The rapid development of mRNA-based therapeutics, especially post-COVID-19, has necessitated the precise characterization of mRNA quality attributes, including sequence integrity. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) has been widely accepted as a reference method for the characterization of small oligonucleotides. Some studies have already investigated the use of IP-RPLC for RNA, but no systematic approach has been developed to assess the impact of ion-pairing agents (IPAs) on the separation of large RNA molecules.
View Article and Find Full Text PDFThe isolation of pure compounds from extracts represents a key step common to all investigations of natural product (NP) research. Isolation methods have gone through a remarkable evolution. Current approaches combine powerful metabolite profiling methods for compounds annotation with omics mining results and/or bioassay for bioactive NPs/biomarkers priorisation.
View Article and Find Full Text PDFIon-pair reversed-phase liquid chromatography (IP-RPLC) is clearly recognized as the gold standard for analyzing therapeutic oligonucleotides (ONs). Recent studies have shown that ONs exhibit an on-off retention behavior in IP-RPLC, meaning that minor changes in acetonitrile (ACN) proportion can significantly impact retention. However, this behavior was initially demonstrated with only a single mobile phase condition.
View Article and Find Full Text PDFThis study presents a systematic approach for developing an innovative hydrophilic interaction liquid chromatography (HILIC) method for collagen peptide mapping analysis. The predominant post-translational modification (PTM) of collagen, proline hydroxylation, introduces polar hydroxyl groups throughout the collagen sequence, making HILIC a promising alternative to classical reversed-phase liquid chromatography (RPLC) approaches. This study employs sixteen model peptides, selected from in silico predicted tryptic peptides with zero missed cleavages and representing diverse physicochemical properties and structural motifs of collagen.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFPressurized 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.
View Article and Find Full Text PDFSteroids 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.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFBiopharmaceutical 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.
View Article and Find Full Text PDFTherapeutic 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).
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFThis 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.
View Article and Find Full Text PDFOnline 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.
View Article and Find Full Text PDFCharge 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.
View Article and Find Full Text PDFWhen 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.
View Article and Find Full Text PDFThe impact of injected sample volume on apparent efficiency has been studied for very short columns in a systematic way. For large molecules such as therapeutic proteins, it was found that relatively large volumes can be injected onto ultra-short RPLC and IEX columns (i.e.
View Article and Find Full Text PDFJ Chromatogr A
September 2023
Today, reverse phase liquid chromatography (RPLC) analysis of proteins is almost exclusively performed on conventional columns (100-150 mm) in gradient elution mode. However, it was shown many years ago that large molecules present an on/off retention mechanism, and that only a very short inlet segment of the chromatographic column retains effectively the large molecules. Much shorter columns - like only a few centimetres or even a few millimetres - can therefore be used to efficiently analyse such macromolecules.
View Article and Find Full Text PDFMonoclonal antibody (mAb)-based therapies have been a major advance in oncology patient care, even though they represent a significant healthcare cost. Biosimilars, launched in Europe in 2004 are an economically attractive alternative to expensive originator biological drugs. They also increase the competitiveness of pharmaceutical development.
View Article and Find Full Text PDFIon-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFThe 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.
View Article and Find Full Text PDFMonitoring the central carbon metabolism (CCM) network using liquid chromatography/mass spectrometry (LC-MS) analysis is hampered by the diverse chemical nature of its analytes, which are extremely difficult to analyze using single chromatographic conditions. Furthermore, CCM-related compounds present non-specific adsorption on metal surfaces, causing detrimental chromatographic effects and sensitivity loss. In this study, polar reversed-phase, mixed-mode (MMC), and zwitterionic hydrophilic interaction chromatography (HILIC) featuring low-adsorption hardware were investigated towards untargeted analysis of biological samples with a focus on energy metabolism-related analytes.
View Article and Find Full Text PDFIn 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.
View Article and Find Full Text PDFIn 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.
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