Characterization of Critical Reagents in Ligand-Binding Assays: Enabling Robust Bioanalytical Methods and Lifecycle Management

Geist, Brian; Egan, Adrienne Clements; Yang, Tong‐Yuan; Yan, Dong; Shankar, Gopi

doi:10.4155/bio.12.304

Cited by 47 publications

(11 citation statements)

References 52 publications

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“…61,[64][65][66] So-called signal generating reagents (SGR) 67 are analytebinding molecules tagged to some sort of reporter, such as an enzyme, a dye, fluorophore, or hapten group to enable downstream detection of the binding event. 69,70 Reduction of background signals can be obtained by minimization of non-specific SGR binding through careful choice of reagents, extensive blocking, and, in some cases, by sample dilution (see below). For example, it has been demonstrated that PCR-based signal amplification enabled the use of weak binders which were not well suited for conventional ELISA.…”

Section: General Aspectsmentioning

confidence: 99%

Highly sensitive ligand-binding assays in pre-clinical and clinical applications: immuno-PCR and other emerging techniques

Spengler¹,

Adler²,

Niemeyer

2015

Analyst

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Recombinant DNA technology and corresponding innovations in molecular biology, chemistry and medicine have led to novel therapeutic biomacromolecules as lead candidates in the pharmaceutical drug development pipelines. While monoclonal antibodies and other proteins provide therapeutic potential beyond the possibilities of small molecule drugs, the concomitant demand for supportive bioanalytical sample testing creates multiple novel challenges. For example, intact macromolecules can usually not be quantified by mass-spectrometry without enzymatic digestion and isotopically labeled internal standards are costly and/or difficult to prepare. Classical ELISA-type immunoassays, on the other hand, often lack the sensitivity required to obtain pharmacokinetics of low dosed drugs or pharmacodynamics of suitable biomarkers. Here we summarize emerging state-of-the-art ligand-binding assay technologies for pharmaceutical sample testing, which reveal enhanced analytical sensitivity over classical ELISA formats. We focus on immuno-PCR, which combines antibody specificity with the extremely sensitive detection of a tethered DNA marker by quantitative PCR, and alternative nucleic acid-based technologies as well as methods based on electrochemiluminescence or single-molecule counting. Using case studies, we discuss advantages and drawbacks of these methods for preclinical and clinical sample testing.

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Section: General Aspectsmentioning

confidence: 99%

Highly sensitive ligand-binding assays in pre-clinical and clinical applications: immuno-PCR and other emerging techniques

Spengler¹,

Adler²,

Niemeyer

2015

Analyst

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“…Reagent characteristics can include but are not limited to the following: identity, source, purity, concentration (or titer), binding affinity, isotype (monoclonal/polyclonal antibody), molecular weight, specificity, incorporation ratio, and aggregation level. Several recent publications discuss in detail critical reagent characterization and testing methods (5,6,10).…”

Section: Characterization and Qualification Of Critical Reagentsmentioning

confidence: 99%

Ligand Binding Assay Critical Reagents and Their Stability: Recommendations and Best Practices from the Global Bioanalysis Consortium Harmonization Team

King

Farley

Imazato

et al. 2014

AAPS J

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Abstract. The L4 Global Harmonization Team on reagents and their stability focused on the management of critical reagents for pharmacokinetic, immunogenicity, and biomarker ligand binding assays. Regulatory guidance recognizes that reagents are important for ligand binding assays but do not address numerous aspects of critical reagent life cycle management. Reagents can be obtained from external vendors or developed internally, but regardless of their source, there are numerous considerations for their reliable long-term use. The authors have identified current best practices and provided recommendations for critical reagent lot changes, stability management, and documentation.

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“…Reagent screening/pairing The quality of critical reagents, for example, specificity, selectivity and consistency between batches, is essential for the good performance of all LBA-based bioanalytical methods for supporting biologics [8][9][10][11]. The rapid generation and selection of these reagents such as anti-idiotypic ('anti-Id') antibodies often form the primary start point of the critical path of method development.…”

Section: Applications For Laboratory Automationmentioning

confidence: 99%

Application of Automated Liquid Handling in Ligand-Binding Assay-Based Bioanalytical Method Development: the Practical Considerations

et al. 2018

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The ligand-binding assay (LBA) is the standard bioanalytical methodology to support preclinical and clinical development of biologics. LBA methods are based on molecular interactions of specific and selective critical reagents, which are large proteins themselves, with intended analyte in often a complex biological matrix such as serum. There are several benefits of automation in LBA method development. Primarily the replacement of manual with automated liquid handling will result in improved accuracy and precision of the assay [1], reduced human errors and lowering of the physical stress due to manual steps conducted by bench scientists. The decreased variation in pipetting that will then be achieved leads to the generation of higher quality bioanalytical data having greater reproducibility within studies and therefore more comparability across studies over time. This commentary will highlight our experience in applying automated liquid handling to each of these critical components of LBA method development process in bioanalytical laboratories conducting pharmacokinetic (PK), anti-drug antibody (ADA) and target engagement (TE) method development using LBAs, from start of critical reagent screening/pairing, to reagent addition, to sample/quality control (QC) dilutions, preparation of standard calibrators and QC samples with a focus on implementation in regulated bioanalysis. Selection of automation platformsThere is a broad variety of automated liquid handling systems available in the market today. They mainly are either air-based system or liquid-filled system for pipetting with either/or a combination of fixed and disposable tips. Automation platforms with liquid detection system are used to not only provide a record of action of aspiration but also to aid when the volumes of bioanalytical samples are low.Numerous publications have promoted using automated robotic systems in bioanalytical laboratories with different platforms and logistics of set-up [2][3][4][5][6][7]. The 'total laboratory automation (TLA)' proposed for routine clinical chemistry laboratories [4] is not always the best strategy for the application of automation to the development of PK, ADA and TE methods based on LBA methods. Most of the leading manufacturers of integrated robotic and automated liquid handling systems have produced LBA-based systems and have the flexibility to work with multiple assay platforms, run multiple methods in a single operation and are designed to take advantage of the vertical space within a laboratory to achieve the capacity of functionality required.In our laboratory, we took the initial path of purchasing modular automated liquid handling systems which can serve several benefits to our bioanalytical operation. Focus upon the accuracy and precision of liquid handling and its laboratory-wide effect upon flexible operation and reproducibility for LBA methods that will be routinely employed over several years was one of the major reasons for choosing modular platforms over TLA. The breakdown of the preparation of ...

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Characterization of Critical Reagents in Ligand-Binding Assays: Enabling Robust Bioanalytical Methods and Lifecycle Management

Cited by 47 publications

References 52 publications

Highly sensitive ligand-binding assays in pre-clinical and clinical applications: immuno-PCR and other emerging techniques

Highly sensitive ligand-binding assays in pre-clinical and clinical applications: immuno-PCR and other emerging techniques

Ligand Binding Assay Critical Reagents and Their Stability: Recommendations and Best Practices from the Global Bioanalysis Consortium Harmonization Team

Application of Automated Liquid Handling in Ligand-Binding Assay-Based Bioanalytical Method Development: the Practical Considerations

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