William Nowatzke

Stability of N-Terminal Pro-Brain Natriuretic Peptide after Storage Frozen for One Year and after Multiple Freeze-Thaw Cycles

Cardiac natriuretic peptides are of interest for their potential role in assisting in the diagnosis, prognosis, and monitoring of left ventricular dysfunction and congestive heart failure (1). An electrochemiluminescence immunoassay for the measurement of N-terminal pro-brain natriuretic peptide (NT-proBNP) on the Elecsys® immunoassay analyzer platform (Roche Diagnostics Corporation) has recently received Food and Drug Administration clearance to aid in the diagnosis of congestive heart failure (2). We report here on the stability of the NT-proBNP analyte when measured with the Roche Diagnostic NT-proBNP assay after specimen storage at −80 °C for longer than 1 year and after five freeze-thaw (−80 and 22 °C) cycles. Twenty-five samples were collected from healthy and heart failure clinic adult volunteers under informed consent according to the policies of the Washington University School of Medicine’s Institutional Review Board. All glass collection tubes were from Becton Dickinson. Lithium-heparin plasma (prod. no. 367686) was collected from …

Recommendations on biomarker bioanalytical method validation by GCC

The 5th GCC in Barcelona (Spain) and 6th GCC in San Antonio (TX, USA) events provided a unique opportunity for CRO leaders to openly share opinions and perspectives, and to agree upon recommendations on biomarker bioanalytical method validation.

Unique challenges of providing bioanalytical support for biological therapeutic pharmacokinetic programs

Regulatory recommendations for providing bioanalytical support for biological therapeutics have co-evolved with the increasing success of these unique pharmaceuticals. Immunoassays have been used to quantify biological macromolecules for more than 50 years. These assays rely on the use of antigen-specific antibodies. More recently, LC-MS methods have being adapted to quantitate biologics. LC-MS has attributes that complement the limitations encountered by immunoassays. Whether employing immunoassay or LC-MS methods, compared with traditional chemical-based therapeutics, biological therapeutics present unique analytical challenges to analysts. In this article, we review bioanalytical strategies for supporting biologics and discuss the regulatory and analytical challenges that must be met.

Systematic analytical validation of commercial kits for the determination of novel biomarkers for clinical drug development

The use of biomarkers during clinical drug-development programs may expedite pipeline decision making by adding critical information about the pharmacological mechanism and efficacy of a potential therapeutic agent. Currently, advice for laboratorians conducting method development and analytical validation of biomarker methods is provided by published White Paper recommendations from industry thought leaders. The adaptation of commercial test kits to generate biomarker data to support regulated studies offers unique challenges and limitations. In this perspective, we address these issues, including factors to consider when identifying a kit manufacturer and adapting commercial test kits for use in regulated studies. We offer a logical and systematic approach for defining the extent of analytical validation needed for application of commercial kits based upon the intended use of the biomarker data.

Conference Report: 6th GCC focus on LBA: critical reagents, positive controls and reference standards; specificity for endogenous compounds; biomarkers; biosimilars

The 6th Global CRO Council for Bioanalysis (GCC) Closed Forum was held on 27 March 2012 in San Antonio, TX, USA, the day before the start of the 6th Workshop on Recent Issues in Bioanalysis. The attendance consisted of 45 bioanalytical CRO senior-level representatives on behalf of 37 CRO companies/sites from six countries. In addition to following up on the issue of co-administered drugs stability and on recommendations regarding the European Medicines Agency guideline, this GCC Closed Forum discussed topics of current interest in the bioanalytical field with focus on ligand-binding assays, such as lot changes for critical reagents, positive controls and reference standards, specificity for endogenous compounds, qualification and validation of biomarker assays, approach for biosimilars and criteria for LC–MS assays of small versus large molecules.

8th GCC: Consolidated feedback to US FDA on the 2013 Draft FDA Guidance on Bioanalytical Method Validation

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.

Insights in regulated bioanalysis of human insulin and insulin analogs by immunoanalytical methods

Despite the long and illustrious history of insulin and insulin analogs as important biotherapeutics, the regulated bioanalysis (in this article, regulated bioanalysis refers to the formalized process for generating bioanalytical data to support pharmacokinetic and toxicokinetic assessments intended for development of insulin and insulin analogs as biotherapeutics, as opposed to the analytical process used for measuring insulin as a biomarker) of these peptides remains a challenging endeavor for a number of reasons. Paramount is the fact that the therapeutic concentrations are often low in serum/plasma and not too dissimilar from the endogenous level, particularly in patients with insulin resistance, such as Type 2 diabetes mellitus. Accordingly, this perspective was written to provide helpful background information for the design and conduct of immunoassays to support regulated bioanalysis of insulin and insulin analogs. Specifically, it highlights the technical challenges for determination of insulin and insulin analogs by immunoanalytical methods that are intended to support evaluations of pharmacokinetics and toxicokinetics. In a broader sense, this perspective describes the general bioanalytical issues that are common to regulated bioanalysis of peptides and articulates some of the bioanalytical differences between conventional monoclonal antibodies and peptide therapeutics.

The impact of decreased bead count to determine concentrations of amyloid beta1‐42, total‐tau, and phosphorylated‐tau181 in human cerebrospinal fluid using xMAP technology

Alzheimers disease is the leading cause of human dementia. The lack of diagnostic tests and limited therapeutic options has driven the search for endogenous biomarkers. The INNO-BIA AlzBio3 assay is a multiplex flow-based immunoassay measuring Aβ42, tau, and p-tau in cerebrospinal fluid (CSF). This study assesses assays performance under varying bead count (BC) parameters. Original method validation parameters at 100 BC were acceptable. Reanalyses performed at 3, 10, 25, and 50 BCs were compared to 100 BC data by ANOVA, Bland-Altman analysis, evaluation of concordance correlation coefficients, and frequency distribution of coefficient of variation (CV) ranges. Method validation characteristics were acceptable with 100 BCs. Equivalency for 25 and 50 versus 100 BCs was demonstrated, but not for 3 and 10 BCs. A general trend of decreasing agreement between decreasing BCs and the 100 BC reference resulted in decreases in concordance coefficients ρ(c) . The frequency of CV values greater than 20% increased with decreasing BCs, and CV values of 5% or less decreased with decreased BCs. Statistical analyses demonstrate that BCs of 3 and 10 are not equivalent with the reference and should not be used as a basis for determination of Aβ42, tau, and p-tau concentration in human CSF.

Development and Maintenance of Critical Reagents for Ligand Binding Assays to Support Regulatory-Compliant Bioanalysis

The FDA approved the first biotherapeutic (synthetic insulin) in 1982. Thirty-five years later, this novel class of pharmaceuticals has improved the quality of life for millions of individuals. While drug discovery of biotherapeutics and biosimilars was originally dominated by small biotechnology companies, today nearly every major pharmaceutical company in the world is engaged in this effort. Biotherapeutic programs present unique challenges to drug development, including generating supportive data to demonstrate safety and efficacy in the target population. Bioanalysis of traditional chemical pharmaceutics may be achieved after organic extraction from biological fluids, thus removing potentially interfering substances. Furthermore, strategic decisions based on the structure of the molecule often facilitates the development of an analytical method utilizing sensitive mechanical instrumentation. Biotherapeutics are often composed of amino acids whose functionality depends on complex structure. These compounds are measured via protein-protein interactions requiring unique reagents for each drug program. In this chapter, we describe these interactions and focus on the importance of generating and maintaining high-quality reagents, a process known as reagent lifecycle management .

The Role of Commercial Biomarker Assay Kits in Preclinical and Clinical Trials

The aim of this chapter is to discuss the role of commercial assay kits when quantitatively measuring biomarkers to support preclinical and clinical drug development programs. Research use only and clinical diagnostic kits will be compared and their usefulness addressed in the context of the 2013 FDA draft guidance on bioanalytical method validation. Analytical method performance characteristics will be discussed as well as mechanisms to generate consistent biomarker data throughout the life of the study.