Search

In response to an earlier workshop covering the pros and cons of quantification below the LLOQ (BLQ) the author reviews the topics discussed from the bioanalytical standpoint. Important considerations for estimating concentrations below the LLOQ include: method signal-to-noise, baseline shape and condition, close lying interference peaks (especially for protein methods), matrix effect, adsorption and stability of the analyte at low concentrations and carryover. These methodological issues are discussed as possible contributors to inaccuracy in BLQ estimations, and appropriate cautions are provided via examples. A proposed method for the evaluation of BLQ estimations utilizes extended incurred sample reanalysis analysis where BLQ samples or spiked simulated samples are analyzed with quality controls and standards in addition to those in the original study. Generally, BLQ estimations are discouraged, with the recommendation that any extrapolations should be done in close collaboration between the pharmacokinetic (PK) and bioanalytical scientists in consultation with the regulatory agency.

A refinement of protein LC–MS bioanalysis is to use predigestion immunoaffinity capture to extract the drug from matrix prior to digestion. Because of their increased sensitivity, such hybrid assays have been successfully validated and applied to a number of clinical studies; however, they can also be subject to potential interferences from antidrug antibodies, circulating ligands or other matrix components specific to patient populations and/or dosed subjects. The purpose of this paper is to describe validation experiments that measure immunocapture efficiency, digestion efficiency, matrix effect and selectivity/specificity that can be used during method optimization and validation to test the resistance of the method to these potential interferences. The designs and benefits of these experiments are discussed in this report using an actual assay case study.

This paper highlights the recommendations of a group of industry scientists in validating regulated bioanalytical LC–MS/MS methods for protein therapeutics in a 2015 AAPSJ White Paper. This group recommends that most of the same precision and accuracy validation criteria used for ligand-binding assays (LBAs) be applied to LC–MS/MS-based assays where proteins are quantified using the LC–MS/MS signal from a surrogate peptide after proteolytic digestion (PrD-LCMS methods). PrD-LCMS methods are generally more complex than small molecule LC–MS/MS assays and may often include LBA procedures, leading to the recommendation for a combination of chromatographic and LBA validation strategies and appropriate acceptance criteria. Several key aspects of this bioanalytical approach that are discussed in the White Paper are treated here in additional detail. These topics include selectivity/specificity, matrix effect, digestion efficiency, stability and critical reagent considerations.

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.