Techniques for quantitative LC-MS/MS analysis of protein therapeutics: advances in enzyme digestion and immunocapture.

LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices. Immunocapture, in which a capture agent is used to preferentially bind the protein therapeutics over other proteins, is gaining traction. The protein therapeutics is eluted for digestion and LC-MS/MS analysis. One area of tremendous potential for immunocapture-LC-MS/MS is to obtain quantitative data where ligand-binding assay alone is not sufficient, for example, quantitation of antidrug antibody complexes. Herein, we present an overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation.

Utilizing Internal Standard Responses to Assess Risk on Reporting Bioanalytical Results from Hemolyzed Samples.

Bioanalytical analysis of toxicokinetic and pharmacokinetic samples is an integral part of small molecule drugs development and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the technique of choice. One important consideration is the matrix effect, in which ionization of the analytes of interest is affected by the presence of co-eluting interfering components present in the sample matrix. Hemolysis, which results in additional endogenous components being released from the lysed red blood cells, may cause additional matrix interferences. The effects of the degree of hemolysis on the accuracy and precision of the method and the reported sample concentrations from hemolyzed study samples have drawn increasing attention in recent years, especially in cases where the sample concentrations are critical for pharmacokinetic calculation. Currently, there is no established procedure to objectively assess the risk of reporting potentially inaccurate bioanalytical results from hemolyzed study samples. In this work, we evaluated the effect of different degrees of hemolysis on the internal standard peak area, accuracy, and precision of the analyses of BMS-906024 and its metabolite, BMS-911557, in human plasma by LC-MS/MS. In addition, we proposed the strategy of using the peak area of the stable isotope-labeled internal standard (SIL-IS) from the LC-MS/MS measurement as the surrogate marker for risk assessment. Samples with peak areas outside of the pre-defined acceptance criteria, e.g., less than 50% or more than 150% of the average IS response in study samples, plasma standards, and QC samples when SIL-IS is used, are flagged out for further investigation.

Targeting an acid labile aspartyl-prolyl amide bond as a viable alternative to trypsin digestion to generate a surrogate peptide for LC-MS/MS analysis.

BACKGROUND: FGF21-AdPKE is a fusion protein and functionally inactivated in vivo by cleavage around the C-terminus. It is important to quantify the intact active protein in serum. RESULTS & DISCUSSION: Taking advantage of a uniquely acid-labile aspartyl-prolyl amide bond, we developed an acid hydrolysis procedure based on heating FGF21-AdPKE in dilute formic acid to generate a surrogate peptide encompassing the last 17 amino acids at the C-terminus. The monkey serum samples were extracted with an immunocapture procedure with an antibody specific for AdPKE. The calibration range was 200-50000 ng/ml. The assay accuracy and precision were between 92.8-99.8% and 3.9-14.5%, respectively. The method was applied to analyze incurred serum samples from a cynomolgus monkey toxicokinetic study involving administration of FGF21-AdPKE. CONCLUSION: A method of combining immunocapture and acid hydrolysis to quantify a therapeutic protein in biological fluids was developed.

High-resolution MS in regulated bioanalysis: where are we now and where do we go from here?

While triple quadrupole MS remains the workhorse of bionanalytical laboratories, LC coupled with high-resolution MS (LC-HRMS) is making headway in drug discovery. LC-HRMS is well suited for quantitative bioanalysis with the inherent advantage of post-acquisition data mining, which is not possible with triple quadrupole systems operated in SRM mode. LC-HRMS can, thus, accomplish the core task of a bioanalytical laboratory--accurate determination of a targeted analyte--with the added bonus of being able to monitor other compounds of interest either at the time of sample analysis, or as an afterthought, after sample analysis, with no additional effort in sample preparation, chromatographic optimization or sample reinjection. Despite these advantages, LC-HRMS has not been broadly adopted in regulated bioanalytical laboratories. The slow progress in embracing the technology may be due, in part, to difficulties in replacing an entire fleet of triple quadrupole MS. Additional reasons are that data mining is of less benefit in development, especially late-stage, than in discovery and that the technical and regulatory challenges associated with the change of platform are perceived to be significant. In addition, the current platform of LC-HRMS introduced by instrument companies has not been tailored to the core responsibility of the bioanalytical community. In marketing current LC-HRMS systems, there is a tendency to combine the needs of the bioanalytical community with those of the drug metabolism community, despite their inherent differences. As a result, the current HRMS systems available lack some basic features desired for bioanalysis, but include features that are not important for bioanalysis making the systems unnecessarily complex and expensive. A simple, cost effective, ideal HRMS system for a bioanalytical laboratory would provide HRMS with high resolving power (the higher the better), no MS/MS capability, and with software suitable for quantitative analysis and appropriate for use in regulated laboratories. Under this scenario, one can foresee a future where part of the regulated bioanalytical work will be accomplished using LC-HRMS, reserving triple quadrupole-based LC-MS/MS for assays that require exquisite sensitivity.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Full-scan high resolution accurate mass spectrometry (HRMS) in regulated bioanalysis: LC-HRMS for the quantitation of prednisone and prednisolone in human plasma.

A liquid chromatography-full scan high resolution accurate mass spectrometry (LC-HRMS) method for quantifying prednisone and prednisolone in human plasma using a quadrupole time-of-flight mass spectrometer (Q-TOF) was developed. Plasma samples were extracted using a liquid-liquid extraction procedure. Full scan data were acquired in the TOF only mode and extracted ion chromatograms were generated post-acquisition with the exact masses of the analytes. The calibration range was 5-2500 ng/mL, with a Lower Limit of Quantitation (LLOQ) of 5 ng/mL. The assay accuracy was between 98.4% and 106.3%. The between-run (inter-day) and within-run (intra-day) precision were within 1.7% and 2.9%, respectively. The matrix effect was between 0.98 and 1.10 for the six different lots of human plasma evaluated. Pooled incurred samples were analyzed by the method and the results matched those obtained from an LC-MS/MS method. In addition, qualitative information on phospholipids, and other endogenous components were also extracted from the full-scan data acquired.

Effective screening approach to select esterase inhibitors used for stabilizing ester-containing prodrugs analyzed by LC-MS/MS.

BACKGROUND: Analysis of prodrugs, with their short half-lives, especially ester-containing ones, poses a unique challenge in developing and validating bioanalytical assays for nonclinical and clinical studies. A screening approach is needed to expeditiously select esterase inhibitors for stabilizing them during sample collection and processing. RESULTS: The screening process consisted of three steps. Initially, nine different esterase inhibitors were screened at three different plasma concentrations against an ester prodrug. Four inhibitors were chosen for the next step, in which plasma pH and processing temperature were optimized. Finally, whole-blood stability of the prodrug was evaluated. Three inhibitors with optimized plasma pH and processing temperature were selected for further bioanalytical assay development. CONCLUSION: An effective approach was successfully developed to promptly select suitable esterase inhibitors for stabilizing ester-containing prodrugs.

A computer program for automated data evaluation to support in vitro higher-throughput screening for drug metabolism and pharmacokinetics attributes.

With the advances in analytical techniques, higher-throughput screening for drug metabolism and pharmacokinetics (DMPK) attributes has become an integral part of drug discovery. However, as the number of compounds increases, the volume of data that needs to be processed and evaluated increases exponentially. As a result, a major challenge for the analytical chemist is how to quickly process the vast amount of data so as to keep up with the throughput of the screening assay. We have developed a customized computer program for automated evaluation of the liquid chromatography/tandem mass spectrometric (LC/MS/MS) data generated from the in vitro DMPK screening assays. This program performs automatic data processing and quality control. It identifies analytical anomalies, such as low internal standard intensity and poor reproducibility of replicates. All analytical anomalies for individual compounds are summarized into an 'E-Log' in a color-coded format for reviewing. With the use of this program and other supporting software, data processing and evaluation for up to 100 compounds are accomplished in several minutes.

Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography-tandem mass spectrometry.

A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1-2 compounds using the conventional methods.

Capillary electrophoresis-ion trap mass spectrometry analysis of Ziagen and its phosphorylated metabolites.

A capillary electrophoresis-ion trap mass spectrometry method with a time-segment program was developed to simultaneously analyze Ziagen and its phosphorylated metabolites such as carbovir monophosphate, carbovir diphosphate, and carbovir triphosphate. By using the time-segment program, the positively charged nucleoside analog and negatively charged nucleotides were separated and detected in a single electrophoretic run. The limits of detection were less than 2 micro M for all of the analytes. Calibration curves of the compounds showed excellent linearity over the range of 2-100 micro M. The capability of the method was demonstrated by analyzing Ziagen and its phosphorylated metabolites that were spiked in cellular extracts of human peripheral blood mononuclear cells at 20 micro M levels. Some endogenous nucleotides such as adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate, were also detected in the cellular extracts.

Higher-throughput screening for Caco-2 permeability utilizing a multiple sprayer liquid chromatography/tandem mass spectrometry system.

In the current drug discovery environment, higher-throughput analytical assays have become essential to keep pace with the screening demands for drug metabolism and pharmacokinetics (DMPK) attributes. This has been dictated by advances primarily in chemical procedures, notably combinatorial and parallel syntheses, which has resulted in many-fold increases in the number of compounds requiring DMPK evaluation. Because of its speed and specificity, liquid chromatography/tandem mass spectrometry (LC/MS/MS) has become the dominant technology for sample analysis in the DMPK screening assays. For higher-throughput assays, analytical speed as well as other factors such as method development, data processing, quality control, and report generation, must be optimized. The four-way multiplexed electrospray interface (MUX), which allows for the analysis of four LC eluents simultaneously, has been adopted to maximize the rate of sample introduction into the mass spectrometer. Generic fast-gradient HPLC methods that are suitable for approximately 80% of the new chemical entities encountered have been developed. In-house-written software programs have been used to streamline information flow within the system, and for quality control by automatically identifying analytical anomalies. By integrating these components together with automated method development and data processing, a system capable of screening 100 compounds per week for Caco-2 permeability has been established.