Michael J. Berna

Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.

Recent Advances in use of LC/MS/MS for Quantitative High-Throughput Bioanalytical Support of Drug Discovery

LC/MS/MS based bioanalysis using atmospheric pressure ionization (API)-style interfaces has now been applied for over a decade. This technology, which initially found application for clinical bioanalysis, is now firmly established as the primary bioanalytical tool for ADME studies related to drug discovery and lead optimization (LO). This review focuses on recent advances in LC/MS/MS based bioanalysis in support of drug discovery and LO. The initial part of the article reviews the principal components of LC/MS/MS bioanalysis: sample preparation, chromatography, ionization and mass analysis. In each section, factors affecting high throughput bioanalysis are addressed. Because of the importance of on-line column switching methods to discovery bioanalysis, the section on sample preparation is divided into off-line and on-line approaches. In addition, the discussion of chromatography is limited to reversed phase liquid chromatography with emphasis given to the trend towards high-flow gradient elution techniques. The latter part of the review focuses on considerations for experimental design. In this section, pooling methods such as cassette dosing are discussed along with more highly integrated strategies linking bioanalysis with protocol generation and sample collection. The article concludes by briefly reviewing factors, which affect bioanalytical precision and accuracy, such as ion suppression, analyte stability and metabolite interference.

Current Applications of Liquid Chromatography/Mass Spectrometry in Pharmaceutical Discovery After a Decade of Innovation

Current drug discovery involves a highly iterative process pertaining to three core disciplines: biology, chemistry, and drug disposition. For most pharmaceutical companies the path to a drug candidate comprises similar stages: target identification, biological screening, lead generation, lead optimization, and candidate selection. Over the past decade, the overall efficiency of drug discovery has been greatly improved by a single instrumental technique, liquid chromatography/mass spectrometry (LC/MS). Transformed by the commercial introduction of the atmospheric pressure ionization interface in the mid-1990s, LC/MS has expanded into almost every area of drug discovery. In many cases, drug discovery workflow has been changed owing to vastly improved efficiency. This review examines recent trends for these three core disciplines and presents seminal examples where LC/MS has altered the current approach to drug discovery.

Determination of olanzapine in human blood by liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay was developed and validated to quantitatively determine olanzapine (OLZ) concentrations in human blood. Liquid-liquid extraction, using n-butanol:cyclohexane (3:47, v/v), was used to isolate OLZ and its internal standard, LY170158, from the biological matrix. Chromatographic resolution of OLZ from endogenous interferences and known metabolites was accomplished with a MetaChem Monochrom HPLC column (4.6 x 150 mm, d(p) 5 microm). Detection occurred using a Perkin-Elmer Sciex API III Plus triple quadrupole mass spectrometer using positive ion APCI and multiple reaction monitoring (MRM). The linear dynamic range was from 5 to 500 ng ml(-1) based on a 0.25-ml aliquot of human blood. The inter-day precision (%RSD) and accuracy (%RE) ranged from 3.65 to 10.64 and from -2.14 to 3.07, respectively. Modifications to an existing assay for the determination of OLZ in human plasma were necessary. A different structural analog was used as the internal standard due to instability observed for the original analog when using human blood as the matrix. A second modification was the addition of the anti-oxidant sodium ascorbate to inhibit degradation of OLZ in human blood, as has been noted by other investigators. Upon fortification of human blood with sodium ascorbate (final concentration, 0.33 mM), OLZ was found to be stable for at least 1 week at -70 degrees C as well as through two freeze-thaw cycles. This assay, which will be used to investigate the distribution of OLZ in human blood, grants insight into the proper sample handling conditions needed to perform valid determinations of OLZ in human blood.

Increased Throughput for Low-Abundance Protein Biomarker Verification by Liquid Chromatography/ Tandem Mass Spectrometry

Low-abundance protein quantification has historically been performed using ligand binding techniques. However, due to the time and cost associated with developing enzyme-linked immunosorbent assay (ELISA), mass spectrometric approaches are playing an increasingly important role. Protein quantification at or below the nanogram per milliliter level using liquid chromatography/tandem mass spectrometry (LC/MS/MS) typically utilizes an immunoaffinity (IA) enrichment step such as immunoprecipitation. In order to maximize mass spectrometry (MS) sensitivity, protein enrichment is followed by a proteolytic cleavage step used to generate a surrogate peptide with better mass spectrometric properties. Unlike ELISA, IA-LC/MS/MS is a serial technique that can require up to 3 days for a single batch analysis due to lengthy incubation and digestion steps. This report describes the use of immunoprecipitation in 96-well ELISA format (IPE) and microwave-assisted protein digestion to reduce the time required to perform LC/MS/MS protein analyses to within a single day. The utility of this approach was investigated through its application to previously published LC/MS/MS protein assays from our laboratory for two cardiotoxicity biomarkers, Myl3 and NTproBNP. Using commercially available antibodies, IPE and microwave-assisted digestion were used to repeat intraday validations for these markers, and intraday precision (%CV) and accuracy (%RE) did not exceed 11% or 3% for either assay, respectively. Additionally, lower limits of quantification of 100 pg/mL (NTproBNP) and 0.95 ng/mL (Myl3) were achieved.

Detection of Left Ventricular Hypertrophy in Rats Administered a Peroxisome Proliferator–Activated Receptor α/γ Dual Agonist Using Natriuretic Peptides and Imaging

Chronic treatment with suprapharmacologic doses of peroxisome proliferator-activated receptor (PPAR) agonists has a known potential for causing left ventricular hypertrophy (LVH). The mechanism by which LVH develops is not well understood nor are biomarkers of it well characterized. Natriuretic peptides are important regulators of cardiac growth, blood volume, and arterial pressure and may be useful biomarkers of LVH and hemodynamic changes that precede it. We measured amino-terminal pro-atrial natriuretic peptide (NTproANP), amino-terminal pro-brain natriuretic peptide (NTproBNP), and cardiac troponin I (cTnI) concentrations in serum and plasma, as well as transcripts in left ventricular heart tissue for atrial natriuretic peptide precursor (Nppa), brain natriuretic peptide precursor (Nppb), and myosin heavy chain-beta (Myh7) as potential biomarkers of LVH induced by a PPARalpha/gamma dual agonist in Sprague-Dawley rats. We used magnetic resonance imaging, echocardiography, and hemodynamics to identify structural and functional cardiovascular changes related to the biomarkers. Heart-to-brain weight ratios (HW:BrW) were correlated with NTproANP, NTproBNP, and cTnI concentrations in serum as well as fold change in expression of Nppa and Nppb. LVH was characterized by increased left ventricular wall thickness and inner diameter, increased cardiac output, decreased arterial blood pressure, and increased heart rate. In these studies, each end point contributed to the early detection of LVH, the ability to monitor its progression, and demonstrated the ability of NTproANP concentration in serum to predict LVH and hemodynamic changes.

Quantification of heart fatty acid binding protein as a biomarker for drug‐induced cardiac and musculoskeletal necroses

Heart fatty acid binding protein (Fabp3) is a cytosolic protein expressed primarily in heart, and to a lesser extent in skeletal muscle, brain, and kidney. During myocardial injury, the Fabp3 level in serum is elevated rapidly, making it an ideal early marker for myocardial infarction. In this study, an MS‐based selected reaction monitoring method (LC‐SRM) was developed for quantifying Fabp3 in rat serum. Fabp3 was enriched first through an immobilized antibody, and the protein was digested on beads directly. A marker peptide of Fabp3 was quantified using LC‐SRM with a stable isotope‐labeled peptide standard. For six quality control samples with Fabp3 ranging from 0.256 to 25 ng, the average recovery following the procedure was about 73%, and the precision (%CV) between replicates was less than 7%. The Fabp3 concentrations in rat serum peaked 1 h after isoproterenol treatment, and returned to baseline levels 24 h after the dose. Elevated Fabp3 levels were also detected in rats administered with a PPAR α/δ agonist, which has shown to cause skeletal muscle necrosis. Fabp3 can be used as a biomarker for both cardiac and skeletal necroses. The cross‐validation of the LC‐SRM method with an existing ELISA method is described.

Quantitative characterization of the mechanism of action and impact of a ‘proteolysis-permitting’ anti-PCSK9 antibody

ABSTRACT A recent report described a novel mechanism of action for an anti-proprotein convertase subtilisin-kexin type 9 (PCSK9) monoclonal antibody (LY3015014, or LY), wherein the antibody has improved potency and duration of action due to the PCSK9 epitope for LY binding. Unlike other antibodies, proteolysis of PCSK9 can occur when LY is bound to PCSK9. We hypothesized that this allowance of PCSK9 cleavage potentially improves LY efficiency through two pathways, namely lack of accumulation of intact PCSK9 and reduced clearance of LY. A quantitative modeling approach is necessary to further understand this novel mechanism of action. We developed a mechanism-based model to characterize the relationship between antibody pharmacokinetics, PCSK9 and LDL cholesterol levels in animals, and used the model to better understand the underlying drivers for the improved efficiency of LY. Simulations suggested that the allowance of cleavage of PCSK9 resulting in a lack of accumulation of intact PCSK9 is the major driver of the improved potency and durability of LY. The modeling reveals that this novel ‘proteolysis-permitting’ mechanism of LY is a means by which an efficient antibody can be developed with a total antibody dosing rate that is lower than the target production rate. We expect this engineering approach may be applicable to other targets and that the mathematical models presented herein will be useful in evaluating similar approaches.