Abstract:
The invention relates to a method for electrophoresis in a separation chamber, the method involving introduction of one or more counterflow media of at least two differing properties through multiple counterflow medium inlets into the separation chamber, to provide improved collection of fractionated samples.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The invention relates to a method and apparatus for continuous, carrier-free deflection electrophoresis involving counterflow media. 
         [0003]    2. Description of Related Art 
         [0004]    Since the principle of the method known as free-flow electrophoresis (FFE) was described in DE 805 399, Barrolier, J. et al. in Z. Naturforschung, 1958, 13B, pages 754 to 755, Hannig, K. in Zeitschrift der Analytischen Chemie, 1961, 181, pages 244 to 254 and Roman, M. et al. in Journal of Chromatography 1992, 592, pages 3 to 12 this technique has found a permanent position among efficient analytical and preparative methods used in industry and chemistry. Although both small ions as well as large particles can be separated using this technique, a major application is the fractionation of proteins, especially in the biotechnological production of enzymes and other biologically active proteins, membrane particles and even viable cells. In comparison to other methods enabling isolation of separated sample components, FFE offers two main advantages: (i) the separation may be performed continuously and enables one to obtain as much as hundreds of milligrams or even gram amounts of pure substances per hour and (ii) the separation is gentle and preserves enzymatic activity of the separated components. The technology of FFE is particularly useful in the separation and fractionation of complex proteins, and is thus applicable to the emergent field of proteomics, which is growing increasingly important in the academic research, pharmaceutical, biotechnology and clinical diagnostic markets. For example, as proteomic research has grown, there has been an increased demand in the improvement of protein separation performance, especially relative to resolution process reliability, and a universal front-end. 
         [0005]    Generally, free-flow separation methods are suitable for separating ions of any molecular weight as well as bioparticles. It generally does not matter whether the sample to be separated is itself electrically charged or whether the charge is generated by the adsorption or sorption of ions. The process of continuous deflection electrophoresis and its improvement by way of stabilization media and counterflow media is reflected, for example, in U.S. Pat. No. 5,275,706. According to this patent, the counterflow medium is introduced into the separation space counter to the flow direction of the separation medium. Both media are discharged through fractionation outlets, resulting in a fractionation having a low void volume and, additionally, maintaining a laminar flow of the media in the region of the fractionation outlets, e.g., with very low turbulence. A discussion of various modes of free flow electrophoresis can be found, for example, in U.S. patent application 2004/0050697, the disclosure of which is hereby incorporated by reference. 
         [0006]    Isoelectric focusing is an electrophoretic technique that adds a pH-gradient to the buffer solution and together with the electric field focuses most biological materials that are amphoteric. Amphoteric biomaterials such as proteins, peptides, and, viruses, are positively charged in acidic media and negatively charged in basic media. During IEF, these materials migrate in the pH-gradient that is established across, i.e., transverse to the flow-direction, to their isoelectric point (pI) where they have no net charge and form stable, narrow zones. At this point the materials stop migrating transversely and they become focused. In this technique, there is no voltage dependence. Isoelectric focusing yields such high resolution bands because any amphoteric biomaterial which moves away from its isoelectric point due to diffusion or fluid movement will be returned by the combined action of the pH gradient and electric field. The focusing process thus purifies and concentrates the samples into bands that are relatively stable. This is a powerful concept that has yielded some of the highest resolution separations, especially when coupled with electrophoresis in two-dimensional gels. In IEF, however, the rate of electrophoretic migration of each charged species tends to decrease progressively as it approaches its isoelectric point and long residence times are therefore typically required for high resolution. Moreover, proteins have reduced solubility and a loss of biological activity at their isoelectric point. 
         [0007]    One proposed solution to these challenges is reflected in U.S. Pat. No. 6,660,146. The patent discloses an apparatus and method involving opposing the electrophoretic sample velocity with a uniform fluid-flow transverse to the direction of carrier flow through the chamber. This is achieved by using a combination of electrode arrays and insulated screens to provide the electric field gradient and uniform transverse flow necessary for focusing. This approach does not appear to be practical however. 
         [0008]    Zone electrophoresis is another separation mode that can be used in FFE. Zone electrophoresis separates bioparticles primarily based on charge, and to a lesser extent form and size. A further mode than can be used in FFE is isotachophoresis (ITP). ITP FFE involves use of a non-homogeneous separation media. When components separate from the main band of the initial sample, the components enter an area when they are accelerated or decelerated transverse to the bulk sample flow, based on the local conditions. This so-called focusing effect is then used to fractionate the desired components from the bulk sample. 
         [0009]    Other methods for improved separation are desired. 
       SUMMARY OF THE INVENTION 
       [0010]    The invention provides, among other things, methods for improved separation, including improvement to some typical problems found in IEF processes. 
         [0011]    In one embodiment, the invention provides a method comprising the steps of:
       (a) providing a separation chamber comprising a first end wall, a second end wall, a first sidewall, a second side wall, and two plates, wherein the end walls, sidewalls and plates define a separation space; a first electrode and a second electrode located in the separation chamber in proximity to the first sidewall and the second sidewall, respectively; at least one sample inlet located in proximity to the first end wall; at least one separation medium inlet located in proximity to the first end wall; a plurality of collection outlets located in proximity to the second end wall; and a plurality of counterflow inlets located in proximity to the second end wall;   (b) generating an electric field in the chamber via the first and second electrodes;   (c) introducing a separation medium through the at least one separation medium inlet into the separation chamber;   (d) introducing a sample to be separated through the at least one sample inlet into the separation chamber;   (e) introducing one or more counterflow media through the plurality of counterflow medium inlets into the separation chamber, wherein at least two of the counterflow medium inlets have introduced therethrough counterflow media of differing properties; and   (f) collecting separated portions of the sample through the plurality of collection outlets.       
 
         [0018]    (One or more of the introducing and generating steps may be performed simultaneously, or in a different order than as presented above.) 
         [0019]    The differing properties of the counterflow media can be, for example, difference in chemical properties of the compounds, difference in physical properties of the compounds, or difference in flow properties, e.g., linear flow velocity in the proximity of the, of a single compound. This individual control among inlets is able to, for example, alter conditions near collection outlets to better maintain biological activity and solubility, during the relatively long residence times sometimes required in electrophoretic processes. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  is a schematic view of a conventional FFE-separation chamber according to the prior art. 
           [0021]      FIG. 2  is a schematic view of a separation chamber according to an embodiment of the invention. 
           [0022]      FIG. 3A  demonstrates the methodology according to an embodiment of the invention using counterflow media of different properties. 
           [0023]      FIG. 3B  demonstrates the methodology according to an embodiment of the invention in which the flow rate of the counterflow medium differs. 
           [0024]      FIG. 3C  demonstrates the methodology according to an embodiment of the invention in which the flow rate of the counterflow medium differs. 
       
    
    
     DETAILED DESCRIPTION 
       [0025]      FIG. 1  is a schematic view of a separation chamber  1  according to the prior art which, in proximity to its lower end wall  14  has, as an example, nine inlets  20 ,  22 ,  24 , typically having ports (not shown) which are connected by tubes, such as flexible tubes, to the feed channels of a multi-channel pump, such as a peristaltic pump. Inlets  20  are supplied with the separation medium. Inlets  22  and  24  are supplied with the electrodic stabilization medium for the anode  36  and cathode  32 , respectively. The sample containing the analytes is injected into the separation space through sample inlet  26 . All media flow through the separation space under laminar flow conditions. (The direction of flow from the various inlets is shown by arrows.) The electrodes  32  and  36  are arranged in parallel on both sides of the separation chamber, proximate the side walls  16  and  18  to generate an electric field, and are typically purged in a circular flow at a high flow rate using a pump. Membranes  34  and  38 , which are electrically conductive, separate the electrode space from the separation space and inhibit any exchange of media caused by hydro-dynamic flow. A counterflow medium is introduced into the separation chamber through counterflow element  30 . The counterflow enhances separation by allowing adjustment and control of the flow and pressure conditions at the collection outlets  28 . 
         [0026]    As the sample flows through the chamber, the field created by the electrodes induces separation of components within the sample. The separated sample, e.g., analytes of interest, are collected through collection outlets  28  that are arranged along a line, generally in proximity to and parallel to the separation chamber upper or exit wall  12 . The selected sample component collected is generally led through tubing to a collection vessel and may be used for analytic or preparative purposes. Suitable collection vessels are, for instance, micro-titer wells. Extraneous sample components that are not collected through the collection outlets through which the desired sample is withdrawn or which are collected through different collection outlets can be discarded. 
         [0027]    One embodiment of the invention, reflected in  FIG. 2 , is a method for continuous, carrier-free deflection electrophoresis. The method is performed in a device  100  comprising a rectangular separation chamber having a first (lower) wall  114  and a second (upper) wall  112 , two sidewalls  116  and  118 , and two superimposed parallel flat plates (not shown). Together, these elements form the sealed separation chamber. Two electrodes  132  and  136  capable of and designed for generating a high-voltage electric field are located in the chamber and help define a separation space. The separation chamber, at or near (i.e., in proximity to) the first end, contains at least one sample inlet  126  for the injection of the sample and at least one separation medium inlet  120  for injection of the separation medium. (Note that in the case of multiple inlets  120 , more than one separation medium can be provided.) In proximity to the second end is located a plurality of sample collection outlets  128  and a plurality of counterflow media inlets  130 . Both the collection outlets and the counterflow media inlets are typically arranged along a line perpendicular to the flow direction. 
         [0028]    The individual analytes exit the separation chamber through the multiple collection outlets  128  and are generally led through individual tubing to individual collection vessels of any suitable type. In the collection vessels, the analyte is collected together with the separation medium and counterflow medium. The distance between the individual collection outlets  128  of the array of collection outlets should generally be as small as possible in order to provide for a suitable fractionation/separation. The distance between individual collection outlets, measured from the centers of the collection outlets, can be from about 0.1 mm to about 2 mm, more typically from about 0.3 mm to about 1.5 mm. 
         [0029]    At least two of the counterflow medium inlets  130  have counterflow media of at least two different properties introduced therein. Flow is in the direction shown, which is counter to the flow of the separation medium from inlets  120 . 
         [0030]    In various embodiments, the number of separation medium inlets  120  ranges from 1 to 9, or from 1 to 7; the number of sample inlets  126  ranges from 1 to 5, or from 1 to 3, the number of collection outlets  128  ranges from 3 to 384, or from 3 to 96; and the number of counterflow media inlets  130  ranges from 2 to 9, or from 3 to 7. The number of provided inlets and outlets generally depends from the dimensions of the separation device. 
         [0031]    According to one embodiment, a separation chamber  100  contains separation medium inlet  120 , three sample inlets  126  for the injection of the sample, three to ninety-six collection outlets  128  and seven counterflow media inlets  130 . 
         [0032]    According to embodiments of the invention, the method can be combined with variations of free flow electrophoresis processes and devices. For example, multiple devices can be used, which are then arranged in parallel and/or in series. Alternatively, the component flow collected at the collection outlets of a device may be recycled back to the corresponding sample inlets to form a recycling process which can improve resolution by increasing sample component residence time. In addition, different FFE modes are possible, for example IEF and Zone. 
         [0033]    In the case of IEF, it is useful to select media with specific pH values, to facilitate separation of the component of interest. Generally, the pH values are selected based on the isoelectric point of the component or components of interest, to ensure the component(s) are properly separated. The counterflow media is typically selected to be capable of modifying or surpassing the buffering capacity of the separation medium approaching the fractionation outlets, and may therefore be a material having the same viscosity and density but differing in conductivity and/or pH-value and/or their chemical ingredients. In one embodiment, the pH value of the counterflow medium or media is/are greater than the pH value of the separation medium or media. 
         [0034]    Typical counterflow and separation media are selected from the same group of media, and typically contain components such as urea, glycerol, carbohydrates glucose, and similar compounds. Such media are commercially available from several sources, e.g., Immobiline™, Ampholine™ and Pharmalyte™ from General Electric, Servalyt™ from SERVA Electrophoresis GmbH, and similar materials from Becton, Dickinson and Company. 
         [0035]    According to embodiments of the invention, the counterflow media that are introduced into the counterflow medium inlets  130  are not of the same properties through the entire array of counterflow medium inlets but differ from inlet to inlet and/or between group of inlets in their properties, e.g., media having differing chemical and/or physical properties and/or media of different flow characteristics. When varying the counterflow media between groups of inlets, a group that forms a segment of identical properties is, in one embodiment two to nine individual inlets, or three to about seven inlets in another embodiment. For example, a counterflow medium (A) is injected through a first counterflow medium segment (inlets  1  to  10 ), a second counterflow medium (B), of different flow characteristics or different chemical and/or physical properties compared to medium (A), is injected through a second segment (inlets  11  to  20 ), and a third counterflow medium (C) of different properties from medium (B) is injected through a third segment (inlets  21  to  30 ). 
         [0036]    Chemical and physical distinctions include, but are not limited to, differences in pH-value, viscosity, or conductivity. An example where such differing counterflow media are used is reflected in  FIG. 3A . In  FIG. 3A , a chamber  200  is provided with multiple counterflow inlets  230   a  to  230   g , and inlets  230   a  through  230   c  are provided with a first medium, while inlets  230   d  through  230   g  are provided with a different medium. The counterflow media, noted above, are selected to enhance solubility and preserve biological activity. Thus, for example, based on the analytes present in the adjacent sample paths  240  and  242 , the counterflow media are chosen to modify the pH and/or conductivity of the separation medium, on a local scale, in order to achieve the solubility and biological activity improvements. (For clarity, no other elements of the chamber  200  are shown. This is also the case in  FIGS. 3B and 3C .) 
         [0037]    Differences in other properties, but where there exist no chemical or physical distinctions in the counterflow medium may include flow rate. For example, according to embodiments of the invention, the linear flow rate of the individual counterflow media introduced through the individual counterflow inlets is different. This results in differing flow profiles in the region of the collection outlets which can, in some cases, cause an increase of the geometrical distances between adjacent flow paths of separated analytes or, in other cases, a decrease of the geometrical distances between adjacent paths of analytes. 
         [0038]    An example of increased geometrical distances between adjacent paths of separated analytes is shown in  FIG. 3B . In this embodiment, a single counterflow medium is provided to inlets  330   a  to  330   g . However, the flow rate from inlet  330   c  is higher than the other inlets. This increased flow rate causes increased spacing between adjacent paths  340  and  342  of separated components of the sample. This approach may be useful, for example, in cases where the analyte of interest tends to be spaced closely with other components, and collection of a pure sample is therefore difficult. 
         [0039]    An example of decreased geometrical distances between adjacent paths of separated analytes is shown in  FIG. 3C . In this embodiment, a single counterflow medium is provided to inlets  430   a  to  430   g . However, the linear flow rate from inlets  430   b  and  430   d  is higher than the linear flow rate from inlet  430   c  located between them. This increased flow rate around inlet  430   c  causes a reduction in the spacing between adjacent paths  440  and  442  of separated components of the sample. This approach may be useful, for example, in cases where an analyte or analytes of interest tend to be spaced somewhat widely, and it is desired to focus the wide band or bands toward a single or a few collection outlets, or where certain components precipitate and it is desired to collect such precipitates in one area. 
         [0040]    In case of different linear flow rates of counterflow media introduced through different counterflow media inlets or groups of inlets, the ratio of the flow rate of such an inlet or group of inlets relative to an adjacent inlet or a group of inlets typically is about 5:1 or less, or 3:1 or less. 
         [0041]    It is also possible to control flow using both counterflow media having differing chemical and/or physical properties, and differences in flow rates of those differing media. 
         [0042]    A useful ratio of the flow-rate of the separation medium to the flow rate of the counterflow medium is about 1:10 to about 10:1, more typically about 1:3 to about 3:1. The actual flow rates for the separation medium and the counterflow media depend on a variety of considerations, including the geometrical dimensions of the instrument, the particular separation mode used (which may vary the required transit time), the sample to be separated, the separation medium used and the counterflow medium or media used in order to obtain an optimum separation of the analytes. Typical flow rates of all media into the system (stabilization and counterflow) may therefore range widely from 0.3 mL/hour to 3000 mL/hour. 
         [0043]    An apparatus according to embodiments of the invention further contains a multi-channel pump for the separation medium, a multi-channel pump for the sample, and a multi-channel pump for the counterflow medium/media. The device further contains a fraction collector outlet and outlet tubes. Typically, the pumps are multi-channel peristaltic pumps. 
         [0044]    The bottom plate and the upper plate of the separation chamber can independently be made of glass, or suitable plastics, such as PVC, polyolefins, polycarbonate, plexiglass, polyhalohydrocarbons, or Lucite® (an acrylic resin consisting essentially of polymerized methyl methacrylate), with polymer coated glass being preferred. The top and bottom plates are typically separated by spacers that act as gaskets or seals. 
         [0045]    The electrodes are typically composed of a metal such as platinum that will not easily be oxidized in the electric field. The electrodes are typically separated from the separation chamber by ion-exchange, nylon or cellulose acetate membranes. The electrodes are typically washed constantly by a salt or buffer solution to remove electrolysis products that are created during the process. 
         [0046]    The separation space (space between plates) usually has a thickness of about 0.1 to about 1.5 mm, preferably between about 0.3 and about 1.0 mm. 
         [0047]    Temperature control is also useful according to embodiments of the invention. When current passes through an electrolyte solution, the temperature of the conduction medium increases, according to the phenomenon known as Joule heating. To reduce disturbances to the laminar flow profile of the flowing medium caused by such heating, it is generally desirable to dissipate the Joule heat to the surroundings. In one embodiment, the separation chamber is arranged with its bottom plate on a metal support that contains fluid flow channels connected to a temperature control device, such as a thermostat system for controlling the temperature of the separating chamber. Useful temperature ranges are about 2° C. and about 35° C., more typically about 5° C. to room temperature (about 25° C.). 
         [0048]    The invention is particularly suitable for, but not limited to, the analysis and preparative separation of ions, peptides, biopolymers, bioparticles as well as synthetic polymers and particles. 
         [0049]    The above descriptive embodiments are only exemplary of the present invention, and there are numerous modifications and alternative modes that would fall within the scope of the invention, which is set forth in the claims appended hereto.