Free-flow Isoelectric Focusing Research Articles

Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.

Separation and purification of active ingredients in tobacco by free-flow electrophoresis.

The active ingredients from tobacco extracts were continuously separated and purified using a homemade free-flow electrophoresis apparatus. A rectangular free flow electrophoresis device was constructed for the continuous separation and preparation, and the operating conditions of the device were optimized. The fractions obtained from the free-flowing component collection unit were then detected by HPLC and GC-MS. The results showed that a 90% methanol-water solution could maximize the extraction of the active components from tobacco. Chlorogenic acid and nicotine were enriched in three and four of 24 fractions, respectively, after free-flow isoelectric focusing electrophoresis. 2-Hydroxy-2-cyclopentene-1-one, 1-(2-methyl-1,3-oxathiolan-2-yl) ethanone, nornicotine, cotinine, and scopolamine were separated and enriched synchronously. Overall, the use of free-flow electrophoresis technology for the separation and purification of the active substances in tobacco can improve the comprehensive utilization rate of tobacco.

Multi-channel contactless conductivity detection device for online detection of free-flow electrophoresis separation

现有自由流电泳(FFE)装置因不具备在线检测功能,其实用性仍然存在明显不足。针对这一问题,该工作发展了一种多通道电容耦合式非接触电导检测(MC-C4D)装置并开发了自动测量软件。MC-C4D装置采用了并行分时的非接触电导检测技术,即由多个同样的非接触电导检测模块并行排列,而单个电导检测模块又由多个非接触电导检测池组成,采用模拟开关切换这些检测池,能够分时检测流经相应检测池溶液的电导率。多个电导检测模块的检测池总数等于FFE的组分数,它们分别串行接入到FFE各流路中,这样MC-C4D装置就可在线并行分时在线测量各组分溶液的电导率。为验证所设计MC-C4D装置的检测性能,采用配制的氯化钾标准溶液作为检测对象对MC-C4D装置进行了标定和测试。实验数据表明,MC-C4D装置电导率检测范围为0.015~2.5 mS/cm,检出限(LOD)为0.002 mS/cm,日内相对标准偏差(RSD, n=3)为2.31%,测量相对误差(RE)为3.03%和通道间测量相对偏差为1.60%,这些参数表明该装置检测范围较大,LOD低,重复性好,准确性高,通道间测量相对偏差小。另外,将MC-C 4D装置应用于往复式自由流等电聚焦电泳(RFFIEF)在蛋白质聚焦过程中对各组分溶液电导率进行实时在线检测,结果表明,所开发的MC-C4D装置不仅可实现对FFE各组分溶液电导率的实时在线检测,而且还可在RFFIEF实验中辅助掌握分离的实验进度,提高FFE装置的实用性。

In-Depth Metaproteomics Analysis of Oral Microbiome for Lung Cancer.

The human oral microbiome correlates with numerous diseases, including lung cancer. Identifying the functional changes by metaproteomics helps understand the disease-related dysbiosis, yet characterizing low-abundant bacteria is challenging. Here, we developed a free-flow isoelectric focusing electrophoresis-mass spectrometry- (FFIEF-MS-) based metaproteomics strategy to reduce host interferences and enrich low-abundant bacteria for in-depth interpretation of the oral microbiome. With our method, the number of interfering peptides decreased by 52.87%, whereas the bacterial peptides and species increased by 94.97% and 44.90%, respectively, compared to the conventional metaproteomics approach. We identified 3647 bacterial proteins, which is the most comprehensive oral metaproteomics study to date. Lung cancer-associated bacteria were validated among an independent cohort. The imbalanced Fusobacterium nucleatum and Prevotella histicola and their dysregulated functions in inhibiting immune response and maintaining cell redox homeostasis were revealed. The FFIEF-MS may serve as a valuable strategy to study the mechanisms between human diseases and microbiomes with broader applications.

Reciprocating free-flow isoelectric focusing with online array ultraviolet detector for process monitoring of protein separation

Free-flow isoelectric focusing (FFIEF) is a useful tool for separating and purifying proteins, DNA, cells, and organelles, etc. However, the online monitoring of each fraction during an FFIEF run has not been achieved yet, resulting in a lack of process monitoring of FFIEF. Herein, an online array ultraviolet (UV) detection system was developed for the easy assay of FFE fractions. The detector was integrated with an apparatus of FFIEF with 32 fractions to show the online monitoring, and bovine serum albumin (BSA) and lysozyme were chosen as the model proteins for manifesting the UV detector performance. The experiments revealed that (i) all the fluidic cells had good linearity from 0.03 to 10 mg/mL BSA and fair limits of detection (LODs) of 0.01 mg/mL; (ii) all the cells had good uniformity of UV absorbance; and (iii) the deviations of intra-day and inter-day of UV detector were respectively 3.8% and 5.8%, indicating the fair stability of the UV detector. The UV detector could be well used for the process monitoring of two model proteins through the whole FFIEF run, and the online absorbance assay of proteins at the end of FFIEF. The UV detector herein had the evident potential for rapid and convenient assay of protein fraction in FFIEF as well as other FFE modes.

ElectroMicroTransport v2107: Open-source toolbox for paper-based electromigrative separations

electroMicroTransport v2107: Open-source toolbox for paper-based electromigrative separations

Numerical simulations of paper-based electrophoretic separations with open-source tools.

A new tool for the solution of electromigrative separations in paper-based microfluidics devices is presented. The implementation is based on a recently published complete mathematical model for describing these types of separations, and was developed on top of the open-source toolbox electroMicroTransport, based on OpenFOAM® , inheriting all its features as native 3D problem handling, support for parallel computation, and a GNU GPL license. The presented tool includes full support for paper-based electromigrative separations (including EOF and the novel mechanical and electrical dispersion effects), compatibility with a well-recognized electrolyte database, and a novel algorithm for computing and controlling the electric current in arbitrary geometries. Additionally, the installation on any operating system is available due to its novel installation option in the form of a Docker image. A validation example with data from literature is included, and two extra application examples are provided, including a 2D free-flow IEF problem, which demonstrates the capabilities of the toolbox for dealing with computational and physicochemical modeling challenges simultaneously. This tool will enable efficient and reliable numerical prototypes of paper-based electrophoretic devices to accompany the contemporary fast growth in paper-basedmicrofluidics.

Free-Flow Isoelectric Focusing for Comprehensive Separation and Analysis of Human Salivary Microbiome for Lung Cancer.

Human microbiome contains billions of microorganisms that play important roles in the biological system and different diseases. Due to its complexity, conventional culture-independent technology may underestimate the value of low-abundance bacteria, which calls for a highly efficient method for its enrichment and comprehensive analysis. In this study, we developed a recycling free-flow isoelectric focusing (RFFIEF) method-based electrophoresis method to separate salivary microbiome. First, we used Escherichia coli (DH5α) as a model for RFFIEF method development, which was focused in a narrow pH range (0.38 pH unit). The recovery rate was 80.81% with 5.85% relative standard deviation (n = 5). The optimized method was then adopted to separate the human salivary microbiome into 32 fractions, followed by 16S rRNA gene sequencing and metaproteomics analysis. After RFFIEF fractionation, we identified 508 bacterial genera, which increased by 225% on average (n = 3) when compared to the results before fractionation. We further compared the compositional change of microbiome in the saliva of lung cancer group (n = 22) and control group (n = 21) through RFFIEF. Quantitative results demonstrated that six bacterial genera were upregulated dramatically in the lung cancer group, while two genera were downregulated. Through qPCR verification in an independent sample set (n = 48), we confirmed that genus Granulicatella was significantly upregulated in the lung cancer group, whereas Pseudomonas was remarkably downregulated (p < 0.001). RFFIEF is an efficient and reproducible technology to fractionate the microbiome for its comprehensive analysis, which can be further applied to the in-depth study of the complex microbiomes and contribute to the discovery of disease-associated bacteria.

Carrier ampholyte-free free-flow isoelectric focusing for separation of protein.

Free-flow isoelectric focusing (FFIEF) has the merits of mild separation conditions, high recovery and resolution, but suffers from the issues of ampholytes interference and high cost due to expensive carrier ampholytes. In this paper, a home-made carrier ampholyte-free FFIEF system was constructed via orientated migration of H+ and OH- provided by electrode solutions. When applying an electric field, a linear pH gradient from pH 4 to 9 (R2 = 0.994) was automatically formed by the electromigration of protons and hydroxyl ions in the separation chamber. The carrier ampholyte-free FFIEF system not only avoids interference of ampholyte to detection but also guarantees high separation resolution by establishing stable pH gradient. The separation selectivity was conveniently adjusted by controlling operating voltage and optimizing the composition, concentration and flow rate of the carrier buffer. The constructed system was applied to separation of proteins in egg white, followed by MADLI-TOF-MS identification. Three major proteins, ovomucoid, ovalbumin and ovotransferrin, were successfully separated according to their pI values with 15mmol/L Tris-acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9mL/min.

Microfluidic Free-Flow Isoelectric Focusing with Real-Time pI Determination.

Free-flow electrophoresis (FFE) may be used for continuous and preparative separation of a wide variety of biomolecules. Isoelectric focusing (IEF) provides for the separation of compounds according to their isoelectric point (pI). Here we describe a microfluidic chip-based protocol for the fabrication, application, and optical monitoring of free-flow isoelectric focusing (FFIEF) of proteins and peptides on the microscale with optical surveillance of the microscopic pH gradient provided by an integrated pH sensing layer. This protocol may be used with modifications also for the FFIEF of other biomolecules and may serve as template for the fabrication of microfluidic chips with integrated fluorescent or luminescent pH sensor layers for FFE and other applications.

Preparation of intact mitochondria using free-flow isoelectric focusing with post-pH gradient sample injection for morphological, functional and proteomics studies

Mitochondria play essential roles in both energy metabolism and cell signaling, which are critical for cell survival. Although significant efforts have been invested in understanding mitochondrial biology, methods for intact mitochondria preparation are technically challenging and remain to be improved. New methods for heterogeneous mitochondria purification will therefore boost our understanding on their physiological and biophysical properties. Herein, we developed a novel recycling free-flow isoelectric focusing (RFFIEF) with post-pH gradient sample injection (post-PGSI) for preparative separation of mitochondria. Crude mitochondria of rabbit liver obtained from differential centrifugation were purified by the developed method according to their pI values as six fractions. Transmission electron microscope images revealed that intact mitochondria existed in two fractions of pH 6.24 and 6.61, degenerative mitochondria were in two fractions of pH 5.46 and 5.72, and inner membrane vesicles (IMVs) appeared in the fractions of pH 4.70 and 5.04. Membrane potential measurement proved a dramatic difference between intact mitochondria and IMVs, which reflected the bioactivity of obtained populations. Particularly, proteomics analyses revealed that more number of proteins were identified in the intact fractions than that of IMVs or crude mitochondria, which demonstrated that RFFIEF could be powerful tool for the preparation of intact organelle as well as their proteomic and in-depth biological analysis.

Broadening of analyte streams due to a transverse pressure gradient in free-flow isoelectric focusing

Pressure-driven cross-flows can arise in free-flow isoelectric focusing systems (FFIEF) due to a non-uniform electroosmotic flow velocity along the channel width induced by the pH gradient in this direction. In addition, variations in the channel cross-section as well as unwanted differences in hydrostatic heads at the buffer/sample inlet ports can also lead to such pressure-gradients which besides altering the equilibrium position of the sample zones have a tendency to substantially broaden their widths deteriorating the separations. In this situation, a thorough assessment of stream broadening due to transverse pressure-gradients in FFIEF devices is necessary in order to establish accurate design rules for the assay. The present article describes a mathematical framework to estimate the noted zone dispersion in FFIEF separations based on the method-of-moments approach under laminar flow conditions. A closed-form expression has been derived for the spatial variance of the analyte streams at their equilibrium positions as a function of the various operating parameters governing the assay performance. This expression predicts the normalized stream variance under the chosen conditions to be determined by two dimensionless Péclet numbers evaluated based on the transverse pressure-driven and electrophoretic solute velocities in the separation chamber, respectively. Moreover, the analysis shows that while the stream width can be expected to increase with an increase in the value of the first Péclet number, the opposite trend will be followed with respect to the latter. The noted results have been validated using Monte Carlo simulations that also establish a time/length scale over which the predicted equilibrium stream width is attained in the system.

Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing.

Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free-flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free-flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins.

Continuous on-chip fluorescence labelling, free-flow isoelectric focusing and marker-free isoelectric point determination of proteins and peptides.

We present a microfluidic platform that contains a micro flow reactor for on-chip biomolecule labelling that is directly followed by a separation bed for continuous free-flow electrophoresis and has an integrated hydrogel-based near-infrared fluorescent pH sensor layer. Using this assembly, labelling of protein and peptide mixtures, their separation via free-flow isoelectric focusing and the determination of the isoelectric point (pI) of the separated products via the integrated sensor layer could be carried out within typically around 5 minutes. Spatially-resolved immobilization of fluidic and sensing structures was carried out via multistep photolithography. The assembly was characterized and optimized with respect to their fluidic and pH sensing properties and applied in the IEF of model proteins, peptides and a tryptic digest from physalaemine. We have therefore realized continuous sample preparation and preparative separation, analyte detection, process observation and analyte assignment capability based on pI on a single platform the size of a microscope slide.

Reciprocating free-flow isoelectric focusing device for preparative separation of proteins

The traditional recycling free-flow isoelectric focusing (RFFIEF) suffered from complex structure, tedious operations and poor extensibility as well as high cost. To address these issues, a novel reciprocating free-flow isoelectric focusing device (ReFFIEF) was developed for proteins or peptides pre-fractionation. In the new device, a reciprocating background flow was for the first time introduced into free flow electrophoresis (FFE) system. The gas cushion injector (GCI) used in the previous continuous free-flow electrophoresis (CFFE) was redesigned for the reciprocating background flow. With the GCI, the reciprocating background flow could be achieved between the GCI, separation chamber and transient self-balance collector (tSBC). In a run, process fluid flowed to and from, forming a stable reciprocating fluid flow in the separation chamber. A pH gradient was created within the separation chamber, and at the same time proteins were focused repeatedly when passing through the chamber under perpendicular electric field. The ReFFIEF procedure was optimized for fractionations of three model proteins, and the optimized method was further used for pre-fractionation of model human serum samples. As compared with the traditional RFFIEF devices developed about 25 years ago, the new ReFFIEF system showed several merits, such as simple design and structure, user-friendly operation and easy to extend as well as low cost.

Application of Optical pH Sensors in the Microfluidic Free-flow Isoelectric Focusing of Biomolecules

We present methods for fabrication and application of microfluidic chips for free-flow electrophoresis (FFE) that allow integrated pH sensing. Monitoring of the pH gradient in microfluidic free-flow isoelectric focusing (FFIEF) allows on-line adjustment of the parameters for electrophoresis and evaluation of the isoelectric point (pI) of focused analyte bands almost in real time. The fabrication of these assemblies is demonstrated via multistep photopolymerization or a laser cutting and lamination technique. Integration of pH sensing layers is accomplished via photopolymerization or inkjet printing of hydrogels that contain covalently attached pH probes. These microchips were characterized and employed for the FFIEF of proteins, peptides and plasma. Multispectral microscopic imaging was used to separate the pH sensor and the biomolecule fluorescence. We also show pH sensing in FFE based on time-domain dual lifetime referencing (t-DLR), a microreactor for integrated labelling prior to the FFIEF and the isoelectric focusing of unlabeled compounds.

Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.

We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and β-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.

Rapid isoelectric point determination in a miniaturized preparative separation using jet-dispensed optical pH sensors and micro free-flow electrophoresis.

Herein, the fabrication, characterization, calibration, and application of integrated microfluidic platforms for fast isoelectric point (pI) determinations via free-flow electrophoresis with integrated inkjet-printed fluorescent pH sensor microstructures are presented. These devices allow one to determine the pI of a biomolecule from a sample mixture with moderately good precision and without addition of markers in typically less than 10 s total separation and analysis time. Polyhydroxyethyl methacrylate (pHEMA) hydrogels were covalently coupled with fluorescein and hydroxypyrene trisulfonic acid (HPTS)-based pH probes. These were piezoelectrically jet-dispensed onto acrylate-modified glass as pH sensor microarrays with a diameter of 300-600 μm and thicknesses of 0.4-2.4 μm with high spatial accuracy. Microchip fabrication and integration of these pH sensor arrays was realized by multistep liquid-phase photolithography from oligoethylene glycol precursors resulting in glass-based microfluidic free-flow isoelectric focusing (μFFIEF) chips with integrated pH observation capabilities. The microchips were characterized with regard to pH sensitivity, response times, photo-, and flow stability. Depending on the sensor matrix, they allowed IEF within a pH range of roughly 5.5-10.5 with good sensitivity and fast response times. These microchips were used for FFIEF of small molecule markers and several protein mixtures with simultaneous monitoring of local pH. This allowed the determination of their pI via multispectral imaging of protein and pH sensor fluorescence without addition of external markers. Obtained pI's were generally in good agreement with known data, demonstrating the applicability of the method for pI determination in micropreparative procedures within a time frame of a few seconds only.

Mathematical and numerical model to study two-dimensional free flow isoelectric focusing.

Even though isoelectric focusing (IEF) is a very useful technique for sample concentration and separation, it is challenging to extract separated samples for further processing. Moreover, the continuous sample concentration and separation are not possible in the conventional IEF. To overcome these challenges, free flow IEF (FFIEF) is introduced in which a flow field is applied in the direction perpendicular to the applied electric field. In this study, a mathematical model is developed for FFIEF to understand the roles of flow and electric fields for efficient design of microfluidic chip for continuous separation of proteins from an initial well mixed solution. A finite volume based numerical scheme is implemented to simulate two dimensional FFIEF in a microfluidic chip. Simulation results indicate that a pH gradient forms as samples flow downstream and this pH profile agrees well with experimental results validating our model. In addition, our simulation results predict the experimental behavior of pI markers in a FFIEF microchip. This numerical model is used to predict the separation behavior of two proteins (serum albumin and cardiac troponin I) in a two-dimensional straight microchip. The effect of electric field is investigated for continuous separation of proteins. Moreover, a new channel design is presented to increase the separation resolution by introducing cross-stream flow velocity. Numerical results indicate that the separation resolution can be improved by three folds in this new design compare to the conventional straight channel design.

Switchable pH actuators and 3D integrated salt bridges as new strategies for reconfigurable microfluidic free-flow electrophoretic separation

We present novel strategies for reconfigurable, high-throughput microfluidic free-flow electrophoretic separation using electrically switchable pH actuators and 3D integrated salt bridges to allow rapid formation of stable pH gradients and efficient electrophoresis. The pH actuator is achieved by microfluidic integration of bipolar membranes which change electrolyte pH by injecting excess H(+) or OH(-) ions produced by a field-enhanced water dissociation phenomenon at the membrane junction upon voltage bias. The technique does not require conventional multiple buffer inflows and leaves no gas production as experienced in electrolysis, thus providing stable pH gradients for isoelectric focusing (IEF) separation. With the pH actuator inactivated, the platform can perform zone electrophoretic (ZE) separation in a medium of constant pH. We also describe the use of 3D integrated ion conductive polymers that serve as salt bridges for improving the voltage efficiency of electrophoresis and to allow high throughput. The proof of concept was successfully demonstrated for free-flow IEF and ZE separation of protein mixtures showing the potential and the simplicity of the platform for high-throughput and high-precision sample separation.