Immunoaffinity Purification Using Anti-PEG Antibody Followed by Two-Dimensional Liquid Chromatography/Tandem Mass Spectrometry for the Quantification of a PEGylated Therapeutic Peptide in Human Plasma

Abstract

Quantification of a PEGylated peptide in human plasma using LC-MS/MS to support clinical studies presented challenges in terms of assay sensitivity, selectivity, and ruggedness. To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by two-dimensional (2D) LC-MS/MS was developed for MK-2662, an investigational peptide containing 38 amino acids with a 40 kDa branched PEG [poly(ethylene glycol)] at C-terminus. Biotinylated anti-PEG antibody, bound to streptavidin-coated magnetic beads, was used to capture MK-2662 and its stable-isotope-labeled internal standard from human plasma. After on-bead digestion with trypsin, the supernatant was injected on a 2D high-performance liquid chromatography (HPLC) system constructed with strong cation-exchange and reversed-phase columns, followed by MS/MS detection of the surrogate N(1-12)-mer of MK-2662 on an API5000. The assay ruggedness was improved by optimizing the trypsin digestion and sample storage conditions. The intraday validation, conducted in parallel with protein precipitation (PPT) assay, demonstrated 94.8-105.8% accuracy with <9.76% coefficient of variation (CV) for IAP, and 99.0-101.0% accuracy with <3.43% CV for PPT, over a dynamic range of 2-200 nM and 1-1000 nM, respectively. A cross comparison, performed using clinical samples, showed that the values obtained from IAP assay were about 15-30% lower than those from PPT method, which supports more specific PEG recognition provided by IAP.

Direct Quantitative Analysis of a 20 kDa PEGylated Human Calcitonin Gene Peptide Antagonist in Cynomolgus Monkey Serum Using In-Source CID and UPLC-MS/MS

“…As a result, there have been no published reports describing the direct quantitative analysis of PEGylated peptides by LC-MS/MS. Recently, three quantitative LC-MS/ MS methods for PEGylated drugs [27][28][29] have been reported. However, these methods were accomplished by using tryptic digestion prior to LC-MS/MS to generate non-PEGylated signature peptides that could be used as surrogates for the quantitative analysis of the target PEGylated drugs.…”

LC‐MS in Drug Metabolism and Pharmacokinetics: A Pharmaceutical Industry Perspective

“…One such example is PEGylated (polyethylene glycolyated) proteins and their de -PEGylated components. In one study, a full-length PEGylated protein (conjugated with PEG at C -terminus) was analyzed by immunocapture using an anti -PEG antibody, followed by trypsin digestion, and LC -MS/MS analysis of a signature peptide located at the N -terminus [146] .…”

Sequential immunoaffinity-LC/MS assay for quantitation of a therapeutic protein in monkey plasma

“…After washing, the biotherapeutic is eluted from the beads by the addition of acid, followed by digestion and subsequent LC/ MS analysis. As expected, the immunocapture sample cleanup produces a clean, enriched extract and greatly reduces matrix effect on the LC/MS analysis resulting in improved assay sensitivity [10,13]. In an early example of such applications, a lower limit of quantitation (LLOQ) of low ng/mL was achieved for biotherapeutics such as Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer [18].…”

“…The vast majority of LC/MS-based protein quantifications are performed at peptide levels, mainly due to consideration of assay sensitivity [7][8][9][10]. A typical procedure for LC/MS-based quantification includes enzyme digestion and quantification of the target proteins based on selected surrogate peptides derived from the target [8,9].…”

“…A typical procedure for LC/MS-based quantification includes enzyme digestion and quantification of the target proteins based on selected surrogate peptides derived from the target [8,9]. Recently, immunocapture sample purification and enrichment prior to digestion and subsequent LC/MS analysis has been implemented [10][11][12][13][14][15][16]. The combination of immunocapture with LC/MS not only can address the selectivity issues affecting LBA, but also add significant benefits such as multiplexing (multiple analytes in one assay; same assay for multiple species/matrices), fast method development, wide dynamic range (up to 4 orders of magnitude), and good robustness and reproducibility.…”