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WO2003070360A1 - Dispositif de separation de petits volumes - Google Patents

Dispositif de separation de petits volumes Download PDF

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Publication number
WO2003070360A1
WO2003070360A1 PCT/AU2003/000209 AU0300209W WO03070360A1 WO 2003070360 A1 WO2003070360 A1 WO 2003070360A1 AU 0300209 W AU0300209 W AU 0300209W WO 03070360 A1 WO03070360 A1 WO 03070360A1
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WO
WIPO (PCT)
Prior art keywords
sample
electrolyte
chamber
ion
chambers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
PCT/AU2003/000209
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English (en)
Inventor
Thomas Turton
David Ogle
Kailing Wang
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Life Therapeutics Ltd
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Gradipore Ltd
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Filing date
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Priority to AU2003246607A priority Critical patent/AU2003246607A1/en
Publication of WO2003070360A1 publication Critical patent/WO2003070360A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
    • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44769—Continuous electrophoresis, i.e. the sample being continuously introduced, e.g. free flow electrophoresis [FFE]

Definitions

  • the present invention is directed to a membrane-based electrophoresis apparatus capable of processing small volumes and methods of using the apparatus.
  • Membrane-based electrophoresis is a new technology originally developed for the separation of macromolecules such as proteins, nucleotides and complex sugars.
  • This unique preparative electrophoresis technology originally developed for macromolecule separation utilises tangential flow across a polyacrylamide membrane when an electric field or potential is applied across the membranes.
  • the general design of the system facilitates the purification of proteins and other macromolecules under near native conditions. This results in higher yields and excellent recovery.
  • the process provides a high purity, scalable separation that is faster, cheaper and higher yielding than current methods of macromolecule separation.
  • the technology offers the potential to concurrently purify and detoxify macromolecule solutions. Apparatus presently developed can process large volumes of materials for commercial production of compounds or very small volumes for analytical purposes.
  • the present inventors have now developed a membrane-based electrophoresis apparatus adapted to process small sample volumes.
  • the present invention provides an electrophoresis apparatus suitable for processing small sample volumes comprising:- (a) an electrolyte reservoir;
  • a separation unit having a first electrolyte chamber in fluid communication with the electrolyte reservoir, a second electrolyte chamber in fluid communication with the electrolyte reservoir, a first sample chamber disposed between the first electrolyte chamber and the second electrolyte chamber, a second sample chamber disposed adjacent to the first sample chamber and between the first electrolyte chamber and the second electrolyte chamber, the first sample chamber being in fluid communication with the first sample reservoir, and the second sample chamber being in fluid communication with the second sample reservoir, wherein the at least one of the first or second sample chambers has a volume of less than about 2 ml;
  • a first ion-permeable barrier positioned between the first sample chamber and the second sample chamber, the first ion-permeable barrier prevents substantial convective mixing of contents of the first and second sample chambers;
  • a second ion-permeable barrier positioned between the first electrolyte chamber and the first sample chamber, the second ion-permeable barrier prevents substantial convective mixing of contents of the first electrolyte chamber and the first sample chamber;
  • a third ion-permeable barrier positioned between the second sample chamber and the second electrolyte chamber, the third ion-permeable barrier prevents substantial convective mixing of contents of the second electrolyte chamber and the second sample chamber;
  • (j) means for supplying sample or liquid from at least the first sample reservoir to the first sample chamber, or from the second sample reservoir to the second sample chamber.
  • small sample volumes it is meant to cover volumes processed to less than about 10 ml, preferably less than about 5 ml. Although at least one of the sample chambers has a small dead volume, when a sample is passed through the chamber, larger sample volumes can be processed but only the material in the sample chamber will be treated at any time by electrophoresis.
  • both sample chambers have a volume of less than about 2 ml.
  • the volume of at least one of the sample chambers can be 1 ml or less.
  • the first sample chamber has a flowing volume of about 1.5 to 100 ml while the second sample chamber has a static volume of about 0.1 to 2 ml.
  • the second sample chamber has a flowing volume of about 1.5 to 100 ml while the first sample chamber has a static volume of about 0.1 to 2 ml.
  • At least one of the ion-permeable barriers is a ion- permeable membrane having a characteristic average pore size and pore size distribution. In one form, all the ion-permeable barriers are ion-permeable membranes having a characteristic average pore size and pore size distribution. This configuration of the apparatus is suitable for separating compounds on the basis of charge and or size.
  • the membranes having a characteristic average pore size and pore size distribution preferably comprise polyacrylamide and have a molecular mass cut-off between about 1 kDa to 3000 kDa.
  • At least one membrane forming an ion-permeable barrier is capable of controlling substantial bulk movement of liquid under the influence of an electric field and is an inducible electro-endo-osmotic membrane.
  • the inducible electro- endo-osmotic membrane is preferably a cellulose tri-acetate (CTA) membrane. It will be appreciated that the inducible electro-endo-osmotic membrane can be formed from any other suitable membrane material such as poly(vinyl alcohol) cross-linked with glutaraldehyde (PVAI+glut).
  • CTA membrane having a nominal molecular mass cut-off of 5, 10 or 20 kDa is particularly suitable for use in the apparatus according to the present invention. It will be appreciated that other molecular mass cutoffs would also be suitable for the present invention.
  • the first ion-permeable barrier is an isoelectric membrane having a characteristic pH value.
  • the isoelectric membrane has a pH value in a range of about 2 to 12.
  • the second and third ion-permeable barriers are membranes having a characteristic average pore size and pore-size distribution.
  • At least one of the second or third ion-permeable barriers is an isoelectric membrane having a characteristic pH value.
  • the at least one isoelectric membrane has a pH value in a range of about 2 to 12.
  • both the second and third ion-permeable barriers are isoelectric membranes each having a characteristic pH value.
  • the isoelectric membranes have a pH value in a range of about 2 to 12.
  • the membranes can have the same or different characteristic pH values.
  • the isoelectric membranes are preferably immobiline polyacrylamide membranes. It will be appreciated, however, that other isoelectric membranes would also be suitable for the present invention.
  • Suitable isoelectric membranes can be produced by copolymerizing acrylamide, N,N'- methylene bisaccrylamide and appropriate acrylamide derivatives of weak electrolytes - yielding isoelectric membranes with pH values in the 2 to 12 range, and average pore sizes that either facilitate or substantially prevent trans-membrane transport of components of selected sizes.
  • the electrolyte and sample chambers are formed by four spacer elements positioned between or adjacent the respective ion-permeable barriers.
  • the spacer elements preferably comprise an elongate rectangular cut-out portion, preferably disposed centrally and longitudinally, defining the volume of the chambers.
  • at least one of the spacer elements adjacent the first ion-permeable barrier has a cut-out portion having a volume less than the other spacer elements.
  • Electrode buffers or electrolytes and sample buffers or diluents can be any suitable buffer or electrolyte. Examples include but not limited to Tris/Borate,
  • the apparatus comprises:
  • (k) means for circulating electrolyte from electrolyte reservoir through the respective first and second electrolyte chambers forming first and second electrolyte streams in the respective electrolyte chambers;
  • means (I) means for circulating contents from each of the first and second sample reservoirs through the respective first and second sample chambers forming first and second sample streams in the respective sample chambers.
  • means (k) and (I) are pump arrangements separately controllable for independent movement of the electrolyte streams and the sample streams.
  • the apparatus may further include:
  • the apparatus may further include:
  • the apparatus may further include:
  • the separation unit is provided as a cartridge or cassette fluidly connected to the electrolyte reservoir(s) and the sample reservoirs.
  • the apparatus according to the present invention is suitable for electrophoresis, isoelectric focusing and electrodialysis of molecules, particularly biomolecules including proteins, peptides, glycoproteins, nucleic acid molecules, recombinant molecules, metabolites, drugs, neutraceuticals, pharmaceuticals, microorganisms including viruses, bacteria, fungi and yeasts, and prions.
  • the present invention is suitable for the separation or treatment of any compound capable of having a charge or a defined molecular mass. Examples include, but are not limited, to biological compounds such as peptides, proteins, nucleic acids, and the like.
  • the apparatus is particularly suitable for treating biological samples to separate infectious agents such as prions or concentrate samples for further analysis.
  • a sample to be treated is placed in the first and/or second sample reservoirs and provided to, or circulated through, the first and/or second chambers.
  • Electrolyte is placed in the electrolyte reservoirs and passed to, or circulated through, the respective first and second electrolyte chambers.
  • Electrolyte or other liquid can be placed in the first and/or second sample reservoirs if required.
  • An electric potential is applied to the electrodes wherein one or more components in the first and/or second sample chamber are caused to move through an ion-permeable barrier to the second and/or first sample chamber, or to the first and/or second reservoir chambers.
  • Treated sample or product can be collected in the second and/or first sample reservoir.
  • the present invention provides a method for separating or removing at least one component from a sample in a small volume by electrophoresis, the method comprising the steps of:
  • small sample volumes it is meant to cover volumes processed to less than about 10 ml, preferably less than about 5 ml. Although at least one of the sample chambers has a small dead volume, when a sample is passed through the chamber, larger sample volumes can be processed but only the material in the sample chamber will be treated at any time by electrophoresis.
  • At least one sample component has a defined charge, or size.
  • electrolyte from electrolyte reservoir is circulated through the first or second electrolyte chamber forming a first or second electrolyte stream.
  • electrolye in the electrolyte chamber will depend on the type of separation required. In some instances, the choice may be related to the pi of compound or compounds to be treated, separated or transferred from a sample chamber to the other sample chamber, or one or both of the electrolyte chambers. Similarly, the choice of the pH of the isoelectric membrane(s), if used, will also depend on the pi of compound or compounds to be treated, separated or transferred form a given sample.
  • content of the first or second sample reservoir is circulated through the first or second sample chamber forming a first or second sample stream through the first or second sample chamber.
  • at least one of the sample chambers can remain static so that a sample is applied to the chamber and that sample is not circulated between the sample reservoir and sample chamber.
  • content of both the first and second sample reservoirs are circulated through the first and second sample chambers forming first and second sample streams through the first and second sample chambers.
  • sample or liquid in the first or second sample reservoir is removed and replaced with fresh sample or liquid.
  • step (g) is maintained until at least one desired component reaches a desired purity level in the first or second sample chamber or in the first or second sample reservoirs.
  • the present invention provides an electrophoresis separation unit comprising: (a) a first electrolyte chamber;
  • a second ion-permeable barrier positioned between the first electrolyte chamber and the first sample chamber, the second ion-permeable barrier prevents substantial convective mixing of contents of the first electrolyte chamber and the first sample chamber;
  • a third ion-permeable barrier positioned between the second sample chamber and the second electrolyte chamber, the third ion-permeable barrier prevents substantial convective mixing of contents of the second electrolyte chamber and the second sample chamber;
  • a plurality of substantially planar spacer elements disposed adjacent the ion- permeable barriers and defining the chambers; and
  • electrodes positioned in the first and second electrolyte chambers.
  • small sample volumes it is meant to cover volumes processed to less than about 10 ml, preferably less than about 5 ml. Although at least one of the sample chambers has a small dead volume, when a sample is passed through the chamber, larger sample volumes can be processed but only the material in the sample chamber will be treated at any time by electrophoresis.
  • both sample chambers have a volume of less than about 2 ml.
  • the volume of at least one of the sample chambers can be 1 ml or less.
  • the electrolyte and sample chambers are formed by four spacer elements positioned between or adjacent the respective ion-permeable barriers.
  • the spacer elements preferably comprise an elongate rectangular cut-out portion, preferably disposed centrally and longitudinally, defining the volume of the chambers.
  • at least one of the spacer elements adjacent the first ion-permeable barrier has a cut-out portion having a volume less than the other spacer elements.
  • At least one of the membrane barriers is a ion-permeable membrane having a characteristic average pore size and pore size distribution. In one form, all the membrane barriers are ion-permeable membranes having a characteristic average pore size and pore size distribution. This configuration of the apparatus is suitable for separating compounds on the basis of charge and or size.
  • the membranes having a characteristic average pore size and pore size distribution are preferably comprise polyacrylamide and have a molecular mass cut-off between about 1 kDa to 3000 kDa.
  • At least one membrane forming the barrier is capable of controlling substantial bulk movement of liquid under the influence of an electric field an inducible electro-endo-osmotic membrane.
  • the inducible electro-endo-osmotic membrane is preferably a cellulose tri-acetate (CTA) membrane. It will be appreciated that the inducible electro-endo-osmotic membrane can be formed from any other suitable membrane material such as poly(vinyl alcohol) cross-linked with glutaraldehyde (PVAI+glut).
  • CTA membrane having a nominal molecular mass cut-off of 5, 10 or 20 kDa is particularly suitable for use in the apparatus according to the present invention. It will be appreciated that other molecular mass cutoffs would also be suitable for the present invention.
  • the first ion-permeable barrier is an isoelectric membrane having a characteristic pH value.
  • the isoelectric membrane has a pH value in a range of about 2 to 12.
  • the second and third ion-permeable barriers are membranes having a characteristic average pore size and pore-size distribution.
  • At least one of the second or third ion-permeable barriers is an isoelectric membrane having a characteristic pH value.
  • the at least orie isoelectric membrane has a pH value in a range of about 2 to 12.
  • both the second and third ion-permeable barriers are isoelectric membranes each having a characteristic pH value.
  • the isoelectric membranes have a pH value in a range of about 2 to 12.
  • the membranes can have the same or different characteristic pH values.
  • the isoelectric membranes are preferably Immobiline polyacrylamide membranes. It will be appreciated, however, that other isoelectric membranes would also be suitable for the present invention.
  • Suitable isoelectric membranes can be produced by copolymerizing acrylamide, N,N'-methylene bisaccrylamide and appropriate acrylamide derivatives of weak electrolytes yielding isoelectric membranes with pH values in the 2 to 12 range, and average pore sizes that either facilitate or substantially prevent trans-membrane transport of components of selected sizes.
  • Electrode buffers or electrolytes and sample buffers can be any suitable buffer or electrolyte. Examples include but not limited to Tris/Borate, Hepes/lmidazole, GABA/Acetic acid and Hepes/Histidine buffers.
  • the present invention provides a method for separating or removing at least one component from a sample in a small volume by electrophoresis, the method comprising the steps of:
  • substantially all trans-membrane or trans-barrier migration occurs upon the application of the electric potential.
  • At least one sample component has a pi value.
  • step (e) is maintained until at least one desired component reaches a desired purity level in the first or second sample chamber.
  • the distance between the electrodes has an effect on the separation or movement of sample constituents through the membranes.
  • a distance of about 3 - 4 mm has been found to be particularly suitable for a the present apparatus.
  • the distance will also relate to the voltage applied to the apparatus.
  • the effect of the electric field is based on the equation:
  • the distance between the electrodes should decrease in order to increase electric field strength.
  • Flow rate of sample/buffer/fluid can have an influence on the separation of constituents. Rates of millilitres per minute up to litres per minute are used depending on the configuration of the apparatus and the nature of the sample to be separated. Currently in the present apparatus, the preferred flow rate for the sample streams is about 1 to 2 ml/min.
  • the buffer flow rate in the electrode chambers is usually about 1 to 3 l/min. The selection of the flow rate of samples and electrode buffers is dependent on the product to be transferred, efficiency of transfer, pre- and post- positioning with other separation applications.
  • the voltage and/or current applied varies depending on the separation. Typically up to several thousand volts are used but choice and variation of voltage will depend on the configuration of the apparatus, buffers and the sample to be separated. In a laboratory scale instrument, the preferred voltage is about 250 V.
  • the electric potential may be periodically stopped and/or reversed to cause movement of a constituent having entered a membrane to move back into the volume or stream from which it came, while substantially not causing any constituents that have passed completely through a membrane to pass back through the membrane.
  • Reversal of the electric potential is an option but another alternative is a resting period. Resting (a period without an electric potential being applied) is an optional step that can replace or be included before or after an optional electrical potential reversal. This resting technique can be often practised for specific separation applications as an alternative or adjunct to reversing the potential.
  • the present invention provides use of the apparatus according the first aspect of the present invention to alter composition of a sarinple having a small volume and containing at least one compound.
  • the present invention provides a product obtained by the method according to the second aspect of the present invention.
  • the present invention provides use of the electrophoresis unit according the third aspect of the present invention to alter composition of the sample having a small volume and containing at least one compound.
  • the present invention provides a product obtained by the method according to the fourth aspect of the present invention.
  • the present invention provides use of the electrophoresis apparatus according the first aspect of the present invention in a diagnostic test.
  • the present invention provides use of the electrophoresis unit according the third aspect of the present invention in a diagnostic test.
  • An advantage of the present invention is that the apparatus and method can effectively and efficiently process and separate charged molecules and other components in samples having small volumes.
  • Yet another advantage of the present invention is that the apparatus and method have improved yields of the separation component, minimal or no inactivation of the separated or isolated component or other components in the sample, and improved purity of the separated component.
  • the first sample chamber is called the stream 1 and the second sample chamber called the stream 2.
  • sample is placed in the first sample chamber (stream 1) and constituents caused to move through the first membrane into the second sample chamber (stream 2). It will be appreciated, however, that the order can be reversed where sample is placed in the second sample chamber and constituents caused to move through the first membrane into the first sample chamber.
  • GradiflowTM is a trade mark of Gradipore Limited, Australia for its membrane- based electrophoresis apparatus.
  • Figure 1 is a schematic diagram of a separation unit for use in the present invention.
  • Figure 2 is an exploded view of a cartridge which may be used with the separation unit of Figure 1.
  • Figure 3 is a plan view of a grid element which may be incorporated as a component of a cartridge of a separation unit.
  • Figure 4 is a PAGE gel showing the separation and concentration of albumin using an apparatus according to the present invention.
  • ion-permeable barriers that substantially prevent convective mixing between the adjacent chambers of the apparatus or unit are placed in an electric field and components of the sample are selectively transported through the ion-permeable barriers.
  • the particular ion-permeable barriers used will vary for different applications and generally have characteristic average pore sizes and pore size distributions and/or isoelectric points allowing or substantially preventing passage of different components.
  • Separation unit 2 comprises first electrolyte inlet 4, and second electrolyte inlet 6, first sample inlet 8, and second sample inlet 10, first electrolyte outlet 12, and second electrolyte outlet 14, and first sample outlet 16 and second sample outlet 18. Between first electrolyte inlet 4 and first outlet 12 is first electrolyte chamber 22. Likewise, between second electrolyte inlet 6 and second electrolyte outlet 14 is second electrolyte chamber 24. First sample and second sample inlets and outlets also have connecting chambers. First sample chamber 26 running adjacent to first electrolyte chamber 22 connects first sample inlet 8 to first sample outlet 16.
  • first sample chamber 28 running adjacent to second electrolyte chamber 24 connects second sample inlet 10 to second sample outlet 18.
  • Membrane barriers 30 and 32 separate electrolyte chambers 22 and 24 from first sample and second sample chambers 26 and 28, respectively. Between first sample and second sample chambers 26 and 28 is membrane barrier 34.
  • first and second electrolyte 36 and 38 occupy first and second electrolyte chambers 22 and 24. It should be understood that during operation, first and second electrolyte 36 and 38, as well as first and second sample may be stagnant, or flow through, in the respective chambers.
  • FIG. 2 is an exploded view of cartridge 100 which is preferably a modular component of separation unit 2.
  • cartridge 100 When configured as a modular unit, cartridge 100 preferably comprises housing 102 for holding in place or encasing the component parts of cartridge 100.
  • cartridge 10.0 is generally elongated and has side walls 104 which are generally parallel to one another and the longitudinal axis A of cartridge 100.
  • the cartridge is suitably generally octagonal, hexagonal, or ovular.
  • cartridge 100 has three end walls 106 on each side of side walls 104 forming an octagon.
  • two end walls on each side 106 are suitably used to form a hexagon, or one curved end wall 106 on each side is suitably used to form a generally ovular shape.
  • end walls 106 are suitably either straight or generally curved.
  • a small flange 108 Extending around the base of side walls 104 and end walls 106 is a small flange 108 that is generally perpendicular to side walls 104 and end walls 106 and projects inward toward the center of cartridge 100.
  • Flange 108 is preferably configured to interact with lower gasket 112 (if gaskets are used).
  • lower gasket 112 is generally planar and configured to fit inside walls 104 and 106 of cartridge 100.
  • lower gasket 112 is made from silicon rubber. Lower gasket 112 may be configured so that it has an aperture 114 extending in an elongated manner through the center of lower gasket 112.
  • alignment holes 116 are also extending through and adjacent each end of lower gasket 112.
  • alignment holes 116 are circular, forming generally cylindrical channels through lower gasket 112.
  • alignment holes 116 are suitably triangular, square, rectangular, hexagonal, octagonal, or similarly shaped.
  • a generally planar lower ion-permeable barrier typically in the form of a membrane 32.
  • the external shape of ion-permeable barrier 32 is generally the same as that of lower gasket 112 and the interior of cartridge 100 so that membrane barrier 32 is configured to fit inside cartridge 100.
  • ion- permeable barrier 32 preferably has two alignment holes of the same location and configuration as alignment holes 116 in lower gasket 112. Ion-permeable barrier 32 substantially prevents convective mixing of the contents of second electrolyte chamber 24 and second sample chamber 28, while permits selective trans-barrier transport of selected constituents upon application of the electric potential.
  • ion-permeable barrier 32 is formed from an ion-permeable membrane with a characteristic average pore size and pore-size distribution. The average pore size and pore size distribution of the membrane is selected to facilitate trans-membrane transport of certain constituents, while substantially preventing trans- membrane transport of other constituents.
  • ion-permeable barrier 32 is an endo-osmotic barrier, such as an inducible electro-endo-osmotic membrane that substantially prevents convective mixing of the contents of second electrolyte chamber 24 and second sample chamber 28, which permits trans-barrier transport of water upon application of the electric potential.
  • the inducible electro-endo-osmotic membrane is preferably a cellulose tri- acetate (CTA) membrane. It will be appreciated that the inducible electro-endo-osmotic membrane can be formed from any other suitable membrane material such as poly(vinyl alcohol) (PVA) cross-linked with glutaraldehyde (PVAI+glut).
  • CTA membrane having a nominal molecular mass cut-off of 5, 10 or 20 kDa is particularly suitable for use in the apparatus according to the present invention. It will be appreciated that other molecular mass cut-offs would also be suitable for the present invention.
  • ion-permeable barrier 32 is an isoelectric ion-permeable barrier, such as an isoelectric membrane that substantially prevents convective mixing of the contents of second electrolyte chamber 24 and second sample chamber 28, which permits selective trans-barrier transport of selected constituents upon application of the electric potential.
  • Suitable isoelectric membranes can be produced by copolymerizing acrylamide, N,N'-methylene bisacrylamide and appropriate acrylamide derivatives of weak electrolytes yielding isoelectric membranes with pH values in the 2 to 12 range, and average pore sizes that either facilitate or substantially prevent trans-membrane transport of components of selected sizes.
  • lower grid element 118 that is generally planar and also shaped like lower gasket 112 and the interior of cartridge 100 so that lower spacer element 118 is configured to fit inside cartridge 100.
  • One of the functions of lower spacer element 118 is to separate lower ion-permeable barrier 32 from ion- permeable barrier 34.
  • Another function of lower grid element 118 is to provide a flow path for second sample 28.
  • lower spacer element 118 suitably also has alignment holes 116.
  • ion-permeable barrier 34 Above lower grid element 118 is generally planar ion-permeable barrier 34.
  • the external shape of ion-permeable barrier 34 is generally the same as that of lower gasket 112 and the interior of cartridge 100 so that ion-permeable barrier 34 is configured to fit inside cartridge 100.
  • Ion-permeable barrier 34 substantially prevents convective mixing of the contents of first sample chamber 26 and second sample chamber 28, while permits selective trans-barrier transport of selected constituents upon application of the electric potential.
  • ion-permeable barrier 34 is formed from a ion-permeable membrane with a characteristic average pore size and pore-size distribution.
  • ion-permeable barrier 34 is an endo-osmotic barrier, such as an inducible electro-endo-osmotic membrane that substantially prevents convective mixing of the contents of second electrolyte chamber 24 and second sample chamber 28, which permits trans-barrier transport of water upon application of the electric potential.
  • the inducible electro-endo-osmotic membrane is preferably a cellulose tri-acetate (CTA) membrane.
  • the inducible electro-endo- osmotic membrane can be formed from any other suitable membrane material such as poly(vinyl alcohol) cross-linked with glutaraldehyde (PVAI+glut).
  • PVAI+glut poly(vinyl alcohol) cross-linked with glutaraldehyde
  • the present inventors have found that a CTA membrane having a nominal molecular mass cut-off of 5, 10 or 20 kDa is particularly suitable for use in the apparatus according to the present invention. It will be appreciated that other molecular mass cut-offs would also be suitable for the present invention.
  • ion-permeable barrier 34 is an isoelectric ion-permeable barrier, such as an isoelectric membrane that substantially prevents convective mixing of the contents of first sample chamber 26 and second sample chamber 28, while permits selective trans-barrier transport of selected constituents upon application of the electric potential.
  • Suitable isoelectric membranes can be produced by copolymerizing acrylamide, N,N'-methylene bisacrylamide and appropriate acrylamide derivatives of weak electrolytes yielding isoelectric membranes with pH values in the 2 to 12 range, and average pore sizes that either facilitate or substantially prevent trans-membrane transport of components of selected sizes.
  • ion-permeable barrier 34 Above ion-permeable barrier 34 are three upper components: upper spacer element 120, upper ion-permeable barrier 38, and upper gasket 124. These three components are placed so that upper spacer element 120 is immediately above ion- permeable barrier 34, ion-permeable barrier 38 is immediately above upper spacer element 120, and upper gasket 124 is immediately above ion-permeable barrier 38.
  • the configuration of the three upper components suitably mirrors that of the lower three components. The combination of these components forms the first sample chamber 26.
  • ion-permeable barrier 34 having alignment holes 116 may be connected together with a fastener, which is any type of connector configured to interact with alignment holes 116 and facilitate through flow of second sample.
  • a fastener any type of connector configured to interact with alignment holes 116 and facilitate through flow of second sample.
  • components above ion-permeable barrier 34 having alignment holes 116 may be connected together with a fastener, which is any type of connector configured to interact with alignment holes 116 and facilitate through flow of the first sample.
  • Ion-permeable barrier 38 substantially prevents convective mixing of the contents of first electrolyte chamber 22 and first sample chamber 26, while permits selective trans- barrier transport of selected constituents upon application of the electric potential.
  • ion-permeable barrier 38 is an endo-osmotic barrier, such as an inducible electro-endo-osmotic membrane that substantially prevents convective mixing of the contents of first electrolyte chamber 22 and first sample chamber 26, which permits trans-barrier transport of water upon application of the electric potential.
  • the inducible electro-endo-osmotic membrane is preferably a cellulose tri-acetate (CTA) membrane. It will be appreciated that the inducible electro-endo- osmotic membrane can be formed from any other suitable membrane material such as poly(vinyl alcohol) cross-linked with glutaraldehyde (PVAI+glut).
  • ion-permeable barrier 38 is formed from an ion-permeable membrane with a characteristic average pore size and pore-size distribution. The average pore size and pore size distribution of the membrane is selected to facilitate trans-membrane transport of certain constituents, while substantially preventing trans- membrane transport of other constituents.
  • ion-permeable barrier 38 is an isoelectric ion-permeable barrier, such as an isoelectric membrane that substantially prevents convective mixing of the contents of first electrolyte chamber 22 and first sample chamber 26, while permits selective trans-barrier transport of selected constituents upon application of the electric potential.
  • Suitable isoelectric membranes can be produced by copolymerizing acrylamide, N,N'-methylene bisacrylamide and appropriate acrylamide derivatives of weak electrolytes yielding isoelectric membranes with pH values in the 2 to 12 range, and average pore sizes that facilitate or substantially prevent trans-membrane transport of components of selected sizes.
  • Preferred spacer elements 118 and 120 are shown in more detail in Figure 3.
  • Figure 3 shows a plan view of a preferred spacer element which is incorporated as a component of cartridge 100 for separation unit 2.
  • An elongate rectangular cut-out portion 128 is defined in the center of the spacer element.
  • an alignment hole 116 is suitably provided for alignment with the other components of cartridge 100. .
  • the thickness of the spacer element is preferably relatively small. In one presently preferred embodiment, exterior areas of the element are 0.3 mm thick.
  • the relative thinnest of the spacer provides several advantages. First, it results in a more even distribution of liquid over ion-permeable barrier 34 or 38 and assists in inhibiting its fouling by macromolecules.
  • volume of liquid required is decreased by the use of a relatively thin spacer which enables relatively small sample volumes to be used for laboratory-scale separations, a significant advantage over prior art separation devices.
  • the apparatus typically includes a cartridge which housed a number of membranes forming two chambers, cathode and anode connected to a suitable power supply, reservoirs for samples, buffers and electrolytes, pumps for passing samples, buffers and electrolytes, and cooling means to maintain samples, buffers and electrolytes at a required temperature during electrophoresis.
  • the cartridge contains three substantially planar membranes positioned and spaced relative to each other to form two chambers through which sample or solvent can be passed.
  • a separation membrane was positioned between two outer membranes (termed restriction membranes as their molecular mass cut-offs are usually smaller than the cut-off of the separation membrane).
  • restriction membranes were located adjacent to an electrode.
  • the cartridge is described in AU 738361. Description of membrane-based electrophoresis can be found in US 5039386 and US 5650055 in the name of Gradipore Limited.
  • a modified apparatus according to the present invention has been constructed and tested.
  • This device can process flowing volumes of less than 2 ml and one stream can be static with a volume as low as 100 ⁇ l. It has been found that this device is very comparable to the standard laboratory scale electrophoresis apparatus in terms of purification ability but overcomes the problems of the prior art apparatus regarding processing small volumes.
  • EXPERIMENTAL One major application of the apparatus according to the present invention is the processing of samples for diagnostics, where target molecules or entities are required to be separated and concentrated.
  • 0.5 - 20 ml of blood is usually taken from a patient, yielding around 0.2 - 10 ml of plasma.
  • a 0.5 -20 ml specimen is therefore the approximate starting sample expected to be processed by any separation or concentration process.
  • Indicators of disease such as antibodies, proteins, infectious agents (abnormal prion/virus etc), are often found in extremely small amounts in the blood stream (especially abnormal prion). As a result of this any such indicators isolated from blood may have to be concentrated significantly in order that they can be detected/analysed.
  • the target is either transferred to a second stream of a significantly lower volume or the volume of the first stream must be reduced during or after purification (if the target is to remain in stream 1 ).
  • a device is required that has chamber and streams of variable volume with very low minimum volumes. The current devices do not fulfil this requisite.
  • Standard PAGE methods were employed as set out below.
  • Reagents 10x SDS Glycine running buffer (Gradipore Limited, Australia), dilute using Milli-Q water to 1x for use; 1x SDS Glycine running buffer (29 g Trizma base, 144 g Glycine, 10 g SDS, make up in RO water to 1.0 I); 10x TBE II running buffer (Gradipore), dilute using Milli-Q water to 1x for use; 1x TBE II running buffer (10.8 g Trizma base, 5.5 g Boric acid, 0.75 g EDTA, make up in RO water to 1.0 I); 2x SDS sample buffer (4.0 ml, 10% (w/v) SDS electrophoresis grade, 2.0 ml Glycerol, 1.0 ml 0.1 % (w/v) Bromophenol blue, 2.5 ml 0.5M Tris-HCI, pH 6.8, make up in RO water up to 10 ml); 2x Native sample buffer (10% (v/
  • SDS PAGE e.g. Sigma wide range
  • Western Blotting e.g. color / rainbow markers
  • 2x SDS sample buffer was added to sample at a 1 : 1 ratio (usually 50 ⁇ l / 50 ⁇ l) in the microtiter plate wells or 1.5 ml tubes. The samples were incubated for 5 minutes at approximately 100°C. Gel cassettes were clipped onto the gel support with wells facing in, and placed in the tank. If only running one gel on a support, a blank cassette or plastic plate was clipped onto the other side of the support
  • Sufficient 1x SDS glycine running buffer was poured into the inner tank of the gel support to cover the sample wells.
  • the outer tank was filled to a level approximately midway up the gel cassette.
  • the sample wells were rinsed with the running buffer to remove air bubbles and to displace any storage buffer and residual polyacrylamide.
  • the gel cassette was opened to remove the gel which was placed into a container or sealable plastic bag.
  • the gel was thoroughly rinsed with tap water, and drained from the container.
  • Coomassie blue stain (approximately 100 ml GradipureTM, Gradipore Limited, Australia)) was added and the container or bag sealed. Major bands were visible in 10 minutes but for maximum intensity, stained overnight.
  • To de-stain the gel the stain was drained off from the container.
  • the container and gel were rinsed with tap water to remove residual stain.
  • 6% acetic acid (approximately 100 ml) was poured into the container and sealed. The de- stain was left for as long as it takes to achieve the desired level of de-staining (usually 12 hours). Once at the desired level, the acetic acid was drained and the gel rinsed with tap water.
  • Protein concentrations were performed using albumin as a model separation target. Albumin was concentrated successfully by a factor of 10. Samples (20 ml of 0.5 mg/ml albumin) were completely transferred from stream 1 to only 2 ml in stream 2 (5 mg/ml) within 120 min (see Figure 4).
  • This apparatus included specialised spacers that produce a reduced separation area and therefore a smaller volume.
  • the separation area in the low volume device spacers was reduced to a width of 1 mm from 12 mm in the normal grid form of spacer.
  • the length was substantially unchanged.
  • the depth of normal grids is 1 mm whereas the low volume device spacers had depths of 0.35 mm and 1.6 mm.
  • These have been constructed from reinforced silicon rubber and silicon rubber respectively. A range materials and depths for the spacers may be used. Due to the "soft" nature of these spacers, gaskets were not required. Further reduction in running volumes was obtained by reducing the diameter and length of the tubing and heat exchangers and by reducing the volume of the separation unit channels.
  • Concentration where the target compound or macromolecule is not transferred from a large volume to small volume, may be performed by use of endo-osmotic membranes. This method works by transferring the contaminants to the second sample chamber substantially simultaneously with volume reduction by electro-endo-osmotic membranes.
  • Flow rate used was about proportional to separation area in order to optimise many separations or concentrations. Separation area of the apparatus was one twelfth that of standard separation system and therefore the flow rates can be reduced by approximately a factor of 12.
  • Tubing internal diameter was reduced from 1.6 mm to 0.8 mm (by a factor of 2). Therefore volume decreased by a factor 4.
  • the apparatus according to the present invention is particularly suitable for: front-end application for diagnostics; purification/concentration of molecules such as antibodies, coagulation factors, signalling proteins, viruses etc; fast pre-fractionation/concentration of molecules for proteomics; small volume runs where samples are in short supply or costly. One-fifth the volume (2 ml instead of 10 ml or more) could be carried out.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Environmental & Geological Engineering (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)

Abstract

L'invention concerne un dispositif d'électrophorèse convenant pour traiter de petits volumes d'échantillon, qui comprend : (a) un réservoir d'électrolyte ; (b) un premier réservoir d'échantillon et un deuxième réservoir d'échantillon ; (c) une unité de séparation comportant une première chambre d'électrolyte, une deuxième chambre d'électrolyte, une première chambre d'échantillon, une deuxième chambre d'échantillon, la première et/ou la deuxième chambre d'échantillon présentant un volume inférieur à environ 2 ml ; (d) une première barrière perméable aux ions ; (e) une deuxième barrière perméable aux ions ; (f) une troisième barrière perméable aux ions ; (g) une pluralité d'éléments espaceurs sensiblement planes, placés de manière adjacente aux barrières perméables aux ions définissant les chambres ; (h) des électrodes; (h) des moyens d'apport d'électrolyte ; et des (i) moyens d'apport d'échantillon ou de liquide.
PCT/AU2003/000209 2002-02-19 2003-02-19 Dispositif de separation de petits volumes Ceased WO2003070360A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003246607A AU2003246607A1 (en) 2002-02-19 2003-02-19 Low volume separation apparatus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPS0630A AUPS063002A0 (en) 2002-02-19 2002-02-19 Low volume electrophoresis apparatus
AUPS0630 2002-02-19

Publications (1)

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WO2003070360A1 true WO2003070360A1 (fr) 2003-08-28

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001078878A1 (fr) * 2000-04-18 2001-10-25 Gradipore Limited Appareil de separation pour petits volumes
US6328869B1 (en) * 1998-09-07 2001-12-11 Gradipore Limited Apparatus for macromolecule purification
WO2002049744A1 (fr) * 2000-12-21 2002-06-27 Gradipore Limited Appareil et procede pour la separation de molecules et le deplacement de fluides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6328869B1 (en) * 1998-09-07 2001-12-11 Gradipore Limited Apparatus for macromolecule purification
WO2001078878A1 (fr) * 2000-04-18 2001-10-25 Gradipore Limited Appareil de separation pour petits volumes
WO2002049744A1 (fr) * 2000-12-21 2002-06-27 Gradipore Limited Appareil et procede pour la separation de molecules et le deplacement de fluides

Also Published As

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