Perspective
Bioanalytical method validation considerations for LC–MS/MS assays of therapeutic proteins
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.
Over the past several years, there has been an increasing focus on bioanalytical methods for protein therapeutics using LC–MS tech- nology. Thus far, the majority of assays have involved indirect quantification by LC–MS/ MS analysis of a surrogate peptide follow- ing proteolytic digestion of the protein [1,2]. In these methods, the targeted protein drug is subjected to digestion by specific protease enzyme(s) to yield a digest containing one or more signature peptide(s) that are unique to that protein and not subject to interference from the matrix. The resulting digest is then subjected to LC–MS/MS, and the SRM signal of the signature peptide is measured, usually along with that of an IS. Although there may be affinity or immunoaffinity purification steps applied before or after the proteolytic digestion [3–5], the general technique is the same and has been called PrD-LCMS, signifying protein digestion followed by LC–MS/MS analysis [6]. With the increased use of
these assays
comes the need to apply them to both clini- cal and nonclinical bioanalytical studies in the regulated environment. This means that clear criteria must be established for
10.4155/BIO.15.69 © 2015 Future Science Ltd
the validation of such methods. Currently, regulatory guidances exist for the validation of protein ligand-binding (LBA) and small molecule (chromatographic) bioanalytical methods [7–9] but not for PrD-LCMS meth- ods. The recently updated draft US FDA guidance for bioanalytical method validation also addresses validation criteria for LBAs and small molecule methods, but makes no mention of PrD-LCMS methods [10]. Here, we will summarize the consensus
of a group of industry practitioners in PrD- LCMS who formed a consortium under the auspices of the AAPS bioanalytical focus group. We discussed these validation param- eters at length and have generated a White Paper that suggests validation considerations and recommended acceptance criteria for this methodology. This article was recently pub- lished in the AAPS Journal as a consensus report [11].
General consensus While the validation criteria for acceptable precision and accuracy and other param- eters that would be required for PrD-LCMS methods have not yet been defined in
Bioanalysis (2015) 7(11), 1389–1395 ISSN 1757-6180
Jeffrey X Duggan*,1 Vazvaei2
1 , Faye & Rand Jenkins3
Bioanalysis & Metabolite Profiling, Drug Metabolism & Pharmacokinetics, Boehringer-Ingelheim Pharmaceuticals,
Inc., Ridgefield, CT 06877, USA 2
Roche Pharma Research & Early
Development, Pharmaceutical Sciences, Global DMPK & Bioanalytical R&D, Roche Innovation Center New York, 430 East
29th Street, New York, NY 10016, USA 3
Chromatographic Sciences, PPD
Bioanalytical Laboratories, 2244 Dabney Road, Richmond, VA 23230, USA *Author for correspondence: Tel: 203 791 6320
jeffrey.duggan@boehringer-ingelheim.com
part of
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