LC–MS Quantification of Protein Drugs: Validating Protein LC–MS Methods with Predigestion Immunocapture

Abstract

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.

Quantification Below the Lloq in Regulated Lc–Ms/Ms Assays: A Review of Bioanalytical Considerations and Cautions

“…Beyond the quantitative calibration range supported by method's standards and QCs, the validation verifies the selectivity of the method with different lots of matrix, explores the numeric value of the matrix effect, the stability of the analyte in matrix, during bench top processing and extracted storage, the accuracy of dilutions, the precision and accuracy of the assay over multiple batches, as well as a number of other important parameters [8,9]. One example of a key validation component is that of chromatographic selectivity testing used for protein LC-MS/MS methods depicted in Table 1 [10]. In this example 20 individual blanks from both control and disease state matrices are spiked at the assay LLOQ, then they are analyzed in a full assay run.…”

“…In addition to quantitative 'pass/fail' information, inspection of individual blank and LLOQ chromatograms can reveal details such as poor peak shape, co-eluting or close-lying interference peaks and undesirable baseline characteristics that might adversely affect integration [10].…”

“…Hybrid protein LC-MS/MS assays (IC PrD-LC-MS) can be particularly susceptible to interferences by plasma components that block or inhibit the analyte capture step [10,35,36]. These assays work by first purifying the analyte from matrix using immunocapture followed by proteolytic digestion and LC-MS/MS analysis of surrogate peptide for quantification.…”

“…Spike-recovery experiments such as those described below (Table 3) can measure specific recovery of the analyte in the immunocapture step of such assays, and the relative recoveries can be compared between different matrices, additives and putative interfering ligands [10]. In these experiments the matrix samples, with or without putative interferents, are spiked with the analyte protein either before or after the immunocapture step, then separately processed and quantified.…”

2017 White Paper on Recent Issues in Bioanalysis: Rise of Hybrid LBA/LCMS Immunogenicity Assays (Part 2: Hybrid LBA/LCMS Biotherapeutics, Biomarkers & Immunogenicity Assays and Regulatory Agencies’ Inputs)

“…BMV for hybrid LBA/LCMS assays has been discussed multiple times in the past and recommendations were provided [8,11,14,32]. However, to date, there is no regulatory guidance/guideline regarding experiments that should be conducted during method validation for hybrid LBA/LCMS methods.…”