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The pharmaceutical industry is rapidly advancing toward new drug modalities, necessitating the development of advanced analytical strategies for effective, meaningful, and reliable assays. Hydrophilic Interaction Chromatography (HILIC) is a powerful technique for the analysis of polar analytes. Despite being a well-established technique, HILIC method development can be laborious owing to the multiple factors that affect the separation mechanism, such as the selection of stationary phase chemistry, mobile phase eluents, and optimization of column equilibration time. Herein, we introduce a new automated multicolumn and multi-eluent screening workflow that streamlines the development of new HILIC assays, circumventing the existing tedious 'hit-or-miss' approach. A total of 12 complementary columns packed with sub-2 µm fully porous and 2.7 µm superficially porous particles operated on readily available ultra-high pressure liquid chromatography (UHPLC) instrumentation across a diverse set of commercially available polar stationary phases were investigated. Different mobile phases with pH ranging from pH 3 to 9 were evaluated using different organic modifiers. The gradient and column re-equilibration were judiciously set to ensure a reliable assay screening framework that indicates promising conditions for subsequent method optimization to achieve resolution of challenging mixtures. This UHPLC screening system is coupled with a diode array and charged aerosol detectors (DAD, CAD and mass spectrometry) to ensure versatile detection for a variety of compounds. This fast-screening platform lays the foundation for a convenient generic workflow, accelerating the pace of HILIC method development and transfer across both academic and industrial sectors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Steroids can be used as biomarkers in clinical metabolomics and other fields related to human toxicology. This chemical group is known for its complexity, considering its number of isobaric compounds and the wide variety of phases I and II metabolic pathways that parent compounds can undergo. For a successful analysis of steroids in biological samples, liquid chromatography separation must be finely tuned. It is especially challenging for glucuronidated and sulfated steroids derivatives that bear polar heads and can be affected by non-specific adsorption. The benefits of a biphenyl stationary phase chemistry for the selectivity of the separation of steroids and their phase II metabolites and the extent to which nonspecific adsorption phenomena could degrade chromatographic performance were investigated. Replacing a conventional hardware by a passivated hardware allowed to considerably reduce peaks width and asymmetry of sulfated species. The addition of weak ion pairing agents in the mobile phase could also help to reduce non-specific adsorption but are detrimental to mass spectrometry detection. As confirmed by the successful detection of 52 steroids in plasma, the use of a biphenyl stationary phase complemented by a passivated column hardware is of great help for a successful biomedical analysis of steroids and their phase II metabolites., (© 2024 The Author(s). Journal of Separation Science published by Wiley‐VCH GmbH.)

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a new therapeutic approach for patients with peritoneal cancer. So far, most published studies investigated the administration of established cytostatic agents through PIPAC. This study aimed to evaluate the effect of PIPAC on two breakthrough anti-cancer agents, specifically anti-PD1 pembrolizumab, and anti-HER2 antibody-drug conjugate (ADC) - trastuzumab-deruxtecan. We conducted systematic analyses on samples of pembrolizumab and trastuzumab-deruxtecan at clinically relevant concentrations before and after PIPAC administration using an experimental setup of a hermetic container system, mimicking the abdominal cavity and using identical features as in clinical use. We utilized a range of chromatographic and spectroscopic techniques to explore potential alterations in the primary, secondary, and tertiary structures of the drugs, focusing on post-translational modifications resulting from the aerosolization. Our findings indicate that PIPAC did not compromise the integrity of tested biopharmaceuticals. The size variants of both drugs, assessed by size exclusion chromatography (SEC), remained unchanged. Reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC) revealed no significant differences in hydrophobicity variants, the average drug-to-antibody ratio (DAR), or DAR distribution before and after PIPAC treatment. Circular dichroism (CD) spectroscopy confirmed that the secondary and tertiary structures were preserved. While pembrolizumab showed no change in charge variants post-PIPAC, trastuzumab-deruxtecan exhibited a non-negligible change in the quantity of charge variants on the monoclonal antibody itself, while the payload remained unchanged. This shift could possibly be related to the metallic composition of the CapnoPen® device (made of nickel and chromium) used in PIPAC and for these experiments. Together, our results suggest that PIPAC does not alter the structure of pembrolizumab and trastuzumab-deruxtecan, paving the way for future clinical trials., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. The authors declare the following competing financial interests: Mateusz Imiołek is an employee of Waters (Milford, MA, USA), a manufacturer of chromatography systems and consumables. ACQUITY, UPLC, Empower and BioResolve are trademarks of Waters Technologies Corporation. Capnopen® is a trademark of Capno pharm GmbH. Milli-Q is a trademark of Merck KGaA. Keytruda is a trademark of Merck Sharp & Dohme, LLC. Enhertu is a trademark of Daiichi Sankyo Company Limited. Accutron is a trademark of Medtron AG. AdvanceBio is a trademark of Agilent Technologies Inc. MAbPac is a trademark of Thermo Fisher Scientific AG. Excel is a trademark of Microsoft Corporation. All other trademarks are the property of their respective owners., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The first step in the detection of testosterone (T) doping is to measure the urinary steroid profile for the athlete biological passport (ABP). To harmonise the analysis between anti-doping laboratories, urinary steroid profiling is parametrised in deep detail and shall be performed by gas chromatography hyphenated to mass spectrometry (GC-MS). However, due to its requirement for extensive sample preparation, alternatives to GC-MS are being actively pursued. The aim of this study was the evaluation of Ultra-High-Performance Supercritical Fluid Chromatography hyphenated to tandem Mass Spectrometry (UHPSFC-MS/MS) as an alternative for the quantification of endogenous urinary steroids. In this context, we developed a high throughput sample extraction method, followed by a novel UHPSFC-MS/MS method for the analysis of 10 endogenous urinary steroids which are relevant for doping control analysis. Depending on the steroid, the herein presented method is capable of quantification from 0.5 ng/mL up to 10 µg/mL. After validation, the applicability of the method was evaluated by analysing 132 authentic urine samples, which demonstrated results similar to classical GC-MS analysis. Steroid concentrations determined by UHPSFC-MS/MS were slightly overestimated in comparison with GC-MS, but the ratios had <10 % difference between the two methods. As the ABP considers the steroid ratios for passport evaluation, the herein presented method could be used for steroid profiling without reducing the sensitivity of the ABP. Thus, we would propose to consider UHPSFC-MS/MS as an alternative to GC-MS after more tests would have been performed to support our findings. Furthermore, we have also investigated the potential of this technology for sample purification prior to Isotope Ratio Mass Spectrometry (IRMS) for the differentiation between exogenous and endogenous origin of T and its metabolites. While the achieved separation was sufficient to purify urine samples for IRMS analysis in our proof-of-concept study, the instrumental parameters should be further refined for future use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Therapeutic oligonucleotides (ONs) commonly incorporate phosphorothioate (PS) modifications. These introduce chiral centers and generate ON diastereomers. The increasing number of ONs undergoing clinical trials and reaching the market has led to a growing interest to better characterize the ON diastereomer composition, especially for small interfering ribonucleic acids (siRNAs). In this study, and for the first time, we identify higher-order structures as the major cause of ON diastereomer separation in hydrophilic interaction chromatography (HILIC). We have used conformational predictions and melting profiles of several representative full-length ONs to first analyze ON folding and then run mass spectrometry and HILIC to underpin the link between their folding and diastereomer separation. On top, we show how one can either enhance or suppress diastereomer separation depending on chromatographic settings, such as column temperature, pore size, stationary phase, mobile-phase ionic strength, and organic modifier. This work will significantly facilitate future HILIC-based characterization of PS-containing ONs; e.g., enabling monitoring of batch-to-batch diastereomer distributions in full-length siRNAs, a complex task that is now for the first time shown as possible on this delicate class of therapeutic double-stranded ONs.

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.

The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare the following competing financial interests: Mateusz Imiołek, Colette Quinn, Abraham Finny, Matthew Lauber and Szabolcs Fekete are employees of Waters (Milford, MA, USA), a manufacturer of chromatography systems and consumables. BEH, XBridge, and IonHance are trademarks of Waters Technologies Corporation. Comirnaty is a trademark of BioNTech SE. Spikevax is a trademark of ModernaTx, Inc. DryLab is a trademark of Molnar, IMRE. Fusion QbD is a trademark of S-Matrix Corporation. ChromSwordAuto is a trademark of ChromSword. AutoChrom is a trademark of Advanced Chemistry Development, Inc. Yarra is a trademark of Phenomenex, Inc. NanoMate is a trademark of Advion, Inc. Sephacryl is a trademark of Cytiva Bioprocess R&D AB. All other marks are the property of their respective owner., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Online comprehensive two-dimensional liquid chromatography (online LC x LC) has become increasingly popular. Among the different chromatographic modes that can be combined, hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) are particularly interesting because they offer a high degree of orthogonality. However, this combination remains complex due to the incompatibility of the solvents in the two dimensions. To avoid this problem, it is possible to dilute the first dimension ( 1 D) effluent with (z dilution -1) volumes of a weaker solvent added to one volume of 1 D-effluent, where z dilution represents the extent to which the fraction volume has been multiplied. This can be done using either active solvent modulation technology or an additional pump, prior to the second dimension analysis. The objective of this study was to develop theoretical models to predict whether or not dilution can be effective, and, if so, what is the minimum z dilution value required. This approach is based on the calculation of the ratio (called x dilution ) between the peak standard deviation due to the injection process and the peak standard deviation in the absence of extra-column dispersion. x dilution was calculated from theoretical relationships and plotted as a function of z dilution , to predict the value required to obtain good peak shapes for the compound of interest. The maximum x dilution value was found to be of the order of 1 for chromatographically acceptable peak shapes. The proposed theoretical approach was experimentally validated on a number of representative small molecules and peptides. Agreement between experimental results and theoretical models was very high, especially for small molecules. Finally, it is shown that this approach helps to predict the most appropriate set of conditions in HILIC x RPLC, depending on the compounds to be separated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

This review provides an overview of the latest advancements and applications in multi-dimensional liquid chromatography coupled with mass spectrometry (mD-LC-MS), covering aspects such as inter-laboratory studies, digestion strategy, trapping column, and multi-level analysis. The shift from an offline to an online workflow reduces sample processing artifacts, analytical variability, analysis time, and the labor required for data acquisition. Over the past few years, this technique has demonstrated sufficient maturity for application across a diverse range of complex products. Moreover, there is potential for this strategy to evolve into an integrated process analytical technology tool for the real-time monitoring of monoclonal antibody quality. This review also identifies emerging trends, including its application to new modalities, the possibility of evaluating biological activity within the mD-LC set-up, and the consideration of multi-dimensional capillary electrophoresis as an alternative to mD-LC. As mD-LC-MS continues to evolve and integrate emerging trends, it holds the potential to shape the next generation of analytical tools, offering exciting possibilities for enhanced characterization and monitoring of complex biopharmaceutical products., (© 2024 The Authors. Journal of Separation Science published by Wiley-VCH GmbH.)

When therapeutic proteins are analysed under hydrophilic interaction liquid chromatography (HILIC) conditions, there is an inherent mismatch between the sample diluent (proteins must be solubilised in aqueous media) and the mobile phase, which is mostly composed of aprotic solvent (acetonitrile). This difference in eluent strength between sample diluent and mobile phase is responsible for severe analyte breakthrough and peak distortion. As demonstrated with therapeutic proteins of different sizes (insulin of 6 kDa, anakinra of 17 kDa and rituximab subunits of 25 and 50 kDa), only very small volumes of 0.1-0.2 µL can be injected without breakthrough effects, when performing rapid analysis on short HILIC columns of 20-50 mm, leading to poor sensitivity. In order to avoid the undesired effect of the strong sample diluent, a special injection program should be preferred. This consists in the addition and automatic injection of a defined volume of weak solvent (acetonitrile) along with the sample to increase retention factors during sample loading. Various injection programs were tested, including the addition of a pre-injection or post-injection or both (bracketed injection) of acetonitrile plugs. Several weak to strong injection solvent ratios of 1:1, 1:2, 1:4 and 1:10 were tested. Our work proves that the addition of a pre-plug solvent with a weak vs. strong injection solvent ratio of 1:10 is a valuable strategy to inject relatively large volumes of proteins in HILIC, regardless of column dimensions, thus maximising sensitivity. No peak deformation or breakthrough was observed under these conditions. However, it is important to note that peak broadening (40 % larger peaks) was observed when the injection program increased the injection solvent ratio from 1:1 to 1:10. Finally, this strategy was applied to a wide range of therapeutic mAb products with different physico-chemical properties. In all cases, relatively large volumes can be successfully injected onto small volume HILIC columns using a purely aqueous sample diluent, as long as an appropriate (weak) solvent pre-injection is applied., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Waters, ACQUITY, UPLC, Empower, and BEH are trademarks of Waters Technologies Corporation. Excel is a trademark of Microsoft Corporation. Statistica is a trademark of Cloud Software Group, Inc., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.