Publications by authors named "Steven Piccoli"

23 Publications

  • Page 1 of 1

Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues.

Sci Rep 2020 12 17;10(1):22155. Epub 2020 Dec 17.

Epigenetics, Oncology R&D, GSK, Collegeville, USA.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.
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http://dx.doi.org/10.1038/s41598-020-78800-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746746PMC
December 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

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.

F Hoffmann-La Roche, Basel, Switzerland.

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.

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

2018 White Paper on Recent Issues in Bioanalysis: 'A global bioanalytical community perspective on last decade of incurred samples reanalysis (ISR)' (Part 1 - small molecule regulated bioanalysis, small molecule biomarkers, peptides & oligonucleotide bioanalysis).

Bioanalysis 2018 Nov 29;10(22):1781-1801. Epub 2018 Nov 29.

UK MHRA, London, UK.

The 2018 12 Workshop on Recent Issues in Bioanalysis (12th WRIB) 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 LC-MS, hybrid ligand binding assay (LBA)/LC-MS 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 1) covers the recommendations for LC-MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.
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http://dx.doi.org/10.4155/bio-2018-0268DOI Listing
November 2018

Biomarker assay validation.

Bioanalysis 2018 Jun;10(12):889-891

Angelini Pharma, Rome, Italy.

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http://dx.doi.org/10.4155/bio-2018-0127DOI Listing
June 2018

Surface plasmon resonance as a tool for ligand-binding assay reagent characterization in bioanalysis of biotherapeutics.

Bioanalysis 2018 Apr 27;10(8):559-576. Epub 2018 Apr 27.

Bioanalytical Sciences, Analytical & Bioanalytical Operations, Bristol-Myers Squibb, Princeton, NJ 08543, USA.

Ligand-binding assay (LBA) performance depends on quality reagents. Strategic reagent screening and characterization is critical to LBA development, optimization and validation. Application of advanced technologies expedites the reagent screening and assay development process. By evaluating surface plasmon resonance technology that offers high-throughput kinetic information, this article aims to provide perspectives on applying the surface plasmon resonance technology to strategic LBA critical reagent screening and characterization supported by a number of case studies from multiple biotherapeutic programs.
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http://dx.doi.org/10.4155/bio-2017-0271DOI Listing
April 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

Characterization of labeled reagents in ligand-binding assays by a surface plasmon resonance biosensor.

Bioanalysis 2017 Jan;9(2):193-207

BioAnalytical Sciences, Analytical & Bioanalytical Operations, Bristol-Myers Squibb, Princeton, NJ 08543, USA.

Aim: Ligand-binding assay (LBA) reagent labeling may change the binding characteristics of the reagent to its target and degrade its performance in LBAs.

Results: A surface plasmon resonance (SPR) biosensor was used to evaluate the impact of the biotin labeling process on reagent-binding kinetics and affinity for a specific target. The SPR results demonstrate that the biotin molar challenge ratio affects both association and dissociation rates for the labeled reagent binding to its target. The SPR results also predict the labeled reagent performance in LBAs.

Conclusion: The methodology used in this study provides an example of using an SPR biosensor as an efficient way to analytically and functionally characterize critical reagents and to understand their performance postmodification in LBAs.
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http://dx.doi.org/10.4155/bio-2016-0204DOI Listing
January 2017

Real-time observation of protein aggregates in pharmaceutical formulations using liquid cell electron microscopy.

Lab Chip 2017 01;17(2):315-322

Virginia Tech Carilion Research Institute, Virginia Tech, Roanoke, VA 24016, USA.

Understanding the properties of protein-based therapeutics is a common goal of biologists and physicians. Technical barriers in the direct observation of small proteins or therapeutic agents can limit our knowledge of how they function in solution and in the body. Electron microscopy (EM) imaging performed in a liquid environment permits us to peer into the active world of cells and molecules at the nanoscale. Here, we employ liquid cell EM to directly visualize a protein-based therapeutic in its native conformation and aggregate state in a time-resolved manner. In combination with quantitative analyses, information from this work contributes new molecular insights toward understanding the behaviours of immunotherapies in a solution state that mimics the human body.
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http://dx.doi.org/10.1039/c6lc01160hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507349PMC
January 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

Development and Validation of Electrochemiluminescence Assays to Measure Free and Total sSLAMF7 in Human Serum in the Absence and Presence of Elotuzumab.

AAPS J 2016 07 26;18(4):989-99. Epub 2016 Apr 26.

Bioanalytical Science-Biologics, Bristol-Myers Squibb, L14-03, Route 206 & Province Line Rd, Princeton, New Jersey, 08543, USA.

Elotuzumab is a first in class humanized IgG1 monoclonal antibody for the treatment of multiple myeloma (MM). Elotuzumab targets the glycoprotein signaling lymphocyte activation molecule family 7 (SLAMF7, also described as CS1 or CRACC) which is expressed on the surface of myeloma cells and a subset of immune cells, including natural killer cells. A soluble version of SLAMF7 (sSLAMF7) has also been reported in MM patients but has not been evaluated as a potential biomarker following therapeutic intervention. In order to measure serum levels of sSLAMF7, two immunoassays were developed to monitor changes in circulating sSLAMF7 before and after elotuzumab treatment. Free (drug-unbound) and total (drug-bound and unbound) electrochemiluminescence (ECL) ELISA assays were developed and validated following a fit for purpose (FFP) methodology. Both assays met analytical acceptance criteria for precision, drug interference, dilution linearity, spike recovery, parallelism, and stability. Both exhibited the range and sensitivity necessary to measure clinical samples with an LLOQ of 51.2 pg/mL and ULOQs of 160 (free) and 800 ng/mL (total). Previously described assays were unable to detect sSLAMF7 in healthy individuals. However, due to the increased sensitivity of these new assays, low but measurable sSLAMF7 levels were detected in all normal healthy sera evaluated and were significantly elevated in MM patients. Cohort statistics revealed a significant increase of circulating sSLAMF7 in MM patients versus normal controls and both significant decreases in free and increases in total levels of protein post-elotuzumab treatment.
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http://dx.doi.org/10.1208/s12248-016-9912-3DOI Listing
July 2016

Development and characterization of antibody reagents to assess anti-PEG IgG antibodies in clinical samples.

Bioanalysis 2015 ;7(15):1869-83

Bristol-Myers Squibb Company, Rt 206 & Province Line Road, Princeton, NJ 08543, USA.

Background: Polyethylene glycol (PEG) is a polymer that can be conjugated with therapeutic proteins. Monitoring anti-PEG antibodies in human subjects may be required as part of immunogenicity assessment. The lack of well-characterized anti-PEG reagents have limited our understanding of anti-PEG humoral response.

Results: Antibodies reactive to PEG were engineered with a human IgG1 Fc. Surface plasmon resonance and plate-based methods demonstrated that their binding was dependent on molecular weight (MW) of PEG. Specificity experiments using chemical analogs identified their specificity.

Conclusion: Affinity, specificity and MW of PEG are critical characteristics that impact interactions of anti-PEG antibodies with PEG. These attributes especially MW of PEG and the assay formats may impact the ability to detect anti-PEG antibodies.
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http://dx.doi.org/10.4155/bio.15.112DOI Listing
May 2016

An integrated multiplatform bioanalytical strategy for antibody-drug conjugates: a novel case study.

Bioanalysis 2015 ;7(13):1569-82

Bioanalytical Sciences-Biologics, Bristol-Myers Squibb, Lawrenceville, NJ 08648, USA.

Background: The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker.

Methods: The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC.

Results & Conclusion: Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.
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http://dx.doi.org/10.4155/bio.15.80DOI Listing
April 2016

Anti-PEG antibody bioanalysis: a clinical case study with PEG-IFN-λ-1a and PEG-IFN-α2a in naive patients.

Bioanalysis 2015 ;7(9):1093-106

3LabCorp, 115 Silvia Street, West Trenton, NJ 08628, USA.

Background: Extensive use of polyethylene glycol (PEG) in consumer products necessitates the assessment of anti-PEG antibodies (APAb).

Methods: In clinical trials comparing PEG-IFN-λ to PEG-IFN-α, conventional bridge and direct assays were assessed.

Results & Conclusion: The bridge assay detected IgM and IgG APAb reactive with common PEG sizes and derivatives at sufficient sensitivity, 15-500 ng/ml. Of subjects evaluated, 6% of PEG-IFN-λ and 9% of PEG-IFN-α subjects had persistent APAb while 60% of PEG-IFN-λ and 33% of PEG-IFN-α subjects had persistent anti-interferon antibodies (AIAb). Pre-existing APAb and AIAb prevalence was comparable (approximately 10% of subjects). APAb were earlier onset, less frequent, less persistent and lower titer than AIAb. No associated hypersensitivity events were reported.
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http://dx.doi.org/10.4155/bio.15.36DOI Listing
February 2016

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

AAPS J 2015 Jul 1;17(4):976-87. Epub 2015 May 1.

Bioanalytical Science-Biologics, Bristol-Myers Squibb, L4.016B, Route 206 & Province Line Rd, Princeton, New Jersey, 08543, USA,

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.
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http://dx.doi.org/10.1208/s12248-015-9762-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476994PMC
July 2015

Targeting an acid labile aspartyl-prolyl amide bond as a viable alternative to trypsin digestion to generate a surrogate peptide for LC-MS/MS analysis.

Bioanalysis 2014 ;6(22):2985-98

R&D, Bristol-Myers Squibb Co., Route 206/Province Line Road, Lawrenceville, NJ 08543, USA.

Background: FGF21-AdPKE is a fusion protein and functionally inactivated in vivo by cleavage around the C-terminus. It is important to quantify the intact active protein in serum.

Results & Discussion: Taking advantage of a uniquely acid-labile aspartyl-prolyl amide bond, we developed an acid hydrolysis procedure based on heating FGF21-AdPKE in dilute formic acid to generate a surrogate peptide encompassing the last 17 amino acids at the C-terminus. The monkey serum samples were extracted with an immunocapture procedure with an antibody specific for AdPKE. The calibration range was 200-50000 ng/ml. The assay accuracy and precision were between 92.8-99.8% and 3.9-14.5%, respectively. The method was applied to analyze incurred serum samples from a cynomolgus monkey toxicokinetic study involving administration of FGF21-AdPKE.

Conclusion: A method of combining immunocapture and acid hydrolysis to quantify a therapeutic protein in biological fluids was developed.
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http://dx.doi.org/10.4155/bio.14.182DOI Listing
July 2015

Measuring biotherapeutics with endogenous counterparts and pre-existing antibodies: an interferon case study.

Bioanalysis 2014 Apr;6(8):1113-22

Bristol-Myers Squibb, Route 206 & Province Line Rd, Princeton, NJ 08543, USA.

Background: This article presents case study data demonstrating the importance of having a thorough understanding of matrix-interference in support of global clinical trials.

Methods/results: A ligand-binding assay used in the measurement of interferon lambda in human serum was transferred from the reference laboratory to a US-based comparator laboratory and then to a China-based comparator laboratory. The method was successfully validated at each laboratory, however, during cross-validation, there were notable differences, including 30-60% difference in incurred study sample results. The differences were attributed to matrix factors included in the serum pool used to prepare standards and quality controls. Newly procured serum (n = 75 individuals) was tested for assay interference. 12% contained either pre-existing antibodies (auto-antibodies) or were identified as pharmacokinetic assay outliers.

Conclusion: Prescreening of serum to exclude reactive individuals resulted in successful cross-validation and the establishment of a high integrity pharmacokinetic assay in support of global clinical trials.
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http://dx.doi.org/10.4155/bio.14.37DOI Listing
April 2014

Conjugated critical reagent characterization for ligand-binding assays: using MALDI-TOF-MS as an orthogonal tool to assess assay performance.

Bioanalysis 2014 Apr;6(7):983-92

Bristol-Myers Squibb Company, Rt 206 & Province Line Rd, Princeton, NJ 08543, USA.

Large-molecule biotherapeutics are forming an increasingly large percentage of emerging pharmaceutical pipelines. These molecules present specific challenges to the bioanalysts charged with measuring in vivo concentrations of the biotherapeutic. The challenges are typically met using ligand-binding assays in support of pharmacokinetic, pharmacodynamic and immunogenicity assays. Ligand-binding assays employ complex biological molecules that specifically recognize the biotherapeutic for quantitation. Generally, a minimum of one of these critical reagents must be chemically modified to generate a signal that is measured in the assay. Once chemically modified it is necessary to characterize the reagent prior to use in an assay. The concentration, purity and molar incorporation ratio of chemical modification are key characteristics. This article presents mass spectral techniques for determining the molar incorporation ratio. Case studies are provided to demonstrate the time and cost savings that can be realized with timely and detailed characterization of critical reagents for ligand-binding assays.
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http://dx.doi.org/10.4155/bio.14.65DOI Listing
April 2014

Roux-en-Y gastric bypass corrects hyperinsulinemia implications for the remission of type 2 diabetes.

J Clin Endocrinol Metab 2011 Aug 18;96(8):2525-31. Epub 2011 May 18.

Department of Exercise and Sport Science, Brody Medical School, East Carolina University, Greenville, North Carolina 27834, USA.

Context: Roux-en-Y gastric bypass (RYGB) has been shown to induce rapid and durable reversal of type 2 diabetes.

Objective: The aim of the study was to investigate a possible mechanism for the remission of type 2 diabetes after RYGB.

Design: A cross-sectional, nonrandomized, controlled study was conducted. Surgery patients were studied before RYGB and 1 wk and 3 months after surgery.

Setting: This study was conducted at East Carolina University.

Subjects: Subjects were recruited into three groups: 1) lean controls with no surgery [body mass index (BMI) < 25 kg/m²; n = 9], 2) severely obese type 2 diabetic patients (BMI > 35 kg/m²; n = 9), and 3) severely obese nondiabetic patients (BMI > 35 kg/m²; n = 9).

Intervention: Intervention was RYGB.

Results: One week after RYGB, diabetes was resolved despite continued insulin resistance (insulin sensitivity index was approximately 50% of lean controls) and reduced insulin secretion during an iv glucose tolerance test (acute insulin response to glucose was approximately 50% of lean controls). Fasting insulin decreased and was no different from lean control despite continued elevated glucose in the type 2 diabetic patients compared with lean.

Conclusions: After RYGB, fasting insulin decreases to levels like those of lean control subjects and diabetes is reversed (fasting blood glucose < 125 mg/dl). This leads us to propose that 1) exclusion of food from the foregut corrects hyperinsulinemia and 2) fasting insulin is dissociated from the influence of fasting glucose, insulin resistance, and BMI. The mechanisms for reversal of diabetes in the face of reduced insulin remain a paradox.
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http://dx.doi.org/10.1210/jc.2011-0165DOI Listing
August 2011

Optimization of Rolling-Circle Amplified Protein Microarrays for Multiplexed Protein Profiling.

J Biomed Biotechnol 2003 ;2003(5):299-307

Protein microarray-based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions. The development of high-throughput, sensitive, convenient, and cost-effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes. In this paper, we describe the generation of highly multiplexed, antibody-based, specific, and sensitive protein microarrays coupled with rolling-circle signal amplification (RCA) technology. A total of 150 cytokines were simultaneously detected in an RCA sandwich immunoassay format. Greater than half of these proteins have detection sensitivities in the pg/ml range. The validation of antibody microarray with human serum indicated that RCA-based protein microarrays are a powerful tool for high-throughput analysis of protein expression and molecular diagnostics.
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http://dx.doi.org/10.1155/S1110724303209268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC521503PMC
January 2003

Clinical evaluation of the Elecsys total prostate-specific antigen assay on the Elecsys 1010 and 2010 systems.

Clin Chem 2002 Jun;48(6 Pt 1):944-7

The James Buchanan Brady Urological Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

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June 2002