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Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl 2 ) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.

Charge heterogeneity among therapeutic monoclonal antibodies (mAbs) is considered an important critical quality attribute and requires careful characterization to ensure safe and efficacious drug products. The charge heterogeneity among mAbs is the result of chemical and enzymatic post-translational modifications and leads to the formation of acidic and basic variants that can be characterized using cation exchange chromatography (CEX). Recently, the use of mass spectrometry-compatible salt-mediated pH gradients has gained increased attention to elute the proteins from the charged stationary phase material. However, with the increasing antibody product complexity, more and more selectivity is required. Therefore, in this study, we set out to improve the selectivity by using a solvent-enriched mobile phase composition for the analysis of a variety of mAbs and bispecific antibody products. It was found that the addition of the solvents to the mobile phase appeared to modify the hydrate shell surrounding the protein and alter the retention behavior of the studied proteins. Therefore, this work demonstrates that the use of solvent-enriched mobile phase composition could be an attractive additional method parameter during method development in CEX.

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization.

The aim of the present work is to evaluate the possibilities and limitations of reversed hydrophilic interaction chromatography (revHILIC) mode in liquid chromatography (LC). This chromatographic mode consists of combining a highly polar stationary phase (bare silica) with a gradient varying from very low (1-5%) to high (40%) acetonitrile content (reversed gradient compared to HILIC). The retention behavior of revHILIC was first compared with that of reversed-phase LC (RPLC) and HILIC using representative mixtures of peptides and pharmaceutical compounds. It appears that the achievable selectivity can be ranked in the order RPLC > revHILIC > HILIC with the two different samples. Next, two-dimensional liquid chromatography (2D-LC) conditions were evaluated by combining RPLC, revHILIC, or HILIC with RPLC in an on-line comprehensive (LC × LC) mode. evHILIC × RPLC not only showed impressive performance in terms of peak capacity and sensitivity, but also provided complementary selectivity compared to RPLC × RPLC and HILIC × RPLC. Indeed, both the elution order and the retention time range differ significantly between the three techniques. In conclusion, there is no doubt that revHILIC should be considered as a viable option for 2D-LC analysis of small molecules and also peptides.

The objective of this work was to provide an unbiased comparison of one-dimensional reversed-phase liquid chromatography (1D-RPLC) and comprehensive two-dimensional RPLC (RPLC × RPLC), through calculations and experimental verifications. For this purpose, various quality descriptors were evaluated, including peak capacity, analysis time, dilution factor, number of runs in the second dimension, and injection volume. The same strategy was applied to small pharmaceuticals and peptides. Whatever the analysis time between 30 and 200 min, short columns of only 30 × 2.1 mm packed with sub-2-µm particles should be selected in both dimensions of the 2D-LC setup to obtain the best compromise in terms of peak capacity and sensitivity. The peak capacity in RPLC × RPLC vs. RPLC was significantly improved for analysis times beyond 5 min. However, extra-column volume located after the second-dimension column was found to be particularly critical for peptides, and up to 50% lower peak capacity was observed with MS vs. UV detection. Contrary to common belief, higher dilution is not always observed in RPLC × RPLC. With adequate analytical conditions, better sensitivity (in theory fivefold and in practice three- to fivefold) could be achieved in RPLC × RPLC compared to 1D-RPLC, regardless of the analysis time., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC-MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC-MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC-MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC-MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC-MS platforms for high-throughput determination of major CQAs in a regulated environment.

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.

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, 2.7 µm particle column. However, compared to acetonitrile, the selectivity was not improved, at least for the selected biopharmaceutical products. Importantly, very fast separations (sub-1 min) of high quality were systematically obtained for the different samples when using a spectroscopic detector, but a severe loss of performance was observed with mass spectrometry (MS) detection due to dispersion effects. Based on these results, there is a clear need to improve the interfacing between LC and MS (shorter/thinner tubing) to mitigate band broadening.

Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids' full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an "on/off" retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method.

Despite many promising results obtained in previous preclinical studies, the clinical development of encapsulated cell technology (ECT) for the delivery of therapeutic proteins from macrocapsules is still limited, mainly due to the lack of an allogeneic cell line compatible with therapeutic application in humans. In our work, we generated an immortalized human myoblast cell line specifically tailored for macroencapsulation. In the present report, we characterized the immortalized myoblasts and described the engineering process required for the delivery of functional therapeutic proteins including a cytokine, monoclonal antibodies and a viral antigen. We observed that, when encapsulated, the novel myoblast cell line can be efficiently frozen, stored, and thawed, which limits the challenge imposed by the manufacture and supply of encapsulated cell-based therapeutic products. Our results suggest that this versatile allogeneic cell line represents the next step toward a broader development and therapeutic use of ECT., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. E.C., A.E., and J.G. are employees of MaxiVAX SA. A.L. is a former employee of MaxiVAX SA and a current scientific advisor. N.M is founder and minority shareholder of MaxiVAX SA. MaxiVAX SA is a Geneva-based biotech company involved in cell encapsulation technology., (© 2022 The Authors.)

Bioprocess development of increasingly challenging therapeutics and vaccines requires a commensurate level of analytical innovation to deliver critical assays across functional areas. Chromatography hyphenated to numerous choices of detection has undeniably been the preferred analytical tool in the pharmaceutical industry for decades to analyze and isolate targets (e.g., APIs, intermediates, and byproducts) from multicomponent mixtures. Among many techniques, ion exchange chromatography (IEX) is widely used for the analysis and purification of biopharmaceuticals due to its unique selectivity that delivers distinctive chromatographic profiles compared to other separation modes (e.g., RPLC, HILIC, and SFC) without denaturing protein targets upon isolation process. However, IEX method development is still considered one of the most challenging and laborious approaches due to the many variables involved such as elution mechanism (via salt, pH, or salt-mediated-pH gradients), stationary phase's properties (positively or negatively charged; strong or weak ion exchanger), buffer type and ionic strength as well as pH choices. Herein, we introduce a new framework consisting of a multicolumn IEX screening in conjunction with computer-assisted simulation for efficient method development and purification of biopharmaceuticals. The screening component integrates a total of 12 different columns and 24 mobile phases that are sequentially operated in a straightforward automated fashion for both cation and anion exchange modes (CEX and AEX, respectively). Optimal and robust operating conditions are achieved via computer-assisted simulation using readily available software (ACD Laboratories/LC Simulator), showcasing differences between experimental and simulated retention times of less than 0.5%. In addition, automated fraction collection is also incorporated into this framework, illustrating the practicality and ease of use in the context of separation, analysis, and purification of nucleotides, peptides, and proteins. Finally, we provide examples of the use of this IEX screening as a framework to identify efficient first dimension ( 1 D) conditions that are combined with MS-friendly RPLC conditions in the second dimension ( 2 D) for two-dimensional liquid chromatography experiments enabling purity analysis and identification of pharmaceutical targets., (© 2022. Merck & Co., Inc., Kenilworth, NJ, USA and its affiliates and Davy Guillarme under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Untargeted metabolomics is now widely recognized as a useful tool for exploring metabolic changes taking place in biological systems under different conditions. In this article, we aim to provide a short overview of the liquid-phase separation methods hyphenated to MS to perform untargeted metabolomics of biological samples. Each approach is complemented by up-to-date literature to guide readers, as well as practical information for avoiding or fixing some of the most frequently encountered pitfalls. This article covers mainly data acquisition, but a short discussion is provided regarding signal processing and data treatment, as well as data analysis and its biological interpretation in the context of metabolomic studies., (Copyright 2022 Serge Rudaz, Elena Tobolkina, Víctor González-Ruiz, Isabel Meister, Miguel De Figueiredo, Davy Guillarme, Julien Boccard. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N -glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade ® ) was assessed and compared to two of its commercially available biosimilars (Remsima ® and Inflectra ® ). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm Astrocaryum sciophilum . The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as ( R )-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pellissier, Koval, Marcourt, Ferreira Queiroz, Lecoultre, Leoni, Quiros-Guerrero, Barthélémy, Duivelshof, Guillarme, Tardy, Eparvier, Perron, Chave, Stien, Gindro, Katanaev and Wolfender.)

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

The aim of this study was to assess the interlaboratory reproducibility of ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry method for routine antidoping analyses. To do so, a set of 21 doping agents, spiked in urine and analyzed after dilute and shoot treatment, was used to assess the variability of their retention times between four different laboratories, all equipped with the same chromatographic system and with the same ultra-high performance supercritical fluid chromatography stationary phase chemistry. The average relative standard deviations (RSD%) demonstrated a good reproducibility of the retention times for 19 out of 21 analytes, with RSD% values below 3.0%. Only for two substances, namely fenbutrazate and niketamide, the retention was not repeatable between laboratories, with RSD% of approximately 15% in both cases. This behaviour was associated with (a) the low organic modifier percentage (around 2-4%) in the mobile phase at the corresponding retention times, and (b) the influence of the system volume on poorly retained analytes. An analysis on seven "blind" urines was subsequently carried out in the same four laboratories. In these blind samples, either one, two, or none of the 21 doping agents previously analyzed were present at an unknown concentration. Each laboratory had to perform the identification of the compounds in the samples and estimate their concentrations. All laboratories assigned all target analytes correctly in all blind urine samples and provide a comparable estimation of their concentrations., (© 2020 The Authors. Analytical Science Advances published by Wiley‐VCH GmbH.)

Competing Interests: The authors declare that there is no conflict of interest.

Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is a powerful and essential technique for metabolite annotation in untargeted metabolomic applications. The aim of this study was to evaluate the performance of diverse tandem MS (MS/MS) acquisition modes, i.e., all ion fragmentation (AIF) and data-dependent analysis (DDA), with and without ion mobility spectrometry (IM), to annotate metabolites in human plasma. The influence of the LC separation was also evaluated by comparing the performance of MS/MS acquisition in combination with three complementary chromatographic separation modes: reversed-phase chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) with either an amide (aHILIC) or a zwitterionic (zHILIC) stationary phase. RPLC conditions were first chosen to investigate all the tandem MS modes, and we found out that DDA did not provide a significant additional amount of chemical coverage and that cleaner MS/MS spectra can be obtained by performing AIF acquisitions in combination with IM. Finally, we were able to annotate 338 unique metabolites and demonstrated that zHILIC was a powerful complementary approach to both the RPLC and aHILIC chromatographic modes. Moreover, a better analytical throughput was reached for an almost negligible loss of metabolite coverage when IM-AIF and AIF using ramped instead of fixed collision energies were used.

Biosimilars marketing authorization requires a strict demonstration of similarity with the reference antibody, through preclinical and clinical studies. This article reviews the panel of in vitro physicochemical and functional analyses, which are performed prior to clinical studies. For each critical attribute of the antibody, we detail the commonly used analytical techniques, their working principle and the type of information they may give., (© 2019 médecine/sciences – Inserm.)

In the present analytical workflow, chromatographic methods have been developed and hyphenated to mass spectrometry (MS) for the characterization of protein size, charge, hydrophobic, and hydrophilic variants of daratumumab. Multiple critical quality attributes (CQAs) were characterized in forced degraded daratumumab sample, using size exclusion, ion exchange (IEX), and hydrophobic interaction (HIC) chromatography coupled to fluorescence detection for relative quantification and fractionation. Mass assignment was performed by using a fast, non-denaturing and universal size exclusion chromatography (SEC) method prior to native MS analysis of the collected fractions (off-line approach). This allowed the identification of N-terminal lysine clipping, and the extent of glycation and oxidation at intact protein level. Finally, middle-up analysis of daratumumab was performed using reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography coupled to MS to obtain a comprehensive overview of all PTMs after the forced stressed conditions and a fine characterization of the glycosylation profile. Conveniently, the presented workflow maintains the established golden standard non-denaturing chromatography techniques and additionally introduces a straightforward and automated desalting procedure prior to MS analysis. Therefore, it is expected that the off-line coupling of SEC, IEX, and HIC to SEC-MS has great potential to be implemented in routine characterization of mAbs. Graphical abstract ᅟ.

As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.

Multi-target screening of biological fluids is a key tool in clinical and forensic toxicology. A complete toxicological analysis encompasses the sample preparation, the chromatographic separation and the detection. The present review briefly covers the new trends in sample preparation and detection and mainly focuses on the chromatographic stage, since a lot of technical improvements have been proposed over the last years. Among them, columns packed with sub-2 μm fully porous particles and sub-3 μm core-shell particles allow for significant improvements of resolution and higher throughput. Even if reversed-phase LC remains the most widely used chromatographic mode for toxicological screening, hydrophilic interaction chromatography and supercritical fluid chromatography appear as promising alternatives for attaining orthogonal selectivity, retention of polar compounds, and enhanced MS sensitivity.

Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ~250 Å and macropores of ~ 1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ~300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene-divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.

Since the first anti-doping tests in the 1960s, the analytical aspects of the testing remain challenging. The evolution of the analytical process in doping control is discussed in this paper with a particular emphasis on separation techniques, such as gas chromatography and liquid chromatography. These approaches are improving in parallel with the requirements of increasing sensitivity and selectivity for detecting prohibited substances in biological samples from athletes. Moreover, fast analyses are mandatory to deal with the growing number of doping control samples and the short response time required during particular sport events. Recent developments in mass spectrometry and the expansion of accurate mass determination has improved anti-doping strategies with the possibility of using elemental composition and isotope patterns for structural identification. These techniques must be able to distinguish equivocally between negative and suspicious samples with no false-negative or false-positive results. Therefore, high degree of reliability must be reached for the identification of major metabolites corresponding to suspected analytes. Along with current trends in pharmaceutical industry the analysis of proteins and peptides remains an important issue in doping control. Sophisticated analytical tools are still mandatory to improve their distinction from endogenous analogs. Finally, indirect approaches will be discussed in the context of anti-doping, in which recent advances are aimed to examine the biological response of a doping agent in a holistic way., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MS(E) with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MS(E) analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.

Recent developments in chromatographic supports and instrumentation for liquid chromatography (LC) are enabling rapid and highly efficient separations. Various analytical strategies have been proposed, for example the use of silica-based monolithic supports, elevated mobile phase temperatures, and columns packed with sub-3 microm superficially porous particles (fused core) or with sub-2 microm porous particles for use in ultra-high-pressure LC (UHPLC). The purpose of this review is to describe and compare these approaches in terms of throughput and resolving power, using kinetic data gathered for compounds with molecular weights ranging between 200 and 1300 g mol(-1) in isocratic and gradient modes. This study demonstrates that the best analytical strategy should be selected on the basis of the analytical problem (e.g., isocratic vs. gradient, throughput vs. efficiency) and the properties of the analyte. UHPLC and fused-core technologies are quite promising for small-molecular-weight compounds, but increasing the mobile phase temperature is useful for larger molecules, for example peptides.

Ultra-high-pressure liquid chromatography (UHPLC) systems able to work with columns packed with sub-2 microm particles offer very fast methods to determine the lipophilicity of new chemical entities. The careful development of the most suitable experimental conditions presented here will help medicinal chemists for high-throughput screening (HTS) log P(oct) measurements. The approach was optimized using a well-balanced set of 38 model compounds and a series of 28 basic compounds such as beta-blockers, local anesthetics, piperazines, clonidine, and derivatives. Different organic modifiers and hybrid stationary phases packed with 1.7-microm particles were evaluated in isocratic as well as gradient modes, and the advantages and limitations of tested conditions pointed out. The UHPLC approach offered a significant enhancement over the classical HPLC methods, by a factor 50 in the lipophilicity determination throughput. The hyphenation of UHPLC with MS detection allowed a further increase in the throughput. Data and results reported herein prove that the UHPLC-MS method can represent a progress in the HTS-measurement of lipophilicity due to its speed (at least a factor of 500 with respect to HPLC approaches) and to an extended field of application.

The measurement of physicochemical properties at an early phase of drug discovery and development is crucial to reduce attrition rates due to poor biopharmaceutical properties. Among these properties, ionization, lipophilicity, solubility and permeability are mandatory to predict the pharmacokinetic behavior of NCEs (new chemical entities). Due to the high number of NCEs, the analytical tools used to measure these properties are automated and progressively adapted to high-throughput technologies. The present review is dedicated to experimental methods applied in the early drug discovery process for the determination of solubility, ionization constants, lipophilicity and permeability of small molecules. The principles and experimental conditions of the different methods are described, and important enhancements in terms of throughput are highlighted.