Highly sensitive ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method for the multiplex analysis of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma.

Levosimendan is a positive inotrope and vasodilator used in patients with acute and chronic decompensated heart failure. It is metabolized into OR-1855 (inactive metabolite), which is further acetylated into OR-1896 (active metabolite having a prolonged half-life, hence a sustained effect). Levosimendan represents a valuable alternative to traditional inotropes with broad clinical applications in critically ill patients with cardiogenic shock, advanced heart failure and post-cardiac surgery.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods for the therapeutic drug monitoring of cytotoxic anticancer drugs: An update.

In the era of precision medicine, there is increasing evidence that conventional cytotoxic agents may be suitable candidates for therapeutic drug monitoring (TDM)- guided drug dosage adjustments and patient's tailored personalization of non-selective chemotherapies. To that end, many liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assays have been developed for the quantification of conventional cytotoxic anticancer chemotherapies, that have been comprehensively and critically reviewed. The use of stable isotopically labelled internal standards (IS) of cytotoxic drugs was strikingly uncommon, accounting for only 48 % of the methods found, although their use could possible to suitably circumvent patients' samples matrix effects variability.

Ultra-Fast Middle-Up Reversed Phase Liquid Chromatography Analysis of Complex Bispecific Antibodies Obtained in Less Than One Minute.

This work illustrates the benefits and limitations of using ultra-short reversed phase liquid chromatography (RPLC) columns for the characterization of various complex bispecific antibodies after prolonged thermal stress at the middle-up level of analysis. First, we have demonstrated that alternative organic modifiers, such as isopropanol, can be used in RPLC mode without generating excessive pressure, thanks to the prototype 10 × 2.1 mm, 2.

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products.

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic-inorganic silica surfaces called hybrid surface technology.

The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry.

The goal of the study was to evaluate the possibilities offered by a new generation of metal-free SEC column to perform direct SEC-MS of protein biopharmaceuticals using ammonium acetate as the main mobile phase additive. The prototype metal-free SEC column hardware used in this work was a polyether ether ketone (PEEK) infused stainless steel tube including PEEK frits. This PEEK-lined column provides a fully bioinert and metal-free fluidic path, while maintaining the stability of the metal hardware, and could be a good solution to limit possible undesired interactions between proteins and column wall/frits.

Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins.

The purpose of this work was to study the potential of recently developed ultra-short column hardware for ion exchange chromatography (IEX). Various prototype and commercial columns having lengths of 5, 10, 15, 20 and 50 mm and packed with non-porous 3 µm particles were systematically compared. Both pH and salt gradient modes of elution were evaluated.

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies.

This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested.

Empirical correction of non-linear pH gradients and a tool for application to protein ion exchange chromatography.

This concept article reports a practical solution to improve the linearity of effluent pH response as observed in pH gradient cation exchange chromatography (CEX). When performing pH gradient CEX, it is not easy to develop buffer systems that will universally provide pH response proportional with the mobile phase (buffer) composition. It is an especially challenging pursuit when exploring MS compatible buffers (e.

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems.

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.

Aptamer-based immunoaffinity LC-MS using an ultra-short column for rapid attomole level quantitation of intact mAbs.

Quantification of proteins in biofluids has largely involved either traditional ligand binding assays or "bottom-up" mass spectrometry. Recently, top-down mass spectrometry using reversed-phase liquid chromatography (RPLC) paired with high-resolution mass spectrometry (HRMS) has emerged as a promising technique, due to the potential of better identification of post-translational modifications (PTMs), lack of downstream interferences, and less time-consuming sample preparation and analysis times. However, it can be difficult with this approach to robustly obtain high-fidelity MS data, especially when pushing for low limits of detection.

New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies.

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH).

Use of Ultra-short Columns for Therapeutic Protein Separations, Part 2: Designing the Optimal Column Dimension for Reversed-Phase Liquid Chromatography.

In the first part of the series, it was demonstrated that very fast (<30 s) separations of therapeutic protein species are feasible using ultra-short (5 × 2.1 mm) columns. In the second part, our purpose was to find the appropriate column length; therefore, a systematic study was performed using various custom-made prototype reversed-phase liquid chromatography (RPLC) columns ranging from 2 to 50 mm lengths.

Negative gradient slope methods to improve the separation of closely eluting proteins.

In the present work, we describe the fundamental and practical advantages of a new strategy to improve the resolution of very closely eluting peaks within therapeutic protein samples. This approach involves the use of multiple isocratic steps, together with the addition of a steep negative gradient segment (with a decrease in mobile phase strength) to "park" a slightly more retained peak somewhere along the column (at a given migration distance), while a slightly less retained compound can be eluted. First, some model calculations were performed to highlight the potential of this innovative approach.

Therapeutic Fc-fusion proteins: Current analytical strategies.

Fc-Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc-Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps.

Determination of size variants by CE-SDS for approved therapeutic antibodies: Key implications of subclasses and light chain specificities.

In the present work, a generic non-reducing capillary electrophoresis sodium dodecyl sulphate (nrCE-SDS) method was tested for a wide range of 26 FDA and EMA approved monoclonal antibodies (mAbs) and 2 antibody drug conjugates (ADCs) as well as for the NISTmab, in a QC environment (e.g. testing quality requirements for batch manufacturing or batch release).

Practical considerations on the particle size and permeability of ion-exchange columns applied to biopharmaceutical separations.

The goal of this study was to better understand the possibilities and limitations of modern cation exchange chromatography (CEX) columns for the separation of protein biopharmaceuticals (typically mAbs and related products). Several commercial and research columns consisting of a non-porous polymeric core particle with a thin hydrophilic coating and grafted ion-exchanger sulfonate groups, were compared. The impact of particle size, porosity and packing pressure on the separation of therapeutic proteins was evaluated in a systematic way.

Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 2: Evaluation of recent stationary phases.

In this second part of the series, recently commercialized cation exchanger stationary phases were systematically investigated for their capabilities to separate therapeutic monoclonal antibodies. It was demonstrated that the different combinations of stationary and mobile phases result in diverse retention, selectivity and efficiency. Hence, the whole phase system (combination of stationary and mobile phase) should be considered when developing a method.

Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation.

Cation exchange chromatography (CEX) of therapeutic monoclonal antibodies is generally performed with either salt gradient (MES buffer + NaCl) or using commercial pH gradient buffer. The goal of this study was to find out some alternative buffer systems for CEX separation of mAbs, which may offer alternative selectivity, while maintaining similar peak shapes. Among the new buffers that were tested, (N-morpholino)ethanesulfonic acid (MES) / 1,3-diamino-2-propanol (DAP), and citric acid / 2-(cyclohexylamino)ethanesulfonic acid (CHES) systems were particularly promising, especially when combining them with a moderate salt gradient of NaCl.