Perspective on LC-MS(/MS) for biotherapeutic and biomarker proteins in research and regulated Bioanalysis: a consolidation of more than a decade of experience across the European Bioanalysis Forum community (Part 1: "The What").

Following up on our most recent discussion paper focusing on the continued regulatory challenges for bioanalysis of biotherapeutic and biomarker proteins with LC-MS/MS, the European Bioanalysis Forum reports back on their internal discussions on and experience with method development for biotherapeutic and biomarker proteins in research and regulated bioanalysis. Due to the broad array of topics discussed, this information is spread over two research papers, where one focusses on the fundamental principles on which the technology is built (i.e.

Transporter tandems: precise tools for normalizing active transporter in the plasma membrane.

The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC-MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious.

Bypassing nonparallelism of a monoclonal antibody ligand-binding assay by employment of alternative assay formats.

Determination of concentration-time profiles in cynomolgus monkeys of a therapeutic monoclonal antibody against a soluble target revealed a substantial discrepancy between a generic anti-human IgG capture/detection and target bridging assay with the target bridging assay leading to dose- and time-dependent underquantification of drug concentrations, lack of parallelism and subsequently different pharmacokinetic parameters. In contrast, plasma levels derived from a target capture and an anti-idiotypic antibody bridging assay were in close concordance with the generic assay and demonstrated parallelism with high precision across several dilutions. The results provide a practical attempt to overcome nonparallelism by employing alternative assay formats utilizing tailored assay reagent combinations in order to obtain unbiased pharmacokinetic data.

Determination of nifurtimox in dog plasma by stable-isotope dilution LC-MS/MS.

Background: Nifurtimox is a 5-nitrofuran derived antiprotozoal drug used to treat diseases caused by trypanosomes including Chagas' disease and sleeping sickness (African trypanosomiasis). Available methods for the determination of nifurtimox in plasma are tedious and of low sensitivity. For the first time, an isotope dilution HPLC/MS/MS method for the sensitive quantitation of nifurtimox down to 10.

Determination of riociguat and its major human metabolite M-1 in human plasma by stable-isotope dilution LCMS/MS.

Background: Riociguat (BAY 63-2521) is an oral NO-independent as well as NO-synergistic stimulator of soluble guanylate cyclase (sGC) for the treatment of pulmonary hypertension. BAY 60-4552 (M-1) is its pharmacologically active major metabolite. An isotope dilution LC-ESI-MS/MS method has been developed and validated for the simultaneous determination of riociguat and M-1 in lithium heparinized human plasma.

Pharmacokinetics of the soluble guanylate cyclase activator cinaciguat in individuals with hepatic impairment.

Cinaciguat is intended for use in patients with acute decompensated heart failure. The drug is eliminated predominantly via the liver and, therefore, the potential impact of hepatic impairment on cinaciguat pharmacokinetics needs to be determined. This nonrandomized, open-label, observational study investigated the pharmacokinetics of cinaciguat in individuals with mild (Child-Pugh A; n = 8) or moderate (Child-Pugh B; n = 8) hepatic impairment and matched healthy volunteers (n = 16).

In vitro and in vivo P-glycoprotein transport characteristics of rivaroxaban.

Rivaroxaban, an oral, direct factor Xa inhibitor, has a dual mode of elimination in humans, with two-thirds metabolized by the liver and one-third renally excreted unchanged. P-glycoprotein (P-gp) is known to be involved in the absorption, distribution, and excretion of drugs. To investigate whether rivaroxaban is a substrate of P-gp, the bidirectional flux of rivaroxaban across Caco-2, wild-type, and P-gp-overexpressing LLC-PK1 cells was investigated.

Volume to dissolve applied dose (VDAD) and apparent dissolution rate (ADR): tools to predict in vivo bioavailability from orally applied drug suspensions.

Low solubility of drug candidates generated in research contributes to their elimination during subsequent development due to insufficient oral bioavailability (BA) of crystalline compound. Therefore, the purpose of the study was to identify critical in vitro solubility and dissolution parameter that would predict critical in vivo dissolution by means of in vitro-in vivo correlation. Thermodynamic solubility and apparent dissolution rate (ADR) were determined using the shake-flask method and mini-flow-through-cell, respectively.

A novel PDE2A reporter cell line: characterization of the cellular activity of PDE inhibitors.

We report here the generation and pharmacological characterization of a phosphodiesterase 2A (PDE2A) reporter cell line. Human PDE2A was stably transfected in a parental cell line expressing the atrial natriuretic peptide (ANP) receptor and the cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the biosensor for intracellular cGMP. In this reporter cell line, cGMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel.

Characterization of substrates and inhibitors for the in vitro assessment of Bcrp mediated drug-drug interactions.

Purpose: In vitro assessment of drug candidates' affinity for multi-drug resistance proteins is of crucial importance for the prediction of in vivo pharmacokinetics and drug-drug interactions. To have well described experimental tools at hand, the objective of the study was to characterize substrates and inhibitors of Breast Cancer Resistance Protein (BCRP) and P-glycoprotein (P-gp).

Methods: Madin-Darbin canine kidney cells overexpressing mouse Bcrp (MDCKII-Bcrp) were incubated with various Bcrp substrates, or a mixture of substrate and inhibitor to either the apical (A) or basolateral (B) compartment of insert filter plates.

Activation of soluble guanylate cyclase reverses experimental pulmonary hypertension and vascular remodeling.

Background: Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3(-/-)) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling.

Methods And Results: Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system.