WO2009133153A1

WO2009133153A1 - Methods and systems for the separation and analysis of analytes using ffe

Info

Publication number: WO2009133153A1
Application number: PCT/EP2009/055224
Authority: WO
Inventors: Mikkel Nissum; Gerhard Weber
Original assignee: Becton, Dickinson And Company
Priority date: 2008-04-29
Filing date: 2009-04-29
Publication date: 2009-11-05
Also published as: EP2294395A1

Abstract

The present invention provides novel and advantageous methods, compositions, kits, and devices for separating analytes by free flow electrophoresis. The methods are particularly suitable for simultaneously separating analytes of interest and transferring at least one analyte of interest into a suitable buffer medium that is compatible with downstream analysis methods. Also provided are, kits comprising such buffer systems, and devices for carrying out the free flow electrophoretic separation methods of the present invention.

Description

METHODS AND SYSTEMS FOR THE SEPARATION AND ANALYSIS OF ANALYTES

USING FFE

Field of the Invention

[0001] The invention relates to systems and methods for carrier-free deflection electrophoresis (also known as free flow electrophoresis or FFE) involving separation conditions and media that enable the separation and subsequent analysis of certain analytes. Background of the Invention

[0002] Electrophoresis is a well-established technology for separating particles based on the migration of charged particles under the influence of a direct electric current. Several different operation modes such as isoelectric focusing (IEF), zone electrophoresis (ZE) and isotachophoresis (ITP) have been developed as variants of the above separation principle and are generally known to those of skill in the art.

[0003] IEF (isoelectric focusing), one of the above general operation modes of electrophoresis, including free flow electrophoresis, is a technique commonly employed, e.g., in protein characterization as a mechanism to determine a protein's isoelectric point (see, e.g., Analytical Biochemistry, Addison Wesley Longman Limited-Third Edition, 1998) or to separate analytes according to their isoelectric point (pi). IEF is discussed in various texts such as Isoelectric Focusing by P. G. Righetti and J. W. Drysdale (North Holland Publ., Amsterdam, and American Elsevier Publ., New York, 1976). Zone electrophoresis (ZE) is another alternative operation mode based on the difference between the electrophoretic mobility value of the particles to be separated and the charged species of the separation medium employed.

[0004] Isotachophoresis (ITP) is a more recent variant of electrophoresis wherein the separation is carried out in a discontinuous buffer system. Sample material to be separated is inserted between a "leading electrolyte" and a "terminating electrolyte", the characteristics of buffers being that the leader will comprise ions having a net electrophoretic mobility higher than those of the sample ions, while the terminator must comprise ions having a net electrophoretic mobility lower than those of the sample ions. In such a system, sample components sort themselves from leader to terminator in accordance with their decreasing mobilities in a complex pattern governed by the so-called Kohlrausch regulating function. The process has been described in the art, for instance, in Bier and Allgyer, Electrokinetic Separation Methods 443-69 (Elsevier/North-Holland 1979).

[0005] Among electrophoretic technologies, free flow electrophoresis (FFE) is one of the most promising [Krivanova L. & Bocek P. (1998), "Continuous free-flow electrophoresis", Electrophoresis 19: 1064-1074]. FFE is a technology wherein the separation of the analytes occurs in a carrier-free medium, i.e., a liquid (aqueous) medium in the absence of a stationary phase (or solid support material) to minimize sample loss by adsorption. FFE is often referred to as carrier-less deflection electrophoresis or matrix-free deflection electrophoresis. [0006] In the field of proteomics, FFE is the technology of choice for the defined pre- separation of complex protein samples in terms of their varying isoelectric point (pi) values. Using FFE, a great variety of analytes, including organic and inorganic molecules, bioparticles, biopolymers and biomolecules can be separated on the basis of their electrophoretic mobility. The corresponding principles have already been described [e.g. Bondy B. et al. (1995), "Sodium chloride in separation medium enhances cell compatibility of free-flow electrophoresis", Electrophoresis 16: 92-97].

[0007] The process of FFE has been improved, e.g., by way of stabilization media and counter-flow media. This is reflected, for example, in U.S. Patent 5,275,706, the disclosure of which is hereby incorporated by reference in its entirety. [0008] A particular FFE technique referred to as interval FFE is disclosed, for example, in U.S. Patent 6,328,868. In this patent, the sample and separation medium are both introduced into an electrophoresis chamber, and the analytes in the sample are separated using an electrophoresis mode such as ZE, IEF or ITP, and are finally expelled from the chamber through fractionation outlets. Embodiments of the '868 patent describe the separation media and sample movement to be unidirectional, traveling from the inlet end towards the outlet end of the chamber, with an effective voltage applied causing electrophoretic migration to occur while the sample and media are not being fluidically driven from the inlet end towards the outlet end, in contrast to the technique commonly used in the art wherein the sample and media pass through the apparatus while being separated in an electrical field (commonly referred to as continuous FFE).

[0009] International patent application WO 02/50524 A and U.S. patent application 2004/050698 and International patent application PCT/EP2007/061840 which is hereby incorporated in its entirety disclose FFE apparatus.

[0010] A number of separation media for the separation of analytes such as bioparticles and biopolymers are known in the art. For example, the book "Free-flow Electrophoresis", published by K. Hannig and K. H. Heidrich, (ISBN 3-921956-88-9) reports a list of separation media suitable for FFE and in particular for free flow ZE (FF-ZE).

[0011] U.S. patent 5,447,612 discloses another separation medium which is a pH buffering system for separating analytes by isoelectric focusing by forming functionally stable pre-cast narrow pH zone gradients in free solution. It employs buffering components in complementary buffer pairs.

[0012] U.S. Pending Provisionals Ser. # 60/945,246 and # 60/987,208 refer to volatile buffer systems suitable for FFE. The volatile buffer systems offer the advantage that they can be easily removed subsequent to a FFE step and prior to a downstream analysis such as MS, or do not disturb a downstream analysis.

[0013] International patent application PCT/EP2007/061840 refers to media combinations for enhanced free flow ITP.

[0014] Proteomics research combines high-resolution separation techniques (e.g., electrophoresis), applied to complex protein mixtures with state-of-the-art identification methods such as mass spectrometry (MS). It is generally agreed that none of the existing separation and identification methodologies on its own can give a full account of the protein composition or the protein expression in complex mixtures, (e.g. biological fluids such as serum, plasma, synovial fluid, cerebrospinal fluid, urine, whole cells, cell fractions, or tissue extracts). [0015] Many protein purification and separation media comprise additives such as denaturants to yield suitable conditions for these methods such as denaturing conditions. In those methods, wherein, e.g., denaturants are commonly used, the denaturants must often be removed prior to a subsequent analysis such as MS because these denaturants interfere with the sensitivity of such analytical methods. In order to address this problem, classical methods to remove such disturbing substances are normally used subsequent to a successful electrophoresis and prior to, e.g., a downstream analysis. These extensive and often difficult cleaning and/or purification procedures cause an increase in the overall length of time required for the analysis and typically result in a loss of sample. Furthermore, these laborious procedures represent an obstacle for automation. Therefore, sample preparation is one critical, and often technically challenging task in a successful biomolecule analysis project today.

[0016] The importance of the compatibility of buffer media to, e.g., downstream analytical techniques or subsequent storage cannot be underestimated when developing new separation methodologies. In view of the above, there remains a need in the art for highly efficient as well as economical separation methods for, e.g., proteins. Summary of the Invention

[0017] The present inventors have found that the methods and kits and devices provided herein can be successfully used for the separation of analytes and at the same time for the transfer of analytes of interest into buffer media which are, e.g., suitable for further subsequent analysis techniques or storage. The methods according to embodiments of the present invention therefore, e.g., reduce the need of a buffer exchange step after a free flow electrophoresis and are thus particularly useful for high-throughput applications comprising the direct transfer of analytes of interest into buffer media that are compatible with downstream analysis methods. [0018] Accordingly, one aspect of the present invention relates to an electrophoretic separation method for separating an analyte from a sample by free flow electrophoresis comprising: a) forming within a free flow electrophoresis (FFE) chamber a separation zone between an anode and a cathode that comprises a zone I formed by at least one separation buffer medium (SBM) type I and a zone Il formed by at least one SBM type II,

wherein the separation buffer medium of type I comprises at least one additive at an effective concentration; and

wherein the separation buffer medium of type Il does not contain the additive(s) at an effective concentration; and

b) introducing a sample into the separation zone; and

c) transferring at least one analyte to be separated from a separation medium type I into a separation medium type Il or transferring at least one analyte to be separated from a separation medium type Il into a separation medium type I during the free flow electrophoretic separation.

[0019] Another aspect of the present invention is a method for separating an analyte from a sample by free flow electrophoresis comprising: a) forming within a free flow electrophoresis (FFE) chamber a separation zone between an anode and a cathode that comprises a zone I formed by at least one separation buffer medium (SBM) type I that yields denaturing separation conditions and a zone Il formed by at least one SBM type Il that yields non-denaturing separation conditions; and b) introducing a sample into the separation zone; and

c) transferring at least one analyte to be separated from a SBM type I into a SBM type Il or transferring at least one analyte to be separated from a SBM type Il into a SBM type I during the free flow electrophoretic separation.

[0020] In certain embodiments, methods are provided that are suitable to perform the separation of at least two analytes to be separated comprising: introducing together with the sample or with at least one SBM type I an interaction partner of at least two of the analytes;

wherein the presence of at least one additive at an effective concentration in SBM type I suppresses the interaction between at least one of the analytes of interest and the interaction partner and wherein at least one other analyte interacts with the interaction partner under the same conditions thereby forming an analyte-interaction partner-complex;

and further wherein the analyte-interaction partner-complex(s) has a different pi compared to the pi of the analyte(s) of interest;

and wherein the analyte-interaction partner-complex(s) remains in a SBM type I, whilst at least one of the analytes of interest is transferred into a SBM type Il during the free flow electrophoretic separation.

[0021] Furthermore, a method for analyzing analytes from a composition of analytes is provided which comprises the steps of: conducting a free flow electrophoresis according to embodiments of the present invention; and

eluting the analytes in a multiplicity of fractions from the FFE chamber; and

subsequently analyzing one or more fractions of SBM type I and/or one or more fractions of SBM type II.

[0022] To carry out an electrophoretic separation method according to embodiments of the present invention, kits are provided herein comprising at least one additive and at least those buffer compounds necessary for preparing at least one buffer system for the preparation of at least one SBM type I and at least one SBM type II. [0023] Finally, a further aspect of the present invention relates to an FFE apparatus for carrying out an electrophoretic separation method according to embodiments of the present invention and the use thereof. The FFE apparatus according to this aspect comprises: an electrophoresis chamber comprising a set of electrodes, wherein at least one of the electrodes is a cathode and at least one of the electrodes is an anode, and a separation zone interposed therebetween, wherein the apparatus further contains means for forming a zone I formed by at least one SBM type I; and means for forming a zone Il formed by at least one SBM type II; and optionally, means for forming stabilizing media within the separation zone.

Brief Description of the Figures

[0024] Fig. 1 shows a schematic view of a free flow electrophoresis apparatus comprising a separation zone that comprises a zone I formed by separation buffer media of type I and a zone Il formed by separation buffer media of type II. The positioning and breadth of zones I and Il may be different from the set-up shown in Fig. 1.

[0025] Fig. 2 shows the fractional separation of an insulin Detemir standard between anode (left) and cathode (right) (96 fractions) and indicates the pH of the fractions. Colored pl-markers were separated to evaluate the separation performance of the system. The absorbance of each fraction at λ=420nm, 515nm and 595nm which represent the absorbance of the respective pl-markers are also reported in FIG. 2.

[0026] Fig. 3 shows the corresponding silver stained SDS-PAGE gel obtained for the various fractions of the FFE separation. The gel demonstrates the elution profile of insulin Detemir.

[0027] Fig. 4 shows the corresponding silver stained SDS-PAGE gel obtained for the various fractions of a sample comprised of insulin Detemir that was spiked into a human blood plasma sample. The sample was introduced into a zone yielding denaturing conditions. Insulin Detemir was separated from albumin and transferred into a zone yielding non- denaturing conditions. The gel demonstrates the essential depletion of albumin from fractions comprising insulin Detemir. Detailed Description

[0028] Aspects of the present invention relate to a novel concept of media for free flow electrophoresis, (hereinafter FFE) combining two types of separation buffer media wherein one type comprises a specific additive, whereas the other type does not. During free flow electrophoretic separation, at least one analyte of interest is transferred from one separation buffer media type into another separation buffer media type. This improves the compatibility of the FFE separation media comprising the analytes eluted from the separation zone with downstream analytical techniques, i.e., the methods avoid a buffer exchange step subsequent to the separation step and prior to a further analytical step compared to protocols that represent the current state-of-the-art.

[0029] An enrichment of specific peptides or proteins can be obtained in one or multiple FFE fractions to overcome dilution effects thereby increasing the sensitivity of the separation system by using focusing media formed by a separation buffer medium type according to an embodiment of the present invention. It also allows the user to investigate interactions between particles that would otherwise be difficult or impossible to be investigated by the combination of, e.g., denaturing and non-denaturing media types in the same FFE separation.

[0030] In one aspect of the present invention, the method relates to the separation of analytes of interest and the simultaneous transfer from a separation buffer medium exhibiting denaturing conditions into a separation buffer medium exhibiting non-denaturing, or even native conditions which, e.g., offer a better compatibility for downstream analytic methods. This method also allows for the separation of analytes such as peptides and proteins that do not interact with e.g. albumin under denaturing conditions from analytes that interact with albumin under denaturing condition and isolating them in a non-denaturing medium, wherein the non-denaturing medium may preferably be a focusing medium, thereby resulting in focusing the at least one analyte of interest into one or a limited number of FFE fractions. In other words, in this way potential biomarkers can be investigated in ways currently not possible, or at least which would otherwise be extremely difficult to analyze with currently existing techniques. Albumin is known to non-specifically bind or rather act as a "sponge" for proteins and peptides in plasma or serum. Conventionally, the isolation of certain proteins and peptides is therefore difficult and may only be performed without getting rid of large, contaminating amounts of albumin. In contrast, the present invention enables the separation of the analytes of interest from albumin under denaturing conditions which suppress interactions of the analytes with albumin, and concomitant transfer into a medium exhibiting native conditions that are compatible with downstream applications. Further, the analytes may be focused into one or a limited number of fractions by the appropriate choice of the medium exhibiting native conditions. This is an important advantage since the species under investigation may be low abundant proteins or peptides that require enrichment in order to be detected in the subsequent analysis of the FFE fractions using, e.g. mass spectrometry (MS) or immuno assays such as ELISA. In particular this applies to the biomarkers or analytes of interest used for research and/or possible clinical diagnostic applications.

[0031] In another example, the methods described herein offer the further advantage of reducing the required amount of expensive additives, such as so-called "cleavable surfactants". These additives are of course only required in fractions containing an analyte of interest, but not in other fractions of the FFE separation. Thus, separation buffer media that do not contain an analyte of interest after the FFE separation may contain a surfactant that is not necessarily compatible with a subsequent MS analysis (i.e. which would negatively impact the MS analyzer and/or the resulting analysis).

[0032] Typical analytes to be separated by an FFE method according to embodiments of the present invention may include inorganic and organic molecules, and preferably bioparticles, biopolymers, biomolecules, including biomarkers such as proteins, protein aggregates, peptides, DNA-protein complexes, DNA, membranes, membrane fragments, lipids, saccharides, polysaccharides, hormones, liposomes, cells, cell organelles, viruses, virus particles, antibodies, chromatin, and the like. Inorganic or organic molecules which can be separated in accordance with certain embodiments of the invention are surface charge- modified polymers and particles such as melamine resins, latex paint particles, polystyrenes, polymethylmethacrylates, dextranes, cellulose derivatives, polyacids, illicit drugs, explosives, toxins, pharmaceuticals, carcinogens, poisons, allergens, infectious agents and the like.

[0033] As used herein, "biomarker" refers to naturally occurring or synthetic compounds which are regarded as a marker of a disease state or of a normal or pathologic process that occurs in an organism (e.g., drug metabolism).

[0034] The term "protein", as used herein, means any protein, including, but not limited to peptides, enzymes, glycoproteins, hormones, receptors, antigens, antibodies, growth factors, etc., without limitation, with about 20 or more amino acids. Proteins include those comprised of greater than about 20, 50, 100, 200, or greater than about 400 amino acid residues. Also comprised are recombinant proteins and protein derivatives comprising covalent (often post-translational) modifications such as glycosylation, phosphorylation and the like.

[0035] The term "peptide" as used herein refers to any entity comprising at least one peptide bond, and can comprise either D and/or L amino acids. A peptide can have about 2 to about 150, preferably about 2 to about 100, more preferably about 2 to about 50 and most preferably about 2 to about 20 amino acids.

[0036] In the context of the present application, the terms "to separate" and "separation" are intended to mean any spatial partitioning of a mixture of two or more analytes based on their different behavior in an electrical field (caused, e.g., by their different pi or different electrophoretic mobility). Separation therefore includes, but is not limited to fractionation as well as to a specific and selective enrichment or depletion, concentration and/or isolation of certain fractions or analytes contained in a sample. Thus, whenever the application refers to the terms "to separate" or "separation", they are intended to include at least one of the foregoing meanings.

[0037] The terms "analyte to be separated" or "analyte of interest" refer to an analyte or to multiple analytes which should be spatially partitioned by FFE from further analytes contained in a sample. For example, at least one analyte of interest is transferred from one SBM type to another SBM type when, for example, subjected to a method according to embodiments of the present invention.

[0038] The term "interaction partner" as used herein refers to an analyte that is not an analyte of interest and that interacts under certain conditions via a at least one covalent bond, ionic interactions, an interaction caused by van der Waals forces, a coordinative bond, any other affinity or any combination thereof with at least one analyte of interest, thereby forming an analyte-interaction partner-complex that results in a different behavior, e.g., a different charge and/or a different electrophoretic mobility compared to the free "analyte of interest" or the essentially non-complexed analyte of interest.

[0039] The term "analyte-interaction partner-complex" as used herein refers to a fusion of an analyte of interest with an interaction partner. The interaction between the analyte of interest and the interaction partner can be a covalent bond, although the interaction partners are typically held together by ionic interactions, polar interactions, an interaction caused by van der Waals forces, a coordinative bond, any other affinity or any combination thereof. In the alternative, the interaction between the analyte of interest and the interaction partner may be of a specific or non-specific nature. [0040] The term "free analyte" as used herein refers to an analyte of interest which does not interact with an interaction partner of an analyte-interaction partner-complex, or which does not interact under specific separation conditions (e.g., of a SBM medium type I) with an interaction partner of an analyte-interaction partner-complex, or which would interact with an interaction partner if the interaction partner is present. In the latter case, the free analyte is the analyte of a possible analyte-interaction partner-complex without the interaction partner. [0041] The term "sample" as used herein comprises at least two analytes which are sufficiently soluble in an FFE separation medium according to various embodiments of the present invention. The samples employed in the methods, compositions, and devices of the present invention may be derived from, but are not limited to protein mixtures (which may additionally comprise other compounds), other reaction mixtures, or from natural sources such as biological fluids (e.g., blood, plasma, urine, and the like).

[0042] A "fractionated sample" in the context of the present invention means a sample wherein the various analytes in the sample are separated during an FFE step and wherein the sample can thus be divided into several fractions during and/or after the FFE separation step. Those of skill in the art will understand how a fractionated sample may be fractionated within an electrophoresis chamber or alternatively collected into individual fractions upon elution from the separation chamber of an apparatus suitable for FFE. Typically, elution from the separation chamber may be carried out through multiple collection outlets and are generally led through individual tubings to individual collection vessels of any suitable type (e.g., 96 well plates, microtiter plates, and plates of different sizes and vessel quantities, e.g., 6, 24, 384, or even 144, 288, 576 or other quantities of wells).

[0043] Free flow electrophoresis (FFE) as used herein refers to a technology wherein the separation of the analytes occurs in liquid medium in the absence of a stationary phase (or solid support material), e.g., to minimize sample loss by adsorption. FFE is often referred to as carrier-less deflection electrophoresis or matrix-free deflection electrophoresis. The FFE separation may principally be carried out in a preparative manner so that certain fractions are subsequently collected, or may merely be carried out analytically, where the analyte of interest or its presence in a certain fraction is merely detected by suitable means, but not collected or extracted from the electrophoresis chamber, e.g., for further use. [0044] A "separation zone" as used herein should be understood to be located between two electrodes of an apparatus suitable to perform a free-flow electrophoretic separation. A separation zone is formed by at least two separation buffer media (herein SBM), whereof at least one SBM is of a type I and at least one SBM is of a type II. A typical separation zone comprises buffer separation media (SBM), whereof, optionally, at least one SBM may act as a focusing medium. Optionally, a separation zone may additionally comprise stabilizing media. In some embodiments, a focus medium acts as a stabilizing medium. Typically, a separation zone is flanked on each side by an electrode medium (see Figure 1 ). In certain specific embodiments, a stabilizing medium may also act as an electrode medium. In the latter case, a separation zone also encompasses an electrode medium. [0045] A "separation buffer medium" (SBM) as used herein refers to a mixture of mono, di- or tri- protic/basic compounds (buffer compounds) which are able to maintain a solution at an essentially constant pH value upon addition of small amounts of acid or base, or upon dilution. At least two buffer compounds form a buffer system within a medium. The combination of separation buffer media within a separation zone is suitable to perform a free flow electrophoretic method. Such a method may be carried out in an operation method selected from but not limited to zone electrophoresis (ZE), isoelectric focusing (ITP), combinations thereof, comprising, e.g., pH plateaus, as described in International patent application WO 2007/147862, which is herein incorporated by reference in its entirety, or isotachophoresis (ITP). Depending on the FFE separation method, the separation of at least one analyte to be separated relies on the pi (isoelectric point), net charge, or its electrophoretic mobility. Non-limiting examples for suitable categories of buffer systems are selected from commercially available ampholytes, complementary multi-pair buffer systems, volatile buffer systems or binary buffer systems (A/B buffer systems). One skilled in the art will appreciate and understand that the buffer system category may be the same or optionally may differ between the different SBM types used in methods of embodiments of the present invention, provided they are compatible with each other under the chosen experimental conditions.

[0046] The isoelectric point (pi) is the pH at which a particular molecule or surface carries no net electrical charge. Amphoteric molecules called zwitterions contain both positive and negative charges depending on the functional groups present in the molecule.

They are affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+). A molecule's pi can affect its solubility at a certain pH. In general, the pi of a certain analyte must be either known or must be identified by means known to those of skill in the art. One possible way to determine the pi of a given analyte of course includes the determination by a suitable FF-IEF technique.

[0047] The electrophoretic mobility EM (or μ_e) is defined as the coefficient between particle speed (V) and electric field strength (E): υ μ^{* =} E The determination of the electrophoretic mobility EM can be easily carried out using techniques that are well-known to those skilled in the art.

[0048] The terms "zone I" and "zone II" as used herein refer to areas within a separation zone which are formed by at least one SBM type I or type II, respectively. [0049] The term "transferring" as used in the context of the present invention refers to the fact that at least one analyte of interest is introduced together with a SBM type I or into a zone I formed by at least one SBM type I, and moves during an FFE separation into a zone Il formed by at least one SBM of type Il or vice versa (i.e. from a SBM type ll/zone Il into a SBM type I/zone I).

[0050] A "SBM type I" as used herein refers to a SBM comprising at least one additive at an effective concentration which is not present in a "SBM type II".

[0051] Accordingly, a "SBM type II" refers to a SBM which does not contain an additive at an effective concentration that is comprised in a SBM type I, i.e., the additive may be present in a SBM type Il below an effective concentration and will preferably not be present in a SBM type II, i.e., a SBM is typically produced without the additive.

[0052] In certain embodiments, as will be understood by the skilled person, minor contaminants of the additive may move from a SBM type I into a SBM type Il during electrophoretic separation, depending on diffusion, the charge or electrophoretic mobility of the additive. Such minor contaminants below the effective concentration may still be included in a SBM type Il as defined herein.

[0053] In certain embodiments, the additive is absent in a fraction comprising at least one analyte of interest which is transferred during FFE separation from one SBM type into another SBM type, i.e., the concentration of the additive in a fraction comprising at least one analyte of interest and a SBM type Il is zero or at least below the detection limit.

[0054] Where an additive at an effective concentration in a SBM type I is present in a minor concentration in a SBM type Il fraction subsequent to an FFE separation, the concentration ratio between the additive in the fraction and the concentration of the additive in the SBM type I prior to the FFE separation method is <0.5, <0.2, <0.1 , <0.05, preferably <0.01 , more preferably <0.001 and most preferably <0.0001.

[0055] In certain embodiments, the additive may be present in a SBM type Il before an FFE step, but then in a concentration below the effective concentration. In such a case, the concentration is in a range wherein the effect caused by the effective concentration of the additive is not exhibited in a SBM type Il compared to a SBM type I. The concentration ratio will be again as described in the preceding paragraph.

[0056] As an example, an "additive at an effective concentration" may be a denaturant. In a SBM type I, the denaturant is present in an effective concentration to exhibit denaturing conditions in regard of at least one analyte of interest, whilst the denaturant is present in a SBM type Il in a concentration that essentially does not lead to the disruption of the tertiary and/or quaternary structure of the at least one analyte of interest. The skilled person knows how to determine the tertiary and/or quaternary structure of a protein using, e.g., circular dichroism. In the latter case, it is irrelevant whether the minor concentration of the denaturant was present in SBM type Il before the SBM type Il entered the separation chamber of an FFE apparatus or whether the minor concentration stems from a migration of the denaturant from a SBM type I into a SBM type Il during the FFE separation.

[0057] The skilled person will understand that additives are present in a SBM to provide a desired effect, e.g., denaturing conditions/native condition, enhanced/reduced viscosity, enhanced/reduced surface tension, inhibition of an enzyme activity etc. It will be appreciated that the effect is caused by the presence of an additive that must be present at an effective concentration. The skilled person will be well aware of desired/undesired effects that are caused by the absence (or presence below an effective concentration)/presence of the additive an effective concentration.

[0058] As already stated above, the term "additive at an effective concentration" relates to an additive that is present in a SBM type I at an effective concentration and that is absent or below the effective concentration in a SBM type II. One goal achieved by the methods of the present invention is the provision of a fraction comprising an analyte, wherein the SBM of the fraction after a FFE separation exhibits a desired effect/advantageous property compared to the SBM of a different type. [0059] It will be understood in the context of the present invention that the additive is not part of the buffer system forming the separation buffer medium and is not a strong acid or a strong base. For example, a method according to embodiments of the present invention therefore allows avoiding a buffer exchange step subsequent to the FFE step and prior to further analysis steps or prior to storage by combining a separation and a buffer exchange step.

[0060] A desired effect caused by the additive depends in general on the concentration, i.e., the "effective concentration", of the additive within a medium. Furthermore, the common knowledge of a skilled person enables the skilled person to determine the concentration of an additive at which a desired effect occurs. The skilled person is well aware of methods how to determine the effective concentration of an inhibitor (e.g., the "additive at an effective concentration" is an inhibitor such as a protease inhibitor present in a SBM type I and not present at all in a SBM type II, or merely present in a SBM type Il in minor concentrations caused by its movement in an electrical field or by migration from SBM type I into SBM type Il during the FFE separation, or present in a concentration below the effective concentration in an SBM type Il before the FFE separation) for example via state-of-the-art enzymatic activity tests, or how to determine the effective concentration of a denaturant in view of at least one analyte of interest (e.g., the "additive at an effective concentration" is a denaturant such as urea or thiourea present in a SBM type I and not present at all in a SBM type II, or merely present in a SBM type Il in minor concentrations caused by its movement in an electrical field or by migration from SBM type I into SBM type Il during the FFE separation, or present in a concentration below the effective concentration in an SBM type Il before the FFE separation) for example via circular dichroism.

[0061] The term "strong acid" as used herein, refers to an acid that ionizes completely in an aqueous solution (in case of diprotic or triprotic acids, at least the first proton is completely ionized), or in other terms, with a pK_a < -1.74. This generally means that in aqueous solution at standard temperature and pressure, the concentration of hydronium ions is equal to the concentration of strong acid introduced to the solution.

[0062] The term "strong base" as used herein refers to a basic chemical compound that is able to deprotonate very weak acids in an acid-base reaction. Compounds with a pKa of more than about 13 are called strong bases. Common examples of strong bases are the hydroxides of alkali metals and alkaline earth metals like NaOH and Ca(OH)₂.

[0063] The skilled person will understand that the presence of an additive at an effective concentration in a SBM type can change the conductivity and/or viscosity of the medium. Accordingly, it may be necessary in certain methods to add a compound into the other SBM type to equilibrate the conductivity/viscosity of the different media types.

[0064] In certain preferred embodiments, a fraction comprising a separated analyte does not contain an additive at an effective concentration, or most preferably does not contain the additive at all. Such an additive at an effective concentration is, e.g., a denaturant that can exhibit denaturing condition. Therefore, a method according to embodiments of the present invention comprises a zone I formed by at least one SBM type I comprising a denaturant and exhibiting a pH value/pH values that is/are different from the pi of at least one analyte of interest and a zone Il formed by at least one SBM type II, wherein at least one analyte of interest is transferred during the FFE separation from a SBM type I into a SBM type Il and is eluted from the separation zone in a fraction formed by a SBM type Il that does not comprise the denaturant or comprises the denaturant in a concentration range wherein the SBM type Il exhibits non-denaturing conditions. Depending on the FFE technique, the pH of zone Il encompasses the pi of the at least one analyte or exhibits a conductivity and/or pH value that allows the analyte of interest to enter the zone I but reduces its movement towards an electrode to essentially zero, i.e. zone I acts as a focus medium. [0065] The term "denaturing", as used herein, refers to a process in which the native conformation of an analyte (three-dimensional structure) is changed but the primary structure (e.g., amino acid chain, peptide links) of the analyte remains unchanged, i.e., "denaturing conditions" refer to those conditions that disrupt the tertiary and/or quaternary structure of the target molecule.

[0066] The term "denaturant", as used herein, refers to an agent, in the presence of which (normally in solution) the native conformation of an analyte is not preserved. Biological activity of, e.g., proteins in the presence of denaturants is changed and is not preserved.

[0067] The term "non-denaturing conditions" as used herein refers to conditions such that denaturing of an analyte to be separated does not occur. These conditions refer to e.g., conditions where no denaturant is present or is present below denaturing concentrations.

[0068] The term "native conditions" as used herein refers to conditions in which an analyte to be separated can preserve the native conformation and in the case of a protein the biological activity, i.e., "native conditions" refer to those conditions under which an analyte to be separated maintains its normal tertiary and quaternary structure.

[0069] The terms "surfactant", "detergent", "wetting agent" and "emulsifier" may be used interchangeably herein and all refer to molecules or compositions which are capable of reducing the surface tension in water or water-based solutions. For example, a surfactant promotes keeping a hydrophobic peptide or protein in an aqueous solution. [0070] A "chaotropic agent', also known as chaotropic reagent and chaotrope, is a substance which disrupts the three dimensional structure in macromolecules such as proteins, DNA, or RNA. Chaotropic agents interfere with stabilizing intra-molecular interactions mediated by non-covalent forces such as hydrogen bonds, Van der Waals forces, and hydrophobic effects. Often structural features as detected by means such as circular dichroism can be titrated in a chaotrope concentration-dependent fashion.

[0071] With the methods of the present invention, it is possible to transfer an analyte of interest from a SBM comprising a "conventional" surfactant or denaturant into a SBM containing an MS-compatible zwitterionic or nonionic surfactant which does not interfere with subsequent MS analysis. Since these MS-compatible surfactants are typically rather expensive, it is readily apparent that a transfer of an analyte of interest into one or a limited number of fractions during the FFE separation offers economical, as well as preparative advantages. Thus, in these instances the analyte will be transferred from a SBM type Il (which may nevertheless comprise a conventional denaturant such as urea) into a SBM type I containing the MS-compatible zwitterionic or nonionic surfactant and optionally comprising a volatile buffer system which can be directly used in a subsequent analysis (e.g., MS). [0072] The term "MS-compatible zwitterionic or nonionic surfactant" as used herein means MS-compatible surfactants that can be zwitterionic or nonionic. In some embodiments, a zwitterionic or nonionic surfactant may be in sum negatively or positively charged depending on the pH of a distinct area between two electrodes, but a nonionic, MS- compatible surfactant is in any event not charged within the pH range, wherein an analyte of interest is inserted into and is eluted from an apparatus suitable for free-flow electrophoresis. Furthermore, it is to be understood that the isoelectric point of a zwitterionic, MS-compatible surfactant as used in the present invention is generally within the pH range of the separation zone. The term "MS-compatible surfactant" and "MS-compatible zwitterionic or nonionic surfactant" as used herein may be used interchangeably since a surfactant suitable for FFE must be either zwitterionic or nonionic within the pH range of the separation zone.

[0073] The term "zwitterionic" as used herein in the context of surfactants refers to a compound that is electrically neutral but carries formal positive and negative charges on different atoms. Examples, which are not to be understood as limiting, are, e.g., betaine derivatives, preferably sulfobetaines such as 3-(trimethylammonium)-propylsulfonat or phosphobetaines. Typically, the isoelectric point of a zwitterionic surfactant as used in the present invention is within the pH range of the separation zone.

[0074] The term "nonionic" as used herein in the context of surfactants refers to (bi)polar compounds. Examples include but are not limited to saccharide derivatives. Typically, a nonionic surfactant is uncharged within the pH range of the separation zone. However, depending on the pH range of the zone, it may happen that a nonionic compound nevertheless becomes charged at a certain pH outside the pH range used to separate an analyte of interest.

[0075] The term "MS-compatible" as used herein denotes surfactants that can be used in MS analyses. The term "MS-compatible surfactants" encompasses surfactants that are per se suitable for MS analysis, i.e. without modification, and also encompasses "cleavable" surfactants which are not MS-compatible in their non-cleaved state but which can be cleaved at least one position into at least two moieties. The moieties can be MS-compatible or non- MS-compatible. A non MS-compatible moiety of a cleavable surfactant as described herein can be easily removed by, e.g., centrifugation, filtration or evaporation, whereas an MS- compatible moiety may stay in solution and may be present during a downstream analysis or may under certain conditions likewise be removed by centrifugation, filtration or evaporation. In a preferred embodiment, more than one resulting moiety is MS-compatible. Such MS- compatible cleavable surfactants are suitable, e.g., in methods comprising a protein digestion step. A protein may be insoluble in water but its fragments or part of the fragments resulting from the digest may be soluble and can be analyzed by, e.g., MS. [0076] As a non-limiting example for the advantages provided by the cleavable surfactants described herein, the sensitivity of a mass spectrometric detection of an analyte in the presence of a suitable, MS-compatible surfactant is much greater than the sensitivity of a mass spectrometric detection of an analyte in the presence of, e.g., SDS. In most cases, a mass spectrum of a sample comprising SDS exhibits no signals at all or only weak signals due to an analyte treated with SDS or break down products of the analyte. In contrast, a sample that comprises the analyte and that is subjected to a mass spectrometric analysis in the presence of an MS-compatible surfactant instead of SDS exhibits signals related to the analyte and to break-down products of the analyte. [0077] Accordingly, an MS-compatible surfactant can be understood as a surfactant whose presence in a sample comprising a soluble control analyte having a defined concentration (S sample) that is subjected to a mass spectrometric analysis leads to mass spectra comprising essentially at least the same mass peaks (at similar or even higher intensity) compared to a mass spectrum of a sample comprising the control analyte in the same defined concentration, but without a surfactant (C (control) sample), i.e. the mass spectra are essentially identical. In some embodiments, an MS-spectrum derived from an S sample may even comprise more mass peaks due to break down products of the control analyte compared to an MS-spectrum derived from a C sample, e.g., when a control analyte is digested prior to mass spectrometric analysis and break down products are hydrophobic and precipitate in a C sample prior to mass spectrometric analysis.

[0078] A suitable procedure to identify MS-compatible surfactants is for example described in WO 2006/047614. BSA, a commonly utilized test protein can be used as an exemplary intact protein and a tryptic digest of β-galactosidase (t-beta-gal) can be used as an exemplary peptide mixture. The β-galactosidase tryptic fragments have a range of solubility's from hydrophilic to hydrophobic. Moreover, many other substances can also act as control analytes as long as they are soluble enough in water so as to yield an MS- spectrum.

[0079] As a non-limiting example, a MALDI-TOF analysis of a β-galactosidase S-sample can be compared with a MALDI-TOF analysis of an equivalent C sample. The ionization suppression in the 900-3700 m/z range can be determined by comparing the matches of the mass-ions identified in the S and the C sample. The skilled person will know how to perform a useful MALDI-TOF analysis.

[0080] Preferably, the intensity of each of the aligned mass peaks of the S sample is not less than 25% compared to the intensity of the identical mass peak of the C sample, more preferably it is essentially the same or, most preferably, it is even higher than the intensity of the same peak of the C sample.

[0081] In respect of merely slightly soluble or insoluble analyte(s) or digestion products of a (control) analyte, it is preferred that the intensity of mass peaks within a mass spectrum of a sample comprising the merely slightly soluble or insoluble analyte/digestion product and an MS-compatible surfactant is at least a factor 1 , 1.5, 3 5, 10, 100 or 1000 times higher than the intensity of identical mass peaks of a mass spectrum obtained for a sample containing no surfactants at all.

[0082] "Essentially identical" as used herein means that at least 60%, at least 70%, preferably at least 80%, more preferably at least 90% and most preferably about 100% of the mass peaks due to the break-down products of the control analyte of the C sample are also present in the spectra of the S sample. Search engines such as MASCOT^® can be used to compare an MS-spectrum of, e.g., digested t-beta-gal or BSA with a theoretical MS-spectrum of a digest of t-beta-gal or a theoretical MS-spectrum of BSA. For the purpose of the present invention, the range from 900 to 2600 m/z should typically be considered.

[0083] In other words, a mass spectrum obtained in the presence of an MS-compatible zwitterionic or nonionic surfactant of the present invention comprises at least 60%, at least 70%, preferably at least 80%, more preferably at least 90% and most preferably 100% of the mass peaks due to the break-down products of a control analyte of a C sample. [0084] The mass difference between a mass signal of the C sample and the identical mass signal of the S sample may vary within the error of measurement depending from the used method or apparatus. A skilled person will understand how to determine such error of measurement. For example, the mass measurement accuracy of an ion trap mass spectrometer is typically calculated between 0.5 and 2.5 dalton, whereas the mass measurement accuracy with errors less than 50 ppm or even less than 25 ppm can be achieved by measuring mass signals ranging from around 900 to 3700 dalton with MALDI- TOF applications.

[0085] Regardless of the compatibility of the surfactants of the invention, it will be understood that the concentration of a surfactant in free-flow electrophoresis and a subsequent analysis (such as MS) should be nevertheless as low as possible, preferably around its critical micelle concentration (CMC). Suitable methods in the art to determine the CMC of a surfactant are known to a person skilled in the art. Furthermore, for many surfactants, the CMC is already known.

[0086] The MS-compatible surfactants are typically used in concentrations below 100 mM. Depending on the surfactant, concentrations of below 50 mM, below 30 mM, below 15, below 5, below 1 and even below 0.1 mM may be suitable. For example, the amount of the cleavable surfactant PPS within a sample subjected to a free-flow electrophoresis as used in the present invention was 0.1% (w/v). This amount corresponds to a concentration of between 2 and 10 mM (depending on the alkyl chain combination of PPS). [0087] A skilled person can easily identify a typical MS-compatible surfactant as described herein by comparing the mass spectra of a C sample and an S sample each comprising a control analyte with a distinct concentration. This method allows a skilled person to determine whether a surfactant is MS-compatible or not. Notably, it is to be expected that analytes which are nearly insoluble or insoluble in water (without a surfactant) would hardly give an analyzable mass spectrum at all when the sample preparation does not include the use of a surfactant. Therefore, a separation of an analyte of interest by free-flow electrophoresis in the presence of an MS-compatible surfactant yields samples that are suitable for identifying and characterizing such analytes in a downstream analysis. The downstream analysis can be mass spectrometry or any other suitable analysis method known in the art.

[0088] In some embodiments, the addition of surfactants in volatile buffer systems and methods of the present invention may be necessary. In the latter case it is most preferred that such a surfactant is a MS-compatible zwitterionic or nonionic surfactant. It will be understood that an MS-compatible zwitterionic or nonionic surfactant as described herein may be comprised in a sample medium and/or within at least one separation medium. In other words, a method for separating analytes from a sample by free-flow electrophoresis according to embodiments of the present invention may comprise the use of at least one MS- compatible zwitterionic or nonionic surfactant, wherein the surfactant is present in the sample medium and/or in at least one separation medium. Although it will be understood that the presence of merely one MS-compatible zwitterionic or nonionic surfactant in a sample medium or in a separation medium is preferred, any combination of multiple MS-compatible zwitterionic or nonionic surfactants within a sample medium and/or a separation medium is possible. When a surfactant or surfactants have to be present in at least one medium of the present invention, it will be advantageous if all surfactants are MS-compatible zwitterionic or nonionic surfactants. A person skilled in the art will understand that each of the surfactants can be present within a sample medium and/or at least one separation medium.

[0089] Furthermore, an MS-compatible surfactant as described herein can be MS- compatible per se during the free-flow electrophoretic separation, or it can become MS- compatible through the cleavage of the surfactant. In the latter case an MS-compatible surfactant is an MS-compatible cleavable surfactant. When a method according to embodiments of the present invention has to be carried out in the presence of a surfactant, it may be preferred that at least one MS-compatible zwitterionic or nonionic surfactant is cleavable, although other MS-compatible zwitterionic or nonionic surfactants may be present. In some embodiments it may be advantageous that all MS-compatible surfactants within a sample medium and/or a separation medium are cleavable surfactants. [0090] The terms "MS-compatible zwitterionic or nonionic cleavable surfactant", "MS- compatible cleavable surfactant" or "cleavable surfactant" are used interchangeably herein and refer to surfactants that can be cleaved into at least two moieties under particular conditions. In one embodiment, at least one of the cleaved moieties is MS-compatible as defined above. Such an MS-compatible moiety can be present during mass spectrometric analysis or absent, e.g., evaporated prior to MS-analysis. Non-MS-compatible moieties precipitate after the cleavage or can be evaporated prior to MS analysis.

[0091] As will be explained below, it will be understood that more than two moieties may result from a cleaving step. As an example that is not to be understood as a limitation for the cleavable surfactants suitable for the methods of the present invention, an MS-compatible cleavable surfactant can be cleaved into a hydrophilic head group that is MS-compatible and remains in solution, and a hydrophobic, non-MS-compatible tail that can be easily removed from the sample by centrifugation or filtration. Accordingly, in a preferred embodiment, a method according to embodiments of the present invention may comprise the use of at least one MS-compatible cleavable zwitterionic or nonionic surfactant from which at least one moiety can be removed from a sample or a fractionated sample by filtration, centrifugation and/or by evaporation after a cleavage.

[0092] Any surfactant comprising a bond that combines a hydrophobic moiety (tail) with a hydrophilic moiety (head group) that can be broken down by a cleaving agent under conditions, preferably wherein the analyte of interest is essentially stable and wherein all resulting non-MS-compatible moieties can be easily removed by centrifugation, filtration or evaporation, is suitable as an MS-compatible cleavable surfactant. In accordance with the present invention, such a bond will be referred to as a cleavable bond. Preferably, such a bond is cleaved under conditions wherein an analyte of interest is essentially stable. An essentially stable analyte under conditions suitable to cleave a cleavable surfactant is to be understood as an analyte of interest, whereof at least about 80%, about 90%, preferably about 97%, more preferably about 99% and most preferably 100% of the amount of the analyte present during a cleavage step is unmodified after the cleavage step, i.e., the analyte is mainly, preferably completely, inert to a chemical reaction under the specific conditions during the cleavage step. Inert to a chemical reaction in this context means that no covalent bond within the analyte is broken or established during the cleavage step of the surfactant. [0093] A "cleaving agent" as used herein refers to any instrument or compound or mixture of compounds in any form suitable to selectively cleave a bond within a cleavable surfactant. Non-limiting examples for compounds suitable to selectively cleave a cleavable surfactant would be acids or bases or a solution/mixture thereof to selectively cleave a acid or base labile bond within a cleavable surfactant. This and further examples are described in more detail below. Furthermore, the term "cleaving agent" encompasses instruments suitable to selectively cleave a bond within a cleavable surfactant. Such an instrument can be, e.g., a light emitting instrument that emits light of a discrete wavelength to cleave a photo labile, cleavable surfactant. [0094] The term "solution for cleaving a cleavable surfactant" as used herein refers to any solution comprising an agent or a composition suitable to selectively cleave one or more bonds between a linker and a moiety within a cleavable surfactant resulting in at least two moieties wherefrom moieties which are non-MS-compatible can be easily removed from the sample by centrifugation, filtration or evaporation and MS-compatible moieties may stay in solution or may likewise be removed by centrifugation, filtration or evaporation.

[0095] An MS-compatible cleavable surfactant may comprise more than one cleavable bond, e.g., two cleavable bonds resulting in three moieties from one or more cleaving steps. Each cleavable bond can be independently selected from the group consisting of a covalent bond, an ionic bond, a hydrogen bond, or a complex bond. One or more covalent bonds are preferred in the context of the present invention.

[0096] In a preferred embodiment, at least one cleavable zwitterionic or nonionic surfactant of at least one fraction of a sample separated by a free-flow electrophoretic separation according to embodiments of the present invention is cleaved after the electrophoretic separation, i.e., at least one MS-compatible zwitterionic or nonionic surfactant is cleavable into at least one MS-compatible moiety and a moiety that can be easily removed by filtration, evaporation or centrifugation. Again, it is noted that an MS-compatible moiety might be also removed by evaporation prior to a subsequent analysis, i.e., a non-MS- compatible moiety resulting from a cleavage step is not subjected to the downstream analysis, whereas an MS-compatible moiety might be present or, optionally, absent in a downstream analysis.

[0097] MS-compatible cleavable surfactants may comprise at least one acid labile bond, i.e., the surfactant is acid labile, or at least one base labile bond, i.e., the surfactant is base labile, or at least one photo labile bond, i.e., the surfactant is photo labile, or at least one chemo reactive bond, i.e., the surfactant is chemo reactive. [0098] Acid and base labile cleavable surfactants may be cleaved by changing the pH of at least part of a fractionated sample/fraction, e.g., by acidifying or alkalifying of least part of a fractionated sample/fraction comprising an acid or base labile cleavable surfactant. Photo labile cleavable surfactants may be cleaved by irradiation, i.e. the cleavage of a cleavable surfactant is carried out by subjecting at least part of a fractionated sample/fraction comprising at least one photo labile cleavable surfactant to irradiation with light comprising or consisting of a defined wavelength suitable to selectively break the bond between a linker and a moiety of the surfactant. Chemo reactive cleavable surfactants may be cleaved by adding reactive agents, i.e. the cleavage of a cleavable surfactant is carried out by adding a reagent to at least part of a fractionated sample/fraction that is capable of breaking a bond within a chemo reactive surfactant. For example, a suitable reactant to cleave disulfide bonds and the like is DTT (dithiothreitiol) or a suitable reactant to cleave silane compounds of the general formula:

wherein R1 is selected from C7-C20 alkyl or C7-C30 alkyl aryl

R2, R3, R4, R5 and R6 are independently C1-C5 alkyl A is N or P X- is halide n is 1-5. [0099] One preferred chemo active cleavable surfactant for use in a FFE separation according to embodiments of the present invention is {2-[(dimethyl-octyl-silanyl)-ethoxy]-2- hydroxy-ethylj-trimethyl ammonium bromide.

[0100] A group of photo labile surfactants are, e.g., cinnamate esters such as 3-(2,4,6- trihydroxyphenyl) acryl acid octyl ester. [0101] A non-limiting example for an acid labile, cleavable surfactant is 3-[3-(1 ,1- bisalkoxyethyl)pyridine-1 -yl]propane-1 -sulfonate (PPS).

[0102] For chemo active cleavable surfactants and especially for acid or base labile cleavable surfactants, the FFE methods of the present invention provide distinct advantages over other electrophoretic methods/techniques. In fact, FFE allows using a wide variety of cleavable surfactants, which is not possible with other electrophoresis techniques. For example, acid labile cleavable surfactants such as PPS are extremely hygroscopic and are cleaved slowly by water at neutral pH, and at an accelerated rate at acidic or basic pH. According to Protein Discovery, the manufacturer of PPS, it is advised that once the package is opened to air, the contents should be immediately reconstituted in aqueous buffer (pH 7 - 8), protected from elevated temperatures and used within 12 hours. This means that especially pH labile cleavable surfactants can only be used for electrophoresis if the duration of the experiment is relatively short. The maximum duration of the experiment is even lower when the pH is decreased or increased. Therefore, at non-neutral pH, the electrophoretic experiment must be carried out within an even shorter timeframe. The advantage of FFE is that an electrophoretic separation, e.g. free-flow IEF, can be performed within this short time frame required to maintain the stability of the surfactant. In contrast, IEF as performed in the first dimension of 2D-gel electrophoresis (or in the off-gel instrument) typically requires experiment times of 5 hours or more, or even longer (up to 7-9 hours or more). Thus, the cleavable surfactant would be degraded to a larger extent, especially at very low or very high pH. [0103] Furthermore, free-flow (interval-) zone electrophoresis for separating analytes can be performed at a generally constant pH wherein the surfactant is stable for a sufficiently long time.

[0104] In addition, the use of counter flow media as described in the present invention can stabilize the cleavable surfactant immediately after the separation has taken place. This allows a separation of analytes at highly acidic or basic pHs in a very short time frame (e.g., down to around 5 min) followed by immediately adjusting the pH through the counter flow. Accordingly, one embodiment of the present invention relates to a FFE method, wherein a counter flow medium is used to adopt the medium conditions so as to stabilize a cleavable surfactant comprised therein after the free-flow electrophoresis, e.g., by adjusting the pH of a distinct fraction subsequent to a free-flow electrophoretic separation step.

[0105] It will be understood that these principles as described in the above non-limiting example can be extended to other types of cleavable surfactants that are stable under certain separation conditions for only a limited amount of time.

[0106] The counter-flow media can also be used in a different way, e.g., to introduce a cleaving agent that cleaves the surfactant for immediate further processing of the FFE fractions.

[0107] Accordingly, another embodiment of the present invention relates to a free flow electrophoretic method wherein a counter flow medium comprising a cleaving agent is used to provide the cleaving agent to a sample or a fraction thereof after free-flow electrophoretic separation that comprises a cleavable surfactant to cleave the cleavable surfactant. [0108] It will be understood that the use of MS-compatible surfactants is not limited to

MS applications but the MS-compatible surfactants may also be present in other analytic applications subsequent to any of the free-flow electrophoretic methods of the present invention. [0109] Hence, a method for analyzing analytes according to embodiments of the present invention may comprise an FFE separation for separating analytes according to embodiments of the present invention and a subsequent downstream analysis.

[01 10] In case the analyte of interest is a protein or polypeptide, a digestion step of the protein or polypeptide may be carried out prior or subsequent to the free-flow electrophoresis step. Those of skilled in the art know how to carry out a protein digestion step, e.g., using trypsin. There is also no need to remove the MS-compatible surfactants used in the free-flow electrophoresis to perform the digestion step. To the contrary, the presence of the surfactants may even improve the digestion, whereas, e.g., urea has to be at least partially removed prior to the digestion step. [01 11] In certain embodiments, the protein digestion step is carried out in at least one fraction collected from the free-flow electrophoresis step prior or subsequent to the cleavage step of a cleavable surfactant as described herein.

[01 12] Typically, the removal of non-MS-compatible moieties is easily achieved by well-known methods leading to no or essentially no sample loss. A purification step according to embodiments of the present invention is typically selected from the group consisting of evaporation, filtration and centrifugation to remove a precipitated moiety of a cleavable surfactant.

[01 13] The term "essentially no sample loss" as used herein means that less than 5% of an analyte of interest, preferably less than 1%, more preferably less than 0.2% and most preferably less than 0.1% may, e.g., stick on a filter used to remove a precipitated moiety of a cleaved surfactant or may remain within the pellet of a precipitated moiety of a cleavable surfactant that is removed by centrifugation, or may vaporize together with a moiety of a cleavable surfactant or a volatile buffer compound.

[01 14] In any case, for good results in downstream analysis methods, particularly mass spectrometric applications, additives such as those mentioned hereinabove should or at times must preferably be avoided, not the least because most additives are known to be non- compatible with mass spectrometry in general, at least if present above certain threshold levels which are generally known in the art. [01 15] The presence of MS-compatible surfactants which are MS-compatible per se or which can be cleaved to yield at least one MS-compatible moiety and, optionally, a non-MS- compatible moiety that can be easily removed, is advantageous since purification steps that are time consuming and/or lead to sample-loss are not required. Accordingly, a method according to embodiments of the present invention that comprises the use of MS-compatible surfactants as described herein does not require a purification step to remove surfactants selected from the group consisting of dialysis, chromatography, reversed phase chromatography, ion exchange chromatography, surfactant exchange, protein precipitation, affinity chromatography, electro blotting, liquid-liquid phase extraction, and solid-liquid phase extraction. In other words, it is not necessary to subject a fraction obtained from a FFE separation according to embodiments of the present invention to such a purification step prior to a downstream analysis.

[01 16] A combination of the volatile buffer compounds and MS-compatible surfactants as described herein offers the advantage of a notably reduced sample preparation of a fraction of a sample separated by FFE for a subsequent analysis. .

[01 17] It will be apparent to those skilled in the art that most electrophoresis applications will advantageously employ an ensemble of separation media and stabilizing media that are adapted to the specific application and apparatus used for the separation / fractionation problem. However, certain embodiments of the present invention may also be used in concert with commercially available proprietary stabilizing media (e.g., available from BD GmbH, Germany).

[01 18] The term "focus medium" as used herein refers to a SBM comprising an acid for an anodic focus medium or a base for a cathodic focus medium which form a conductivity step and, optionally, a pH step regarding the adjacent SBM. A focus zone formed by at least one SBM type I or at least one SBM type II, respectively, reduces the movement of analytes towards the anode or cathode essentially to zero due to a conductivity step. Such a conductivity step can be achieved by adding a strong acid or strong base to the SBM forming the focus zone. The concentration of the acid and base will be chosen so as to be sufficient to increase the conductivity of the at least one SBM focus medium, preferably by a factor of at least 2, and more preferably of at least 3, at least 5, or even more with regard to an adjacent SBM. This abrupt increase in the electrical conductivity of the medium is useful to accumulate analytes with a different pi as the pH range of the SBMs at the border of the two media having different conductivity values since the mobility of analytes moving to the anode or cathode, respectively is reduced to essentially zero. The principles of "focus media" are described in, e.g., International patent application PCT/EP2008/050597 and U.S. Pending Provisionals Ser. # 60/945,246 and 60//987,208, which are incorporated herein by reference in their entirety. A focus zone is generally formed by one focus medium, i.e., by one SBM but can also be formed by more than one SBM. A focus zone formed by a SBM type I may be adjacent to a SBM type I or adjacent to a SBM type II. Accordingly, a focus zone formed by a SBM type Il may be adjacent to a SBM type Il or adjacent to a SBM type I. [01 19] In certain embodiments, the pKa value of the acid in the anodic focus medium will be selected to be lower than the pKa value of the acid employed in the adjacent pH function, pH gradient or pH separation plateau (i.e. a stronger acid is selected for the anodic focus medium). In certain embodiments of the present invention, the pKa difference is greater than about 1 pH unit, preferably greater than about 2 pH units, and most preferably even greater than about 3 pH units. Suitable examples for an acid used to increase the conductivity is selected from, but not limited to the group consisting of sulfuric acid, pyridine- ethanesulfonic acid, hydrochloric acid, phosphoric acid, trifluoroacetic acid, trichloroacetic acid, and formic acid. An anodic focus medium may comprise the acid responsible for the increased conductivity and additionally the buffer compounds of an adjacent separation buffer medium, a volatile buffer system, a binary buffer acid/buffer base system (A/B medium), a commercially available ampholytes, a complementary multi pair buffer system (CMPBS) and/or a weak base to regulate the pH of the focus medium. A weak base should be understood to have a pKa that is lower than the pKa of the base used in the adjacent separation buffer medium. [0120] The same principles apply mutatis mutandis to the selection criteria for the base in the cathodic focus medium. Accordingly, the pKa value of the base in the cathodic focus medium will be selected to be higher than the pKa value of the base employed in the adjacent pH function, pH gradient or pH separation plateau (i.e. a stronger base is selected for the cathodic focus medium). In certain embodiments of the present invention, the pKa difference is greater than about 1 pH unit, preferably greater than about 2 pH units, and most preferably even greater than about 3 pH units. Suitable examples for a base used to increase the conductivity is selected from, but not limited to the group consisting of alkali or earth alkali hydroxides such as sodium hydroxide, 3-morpholino-2-hydroxy-propansulfonic acid, Tris, and the like. A cathodic focus medium may comprise the base responsible for the increased conductivity and additionally the buffer compounds of an adjacent separation buffer medium, a volatile buffer system, a binary buffer acid/buffer base system (A/B medium), a commercially available ampholytes, a complementary multi pair buffer system (CMPBS) and/or a weak acid to regulate the pH of the focus medium.

[0121] By virtue of its high electrical conductivity and its composition, a focus medium may also act as a stabilizing medium. [0122] "Stabilizing media" used for methods of the present invention have been described in co-pending PCT application PCT/EP2008/050597, which is incorporated herein by reference in its entirety. The stabilizing media are useful and suitable for stabilizing the conditions within the separation zone. A suitable stabilizing medium thus also acts as a "reservoir" supplying or replacing the ions in the separation zone.

Methods

[0123] The present invention provides methods for separating analytes which at the same time allow a buffer medium exchange, i.e., at least one analyte of interest is transferred during separation from or into a buffer medium type which comprises at least one additive at an effective concentration,.

[0124] Accordingly, one main aspect of the present invention relates to a method for separating an analyte from a sample by free flow electrophoresis comprising: a) forming within a free flow electrophoresis (FFE) chamber a separation zone between an anode and a cathode that comprises a zone I formed by at least one separation buffer medium type I and a zone Il formed by at least one separation buffer medium type II, wherein a separation buffer medium of type I comprises at least one additive at an effective concentration; and wherein a separation buffer medium of type Il does not contain the additive(s) at an effective concentration; and b) introducing a sample into the separation zone; and c) transferring at least one analyte to be separated from a separation medium type I into a separation medium type Il or transferring at least one analyte to be separated from a separation medium type Il into a separation medium type I during the free flow electrophoretic separation.

[0125] Another aspect relates to a method for separating an analyte from a sample by free flow electrophoresis comprising: a) forming within a free flow electrophoresis (FFE) chamber a separation zone between an anode and a cathode that comprises a zone I formed by at least one SBM type I that yields denaturing separation conditions and a zone Il formed by at least one SBM type Il that yields non-denaturing separation conditions; and b) introducing a sample into the separation zone; and c) transferring at least one analyte to be separated from a SBM type I into a SBM type Il or transferring at least one analyte to be separated from a SBM type Il into a SBM type I during the free flow electrophoretic separation.

[0126] A third aspect relates to a method wherein at least two analytes are separated comprising: introducing together with the sample or with at least one SBM type I an interaction partner of at least two of the analytes; wherein the presence of at least one additive at an effective concentration in SBM type I suppresses the interaction between at least one of the analytes of interest and the interaction partner and wherein at least one other analyte interacts with the interaction partner under the same conditions thereby forming an analyte-interaction partner-complex; and further wherein the analyte-interaction partner-complex(s) has a different pi compared to the pi of the analyte(s) of interest; and wherein the analyte-interaction partner-complex(s) remains in a SBM type I, whilst at least one of the analytes of interest is transferred into a SBM type Il during the free flow electrophoretic separation.

[0127] As a non-limiting example, the method is useful to separate peptides and proteins that interact and/or bind to an interaction partner such as albumin under non- denaturing conditions, but do not interact and/or bind to the interaction partner under denaturing conditions, whereas other peptides/proteins may interact / bind to the interaction partner under denaturing conditions.

[0128] Accordingly, a preferred embodiment of the present aspect relates to a method wherein the separation conditions of SBM type I are denaturing conditions and the conditions of SBM type Il are non-denaturing conditions.

[0129] This method is suitable to even separate analytes which have a similar or essentially identical pi such as isoforms and/or analogs of proteins, e.g., insulin, insulin isoforms or insulin analogs.

[0130] In certain embodiments, the pi of at least one analyte of interest that is transferred from a SBM type I into a SBM type Il during FFE separation differs at most 0.5, at most 0.2, at most 0.1 and at most 0.05 pi units from the pi of another analyte that forms an analyte-interaction partner-complex with an interaction partner and remains in zone I during the FFE separation. [0131] The present invention relates in another aspect to a method for analyzing analytes obtained from a composition of analytes comprising the steps of: conducting a free flow electrophoresis according to embodiments of the present invention; and eluting the analytes in a multiplicity of fractions from the FFE chamber; and subsequently analyzing one or more fractions of SBM type I and/or one or more fractions of SBM type Il by an analysis method.

[0132] A subsequent analysis method may be selected from, but is not limited to, the group consisting of free flow electrophoresis, gel electrophoresis, 1 D- and 2D-PAGE, MS, MALDI, ESI, SELDI, LC-MS(/MS), MALDI-TOF-MS(/MS), chemiluminescence, HPLC, Edman sequencing, NMR spectroscopy, IR-spectroscopy, UV-spectroscopy, X-ray diffraction, nucleic acid sequencing, electroblotting, amino acid sequencing, flow cytometry, circular dichroism, immuno detection, radio immuno detection, ELISA and any combination thereof. [0133] In most preferred embodiments, the subsequent analysis is performed without a buffer exchange step subsequent to the FFE separation and prior to the subsequent analysis. Notably, a buffer exchange during FFE separation (the transfer of an analyte of interest from one SBM type into the other SBM type during FFE separation) is not encompassed by the term buffer exchange step. A buffer exchange step means a step subsequent to an FFE separation according to embodiments of the present invention to exchange the buffer medium of at least one fraction comprising at least one transferred and separated analyte of interest. Such additional buffer exchange step that can be avoided are, e.g., gel filtration chromatography, dialysation, ultrafiltration and the like.

[0134] In preferred embodiments of all aspects of the present invention, at least one additive at an effective concentration in a SBM type I is selected from but not limited to the group consisting of a chaotropic agent (e.g. urea, guanidine hydrochloride, or lithium perchlorate), a denaturant (e.g., urea, thiourea, or guanidine hydrochloride), a reducing agent, a surfactant and an inhibitor.

[0135] Further preferred embodiments of the present invention relate to methods wherein the at least one additive at an effective concentration in a SBM type I is not present in a SBM type Il prior to the FFE separation, i.e., the SBM type Il is prepared and introduced into the FFE separation chamber without the additive.

[0136] Furthermore, most preferred embodiments of the present invention relate to

FFE methods wherein the concentration of at least one or even all additive(s) at an effective concentration in a SBM type I is/are below the detection limit, preferably zero, in a SBM type Il fraction comprising an analyte of interest, or wherein the concentration of at least one or even all additives at an effective concentration in a SBM type I is/are below the detection limit, preferably zero, in a SBM type II. [0137] In certain embodiments wherein the additive at an effective concentration is a surfactant, it is preferred that the denaturant can be an MS-compatible zwitterionic or nonionic surfactant. In such embodiments, an analyte of interest can be transferred from a SBM type Il into a SBM type I, which comprises a MS-compatible zwitterionic or nonionic surfactant, whereas the SBM type Il may contain a non-MS-compatible surfactant. This offers the advantage of reducing the costs of, e.g., combined FFE-MS. MS-compatible zwitterionic or nonionic surfactants are expensive compared with non-MS-compatible denaturants such as urea. According to this aspect of the present invention, merely one or more fractions of an FFE separation method which comprises a SBM type I need to contain the MS-compatible zwitterionic or nonionic surfactant although the denaturing conditions are maintained over the whole breadth of the separation zone.

[0138] In other preferred embodiments, a SBM type I yields denaturing conditions and a SBM type Il yields non-denaturing conditions, more preferably native conditions.

[0139] In further preferred embodiments, a FFE separation according to embodiments of the present invention comprises a focus medium zone formed by one SBM type, i.e., wherein at least one SBM type I or at least one SBM type II, respectively, forms the focus medium zone and at least one analyte of interest is transferred from a SBM type I or II, respectively, into the focus medium zone formed by a SBM type Il or I, respectively .

[0140] In certain embodiments, the focus zone is formed by at least one SBM type I or type II, respectively, and is adjacent to a SBM zone Il or SBM zone I, respectively, i.e., one SBM type forms a focus medium zone adjacent to the other SBM type, in other embodiments, a focus medium zone is comprised in a zone I or II, respectively, but the focus medium zone is not at the zone I/zone Il border. The zone I/zone Il border is the contact area of the two media types within an FFE chamber.

[0141] In preferred embodiments, the conductivity of the SBM type forming the focus medium zone is at least about 2-fold, at least 3-fold and preferably at least 5-fold higher than the conductivity of the adjacent SBM, which can in certain embodiments be a SBM of the same or of the other type.

[0142] Depending on the operation method, e.g., IEF, a method according to embodiments of the present invention comprises a pH gradient within a separation zone. [0143] Accordingly, in some embodiments of the present invention, a method comprises a pH gradient within a separation zone wherein the pH gradient is formed by at least one SBM type Il or SBM type I, respectively. In other embodiments, the methods comprise a spacer zone wherein the spacer zone is formed by a SBM type Il or type I, respectively.

[0144] The skilled person will understand that a sample for a FFE separation can be provided into a FFE separation chamber via various ways.

[0145] Accordingly, in one preferred embodiment of the present invention, a sample is introduced through an individual sample inlet into a FFE separation chamber. A sample inlet is located to be in fluid communication with the separation zone between the two electrodes, and additionally located longitudinally (parallel to the electrodes) between the media inlets and outlets, preferably closer to the media inlet ports than the outlet ports (see, e.g., Figure 1 ). In general, a sample inlet may be positioned at any desired position between the anode and the cathode of the FFE apparatus. [0146] In further preferred embodiments, a sample is introduced through at least one media inlet, typically but not necessarily together with at least one SBM type I or with at least one SBM type II, respectively. The skilled person will understand how to determine whether it is suitable to introduce the sample together with an SBM type I that comprises an additive at an effective concentration, or with an SBM type Il that does not comprise an additive at an effective concentration. The skilled person will understand that a sample can be introduced through merely one separation medium inlet or a plurality of separation medium inlets. Additionally, the sample can be introduced through at least one medium inlet and at least one sample inlet.

[0147] In certain preferred embodiments of the present invention, it is preferred that the analytes are eluted from the FFE chamber after the FFE separation in a plurality of fractions.

[0148] In further preferred embodiments, a method according to embodiments of the present invention comprises the use of an anodic and a cathodic stabilizing medium.

[0149] An FFE separation method according to embodiments of the present invention can be carried out in an operation mode selected from but not limited to the group consisting of continuous mode, interval mode and cyclic interval mode.

Suitable buffer systems

[0150] In certain embodiments of the present invention, it may be advantageous to use specific buffer systems to prepare each SBM type I and II, respectively. As non-limiting examples, a SBM may comprise commercial ampholytes as a buffer system, a volatile buffer system, a binary buffer acid/buffer base system (A/B medium), or a complementary multi pair buffer system (CMPBS).

Volatile buffer systems

[0151] In certain embodiments of the present invention, volatile buffer systems can be used as a buffer system of at least on separation buffer medium. These buffer systems are disclosed in U.S. Pending Provisional Ser. # 60/945,246 and US 60/987,208 and offer the particular advantage that they can be removed residue-free from the recovered fractionated sample after an FFE separation step or are MS-compatible per se and can remain in the sample.

[0152] A volatile buffer system comprises at least one buffer acid and at least one buffer base, wherein all of the buffer compounds are volatile. Optionally, at least one of the buffer compounds may be capable of functioning as a (volatile) matrix for mass spectrometry, particularly in MALDI applications. [0153] The term "volatile" used in connection with the buffer compounds herein should be understood to refer to the buffer compound's ability to be completely removable from an aqueous sample under suitable conditions, i.e., the buffer compound can be evaporated without leaving behind any residual compound (e.g., a salt), i.e. residue-free. In its broadest meaning, a volatile buffer compound can be removed residue-free under conditions selected from, but not limited to, the group of reduced atmospheric pressure, increased temperature, supply of energy by irradiation (e.g. UV light, or by applying a laser light), or any combination thereof, although it will be appreciated that a volatile buffer compound must essentially be non-volatile under FFE working conditions (i.e., atmospheric pressure and temperature ranges of typically between 0 and 40 ⁰C as explained hereinabove). [0154] In this context, the skilled person will understand that in one embodiment of the invention, the analyte(s) that is (are) present in a sample comprising volatile buffer compounds will be non-volatile under the afore-mentioned conditions, i.e., the analyte(s) is (are) essentially not modified (e.g., by fragmentation or oxidation) and remain(s) in solution or in its (their) solid state. In certain embodiments, particularly under mass spectrometric working conditions, the analyte(s) will also be volatile and will be ionizable (required for detection by MS).

[0155] The term "non-volatile under FFE working conditions" as used herein means a volatility of a buffer compound leading to a concentration reduction of the respective buffer compound in the separation medium of less than 5% w/v or, preferably less than 2% w/v under working conditions and during the separation period of FFE. Most preferably, no concentration reduction will be observed at all under working conditions and the separation period of FFE.

[0156] The term "residue-free" as used herein means that the volatile compound itself evaporates completely, but that residues caused, e.g., by an impurity of the used substances, may be non-volatile. However, it is well known to those of skill in the art that only compounds having the highest purity grade available should be used for analytic purposes, and particularly so for mass spectrometric analysis.

[0157] Removal of the solvent and buffer compounds by "evaporation" as used herein should be understood to refer to a removal from the analytes of interest through transferring the compounds into the gas phase and subsequent elimination of the gas phase by suitable means. Thus, evaporation as defined herein is different from eliminating the buffer compounds by techniques commonly referred to as buffer exchange (sometimes also referred to as "desalting"), including column chromatography, dialysis or cut-off filtration methods, or techniques known as solid phase extraction or analyte precipitation. Alternatively, in certain applications that are not included under the term evaporation, the buffer compounds present in salt form are simply washed away with water, although this obviously leads to an undesirable loss of sample material and, moreover, non-quantitative removal of the buffer compounds. Those of skill in the art will appreciate that the volatile buffer compounds as defined herein could, at least in principle, likewise be removed by such buffer exchange or solid phase extraction techniques, although this would of course neglect the distinct advantage offered by the volatility of the buffers (and makes no sense in view of the potential problems connected with buffer exchange techniques, e.g., difficult handling and low sample recovery). [0158] Suitable exemplary techniques for removing the solvent and the volatile buffer compounds from a sample collected from an FFE separation step by evaporation include, but are not limited to, vacuum centrifugation using suitable devices such as a centrifugal evaporator or a vacuum centrifuge known for example under the name SpeedVac^®, by lyophilization or by a (gentle) heating of the aqueous sample. Other possibilities to evaporate the solvent and the buffer compounds include evaporation by subjecting the sample to reduced pressure conditions, e.g., applying a vacuum to the sample placed on a target plate used in mass spectrometric analysis. Those of skill in the art will appreciate that most mass spectrometric methods operate under vacuum conditions (for example vacuum MALDI) so that the volatile buffer compounds are conveniently removed after the introduction of the sample into the MS instrument, but prior to ionization. [0159] Preferably, the volatile buffer compounds are removable under conditions of reduced pressure and/or increased temperature. Moreover, in other embodiments, the volatile buffer compounds may even be evaporated under ambient temperature and atmospheric pressure conditions, particularly if the volatile buffer-containing sample is present in a small volume (e.g., for mass spectrometric analysis). However, in most cases at least some buffer solution will not evaporate readily under those conditions. In yet other embodiments, the volatile buffer compounds can only be removed under harsher conditions (e.g., in vacuum and/or high temperatures, optionally with irradiation, such as under mass spectrometric working conditions). [0160] In certain embodiments of the present invention, the FFE separation media comprise volatile buffer compounds wherein at least one of the volatile buffer compounds may act as a (volatile) matrix for mass spectrometric analysis, i.e., the compound can only be removed under mass spectrometric working conditions. It will be understood that the term matrix in the context of mass spectroscopy (MS) as used herein is different from the term "matrix" used in the context of electrophoresis (e.g., polyacrylamide or agarose). Therefore, in some embodiments wherein the downstream analysis is for example a MALDI application, a matrix component for MALDI analysis is added to the analyte buffer solution prior to mass spectrometric analysis.

[0161] Examples for volatile buffer systems include, but are not limited to combinations of TRIS / acetic acid, diethanolamine / picolinic acid, dimethylamino-proprionitril / acetic acid, 2-pyridine ethanol / picolinic acid, benzylamine / 2-hydroxypyridine, tri-n-propylamine / trifluoroethanol, and the like.

Complementary multi-pair buffer systems (CMPBS)

[0162] In certain embodiments of the invention, a buffer mixture used to generate the pH gradient may be comprised of carefully matched acids and bases such that the mixture may provide a smooth pH gradient when current flows through the buffer system. A mixture of low molecular weight organic acids and bases are chosen that enable an increased buffering capacity compared to commercially available high molecular weight ampholytes.

These mixtures of carefully matched acids and bases are extremely well characterized in terms of molecular weight, pi, purity, and toxicity. Generally, the acids and bases have a smaller molecular weight than those of commercial ampholytes. Suitable complementary multi-pair buffer systems are known in the art. Specifically, a mixture with a pH range from 3 to 5 is sold as BD FFE Separation medium 1 while a mixture with a pH range from 5 to 8 is sold as BD FFE Separation medium 2 by BD GmbH Germany. These buffer systems have, for example, been described in general form in US patent application US 2004/0101973 and in EP 1 320 747 which are incorporated herein by reference in their entirety. Complementary multi-pair buffer systems as described above are referred herein as "CMPBS" or "CMPBS media".

Binary buffer systems (A/B buffer system)

[0163] Binary buffer systems as defined below are referred to herein as "A/B buffer systems" and are disclosed in detail in International patent application PCT/EP2008/050597, which is incorporated herein by reference in their entirety. The buffer system comprises at least one buffer acid and at least one buffer base, with the proviso that the pKa value of the buffer acid must be higher than the pH of the SBM and the pKa of the buffer base is lower than the pH of the SBM. Put another way, the pKa of the buffer acid will be higher than the pKa of the buffer base.

[0164] The pH profile exhibited by the A/B SBM may be essentially linear (i.e., without any major pH steps during electrophoretic separation). Depending on the stabilizing media employed as well as the pKa differences between the buffer acid and the buffer base, the A/B SBM according to this aspect of the invention will offer an essentially constant (i.e., flat) pH profile, or a rather gentle/flat pH gradient within the separation chamber. It will be appreciated that the separation media providing a zone with an essentially constant pH in the separation chamber between the electrodes are particularly useful for the creation of pH separation plateaus in accordance with the methods described herein. However, since the A/B SBM may also form flat- or ultra flat pH gradients, they can also be used for the creation of pH functions or pH gradients as defined herein.

[0165] Preferably, the A/B SBM employing at least one buffer acid and one buffer base in the above aspect of the present invention are characterized by a pKa difference between the at least one buffer acid and the at least one buffer base of between about 0.5 and 4 pH units, wherein the pKa of the acid must be higher than the pKa of the base as explained above. In preferred embodiments, the ΔpKa is between 1.2 and 1.8, which is particularly useful for pH separation plateaus having a constant pH within the separation chamber of an FFE apparatus. In other preferred embodiments, the ΔpKa will be between about 2.5 and 3.3, the latter being particularly suitable for flat pH-gradients. [0166] One characteristic of the A/B SBM is that the electrical conductivity of the medium is relatively low, although it will be appreciated that the conductivity must be sufficiently high to achieve acceptable separation of the analytes in a reasonable amount of time. Thus, the conductivity of the A/B SBM is typically between 50 and 1000 μS/cm, and more preferably between 50 and 500 μS/cm, although those of skill in the art will be aware that the exact conductivity in the separation medium will of course depend on the specifics of the separation / fractionation problem, the presence of other charged species in the medium (e.g., ions required for sample/analyte stability) and the electrochemical properties of the analyte. [0167] Preferably, the A/B SBM comprise only one buffer acid and one buffer base. In other words, such separation media represent binary media wherein one acid function of a compound and one base function of the same or another compound essentially serve to establish a separation medium with the desired pH and conductivity profile. While good results may also be achieved with two or more buffer acids and buffer bases in the separation medium, it is typically advantageous to use as few components as possible, not only because it is easier to prepare and possibly cheaper to use, but also because the electrochemical properties of the medium will become more complex if the number of charged species present in the separation chamber is increased.

[0168] Suitable buffer bases in this context are, for example, taurine, glycine, 2-amino- butyric acid, glycylglycine, β-alanine, GABA, EACA, creatinine, pyridine-ethanol, pyridine- propanol, histidine, BISTRIS, morpholinoethanol, triethanolamine, TRIS, ammediol, benzylamine, diethylaminoethanol, trialkylamines, and the like. Suitable buffer acids are, for example, HIBA, acetic acid, picolinic acid, 4-pyridineethanesulfonic acid (PES), MES, ACES,

MOPS, HEPES, EPPS, TAPS, AMPSO, CAPSO, α-alanine, GABA, EACA, A- hydroxypyridine, 2-hydroxypyridine, and the like, provided the pKa relationships between the buffer acid and buffer base as described above is met.

[0169] Furthermore, in the methods of the present invention binary buffer systems as disclosed in, e.g., U.S. patent 5,447,612 for separating analytes by FFE can also be employed. These binary media may be suitable for forming relatively flat pH gradients of between 0.4 to 1.25 pH units.

FFE methods and modes

[0170] The methods according to the present invention can be carried out using, e.g., one of the following operation methods.

[0171] Several FFE operation methods are known to those skilled in the art and are contemplated in the context of the present invention. For example, in certain embodiments of the present invention a sample can be separated according using a electrophoretic separation method selected from isoelectric focusing (IEF), zone electrophoresis (ZE), combinations thereof, which are, e.g., described in WO 2007/147862, which is herewith incorporated in its entity, or isotachophoresis (ITP)). [0172] Additionally, several FFE operation modes are known to those of skill in the art and are contemplated in the context of the present invention. For example, the sample and separation medium may be continuously driven towards the outlet end while applying an electrical field between the anode and the cathode of an FFE apparatus ("continuous mode"). Continuous mode in the context of FFE should be understood to mean that the injection step as well as the separation step occurs continuously and simultaneously. The electrophoretic separation occurs while the medium and the analytes pass through the electrophoresis chamber where the different species are being separated according to their pi (IEF), net charge density (ZE) or electrophoretic mobility (ITP). Continuous mode FFE allows continuous injection and recovery of the analytes without the need to carry out several independent "runs" (one run being understood as a sequence of sample injection, separation and subsequent collection and/or detection). It will be appreciated that continuous mode FFE includes separation techniques wherein the bulk flow rate is reduced (but not stopped) compared to the initial bulk flow rate while the analytes pass the separation space between the electrodes in order to increase the separation time. In the latter case, however, one can no longer speak of a true continuous mode because the reduction of the bulk flow rate will only make sense for a limited amount of a sample.

[0173] Another FFE operation mode known as the so-called "interval mode" or "static interval mode" in connection with FFE applications has also been described in the art. For example, a process of non-continuous (i.e. interval) deflection electrophoresis is shown in U.S. patent 6,328,868, the disclosure of which is hereby incorporated by reference. In this patent, the sample and separation medium are both introduced into an electrophoresis chamber, and then separated using an electrophoresis mode such as zone electrophoresis, isotachophoresis, or isoelectric focusing, and are finally expelled from the chamber through fractionation outlets. Embodiments of the '868 patent describe the separation media and sample movement to be unidirectional, traveling from the inlet end towards the outlet end of the chamber. This direction, unlike in traditional capillary electrophoresis, is shared by the orientation of the elongated electrodes. In the static interval mode described, e.g., in the '868 invention, acceleration of the sample between the electrodes caused by a pump or some other fluidic displacement element only takes place when the electrical field is off or at least when the voltage is ineffective for electrophoretic migration, i.e., when no part of the sample is being subjected to an effective electric field.

[0174] In other words, the interval process is characterized by a loading phase where the sample and media are introduced into the separation chamber of the electrophoresis apparatus, followed by a separation process where the bulk flow of the medium including the sample is halted while applying an electrical field to achieve separation. After separation/fractionation of the sample, the electrical field is turned off or reduced to be ineffective and the bulk flow is again turned on so that the fractionated sample is driven towards the outlet end and subsequently collected/detected in a suitable container, e.g., in a micro titer plate. [0175] The so-called cyclic or cyclic interval mode in the context of FFE as used herein has been described in International application WO/2008/025806, hereby incorporated by reference in its entity. In sum, the cyclic interval mode is characterized by at least one, and possible multiple reversals of the bulk flow direction while the sample is being held in the electrophoretic field between the elongated electrodes. In contrast to static interval mode, the sample is constantly in motion thereby allowing higher field strength and thus better (or faster) separation. Additionally, by reversing the bulk flow of the sample between the elongated electrodes, the residence time of the analytes in the electrical field can be increased considerably, thereby offering increased separation time and/or higher separation efficiency and better resolution. The reversal of the bulk flow into either direction parallel to the elongated electrodes (termed a cycle) can be repeated for as often as needed in the specific situation, although practical reasons and the desire to obtain a separation in a short time will typically limit the number of cycles carried out in this mode.

Further additives

[0176] In addition to the at least one additive that is present at an effective concentration in a SBM type I but absent or below an effective concentration in a SBM type Il as defined herein , the SBMs of the present invention may comprise one or more further additives which bring about a further effect unrelated to the first additive. Thus, the further additives can typically be present in all SBMs.

[0177] Further additives in accordance with the present invention are compounds or ions that do not (or at least not significantly) contribute to the buffering capacity provided by the buffer acids and the buffer bases. Generally, the number and concentration of additives should be kept to a minimum, although it will be appreciated that certain analytes or separation problems require the presence of additional compounds either for maintaining analyte integrity or for achieving the desired properties of the medium (e.g., viscosity adaptation between various separation media, etc.).

[0178] Possible additives are preferably selected from other acids and/or bases, so- called "essential" mono- and divalent anions and cations, viscosity enhancers, protein solubilizing agents, affinity ligands, reducing agents, and the like. [0179] As apparent from the foregoing explanations, other acids or base may be present in the separation media of the invention, provided the pKa of their acid or base function is sufficiently far-removed from the pH or pH range of the separation medium to avoid contributing to the buffering capacity of the solution (although they may of course contribute to the electrical conductivity in the medium). Examples for possible acids and bases include small amounts of strong acids or bases (e.g., NaOH, HCI, etc.) that are completely dissociated in solution, or very weak acids or bases that are present as essentially undissociated species in the medium (i.e. having a pKa that is more than about 4 units away from the pH of the medium). [0180] Essential mono- and divalent anions and cations in the sense of the present application are ions that may be needed for maintaining the structural and/or functional integrity of the analytes in the sample. Examples for such essential anions and cations include, but are not limited to magnesium ions, calcium ions, zinc ions, Fe(II) ions, chloride ions, sulfate ions, phosphate ions or complexing agents such as EDTA or EGTA, or azide ions (e.g., for avoiding bacterial contamination), and the like.

[0181] Viscosity enhancers commonly used in the separation media may include polyalcohols such as glycerol or the various PEGs, hydrophilic polymers such as HPMC and the like, carbohydrates such as sucrose, hyaluronic acid, and the like. Viscosity enhancers may be required to adapt the viscosity of the separation medium to the viscosity of the sample introduced into the separation space, or to the viscosity of other separation and/or stabilizing media within the separation chamber in order to avoid turbulences created by the density or viscosity differences between sample and medium or between different adjacent media.

[0182] Additional additives that may be present include chiral selectors such as certain dextrins including cyclodextrins, or affinity ligands such as lectins and the like.

[0183] In certain cases, it may be required to add reducing agents to prevent the oxidation of an analyte in the solution. Suitable reducing agents that may be added to the sample and/or the separation medium includes mercaptoethanol, mercaptopropanol, dithiothreitol (DTT), ascorbic acid, sodium or potassium metabisulfite, and the like. [0184] In any event, because many of the aforementioned additives are electrically charged, their concentration should be kept as high as needed but at the same time as low as possible so as to maintain the electrical conductivity of the separation medium within the desired (low) range. Kits and Electrophoretic Media Compositions

[0185] It will be apparent to those skilled in the art that the SBM types contemplated herein may be selected, prepared and used alone, or, alternatively, together with other stabilizing media, focus media and SBM, respectively. [0186] Accordingly, another aspect of the present invention also relates to a kit for carrying out the FFE methods according to embodiments of the present invention, wherein the kit comprises at least buffer compounds for at least one buffer system and at least one additive for the preparation of at least one SBM type I and at least one SBM type Il to carry out the desired method. The skilled person will understand that, depending on the desired method (e.g., IEF) more than one SBM of one type can be provided to ensure, e.g., a linear gradient). Therefore, the number of buffer systems and therefore the number of different SBMs can also be between 2 and 15, preferably between 3 and 12, and more preferably between 3 and 7. Notably, at least one of the SBMs is a SBM type I. The skilled person will understand that also other numbers of SBM such as up to 20, 30 or 40 are possible. In this context, it is noted that SBM may also have properties as stabilizing and/or focus media.

[0187] Since anodic and cathodic stabilization are both particularly useful for successful electrophoretic applications, particularly in FFE, the kit will in addition to the SBM preferably comprise one anodic and/or one cathodic stabilizing medium as defined herein. They are generally located between the anode and/or cathode, respectively, and further adjacent SBMs of the separation zone.

[0188] The kit may comprise at least one, several or all SBM type I and/or type Il as aqueous solutions that are ready to be used in the FFE separations described herein(i.e., all components are present in the desired concentration for the electrophoretic separation problem), or it may contain one, several or all of the SBM type I and/or type Il in the form of a concentrated aqueous stock solution that is to be diluted with a p re-determined amount of solvent to the appropriate concentration prior to their use. Alternatively, the kit may comprise one, several or all components of the kit in dry form or lyophilized form wherein the components are to be dissolved with a predetermined amount of solvent, e.g. water or the buffer system in water, prior to their use in an electrophoretic separation process. [0189] It will be understood that each component such as each dried component and/or each stock solution and/or each solution ready for use may each be separately placed in a sealed container as appropriate. [0190] It will be further understood that all of the preferred SBM described herein, as well as the preferred cathodic and/or anodic stabilizing media, may be included in the kits of the present invention.

[0191] It is generally preferred that each medium (SBM, cathodic stabilizing medium, anodic stabilizing medium, counter flow medium) will be present in a separate container, although it will be apparent to those of skill in the art that other combinations and packaging options may be possible and useful in certain situations. For example, it has been mentioned above that the separation media for IEF applications may consist of a distinct number of "sub-fractions" having different concentrations of the ingredients (and thereby a different pH) in order to create a pre-formed pH gradient within the electrophoresis apparatus. In one embodiment, the pH of each SBM used to form the gradient is different. The number of sub- fractions employed in IEF applications will depend on the separation problem, the desired pH span achieved with the SBM and the electrophoresis apparatus used for the separation. In FFE applications, the apparatus will typically comprise several media inlets (e.g., N=7, 8 or 9 inlets), so that the sub-media creating the separation space within the apparatus may be introduced into at least one to a maximum of N-2 inlets (at least one inlet on each side is usually reserved for a stabilizing medium, if present). The number of SBM, which can be inserted into an apparatus suitable for FFE, is thus typically between 2 and 15, or between 3 and 12, or between 4 and 9. [0192] In particularly preferred embodiments, the separation media in the kit will be formed by binary buffer systems, comprising only one buffer acid and one buffer base. It is contemplated that all of the separation media described herein, be they preferred or not, may be included in the kits of the present invention.

[0193] In further preferred embodiments, a kit further comprises at least one product manual that describes one or more experimental protocols to carry out a free flow electrophoretic separation according to embodiments of the present invention, and optionally storage conditions for the components.

Apparatus for carrying out FFE methods according to the present invention

[0194] An FFE instrument or apparatus for carrying out a method according to embodiments of the present invention comprises: an electrophoresis chamber comprising a set of electrodes, wherein at least one of the electrodes is a cathode and at least one of the electrodes is an anode, and a separation zone interposed therebetween, wherein the apparatus further contains means for forming a zone I formed by at least one SBM type I; and means for forming a zone Il formed by at least one SBM type II; and optionally, means for forming stabilizing media within the separation zone.

[0195] In an alternative embodiment, an FFE instrument or apparatus for carrying out an FFE method as described herein comprises: an electrophoresis chamber comprising a set of electrodes, wherein at least one of the electrodes is a cathode and at least one of the electrodes is an anode, and a separation zone interposed therebetween, wherein the separation zone comprises at least one zone I; at least one zone II; wherein zone I comprises at least one SBM type I and zone Il comprises at least one SBM type II; and a sample inlet capable of fluid communication with the separation zone.

[0196] Accordingly, the use of an apparatus as defined above for performing a separation of at least one analyte of interest from a composition of analytes by free flow electrophoresis is also an embodiment of the present invention.

[0197] It will be apparent to those of skill in the art that many modifications and variations of the embodiments described herein are possible without departing from the spirit and scope of the present invention. The present invention and its advantages are further illustrated in the following, non-limiting examples.

Examples

EXAMPLE 1 : Separation of insulin Detemir and albumin under denaturing conditions (in SBM type I) and elution of insulin Detemir under native conditions (in SBM type II)

[0198] Insulin Detemir is a long-acting human insulin analogue for maintaining the basal level of insulin. It is an insulin analogue in which to the lysine amino acid at position B29 a fatty acid (myristic acid) is bound. It strongly interacts under native conditions with albumin through the fat acid at position B29. [0199] The separation medium and stabilizing media were tested on a BD™ Free Flow

Electrophoresis System in FF-IEF mode using a quality control solution. The apparatus was set up comprising seven media inlets (E1-E7) and four sample inlets (S1-S4). Anodic stabilizing medium was introduced into inlet E1. The cathodic stabilizing medium was introduced into inlet E7 and the sample was introduced via sample inlet S4. The total time of electrophoresis was approximately 10 minutes. The voltage applied was 1200V and the current was 37 mA. The sample and the media were introduced at a flow rate of 1.5 ml/h and 120 ml/h, respectively.

[0200] After the test of the separation medium and stabilizing media, an insulin Detemir standard was run to evaluate fractions comprising the analyte of interest.

[0201] Subsequently, insulin Detemir was spiked into a blood plasma sample and the sample was run under the same separation conditions.

[0202] All samples were introduced into a zone I (E4) yielding denaturing conditions that suppress or inhibit an insulin Detemir-albumin interaction. Insulin Detemir was eluted in fractions containing SBM type Il yielding non-denaturing conditions.

[0203] Anodic stabilizing medium: 10OmM H₂SO₄ 5OmM HAc 20OmM 2-Ami.Butt.

3OmM Glycylglycin (pH = 2.16; conductivity: 5650 μS/cm) (E1 );

[0204] Cathodic stabilizing medium: 15OmM NaOH 30OmM β-Alanin 3OmM ethanolamine (pH = 10.09; conductivity: 3200 μS/cm) (E9);

[0205] Separation medium:

[0206] [0207] The pH of each of the FFE fractions was determined using a pH electrode and is presented by the graph in FIG. 2. Colored pl-markers were separated to evaluate the separation performance of the system. In addition, the absorbance of the fraction at λ = 420 nm, 515 nm, and 595 nm which represent the absorbance of the respective pl-markers are reported in FIG. 2. [0208] The insulin Detemir standard eluted into fractions 42 to 45 (see FIG. 3).

[0209] FIG. 4 depicts a silver stained gel showing the successful separation of insulin

Detemir from albumin, insulin Detemir eluted into fractions of SBM type Il (yielding non- denaturing conditions), whereas albumin retained in fractions formed by SBM's of type I (yielding denaturing conditions).

Claims

1. An electrophoretic separation method for separating an analyte from a sample by free flow electrophoresis comprising: a) forming within a free flow electrophoresis (FFE) chamber a separation zone between an anode and a cathode that comprises a zone I formed by at least one separation buffer medium type I and a zone Il formed by at least one separation buffer medium type II, wherein the separation buffer medium of type I comprises at least one additive at an effective concentration; and wherein the separation buffer medium of type Il does not contain the additive(s) at an effective concentration; and b) introducing a sample into the separation zone; and c) transferring at least one analyte to be separated from a separation medium type I into a separation medium type Il or transferring at least one analyte to be separated from a separation medium type Il into a separation medium type I during the free flow electrophoretic separation.

2. The method according to claim 1 , wherein the electrophoretic separation method comprises isoelectric focusing (IEF).

3. The method according to claim 1 , wherein the electrophoretic separation method comprises zone electrophoresis (ZE).

4. The method according to claim 1 , wherein the electrophoretic separation method comprises isotachophoresis (ITP).

5. The method according to any one of claims 1 to 4, wherein the analytes are eluted from the FFE chamber into a plurality of fractions.

6. The method according to any one of claims 1 to 5, wherein at least one additive at an effective concentration in a SBM type I is a chaotropic agent, a denaturant, a reducing agent, a surfactant or an inhibitor.

7. The method according to any one of claims 1 to 6, wherein a SBM type I yields denaturing conditions and a SBM type Il yields non-denaturing conditions.

8. The method according to claim 7, wherein SBM type Il yields native conditions.

9. The method according to any one of claims 1 to 8, wherein the analyte maintains its native state while in the presence of SBM type Il and is denatured while in the presence of SBM type I.

10. The method according to any one of claims 1 to 9, wherein the additive at an effective concentration is an MS-compatible zwitterionic or nonionic surfactant.

1 1. The method according to any one of claims 1 to 3 and 5 to 10, wherein one SBM type forms a focus medium zone.

12. The method of claim 1 1 , wherein the focus medium zone is adjacent to the other SBM type. (ZE and IEF)

13. The method of claim 11 or claim 12, wherein the conductivity of the SBM type forming the focus medium zone is at least about 2-fold, at least 3-fold and preferably at least 5 fold higher than the conductivity of the adjacent SBM.

14. The method according to claims 11 to 13, wherein the focus medium is formed by a SBM type I.

15. The method according to claims 11 to 13, wherein the focus medium is formed by a SBM type II.

16. The method according to any one of claims 1 , 2 or 5 to 15, wherein a pH gradient within the separation zone is formed by at least one SBM type II.

17. The method according to claim 4, wherein a separation buffer medium type Il forms a spacer zone. (ITP)

18. The method according to any one of claims 1 , 2 or 5 to 15, wherein a pH gradient within the separation zone is formed by at least one SBM type I.

19. The method according to any one of claims 1 to 18, wherein the sample is introduced through an individual sample inlet.

20. The method according to any one of claims 1 to 18, wherein the sample is introduced together with at least one SBM type I or with at least one SBM type II, respectively.

21. The method according to any one of 1 to 20, wherein the sample is introduced through a plurality of separation medium inlets.

22. The method according to any one of claims 1 to 21 , wherein at least one analyte to be separated is separated into a SBM type II.

23. The method according to any one of claims 1 to 21 , wherein at least one analyte to be separated is separated into a SBM type I.

24. The method according to any one of claims 1 to 23, wherein the method comprises an anodic and a cathodic stabilizing medium.

25. The method of any one of claims 1 to 24, wherein the buffer systems comprised in the SBM type I and the SBM type Il are each individually selected from the group consisting of commercial ampholytes, CMPBS media, volatile buffer systems and binary buffer acid/buffer base systems (A/B media).

26. The method of any one of claims 1 to 25, wherein the concentration ratio between the additive in a SBM type Il fraction and the concentration on the SBM type I prior to the FFE separation method is <0.5, <0.2, <0.1 , <0.05, <0.01 , <0.001 , ≤O.OOOL

27. The method of any one of claims 1 to 26, wherein the concentration ratio between the additive in a SBM type Il prior to the FFE separation method and the concentration on the SBM type I prior to the FFE separation method is <0.5, <0.2, <0.1 , <0.05, <0.01 , <0.001 , <0.0001.

28. The method of any one of claims 1 to 27, wherein the free flow electrophoresis is operated in continuous mode, interval mode or cyclic interval mode.

29. The method of any one of claims 1 to 28, wherein at least one analyte to be separated is selected from the group consisting of proteins, protein aggregates, peptides, DNA-protein complexes, DNA, membranes, membrane fragments, lipids, saccharides, polysaccharides, hormones, liposomes, cells, cell organelles, viruses, virus particles, antibodies, chromatin, melamine resins, latex paint particles, polystyrenes, polymethylmethacrylates, dextranes, cellulose derivatives, polyacids, illicit drugs, explosives, toxins, pharmaceuticals, carcinogens, poisons, allergens, infectious agents.

30. The method of any one of claims 1 to 29, wherein at least one analyte to be separated is a protein.

31. The method of any one of claims 1 to 30, wherein the concentration of at least one additive at an effective concentration in a SBM type I is below the detection limit in a SBM type Il fraction comprising an analyte of interest.

32. A method for separating an analyte from a sample by free flow electrophoresis comprising: a) forming within a free flow electrophoresis (FFE) chamber a separation zone between an anode and a cathode that comprises a zone I formed by at least one SBM type I that yields denaturing separation conditions and a zone Il formed by at least one SBM type Il that yields non-denaturing separation conditions; and b) introducing a sample into the separation zone; and c) transferring at least one analyte to be separated from a SBM type I into a SBM type Il or transferring at least one analyte to be separated from a SBM type Il into a SBM type I during the free flow electrophoretic separation.

33. The method according to claim 32, wherein the non-denaturing conditions are native conditions.

34. The method according to any one of claims 1 to 33, wherein an analyte of interest is separated from an analyte that is an interaction partner of the analyte comprising: introducing a sample comprising the analyte and the interaction partner thereof into a zone I; wherein the presence of at least one additive at an effective concentration in zone I suppresses the interaction between the analyte and the interaction partner; wherein the interaction partner remains in zone I, whereas the analyte is transferred into a zone II.

35. The method according to claim 34, wherein zone I comprises a pH plateau.

36. The method according to any one of claims 1 to 33, wherein at least two analytes to be separated are separated comprising: introducing together with the sample or with at least one SBM type I an interaction partner of at least two of the analytes; wherein the presence of at least one additive at an effective concentration in SBM type I suppresses the interaction between at least one of the analytes of interest and the interaction partner and wherein at least one other analyte interacts with the interaction partner under the same conditions thereby forming an analyte-interaction partner-complex; and further wherein the analyte-interaction partner-complex(s) has a different pi compared to the pi of the analyte(s) of interest; and wherein the analyte-interaction partner-complex(s) remains in a SBM type I, whilst at least one of the analytes of interest is transferred into a SBM type Il during the free flow electrophoretic separation.

37. The method according to any one of claims 34 to 36, wherein the pi difference of at least two analytes to be separated is at most 0.5, at most 0.3, at most 0.1 , or at most 0.05.

38. The method according to any one of claims 34 to 37, wherein the interaction partner is albumin.

39. The method according to any one of claims 34 to 38, wherein at least one analyte to be separated is insulin or an insulin analog.

40. The method according to claim 34 or claim 39, wherein the separation conditions of SBM type I are denaturing conditions and the conditions of SBM type Il are non- denaturing conditions.

41. A method for analyzing analytes from a composition of analytes comprising the steps of: conducting a free flow electrophoresis according to any one of claims 1 to 40; and eluting the analytes in a multiplicity of fractions from the FFE chamber; and subsequently analyzing one or more fractions of SBM type I and/or one or more fractions of SBM type II.

42. The method of claim 41 , wherein a subsequent analysis includes a technique chosen from the group of free flow electrophoresis, gel electrophoresis, 1 D- and 2D-PAGE, MS, MALDI, ESI, SELDI, LC-MS(/MS), MALDI-TOF-MS(/MS), chemiluminescence,

HPLC, Edman sequencing, NMR spectroscopy, IR-spectroscopy, UV-spectroscopy, X-ray diffraction, nucleic acid sequencing, electroblotting, amino acid sequencing, flow cytometry, circular dichroism, immuno detection, radio immuno detection, ELISA, and any combination thereof.

43. The method according to claim 41 or claim 42, wherein the subsequent analysis is performed without a buffer exchange step.

44. A kit for carrying out a free flow electrophoretic separation according to any one of claims 1 to 43, comprising at least buffer compounds for one buffer system and at least one additive for the preparation of at least one SBM type I and at least one SBM type II.

45. The kit of claim 44, wherein the number of different SBM is between 2 and 15, preferably between 3 and 12, and most preferably between 3 and 7 and wherein at least one SBM is a SBM type I.

46. The kit of any one of claims 44 to 45, further comprising one anodic and/or one cathodic stabilizing medium.

47. The kit of any one of claims 44 to 46, wherein at least one SBM type I or Il is present as aqueous solution ready for use in free-flow electrophoresis applications.

48. The kit of any one of claims 44 to 47, wherein the components of at least one SBM type I and/or Il are present as concentrated aqueous stock solutions that is to be diluted to the appropriate concentration for use in free-flow electrophoresis applications.

49. The kit of any one of claims 44 to 48, wherein at least one component of the kit is present in dried or lyophilized form that is to be dissolved with solvent to the appropriate concentration for use in free-flow electrophoresis applications.

50. The kit according to any one of claim 44 to 49, further comprising at least one product manual that describes one or more experimental protocols to carry out a free flow electrophoretic separation according to any one of claims 1 to 43, and optionally storage conditions for the components.

51. The kit according to any one of claims 44 to 50, wherein each component such as each dried component and/or each stock solution and/or each solution ready for use may each be separately placed in a sealed container.

52. An FFE apparatus comprising:

an electrophoresis chamber comprising a set of electrodes, wherein at least one of the electrodes is a cathode and at least one of the electrodes is an anode, and a separation zone interposed therebetween, wherein the separation zone comprises at least one zone I; at least one zone II; wherein zone I comprises at least one SBM type I and zone Il comprises at least one SBM type II; and a sample inlet capable of fluid communication with the separation zone.

53. An FFE apparatus comprising: an electrophoresis chamber comprising a set of electrodes, wherein at least one of the electrodes is a cathode and at least one of the electrodes is an anode, and a separation zone interposed therebetween, wherein the apparatus further contains means for forming a zone I formed by at least one SBM type I; and means for forming a zone Il formed by at least one SBM type II; and optionally, means for forming stabilizing media within the separation zone.

54. The apparatus according to claim 52 or claim 53, wherein the apparatus is adapted for performing a method according to anyone of claims 1 to 43.

55. The apparatus according to claim 54 for performing a separation of at least one analyte of interest from a composition of analytes by free flow zone electrophoresis, free flow isoelectric focusing, combinations thereof, or free flow isotachophoresis.

56. Use of an apparatus as defined in any one of claims 52 to 55 for the separation of at least one analyte of interest from a composition of analytes by a method according to any one of claims 1 to 43.