ELECTROPHORESIS最新文献

A holistic understanding of the charge heterogeneity in monoclonal antibodies (mAbs) is paramount for ensuring acceptable product quality. Hence, biotherapeutic manufacturers are expected to thoroughly characterize their products via advanced analytical techniques. Recently, two-dimensional liquid chromatography (2DLC) methods have gained popularity for resolving complex charged species. Capillary electrophoresis (CE) is regarded as a sensitive and faster tool for charged species estimation in biotherapeutics. In this study, we aim to combine the separation power of chromatographic and electrophoretic tools (liquid chromatography [LC]-CE) so as to achieve maximum resolution of mAb charge variants. Hydrophobic interaction chromatography (HIC) has been used as the preferred LC mode with CE for achieving successful separation of both charge and hydrophobic variants for two of the mAbs (trastuzumab and rituximab). The standalone HIC and capillary zone electrophoresis (CZE) methods separated 4 hydrophobic variants and 7 charge variants for each mAb, whereas the 2DLC method separated 10 and 11 variants for mAbs A and B. On the other hand, the HIC-CZE-UV method resolved 29 variants in mAb A and 23 variants in mAb B. The reproducibility of the HIC-CZE-UV method was demonstrated by % change in values of retention time (RT) and peak area as <5% (mAb A), <3% (mAb B), and <12% (for both mAbs), respectively. Thus, the utility of the proposed LC-CE method for characterization of mAb charge variants has been displayed.

The analysis of biopharmaceuticals for charge variants occurs from early-stage samples through formulation and process-development optimization. Higher throughput methods allow increased analysis of these samples to facilitate greater understanding of the samples and to better optimize their production and formulation. To enable higher throughput charge variant analysis, a new, rapid platform imaged capillary isoelectric focusing (icIEF) method was optimized to be two to three times faster than standard methods.

Osteopontin (OPN) in milk plays an important role in intestinal and brain development in early infancy, and great attention has been focused on OPN isolation to add extra OPN in infant formula. However, large-scale OPN isolation is limited by the low efficiency of sample pretreatment. Herein, we utilized preparative reciprocating free-flow isoelectric focusing (RFFIEF) to showcase the enrichment of low-abundance OPN in bovine milk, which contained an extremely high concentration of unwanted proteins. The reciprocating IEF format and the design of the multi-channel collector allowed us to enrich OPN in 1 L milk within 6 h. We removed 97.5% of unwanted proteins and obtained an enrichment factor of 11. Thus, our RFFIEF method can be applied to the preparative pretreatment of the large-scale milk sample and potentially improve the efficiency of downstream OPN purification.

Ireland has a successful pharmaceutical industry with over 100 pharmaceutical manufacturing sites across the island. Although this success has many benefits, the irreversible effects emissions from pharmaceutical manufacturing can have on the environment are a major drawback. Although known pollutants are regularly monitored with limits set out by the Environmental Protection Agency, one significant pollutant has been overlooked: pharmaceutical pollution. Detecting these pollutants and ensuring they are at a safe concentration for the environment is of utmost importance. In recent years, capillary electrophoresis is being recognised as a suitable alternative to high-performance liquid chromatography due to its many benefits such as faster analysis, water-based buffers and smaller sample volumes. In this paper, a capillary zone electrophoresis (CZE) method with a preconcentration step of solid-phase extraction was developed for an anti-parasitic active pharmaceutical ingredient (API) called ZB23. The API was successfully detected in a wastewater sample in less than 10 min using the CZE parameters of 25 mM borate buffer with a pH of 10.5, 15% MeOH, 10 kV voltage, 25 mbar for 5 s injection size, an Lt of 40 cm, an Ld of 31.5 cm and a detection wavelength of 214 nm.

Isotope ratio analysis of sulfur in biological samples using inductively coupled plasma-mass spectrometry (ICP-MS) has gained significant interest for applications in quantitative proteomics. Advancements like coupling separation techniques with multicollector ICP-MS (MC-ICP-MS) enhance the throughput of species-specific sulfur isotope ratio measurements, fostering new avenues for studying sulfur metabolism in complex biological matrices. This proof-of-concept study investigates the feasibility of online CE/MC-ICP-MS for directly analyzing sulfur isotope ratios in proteins (albumin). Leveraging our previous work on the applicability of CE/ICP-MS for quantifying sulfur-containing biological molecules, we explore its potential for sulfur isotope analysis. Our results demonstrate that direct analysis of sulfur isotopes in albumin protein using online capillary electrophoresis MC-ICP-MS (CE/MC-ICP-MS) eliminates the need for laborious pretreatment steps, while yielding isotope ratios comparable to the reference values. Although initial precision can be improved through further system optimization and protein injection techniques, this approach paves the way for future analysis of mixtures of various biological compounds in, for example, clinical diagnosis studies.

A novel supplementary non-CODIS STR multiplex assay designated as the "Microreader 23HS Plex ID System" was developed. The Microreader 23HS Plex ID System enables simultaneous profiling of 23 STR loci and the amelogenin locus. The majority of these loci are non-CODIS STRs (D4S2408, D9S2157, D20S161, D3S2459, D18S1364, D13S305, D1S2142, D19S400, D6S1017, D7S1517, D2S1776, D2S1360, D3S1744, D16S3391, D3S1545, D11S4463, D20S85, D1S549, D10S2325, D21S2055), with the exception of three CODIS STRs (D2S441, D12S391, and D22S1045). Followed the recommendations of Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese validation standards, a comprehensive set of validation studies were conducted, encompassing PCR conditions, stutter ratio and peak height balance, sensitivity, precision and accuracy, reproducibility, species specificity, inhibition, as well as mixture testing. The results demonstrated that the Microreader 23HS Plex ID System is a reliable and robust assay, with well-balanced peak heights, high precision and accuracy, species specificity, and resistance to common inhibitors. The sensitivity of the assay was determined to be 0.125 ng of template DNA. In mixture study, all minor alleles were detected in two-sample mixtures across various ratios (1:19, 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 4:1, 9:1, and 19:1). In population study, a total of 500 unrelated individuals of Han ethnicity from East China were genotyped. The allele frequencies and forensic population genetic parameters were calculated, with a cumulative random match probability of 7.757 × 10^-27, and a total power of discrimination exceeding 0.999,999,999,999,999,999,999,999,99. In conclusion, the Microreader 23HS Plex ID System shows promise as a valuable supplementary tool for forensic applications, particularly in addressing complex kinship testing and challenges posed by STR mutation.

Capillary electrophoresis (CE) is a powerful analysis technique with advantages such as high separation efficiency with resolution factors above 1.5, low sample consumption of less than 10 µL, cost-effectiveness, and eco-friendliness such as reduced solvent use and lower operational costs. However, CE also faces limitations, including limited detection sensitivity for low-concentration samples and interference from complex biological matrices. Prior to performing CE, it is common to utilize sample preparation procedures such as solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) in order to improve the sensitivity and selectivity of the analysis. Recently, there have been advancements in the development of novel materials that have the potential to greatly enhance the performance of SPME and LPME. This review examines various materials and their uses in microextraction when combined with CE. These materials include carbon nanotubes, covalent organic frameworks, metal-organic frameworks, graphene and its derivatives, molecularly imprinted polymers, layered double hydroxides, ionic liquids, and deep eutectic solvents. The utilization of these innovative materials in extraction methods is being examined. Analyte recoveries and detection limits attained for a range of sample matrices are used to assess their effects on extraction selectivity, sensitivity, and efficiency. Exploring new materials for use in sample preparation techniques is important as it enables researchers to address current limitations of CE. The development of novel materials has the potential to greatly enhance extraction selectivity, sensitivity, and efficiency, thereby improving CE performance for complex biological analysis.

Circular RNAs (circRNAs) have emerged as pivotal players in RNA therapeutics. Unlike linear counterparts, circRNAs possess a closed-loop structure, conferring them with enhanced stability and resistance to degradation. Ribozyme-based strategy stands out as the predominant method for synthetic circRNA production, by precisely cleaving and promoting the formation of a covalent circular structure. However, there is still a lack of analytical methods that can provide high-throughput and quantitative analysis to facilitate the circRNA vector engineering process. In the report, we detail analytical methods to characterize and evaluate ribozyme-based RNA circularization efficiency. Our approach will capture the attention of researchers interested in optimizing RNA circularization efficiency, as well as those focused on exploring key elements for ribozyme catalytic activity.

Native mass spectrometry (nMS) is a cutting-edge technique that leverages electrospray ionization MS (ESI-MS) to investigate large biomolecules and their complexes in solution. The goal of nMS is to retain the native structural features and interactions of the analytes during the transition to the gas phase, providing insights into their natural conformations. In biopharmaceutical development, nMS serves as a powerful tool for analyzing complex protein heterogeneity, allowing for the examination of non-covalently bonded assemblies in a state that closely resembles their natural folded form. Herein, we present an imaged capillary isoelectric focusing-MS (icIEF-MS) workflow to characterize cysteine-linked antibody-drug conjugate (ADC) under native conditions. Two ADCs were analyzed: a latest generation cysteine-linked ADC polatuzumab vedotin and the first FDA-approved cysteine-linked ADC brentuximab vedotin. This workflow benefits from a recently developed icIEF system that is MS-friendly and capable of directly coupling to a high-sensitivity MS instrument. Results show that the icIEF separation is influenced by both drug payloads and the post-translational modifications (PTMs), which are then promptly identified by MS. Overall, this native icIEF-MS method demonstrates the potential to understand and control the critical quality attributes (CQAs) that are essential for the safe and effective use of ADCs.