Preparative Free-flow Electrophoresis Research Articles

Characterization of the acidic species of a monoclonal antibody using free flow electrophoresis fractionation and mass spectrometry

Antibody charge heterogeneity is one of the major product-related variants in recombinant biopharmaceuticals, which has been commonly monitored by imaged capillary isoelectric focusing (icIEF). Due to the challenges with sample recovery and fractionation, other charge-based analytical approaches have been explored as complementary methods allowing for further detailed charge variant characterization. This study describes the utilization of free flow electrophoresis (FFE) fractionation in combination with other analytical techniques, such as mass spectrometry for monoclonal antibody acidic variants characterization. The preparative FFE technique allowed for continuous sample separation and fluid phase fractionation of antibody charge isoforms. The monoclonal antibody starting material was fractionated by FFE, followed by purification and characterization. icIEF analysis demonstrated the purity of the fractions and comparability of the charge profiles between these two techniques. The intact molecular mass analysis revealed that glycation modification was highly enriched in the acidic fractions. SEC UV/Fluorescence method was developed to assess the levels of aggregation and fluorescent advanced glycation end-products (AGEs). Detailed peptide map was performed and revealed that acidic fractions were enriched in AGEs, methionine, tryptophan, histidine oxidation, asparagine deamidation, lysine glycation, carboxymethyl lysine, glycine to aspartic acid substitution compared to the main peak and starting material. The results indicate that acidic variants can account for a variety of low-level modifications present as very heterogeneous forms.

Recent advances in preparative electromigration techniques for proteins

Preparative electromigration techniques are a category of separation and preparation techniques based on differential electromigration principle. These techniques are widely applied in biomacromolecule and proteome researches. Related techniques mainly include preparative electrophoresis, preparative electrochromatography, preparative isoelectric focusing and free flow electrophoresis and so on. We review each kind of the preparative electromigration techniques on its apparatus designs, applications, advantages and disadvantages in detail. Microscale preparative electrophoresis is a hot spot in recent years. Its high resolution, high recovery and high efficiency make it a more prominent role in the biological sample analysis. In this review, we focus on some advances in microscale preparative electrophoresis and also give an outlook on preparative electromigration techniques in the future.

Size separation of graphene oxide using preparative free-flow electrophoresis

Graphene oxide nanosheets often bear a wide size distribution. However, it is critical to have nanosheets with narrow size distribution for their unique size-dependent physiochemical properties, and nanosheets with a narrow size distribution are the cornerstones for application. Therefore, efficient separation methods of graphene nanosheets have been given considerable attention in many scientific areas recently. Free-flow electrophoresis is extensively used in the separation and purification of biological molecules with continuous flow separation. The charged graphene oxide nanosheets to some extent are very close in size to biological molecules and share similarity in motion behavior in an electric field. Thus, in the present work, we present a new and simple means to separate graphene oxide nanosheets into more mono-dispersed size groups by using the free-flow electrophoresis technique. By optimizing the separation conditions, we were able to obtain graphene oxide sheets with narrow size distribution. The separated samples were characterized by atomic force microscopy, and the size measurements were made by using the software "Image Pro Plus." In addition, a brief discussion is also given into the theoretic background of the separation of graphene oxide according to the size by the technique of preparative free-flow electrophoresis.

Controlling of band width, resolution and sample loading by injection system in a simple preparative free-flow electrophoresis with gratis gravity

In this paper, the controllable band width, resolution and sample loading were investigated by the injection system of free-flow electrophoresis (FFE) with gratis gravity. Two general injection methods were described herein. The first method was the one in which sample injection fluxes were variable with constant background flux, while the second was the one in which the background fluxes were flexible with stable sample flux. With methyl green and crystal violet as two viewable model compounds, a series of experiments were performed, and the experimental results revealed that (1) the sample band width could be under desiring control through the regulation of ratios between sample and background fluxes, (2) the separative resolution could be also adjusted elaborately via the regulation of flux ratios during the separation of methyl green and crystal violet with only one charge disparity, and (3) the sample loading could be conveniently controlled via the flux ratios and an approximate maximum sample loading could be selected under the condition of just completed separation of two adjoining solutes. In addition, it was observed that the flux ratio had soft influence on the separative resolution of two solutes. These results were of significance to the designs on band width, resolution and sample loading in the newly developed FFE device with gratis gravity.

Lipid Transfer Mediated by a Recombinant Pro-Sterol Carrier Protein 2 for the Accurate Preparation of Asymmetrical Membrane Vesicles Requires a Narrow Vesicle Size Distribution: A Free-Flow Electrophoresis Study

We applied protein-mediated lipid transfer using recombinant His-tagged pro-sterol carrier protein 2 (pro-SCP2) to prepare asymmetrical membrane vesicles (AMV) featuring an unequal transmembrane distribution of the negative phospholipid egg-phosphatidylglycerol (EPG). Pure egg-phosphatidylcholine (EPC) vesicles were used as the acceptor and EPC:EPG 90:10 mol % vesicles as the donor populations. The changes in surface charge during EPG transfer were used to quantify the degree of asymmetry by free-flow electrophoresis (FFE). The relative deflection in FFE correlated with EPG content in the outer monolayer (x(EPG)). The initial transfer rates and first order rate constants for the transfer process were determined. The addition of pro-SCP2 at a molar protein-to-lipid ratio R(P/L) of (15-20) x 10(-5) accelerated the EPG transfer to half-times of between 2 and 3 h. Thus, the transmembrane redistribution of EPG by flip-flop, which reduces the degree of asymmetry and occurs at half-times of tens of hours, was minimized during the transfer process. We investigated the influence of membrane curvature on the transfer rate using 50 and 100 nm vesicles with very low size distribution widths (RSD of 13-17%). Transfer occurred with a 55.7% higher initial rate between the smaller vesicles. The use of equally sized acceptor and donor populations of such narrow size distributions was shown to be important for the preparation of AMV with a uniform degree of asymmetry. Under these conditions, AMV were obtained after less than 3 h by preparative FFE separation. In the case of the acceptor vesicles, EPG transfer increased x(EPG) to 3 mol %, whereas it was reduced to 6 mol % in the donor vesicles.

From micro to macro: Conversion of capillary electrophoretic separations of biomolecules and bioparticles to preparative free‐flow electrophoresis scale

This invited contribution in the special issue of Electrophoresis published in celebration of the 30th Anniversary of this journal reflects the impact of our milestone paper [Prusík, Z., Kasicka, V., Mudra, P., Stepánek, J., Smékal, O., Hlavácek, J., Electrophoresis 1990, 11, 932-936] in the area of conversion of microscale analytical and micropreparative CE separations of biomolecules and bioparticles into (macro)preparative free-flow electrophoresis (FFE) scale on the basis of a correlation between CE and FFE methods. In addition to the survey of advances in the relatively narrow field of CE-FFE correlation and CE-FFE conversion, a comprehensive review of the recent developments of micropreparative CE and (macro)preparative FFE techniques is also presented and applications of these techniques to micro- and (macro)preparative separations and purifications of biomolecules and bioparticles are demonstrated. The review covers the period since the year of publication of the above paper, i.e. ca. the last 20 years.

A simple preparative free‐flow electrophoresis joined with gratis gravity: I. Gas cushion injector and self‐balance collector instead of multiple channel pump

This paper describes a novel free-flow electrophoresis (FFE), which is joined with gratis gravity, gas cushion injector (GCI) and self-balance collector instead of multiple channel pump, for the purpose of preparative purification. The FFE was evaluated by systemic experiments. The results manifest that (i) even though one-channel peristaltic pump is used for the driving of background buffer, there is still stable flow in the FFE chamber; (ii) the stable flow is induced by the gravity-induced pressure due to the difference of buffer surfaces in the GCI and self-balance collector; (iii) the pulse flow of background buffer induced by the peristaltic pump is greatly reduced by the GCI with good compressibility of included air; (iv) the FFE can be well used for zone electrophoretic separation of amino acids; (v) up to 20 inlets simultaneous sample injection and up to five to tenfold condensation of amino acid can be achieved by combining the FFE device with the method of moving reaction boundary. To the best of authors' knowledge, FFE has not been used for such separation and condensation of amino acids. The relevant results achieved in the paper have evident significance for the development of preparative FFE.

Preparative free-flow electrofocusing in a vortex-stabilized annulus.

This paper describes the development of an annular chamber designed for preparative free-flow electrophoresis which can operate at voltages up to 20 kV, electric fields up to 65 kV/m, and power densities as high as 10 MW/m(3). This apparatus uses counter-rotating fluid vortices to stabilize the aqueous medium in the annulus against natural convection while improving heat and mass transfer over vortex-free Couette flow. The vortices are generated by rotating the inner surface of the annulus and, if the rotor and stator have complementary shapes, dispersion induced by electrokinetic and electrohydrodynamic flows can be mitigated even at the highest operating voltages. Following a brief overview of contemporary annular free-flow instrumentation, the theoretical principles for momentum and heat transport in the vortices are discussed in some detail and then the results of several electrofocusing experiments are provided to illustrate the resolving power of this instrument.

Up-scaling capillary zone electrophoresis separations of polydisperse anionic polyelectrolytes with preparative free-flow electrophoresis exemplified with a soil fulvic acid.

A scale-up of analytical capillary zone electrophoresis (CZE) to preparative free-flow electrophoresis (FFE) is described. FFE allows fractionations based on charge densities in larger amounts than in CZE, enabling further off-line analysis of the fractions. Model compounds (carboxylic acids and polystyrene sulfonates) showed a similar behavior in FFE as in CZE. Diffusion and electrodynamic distortion effects are more pronounced in FFE than in CZE. A soil fulvic acid was analyzed by CZE and fractionated by FFE. A comparison of the FFE fractions with CZE measurements of the same sample using the effective mobility scale showed good agreement of the two methods.

Band-broadening effects in preparative free-flow zone electrophoresis.

Recently, we proposed a novel preparative free flow zone electrophoresis cell with extremely short residence time, which does not require external cooling (Poggel, M., Melin, T., Electrophoresis 2001, 22, 1008-1015). Within the new cell the smallest chamber dimension is not orientated perpendicular but in direction of the electric field. This alteration provides straight forward scale up opportunities. In this paper, new experimental results are reported, from which the limits of stable flow can be determined. The data suggest that not density differences but electrohydrodynamic effects are responsible for the disturbance of the laminar flow pattern, which is observed above a critical field strength. To demonstrate the efficiency of the new system, a three-component mixture consisting of bovine serum albumin (BSA), myoglobin and cytochrome c is processed, resulting in relatively high recovery and purity values of the different proteins, although a complete separation is not achieved.

Separation of enzymes from microorganism crude extracts by free-flow zone electrophoresis

Continuous, single-step, state-of-the-art preparative separations of enzymes from microorganism crude extracts by free-flow zone electrophoresis are presented. In the first example, the enzymes formate dehydrogenase, formaldehyde dehydrogenase, and methanol oxidase were continuously separated from Candida boidinii crude extract. Yields of 85% to 95% and purification factors between 3 and 7 were obtained along with a simultaneous separation of the finer cell debris from the enzymes. Using multiple injections of sample, a throughput of 46.2 mg protein/h was recorded. In the second example, a fivefold purification of beta-galactosidase from Escherichia coli was achieved along with complete, simultaneous cell debris separation from the enzyme. The yield of the enzyme was greater than 90%. The preparative free-flow zone electrophoresis experiments were run continuously for a period of 12 h and the separations were found to be stable; i.e., the enzymes and the cell debris eluted at their respective fraction numbers during the entire period. In both examples, choice of the type of buffer played a critical role and had to be investigated and optimized experimentally. Scale-up aspects of the separations are also discussed. Recently, by comparison of free-flow zone electrophoresis with ion-exchange chromatography, we have presented evidence that free-flow electrophoresis separations are governed by net surface charge (S. Nath et al., Biotechnol. Bioeng. 1993, 42: 829-835). Here, we offer further confirmation of this evidence by comparison of preparative free-flow zone electrophoresis experiments at various pHs on a mixture of two model proteins with analytical electrophoretic titration curves of the proteins. We are thus in a position to predict separations in free-flow zone electrophoresis. (c) 1996 John Wiley & Sons, Inc.

Application of binary buffer systems to free flow cell electrophoresis.

The applicability of free flow electrophoresis (FFE) was expanded towards processing of sensitive cells. The chamber medium was adjusted to a physiologic pH of 7.35 by a mixture of N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid) (EPPS) and 2,2-bis(hydroxymethyl)-2,2'2"-nitrilotriethanol (BISTRIS). These substances proved to be nontoxic to sensitive cells such as human smooth muscle or thyroid cells. They enhanced the electrical conductivity of the medium only slightly so that a new cell electrophoresis separation medium could be prepared, which contained 30 mM NaCl together with or without 1 mM CaCl2 but did not generate problems of overheating the fluid. Suspended in this medium, human smooth muscle cells as well as human thyroid carcinoma cells remained viable single cells for at least 120 min. After this period they could be recultured to form monolayers. If electrophoresed in the Octopus preparative FFE device, they migrated as single cells and did not clot; therefore, their electrophoretic behavior could be determined exactly.

Cell-Free Transfer of the Vesicular Stomatitis Virus G Protein from an Endoplasmic Reticulum Compartment of Baby Hamster Kidney Cells to a Rat Liver Golgi Apparatus Compartment for Man8-9 to Man5 Processing

We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37°C with wild-type VSV or at the permissive temperature of 34°C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37°C in the presence of nucleoside triphosphate was eightfold that at 4°C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5°C but were able to proceed at the permissive temperature of 34°C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo.

Dynamic computer simulations of the influence of injection conditions on capillary zone electrophoretic stacking of preparative free-flow zone electrophoresis fractions of peptides

Dynamic computer simulations were carried out to investigate the effect of residual acetic acid content on the initial stacking conditions of oligoglycine samples during the analytical CE run. These simulations were carried out with both individual peptides and with their equimolar mixtures. Results indicate that optimum acetic acid conditions were in the range of equimolar values.

Cell-free analysis of Golgi apparatus membrane traffic in rat liver.

Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway.

Separation of endosomes by aqueous two-phase partition and free-flow electrophoresis

We have developed two endosome models to evaluate the separation of endosome populations by aqueous two-phase partition. In the first model, bovine kidney endosomes were used. In the second model, HeLa endosomes were identified in homogenates by means of a latent drug-(capsaicin-)inhibited NADH oxidase (NOX). Endosomes were first isolated by aqueous two-phase partition. To separate early and late endosomes, the endosomes were incubated with ATP to acidify the endosome interiors by activating a proton-translocating ATPase. Thus far, we have been able to resolve the early and late endosomes from any source only by preparative free-flow electrophoresis and not by phase-partition. Previous studies have shown that gravitational forces may be important for separation of endosomes by phase partition. Low-speed centrifugation (≤12.5 g) during phase resolution altered the activity of the latent NADH oxidase used as a marker for HeLa cell endosomes.

Free-flow electrophoresis for fractionation of Arabidopsis thaliana membranes.

Highly purified tonoplast and plasma membrane vesicles were isolated from microsomes of Arabidopsis thaliana by preparative free-flow electrophoresis. The most electronegative fractions were identified as tonoplast using nitrate-inhibited Mg2+-ATPase as enzyme marker. The least electronegative fractions were identified as plasma membrane using glucan-synthase II, UDPG: sterol-glucosyl-transferase, and vanadate-inhibited Mg2+-ATPase as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (endoplasmic reticulum) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane. Immunoblot analysis of membrane fractions by antibodies directed against tonoplast and plasma membrane proteins confirmed the nature and the purity of the isolated membranes. The cytoskeletal protein actin, which was also identified by immunoblotting, was found to be specifically attached to the plasma membrane vesicles. The structural and functional integrity of the isolated membranes from Arabidopsis thaliana is discussed in the light of results obtained for the location of receptors and enzymes, or for the determination of ligand binding activity.

Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants

Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 m M ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.

Analysis of trypanosomal endocytic organelles using preparative free-flow electrophoresis.

In this paper we demonstrate the power of preparative free-flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free-flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time-dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conclusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket-derived membranes from other endosomal and lysosomal compartments of African trypanosomes.

NADH oxidase activity present on both the external and internal surfaces of soybean plasma membranes

Soybean plasma membranes exhibit NADH oxidase activities accessible to NADH supplied to either the external or internal membrane surfaces. Activity at the external surface was demonstrated using plasma membranes of right side-out orientation prepared by aqueous two-phase partition and using intact soybean cells grown in culture. Activity at the internal membrane surface was demonstrated using vesicles of inside-out orientation obtained by preparative free-flow electrophoresis or by aqueous two-phase partition following freeze–thaw or Brij detergent treatment to invert some of the right side-out vesicles. The NADH oxidase activity of both membrane surfaces appeared to be stimulated by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Following the freeze–thaw treatment utilized to evert the vesicles, however, the NADH oxidase activity of right side-out vesicles was greatly reduced. A 2,4-D-stimulated activity of these vesicles could be restored by treatment first with reduced glutathione and then with hydrogen peroxide or oxidized glutathione. Upon treatment with 0.1% Triton X-100, both right side-out and inside-out vesicles exhibited approximately the same specific activity and the same 2,4-D-stimulated component of activity equal to the sum of the activities of the right side-out and inside-out vesicles assayed separately. These findings suggest that a significant NADH oxidase activity of the soybean plasma membrane responsive to 2,4-D resides on the external plasma membrane surface. This latter activity is unlikely to function under physiological conditions in the direct oxidation of NADH and alternative functions, such as in protein disulfide–thiol interchange, would be more appropriate.