bg signaling induced STAT5 phosphorylation and sustained FOXP3 expression. Treg declines were not associated with daclizumab-related clinical benefit or cutaneous adverse events. These results demonstrate that Treg phenotype and lineage stability can be maintained in the face of CD25 blockade.
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http://dx.doi.org/10.4049/jimmunol.1402140DOI Listing
January 2015

New FDA draft guidance on immunogenicity.

AAPS J 2014 May 29;16(3):499-503. Epub 2014 Mar 29.

Mercer University, 3001 Mercer University Drive, Atlanta, Georgia, 30341, USA,

A "Late Breaking" session was held on May 20 at the 2013 American Association of Pharmaceutical Scientists-National Biotech Conference (AAPS-NBC) to discuss the US Food and Drug Administration's (FDA) 2013 draft guidance on Immunogenicity Assessment for Therapeutic Protein Products. The session was initiated by a presentation from the FDA which highlighted several key aspects of the 2013 draft guidance pertaining to immunogenicity risk, the potential impact on patient safety and product efficacy, and risk mitigation. This was followed by an open discussion on the draft guidance which enabled delegates from biopharmaceutical companies to engage the FDA on topics that had emerged from their review of the draft guidance. The multidisciplinary audience fostered an environment that was conducive to scientific discussion on a broad range of topics such as clinical impact, immune mitigation strategies, immune prediction and the role of formulation, excipients, aggregates, and degradation products in immunogenicity. This meeting report highlights several key aspects of the 2013 draft guidance together with related dialog from the session.
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http://dx.doi.org/10.1208/s12248-014-9587-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4012054PMC
May 2014

Daclizumab high-yield process in relapsing-remitting multiple sclerosis (SELECTION): a multicentre, randomised, double-blind extension trial.

Lancet Neurol 2014 May 19;13(5):472-81. Epub 2014 Mar 19.

Biogen Idec, Cambridge, MA, USA.

Background: In the SELECT trial, disease activity was reduced in patients with multiple sclerosis who received daclizumab high-yield process (HYP) for 52 weeks. The primary aim of the SELECTION extension study was to assess the safety and immunogenicity of extended treatment with daclizumab HYP.

Methods: A multicentre, randomised, double-blind, 52-week extension trial was done in 74 centres in the Czech Republic, Germany, Hungary, India, Poland, Russia, Ukraine, and the UK between Feb 13, 2009, and Oct 3, 2012. Eligible patients were aged 18-55 years, had relapsing-remitting multiple sclerosis, and had completed the SELECT study. Patients who received placebo in SELECT were randomly assigned (1:1) to receive 150 mg or 300 mg subcutaneous daclizumab HYP every 4 weeks for 52 weeks (treatment initiation group); those who had received daclizumab HYP were randomly assigned (1:1) to continue their present dose with (washout and re-initiation group) or without (continuous treatment group) a washout period of 20 weeks. All randomisation was done with a centralised, interactive voice-response system. Patients and personnel were masked to treatment assignment, except for the site pharmacist who prepared the study drug but had no interaction with patients. The primary endpoints were the safety and immunogenicity of daclizumab HYP. Analyses were by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00870740.

Findings: 517 (91%) of 567 patients who completed the SELECT trial entered SELECTION, of whom 170 were in the treatment initiation group, 173 in the continuous treatment group, and 174 in the washout and re-initiation group. 11 patients in the treatment initiation group (6%), 13 in the continuous treatment group (8%), and ten in the washout and re-initiation group (6%) had any serious adverse event other than relapse of multiple sclerosis. One patient in the washout and re-initiation group (300 mg daclizumab HYP) died because of autoimmune hepatitis; a contributory role of daclizumab HYP could not be excluded. Seven patients tested positive for neutralising antidrug antibodies: one (1%) of 128 for whom data were available in the continuous treatment group (this patient also tested positive at SELECTION baseline), four (2%) in the treatment initiation group, and two (2%) of 129 in the washout and re-initiation group.

Interpretation: Adverse events and immunogenicity were not increased in the second year of continuous treatment with daclizumab HYP or during treatment washout and re-initiation. These results support further assessment of daclizumab HYP for relapsing-remitting multiple sclerosis.

Funding: Biogen Idec and AbbVie Biotherapeutics.
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http://dx.doi.org/10.1016/S1474-4422(14)70039-0DOI Listing
May 2014

2013 White Paper on recent issues in bioanalysis: 'hybrid'--the best of LBA and LCMS.

Bioanalysis 2013 Dec 10;5(23):2903-18. Epub 2013 Oct 10.

Biogen Idec Inc.,Cambridge, MA, USA.

The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These 'hot' topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science. Such as the previous editions, this 2013 White Paper addresses important bioanalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.
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http://dx.doi.org/10.4155/bio.13.238DOI Listing
December 2013

Safety, tolerability, pharmacokinetics, and pharmacodynamics of anti-TWEAK monoclonal antibody in patients with rheumatoid arthritis.

Clin Ther 2013 Aug 6;35(8):1137-49. Epub 2013 Aug 6.

Biogen Idec, Berkshire, United Kingdom.

Background: Persistent upregulation of signaling by cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) through its receptor fibroblast growth factor-inducible molecule-14 (Fn14) promotes chronic inflammation and tissue destruction.

Objective: The aim of this study was to explore the safety and tolerability of the TWEAK-blocking monoclonal antibody BIIB023 and determine its pharmacokinetics and effects on TWEAK pathway pharmacodynamic markers in rheumatoid arthritis (RA).

Methods: Phase I, first-in-human, 2-part, multicenter, double-blind, dose-escalation study. Patients were randomized to a single dose of BIIB023 (0.03-20 mg/kg) (n = 38) or placebo (n = 15) as an add-on to methotrexate. Three open-label cohorts of RA patients taking background disease-modifying antirheumatic drugs and stable tumor necrosis factor (TNF) inhibitor therapy (n = 12) received a single-dose of BIIB023 of 2, 10, or 20 mg/kg and were assessed over 70 days.

Results: The incidence of treatment-emergent adverse events for the BIIB023 monotherapy cohorts and open-label cohorts of BIIB023 as add-on therapy to TNF inhibitors compared with placebo were 47% and 50% versus 33%, respectively. Serum exposure to BIIB023 increased in a dose-dependent manner from 0.03 to 20 mg/kg, but not in direct proportion to dose level. After administration, the time course of BIIB023 serum concentration was multiphasic and showed expedited elimination when levels decreased to < 10 µg/mL. Serum-soluble TWEAK levels were suppressed at all dose levels by 6 hours post-dose and recovered to baseline between days 7 and 28. A trend toward downward modulation of serum biomarkers of inflammatory response was suggested in monocyte chemoattractant protein 1, inducible protein 10, macrophage inflammatory protein 1β, and tissue inhibitor of metalloproteinase 1 in the BIIB023 group versus placebo.

Conclusions: Single-dose BIIB023 showed a favorable safety and tolerability profile in RA. Suppression of serum-soluble TWEAK for ≤ 28 days was observed and downward trends in serum biomarkers suggested.
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http://dx.doi.org/10.1016/j.clinthera.2013.06.008DOI Listing
August 2013

Adapting dried blood spot sampling for an anti-therapeutic antibody immunogenicity assay.

J Immunol Methods 2013 Jul 18;393(1-2):53-60. Epub 2013 Apr 18.

Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA.

Dried blood spot sampling is a microvolume sampling technique with many potential advantages. It allows for easier handling and less expensive shipment and storage of biological samples. Additionally, it can provide ethical benefits in the pre-clinical setting through a reduction in animal usage by allowing intensive serial sample collection from the same animals. In the clinical setting, ease of sample collection, greater flexibility of sample storage, and shipping are distinct advantages. These advantages can enhance preclinical and clinical data quality, where immunogenicity monitoring plays an important role in the interpretation of pharmacokinetic data. To date, a method for usage of dried blood spot sampling with an immunogenicity assay has not been published. Herein we demonstrate that the measurement of anti-drug antibodies (ADA) using DBS was comparable to traditional methods in terms of reproducibility, assay sensitivity and drug tolerance. The data demonstrate that DBS is a viable sample collection method, and in some cases may be preferred, over classic serum or plasma sampling for antidrug antibody assays.
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http://dx.doi.org/10.1016/j.jim.2013.04.006DOI Listing
July 2013

Novel data analysis methods to overcome cut point challenges and enable comprehensive assessment of antidrug binding activity in confirmatory assays.

J Immunol Methods 2013 Jun 28;392(1-2):38-48. Epub 2013 Mar 28.

Biogen Idec, Inc., Cambridge, MA 02142, USA.

Immunogenicity assessments in response to drug treatment are commonly performed using a tiered approach strategy. All samples are initially tested in a screening assay followed by the evaluation of the screened positive samples in a confirmatory assay. Percent inhibition of signal intensity by the competing unlabeled drug in a confirmatory assay is typically used to measure the specificity of antidrug binding activity in samples, and has been successfully applied to most immunogenicity assays. However, the percent inhibition approach may not be suitable in cases where broadly distributed and high percent inhibition values are observed in drug-naïve subjects or when persistent operator-dependent differences in assay performance are encountered. Herein, we present the case studies faced with such challenges and provide appropriate solutions by introducing two novel data analysis methods: (1) Reference Delta, and (2) Reference Percent Inhibition, - in which relative-to-baseline signal inhibition is calculated for each sample. These novel methods significantly improve the confirmatory assay's ability to detect the samples positive for antidrug antibodies (ADA), especially when challenges are encountered using the traditional percent inhibition approach. Furthermore, both methods can be implemented in parallel with the percent inhibition method, enabling not only confirmation of ADA specificity, but also providing additional insights about the relevance of this antidrug binding activity to drug treatment.
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http://dx.doi.org/10.1016/j.jim.2013.03.008DOI Listing
June 2013

Mini Focus: bioanalysis of biosimilars.

Bioanalysis 2013 Mar;5(5):515-6

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http://dx.doi.org/10.4155/bio.13.26DOI Listing
March 2013

Conference report: 12th Annual University of Wisconsin Land O'Lakes Bioanalytical Conference.

Bioanalysis 2011 Oct;3(19):2171-5

University of Wisconsin, 777 Highland Ave., Madison, WI 53705, USA.

This University of Wisconsin School of Pharmacy bioanalytical conference is presented each year by the Extension Services in Pharmacy, the professional development department within the school. The purpose of this 4-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program was a mixture of lectures, poster sessions, round table discussions and workshops. This article summarizes the presentations at the 12th Annual Conference.
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http://dx.doi.org/10.4155/bio.11.236DOI Listing
October 2011

Paradigm of combination biologics: analytical challenges related to pharmacokinetic assays and interpretation of pharmacokinetic and immunogenicity results.

Bioanalysis 2011 Mar;3(5):487-98

Preclinical & Clinical Development Sciences, Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA.

Background: Combination biologic therapy is an emerging area of clinical development and the physiological and analytical impact of one treatment on the other requires careful assessment. Significant analytical challenges are associated with developing the corresponding pharmacokinetic assays and further challenges arise in interpreting the subsequent in vivo data, which may be confounded by immunogenicity to one or both of the biologics.

Results: A case study of two monoclonal antibody therapeutics, given in combination, is presented where the immunogenicity rates differed significantly when the drug(s) were administered as monotherapy or in combination.

Conclusion: The interpretation of the in vivo data is inextricably linked to an in-depth understanding of the formats and performance attributes of the associated pharmacokinetic and immunogenicity assays.
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http://dx.doi.org/10.4155/bio.10.214DOI Listing
March 2011

Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development.

J Immunol Methods 2011 Feb 1;365(1-2):38-49. Epub 2010 Dec 1.

Biogen Idec, Inc., Cambridge, MA 02142, USA.

Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for labeling antibodies was evaluated and is presented in this manuscript. In addition, simple methods for evaluation and qualification of streptavidin-coated plates and overcoming soluble target interference in solution ELISA have also been investigated and highlights of these investigations are discussed. The selection of the solution ELISA format was based on availability of generic reagents, achievement of optimal drug tolerance and robust assay performance on a platform that is readily available in many laboratories. This approach removed the heavy reliance on specialized equipment sourced from a single vendor and assay conditions described here are broadly applicable to other immunogenicity assays across many biologics both during clinical development setting and in the post-marketing arena.
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http://dx.doi.org/10.1016/j.jim.2010.11.011DOI Listing
February 2011