Multi-dimensional technology - Recent advances and applications for biotherapeutic characterization.

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.

Enhancing Selectivity of Protein Biopharmaceuticals in Ion Exchange Chromatography through Addition of Organic Modifiers.

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.

Erratum: Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

[This corrects the article DOI: 10.1016/j.omtm.2022.07.017.].

Development of setups for real-time analysis of the effluent of a microreactor by mass spectrometry.

Monitoring a synthesis reaction in real time could allow not only the detection of the intermediates involved in the synthesis, to better understand its mechanisms, but also the impurities. Spectroscopic methods could be performed but are not so performant when analyzing complex mixtures and could require specific properties for the detection of the molecules of interest, the presence of a chromophore moiety for example. Mass spectrometry (MS) may overcome these limitations and is able to reach the accuracy and sensitivity required to efficiently detect, quantify, identify, and characterize the reagents and species produced during the synthesis. This is why the hyphenation of a microreactor with MS has already allowed synthesis processes to be monitored, but most of the time it targets a specific reaction or compounds and involves solvents compatible with MS. In this study, a universal setup for the hyphenation of a microreactor with MS and based on two valves has been developed. This two-valve setup has proven itself for the analysis of molecules of different nature and hydrophilicity, soluble in a large number of solvents even in non-MS-compatible ones. The developed setup evidenced a good repeatability and a linear response for the detection of the studied compounds. In addition, the dilution step included in the two-valve setup allows the MS monitoring of compounds initially synthesized at different concentrations. Finally, it was successfully used to study an amination reaction allowing the detection of the reaction products in 4 min with good repeatability as RSD values of MS signals were lower than 17%.

Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches.

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.

Tackling Issues Observed during the Development of a Liquid Chromatography Method for Small Molecule Quantification in Antibody-Chelator Conjugate.

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.

Extending the performance of FcRn and FcγRIIIa affinity liquid chromatography for protein biopharmaceuticals.

Affinity liquid chromatography using FcRn and FcγRIIIa columns can provide important information on the drug effector functions and the unique PK/PD properties of therapeutic mAbs. In this study, we propose a unique strategy to improve the performance of affinity chromatography by applying pH-gradient programs that incorporate multi-isocratic and negative gradient segments. These alternative gradient programs are known to greatly improve the separation of large solutes that follow a "bind-and-elute" type retention behavior. First, judicious optimization of the mobile phase compositions was performed to obtain a linear pH response. Then, with the developed strategy using multi-isocratic analysis conditions, the FcRn affinity separation selectivity for the analysis of oxidized mAb species was greatly improved. Furthermore, the introduction of negative gradient segments after each eluted peak improved the resolution between multiple glycosylated mAb species on the FcγRIIIa column. Therefore, this work provides a new strategy to improve the performance of affinity chromatography with mAb species, and could assist in the development of more accurate binding assays for important critical quality attributes related to FcRn and FcγRIIIa binding.

Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

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.

A simple mathematical treatment for predicting linear solvent strength behavior in gradient elution: Application to biomolecules.

This paper describes an approach to rapidly and easily calculate the linear solvent strength parameters, namely log k0 and S, under reversed-phase liquid chromatography conditions. This approach, which requires two preliminary gradient experiments to determine the retention parameters, was applied to various representative compounds including small molecules, peptides, and proteins. The retention time prediction errors were compared to the ones obtained with a commercial HPLC modeling software, and a good correlation was found between the values. However, two important constraints have to be accounted for to maintain good predictions with this new approach: i) the retention factor at the initial composition of the preliminary gradient series have to be large enough (i.e., log ki above 2.1) and ii) the retention models have to be sufficiently linear. While these two conditions are not always met with small molecules or even peptides, the situation is different with large biomolecules. This is why our simple calculation method should be preferentially applied to calculate the linear solvent strength parameters of protein samples.

Simplified miniaturized analytical set-up based on molecularly imprinted polymer directly coupled to UV detection for the determination of benzoylecgonine in urine.

A simple, selective, and sensitive method involving a miniaturized solid phase extraction step based on a monolithic molecularly imprinted polymer (MIP) directly coupled on-line to UV detection was developed for the determination of benzoylecgonine (BZE) in complex biological samples. Monolithic MIPs were prepared into 100 µm internal diameter fused-silica capillaries either by thermal or photopolymerization. While leading to similar selectivities with respect to BZE, photopolymerization has made it possible to produce monoliths of different lengths that can be adapted to the targeted miniaturized application. The homogeneous morphology of these monolithic MIPs was evaluated by scanning electron microscopy prior to measuring their permeability. Their selectivity was evaluated leading to imprinting factors of 2.7 ± 0.1 for BZE and 4.0 ± 0.6 for cocaine (selected as template for the MIP synthesis) with polymers resulting from three independent syntheses, showing both the high selectivity of the MIPs and the reproducibility of their synthesis. After selecting the appropriate capillary length and the set-up configuration and optimizing the extraction protocol to promote selectivity, the extraction of BZE present in human urine samples spiked at 150, 250, and 500 ng mL-1 was successfully carried out on the monolithic MIP and coupled directly on-line with UV detection. The very clean-baseline of the resulting chromatograms revealing only the peak of interest for BZE illustrated the high selectivity brought by the monolithic MIP. Limits of detection and quantification of 56.4 ng mL-1 and 188.0 ng mL-1 were achieved in urine samples, respectively. It is therefore possible to achieve analytical threshold in accordance with the legislation on BZE detection in urine without the need for an additional chromatographic separation.

Molecularly imprinted polymers in miniaturized extraction and separation devices.

Molecularly imprinted polymers are highly selective and cost-effective materials, which have attracted significant interest in various areas such as sample pretreatment and chromatographic and electrophoretic separations. This review aims to present the state of the art concerning the miniaturization of these materials in order to meet the societal demand for reliable, fast, cheap, and solvent/sample saving analyses. The polymerization route specificities for the production of miniaturized molecularly imprinted polymers in capillaries or chip channels, such as open tubular, packed particles, magnetic nanoparticles, and in situ imprinted monoliths, are investigated. Their performances as selective supports in solid phase extraction and as stationary phases in electrochromatography and liquid chromatography, as well as their possible perspectives are discussed.

Diversity-oriented synthesis of 17-spirosteroids.

A diversity-oriented synthesis (DOS) approach has been used to functionalize 17-ethynyl-17-hydroxysteroids through a one-pot procedure involving a ring-closing enyne metathesis (RCEYM) and a Diels-Alder reaction on the resulting diene, under microwave irradiations. Taking advantage of the propargyl alcohol moiety present on commercially available steroids, this classical strategy was applied to mestranol and lynestrenol, giving a collection of new complex 17-spirosteroids.

Selective extraction of cocaine from biological samples with a miniaturized monolithic molecularly imprinted polymer and on-line analysis in nano-liquid chromatography.

We report the on-line coupling of a monolithic molecularly imprinted polymer to nano-liquid chromatography for the selective analysis of cocaine and its main metabolite, benzoylecgonine, in complex biological samples. After the screening of different synthesis conditions, a monolithic molecularly imprinted polymer was in situ synthesized into a 100 µm internal diameter fused-silica capillary using cocaine as template, methacrylic acid as functional monomer, and trimethylolpropane trimethacrylate as cross-linker. Scanning electron microscopy was used to assess the homogeneous morphology of the molecularly imprinted polymer and its permeability was measured. Its selectivity was evaluated by nano-liquid chromatography-ultraviolet, leading to imprinting factors of 3.2 ±â€¯0.5 and 2.2 ±â€¯0.3 for cocaine and benzoylecgonine, respectively, on polymers resulting from three independent syntheses, showing the high selectivity and the repeatability of the synthesis. After optimizing the extraction protocol to promote selectivity, the monolithic molecularly imprinted polymer was successfully on-line coupled with nano-liquid chromatography-ultraviolet for the direct extraction and analysis of cocaine present in spiked human plasma and saliva samples. The repeatability of the obtained extraction recovery, between 85.4 and 98.7% for a plasma sample spiked at 100 ng mL-1, was high with relative standard deviation values lower than 5.8% for triplicate analyses on each of the three independently synthesized molecularly imprinted polymers. A linear calibration range was achieved between 100 and 2000 ng mL-1 (R2 = 0.999). Limits of quantification of 14.5 ng mL-1 and 6.1 ng mL-1 were achieved in plasma and urine samples, respectively. The very clean-baseline of the resulting chromatogram illustrated the high selectivity brought by the monolithic molecularly imprinted polymer that allows the removal of a huge peak corresponding to the elution of interfering compounds and the easy determination of the target analyte in these complex biological samples.