Multidimensional Liquid Chromatography Research Articles

Advances in Chromatographic Analysis of Phenolic Phytochemicals in Foods: Bridging Gaps and Exploring New Horizons.

Chromatographic analysis of phenolic phytochemicals in foods has significantly advanced over the past decade (2014-2024), meeting increasing demands for precision and efficiency. This review covers both conventional and advanced chromatographic techniques used for detecting phenolic phytochemicals in foods. Conventional methods like High-Performance Liquid Chromatography, Ultra High-Performance Liquid Chromatography, Thin-Layer Chromatography, and Gas Chromatography are discussed, along with their benefits and limitations. Advanced techniques, including Hydrophilic Interaction Liquid Chromatography, Nano-LC, Multidimensional Liquid Chromatography, and Capillary Electrophoresis, are highlighted for their innovations and improved capabilities. The review addresses challenges in current chromatographic methods, emphasizing the need for standardized and validated procedures according to the Food and Drug Administration, European Cooperation for Accreditation of Laboratories, and The International Organization for Standardization guidelines to ensure reliable and reproducible results. It also considers novel strategies for reducing the environmental impact of chromatographic methods, advocating for sustainable practices in analytical chemistry.

Profiling of Antidiabetic Bioactive Flavonoid Compounds from an Edible Plant Kudzu (Pueraria lobata).

Pueraria lobata (Willd.) Ohwi, known as kudzu and used as a "longevity powder" in China, is an edible plant which is rich in flavonoids and believed to be useful for regulating blood sugar and treating diabetes, although the modes of action are unknown. Here, a total of 53 flavonoids including 6 novel compounds were isolated from kudzu using multidimensional preparative liquid chromatography. The flavonoid components were found to lower blood sugar levels, promote urine sugar levels in mice, and reduce the urine volume. Molecular docking and in vitro assays suggested that the antidiabetic effect of kudzu was attributed to at least three targets: sodium-dependent glucose transporter 2 (SGLT2), protein tyrosine phosphatase-1B (PTP1B), and alpha-glucosidase (AG). This study suggests a possible mechanism for the antidiabetic effect that may involve the synergistic action of multiple active compounds from kudzu.

Applications of high performance liquid chromatography-mass spectrometry in proteomics

Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the "bottom-up" approach and the "top-down" approach. The "shotgun" method is a typical "bottom-up" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. "Top-down" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the "bottom-up" "top-down" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.

Insights into nucleoside hydrolase from Leishmania donovani inhibition: A new bioaffinity chromatography-based screening assay and docking studies

Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(β-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(β-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (Ki = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors.

Stereochemical Heterogeneity Analysis of Polylactides by Multidimensional Liquid Chromatography.

A new and robust high-performance liquid chromatography (HPLC) method that separates poly(lactic acid) (PLA) according to its stereochemical composition is presented. Using this method, poly(l-lactide) incorporating trace amounts of meso-lactide resulting from the racemization is separated from the pristine polymer. To prove this aspect in more detail, a representative poly(l-lactic acid) standard, assumed to be highly homogeneous, was separated using this method. The result showed that this was not the case as a fraction incorporating meso-lactide due to racemization occurring during the synthesis is separated. Employing two-dimensional liquid chromatography (2D-LC), the molar mass differences of the separated species were investigated, and fractions with similar molecular sizes were detected, confirming that the LC separation is solely based on stereochemical heterogeneity. The sample was further fractionated by preparative HPLC, followed by an in-depth analysis of the fractions using homonuclear decoupling in proton nuclear magnetic resonance (1H NMR). Convincing results that unveiled significant differences in the stereochemistry of the isolated PLA fractions were obtained. Subsequent analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) also confirmed oligomer series with different end group structures, indicating that the applied HPLC method is very sensitive to minor variations in stereochemistry and end groups. This integrated approach offers detailed insight into the structural characteristics of PLA polymers, contributing to a better understanding of their composition and potential applications.

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.

High throughput multidimensional liquid chromatography approach for online protein removal and characterization of polysorbates and poloxamer in monoclonal antibody formulations

The majority of commercially available monoclonal antibody (mAb) formulations are stabilized with one of three non-ionic surfactants: polysorbate 20 (PS20), polysorbate 80 (PS80), or poloxamer 188 (P188). All three surfactants are susceptible to degradation, which can result in functionality loss and subsequent protein aggregation or free fatty acid particle formation. Consequently, quantitative, and qualitative analysis of surfactants is an integral part of formulation development, stability, and batch release testing. Due to the heterogeneous nature of both polysorbates and poloxamer, online isolation of all the compounds from the protein and other excipients that may disturb the subsequent liquid chromatography with charged aerosol detection (LC-CAD) analysis poses a challenge. Herein, we present an analytical method employing LC-CAD, utilizing a combination of anion and cation exchange columns to completely remove proteins online before infusing the isolated surfactant onto a reversed-phase column. The method allows high throughput analysis of polysorbates within 8 minutes and poloxamer 188 within 12 minutes, providing a separation of the surfactant species of polysorbates (unesterified species, lower esters, and higher esters) and poloxamer 188 (early eluters and main species). Accuracy and precision assessed according to the International Council for harmonisation (ICH) guideline were 96 – 109 % and ≤1 % relative standard deviation respectively for all three surfactants in samples containing up to 110 mg/mL mAb. Subsequently, the method was effectively applied to quantify polysorbate 20 and polysorbate 80 in nine commercial drug products with mAb concentration of up to 180 mg/mL.

Comprehensive two-dimensional liquid chromatography with high resolution mass spectrometry to investigate the photoelectrochemical degradation of environmentally relevant pharmaceuticals and their degradation products in water

The widespread presence of organic micropollutants in the environment reflects the inability of traditional wastewater treatment plants to remove them. In this context, advanced oxidation processes (AOPs) have emerged as promising quaternary wastewater treatment technologies since they efficiently degrade recalcitrant components by generating highly reactive free radicals. Nonetheless, the chemical characterization of potentially harmful byproducts is essential to avoid the contamination of natural water bodies with hazardous substances. Given the complexity of wastewater matrices, the implementation of comprehensive analytical methodologies is required. In this work, the simultaneous photoelectrochemical degradation of seven environmentally relevant pharmaceuticals and one metabolite from the EU Watch List 2020/1161 was examined in ultrapure water and simulated wastewater, achieving excellent removal efficiencies (overall >95%) after 180 min treatment. The reactor unit was linked to an online LC sample manager, allowing for automated sampling every 15 min and near real-time process monitoring. Online comprehensive two-dimensional liquid chromatography (LC × LC) coupled with high resolution mass spectrometry (HRMS) was subsequently used to tentatively identify degradation products after photoelectrochemical degradation. Two reversed-phase liquid chromatography (RPLC) columns were used: an SB-C18 column operated with 5 mM ammonium formate at pH 5.8 (1A) and methanol (1B) as the mobile phases in the first dimension and an SB-Aq column using acidified water at pH 3.1 (2A) and acetonitrile (2B) as the mobile phases in the second dimension. This resulted in a five-fold increase in peak capacity compared to one-dimensional LC while maintaining the same total analysis time of 50 min. The LC x LC method allowed the tentative identification of 12 venlafaxine, 7 trimethoprim and 10 ciprofloxacin intermediates. Subsequent toxicity predictions suggested that some of these byproducts were potentially harmful.This study presents an effective hybrid technology for the simultaneous removal of pharmaceuticals from contaminated wastewater matrices and demonstrates how multidimensional liquid chromatography techniques can be applied to better understand the degradation mechanisms after the treatment of micropollutants with AOPs.

Multi-Dimensional Liquid Chromatography of Pulse Triacylglycerols with Triple Parallel Mass Spectrometry

We analyzed ten pulses (the dried seeds of legumes), i.e., baby lima beans, black beans, black-eyed peas, butter beans, cranberry beans, garbanzo beans, green split peas, lentils, navy beans, and pinto beans, using three-dimensional liquid chromatography (3D-LC) with parallel second dimensions, LC × (LC + LC). We combined non-aqueous reversed-phase (NARP) chromatography as the first dimension separation, 1D, with argentation UHPLC for separation based on degree and location of unsaturation in the first second dimension, 2D(1), and multi-cycle NARP-UHPLC in the second second dimension, 2D(2). Pulses contained 1.9% to 2.7% lipids, except garbanzo beans, which contained 6.2% lipids. High-resolution, accurate-mass (HRAM) orbitrap mass spectrometry (MS) was used to perform lipidomic analysis of the 2D(2) and percent relative quantification, showing that the most abundant average triacylglycerol (TAG) molecular species across all pulses were PLL at 10.67% and PLLn at 10.45%. Common beans (Phaseolus vulgaris) were clustered together using principal component analysis (PCA), showing the highest levels of linolenic acid, C18:3, in molecular species such as PLnLn, LLnLn, and OLLn, with palmitic (P), C16:0, linoleic (L), 18:2, linolenic (Ln), 18:3, and oleic (O), 18:1, FAs. Calibration curves derived from interweaved sets of regioisomer standards allowed the absolute quantification of 1,2- and 1,3-regioisomers for a subset of TAGs.

Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc. However, traditional offline sampling is too infrequent to fully characterize bioprocesses, and the typical time from sample generation to data analysis and reporting can take weeks. To circumvent these limitations, an automated online sampling multidimensional workflow was developed to enable streamlined measurements of mAb concentration, aggregation, and charge variants. This analytical framework also facilitates automated data export for real-time analysis of up to six bioreactors, including feedback-controlling capability using readily available LC technology. This workflow increases the data points per bioreactor, improving the understanding of each experiment while also reducing the data turnaround time from weeks to hours. Examples of effective real-time analyses of mAb critical quality attributes are illustrated, showing substantial throughput improvements and accurate results while minimizing labor and manual intervention.

Right in two: capabilities of ion mobility spectrometry for untargeted metabolomics.

This mini review focuses on the opportunities provided by current and emerging separation techniques for mass spectrometry metabolomics. The purpose of separation technologies in metabolomics is primarily to reduce complexity of the heterogeneous systems studied, and to provide concentration enrichment by increasing sensitivity towards the quantification of low abundance metabolites. For this reason, a wide variety of separation systems, from column chemistries to solvent compositions and multidimensional separations, have been applied in the field. Multidimensional separations are a common method in both proteomics applications and gas chromatography mass spectrometry, allowing orthogonal separations to further reduce analytical complexity and expand peak capacity. These applications contribute to exponential increases in run times concomitant with first dimension fractionation followed by second dimension separations. Multidimensional liquid chromatography to increase peak capacity in metabolomics, when compared to the potential of running additional samples or replicates and increasing statistical confidence, mean that uptake of these methods has been minimal. In contrast, in the last 15years there have been significant advances in the resolution and sensitivity of ion mobility spectrometry, to the point where high-resolution separation of analytes based on their collision cross section approaches chromatographic separation, with minimal loss in sensitivity. Additionally, ion mobility separations can be performed on a chromatographic timescale with little reduction in instrument duty cycle. In this review, we compare ion mobility separation to liquid chromatographic separation, highlight the history of the use of ion mobility separations in metabolomics, outline the current state-of-the-art in the field, and discuss the future outlook of the technology. "Where there is one, you're bound to divide it. Right in two", James Maynard Keenan.

Dwight R. Stoll and Peter W. Carr (Eds.): Multi-dimensional liquid chromatography: principles, practice, and applications.

Dwight R. Stoll and Peter W. Carr (Eds.): Multi-dimensional liquid chromatography: principles, practice, and applications.

A novel strategy on the study of whole intestinal metabolic profiles for Polygalae Radix before and after processing.

Relieving toxicity and enhancing a calming effect after processing Polygalae Radix (PR) are widely known. Aromatic carboxylic acids (ACAs) may be crucial processed products. However, due to the limited detection methods for ACAs, the whole metabolic profiles via intestinal bacteria are still not very clear. Designing a novel strategy for the detection of ACAs and tracking the whole metabolic profiles before and after processing PR. The stable-isotope labelling derivatisation (SILD) method based on multidimensional ultra-high performance liquid chromatography coupled with a mass spectrometer (UHPLC-MS) technology and UNIFI-pathway mode was firstly designed to systematically study the metabolisms of all the drug-derived ingredients ranging from m/z 100 to 2000 in processing PR via intestinal bacteria. Firstly, the SILD with UHPLC coupled with a triple-quadrupole MS technology was designed to trace eight ACA metabolites of the processed PR with intestinal bacteria. Additionally, the UHPLC coupled with a quadrupole time-of-flight MS with UNIFI-pathway mode was adopted to monitor relatively big metabolites. The metabolism mechanism of ACAs (eight kinds) and the relatively big molecular metabolites (98 kinds) were deeply traced in PR, PR with refined honey (HP), and PR with licorice (LP) via the intestinal bacteria. Totally 106 intact metabolic profiles of drug-derived ingredients were presented. Importantly, the influence of LP on the metabolism of compounds after incubation of intestinal bacteria was greater than that of HP. This research provides a comprehensive and systematic guidance for further study on in vivo metabolisms of the processed PR.

Technological development of multidimensional liquid chromatography-mass spectrometry in proteome research

Liquid chromatography-mass spectrometry (LC-MS) is the method of choice for high-throughput proteomic research. Limited by the peak capacity, the separation performance of conventional single-dimensional LC hampers the development of proteomics. Combining different separation modes orthogonally, multidimensional liquid chromatography (MDLC) with high peak capacity was developed to address this challenge. MDLC has evolved rapidly since its establishment, and the progress of proteomics has been greatly facilitated by the advent of novel MDLC-MS-based methods. In this paper, we will review the advances of MDLC-MS-based methodologies and technologies in proteomics studies, from different perspectives including novel application scenarios and proteomic targets, automation, miniaturization, and the improvement of the classic methods in recent years. In addition, attempts regarding new MDLC-MS models are also mentioned together with the outlook of MDLC-MS-based proteomics methods.

Identification of a Bitter Peptide Contributing to the Off-Flavor Attributes of Pea Protein Isolates

The aversive bitter taste of pea protein ingredients limits product acceptability. Compounds contributing to the bitter perception of pea protein isolates were investigated. Off-line multi-dimensional sensory-guided preparative liquid chromatography fractionation of a 10% aqueous PPI solution revealed one main bitter compound that was identified by Fourier transform ion cyclotron resonance mass spectrometry and de novo tandem mass spectrometry (MS/MS) sequencing as the 37 amino acid peptide PA1b from pea albumin and further confirmed by synthesis. Quantitative MS/MS analysis reported that the concentration of the bitter peptide was 129.3 mg/L, which was above the determined bitter sensory threshold value of 3.8 mg/L and in agreement with the perceived bitter taste of the sample.

Rapid analysis of titer, aggregate, and intact mass of antibody therapeutics using automated multi-dimensional liquid chromatography coupled with native mass spectroscopy.

Monoclonal antibodies are tetrameric complex proteins, primarily produced using mammalian cell culture. Attributes such as titer, aggregates, and intact mass analysis are monitored during process development/optimization. In the present study, a novel workflow such that the Protein-A affinity chromatography is performed in the first dimension for purification and titer estimation, whereas size exclusion chromatography is employed in the second dimension to characterize size variants using native mass spectrometry. The present workflow offers a significant advantage over the traditionally used standalone Protein-A affinity chromatography followed by size exclusion chromatography analysis in that it can monitor these four attributes in 8 min while requiring a minimal sample size (10-15μg) and not requiring any manual peak collection. In contrast, the traditional standalone approach requires manual collection of eluted peaks in Protein-A affinity chromatography followed by buffer exchange to a mass-compatible buffer, which can take up to 2-3h with considerable risk of sample loss, degradation, and induced modifications. As the biopharma industry moves to make analytical testing efficient, we believe that the approach proposed here would be of significant interest due to its ability to monitor multiple process and product quality attributes in a single workflow and via rapid analysis.

Comprehensive and heart-cutting multidimensional liquid chromatography-mass spectrometry and its applications in food analysis.

In food analysis, conventional one-dimensional liquid chromatography methods sometimes lack sufficient separation power due to the complexity and heterogeneity of the analyzed matrices. Therefore, the use of two-dimensional liquid chromatography (2D-LC) turns out to be a powerful tool to consider, especially when coupled to mass spectrometry (MS). This review presents the most remarkable 2D-LC-MS food applications reported in the last 10 years, including a critical discussion of the multiple approaches, modulation strategies as well as the importance of the optimization of the different analytical aspects that will condition the 2D-LC-MS performance. The presence of contaminants in food (food safety), the food quality, and authenticity or the relationship between the beneficial effects of food and human health are some of the fields in which most of the 2D-LC-MS applications are mainly focused. Both heart-cutting and comprehensive applications are described and discussed in this review, highlighting the potential of 2D-LC-MS for the analysis of such complex samples.

Novel Multidimensional Liquid Chromatography Workflow with In-Loop Enzymatic Digests of Multiple Heart-Cuts for Fast and Flexible Characterization of Biotherapeutic Protein Variants

Multidimensional liquid chromatography (mD-LC) is becoming a powerful tool for complete characterization of individual peaks and protein variants through separation methods such as nondenaturing ion exchange (IEC) or size-exclusion chromatography coupled to reversed-phase (RP) chromatography. The flexibility of commercially available and customized mD-LC systems is still limited in terms of enzymatic peak processing between chromatographic dimensions. In this regard, only a few column-immobilized proteases are available for detailed peak characterization by mD-LC coupled to mass spectrometry (mD-LC-MS). Here, we present a purpose-built and automated multiple heart-cutting mD-LC design with a novel analytical workflow involving in-loop enzymatic heart-cut digestion between the first-dimensional column and transfer to the second dimension before MS or MS/MS analyses. The setup facilitates the spike-in of any enzyme to multiple heart-cuts for multilevel analysis, for example, for peptide mapping, fragment generation, or deglycosylation, to reduce heterogeneity and provide maximum flexibility in terms of incubation time for optimal peak characterization. We demonstrate the application of IEC coupled to RP-LC-MS and automated in-loop deglycosylation and on-column reduction of an IgG antibody combined with upper hinge region cleavage for Fab generation. We further employ mD-LC-MS and mD-LC-MS/MS to assess post-translational modifications of a bispecific antibody and to support molecule selection by evaluating the best downstream purification strategy. The novel design and automated workflow of the mD-LC system described here offers enhanced flexibility for in-solution processing and real-time monitoring of multiple heart-cuts enabling streamlined characterization of unknown biotherapeutic charge and size variants.

Identification of Hetero-aggregates in Antibody Co-formulations by Multi-dimensional Liquid Chromatography Coupled to Mass Spectrometry.

Antibody combination therapies have become viable therapeutic treatment options for certain severe diseases such as cancer. The co-formulation production approach is intrinsically associated with more complex drug product variant profiles and creates more challenges for analytical control of drug product quality. In addition to various individual quality attributes, those arising from the interactions between the antibodies also potentially emerge through co-formulation. In this study, we describe the development of a widely applicable multi-dimensional liquid chromatography coupled to tandem mass spectrometry method for antibody homo- versus hetero-aggregate characterization. The co-formulation of trastuzumab and pertuzumab was used, a challenging model system, comprising two monoclonal antibodies with very similar physicochemical properties. The data presented demonstrate the high stability of the co-formulation, where only minor aggregate formation is observed upon product storage and accelerated temperature or light-stress conditions. The results also show that the homo- and hetero-aggregates, formed in low and comparable proportions, are only marginally impacted by the formulation and product storage conditions. No preferential formation of hetero-aggregates, in comparison to the already existing pertuzumab and trastuzumab homo-aggregates, was observed.

Less Configuration and More Dimensionality: Preparative Heart-Cut Multidimensional Liquid Chromatography Based on Trapping Arrays.

The resolving power of multiple dimensional liquid chromatography (mD-LC) is multiplicative as it adds dimensions. However, the issue in creating a preparative mD-LC system is that the higher the dimensionality, the more complicated the system configuration. Thus, we presented a new configuration of preparative mD-LC using one set of LC modules and trapping array-based multiple heart-cut interfaces. A preparative two-dimensional liquid chromatography (2D-LC) separation of herbal medicine formulation produced 40 compounds with a purity of >90%. During the separation process, the interface stores the fractions and allocates positions for the fractions from a different dimension; LC draws the fraction from the interface, makes nD separation, and sends isolated fractions to the interface. By repeating this process, we achieved variable dimensionality of LC separations. We also presented a preparative 3D-LC separation of herbal medicines to validate the principle of "less configuration and more dimensionality". Thus, we can explore the higher dimensional preparative separations. The developed preparative mD-LC displayed exceptional power in the isolation of various compounds and has great potential in the application of natural products.