Microfluidic Capillary Electrophoresis Research Articles

Hydrodynamic injection for microchip electrophoresis: Development of an innovative passive system

The world is facing challenges in terms of environmental sustainability and rising costs. In this context, miniaturized equipment such as microfluidic devices can be used to manipulate small volumes of fluids in micrometer-sized channels incorporating multiple components such as pumps, valves and mixers. Due to their high flexibility, these devices can provide sustainable alternatives to traditional platforms. The present study focuses on the design of a user-friendly and reliable microfluidic capillary electrophoresis chip for pharmaceutical applications. To meet pharmaceutical requirements in terms of quantification performance and overcome the injection variability usually observed with such microfluidic systems, a reliable and reproducible design for hydrodynamic injection using passive valves has been developed. It has been successfully used for synthetic cathinone analysis.

Characterization of Therapeutic Antibody Charge Heterogeneity Under Stress Conditions by Microfluidic Capillary Electrophoresis Coupled with Mass Spectrometry

Therapeutic antibodies are a major class of biopharmaceutics that are applied in disease treatment because of their many advantages, including high specificity and high affinity to molecular targets. Between their production and administration, therapeutic antibodies are exposed to multiple stress conditions. Forced degradation and stress stability studies are conducted to simulate the risk of degradation and the effects of these stresses, thereby enhancing understanding of the drug product to support strategies to mitigate the impact from stressed conditions. These types of studies are also routinely conducted to evaluate product comparability when major process changes are implemented during the production. Charge variant analysis helps understand the changes in the electrostatic environment of biotherapeutics and can uncover underlying molecular level alterations associated with charge variants. Herein, we used ZipChip native capillary electrophoresis-mass spectrometry (nCE-MS) to elucidate the changes in charge variant profiles at the molecular level. In two case studies under thermal stress conditions, we observed that charge variants arose from both post-translational modifications (including deamidation, oxidation, and pyroglutamate formation) and sequence truncations at the hinge regions. Under oxidative stress conditions, oxidation was found to be the major contributor to the changes in the charge variant profiles. Under pH stress conditions, the changes in the charge variant profile were due to increased levels of deamidation, oxidation, and pyroglutamate formation. ZipChip nCE-MS analysis enables identification of charge variant species under various stress conditions, thus supporting process and formulation development of biotherapeutics.

Abstract 5026: Microfluidic capillary electrophoresis optimization of high-throughput cfDNA and pDNA measurements for enabling cancer diagnostics and gene therapy development

Abstract Rapid and accurate electrophoresis characterization can be an important tool for cancer researchers and therapy manufacturers. Advances in NGS of low molecular weight cfDNA for diagnostics may benefit from high-throughput checks of cfDNA sample quality with ultra-low quantities of sample. On the other hand, high molecular weight pDNA characterization and separation of supercoiled, linear and open-linear conformations aids in quality control of these payloads for personalized medicine and in particular cancer vaccines and gene therapies. This work shows optimization of established microfluidic capillary electrophoresis to enable the measurement of cfDNA and pDNA. cfDNA study was completed on plasma reformatted from whole blood tubes using Janus G3 Blood iQ workstation. The cfDNA samples were extracted using commercial cfDNA extraction kits. Using a LabChip GX Touch Biologics Characterization system: gel optimization, marker solution dilution, and inclusion of a low molecular weight DNA standard improved the sensitivity of cfDNA detection. This afforded a new electrophoresis assay with linear dynamic range of 50-1000 pg/uL and sensitivity of <25 ng/uL. pDNA, supercoiled DNA and linear DNA standards were prepared according to published procedures and measured on the LabChip GX Touch Biologics Characterization system. A new ladder system was developed along with gel optimization to develop a new High Sensitivity Assay platform capable of baseline resolving each conformation and providing fragment DNA sizing and purity information. Taken together, this work provides researchers new tools to leverage automated microfluidic capillary electrophoresis for DNA analysis in both upstream and manufacturing processes. Citation Format: Dipti Mehta, Gayatri Gautam, Adriana Coll de Pena, Menel Ben Frej, Anubhav Tripathi, James White. Microfluidic capillary electrophoresis optimization of high-throughput cfDNA and pDNA measurements for enabling cancer diagnostics and gene therapy development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5026.

A dual-reference study design for understanding and improving AAV genome size analysis.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89pg/µL (3×1010 GC/mL AAV) for 5.1kb ssDNA fragment, and a turnaround time at ∼100s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

Investigating the Merits of Microfluidic Capillary Zone Electrophoresis-Mass Spectrometry (CZE-MS) in the Bottom-Up Characterization of Larger RNAs.

Established bottom-up approaches for the characterization of nucleic acids (NAs) rely on the strand-cleavage activity of nucleotide-specific endonucleases to generate smaller oligonucleotides amenable to gas-phase sequencing. The complexity of these hydrolytic mixtures calls for the utilization of a front-end separation to facilitate full mass spectrometric (MS) characterization. This report explored the merits of microfluidic capillary zone electrophoresis (CZE) as a possible alternative to common liquid chromatography techniques. An oligonucleotide ladder was initially employed to investigate the roles of fundamental analyte features and experimental parameters in determining the outcome of CZE-MS analyses. The results demonstrated the ability to fully resolve the various rungs into discrete electrophoretic peaks with full-width half-height (FWHH) resolution that was visibly affected by the overall amount of material injected into the system. Analogous results were obtained from a digestion mixture prepared by treating yeast tRNAPhe (75 nt) with RNase T1, which provided several well-resolved peaks in spite of the increasing sample heterogeneity. The regular shapes of such peaks, however, belied the fact that most of them contained sets of comigrating species, as shown by the corresponding MS spectra. Even though it was not possible to segregate each species into an individual electrophoretic peak, the analysis still proved capable of unambiguously identifying a total of 29 hydrolytic products, which were sufficient to cover 96% of the tRNAPhe's sequence. Their masses accurately reflected the presence of modified nucleotides characteristic of this type of substrate. The analysis of a digestion mixture obtained from the 364 nt HIV-1 5'-UTR proved to be more challenging. The electropherogram displayed fewer well-resolved peaks and significantly greater incidence of product comigration. In this case, fractionating the highly heterogeneous mixture into discrete bands helped reduce signal suppression and detection bias. As a result, the corresponding MS data enabled the assignment of 248 products out of the possible 513 predicted from the 5'-UTR sequence, which afforded 100% sequence coverage. This figure represented a significant improvement over the 36 total products identified earlier under suboptimal conditions, which afforded only 57% coverage, or the 83 observed by direct infusion nanospray-MS (72%). These results provided a measure of the excellent potential of the technique to support the bottom-up characterization of progressively larger NA samples, such as putative NA therapeutics and mRNA vaccines.

Rapid Intact Mass Analysis and Evaluation of the Separation Potential of Microfluidic Capillary Electrophoresis Mass Spectrometry for Oligonucleotides.

Oligonucleotide characterization is a rapidly advancing field in the biopharmaceutical industry. Understanding critical quality attributes, such as intact mass and impurities, requires a toolbox of analytical techniques, which commonly includes liquid chromatography-mass spectrometry (LC-MS). Oligonucleotide LC-MS analysis frequently requires sample run times upward of 15 min to achieve separation of multiple oligonucleotide species. Additionally, LC methods frequently employ mobile phase additives such as triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol that are not always desired for use in MS instrumentation. Here, microfluidic capillary electrophoresis mass spectrometry (CE-MS) via ZipChip technology was employed to enable rapid intact mass analysis of oligonucleotide single strands. Baseline separation of equal length oligonucleotides was achieved in less than 4 min. Additionally, the potential of the ZipChip platform for separation of oligonucleotide full-length products (FLPs) and their impurities was evaluated.

Characterization of bispecific antigen-binding biotherapeutic fragmentation sites using microfluidic capillary electrophoresis coupled to mass spectrometry (mCZE-MS).

Fragmentation of therapeutic proteins is a potential critical quality attribute (CQA) that can occur in vivo or during manufacturing or storage due to enzymatic and non-enzymatic degradation pathways, such as hydrolysis, peroxide mediation, and acid/metal catalysis. Characterization of the fragmentation pattern of a therapeutic protein is traditionally accomplished using capillary gel electrophoresis with UV detection under both non-reducing and reducing conditions (nrCGE and rCGE). However, such methods are incompatible with direct coupling to mass spectrometry (MS) due to the use of anionic surfactants, e.g., sodium dodecyl sulfate (SDS). Here, we present a novel method to characterize size-based fragmentation variants of a new biotherapeutic kind using microfluidic ZipChip® capillary zone electrophoresis (mCZE) system interfaced with mass spectrometry (MS) to determine the molecular masses of fragments. A new modality of immuno-oncology therapy, bispecific antigen-binding biotherapeutic, was chosen to investigate its fragmentation pattern using mCZE-MS for the first time, according to our knowledge. Bispecific antigen-binding biotherapeutic samples from different stages of downstream column purification and forced degradation conditions were analyzed. The results were cross-validated with denaturing size-exclusion chromatography-mass spectrometry and conventional rSDS-CGE. In this study, we demonstrated that mCZE-MS could separate and characterize 12-40 kDa bispecific antigen-binding biotherapeutic fragments rapidly (within ≤12 minutes), with higher resolution and better sensitivity than traditional LC-MS methods.

Cell-Free DNA Fragmentation Patterns in a Cancer Cell Line.

Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed (hepta-nucleosomes were detectable with manual agarose gel electrophoresis), while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1–3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing.

Using multi-material fused deposition modeling (FDM) for one-step 3D printing of microfluidic capillary electrophoresis with integrated electrodes for capacitively coupled contactless conductivity detection

For the first time, the fused deposition modeling (FDM) 3D printing method was used to fabricate a microchip capillary electrophoresis (MCE) with channel dimensions (58 × 65 µm) below 100 µm. Moreover, electrodes to conduct capacitively coupled contactless conductivity detection (C4D) were integrated into the MCE using three conductive thermoplastic filaments containing graphene, carbon black, and copper. After optimizing the parameters for multi-material printing, the MCE-C4D device was fabricated in a single (one step) 3D printing process using an FDM 3D printer equipped with two extrusion nozzles. An optimization design method allowed finding that the distance (gap) between the electrodes of around 1 mm provided the maximum response of the C4D. The optimized frequency of the excitation signal applied in the C4D electrodes depended on the composition of the conductive filament used to print the electrodes. The optimized frequencies for the electrodes printed with thermoplastic polymers containing copper and carbon black were 268 and 384 kHz, respectively. The MCE-C4D successfully separated and detected a mixture of K+, Na+, and Li+ with good resolution, efficiency, precision, linearity, and limits of detections (LOD) of 10.2, 11.6, and 14.1 µmol L-1, respectively.

Fast Discrimination of Sialylated N-Glycan Linkage Isomers with One-Step Derivatization by Microfluidic Capillary Electrophoresis-Mass Spectrometry.

Linkage isomers (α-2,3- or α-2,6-linkage) of sialylated N-glycans are involved in the emergence and progression of some diseases, so they are of great significance for diagnosing and monitoring diseases. However, the qualitative and quantitative analysis of sialylated N-glycan linkage isomers remains challenging due to their low abundance and limited isomeric separation techniques. Herein, we developed a novel strategy integrating one-step sialic acid derivatization, positive charge-sensitive separation and highly sensitive detection based on microfluidic capillary electrophoresis-mass spectrometry (MCE-MS) for fast and specific analysis of α-2,3- and α-2,6-linked sialylated N-glycan isomers. A kind of easily charged long-chain amino compound was screened first for one-step sialic acid derivatization so that only α-2,3- and α-2,6-linked isomers can be quickly and efficiently separated within 10 min by MCE due to the difference in structural conformation, whose separation mechanism was further theoretically supported by molecular dynamic simulation. In addition, different sialylated N-glycans were separated in order according to the number of sialic acids, so that a migration time-based prediction of the number of sialic acids was achieved. Finally, the sialylated N-glycome of human serum was profiled within 10 min and 6 of the 52 detected sialylated N-glycans could be potential diagnostic biomarkers of cervical cancer (CC), whose α-2,3- and α-2,6-linked isomers were distinguished by α-2,3Neuraminidase S.

Using Multi-Material Fused Deposition Modeling (Fdm) for One-Step 3d Printing of a Microfluidic Capillary Electrophoresis Device with Integrated Electrodes for Capacitively Coupled Contactless Conductivity Detection

Using Multi-Material Fused Deposition Modeling (Fdm) for One-Step 3d Printing of a Microfluidic Capillary Electrophoresis Device with Integrated Electrodes for Capacitively Coupled Contactless Conductivity Detection

Use of Microfluidic Capillary Electrophoresis for the Determination of Multi-Component Protein Adsorption Isotherms: Application to High-Throughput Analysis for Hydrophobic Interaction Chromatography.

Chromatography is a widely used separation process for purification of biopharmaceuticals that is able to obtain high purities and concentrations. The phenomena that occur during separation, mass transfer and adsorption are quite complex. To better understand these phenomena and their mechanisms, multi-component adsorption isotherms must be investigated. High-throughput methodologies are a very powerful tool to determine adsorption isotherms and they waste very small amounts of sample and chemicals, but the quantification of component concentrations is a real bottleneck in multi-component isotherm determination. The behavior of bovine serum albumin, Corynebacterium diphtheriae CRM197 protein and lysozyme, selected as model proteins in binary mixtures with hydrophobic resin, is investigated here. In this work we propose a new method for determining multi-component adsorption isotherms using high-throughput experiments with filter plates, by exploiting microfluidic capillary electrophoresis. The precision and accuracy of the microfluidic capillary electrophoresis platform were evaluated in order to assess the procedure; they were both found to be high and the procedure is thus reliable in determining adsorption isotherms for binary mixtures. Multi-component adsorption isotherms were determined with a totally high-throughput procedure that turned out to be a very fast and powerful tool. The same procedure can be applied to every kind of high-throughput screening.

Combining First and Second-Tier Newborn Screening in a Single Assay Using High-Throughput Chip-Based Capillary Electrophoresis Coupled to High-Resolution Mass Spectrometry.

Most first-tier newborn screening (NBS) biomarkers are evaluated by a 2-min flow injection analysis coupled to tandem mass spectrometry (FIA-MS/MS) assay. The absence of separation prior to MS/MS analysis can lead to false positives and inconclusive results due to interferences by nominal isobars and isomers. Therefore, many presumptive positive specimens require confirmation by a higher specificity second-tier assay employing separations, which require additional time and resources prior to patient follow-up. A 3.2-mm punch was taken from dried blood spot (DBS) specimens and extracted using a solution containing isotopically labeled internal standards for quantification. Analyses were carried out in positive mode using a commercially available microfluidic capillary electrophoresis (CE) system coupled to a high-resolution mass spectrometer (HRMS). The CE-HRMS platform quantified 35 first- and second-tier biomarkers from a single injection in <2-min acquisition time, thus, successfully multiplexing first- and second-tier NBS for over 20 disorders in a single DBS punch. The CE-HRMS platform resolved problematic isobars and isomers that affect first-tier FIA-MS/MS assay specificity, while achieving similar quantitative results and assay linearity. Our CE-HRMS assay is capable of multiplexing first- and second-tier NBS biomarkers into a single assay with an acquisition time of <2 min. Such an assay would reduce the volume of false positives and inconclusive specimens flagged for second-tier screening.

PgHMA: Application of the heteroduplex mobility assay analysis in phylogenetics and population genetics.

The heteroduplex mobility assay (HMA) has proven to be a robust tool for the detection of genetic variation. Here, we describe a simple and rapid application of the HMA by microfluidic capillary electrophoresis, for phylogenetics and population genetic analyses (pgHMA). We show how commonly applied techniques in phylogenetics and population genetics have equivalents with pgHMA: phylogenetic reconstruction with bootstrapping, skyline plots, and mismatch distribution analysis. We assess the performance and accuracy of pgHMA by comparing the results obtained against those obtained using standard methods of analyses applied to sequencing data. The resulting comparisons demonstrate that: (a) there is a significant linear relationship (R2 =.992) between heteroduplex mobility and genetic distance, (b) phylogenetic trees obtained by HMA and nucleotide sequences present nearly identical topologies, (c) clades with high pgHMA parametric bootstrap support also have high bootstrap support on nucleotide phylogenies, (d) skyline plots estimated from the UPGMA trees of HMA and Bayesian trees of nucleotide data reveal similar trends, especially for the median trend estimate of effective population size, and (e) optimized mismatch distributions of HMA are closely fitted to the mismatch distributions of nucleotide sequences. In summary, pgHMA is an easily-applied method for approximating phylogenetic diversity and population trends.

Pharmacokinetic and metabolic analysis of an Alzheimer's disease therapeutic in rat serum via microfluidic CZE–MS

Sensitive, high-throughput methods for pharmacokinetic (PK) profiling are essential for potential therapeutics during critical stages of clinical trials. The application of a microfluidic capillary zone electrophoresis mass spectrometry (CZE-MS) method for PK profiling allows for rapid, sensitive and in-depth analysis of multiple samples within a short timeframe. Here, a CZE-MS approach for PK analysis was compared with a traditional UHPLC-MS approach when analyzing serum extracts from rats treated with a potential Alzheimer's disease therapeutic, BNC-1. Resulting PK data generated from both methods displayed statistical similarities. Additionally, the separation efficiency attributed to the use of the CZE-MS method provided substantial metabolic regulation data that was not apparent in the UHPLC-MS method. Additionally, the coupling of the CZE-MS method to the data processing software, MZmine2, was used to monitor changes in metabolism and observe putative BNC-1-derived metabolites. The ability to perform fast analyses without sacrificing sensitivity or metabolic information suggests that this CZE-MS method is ideal for metabolomics-inclusive, high-throughput PK profiling.

On the Feasibility of Informative Biosignature Measurements Using an Enceladus Plume Organic Analyzer

Abstract The icy moons of Saturn and Jupiter are high priority locations in which to search for biomarkers of life elsewhere in our solar system. In particular, the ice plumes that jet out through the ice surface at Enceladus provide an enticing opportunity to sample the organic components of its subsurface ocean for possible biosignatures while ameliorating challenges of planetary protection. Extensive high- and hypervelocity light gas gun experiments, the first to model the Enceladus ice plume encounter accurately, have demonstrated that organic-rich ice particles can be efficiently captured using plume transits at and below 3 km s−1 without significant organic destruction. Realistic scenarios for the amount of ice captured and for the ability of organic chemical analysis instruments, especially microfluidic capillary electrophoresis with laser-induced fluorescence, to produce meaningful detection levels for biosignatures are then presented and compared with results from terrestrial models. This analysis indicates that an Enceladus plume fly-by mission carrying microfluidic instrumentation is capable of biosignature measurements with a sensitivity and analytical capability that is highly informative about the extent of extraterrestrial biological processes.

Rapid and comprehensive monoclonal antibody Characterization using microfluidic CE-MS

The identification and control of monoclonal antibody (mAb) critical quality attributes (CQAs) is a key component of quality by design (QbD). In this work, rapid peptide mapping and native intact charge variants analysis have been developed to comprehensively characterize and monitor mAb CQAs using a microfluidic capillary electrophoresis - mass spectrometry (CE-MS) platform.The ultrafast peptide mapping simultaneously analyzed multiple CQAs, including protein primary structure, oxidation, deamidation, succinimide, C-terminal lysine (Lys) clipping, N-terminal cyclization, and glycosylation. The microfluidic CE-MS based peptide mapping acquired results comparable to conventional but lengthy liquid chromatography – MS (LC-MS) based approach.The native intact analysis resolved mAb charge variants with a comparable resolution as commonly achieved using capillary isoelectric focusing (cIEF). Charge variants’ identities were assigned based on characteristic mass shifts, knowledge learned from peptide mapping, and changes in electrophoretic mobility. Major mAb glycoforms of each charge variants were resolved and identified in the deconvoluted mass spectra.Furthermore, a model simulation was performed to reconstruct intact deconvoluted mass spectra using peptide mapping results. The reconstructed and experimentally determined intact deconvoluted mass spectra were highly correlated, suggesting that our data collected at the peptide level and intact level were consistent and highly comparable.Overall, the microfluidic CE-MS based peptide mapping and native intact charge variants analysis are high-throughput methods that have great potential to support biopharmaceutical development.

Specific Analysis of α-2,3-Sialylated N-Glycan Linkage Isomers by Microchip Capillary Electrophoresis-Mass Spectrometry.

Sialylated N-glycan isomers with α-2,3 and α-2,6 linkages play crucial and distinctive roles in diverse physiological and pathological processes. Changes of α-2,3-linked sialic acids in sialylated N-glycans are especially important in monitoring the initiation and progression of diseases. However, the specific analysis of α-2,3-sialylated N-glycan linkage isomers remains challenging due to their extremely low abundance and technical limitations in separation and detection. Herein, we designed an integrated strategy that combines linkage-specific derivatization and a charge-sensitive separation method based on microfluidic chip capillary electrophoresis-mass spectrometry (microchip CE-MS) for specific analysis of α-2,3-sialylated N-glycan linkage isomers for the first time. The α-2,6- and α-2,3-sialic acids were selectively labeled with methylamine (MA) and N,N-dimethylethylenediamine (DMEN), respectively, which selectively makes α-2,3-sialylated N-glycans positively charged and realizes online purification, concentration, and discrimination of α-2,3-sialylated N-glycans from other N-glycans in microchip CE-MS. This new approach was demonstrated with standard multisialylated N-glycans, and it was found that only the α-2,3-sialylated N-glycans migrated and were detected in order according to the number of α-2,3-sialic acids. Finally, this strategy was successfully applied in highly sensitive profiling and reproducible quantitation of the serum α-2,3-sialylated N-glycome from ovarian cancer (OC) patients, where 7 of 33 detected α-2,3-sialylated N-glycans significantly changed in the OC group compared with healthy controls.

Chip-Based Capillary Zone Electrophoresis Mass Spectrometry for Rapid Resolution and Quantitation of Critical Quality Attributes in Protein Biotherapeutics.

The aspiration of the multi-attribute method (MAM) is to utilize a single mass spectrometry-based method that can measure multiple attributes simultaneously, thus enabling data-driven decisions more quickly and efficiently. However, challenges associated with identifying and quantitating critical quality attributes such as asparagine deamidation and isoaspartic acid using conventional ultrahigh-pressure liquid chromatography (UHPLC) coupled to mass spectrometry have necessitated long gradients to ensure sufficient separation for quantitation. Microfluidic chip-based capillary zone electrophoresis mass spectrometry (CZE-MS) shows potential to enable rapid charge-based separation of peptide mixtures, and this approach was evaluated using multipeptide mixtures of synthetic peptides as well as digested protein therapeutics. In these experiments, repeatability, linearity, and peak-to-peak resolution of several peptide families containing asparagine deamidation and/or isoaspartic acid were demonstrated. In addition, a comparison of peptide map results acquired with both UHPLC-MS and CZE-MS for two enzymatically digested biological therapeutics showed comparable sequence coverage and quantitation results between the two approaches. As MAM becomes increasingly utilized for analysis of biological therapeutics, MS instrument demand will rapidly increase, resulting in a bottleneck. A CZE-based separation shows potential to alleviate this bottleneck by drastically increasing MAM throughput while providing results comparable to those acquired using conventional UHPLC separations.

Genotyping of Duffy Blood Group by Microchip Capillary Electrophoresis

To explore a method for rapidly screening the Duffy blood group genotypes and to establish an information bank of rare blood type donors. The microfluidic capillary electrophoresis system and PCR-SSP method were used to analyze the Duffy genotype of 3 936 unrelated O-type blood donors in our center from December 2014 to September 2018. The serologic identification and typing of other blood type system phenotypes for FYa-negative specimen were performed. The Fy (a-b-) specimen was sequenced for genotyping. The phenotypes of FYa-negative specimens were consistent with the genotyping results of the microfluidic capillary electrophoresis system. The results of Duffy phenotyping in 3 936 specimens were follows: Fy (a+b-) 3 564 (90.54%), Fy(a+b+) 353 (8.97%), Fy(a-b+) 18 (0.46%), Fy (a-b-) 1 (0.03%). The gene frequency was FY*A (95.03%), FY*B (4.94%), and FY*Null (0.03%). The bank of rare blood types of 19 FYa-negative specimens was established. Microfluidic capillary electrophoresis system is suitable for Duffy blood group genotyping screening. It can be used to establish a bank of rare blood type, so as to solve the problem of urgent blood transfusion in patients with rare blood type, and to improve blood transfusion safety.