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Protein therapeutics are usually produced in heterogeneous forms during bioproduction and bioprocessing. Heterogeneity results from post-translational modifications that can yield charge variants and require characterization throughout product development and manufacturing. Isoelectric focusing (IEF) with UV detection is one of the most common methods to evaluate protein charge heterogeneity in the biopharmaceutical industry. To identify charge variant peaks, a new imaged microfluidic chip-based isoelectric focusing (icIEF) system coupled directly to mass spectrometry was recently reported. Bridging is required to demonstrate comparability between existing and new technology. As such, here we demonstrate the comparability of the pI value measurement and relative charge species distributions between the icIEF-MS system and the control data from a frequently utilized methodology in the biopharmaceutical industry for several blinded development-phase biopharmaceutical monoclonal antibodies across a wide pI range of 7.3-9.0. Hyphenation of the icIEF system with mass spectrometry enabled direct and detailed structural determination of a test molecule, with masses suggesting acidic and basic shifts are caused by sialic acid additions and the presence of unprocessed lysine residues. In addition, MS analysis further identified several low-abundance glycoforms. The icIEF-MS system provides sample quantification, characterization, and identification of mAb proteoforms without sacrificing icIEF quantification comparability or speed.

Free-flow electrophoresis (FFE) enables the continuous separation and collection of charged solutes, and as a result, it has drawn interest as both a preparative and an analytical tool for biological applications. Recently, a free-flow counterflow gradient focusing (FF-CGF) mechanism has been proposed with the goal of improving the resolution and versatility of FFE. To realize this potential, the factors that influence solute dispersion deserve further attention, including the gradient strength and the parabolic profile of the counterflow. Therefore, the goal of this work is to develop a theoretical model to study the interplay between these factors and molecular diffusion. Overall, an asymmetric solute distribution emerges for a wide range of parameters, and this behavior can be characterized with an exponentially modified Gaussian function. Results show that FF-CGF can achieve high-resolution separations, with the potential for high-throughput protein purification. Moreover, this work provides a practical guide for optimizing experimental conditions, as well as a strong framework for understanding and developing FF-CGF further.

The review provides a comprehensive overview of developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, microscale isolation, and physicochemical characterization of peptides from 2019 up to approximately the middle of 2021. Advances in the investigation of electromigration properties of peptides and in the methodology of their analysis, such as sample preparation, sorption suppression, EOF control, and detection, are presented. New developments in the individual CE and CEC methods are demonstrated and several types of their applications are shown. They include qualitative and quantitative analysis, determination in complex biomatrices, monitoring of chemical and enzymatic reactions and physicochemical changes, amino acid, sequence, and chiral analyses, and peptide mapping of proteins. In addition, micropreparative separations and determination of significant physicochemical parameters of peptides by CE and CEC methods are described.

An online method involving transient electrokinetic dosing and ITP with neutralization reaction boundary (NRB) and/or carrier ampholyte-free isoelectric focusing (CAF IEF) was developed for the preconcentration, preseparation, and analytical determination of glyphosate in aqueous samples containing low concentrations of the analyte of interest. Various parameters were investigated in the framework of an optimization study with the aim of achieving the maximum concentration limit of detection (cLOD) decrease in minimum time. The proposed method used CAF IEF and/or ITP with NRB. The sample was dosed to the column on the stationary reaction boundary (CAF IEF) and/or moving reaction boundary (ITP with NRB), whereat a sharp pH step exists. Here, charge reversal was due to the ampholytes, and/or acid accumulation occurred because of charge loss. Similarly, the accumulated sample was mobilized with TE and analyzed using classical ITP in the second analytical column. Glyphosate (GLY), the analyte of interest, was chosen as a model substance for ITP with NRB and preconcentration as well as focusing preconcentration and CAF IEF using the asymmetric purpose-built NRB. On one side of the asymmetric boundary was the zone of acidic pH; while the opposite side comprised a neutral/basic non-conductive zone of the ampholyte-in this case, GLY. Such an arrangement enables the use of a lower pH on the acidic side, which allows the focusing of strongly acidic ampholytes and the accumulation of weak acids. The electrolyte composition and the dosing time were optimized, and a 14-fold accumulation was achieved in 25 min compared to that by classical ITP and a 180-fold accumulation was achieved through CAF IEF and preconcentration with a glyphosate sample. Both methods are simple and can be conducted using all commercial ITP systems.

Capillary electrophoresis (CE), after being introduced several decades ago, has carved out a niche for itself in the field of analytical characterization of biopharmaceutical products. It does not only offer fast separation, high resolution in miniaturized format, but equally importantly represents an orthogonal separation mechanism to high-performance liquid chromatography. Therefore, it is not surprising that CE-based methods can be found in all major pharmacopoeias and are recommended for the analysis of biopharmaceutical products during process development, characterization, quality control, and release testing. Different separation formats of CE, such as capillary gel electrophoresis, capillary isoelectric focusing, and capillary zone electrophoresis are widely used for size and charge heterogeneity characterization as well as purity and stability testing of therapeutic proteins. Hyphenation of CE with MS is emerging as a promising bioanalytical tool to assess the primary structure of therapeutic proteins along with any impurities. In this review, we confer the latest developments in capillary electrophoresis, used for the characterization of critical quality attributes of biopharmaceutical products covering the past 6 years (2015-2021). Monoclonal antibodies, due to their significant share in the market, have been given prioritized coverage.

In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor‐binding domain (RBD) with His‐ and Fc‐tag, the S1 subunit with His‐tag, the S1/S2 subunits with His‐tag and the human angiotensin‐converting enzyme 2 (hACE2) with His‐tag. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The determined pI range for the RBD/Fc is 8.24–9.32 (theoretical pI: 8.55), for the RBD/His it is 7.36–9.88 (8.91) and for the S1/His it is 7.30–8.37 (7.80). The pI range of the S1/S2/His is 4.41–5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19–6.11 (5.60) for all experimental conditions chosen. All theoretically derived values were found within these ranges, usually close to the center. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS‐CoV‐2 proteins. The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes.

Electrokinetic processes that lead to pH gradient instabilities in carrier ampholyte-based IEF are reviewed. In addition to electroosmosis, there are four of electrophoretic nature, namely (i) the stabilizing phase with the plateau phenomenon, (ii) the gradual isotachophoretic loss of carrier ampholytes at the two column ends in presence of electrode solutions, (iii) the inequality of the mobilities of positively and negatively charged species of ampholytes, and (iv) the continuous penetration of carbonate from the catholyte into the focusing column. The impact of these factors to cathodic and anodic drifts was analyzed by simulation of carrier ampholyte-based focusing in closed and open columns. Focusing under realistic conditions within a 5 cm long capillary in which three amphoteric low molecular mass dyes were focused in a pH 3-10 gradient formed by 140 carrier ampholytes was investigated. In open columns, electroosmosis displaces the entire gradient toward the cathode or anode whereas the electrophoretic processes act bidirectionally with a transition around pH 4 (drifts for pI > 4 and pI

A two‐step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non‐denaturing separation conditions. CIEF of the intact mAb was combined with a middle‐down approach analyzing Fc/2 and F(ab´)2 fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8–10.5 with PL 3–10 for variants of intact rituximab and of F(ab´)2 fragments, respectively, whereas PL 6.7–7.7 in combination with PL 3–10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)2. In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C‐terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.

Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpKa = pKamonoanion - pKamonocation

A microfluidic system has been designed that integrates both imaged capillary isoelectric focusing (iCIEF) separations and downstream MS detection into a single assay. Along with the construction of novel instrumentation and an innovative microfluidic chip, conversion to MS‐compatible separation reagents has also been established. Incorporation of 280 nm absorbance iCIEF‐MS analysis not only permits photometric quantitation of separated charge isoforms but also facilitates the direct monitoring of analyte focusing and mobilization in real‐time. The outcome of this effort is a device with the unique ability to allow for both the characterization and identification of protein charge and mass isoforms in under 15 min. Acquisition, quantitation, and identification of highly resolved intact mAb charge isoforms along with their critical N‐linked glycan pairs clearly demonstrate analytical utility of our innovative system. In total, 33 separate molecular features were characterized by the iCIEF‐MS system representing a dramatic increase in the ability to monitor multiple intact mAb critical quality attributes in a single comprehensive assay. Unlike previously reported CIEF‐MS results, relatively high ampholyte concentrations, of up to 4% v/v, were employed without impacting MS sensitivity, observed to be on the order of 1% composition.

A novel analytical protocol was developed for general quality screening of chicken meat based on IEF and protein extraction. To demonstrate the developed protocol, 24 chickens were divided into three groups; each had eight chickens. The chickens in Group 1 were slaughtered by exsanguination, Group 2 asphyxiated in water, and that in Group 3 were infected by new castle disease virus. Proteins were extracted from the meat samples by using pure water as an extractant, separated by IEF, verified by western blot, and quantified via imaging analysis. The relevant experiments demonstrated that two myoglobin (Mb) bands were detected at pI 6.8 and 7.04 for all samples of Groups 1, 2, and 3, but there were additional hemoglobin (Hb) bands at pI 7.09 and 7.13 (P < 0.05) for the samples of Groups 2 and 3. The results implied that Hb bands might be a potential biomarker for the screening of chicken meat quality. The RSD values of two Mb bands (pI 6.8 and 7.04) in Group 1 were respectively 4.08 and 3.63%, the ones of two Hb bands (pI 7.09 and 7.13) in Group 2 were 3.66 and 2.10%, and those in Group 3 were 2.17% and 2.77%, respectively. All the RSD values indicated high stability and reliability of the developed protocol. Additionally, the protocol had a direct readout of protein bands in IEF without staining. However, it was time-consuming and had high cost. Even so, the relevant general method and finding have potential for screening of chicken meat quality.

Capillary electrophoresis (CE) offers fast and high-resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user-friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano-electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE-MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two-dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE-modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.

CE is central to the analysis, process development and approval of therapeutic monoclonal antibodies (mAbs). Recently, imaged capillary isoelectric focusing (icIEF) has emerged as a powerful technique for quantitative protein charge heterogeneity monitoring and characterization, particularly for mAbs. However, icIEF has yet to be validated for therapeutically relevant mAbs adhering to the ICH guideline (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use). Here, for the first time, icIEF technology was validated by 10 laboratories across 8 independent companies using a therapeutic mAb. The parameters of this method validation strictly follow the guideline of the ICH. This guideline includes specificity, precision, accuracy, linearity, range, LOQ and robustness. These results represent a significant step forward in standardizing the use of icIEF methods for the clinical approval of therapeutic mAbs.

Biopharmaceuticals are increasingly present in pharmaceutical therapy. These highly complex molecules are still challenging for development and quality control. Charge heterogeneity is next to size heterogeneity, glycan analysis, or peptide mapping one main aspect of their thorough characterization. This review focuses on the charge-based analysis of biopharmaceuticals through CE. An overview is given about the three separation techniques cIEF, imaged cIEF and CZE for the analyses of charge variants, emphasizing assays and their precision. Especially the developments in the experimental conditions have been given a high priority: sample preparation, capillary properties, anolytes and catholytes, carrier ampholytes, sacrificing agents, solubility enhancement, isoelectric point marker, mobilization techniques, and detection modes have been discussed. CIEF shows a particularly high heterogeneity in its methods. Therefore, a design space is suggested that states the most important adjustable parameters and their range. This enables an adequate design of experiments for individual sample separations, in order to accelerate and simplify method developments.

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.

Isoelectric focusing (IEF) is a powerful separation method, useful for resolving subtle changes in the isoelectric point of unlabeled proteins. While microfluidic IEF has reduced the separation times from the hours in traditional benchtop IEF to minutes, the enclosed devices hinder post-separation access to the sample for downstream analysis. The two-layer open IEF device presented here comprises a photopatterned hydrogel lid layer containing the chemistries required for IEF and a thin polyacrylamide bottom layer in which the analytes are separated. The open IEF device produces comparable minimum resolvable difference in isoelectric point and gradient stability to enclosed microfluidic devices while providing post-separation sample access by simple removal of the lid layer. Further, using simulations, we determine that the material properties and the length of the separation lanes are the primary factors that affect the electric field magnitude in the separation region. Finally, we demonstrate self-indexed photomasks for alignment-free fabrication of multi-domain hydrogels. We leveraged this approach to generate arrayed pH gradients with a total of 80 concurrent separation lanes, which to our knowledge is the first demonstration of multiple IEF separations in series addressed by a single pair of electrodes.

We present a simple method to estimate the isoelectric point (pI) of Tomato Bushy Stunt particles. We demonstrate that the combination of agarose gels with different pH buffers can be used to electrophorese the virus particles and their migration patterns can be compared. This method allows us to estimate the pI of the virus particles (wild type, wt, and genetically modified particles) and to monitor the effect of the pI of modified peptide side chains of the viral capsid subunit on the pI of the whole virus particle.

High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling.

The determination of an analytical solution to find the steady-state protein concentration distribution in IEF is very challenging due to the nonlinear coupling between mass and charge conservation equations. In this study, approximate analytical solutions are obtained for steady-state protein distribution in carrier ampholyte based IEF. Similar to the work of Svensson, the final concentration profile for proteins is assumed to be Gaussian, but appropriate expressions are presented in order to obtain the effective electric field and pH gradient in the focused protein band region. Analytical results are found from iterative solutions of a system of coupled algebraic equations using only several iterations for IEF separation of three plasma proteins: albumin, cardiac troponin I, and hemoglobin. The analytical results are compared with numerically predicted results for IEF, showing excellent agreement. Analytically obtained electric field and ionic conductivity distributions show significant deviation from their nominal values, which is essential in finding the protein focusing behavior at isoelectric points. These analytical solutions can be used to determine steady-state protein concentration distribution for experiment design of IEF considering any number of proteins and ampholytes. Moreover, the model presented herein can be used to find the conductivity, electric field, and pH field.

Two-dimensional (2D) gel electrophoresis is a well-proven proteomic technique; however, sample-specific optimisation can often be necessary in order to get consistent quantitation. In particular, plasma samples are often smeared on 2D gels making spot matching difficult. A variety of different sample preparation and 2D methods were tested by using sheep plasma, and it was found that lowering sample pH prior to precipitation, using a long voltage gradient for isoelectric focusing and the inclusion of carrier ampholytes in the electrode wicks, improved both the quality and consistency of spot resolution. Analysis of the internal standards from two different DIGE experiments, one with conventional methodology and one with the improved method, showed that along with substantially improving the number of spots resolved, the average CV (coefficient of variation) of matched standards was lower with the new method. 428 matched spots were found using the improved method compared to 208 matched spots using conventional methodology. For the 174 spots that were matched between the two DIGE experiments, the average CV's of spot volumes were also significantly lower, at 0.20 for the new method compared to 0.24 for the conventional method (p < 0.001).

This paper comprises a continuation of computer simulation studies dealing with carrier ampholyte based CIEF in presence of narrow pH gradients. With this technique, amphoteric sample components with pI values outside the pH gradient are migrating isotachophoretically toward the cathode or anode whereas components with pI values within the gradient become focused. In order to understand the processes occurring in presence of the electric field, the behavior of both carrier ampholytes and amphoteric sample components is investigated by computer modeling. Characteristics of two pH unit gradients with end components having pI values at or around 7.00 and conditions that lead to the formation of a water zone at neutrality were investigated. Data obtained reveal that a zone of water is formed in focusing with carrier ampholytes when the applied pH range does not cover the neutral region, ends at pH 7.00 or begins at pH 7.00. The presence of additional amphoteric components that cover the neutrality region prevent water zone formation under current flow. This situation is met in experiments with narrow pH gradients that end or begin around neutrality. Simulation data reveal that no water zone evolves when atmospheric carbon dioxide dissolved in the catholyte causes the migration of carbonic acid (in the form of carbonate and/or hydrogen carbonate ions) from the catholyte through the focusing structure. An electrolyte change in the electrode solution does not have an impact on the focusing part but does change the isotachophoretic pattern migrating behind the leading ion.

Lipid nanoparticles (LNPs) have been employed for drug delivery in small molecules, siRNA, mRNA, and pDNA for both therapeutics and vaccines. Characterization of LNPs is challenging because they are heterogeneous mixtures of large complex particles. Many tools for particle size characterization, such as dynamic and static light scattering, have been applied as well as morphology analysis using electron microscopy. CE has been applied for the characterization of many different large particles such as liposomes, polymer, and viruses. However, there have been limited efforts to characterize the surface charge of LNPs and CIEF has not been explored for this type of particle. Typically, LNPs for delivery of oligonucleotides contain at least four different lipids, with at least one being an ionizable cationic lipid. Here, we describe the development of an imaged capillary isoelectric focusing method used to measure the surface charge (i.e., pI) of an LNP‐based mRNA vaccine. This method is capable of distinguishing the pI of LNPs manufactured with one or more different ionizable lipids for the purpose of confirming LNP identity in a manufacturing setting. Additionally, the method is quantitative and stability‐indicating making it suitable for both process and formulation development.

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 15 mmol/L Tris-acetic acid (pH = 6.5) as carrier buffer at a flow rate of 12.9 mL/min.

In the presence of methanol and n-decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3-10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M-IPG). In addition, an online self-built platform for protein separation was established on account of the introduction of a cross-shaped unit and two short-off valves. In this platform, a cross-shaped unit was not only used to join the M-IPG column and a six-way injection valve (1.5 μL sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short-off valve in the tee unit or cross-shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7-proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M-IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A1c have been successfully resolved with the newly developed M-IPG column.

Counterflow gradient electrofocusing uses the bulk flow of a liquid solution to counterbalance the electrophoretic migration of an analyte. When either the bulk velocity or the electrophoretic velocity of the analyte is made to vary across the length of the channel, there exists a unique zero-velocity point for the analyte. This focusing method enables simultaneous separation and concentration of different analytes. The high resolution and sensitivity achieved are similar to that of isoelectric focusing, which separates analytes based on their isoelectric points, but the key difference is that analytes will instead focus based on their electrophoretic mobility. Dynamically changing the applied voltage or the counterflow rate over time will shift the zero-velocity point, and therefore allows the focused analytes to pass through a fixed detection point, or elute from the separation channel. Throughout the review, a number of different counterflow gradient techniques will be discussed, along with their recent advancements and potential applications.

An assessment of fractionated mouse hippocampal peptides was conducted. Protein extract from a single mouse hippocampus was enzymatically digested and fractionated by IEF. Aliquots of fractions were pooled into fewer, more complex samples. The unfractionated lysate, fractions, and pooled fractions were subjected to LC-MS/MS analysis. Samples consisting of many individual fractions had more protein identifications, greater protein sequence coverage, and quantified proteins with more spectral counts than protein extract that was unfractionated or pooled into fewer LC-MS/MS samples. Additionally, prefractionation reduced the median CV for spectral counts as much as 33%. However, the relative gain in proteome resolution was found to saturate with increasing fractionation extent. This study demonstrates how prefractionation by offline IEF can improve the resolution of proteomic investigations of the mouse hippocampus, and that a data-driven pooling methodology can reduce excessive and cost-ineffective fractionation.

IEF on immobilized pH gradient strips is a widespread tool for protein separation, especially as first dimension in commonly utilized 2DE. In the latter arrangement, separations are based on two orthogonal molecular characteristics according to pI in the first and molecular weight in the second dimension. However, the approach is time consuming, quantification is difficult and MS can be applied only offline. Capillary IEF and related IEF techniques in combination with MS provide similar information. The major benefits are high mass resolution and mass accuracy, reproducibility, speed, automation, and quantification by using a high-resolution mass spectrometer. However, online hyphenation of CIEF with MS is interfered by the ampholytes, acids, and bases needed for high-resolution IEF. This review will give an overview about important coupling techniques, like low ampholyte concentration, interim separation by chromatography, or the use of a dialysis interface to separate the analytes from interfering substances. It is focused on strategies which allow sensitive MS detection of CIEF-separated analytes. In addition, proteomic and biopharmaceutical applications of capillary IEF techniques combined with MS are briefly summarized.

Based on their characteristics, we hypothesized that the following parameters, namely collagen IV, glutathione S-transferase, secretory component (SC), and AMP-activated protein kinase α1α2 may be useful serum markers in the detection of comorbidities in treated HIV-infected patients. These parameters were determined in 204 HIV-infected patients and 35 controls by using IEF and densitometry. Collagen IV was undetectable in controls and the majority of HIV-infected patients. Twenty-two HIV-infected patients presented significantly elevated levels of collagen IV, most of them were coinfected with hepatitis C virus and/or hepatitis B virus. SC was undetectable in controls. SC was significantly increased in 81 HIV-infected patients and significantly correlated with aspartate aminotransferase (r = 0.267, p = 0.0049), alkaline phosphatase (r = 0.309, p = 0.0011), and γ-glutamyl-transferase (r = 0.264, p = 0.0054). Glutathione S-transferase levels of HIV-infected patients were significantly higher than the controls (3779 ± 5860 vs. 785 ± 71 DU, p = 0.0007) and significantly correlated with serum urea (r = 0.204, p = 0.0038), triglycerides (r = 0.209, p = 0.0033), and lipase (r = 0.219, p = 0.0025). AMP-activated protein kinase α1α2 levels of HIV-infected patients were significantly higher than the controls (5676 ± 6248 vs. 1189 ± 6248 DU, p = 0.0009). Further studies are needed to demonstrate the relevance of these results to diagnose non-AIDS-related illnesses in HIV-infected patients.

The detection of oligoclonal bands (OCBs) in cerebrospinal fluid is an indicator of intrathecal synthesis of immunoglobulins which is a neurochemical sign of chronic inflammatory brain diseases. Intrathecally synthesized IgGs are typically observed in patients with multiple sclerosis. The current standard protocol for the detection of OCBs is IEF on agarose or polyacrylamide gels followed by immunoblotting or silver staining. These methods are time consuming, show substantial interlaboratory variation and cannot be used in a high throughput-approach. We have developed a new nanoscale method for the detection of OCBs based on automated capillary IEF followed by immunological detection. Evidence for intrathecal IgG synthesis was found in all tested patients (n = 27) with multiple sclerosis, even in two subjects who did not have oligoclonal bands according to standard methods. The test specificity was at 97.5% (n = 19). Our findings indicate that the novel OCB-CIEF-immunoassay is suitable for the rapid and highly sensitive detection of OCBs in clinical samples. Furthermore, the method allows for a higher sample throughput than the current standard methods.

Intracellular binding of cisplatin to proteins has been associated with acquired resistance to chemotherapy. In our previous study we established an analytical method for the identification of intracellular cisplatin-binding proteins. The method used a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analogue (CFDA-cisplatin), two-dimensional gel electrophoresis (2DE) and mass spectrometry, which allows detecting and identifying intracellular CFDA-cisplatin-containing protein adducts in the acidic pH range (pH 4-7). Based on this analytical method we extended the identification of intracellular cisplatin-protein adducts to the alkaline pH range (pH 6-10) giving chance to discover new important binding partners. 2DE analysis of alkaline proteins is challenging due to the difficult separation of basic proteins during the isoelectric focusing (IEF). The establishment of an optimized IEF protocol for basic proteins enabled us to identify several intracellular CFDA-cisplatin-binding proteins including enzymes of the glucose and serine metabolism like alpha enolase and D-3-phosphoglycerate 1-dehydrogenase.

To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non-denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non-denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF-separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme-ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme-ovotransferrin complex, can be effectively separated and extracted using non-denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.

High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling.

The 2DE is a powerful proteomic technique, with excellent protein separation capabilities where intact proteins are spatially separated by pI and molecular weight. 2DE is commonly used in conjunction with MS to identify proteins of interest. Current 2DE workflow requires several manual processing steps that can lead to experimental variability and sample loss. One such step is the transition between first dimension IEF and second-dimension SDS-PAGE, which requires exchanging denaturants and the reduction and alkylation of proteins. This in-solution-based equilibration step has been shown to be rather inefficient, losing up to 30% of the original starting material through diffusion effects. We have developed a refinement of this equilibration step using agarose stacking gels poured on top of the second-dimension SDS-PAGE gel, referred to as in-gel equilibration. We show that in-gel equilibration is effective at reduction and alkylation in SDS-PAGE gels. Quantification of whole-cell extracts separated on 2DE gels shows that in-gel equilibration increases protein retention, decreased intergel variability, and simplifies 2DE workflow.

This paper describes a simple and reusable microfluidic device combining solution IEF (sIEF) with MALDI-TOF MS for rapid proteomic and metabolic analysis of microliter samples. The device contains two glass plates with nanoliter microwell arrays, which can be assembled to form a fluidic path for sIEF separation, and reconfigured for dividing separated bands. One microliter samples can be loaded and separated by sIEF into static bands in 10∼30 min. After a slipping operation, the static IEF bands can be divided into nanoliter droplets in microwells without mobilization, and the device can be opened for in situ MALDI-TOF MS detection without loss of separation resolution. The performance of the device is characterized by separating and identifying intact proteins. The applicability in metabolic analysis is demonstrated by preliminary experiments on profiling small molecular metabolites in cerebrospinal fluid microdialysates from rat brain.

In this paper, we suggest new electrolyte system for fast preparative electrofocusing in wide pH range. It is based on bidirectional ITP with multiple counterions and spacers created by commercially available defined simple buffers. The migration course of proposed focusing model can be simulated in advance by using separation conditions and electrolyte components that are consequently applied during the experiments. The suggested electrolyte system allows high current densities at the initial stages of focusing without danger of local overheating, which strongly reduces the time needed for analysis completion. The performance of the electrolyte system is demonstrated by the focusing of synthetic colored low molecular weight indicators and proteins in the arrangements with both linear narrow strip and nonwoven fabric sheet with continuous flow.

CIEF was developed for the rapid analysis of charge heterogeneity of trastuzumab biosimilar using commercially available fluorocarbon-coated capillary. The CIEF master mix was composed of 0.30% w/v methyl cellulose, 2.3 M urea, 56.8 mM l-arginine, 1.52 mM iminodiacetic acid, 4.5% v/v carrier ampholytes (broad-range pI 3-10 and narrow-range pI 8-10.5 with ratio of 3:1), and 0.45% v/v 10.0, 9.5, 7.0, 5.5, 4.1 pI markers. To get a robust method to analyze charge heterogeneity, some separation parameters, including focusing time and separation temperature, were investigated and optimized. The optimized method gave good precision in estimated pI values of charge variants with RSDs of not more than 0.16% intraday analysis (n = 6) and < 0.18% interday analysis (n = 9). In addition, the applications of this method including purity, stability, lot consistency, peptide N-glycosidase F digest, and C-terminal lysine variants characterization were also investigated.

Polysaccharide based-vaccines have been successful in providing protection in adults from bacterial infections, however they are not as effective in infants or young children. To enhance the immune response in these high risk groups, the polysaccharide is conjugated with a carrier protein such as cross-reacting material 197 (CRM197). The CRM197 protein has been well-characterized biochemically and biophysically using various analytical techniques however, none of these have been CE-based methods. Of the various CE techniques, imaged capillary isoelectric focusing (icIEF) is a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. Applications of icIEF technique using Convergent Bioscience icIEF instrumentation with whole-field imaging technology are presented and discussed in this paper. These applications include rapid method development to establish a CRM197 identity test for product release, a concentration assay for upstream and downstream in-process product development, and CRM197 stability with respect to its charge heterogeneity under accelerated temperature stress. The data presented demonstrates the utility of the icIEF method as a multifunctional assay because it can screen for better product candidates during early stage clonal selection as well as support in-process and final product characterization throughout CRM197 development.

Isoelectric focusing (IEF) is a powerful tool for amphoteric protein separations because of high sensitivity, bio-compatibility, and reduced complexity compared to chromatography or mechanical separation techniques. IEF miniaturization is attractive because it enables rapid analysis, easier adaptation to point of care applications, and smaller sample demands. However, existing small-scale IEF tools have not yet been able to analyze single protein spots from array libraries, which are ubiquitous in many pharmaceutical discovery and screening protocols. Thus, we introduce an in situ, novel, miniaturized protein analysis approach that we have termed surface isoelectric focusing (sIEF). Low volume printed sIEF gels can be run at length scales of ∼300 μm, utilize ∼0.9 ng of protein with voltages below 10 V. Further, the sIEF device platform is so simple that it can be integrated with protein library arrays to reduce cost; devices demonstrate reusability above 50 uses. An acrylamide monomer solution containing broad-range carrier ampholytes was microprinted with a Nano eNablerTM between micropatterned gold electrodes spaced 300 μm apart on a glass slide. The acrylamide gel was polymerized in situ followed by protein loading via printed diffusional exchange. A pH gradient formed via carrier ampholyte stacking when electrodes were energized; the gradient was verified using ratiometric pH-sensitive FITC/TRITC dyes. Green fluorescent protein (GFP) and R-phycoerythrin (R-PE) were utilized both as pI markers and to test sIEF performance as a function of electric field strength and ampholyte concentration. Factors hampering sIEF included cathodic drift and pH gradient compression, but were reduced by co-printing non-ionic Synperonic F-108 surfactant to reduce protein-gel interactions. sIEF gels achieved protein separations in < 10 minutes yielding bands < 50 μm wide with peak capacities of ∼8 and minimum pI differences from 0.12 to 0.14. This new sIEF technique demonstrated comparable focusing at ∼100 times smaller dimensions than any previous IEF. Further, sample volumes required were reduced four orders of magnitude from 20 μL for slab gel IEF to 0.002 μL for sIEF. In summary, sIEF advantages include smaller volumes, reduced power consumption, and microchip surface accessibility to focused bands along with equivalent separation resolutions to prior IEF tools. These attributes position this new technology for rapid, in-situ protein library analysis in clinical and pharmaceutical settings. This article is protected by copyright. All rights reserved