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Patent Issued for Method for Electrophoresis Involving Parallel and Simultaneous Separation

May 27, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- A patent by the inventor Weber, Gerhard (Kirchheim, DE), filed on April 27, 2006, was published online on May 13, 2014, according to news reporting originating from Alexandria, Virginia, by NewsRx correspondents (see also Becton, Dickinson and Company).

Patent number 8721861 is assigned to Becton, Dickinson and Company (Franklin Lakes, NJ).

The following quote was obtained by the news editors from the background information supplied by the inventors: "The present invention relates to an electrophoresis method involving parallel and simultaneous separation.

"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.

"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 counter-flow media is reflected, for example, in U.S. Pat. No. 5,275,706, the disclosure of which is hereby incorporated by reference. According to this patent, the counter-flow 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.

"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.

"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 that 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.

"Typically, FFE methods involve separation, by flowing the sample through a single separation space within a chamber, this space flanked by two electrodes. An improved method, allowing parallel and simultaneous focusing, in a single chamber, is reflected in United States patent application US2004/045826. This patent application describes a single separation chamber with multiple electrodes, to provide multiple separation spaces within a single chamber. Further improvements relating to parallel, simultaneous separation within a single chamber are desired."

In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventor's summary information for this patent: "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 single anode and a single cathode located in the separation chamber in proximity to the first sidewall and the second sidewall, respectively; at least two sample inlets located in proximity to the first end wall; at least two separation medium inlets located in proximity to the first end wall; a first anodic stabilization medium inlet in proximity to the first end wall and in proximity to the anode; a first cathodic stabilization medium inlet in proximity to the first end wall and in proximity to the cathode; and one or more additional anodic stabilization medium inlets and one or more additional cathodic stabilization medium inlets, the additional anodic and cathodic stabilization medium inlets located in proximity to the first end wall and further located between the first anodic stabilization medium inlet and the first cathodic stabilization medium inlet, (b) introducing at least one separation medium through the at least two separation medium inlets into the separation chamber, introducing one or more samples to be separated through the at least two sample inlets into the separation chamber, and (d) introducing an anodic stabilization medium through the first and the additional anodic stabilization medium inlets into the separation chamber and introducing a cathodic stabilization medium through the first and the additional cathodic stabilization medium inlets into the separation chamber, wherein one or more boundaries defining separation sub-spaces are provided by adjacent flow of anodic and cathodic stabilization media through the additional anodic stabilization medium inlets and the additional cathodic stabilization medium inlets.

"In a further 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; an anode and a cathode located in the separation chamber in proximity to the first sidewall and the second sidewall, respectively; at least two sample inlets located in proximity to the first end wall; at least two separation medium inlets located in proximity to the first end wall; a first anodic stabilization medium inlet in proximity to the first end wall and in proximity to the anode; a first cathodic stabilization medium inlet in proximity to the first end wall and in proximity to the cathode; and one or more additional anodic stabilization medium inlets and one or more additional cathodic stabilization medium inlets, the additional anodic and cathodic stabilization medium inlets located in proximity to the first end wall and further located between the first anodic stabilization medium inlet and the first cathodic stabilization medium inlet, (b) introducing at least one separation medium through the at least two separation medium inlets into the separation chamber, introducing one or more samples to be separated through the at least two sample inlets into the separation chamber, and (d) introducing an anodic stabilization medium through the first and the additional anodic stabilization medium inlets into the separation chamber and introducing a cathodic stabilization medium through the first and the additional cathodic stabilization medium inlets into the separation chamber, wherein one or more boundaries defining separation sub-spaces are provided by adjacent flow of anodic and cathodic stabilization media through the additional anodic stabilization medium inlets and the additional cathodic stabilization medium inlets, wherein the anodic stabilization medium comprises a monoprotic acid the anion of which has an electrophoretic mobility less than or equal to about 40 m.sup.2/V/sec, and wherein the cathodic stabilization medium comprises a monobasic base the cation of which has an electrophoretic mobility less than or equal to about 40 m.sup.2/V/sec.

"These embodiments also involve generating an electric field via the anode and cathode. The separation sub-spaces allow parallel and simultaneous separation within the overall device, each sub-space essentially acting as an independent separation chamber. The invention is therefore able to provide, for example, separation of an increased amount of sample via parallel and simultaneous processes, yet without the need for multiple, separate chambers and instruments.

"(In both embodiments above, one or more of the introducing and generating steps may be performed simultaneously, or in a different order than as presented above.)

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1 is a schematic view of a prior art apparatus for performing parallel and simultaneous carrier-free electrophoresis, comprising three separation spaces, each space defined by two electrodes.

"FIG. 2 is a schematic view of an embodiment of the invention.

"FIG. 3 demonstrates the characteristics of the free flow electrophoresis chamber according to an embodiment of the invention."

URL and more information on this patent, see: Weber, Gerhard. Method for Electrophoresis Involving Parallel and Simultaneous Separation. U.S. Patent Number 8721861, filed April 27, 2006, and published online on May 13, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=102&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=5072&f=G&l=50&co1=AND&d=PTXT&s1=20140513.PD.&OS=ISD/20140513&RS=ISD/20140513

Keywords for this news article include: Peptides, Proteins, Amino Acids, Becton Dickinson and Company.

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Source: Life Science Weekly


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