Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics
Graphical abstract
Introduction
Recently, LC–MS technology has been increasingly used for protein and peptide quantitation in biological matrices to complement ligand-binding assays (LBA) in support of toxicokinetic (TK) or pharmacokinetic (PK) studies in drug discovery and development. An LC-MS based assay is capable of analyzing multiple drug related components within a single assay, as well as detecting in vivo modification of protein or peptides in specific regions of interest. A number of review papers have been published in recent years on protein and peptide quantification by LC-MS technology [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11].
Ideally, the concentration of protein or peptide drug would be measured by direct mass spectrometric detection of intact proteins or peptides [12], [13], [14]. However, the analysis of intact protein or peptide molecules is still very challenging due to the wide mass range requirement and low detection sensitivity caused by broad isotope distributions and multiply-charged ions [15]. Consequently, the concentrations of protein or peptide molecules have been predominantly measured by selected reaction monitoring (SRM) detection of one or more surrogate peptides using a triple quadrupole mass spectrometer [16], [17], [18]. The surrogate peptides are obtained via enzymatic digestion or chemical cleavage prior to LC-MS/MS analysis. The concentration of an intact drug molecule is obtained based on the stoichiometric relationship between the surrogate peptide and the intact protein or peptide molecule.
Disulfide-rich proteins are a group of small protein domains commonly found as independent (single-domain) proteins or as domains within larger polypeptides [19]. These proteins play a wide variety of roles, including growth factors (e.g. insulin-like growth factors (IGFs)), toxins, enzyme inhibitors, hormones, pheromones and allergens [19]. PEG-Protein-I, an investigational drug at Bristol-Myers Squibb (BMS), is a PEGylated small (<15 KDa) disulfide-rich protein. Structurally, it comprises multiple peptide chains linked by inter-chain disulfide bonds, with a 20 kDa PEG moiety attached to one of the peptide chains. Generally, LBA is the gold standard platform for protein quantitation in biological samples, and an LBA was previously validated and used to quantify PEG-Protein-I in serum samples in support of all toxicokinetic (TK) studies. However, the LBA only determined the total drug concentrations including the drug plus any modified forms of the drug. Previous reports suggested that the proteolytic degradation products of protein therapeutics may contribute to the systemic metabolic activity and possible side effects [20], [21]. In order to quantify and evaluate the in vivo stability of PEG-Protein-I, a site specific and sensitive LC-MS assay is needed. However, the presence of the PEGylated moiety in PEG-Protein-I makes it difficult to analyze it as the intact drug molecule by LC-MS due to several bioanalytical challenges, such as low mass spectrometric response, poor LC peak shape and potential contamination of the MS detector.
In this paper, we describe a UHPLC-HRMS method to quantify PEG-Protein-I in monkey serum using two tryptic surrogate peptides. One surrogate peptide is a disulfide-containing peptide, DCP(SS), which contains amino acids that are essential for the PEG-Protein-I function but susceptible to potential in vivo cleavage, while the other is a confirmatory peptide, CP, which is a disulfide-free peptide (Table 1). DCP(SS) was generated from an attempt to produce larger peptides that would more closely represent the intact molecule by eliminating the steps of reduction/alkylation before trypsin digestion. DCP(SS) contains an intact disulfide bond connecting two peptide sequences that include the labile amino acids of interest. Each surrogate peptide contains one methionine and can also be useful for evaluating potential in vivo oxidation of PEG-Protein-I. This UHPLC-HRMS assay to quantify PEG-Protein-I has been validated in monkey serum, and applied to a non-clinical toxicokinetic (TK) study in monkeys. To the best of our knowledge, this is the first report of using an intact disulfide-containing peptide as a surrogate peptide for LC-MS bioanalysis of a therapeutic disulfide bond-rich protein.
Section snippets
Materials and reagents
The investigational PEGylated protein drug candidate, PEG-Protein-I, was obtained from Research and Development, Bristol-Myers Squibb (BMS). Its chemical structure cannot be disclosed for proprietary reasons. The sequence and accurate monoisotopic mass information of the tryptic surrogate peptides and their internal standards (IS) used for quantitation of PEG-Protein-I in monkey serum are shown in Table 1. Four pairs of peptides were chemically synthesized by Discovery Chemistry within BMS
UHPLC–MS/MS method development and optimization
It was previously reported that the PEGylation of a protein or peptide changes its physicochemical behavior, making it possible to extract it into selected water-miscible organic solvents [25], [26], [27]. In particular, isopropanol with 0.1% formic acid was shown to efficiently extract a PEGylated protein of molecular weight of 11 KDa [26]. The same solvent has been used for another PEGylated protein drug candidate with consistent recovery of ∼100% [25], [26]. The presence of 0.1% formic acid
Conclusions
A UHPLC-HRMS method was developed and validated to quantify a PEGylated protein in monkey serum using a surrogate peptide containing a disulfide bridge connecting two peptide chains, in addition to another disulfide bond-free peptide as the confirmatory peptide, CP. SRM detection on a triple quadrupole mass spectrometer was not sensitive enough to detect the disulfide-containing peptide, DCP(SS), due to poor fragmentation. The application of UHPLC-HRMS allowed the sensitive detection of this
Acknowledgments
The authors would like to thank Dr. Binodh Desilva (Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08540, USA) for her review of this manuscript.
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