Perspective on high-throughput bioanalysis to support in vitro assays in early drug discovery.

As the desire for a shortened design/make/test/learn cycle increases in early drug discovery, the pressure to rapidly deliver drug metabolism pharmacokinetic data continues to rise. From a bioanalytical standpoint, in vitro assays are challenging because they are amenable to automation and thus capable of generating a high number of samples for analysis. To keep up with analysis demands, automated method development workflows, rapid sample analysis approaches and efficient data analysis software must be utilized. This work provides an outline of how we implemented those three aspects to provide bioanalytical support for in vitro drug metabolism pharmacokinetic assays, which include developing hundreds of mass spectrometry methods and analyzing thousands of samples per week, while delivering a median bioanalytical turnaround time of 1-2 business days.

Antibody Subunit LC-MS Analysis for Pharmacokinetic and Biotransformation Determination from In-Life Studies for Complex Biotherapeutics.

Complex biotherapeutics present challenges from drug discovery, screening, and development perspectives. While monoclonal antibody drugs are not monitored for metabolites in the same manner as small molecules, biotherapeutics such as fusion proteins, antibody-drug conjugates, or bispecific antibodies may undergo biotransformation (such as clipping, deamidation, or oxidation) in vivo, resulting in catabolites that can have a direct impact on drug safety or efficacy. Here antibody subunit LC-MS is utilized for evaluation of two classes of complex biotherapeutics: an antibody-drug conjugate and a mAb-fusion biotherapeutic. Pharmacokinetic concentration, biotransformation, and DAR data are collectively presented using the subunit LC-MS approach for the two molecules, and the methods shared in detail can be applied to any humanized IgG1 mAb biotherapeutic for preclinical study support. Overall, the data generated from antibody LC-MS analyses can provide key information in early phase development and deliver multiple study end points with a single data set.

Biotherapeutic Antibody Subunit LC-MS and Peptide Mapping LC-MS Measurements to Study Possible Biotransformation and Critical Quality Attributes In Vivo.

Biotransformation monitoring involves tracking drug modification occurring during in-life studies. Critical Quality Attribute monitoring from forced degraded drug material or in-life sample sets can provide an in-depth assessment of product quality for support in early- or late-stage drug development. For Critical Quality Attribute analysis, biotherapeutic monoclonal antibody (mAb) subunit analysis and peptide mapping liquid chromatography-mass spectrometry (LC-MS) approaches are used, although typically from an in vitro setting (e.g., formulation buffer) not involving biological samples or material. Here, samples from a high-dose rat study (in vivo) are subjected to analysis by ligand binding assay, mAb subunit LC-MS, and peptide mapping by LC-MS. Taken together, data from the 3 analytical approaches provide information regarding drug concentration in circulation, biotransformation, and biotherapeutic drug product quality. The concept of a multitier workflow for preclinical or clinical sample sets can be applied to other biotherapeutic mAb products such as bispecific mAbs, fusions proteins, or antibody-drug conjugates.

Review of approaches and examples for monitoring biotransformation in protein and peptide therapeutics by MS.

Biotherapeutic drugs have emerged in quantity in pharmaceutical pipelines, and increasingly diverse biomolecules are progressed through preclinical and clinical development. As purification, separation, mass spectrometer detection and data processing capabilities improve, there is opportunity to monitor drug concentration by traditional ligand-binding assay or MS measurement and to monitor metabolism, catabolism or other biomolecular mass variants present in circulation. This review highlights approaches and examples of monitoring biotransformation of biotherapeutics by MS as these techniques are poised to add value to drug development in years to come. The increased use of such approaches, and the successful quantitation of biotherapeutic structural modifications, will provide insightful data for the benefit of both researchers and patients.

Drug monitoring by volumetric absorptive microsampling: method development considerations to mitigate hematocrit effects.

AIM: GSKA is a compound that was in development in clinical trials. A bioanalysis method to quantify GSKA using volumetric absorptive microsampling (VAMS) was developed and hematocrit (HCT) related assay bias was investigated. METHODOLOGY: After accurate sampling of 10 µl blood, VAMS tips were air dried approximately 18 h and desorbed by an aqueous solution containing internal standard. The recovered blood underwent liquid-liquid extraction in ethyl acetate to minimize matrix suppression. Assay accuracy, precision, linearity, carryover, selectivity, recovery, matrix effects, HCT effects and long-term quality control stability were evaluated. CONCLUSION: HCT-related assay bias was minimized in 30-60% blood HCT range, and all validation parameters met acceptance criteria. The method is suitable for quantitative analysis of GSKA in human blood.

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 novel approach to capillary plasma microsampling for quantitative bioanalysis.

BACKGROUND: A novel device and procedure for the collection and isolation of microvolumes of plasma have been developed and two pilot rodent PK studies have been completed. RESULTS: This method involves collection of blood into a plastic-wrapped, EDTA-coated capillary tube, containing a small amount of a thixotropic gel and a porous plug. Following blood collection, the capillary is placed into a secondary labeled container suitable for centrifugation and plasma is generated. During centrifugation, the thixotropic gel isolates the plasma from the red blood cells and creates a physical barrier between the two matrices. The plasma is then dispensed from the capillary tube into a separate container for storage or processing. CONCLUSION: A simple and robust novel approach for the collection of small plasma volumes from rodent TK studies has been demonstrated.

S-nitrosothiols signal hypoxia-mimetic vascular pathology.

NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes. We used the pharmaceutical antioxidant N-acetylcysteine (NAC) as a bait reactant to measure NO transfer reactions in blood and to study the vascular effects of these reactions in vivo. NAC was converted to S-nitroso-N-acetylcysteine (SNOAC), decreasing erythrocytic S-nitrosothiol content, both during whole-blood deoxygenation ex vivo and during a 3-week protocol in which mice received high-dose NAC in vivo. Strikingly, the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia. Moreover, systemic SNOAC administration recapitulated effects of both NAC and hypoxia. eNOS-deficient mice were protected from the effects of NAC but not SNOAC, suggesting that conversion of NAC to SNOAC was necessary for the development of PAH. These data reveal an unanticipated adverse effect of chronic NAC administration and introduce a new animal model of PAH. Moreover, evidence that conversion of NAC to SNOAC during blood deoxygenation is necessary for the development of PAH in this model challenges conventional views of oxygen sensing and of NO signaling.