Quantitative bottom up analysis of infliximab in serum using protein A purification and integrated μLC-electrospray chip IonKey MS/MS technology

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods.

Biotechnology increasingly delivers highly promising protein-based biopharmaceutical candidates to the drug development funnel. For successful biopharmaceutical drug development, reliable bioanalytical methods enabling quantification of drugs in biological fluids (plasma, urine, tissue, etc.) are required to generate toxicokinetic (TK), pharmacokinetic (PK), and bioavailability data. A clear observable trend is that liquid chromatography coupled to (tandem) mass spectrometry (LC-MS(/MS)) is more and more replacing ligand binding assays (LBA) for the bioanalytical determination of protein-based biopharmaceuticals in biological matrices, mainly due to improved selectivity and linear dynamic ranges. Practically all MS-based quantification methods for protein-based biopharmaceuticals traditionally rely on (targeted) proteomic techniques and include "seven critical factors": (1) internal standardization, (2) protein purification, (3) enzymatic digestion, (4) selection of signature peptide(s), (5) peptide purification, (6) liquid chromatographic separation and (7) mass spectrometric detection. For this purpose, the variety of applied strategies for all "seven critical factors" in current literature on MS-based protein quantification have been critically reviewed and evaluated. Special attention is paid to the quantification of therapeutic monoclonal antibodies (mAbs) in serum and plasma since this is a very promising and rapidly expanding group of biopharmaceuticals. Additionally, the review aims to predict the impact of strategies moving away from traditional protein cleavage isotope dilution mass spectrometry (PC-IDMS) toward approaches that are more dedicated to bioanalysis. Copyright © 2013 Elsevier B.V. All rights reserved.

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.