Electrophoretic characterization of LNP/AAV-encapsulated nucleic acids: Strengths and weaknesses.

The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs (>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.

Stronger together: Analytical techniques for recombinant adeno associated virus.

With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC). Here, we evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two modes of capillary electrophoresis (CE) for their ability to analyze the DNA encapsidated by rAAVs. While TEM and AUC are well-established methods for rAAV, capillary gel electrophoresis (CGE) has been just recently proposed for viral genome sizing. The data presented reflect that samples are very complex, with various DNA species incorporated in the virus, including small fragments as well as DNA that is larger than the targeted transgene. CGE provides a good insight in the filling of rAAVs, but the workflow is tedious and the method is not applicable for the determination of DNA titer, since a procedure for the absolute quantification (e.g., calibration) is not yet established. For estimating the genome titer, we propose a simplified capillary zone electrophoresis approach with minimal sample preparation and short separation times (<5 min/run). Our data show the benefits of using the four techniques combined, since each of them alone is prone to delivering ambiguous results. For this reason, a clear view of the rAAV interior can only be provided by using several analytical methods simultaneously.

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20 nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization. Graphical abstract Two-dimensional capillary electrophoresis system hyphenated with mass spectrometry for the characterization of CE (SDS)-separated proteins. As first dimension, a generic and high MS-interfering CE (SDS) separation is performed for size separation. After heart-cut transfer of the unknown CE (SDS) protein peak, via a four-port nanoliter valve to a volatile electrolyte system as second dimension, interference-free mass spectrometric data of separated mAb fragments and soybean proteins are obtained.

Coupling imaged capillary isoelectric focusing with mass spectrometry using a nanoliter valve.

Imaged capillary isoelectric focusing (iCIEF) is a powerful separation technique applied frequently for the analysis of biotherapeutics. However, direct mass spectrometric characterization is usually not possible. Here, focused peaks from an iCIEF system are transferred to a four-port nanoliter valve where peaks of interest are cut and transferred directly (flow injection) or via capillary zone electrophoresis to electrospray mass spectrometry. At first, flow injection coupling was tested with a mixture of peptides showing intraday precision (RSD) of 20.0 and 3.4% in area and 15.2 and 6.3% in intensity for angiotensin I and leucine-enkephalin, respectively. For the analysis of charge variants in an intact mAb, flow injection and CZE as second dimension were compared, demonstrating the usability of flow injection even for large proteins. However, improved spectra quality was achieved when CZE-MS was applied. In this way, accurate masses were obtained not only for the main isoform, but also for the main acidic and basic variants. These results demonstrate the power of iCIEF-CZE-MS for the analysis of biotherapeutics.

Characterization of a nanoflow sheath liquid interface and comparison to a sheath liquid and a sheathless porous-tip interface for CE-ESI-MS in positive and negative ionization.

Coupling of capillary electrophoresis to electrospray mass spectrometry still remains challenging and a topic of research to find the best interface regarding sensitivity, robustness, and ease of use. Here, a nanoflow sheath liquid interface for CE-ESI-MS is presented and compared to both a standard triple-tube sheath liquid and a porous-tip sheathless interface for three groups of analytes. The nanoflow sheath liquid interface with a separation capillary inserted into a glass emitter was initially characterized to facilitate optimization and method development. Implementation of a shut-off valve, syringe pump, and inline filter enabled easy handling and fast analyses, repeatable both in positive and negative modes (intra-day RSD of 6.6 to 12.0%). The same setup was used for sheathless interfacing by exchanging the emitter and using a porous etched tip separation capillary. Both nanoflow interfaces showed similar performance. Average peak areas using the nanoflow sheath liquid interface were a factor of 38 for 6 organic acids in negative mode, 114 and 36 for the light and heavy chain of a monoclonal antibody, and 13 higher for peptides in positive mode compared to the triple-tube interface. This first direct comparison of the three most common interfaces exhibits a strong improvement in sensitivity to the same extent for both nanoflow interfaces, where sheath liquid interfaces offer full flexibility in method development.

In-capillary approach to eliminate SDS interferences in antibody analysis by capillary electrophoresis coupled to mass spectrometry.

Capillary electrophoresis is an important technique for the characterization of monoclonal antibodies (mAbs), especially in the pharmaceutical context. However, identification is difficult as upscaling and hyphenation of used methods directly to mass spectrometry is often not possible due to separation medium components that are incompatible with MS detection. Here a CE-MS method for the analysis of mAbs is presented analyzing SDS-complexed samples. To obtain narrow and intensive peaks of SDS-treated antibodies, an in-capillary strategy was developed based on the co-injection of positively charged surfactants and methanol as organic solvent. For samples containing 0.2% (v/v) of SDS, recovered MS peak intensities up to 97 and 95% were achieved using cetyltrimethylammonium bromide or benzalkonium chloride, respectively. Successful removal of SDS was shown in neutral coated capillaries but also in a capillary with a positively charged coating applying reversed polarity. The usefulness of this in-capillary strategy was demonstrated also for other proteins and for antibodies dissolved in up to 10% v/v SDS solution, and in other SDS-containing matrices, including the sieving matrix used in a standard CE-SDS method and gel-buffers applied in SDS-PAGE methods. The developed CE-MS approaches enable fast and reproducible characterization of SDS-complexed antibodies.

Analysis of antibiotics by CE and their use as chiral selectors: An update.

The widespread use of antibiotics in medicine and as growth-promoting agents has increased the demand for suitable analytical techniques for their analysis. Analytical methods based on CE or miniaturized CE systems have proved over the years their ability for the analysis of antibiotics. Since our last review (Electrophoresis 2014, 35, 28-49) several new CE methodologies have been reported for antibiotic analysis. This review presents an update of the literature published from June 2013 to June 2015 for the analysis of antibiotics by CE. UV continues being the most used detection system for antibiotics analysis by CE. Strategies to improve sensitivity as the use of sensitive detection systems and the application of preconcentration techniques appear to be the major developments. Furthermore, the use of portable and miniaturized devices for antibiotic analysis is presented in detail. Applications of the developed methodologies to the determination of residues of antibiotics in biological, food, and environmental samples are carefully described. Finally, new developments and applications of antibiotics as chiral selectors in CE are also included.

On-line two-dimensional capillary electrophoresis with mass spectrometric detection using a fully electric isolated mechanical valve.

CE is becoming more and more important in many fields of bioanalytical chemistry. Besides optical detection, hyphenation to ESI-MS detection is increasingly applied for sensitive identification purposes. Unfortunately, many CE techniques and methods established in research and industry are not compatible to ESI-MS since essential components of the background electrolyte interfere in ES ionization. In order to identify unknown peaks in established CE methods, here, a heart-cut 2D-CE separation system is introduced using a fully isolated mechanical valve with an internal loop of only 20 nL. In this system, the sample is separated using potentially any non-ESI compatible method in the first separation dimension. Subsequently, the portion of interest is cut by the internal sample loop of the valve and reintroduced to the second dimension where the interfering compounds are removed, followed by ESI-MS detection. When comparing the separation efficiency of the system with the valve to a system using a continuous capillary only a slight increase in peak width is observed. Ultraviolet/visible detection is integrated in the first dimension for switching time determination, enabling reproducible cutting of peaks of interest. The feasibility of the system is successfully demonstrated by a 2D analysis of a BSA tryptic digest sample using a nonvolatile (phosphate based) background electrolyte in the first dimension.

Analysis of glycerophospho- and sphingolipids by CE.

Glycerophospholipids are amphiphilic molecules possessing polar head groups with a glycerol backbone and nonpolar variable long-chain fatty acids. Numerous molecular species are found in a single class of glycerophospholipid, conferring to these lipids a high structural diversity. They are major components of biological membranes and participate in important activities involving cell signaling and substrate transport. Sphingolipids consist of long-chain bases linked by an amide bond to a fatty acid and via the terminal hydroxyl group to complex carbohydrate or phosphorus moieties, constituting a complex family of compounds that also present an enormous structural variability. As important component of neuronal membranes, sphingolipids contribute to cellular diversity and functions and are associated with several neurodegenerative disorders. Moreover, they were studied in several foods due to their sensorial, reological, and antioxidant characteristics. In this work, the most relevant information available on glycerophospholipid and sphingolipid analysis by CE is reviewed. CE is a very promising analytical technique in polar lipid analysis, which provides high efficiency, relatively high resolution, and enormous versatility and requires small amounts of sample and solvent. MEKC and NACE methodologies have been developed as the most useful alternatives for these analyses by CE. Very interesting LODs have been achieved enabling the application of CE to the determination of glycerophospholipids and sphingolipids in several food and biological matrices.

Potential of vancomycin for the enantiomeric resolution of FMOC-amino acids by capillary electrophoresis-ion-trap-mass spectrometry.

The potential of the antibiotic vancomycin (VC) as chiral selector for the enantiomeric separation of amino acids by CE-ESI-MS/MS² was investigated for the first time in this work. Derivatization of amino acids with FMOC-Cl was carried out to enable their interaction with VC as well as the formation of precursor ions with larger m/z which were employed in MS² experiments. The partial filling of a coated capillary was employed to avoid the loss in MS sensitivity originated by the introduction of VC in the ionization source. Under optimized conditions, the simultaneous enantiomeric separation and unequivocal identification of 17 amino acids (two of them being nonprotein amino acids) took place in about 20 min with LODs in the micromolar range.

Proteins in olive fruit and oil.

This paper is a comprehensive review grouping the information on the extraction, characterization, and quantitation of olive and olive oil proteins and providing a practical guide about these proteins. Most characterized olive proteins are located in the fruit, mainly in the seed, where different oleosins and storage proteins have been found. Unlike the seed, the olive pulp contains a lower protein content having been described a polypeptide of 4.6 kDa and a thaumain-like protein. Other important proteins studied in olive fruits have been enzymes which could play important roles in olives characteristics. Part of these proteins is transferred from the fruit to the oil during the manufacturing process of olive oil. In fact, the same polypeptide of 4.6 kDa found in the pulp has been described in the olive oil and, additionally, the presence of other proteins and enzymes have also been described. Protein profiles have recently been proposed as an interesting strategy for the varietal classification of olive fruits and oils. Nevertheless, there is still a lot of knowledge without being explored requiring new studies focused on the determination and characterization of these proteins.

Peanut allergens: an overview.

Peanut is recognized as a potent food allergen producing one of the most frequent food allergies. This fact has originated the publication of an elevated number of scientific reports dealing with peanut allergens and, especially, the prevalence of peanut allergy. For this reason, the information available on peanut allergens is increasing and the debate about peanut allergy is always renewed. This article reviews the information currently available on peanut allergens and on the techniques used for their chemical characterization. Moreover, a general overview on the current biotechnological approaches used to reduce or eliminate peanut allergens is also provided.

Separation of proteins from olive oil by CE: an approximation to the differentiation of monovarietal olive oils.

The separation of proteins from olive oils by CE has been achieved for the first time in this work. Based on the protein profiles obtained using UV detection, a novel approach has also been proposed enabling the differentiation of monovarietal olive oils. An extraction procedure based on the precipitation of proteins in cold acetone and their isolation and solubilization in a hydro-organic medium prior to their in-capillary preconcentration in CE was a critical step in method development since proteins are minor components of olive oils. Then, a CE separation using a basic medium in a dynamically coated capillary originated electrophoretic profiles with multiple peaks from which seven of them were identified as proteins. When different monovarietal olive oils were injected, three different regions (zones I, II, and III) were identified. Zone III, called as differential zone, enabled to distinguish easily between extra virgin olive oils made from the Arbequina variety and the Picual and Hojiblanca varieties. This is the first time that protein profiles have been employed for the differentiation of botanical varieties of olive oils showing an enormous potential as traceability markers for these highly appreciated oils.

Development of chiral methodologies by capillary electrophoresis with ultraviolet and mass spectrometry detection for duloxetine analysis in pharmaceutical formulations.

Two chiral methodologies were developed by capillary electrophoresis (CE) with UV and mass spectrometry (MS) detection to ensure the quality control of the drug duloxetine, commercialized as a pure enantiomer. Both methods were optimized to achieve a high baseline enantioresolution (Rs>2) and an acceptable precision (RSD values <5% for instrumental repeatability and <10% for intermediate precision). In addition to allow the unequivocal identification of duloxetine enantiomers, the CE-MS method improved the sensitivity with respect to the use of CE-UV (LOD 200 ng/mL by CE-UV and 20 ng/mL by CE-MS) enabling to detect 0.02% of duloxetine enantiomeric impurity. This is the lowest LOD value ever reported for this drug, being this work the first one enabling to accomplish with the ICH guidelines requirements. The developed methods were validated and applied for the first time to the analysis of four pharmaceutical formulations. The content of R-duloxetine in all these samples was below the detection limit and the amount of S-duloxetine was in good agreement with the labeled content, obtaining results by the two methods that did not differ significantly (p-values >0.05).

Separation of olive proteins by capillary gel electrophoresis.

Olive proteins are not well known and there are still a lot of unknown information requiring further studies focused on the determination and characterization of these proteins. Despite the widely use of gel electrophoresis, this is the first time that capillary gel electrophoresis (CGE) is applied to separate proteins extracted from olive fruits. Seven common peaks were identified in the twenty olive varieties studied in this work. According to their migration times, these seven peaks could correspond to molecules with molecular masses of 11.0±0.4, 13.9±0.5, 16.3±0.8, 22.1±0.6, 30±1, 48±1, and 53±2 kDa. All of the determined molecular masses could be attributed to proteins and four of them have been previously observed by SDS-PAGE. The electrophoretic profiles were also evaluated for their capability to differentiate olive varieties according to their presumed geographical origin. Results demonstrated that this method could successfully classify the studied olive varieties by its combination with multivariate chemometrics tools.

Molecular characterization of phospholipids by high-performance liquid chromatography combined with an evaporative light scattering detector, high-performance liquid chromatography combined with mass spectrometry, and gas chromatography combined with a flame ionization detector in different oat varieties.

Oat (Avena sativa L.) is an important crop produced in various regions of Europe and North America. Oat lipids are a heterogeneous mixture of acyl lipids and unsaponifiable components. The neutral lipids are mainly triacylglycerols and account for 50-60% of total oat lipids. Oat oil is also rich in polar lipids, that is, phospholipids and glycolipids. Characterization of oat polar lipids has largely been performed by thin-layer chromatography (TLC), but the composition of phospholipid classes has been poorly studied. The aim of our work was the determination of different phospholipids in Romanian oat samples. For that purpose, one commercial sample (Comun) and four pure varieties (Jeremy, Lovrin 1, Lovrin 27-T, and Mures) were used. High-performance liquid chromatography combined with an evaporative light scattering detector results allowed us to establish that phosphatidylethanolamine was the most representative phospholipid in all of the oat samples. In addition, high-performance liquid chromatography combined with electrospray ionization mass spectrometry analysis showed that C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:0, and C20:1 were the fatty acids bound to the glycerol backbone. Using first-preparative TLC and later gas chromatography, it was demonstrated that linoleic acid (C18:2) was the main fatty acid of the phospholipid fraction in all of the samples.

Traceability markers to the botanical origin in olive oils.

This review provides an overview of traceability studies performed to date (April 2009) for olive oils. Special emphasis has been made on the botanical origin because high-quality monovarietal olive oils have been recently introduced on the markets and their quality control requires the development of new and powerful analytical tools as well as new regulations to avoid fraud to consumers. Several parameters with discriminant power have been used for olive oil traceability according to the olive variety used in the production of the oil. They have been considered as traceability markers to the botanical origin and classified, in this work, as compositional and genetical markers.

Separation of olive proteins combining a simple extraction method and a selective capillary electrophoresis (CE) approach: application to raw and table olive samples.

A simple extraction method was developed to extract proteins from olive samples based on chloroform/methanol extraction followed by a protein precipitation with cold acetone. Then, a capillary electrophoresis (CE) method was carried out using an acid buffer (1 M formic acid at pH 2) to ensure a positive net charge for proteins and a neutral charge for potential interferents as polyphenols. The method developed was applied to raw and table olive samples. Interestingly, raw olive samples showed differences in protein profiles depending upon the botanical variety of olives and their geographical region. Protein profiles obtained for table olives also showed differences according to the sample treatment. Thus, a signal reduction in the electropherograms obtained for black olives was observed in comparison to those achieved for treated green olives. In this work, the use of protein profiles was demonstrated to be a powerful tool for studying variations among olive samples.