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

Abstract. Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-forpurpose-based validation, emphasizing issues that are unique to multiplex kit assays.

Section: Solving Challenges With Lot-to-lot Differencesmentioning

confidence: 99%

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development

Jani

Allinson²,

Berisha

et al. 2015

“…In any case, the level of criticality of reagents, their production procedure or procurement options, and any other considerations important to the assay should be documented and described in the analytical method. Since many assays are utilized over several years, the long-term availability and stability of critical reagents should be carefully considered (26).…”

Section: Validation Considerationsmentioning

confidence: 99%

“…It is advisable to monitor for trending that may indicate loss of reagent effectiveness over the duration of its use. Since many assays are utilized over a long period, stability of critical reagents should be carefully managed (26).…”

Section: Critical Assay Reagents Stabilitymentioning

confidence: 99%

Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics

Jenkins¹,

Duggan

Aubry

et al. 2014

151

ABSTRACT. This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group's Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.KEY WORDS: affinity capture mass spectrometry; industry white paper; method validation; protein LC-MS/MS quantification; regulated bioanalysis.

“…Most importantly, with regards to our efforts, the paper acknowledged the gap in expected practices around biomarker assay calibrators, and that specific guidance around selecting and characterizing biomarker calibrator materials is needed. Thus, it was suggested that biomarker calibrators could be treated in the same way as critical reagents in the interim (16).…”

Section: Regulatory Guidance Documents and Recommendations For The Bimentioning

confidence: 99%

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material

Cowan

Amaravadi

Cameron

et al. 2017

Abstract.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 wellcharacterized 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 interchangeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.