Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

Toward best practices in data processing and analysis for intact biotherapeutics by MS in quantitative bioanalysis.

AIM: Typically, quantitation of biotherapeutics from biological matrices by LC-MS is based on a surrogate peptide approach to determine molecule concentration. Recent efforts have focused on quantitation of the intact protein molecules or larger mass subunits of monoclonal antibodies. To date, there has been limited guidance for large or intact protein mass quantitation for quantitative bioanalysis. METHODOLOGY: Intact- and subunit-level analyses of biotherapeutics from biological matrices are performed at 12-25 kDa mass range with quantitation data presented. RESULTS: Linearity, bias and other metrics are presented along with recommendations made on the viability of existing quantitation approaches. CONCLUSION: This communication is intended to start a discussion around intact protein data analysis and processing, recognizing that other published contributions will be required.

A whole-molecule immunocapture LC-MS approach for the in vivo quantitation of biotherapeutics.

AIM: Large-molecule biotherapeutic quantitation in vivo by LC-MS has traditionally relied on enzymatic digestion followed by quantitation of a 'surrogate peptide' to infer whole-molecule concentration. MS methods presented here measure the whole molecule and provide a platform to better understand the various circulating drug forms by allowing for variant quantitation. RESULTS: An immunocapture LC-MS method for quantitation of a biotherapeutic monoclonal antibody from human plasma is presented. Sensitivity, precision and accuracy for each molecular portion are presented along with an example of glycoform variant quantitation. CONCLUSION: The method is presented as a basic platform to be further developed for Good Practice (GxP) applications, critical quality attribute analysis or general understanding of molecular forms present as required for the wide range of drug development processes.

Application of high-resolution MS for development of peptide and large-molecule drug candidates.

BACKGROUND: For quantitative bioanalysis utilizing MS, the instrument of choice is typically a triple quadruple mass spectrometer. However, advances in high-resolution MS have allowed sensitivity and dynamic ranges to approach that of triple quadrupole instruments. RESULTS: A matrix-free protein digest, a digested therapeutic protein and the intact peptide therapeutic liraglutide were each analyzed on high-resolution and triple quadrupole mass spectrometers with data compared. Samples from a mouse PK study with liraglutide were analyzed using the two different instruments, and equivalent PK exposure data were demonstrated. CONCLUSION: High-resolution and triple quadrupole mass spectrometers can generate data resulting in identical PK parameters from an in-life sample set, thus giving confidence in either technique in support of biotherapeutic PK exposure studies.

Comparison of the quantification of a therapeutic protein using nominal and accurate mass MS/MS.

BACKGROUND: The quantification of proteins and peptides in in vivo samples is a critical part of supporting the drug development process for biotherapeutics. LC-MS/MS using tandem quadrupole mass spectrometers is well established as the technology of choice for the quantification of small-molecule drugs and their metabolites in biological fluid. The application of accurate mass MS for quantification in a DMPK environment has attracted considerable interest in recent years. MATERIALS & METHODS: In this article we describe and compare the application of LC-high-resolution MS and LC-selected reaction monitoring (SRM) for the quantification of a therapeutics proteins. RESULTS: The accurate mass instrumentation showed acceptable linearity and sensitivity to quantify the protein therapeutic to the level of 10 ng/ml. The accurate mass instrument was operated in accurate mass SRM using high resolution (SRM-HR), the assay was demonstrated to be linear over three orders of magnitude. By narrowing the mass window from 100 mDa to 40 mDa and then to 20 mDa the assay specificity was significantly improved, hence increasing the S/N and improving the assay sensitivity. CONCLUSION: The high-resolution instrument was demonstrated to be reproducible over the course of the assay. The accurate mass method sensitivity was determined to be within one order of magnitude of that obtained with a tandem quadrupole MS/MS assay.

Absolute quantification of a therapeutic domain antibody using ultra-performance liquid chromatography-mass spectrometry and immunoassay.

BACKGROUND: Domain antibodies (dAbs; ∼10-15 kDa) are made up of the variable heavy chain or the variable light chain of the antibody structure, and retain binding capability. dAbs have proved difficult to detect in plasma using immunoassay without specific antibodies raised against the dAb. RESULTS: A sensitive and selective UPLC-MS/MS method for the absolute quantification of a dAb in monkey plasma was developed (range: 1 to 500 ng/ml) without the need for a specific capture antibody. This method was used to analyze pharmacokinetic studies early on in drug development. Furthermore, an immunoassay was developed and the pharmacokinetic samples were reanalyzed. CONCLUSION: The two assays show good correlation (r(2) = 0.92), giving confidence in using either method for quantification of the dAb.

Application of DBS for quantitative assessment of the peptide Exendin-4; comparison of plasma and DBS method by UHPLC-MS/MS.

BACKGROUND: An investigation was performed in order to establish if dried blood spots (DBS) could be applied to the quantitation of biopharmaceuticals in biological matrices and perform equivalently in terms of accuracy, precision and stability to traditional plasma methods. RESULTS: A method was successfully validated for the peptide Exendin-4 (39 amino acids in length) utilizing DBS technology. The validated DBS method resulted in a more sensitive and simplistic method than an existing monkey plasma method and required tenfold less sample volume. The final DBS method resulted in a 10-2000-ng/ml linear calibration range using approximately 5 µl of dried blood, compared with the plasma method in which 150 µl of plasma coupled with SPE sample preparation resulted in a 20-2000-ng/ml linear calibration range. Although not needed for DBS, SPE was required for the plasma method to reduce endogenous matrix interferences and achieve desired LLOQ. Matrix stability was also enhanced by the implementation of the DBS platform when compared with either plasma or whole blood. CONCLUSION: DBS technology can be utilized for the quantitation of biopharmaceuticals and offer advantages over traditional plasma-based methods.

Utility of dried blood spot sampling and storage for increased stability of photosensitive compounds.

BACKGROUND: Compound stability remains a major point of concern within pharmaceutical development. In attempts to minimize degradation, scientists may utilize acidification of samples prior to storage, dark chambers, decreased freezer temperatures and a variety of other stabilization techniques. All of these steps require additional procedures, increased costs and increased validation steps. Dried blood spots (DBS) are becoming a popular alternative to plasma sampling in many small- and even large-molecule applications. An investigation was performed in order to establish if DBS would provide storage advantages over liquid-based matrices for two light-sensitive compounds, nifedipine and omeprazole, to prevent or minimize photodegradation. RESULTS: Experimental data has shown, through forced and natural photodegradation experiments, that the compounds nifedipine and omeprazole exhibit increased photostability when spotted and stored on various DBS paper, when compared with water, plasma or whole blood. For omeprazole, between 40 and 90% loss was observed in liquid matrices, while photodegradation was negligible when utilizing DBS. Some loss of nifedipine is noted during exposure conditions on DBS; however, photodegradation in liquid matrices is far more severe. CONCLUSION: Within the experimental compound set, DBS technology offers a significant reduction in the photodegradation process when compared with the liquid matrices water, plasma or blood.

Development of an in vivo preclinical screen model to estimate absorption and first-pass hepatic extraction of xenobiotics. II. Use of ketoconazole to identify P-glycoprotein/CYP3A-limited bioavailability in the monkey.

The effect of P-glycoprotein (Pgp) and/or CYP3A on the disposition of xenobiotics has been extensively investigated and is often of interest during drug discovery lead optimization. We have previously described a monkey pharmacokinetic screen to rapidly estimate absorption and first-pass extraction. In the present work, this monkey screen has been expanded to include an assessment of Pgp/CYP3A effects on absorption and first-pass extraction, using ketoconazole as a prototypic dual Pgp/CYP3A inhibitor. To generate a ketoconazole dosing regimen, the pharmacokinetics of ketoconazole were first determined in the monkey and were found to be consistent with that previously described in the rat, dog, and human. Dose-ranging experiments demonstrated that a single 10-mg/kg intraduodenal ketoconazole dose would provide an appropriate exposure; this dose was used throughout subsequent interaction experiments. Next, erythromycin and propranolol were explored as positive and negative control substrates for Pgp/CYP3A interactions, respectively. As anticipated, ketoconazole produced no change in the absorption or first-pass extraction of propranolol but resulted in a substantial increase in absorption and decrease in first-pass extraction of erythromycin. Finally, this ketoconazole-based monkey screen was deployed in a drug discovery setting, and examples of such use are presented. These experiments have allowed a more complete characterization of ketoconazole as a prototypic dual Pgp/CYP3A inhibitor and its use as a tool in a preclinical setting and further demonstrate the use of the monkey to investigate the role of Pgp/CYP3A in limiting the oral bioavailability of new drug candidates.