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Background Reliable analysis of oligonucleotides is essential for therapeutic development and biomarker studies, yet conventional methods often rely on gels or high-salt buffers that hinder mass spectrometry compatibility. MicroDNA and microRNA are commonly analyzed using liquid chromatography or capillary gel electrophoresis (CGE). In a recent study, we introduced a gel-free capillary zone electrophoresis (CZE) method for rapid, high-resolution separation of oligonucleotides at acidic pH. Results Here we optimized a gel-free capillary zone electrophoresis (CZE) method operating at acidic pH using a PVA-coated capillary and malonic acid-ammonia buffer. Adjustments to buffer composition, cation type, and temperature (up to 60 °C) enhanced resolution and reduced current, enabling rapid, high-resolution separation of phosphodiester oligonucleotides ranging from 18 to 25 nucleotides with excellent repeatability (<0.5% mobility variation). Significance This gel-free CZE approach provides a fast and reproducible platform, facilitating detailed characterization of phosphodiester oligonucleotides, microDNA, and microRNA in pharmaceutical and bioanalytical contexts.

The ICH Q14 guideline introduces a structured framework for analytical method development based on Analytical Quality by Design (AQbD) principles, aiming to ensure robust, reliable, and fit-for-purpose methods throughout the product lifecycle. However, implementing ICH Q14 remains challenging due to the lack of complete examples and training resources, making it difficult for organizations to translate theory into practice. Although previous studies have applied AQbD to capillary electrophoresis method development, many have focused only on specific aspects such as the design of experiments (DoEs) or analytical target profile (ATP), leaving a gap in providing comprehensive, practical tools for the entire analytical lifecycle. This manuscript presents a novel, user-friendly approach to implementing ICH Q14 and AQbD, offering ready-to-implement tools and methodologies that simplify the process of method design, optimization, validation, and implementation. Through a stepwise process, the approach provides practical solutions for integrating AQbD principles into everyday workflows, bridging the gap between theoretical concepts and real-world applications. The approach has been thoroughly tested in diverse industrial settings, demonstrating its reliability and effectiveness. This work aims to facilitate the adoption of AQbD in analytical method development by providing structured tools, lessons learned, and best practices that align with ICH Q14 guidelines.

Short DNA and RNA sequences like therapeutic anti-sense oligonucleotides (ASO), microDNA (miDNA) and microRNA (miRNA) are predominantly analyzed using liquid chromatographic techniques or capillary gel electrophoresis. The current demand for improved analysis of these nucleotides gives incentives to reconsider old truths, such as the need for gel-based capillary electrophoresis for DNA and RNA analysis. The present study shows the applicability of capillary zone electrophoresis using acidic background electrolytes for the separation of ASO-, miDNA- and miRNA-sized nucleotides. Using the different pKa of the nucleobases as a foundation, different nucleotides with same size, but varying sequence, has been successfully separated with high efficiency and short analysis time. Furthermore, the ratio of adenosine and cytidine to guanosine and thymidine nucleobase content, AC/GT, is shown as a valid parameter to assess single-stranded nucleotide migration and separation.

Capillary electrophoresis (CE) often offers superior separation relative to chromatography for macromolecules like monoclonal antibodies (mAbs), a major pharmaceutical class. However, electropherogram baselines pose challenges that traditional chromatography algorithms cannot address, requiring complex integration processes. Integration in good manufacturing practice (GMP) laboratories is critically important and has become a focus of data integrity-centric regulatory inspections. Electropherogram integration challenges, the increased use of CE, data systems developed for chromatograms rather than electropherograms, and the increased regulatory scrutiny call for a resolution. This necessity also extends to R&D, clinical, and academic labs. This review examines authoritative sources such as pharmacopoeias, World Health Organization (WHO), Parenteral Drug Association (PDA), and scientific literature. However, none address electropherogram integration. These sources concur that companies should develop integration policies and SOPs. Training programs must ensure analysts are proficient in integration techniques and reviewers are appropriately qualified to assess integrations. Integration parameters must be captured, including slope sensitivity, smoothing factors, and timed events like peak start and stop and baseline correction. Analytical procedures (APs) should include illustrations that define proper integration. Although automatic integration is preferred for its efficiency and objectivity, it is not always accurate. Therefore, manual integration should be permitted under specific conditions, with all settings and iterations documented, justified, and reviewed. Industry collaboration is proposed to create practical integration guidelines specifically for CE.

The ICH guideline Q14 on analytical procedure development underlines the importance of science and risk-based methods for the evaluation of the quality of medicines. Ultimately, a pharmaceutical company, the sponsor, is responsible that the analytical method is fit-for-purpose during routine use throughout its lifecycle. Part of the analytical procedure control strategy is the responsibility to assure availability of critical materials of the analytical method. For capillary zone electrophoresis (CZE) methods, the background electrolyte (BGE) composition is a key and critical material. In this study, we investigated whether key ingredients of the epsilon-aminocaproic acid (eACA) CZE (eACA-CZE) method for monoclonal antibodies can be replaced by structurally related chemicals. The complex heterogeneity patterns are compared, as well as the reportable results as the percentage main, acidic and basic peaks. Overall, the results underline the ruggedness of the eACA-CZE method and provide alternative options to eACA and triethyltetramine (TETA), in case there are quality or supply issues, thus de-risking and safeguarding release and stability studies for therapeutic mAbs.

Capillary zone electrophoresis ultraviolet (CZE-UV) has become increasingly popular for the charge heterogeneity determination of mAbs and vaccines. The epsilon-aminocaproic acid (eACA) CZE-UV method has been used as a rapid platform method. However, in the last years, several issues have been observed, for example, loss in electrophoretic resolution or baseline drifts. Evaluating the role of eACA on the reported issues, various laboratories were requested to provide their routinely used eACA CZE-UV methods, and background electrolyte compositions. Although every laboratory claimed to use the He et al. eACA CZE-UV method, most methods actually deviate from He's. Subsequently, a detailed interlaboratory study was designed wherein two commercially available mAbs (Waters' Mass Check Standard mAb [pI 7] and NISTmAb [pI 9]) were provided to each laboratory, along with two detailed eACA CZE-UV protocols for a short-end, high-speed, and a long-end, high-resolution method. Ten laboratories participated each using their own instruments, and commodities, showing excellence method performance (relative standard deviations [RSDs] of percent time-corrected main peak areas from 0.2% to 1.9%, and RSDs of migration times from 0.7% to 1.8% [n = 50 per laboratory], analysis times in some cases as short as 2.5 min). This study clarified that eACA is not the main reason for the abovementioned variations.

The biopharmaceutical market is one of the fastest growing biotechnology markets. In order to ensure affordable and reliable therapeutics, the biopharmaceutical process has to be closely monitored. Capillary electrophoresis (CE) has proven to be a valuable technique for the analysis of product concentration, critical quality attributes, product and process-related impurities, and nutrients and metabolites in the cell culture medium. Capillary zone electrophoresis, capillary gel electrophoresis, and capillary isoelectric focusing are extensively used for product concentration, and size or charge heterogeneity determination. CE has a number of benefits for the analysis of upstream and downstream process-intermediates, including the ability to handle highly complex matrices found in process-intermediates, high resolving power, little sample preparation requirements, rapid analysis, and low solvent and sample consumption. The small sample volumes (nL range) are especially beneficial for microbioreactor analysis or clone se-lection experiments. The simple setup and the possibility for miniaturisation and automation using microchip CE provides great opportunities for on-site, real-time monitoring of the process. This review discusses CE applications in upstream and downstream processing of the last decade.

This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Watzig H, Degenhardt M, Kunkel A. "Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications," pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.

The rapidly growing, competitive biopharmaceutical market requires tight bioprocess monitoring. An integrated, automated platform for the routine online/at-line monitoring of key factors in the cell culture medium could greatly improve process monitoring. Mono- and disaccharides, as the main energy and carbon source, are one of these key factors. A CE-LIF method was developed for the analysis of several mono- and disaccharides, considering requirements and restrictions for analysis in an integrated, automated monitoring platform, such as the possibility for miniaturization to microchip electrophoresis. Analysis was performed after fluorescent derivatization with 8-aminopyrene-1,3,6-trisulfonic acid. The derivatisation reaction and the separation BGE were optimized using design of experiments. The developed method is applicable to the complex matrix of cell culture medium and proved transferable to microchip electrophoresis.

Protein separation can be achieved with different modes of capillary electrophoresis, such as with capillary gel electroporesis (CGE) or with capillary zone electrophoresis (CZE). CZE protein mapping of peanut extract was approached in four different ways, combining neutral-coated or multilayer-coated capillaries with pHs well over or under the isoelectric point range of the proteins of interest. At acidic pHs, the mobility ranges of the major peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 were identified. Although the pH is a major factor in CZE separation, buffers with different compositions but with the same pH and ionic strength showed significantly different resolutions. Different components of the electrolyte were studied in a multifactorial design of experiment. CE-SDS and CZE proved to be suitable for protein mapping and we were able to distinguish different batches of peanut extract and burned peanut extract.