Sodium dodecyl sulfate-capillary gel electrophoresis with native fluorescence detection for analysis of therapeutic proteins.

A 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection (NFD) of proteins in capillary electrophoresis. The NFD scheme was evaluated in sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) for monoclonal antibody (mAb) characterization. Utilizing a technique by which we filtered the LED emission through a 280 nm bandpass filter, we were able to increase overall concentration sensitivity of SDS-CGE-NFD ~2.3-fold. Under the optimized conditions, the assay linear dynamic range was > 4 orders of magnitude with a correlation coefficient (r2) of 0.9999, and the limit of detection was 8.3 ng/mL. The SDS-CGE-NFD assay was applied to quantitation and purity analysis of Etanercept, a therapeutic protein. Over the range of 50 - 150% of the target concentration, 200 µg/mL, recoveries were in the range of 97.02 - 101.6%. The SDS-CGE-NFD assay allowed for simultaneous quantitation of high- and low-molecular-weight species in Etanercept.

Native fluorescence detection with a laser driven light source for protein analysis in capillary electrophoresis.

While ultraviolet light (UV) absorbance detection is the most widely used detection mode in capillary electrophoresis (CE), it can yield poor concentration sensitivity and has tendencies to exhibit baseline fluctuations. In order to overcome these challenges, alternative detection strategies, including the use of dedicated wavelength lasers, have been applied, resulting in enhancements of concentration sensitivity as well as decreased baseline disturbance. In this work, using a laser driven light source for excitation, we reported a native fluorescence detection (NFD) scheme for use in a commercial CE platform, PA 800 Plus Pharmaceutical Analysis System, for protein analysis. The CE-NFD system was characterized using tryptophan and a reduced IgG. We compared NFD with UV absorbance detection as applied to sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and capillary isoelectric focusing (cIEF). In SDS-CGE, with the reported NFD a non-reduced IgG standard sample yielded a signal-to-noise ratio which was 14.6 times higher than with UV absorbance detection at 214 nm. In cIEF analysis of NISTmAb, Humanized IgG1k, with NFD ∼170 times less sample mass was needed to obtain similar profile quality to that with UV absorbance detection at 280 nm. NFD also eliminated baseline anomalies observed with UV absorbance detection and showed less interference by other absorbing species. These results suggest that CE-NFD is a practical and powerful tool for protein characterization in the biopharmaceutical industry.

On-column and gradient focusing-induced high-resolution separation in narrow open tubular liquid chromatography and a simple and economic approach for pico-gradient separation.

We have recently obtained extraordinarily high efficiencies and sharp peaks using narrow open tubular (OT) columns for liquid chromatographic separations. On-column focusing is commonly observed in liquid chromatography, but this effect alone could not satisfactorily explain the sharpness of these peaks. In this work we investigated the reasons that could have led to the peak sharpness. We hypothesized initially that analytes confined in a narrow OT column might have significantly reduced analyte diffusivities and the reduced diffusivities consequently resulted in the peak sharpness. This hypothesis was invalidated immediately after we measured the diffusion coefficients and did not notice any noticeable diffusivity increases of the analytes inside such columns. We then designed an experiment and revealed a "re-focusing effect". Investigation of this re-focusing effect eventually led us to the observation of a gradient focusing caused by the composition difference between the eluent and the sample matrix. It was this gradient focusing that had contributed primarily to the peak sharpness. On the basis of this insightful understanding, we further developed a simple and economic approach to perform pico-gradient narrow open tubular liquid chromatographic separations.

Cam-based vibration-counter-balanced laser-induced fluorescence scanner for multiplexed capillary detection.

Laser-induced fluorescence (LIF) rotary scanners have been successfully used for multiplexed capillary detection. However, these scanners have a limitation that the capillaries have to be assembled in a circular format, which can be inconvenient for certain applications. A linear LIF scanner works well for flat parallel capillary arrays, but motor accelerations/decelerations (for direction changes) and scanning head vibrations introduce high instrumental noises. The number of capillaries that can be scanned by a linear scanner is limited because of the above constraints. We have constructed a cam-based scanner in an attempt to address these issues. A cam-based scanner eliminates the motor accelerations/decelerations but not the scanning head vibrations. In this work, we attach a second scanning head to the cam on the opposite side of the first scanning head to counter-balance the mechanical vibrations. With this modification, we improve the limit of detection by more than 3 times (from 69 pM to 20 pM fluorescein). We also increase the capillary number capacity by more than 6 times; the total number of capillaries that can be scanned is 426 if 150-µm-o.d. capillaries are used or 320 if 200-µm-o.d. capillaries are used. To demonstrate the utility of this instrument, we assemble a 99-capillary array on one capillary holder and perform capillary electrophoresis of two fluorescent dyes; separations in all capillaries are successfully monitored simultaneously. We also apply it for detecting fluorescently labeled proteins resolved by 24 s-dimension capillaries in a chip-capillary hybrid device; two-dimensional separation results are nicely produced.

Two-dimensional liquid chromatography consisting of twelve second-dimension columns for comprehensive analysis of intact proteins.

A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 µm i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second-dimension columns, while the second-dimension separations were carried out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances.

Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins.

We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem - limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis.

Confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection.

Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-µm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary - hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments.

Simple Means for Fractionating Protein Based on Isoelectric Point without Ampholyte.

In this paper, we develop a simple electrokinetic means for fractionating protein samples according to their pI values without using ampholytes. The method uses inexpensive equipment, and its consumables are primarily ammonium acetate buffers. A key component of its apparatus is a dialysis membrane interface that eliminates electrolysis-caused protein oxidation/reduction and constrains proteins in the desired places. We demonstrate its feasibility for fractionating standard proteins and real-world samples. With the elimination of ampholytes, we can analyze the fractionated proteins directly by a matrix assisted laser desorption/ionization time-of-flight mass spectrometer. Important experimental parameters are also discussed in order to obtain good fractionation results.

Charging YOYO-1 on capillary wall for online DNA intercalation and integrating this approach with multiplex PCR and bare narrow capillary-hydrodynamic chromatography for online DNA analysis.

Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary-hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions.

Simultaneously sizing and quantitating zeptomole-level DNA at high throughput in free solution.

Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zeptomole (zmol) levels of concentration. We have recently developed a new technique, called bare-narrow-capillary/hydrodynamic-chromatography or BaNC-HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC-HDC for measuring the sizes and quantities of DNA fragments at zmol to several-molecule levels of concentration. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA strands of different lengths. BaNC-HDC can be a cost-effective means and an excellent tool for high-throughput DNA sizing and quantitation at extremely low quantity level.

Resolving DNA at efficiencies of more than a million plates per meter using bare narrow open capillaries without sieving matrices.

We report a novel approach for effectively separating DNA molecules in free solution. The method uses a bare narrow open capillary without any sieving matrices to resolve a wide size-range of DNA fragments at efficiencies of more than a million plates per meter routinely.

Miniaturized electroosmotic pump capable of generating pressures of more than 1200 bar.

The pressure output of a pump cannot be increased simply by connecting several of them in series. This barrier is eliminated with the micropump developed in this work. The pump is actually an assembly of a number of fundamental pump units connected in series. The maximum pressure output of this pump assembly is directly proportional to the number of serially connected pump units. Theoretically, one can always enhance the pressure output by adding more pump units in the assembly, but in reality the upper pressure is constrained by the microtees or microunions joining the pump components. With commercially available microtees and microunions, pressures of more than 1200 bar have been achieved. We have recently experimented using open capillaries to build this pump, but many capillaries have to be utilized in parallel to produce an adequate flow to drive HPLC separations. In this paper, we synthesize polymer monoliths inside 75 µm i.d. capillaries, use these monoliths to assemble miniaturized pumps, characterize the performance of these pumps, and employ these pumps for HPLC separations of intact proteins. By tuning the experimental parameters for monolith preparations, we obtain both negatively and positively charged submicrometer capillary channels conveniently. Each monolith in a 75 µm i.d. capillary is equivalent to several thousands of open capillaries.

Pressure-induced transport of DNA confined in narrow capillary channels.

Pressure-induced transport of double-stranded DNA (dsDNA) from 10 base pairs (bp) to 1.9 mega base pairs (Mbp) confined in a 750-nm-radius capillary was studied using a hydrodynamic chromatographic technique and four distinct length regions (rod-like, free-coiled, constant mobility, and transition regions) were observed. The transport behavior varied closely with region changes. The rod-like region consisted of DNA shorter than the persistence length (∼150 bp) of dsDNA, and these molecules behaved like polymer rods. Free-coiled region consisted of DNA from ∼150 bp to ∼2 kilo base pairs (kbp), and the effective hydrodynamic radius R(HD) of these DNA scaled to L(0.5) (L is the DNA length in kbp), a characteristic property of freely coiled polymers. Constant mobility region consisted of DNA longer than ∼100 kbp, and these DNA had a constant hydrodynamic mobility and could not be resolved. Transition region existed between free-coiled and constant mobility regions. The transport mechanism of DNA in this region was complicated, and a general empirical equation was established to relate the mobility with DNA length. Understanding of the fundamental principles of DNA transport in narrow capillary channels will be of great interest in the development of "lab-on-chip" technologies and nongel DNA separations.

Stacking open-capillary electroosmotic pumps in series to boost the pumping pressure to drive high-performance liquid chromatographic separations.

Numerous micropumps have been developed, but few of them can produce adequate flow rate and pressure for high-performance liquid chromatography (HPLC) applications. We have recently developed an innovative hybrid electroosmotic pump (EOP) to solve this problem. The basic unit of a hybrid pump consists of a +EOP (the pumping element is positively charged) and a -EOP (the pumping element is negatively charged). The outlet of the +EOP is then joined with the inlet of the -EOP, forming a basic pump unit, while the anode of a positive high voltage (HV) power supply is placed at the joint. The inlet and outlet of this pump unit are electrically grounded. With this configuration, we can stack many of such pump units in series to boost the pumping power. In this work, we describe in details how an open-capillary hybrid EOP is constructed and characterize this pump systematically. We also show that a hybrid EOP with ten serially stacked pump units can deliver a maximum pressure of 21.5 MPa (∼3100 psi). We further demonstrate the feasibility of using this hybrid EOP to drive eluents for HPLC separations of proteins and peptides.

Protein separation by capillary gel electrophoresis: a review.

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples.

Coupling sodium dodecyl sulfate-capillary polyacrylamide gel electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry via a poly(tetrafluoroethylene) membrane.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS-capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS-CGE has many advantages such as increased separation efficiency, reduced separation time, and automated operation. SDS-CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI-TOF-MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS-CGE-resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS-CGE-separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes.

On-line combination of single-drop liquid-liquid-liquid microextraction with capillary electrophoresis for sample cleanup and preconcentration: a simple and efficient approach to determining trace analyte in real matrices.

In order to improve the sensitivity of capillary electrophoresis (CE) and overcome the deficiency of commercial CE instruments in handling complex matrices directly, we proposed a novel technique which combined single-drop liquid-liquid-liquid microextraction (SD-LLLME) with CE on-line. In this technique, SD-LLLME was realized using a commercial CE instrument and, to further concentrate the target analyte, large-volume sample stacking combined sweeping without polarity switching was utilized. Even though without agitating the donor phase in the extraction process, the model compound, adenine was enriched 550-fold in only 10 min. The enrichment factors were 760 and 1030 when the extraction time was extended to 30 and 60 min, respectively. The relative standard deviations (RSDs) of adenine were 5.24% and 2.29% for peak area and migration time, respectively, which indicated that this method was much more reproducible compared to the existing methods that combined sample-preparation strategies with CE. In addition, this approach was selective while cleaning up target analyte. These mentioned advantages allowed the developed method to be an attractive approach to determining trace target compounds in complex real samples.

In-line preconcentration of oxidized and reduced glutathione in capillary zone electrophoresis using transient isotachophoresis under strong counter-electroosmotic flow.

Transient isotachophoresis (tITP) can improve the sensitivity of capillary electrophoresis (CE). In general, it was carried out under the condition of suppressed electroosmotic flow (EOF). However, some special conditions, such as extreme low pH background electrolyte and coating were needed to achieve the requirements of suppressed EOF. In this work, an approach of tITP under the strong counter-EOF in open system (counter-EOF-tITP) is presented for the rapid and sensitive preconcentrating the reduced glutathione (GSH) and the oxidized glutathione (GSSG) without modifying the capillary and the commercial CE instrument. The parameters of the experimental system, such as the concentration of leading electrolyte, the injected amount of terminating electrolyte and the injected pressure of sample were investigated in detail to understand the mechanism of counter-EOF-tITP. The sensitivity enhancement factors were of 320 for GSH and 280 for GSSG. In addition, the detection limit of 23.4 and 18.0 microg L(-1) for GSH and GSSG was achieved, respectively. The method's applicability was demonstrated by determining GSH and GSSG in tomato and human serum.

Simultaneous enrichment and separation of neutral and anionic analytes through combining large volume sample stacking with sweeping in CE.

In this work, we overcame the deficiencies of large volume sample stacking (LVSS) in separating low-mobility and neutral analytes through combining LVSS with sweeping in CE, and employed this new approach to enrich and separate neutral and anionic analytes simultaneously. This technique was carried out with pressure injection of large-volume sample followed by EOF as a pump pushing the bulk of low-conductivity sample matrix out of the outlet of the capillary while analytes were swept by micelles and separated via MEKC without the electrode polarity switching. Careful optimization of the enrichment and separation conditions allowed the enrichment factors (EFs) of peak height and peak area of the analytes to be in the range of 9-33 and 21-35 comparing with the conventional injection mode, respectively. The five analytes were baseline separated in 15 min and the detection limits ranged from 26.5 to 55.8 ng/mL (S/N = 3). The developed method was successfully applied to determine adenine, caffeine, theophylline, reduced L-glutathione (GSH) and oxidized L-glutathione (GSSG) in two different teas with recoveries that ranged from 84.4 to 105.2%.