U.S. patent application number 10/265798 was filed with the patent office on 2003-02-13 for separation of micromolecules.
Invention is credited to Conlan, Brendon Francis, Gilbert, Andrew Mark, Nair, Chenicheri Hariharan, Ryan, Lucy Jane.
Application Number | 20030029725 10/265798 |
Document ID | / |
Family ID | 25646299 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030029725 |
Kind Code |
A1 |
Conlan, Brendon Francis ; et
al. |
February 13, 2003 |
Separation of micromolecules
Abstract
This invention is directed to an apparatus and method for the
separation of molecules, particularly micromolecules having a
molecular mass of less than 5000 Dalton. The present invention is
directed to an apparatus for separating micromolecules by
electrophoretic separation, the apparatus comprising: (a) an anode;
(b) a cathode disposed relative to the anode so as to be adapted to
generate an electric field in an electric field area therebetween
upon application of a voltage potential between the anode and the
cathode; (c) a separation membrane disposed in the electric field
area; (d) a first restriction membrane disposed between the anode
and the separation membrane so as to define a first interstitial
volume therebetween; (e) a second restriction membrane disposed
between the cathode and the separation membrane so as to define a
second interstitial volume therebetween; and (f) means adapted to
provide a sample constituent in a selected one of the first and
second interstitial volumes; wherein upon application of the
voltage potential, a selected separation product is removed from
the sample constituent, thorough the separation membrane, and
provided to the other of the first and second interstitial volumes,
wherein a micromolecule is capable of being retained in at least
one of the interstitial volumes, and wherein a micromolecule is
capable of being retained in at least one of the interstitial
volumes.
Inventors: |
Conlan, Brendon Francis;
(Lane Cove, AU) ; Gilbert, Andrew Mark; (Eastwood,
AU) ; Ryan, Lucy Jane; (Baulkham Hills, AU) ;
Nair, Chenicheri Hariharan; (Homebush Bay, AU) |
Correspondence
Address: |
BAKER & MCKENZIE
805 THIRD AVENUE
NEW YORK
NY
10022
US
|
Family ID: |
25646299 |
Appl. No.: |
10/265798 |
Filed: |
October 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10265798 |
Oct 7, 2002 |
|
|
|
09834462 |
Apr 13, 2001 |
|
|
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Current U.S.
Class: |
204/543 ;
204/518; 204/544; 204/627 |
Current CPC
Class: |
B01D 57/02 20130101 |
Class at
Publication: |
204/543 ;
204/544; 204/518; 204/627 |
International
Class: |
B01D 063/00; B01D
069/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 2000 |
AU |
PQ 6914 |
Claims
1. An electrophoretic separation apparatus for separating
micromolecules, the apparatus comprising: (a) an anode; (b) a
cathode disposed relative to the anode so as to be adapted to
generate an electric field in an electric field area therebetween
upon application of a voltage potential between the anode and the
cathode; (c) a separation membrane disposed in the electric field
area; (d) a first restriction membrane disposed between the anode
and the separation membrane so as to define a first interstitial
volume therebetween; (e) a second restriction membrane disposed
between the cathode and the separation membrane so as to define a
second interstitial volume therebetween; and (f) means adapted to
provide a sample constituent in a selected one of the first and
second interstitial volumes; wherein upon application of the
voltage potential, a selected separation product is removed from
the sample constituent, thorough the separation membrane, and
provided to the other of the first and second interstitial volumes
and wherein a micromolecule is capable of being retained in at
least one of the interstitial volumes.
2. The apparatus according to claim 1 wherein at least one
restriction membrane is formed as a composite arrangement with at
least two materials.
3. The electrophoretic separation apparatus of claim 1 wherein at
least one of the restriction membranes is comprised of at least two
membranes having distinctive pores sizes.
4. An apparatus for electrophoretic separation of micromolecules,
the apparatus comprising: (a) an anode buffer compartment and a
cathode buffer compartment; (b) electrodes positioned in the buffer
compartments; (c) a first chamber and a second chamber positioned
on either side of an ion-permeable separation membrane having a
defined molecular mass cut-off, the first chamber and the second
chamber being positioned between the anode and the cathode buffer
compartments and separated by an ion-permeable restriction membrane
positioned on at least one side of the separation membrane, the
restriction membrane allowing flow of ions into and out of the
compartments and chambers under the influence of an electric field
but substantially restrict movement of at least one micromolecule
type from the second chamber into the buffer compartment.
5. The apparatus according to claim 4 wherein the ion-permeable
separation membrane has a molecular mass cut-off greater than the
molecular mass of the micromolecule to be separated.
6. The apparatus according to claim 4 wherein at least one buffer
compartment, sample chamber or product chamber is configured to
allow flow of the respective buffer, sample or product solution to
form a stream.
7. The apparatus according to claim 4 wherein at least one
restriction membrane is formed as a composite arrangement with at
least materials.
8. The apparatus according to claim 4 wherein at least one
restriction barrier is formed as a sandwich arrangement with at
least two layers of material.
9. The apparatus according to claim 8 wherein the sandwich
arrangement includes an inner layer comprising a membrane having a
pore size with a molecular mass cut-off less than the about 5000 Da
and an outer layer comprising a membrane having a molecular mass
cut-off of greater than about 5000 Da.
10. The apparatus according to claim 9 wherein the inner layer is
made from an ultrafiltration, electrodialysis or haemodialysis
membrane material and the outer layer is a membrane material made
from polyacrylamide.
11. The apparatus according to claim 10 wherein the ultrafiltration
membrane has a molecular mass cut-off between 100 Da and 5000
Da.
12. The apparatus according to claim 11 wherein the ultrafiltration
membrane has a molecular mass cut-off of about 1 kDa.
13. The apparatus according to claim 4 wherein the ion-permeable
separation membrane is made from polyacrylamide and having a
molecular mass cut-off from 5 to 1000 kDa.
14. A separation cartridge suitable for use in an electrophoretic
separation apparatus for separating micromolecules, the cartridge
comprising: (a) a housing; (b) an ion-permeable separation membrane
having a defined molecular mass cut-off positioned in the housing;
(c) an ion-permeable restriction membrane positioned either side of
the separation membrane in the housing and spaced to form a first
chamber and second chamber on either side of the separation
membrane, wherein the restriction membrane is adapted to allow flow
of ions into and out of the compartments and chambers under the
influence of an electric field but substantially restrict movement
of at least one micromolecule type from the second chamber.
15. The cartridge according to claim 14 further including: (d)
electrodes positioned in the housing on the outer sides of the
restriction barriers.
16. The apparatus according to claim 14 wherein the ion-permeable
separation membrane has a molecular mass cut-off greater than the
molecular mass of a micromolecule to be separated.
17. The cartridge according to claim 14 wherein the separation
membrane is composed of polyacrylamide and having a molecular mass
cut-off from about 5 to 1000 kDa.
18. The cartridge according to claim 14 wherein at least one
restriction membrane is formed as a composite arrangement with at
least two materials.
19. The cartridge according to claim 14 wherein at least one
restriction membrane is formed as a sandwich arrangement of
membranes with at least two layers of material.
20. The cartridge according to claim 19 wherein the sandwich
arrangement includes an inner layer comprising a membrane having a
pore size with a molecular mass cut-off less than the about 5000 Da
and an outer layer comprising a membrane having a molecular mass
cut-off of greater than about 5000 Da.
21. The cartridge according to claim 19 wherein the inner layer is
made from an ultrafiltration, electrodialysis or haemodialysis
membrane material and the outer layer is a membrane material made
from polyacrylamide.
22. The cartridge according to claim 20 wherein the ultrafiltration
membrane has a molecular mass cut-off between 100 Da and 5000
Da.
23. The cartridge according to claim 22 wherein the ultrafiltration
membrane has a molecular mass cut-off of about 1 kDa.
24. The cartridge according to claim 23 wherein the ion-permeable
separation barrier is a membrane made from polyacrylamide and
having a molecular mass cut-off from 5 to 1000 kDa.
25. A method of separating a micromolecule from a liquid sample,
the method comprising: (a) providing an electrophoresis apparatus
according to clam 4; (b) placing the sample in the first chamber of
the apparatus; (c) selecting a solvent for the first chamber having
a pH such that the micromolecule to be separated is charged; (d)
applying an electric potential between the first and second
chambers causing movement of micromolecules in the first stream
through the separation membrane into the second chamber while
unwanted molecules are substantially prevented from entering the
second chamber; (e) optionally, periodically stopping and reversing
the electric potential to cause movement of molecules having
entered the separation membrane to move back into the first
chamber, while substantially not causing any micromolecules that
have entered the second chamber to re-enter first chamber; and (f)
maintaining steps (d) and optionally (e) until the desired amount
of micromolecules are moved to the second chamber.
26. The method according to claim 25 wherein the micromolecule is
selected from the group consisting of biotin, Brilliant Blue FCF
(BB FCF), azorubine, phytoestrogen, digoxigenin, hormones,
cytokines, dyes, vitamins, chemicals, neutraceuticals,
pharmaceuticals food diet supplements, and combinations
thereof.
27. The method according to claim 25 wherein the sample is selected
from the group consisting of crude extracts, microbial cultures,
cell lysates, cellular products, chemical processing mixtures, cell
culture media, plant products or extracts.
28. The method according to claim 25 wherein the solvent is Tris
Borate buffer around pH 9.
29. The method according to claim 28 wherein buffer has a
concentration of 10 mM to 200 mM.
30. The method according to claim 29 wherein the buffer has a
concentration of 20 mM to 80 mM.
31. A micromolecule purified or separated by the method according
to claim 25.
32. The micromolecule according to claim 31 selected from the group
consisting of biotin, Brilliant Blue FCF (BB FCF), azorubine,
phytoestrogen, digoxigenin, hormones, cytokines, dyes, vitamins,
chemicals, neutraceuticals, pharmaceuticals, food supplements, and
combinations thereof.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to apparatus and methods for
the separation of molecules, particularly micromolecules having a
molecular mass of less than about 5000 Dalton.
[0002] There are increasing numbers of micromolecules being used as
food and diet supplements, pharmaceuticals and neutraceuticals.
Increasing numbers of vitamins, co-factors, plant and microbial
extracts are also being developed and used for human and animal
consumption. As many of these compounds are micromolecules,(having
a molecular mass of less than about 5000 Dalton (Da)), there is a
need to develop methods to separate or purify these compounds in a
fast and economical manner. Traditional separation methods for
micromolecules can alter or denature these compounds. Separation
methods for larger molecules typically are not considered suitable
for use in micromolecule separation. Furthermore, traditional
methods can be quite time consuming, expensive and difficult to
scale up commercially.
[0003] In the past, a preparative electrophoresis technology for
macromolecule separation which utilises tangential flow across a
polyacrylamide membrane when a charge is applied across the
membrane was used to separate micromolecules. The general design of
the earlier system facilitated the purification of proteins and
other macromolecules under near native conditions. The technology
is bundled into a cartridge comprising several membranes housed in
a system of specially engineered grids and gaskets which allow
separation of macromolecules by charge and/or molecular weight. The
system can also concentrate and desalt/dialyse at the same time.
The multi-modal nature of the system allows this technology to be
used in a number of other areas especially in the production of
biological components for medical use. The technology isolates
macromolecules using the duality of charge and size. However, the
technology could not be extended to the isolation of molecules
below about 5000 Da. This meant that while molecules smaller than
5000 Da could be removed using at least charge-based separation,
the resulting target molecule could not be captured.
[0004] The separation of micromolecules, molecules deemed to be
less than about 5 kDa, was previously thought not to be possible
using electrophoresis technology devised to separate
macromolecules. This was due to the limit in pore size of membranes
normally used in the systems. For example, the smallest cut-off
produced in polyaccrylamide membranes is about 5 kDa which will
retain any molecule larger than 5 kDa.
[0005] There were several problems encountered in the separation of
micromolecules using an unmodified electrophoresis system.
Difficulty retaining micromolecules in the system has been overcome
with the addition of combinations of membranes. However, these
membranes themselves posed problems in that they are not designed
to retain liquids and can produce large levels of
electro-endo-osmosis. The liquid retention problem has been solved
by backing the membranes with the hydrogel polyacrylamide
membranes, which also helped to reduce the electro-endo-osmosis
levels.
[0006] It is desirable to have a preparative electrophoresis system
which can efficiently and effectively remove micromolecules.
[0007] The subject invention overcomes the above limitations and
others, and teaches an electrophoresis system, which can be scaled
up for preparative applications, which apparatus can efficiently
and effectively separate micromolecules.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention, there is provided
an electrophoresis system which efficiently and effectively
separate micromolecules.
[0009] Further, in accordance with the present invention, there is
provided an apparatus for separating micromolecules by
electrophoretic separation, the apparatus comprising:
[0010] (a) an anode;
[0011] (b) a cathode disposed relative to the anode so as to be
adapted to generate an electric field in an electric field area
therebetween upon application of a voltage potential between the
anode and the cathode;
[0012] (c) a separation membrane disposed in the electric field
area;
[0013] (d) a first restriction membrane disposed between the anode
and the separation membrane so as to define a first interstitial
volume therebetween;
[0014] (e) a second restriction membrane disposed between the
cathode and the separation membrane so as to define a second
interstitial volume therebetween; and
[0015] (f) means adapted to provide a sample constituent in a
selected one of the first and second interstitial volumes;
[0016] wherein upon application of the voltage potential, a
selected separation product is removed from the sample constituent,
thorough the separation membrane, and provided to the other of the
first and second interstitial volume and wherein a micromolecule is
capable of being retained in at least one of the interstitial
volumes.
[0017] Still further, in accordance with the present invention,
there is provided an apparatus for separating micromolecules by
electrophoresis, the apparatus comprising:
[0018] (a) an anode buffer compartment and a cathode buffer
compartment;
[0019] (b) electrodes positioned in the buffer compartments;
[0020] (c) a first chamber and a second chamber positioned on
either side of an ion-permeable separation membrane having a
defined molecular mass cut-off, the first chamber and the second
chamber being positioned between the anode and the cathode buffer
compartments and separated by an ion-permeable restriction membrane
positioned on each side of the separation membrane, the restriction
membrane(s) allowing flow of ions into and out of the compartments
and chambers under the influence of an electric field but
substantially restrict movement of at least one micromolecule type
from the second chamber into the buffer compartment.
[0021] Preferably, the buffer compartments, the first chamber and
the second chamber are configured to allow flow of the respective
buffer, first and second solutions forming streams. In this form,
large volumes can be processed quickly and efficiently. The
solutions are typically moved or recirculated through the
compartments and chambers from respective reservoirs by pumping
means. Peristaltic pumps have been found to be particularly
suitable for moving the fluids.
[0022] Preferably, the ion-permeable separation membrane has a
molecular mass cut-off greater than the molecular mass of the
micromolecule to be separated.
[0023] An advantage of the present invention is that micromolecules
can be separated efficiently and effectively using preparative
electrophoresis under near native conditions which results in
higher yields and excellent recovery.
[0024] Another advantage of the present invention is that the
system is suitably used in a number of other areas, especially in
the production of biological components for medical use.
[0025] Another advantage of the present invention is that the
system can be suitably configured to remove biological contaminants
at the point of separation.
[0026] These and other advantages and benefits of the invention
will be apparent to those skilled in the art upon reading and
understanding of the following detailed description.
[0027] BRIEF DESCRIPTION OF DRAWINGS
[0028] FIG. 1 shows the transfer of BB-FCF through the 5 kDa
separation membrane from the sample stream (US) where it
transiently builds up in the product stream (DS). After 20 minutes
all the BB-FCF had transferred through the bottom restriction
membrane into the buffer stream where it was lost.
[0029] FIG. 2 shows Azorubine transfer using only polyacrylamide
membranes in the Gradiflow system. Transfer from the sample (US) to
the product stream (DS) occurred but the molecules only built up
for a small period of time before moving completely into the buffer
stream.
[0030] FIG. 3 shows that Biotin behaves in a similar manner to the
other tested micromolecules in that it transferred rapidly from the
sample stream (US) and appeared transiently in the product stream
(DS) before eluting into the buffer stream.
[0031] FIG. 4 shoes that phytoestrogen transferred into the product
stream (DS) from the sample stream (US), where it built up and over
time before dissipating into the buffer stream.
[0032] FIG. 5 shows BB-FCF separated with 74% yield in one hour.
The molecules were readily captured using the 1 kDa ultrafiltration
membrane in the cartridge.
[0033] FIG. 6 shows the level of Azorubine in the sample stream
(US) decreased over time and transferred to the product stream
(DS). A total of 83% of the Azorubine was transferred and retained
in 45 minutes.
[0034] FIG. 7 shows Biotin was readily transferred from the sample
stream into the product stream and collected with high yield (84%)
in the product stream.
[0035] FIG. 8 shows phytoestrogen was transferred from the sample
stream into the product stream where it could be collected.
[0036] FIG. 9 shows separation of BB-FCF from Azorubine in a system
adapted to carry out the method according to the present invention.
The BB-FCF was retained in the sample stream whilst the Azorubine
was moved to the product stream.
[0037] FIG. 10 shows BB-FCF was separated from Azorubine using a
size exclusion approach with retention of 74% of the BB-FCF. Only a
small percentage of the Azorubine remained with the BB-FCF after
three hours of separation.
[0038] FIG. 11 is a schematic view of a preferred embodiment of the
separation apparatus of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0039] This invention is directed to an apparatus and method for
the separation of molecules, particularly micromolecules having a
molecular mass of less than about 5000 Dalton. The present
invention is directed to an apparatus for separating micromolecules
by electrophoretic separation, the apparatus comprising:
[0040] (a) an anode;
[0041] (b) a cathode disposed relative to the anode so as to be
adapted to generate an electric field in an electric field area
therebetween upon application of a voltage potential between the
anode and the cathode;
[0042] (c) a separation membrane disposed in the electric field
area;
[0043] (d) a first restriction membrane disposed between the anode
and the separation membrane so as to define a first interstitial
volume therebetween;
[0044] (e) a second restriction membrane disposed between the
cathode and the separation membrane so as to define a second
interstitial volume therebetween; and
[0045] (f) means adapted to provide a sample constituent in a
selected one of the first and second interstitial volumes;
[0046] wherein upon application of the voltage potential, a
selected separation product is removed from the sample constituent,
thorough the separation membrane, and provided to the other of the
first and second interstitial volumes, and wherein a micromolecule
is capable of being retained in at least one of the interstitial
volumes.
[0047] In a preferred embodiment, the present invention is directed
an apparatus for separating micromolecules by electrophoresis, the
apparatus comprising:
[0048] (a) an anode buffer compartment and a cathode buffer
compartment;
[0049] (b) electrodes positioned in the buffer compartments;
[0050] (c) a first chamber and a second chamber positioned on
either side of an ion-permeable separation membrane having a
defined molecular mass cut-off, the first chamber and the second
chamber being positioned between the anode and the cathode buffer
compartments and separated by an ion-permeable restriction membrane
positioned on each side of the separation membrane, the restriction
membrane(s) allowing flow of ions into and out of the compartments
and chambers under the influence of an electric field but
substantially restrict movement of at least one micromolecule type
from the second chamber into the buffer compartment.
[0051] Preferably, the buffer compartments, the first chamber and
the second chamber are configured to allow flow of the respective
buffer, first and second solutions forming streams. In this form,
large volumes can be processed quickly and efficiently. The
solutions are typically moved or recirculated through the
compartments and chambers from respective reservoirs by pumping
means. Peristaltic pumps have been found to be particularly
suitable for moving the fluids.
[0052] Preferably, the ion-permeable separation membrane has a
molecular mass cut-off greater than the molecular mass of the
micromolecule to be separated.
[0053] FIG. 11 shows a preferred embodiment of the apparatus 10 of
the present invention. The apparatus 10 includes an anode buffer
zone or compartment 11 and a cathode buffer zone or compartment 12
separated by an ion-permeable separation barrier 13. Electrodes 14
and 15 are provided inside the buffer zones or compartments so as
to be on opposite sides of the separation membrane 13. It is
understood, however, that in another embodiment, the electrodes are
positioned outside the buffer compartments. The electrodes are used
to apply an electrophoretic potential across the separation
membrane.
[0054] A first chamber 16 is positioned between the anode buffer
compartment 11 and the separation membrane 13. The first chamber is
defined on one side by the separation membrane 13 and on the other
side by a first restriction membrane 18. It is understood, however,
that in another embodiment, the first chamber is positioned between
the cathode buffer compartment and the separation membrane. In one
embodiment, the first restriction membrane is comprised of at least
two membranes 18 and 18b having distinctive pore sizes.
[0055] A second chamber 17 is positioned between the cathode buffer
compartment 12 and the separation barrier 13. The second chamber is
defined on one side by the separation membrane 13 and on the other
side by a second restriction membrane 19 on the other side. It is
understood, however, that in another embodiment, the second chamber
is positioned between the anode buffer compartment and the
separation membrane. In one embodiment, the second restriction
membrane is comprised of at least two membranes 19a and 19b having
distinctive pore sizes.
[0056] The apparatus is further comprised of switch 25 for
selection of the application of a voltage source (such as to turn
the voltage source off or have resting periods), switch 26 to
switch current direction for cathode/anode or to have reversal
periods, and voltage sources 27 and 28.
[0057] The anode buffer compartment and the cathode buffer
compartment are supplied with suitable buffer solutions by any
suitable means. A mixture comprising micromolecules is supplied
directly to the first chamber by any suitable means. The
micromolecules are separated from the second chamber by any
suitable means.
[0058] Preferably, the buffer compartments, the first chamber and
the second chamber are configured to allow flow of the respective
buffer, sample and product solutions forming streams. In this form,
large volumes can be processed quickly and efficiently. The
solutions are typically moved or recirculated through the
compartments and chambers from respective reservoirs by pumping
means. In a preferred embodiment, peristaltic pumps are used as the
pumping means for moving the fluids.
[0059] The buffer, sample or product solutions are cooled by any
suitable means to ensure no inactivation of the micromolecules
occurs during the separation process and to maintained a desired
temperature of the apparatus while in use.
[0060] Preferably, in order to collect and concentrate the
separated micromolecules, solution in the product chamber or stream
is collected and replaced with suitable solvent to ensure that
electrophoresis can continue.
[0061] Preferably, at least one restriction membrane is formed as a
composite or sandwich arrangement with at least two materials.
Preferably, at least one restriction membrane is formed as a
sandwich arrangement with at least two layers of material. In this
preferred form, the sandwich arrangement includes an inner layer
(facing the separation membrane in the first and second solvent
streams, respectively) comprising a membrane having a pore size
with a molecular mass cut-off less than the about 5000 Da and an
outer layer comprising a membrane having a molecular mass cut-off
of greater than about 5000 Da.
[0062] In a preferred form, the inner layer is made from an
ultrafiltration, electrodialysis or haemodialysis material and the
outer layer is made from polyacrylamide. In this preferred
arrangement, the outer layer provides some structural support for
the filtration membrane while preventing unwanted movement of
fluid. The pore size of the filtration membrane is selected
according to the size of the micromolecule to be separated such
that the micromolecule cannot pass through the membrane. Typically,
the molecular mass cut-off of the filtration membrane is between
about 100 Da to 5000 Da. More preferably, the molecular mass
cut-off is around 200 Da.
[0063] Hydrogel ion-permeable separation membranes (an
ultrafiltration, electrodialysis and/or haemodialysis membranes
coated with polyacrylamide) would be an alternative membrane type
suitable for the present invention. Such membranes are possible to
manufacture, but are currently not commercially available.
[0064] Preferably, the ion-permeable separation barrier is a
membrane made from polyacrylamide and having a molecular mass
cut-off from about 5 to 1000 kDa. The size of the separation
membrane cut-off will depend on the sample being processed and the
other molecules in the mixture.
[0065] The restriction barriers or membranes positioned adjacent
the sample and product chambers can have the same molecular mass
cut-off or different cut-offs therefore forming an asymmetrical
arrangement. Typically, the restriction membrane separating the
product chamber from the buffer compartment is formed in a sandwich
configuration.
[0066] The distance between the electrodes can have an effect on
the separation or movement of micromolecules through the barriers.
It has been found that the shorter the distance between the
electrodes, the faster the electrophoretic movement of
micromolecules. A distance of about 6 cm has been found to be
suitable for a laboratory scale apparatus. For scale up versions,
the distance will depend on the number and type of separation
membranes, the size and volume of the chambers for samples, buffers
and separated products. Preferred distances would be in the order
of 6 cm to about 10 cm. The distance will also relate to the
voltage applied to the apparatus. The effect of the electric field
is based on the equation:
e=V/d
[0067] (e=electric field, V=voltage, d=distance)
[0068] Therefore, smaller distances between the electrodes are
preferred. Preferably, the distance between the electrodes should
decrease in order to increase electric field strength, thereby
further improving transfer rates.
[0069] Flow rate of sample/buffer can have an influence on the
separation of micromolecules. Rates of milliliters per hour up to
liters per hour can be used depending on the configuration of the
apparatus and the sample to be separated. Currently in a laboratory
scale instrument, the preferred flow rate is about 20.+-.5 mL/min.
However, flow rates ranging from about 0 to about 50,000 mL/min are
also used across the various separation regimes. In some
embodiments the maximum flow rate is higher, depending on the
pumping means and size of the apparatus. The flow rate is dependent
on the product to be transferred, efficiency of transfer, pre- and
post-positioning with other applications.
[0070] Voltage and/or current applied can vary depending on the
separation. Typically up to several thousand volts may be 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.
However, depending on transfer, efficiency, scale-up and particular
method about 0 to about 5000 are used. Higher voltages may also be
considered, depending on the apparatus and sample to be
treated.
[0071] A number of first and second chambers could be stacked in
the one apparatus for use in a scale-up device.
[0072] A single stream configuration can be produced where the
second chamber forms a buffer chamber. In this configuration,
contaminants would be moved out of the first chamber into the
buffer compartments and the product of interest retained in the
first chamber. In single stream configuration, the membranes can
have either a symmetric or asymmetric arrangements. The present
invention also includes these embodiments.
[0073] In use, a sample containing one or more micromolecules is
added to the first chamber and an electric potential is applied to
cause movement of at least one micromolecule from the sample
through the separation membrane into the second chamber while the
restriction membranes prevent movement of micromolecules from the
first or the second chambers into the respective electrophoresis
buffer chambers. Preferably, the micromolecule is removed and
collected from the product chamber.
[0074] In a second aspect, the present invention provides a
separation cartridge suitable for use in an electrophoresis
apparatus for separating micromolecules, the cartridge
comprising:
[0075] (a) a housing;
[0076] (b) an ion-permeable separation membrane having a defined
molecular mass cut-off positioned in the housing;
[0077] (c) an ion-permeable restriction membrane positioned on
either side of the separation membrane in the housing and spaced to
form a first chamber and second chamber on either side of the
separation membrane, wherein the restriction membrane is adapted to
allow flow of ions into and out of the compartments and chambers
under the influence of an electric field but substantially restrict
movement of at least one micromolecule type from the second
chamber. In a preferred embodiment, the cartridge further
includes:
[0078] (d) electrodes positioned in the housing on the outer sides
of the restriction barriers.
[0079] Preferably, the separation barrier is a membrane composed of
polyacrylamide and having a molecular mass cut-off from about 5 to
1000 kDa.
[0080] Preferably, the ion-permeable separation membrane has a
molecular mass cut-off greater than the molecular mass of the
micromolecule to be separated.
[0081] At least one restriction membrane is preferably formed as a
sandwich or composite arrangement of membranes with at least two
materials. Preferably, the sandwich arrangement includes an inner
layer comprising a restriction membrane having a pore size with a
molecular mass cut-off less than about 5000 Da and an outer layer
comprising a restriction membrane having a molecular mass cut-off
of greater than about 5000 Da.
[0082] In a preferred form, the inner layer is made from an
ultrafiltration, electrodialysis or haemodialysis material and the
outer layer is made from polyacrylamide. In this preferred
arrangement, the outer layer provides some structural support for
the filtration membrane while preventing unwanted movement of
fluid. The pore size of the filtration membrane is selected
according to the size of the micromolecule to be separated such
that the micromolecule cannot pass through the membrane. Typically,
the molecular mass cut-off of the filtration membrane is between
about 100 Da to 5000 Da. More preferably, the molecular mass
cut-off is around 200 Da.
[0083] In a third aspect, the present invention provides a method
of separating a micromolecule from a liquid sample, the method
comprising:
[0084] (a) providing an electrophoresis apparatus according to the
first aspect of the present invention;
[0085] (b) placing the sample in the first chamber of the
apparatus; selecting a solvent for the first chamber having a pH
such that the micromolecule to be separated is charged;
[0086] (c) applying an electric potential between the first and
second chambers causing movement of micromolecules in the first
chamber through the separation membrane into the second chamber
while unwanted molecules are substantially prevented from entering
the second chamber;
[0087] (d) optionally, periodically stopping and reversing the
electric potential to cause movement of molecules having entered
the separation membrane to move back into the first chamber, while
substantially not causing any micromolecules that have entered the
second chamber to re-enter first chamber; and
[0088] (e) maintaining steps (c) and optionally (d) until the
desired amount of micromolecules are moved to the second
chamber.
[0089] The micromolecule can be any micromolecule capable of
receiving or having a charge. Examples include, but not limited to,
biotin, Brilliant Blue FCF (BB FCF), azorubine, phytoestrogen,
digoxigenin, hormones, cytokines, dyes, vitamins, chemicals,
neutraceuticals, pharmaceuticals along with food and diet
supplements.
[0090] The sample can contain one or more micromolecules of
interest. Examples include, but are not limited to, crude extracts,
microbial cultures, cell lysates, cellular products, chemical
processing mixtures, cell culture media, plant products or
extracts.
[0091] Solvent in the form of buffers that have been found to be
particularly suitable for the method according to the present
invention are Tris Borate around pH 9. It will be appreciated,
however, that other buffers or solvents would also be suitable,
depending on the separation. The concentration of the selected
buffers can also influence or effect the movement of micromolecules
through the separation barrier. Typically concentrations of about
10 mM to about 200 mM, more preferably 20 mM to 80 mM, have been
found to be particularly suitable. Almost any buffers and/or
solvents can be used with the present invention. The buffers and/or
solvents that can be used are procedure/method/separation
dependent. The concentration of the buffer and/or solvent is
dependent upon the application/separation/procedure.
[0092] Reversal of current is an option but another embodiment is a
resting period. Resting (a period without an electric potential
being applied, but pumps remain on) is an optional step that can
replace or be included before or after an optional electrical
potential reversal. This reversal technique is often practised for
protein separation work as an alternative to reversing the
potential.
[0093] One benefit of the method according to the present invention
is the possibility of scale-up without denaturing or adversely
altering the physical or biological properties of the
micromolecule.
[0094] In a fourth aspect, the present invention provides a
micromolecule purified or separated by the method according to the
third aspect of the present invention.
[0095] Preferably the micromolecule is less than 5000 Da. Examples
include, but are not limited to, biotin, Brilliant Blue FCF (BB
FCF), azorubine, phytoestrogen, digoxigenin, hormones, cytokines,
dyes, vitamins, chemicals, neutraceuticals, pharmaceuticals along
with food diet supplements, and combinations thereof.
[0096] In a fifth aspect, the present invention relates to use of
the micromolecule according to the fourth aspect of the present
invention in dietary, medical and veterinary applications.
[0097] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element, integer or step, or group of elements, integers or
steps, but not the exclusion of any other element, integer or step,
or group of elements, integers or steps. Any discussion of
documents, acts, materials, devices, articles or the like which has
been included in the present specification is solely for the
purpose of providing a context for the present invention. It is not
to be taken as an admission that any or all of these matters form
part of the prior art base or were common general knowledge in the
field relevant to the present invention as it existed in Australia
before the priority date of each claim of this application.
[0098] In order that the present invention may be more clearly
understood, preferred forms will be described with reference to the
following drawings and examples.
MODES FOR CARRYING OUT THE INVENTION
[0099] The separation of micromolecules, molecules deemed to be
less than about 5 kDa, was previously thought not to be possible
using electrophoresis technology devised to separate
macromolecules. This was due to the limit in pore size of membranes
normally used in the systems. For example, the smallest cut-off
produced in polyaccrylamide membranes is about 5 kDa which will
retain any molecule larger than 5 kDa. The present invention
results from modification of earlier technology to be capable of
separating micromolecules by using some barriers or membranes other
than polyacrylamide membranes traditionally used. It has been found
that when commercially available membranes in the form of
ultrafiltration, electrodialysis and haemodialysis membranes are
used as separation or restriction membranes, the size of molecule
that can be dealt with, is significantly smaller than previously
thought.
[0100] In order to separate and purify micromolecules, hydrogel
polyacrylamide membranes traditionally used in the earlier system's
cartridge were placed as backing to a commercial membrane with the
desired pore size. The polyacrylamide membrane is useful to prevent
unregulated fluid movement across the membranes, whilst the
commercial membrane is used to retain the smaller molecular species
within the sample or product streams or chambers. The membranes
used for this work were Pall Gelman Omega ultrafiltration
membranes. These ultrafiltration membranes are available
commercially with pore sizes ranging from hundreds of kDa down to
1000 Da.
[0101] Another surprising finding was that the relative pore size
of the ultrafiltration membranes was found to be different when
used in the sandwich arrangement than when used in an
ultrafiltration unit. The relative pore size appears to be smaller
than the stated size. Thus it has been shown to be possible to
retain molecules as small as 200 Dalton within the modified
system.
[0102] When an apparatus is operated with the traditional
polyacrylamide membranes used for macromolecule purification or
separation, small molecules under 5 kDa tend to transfer from the
sample stream into the product stream where they remain
transiently. These molecules then move through the bottom
restriction membrane into the buffer stream where they are
lost.
[0103] Experiments were carried out on several model molecules
which have varied structure and function. The use of these
micromolecules demonstrates that a wide variety of molecules could
be used within the system. Two molecules studied were
Bis[4-(N-ethyl-N-3-sulfophenylmethyl)a-
minophenyl]-2-sulfophenylmethylium disodium salt (Brilliant Blue
FCF) which has a molecular mass of 793 Dalton, and Disodium
2-(4-Sulfo-1Napthylazo)-1-Napthol-4-Sulfonate (Azorubine) which has
a molecular mass of 502 Dalton. Two other molecules were also
investigated, one being Vitamin H (Biotin) and the other a small
phytoestrogen separated from red leaf clover (supplied by Novogen,
Sydney Australia). Brilliant Blue FCF and Azorubine are two
chemicals currently used in the food industry as colouring agents.
Biotin has several uses, first as a necessary vitamin in the human
diet and secondly, but not insignificantly, as a labelling agent in
scientific assays. Phytoestrogens are separated commercially from
many sources primarily soy and clover where they are made into
herbal and pharmaceutical medicines.
[0104] Experiments using Brilliant Blue FCF (BB-FCF), Azorubine,
Biotin and a Phytoestrogen all confirmed that standard
polyacrylamide electrophoresis membranes do not retain these
micromolecules during a separation. These molecules are very small
and definitely considered micromolecules; BB-FCF (793 Da),
Azorubine (502 Da), Biotin (244 Da) and a Phytoestrogen (.about.200
Da).
[0105] Experiments using BB-FCF and Azorubine with a pH 9.0 buffer
and a cartridge sandwich of 5-5-5 kDa polyacrylamide membranes
(upper restriction 5 kDa, separation membrane 5 kDa, lower
restriction 5 kDa) showed that complete transfer from the sample
stream occurs in a short period of time. The macromolecules build
slightly in the product stream before passing completely into the
buffer stream where they are diluted so highly that they are lost
to analysis. This is also true for the Phytoestrogen and for
Biotin.
[0106] FIGS. 1, 2, 3, and 4 show the transient build up and
eventual loss of BB-FCF, Azorubine, Biotin, and the Phytoestrogen,
within the an apparatus used for macromolecule separation having
only polyacrylamide membranes. The time taken for the
micromolecules to completely transfer out of the system varies for
each molecule. It is likely that this is due to the difference in
charge to mass ratios between the molecules.
[0107] Use of traditional polyacrylamide membranes was therefore
found not a feasible method for the separation of molecules under
about 5 kDa. This led to the novel step of trialing new membranes,
previously not considered useful for prior art systems. These
commercially available membranes are manufactured to be used under
conditions of high pressure where they act purely as a filter. This
means that the membranes are not always "water tight" when used in
the earlier system. Only those membranes with a very small pore
size retain liquid under the low pressures used in the earlier
system. These ultrafiltration membranes are ideal for use in a
preparative electrophoresis system unless they are backed with a
membrane designed to stop or reduce transfer of liquid but allow
ion and charged molecule transfer under an electric filed.
[0108] It was found that alternative membrane types could be used
when they were backed with a hydrogel membrane to give fluid
retention. This double layering not only prevented fluid leakage
across the membranes but also significantly reduced the levels of
endo-osmosis produced. Endo-osmosis levels are a big consideration
in the design and use of membranes for preparative electrophoresis
systems. Particular chemistries of membranes can produce such large
changes in fluid volumes from one stream to another via
electro-endo-osmosis so cannot be used.
[0109] The separation and retention of very small molecules was
made possible in the apparatus according to the present invention
by using a small pore sized ultrafiltration membrane (Pall Gelman
Omega) as the bottom restriction in combination with a
polyacrylamide membrane. The use of a 1 kDa Omega ultrafiltration
membrane in this position allowed the capture of molecules as small
as 200 Da. Experiments were carried out again using BB-FCF,
Azorubine, Biotin and a Phytoestrogen.
[0110] The following cartridge configuration was used for the
separations:
[0111] 5 kDa polyacrylamide restriction membrane
[0112] Support Grid
[0113] 10 kDa polyacrylamide separation membrane
[0114] Support Grid
[0115] 1 kDa ultrafiltration restriction membrane
[0116] 5 kDa polyacrylamide restriction membrane
[0117] It was possible to move BB-FCF, Azorubine and Biotin using
the above cartridge configuration from the sample stream to the
product stream where they were trapped and collected. The
Phytoestrogen experiments used a 5 kDa ultrafiltration membrane to
retain the molecules in the product stream.
[0118] Brilliant Blue FCF was readily transferred and retained in
the product stream with the use of the 1 kDa Omega ultrafiltration
membrane. This molecule is only 793 Da and so retention with a 1000
Da membrane would not be expected. The BB-FCF separation is
represented by FIG. 5. Analysis of the BB-FCF separation was
carried out using the absorbance at 630 nm.
[0119] Azorubine was moved across the separation membrane and
collected in the product stream. Using a pH 9.0 Tris-Borate buffer
83% of the Azorubine was transferred from the sample stream to the
product stream within 45 minutes. This transfer was measured using
the absorbance of Azorubine at 516 nm. The separation is
illustrated in FIG. 4.
[0120] The Biotin separation utilised a pH 9.0 buffer with the same
cartridge configuration as that used for the BB-FCF and Azorubine.
The transfer of this molecule was monitored using the absorbance at
230 nm. This experiment showed that the 1 kDa ultrafiltration
membrane used could retain molecules as small as 244 Dalton. FIG. 7
shows that over 80% of the Biotin was transferred to the product
stream where it was contained. The phytoestrogen transfer
experiment depicted in FIG. 8 showed the movement and successful
capture of phytoestrogen. The decrease over time after the initial
high levels of phytoestrogen are most likely due to the fact that a
5 kDa ultrafiltration membrane was used as the bottom restriction
membrane. The use of a 1 kDa ultrafiltration membrane would help to
completely retain the small phytoestrogen.
[0121] Not only could Azorubine and BB-FCF be moved across a
separation membrane from sample stream to the product stream, these
two molecules could also be separated from each other. With only
293 Da difference in size, the two compounds were separated from
each other using a size exclusion separation where the largest
molecule was retained in the product stream whilst the smaller
molecule was allowed to transfer through into the buffer stream.
The following cartridge configuration was used for the
separation:
[0122] 5 kDa polyacrylamide restriction membrane
[0123] Support Grid
[0124] 3 kDa ultrafiltration separation membrane
[0125] 5 kDa polyacrylamide separation membrane
[0126] Support Grid
[0127] 1 kDa ultrafiltration restriction membrane
[0128] 5 kDa polyacrylamide restriction membrane
[0129] BB-FCF was separated from Azorubine. By allowing Azorubine
to pass through the 3 kDa ultrafiltration membrane whilst retaining
the BB-FCF in the sample stream, an adequate separation was
achieved. The concentration of Azorubine decreased significantly
from the sample stream but did not build up substantially in the
product stream. This was due to loss into the buffer stream over
time. The BB-FCF can pass through a 3 kDa membrane but only very
slowly so separation of the two molecules was achieved. The
movement of both molecules was monitored using 603 nm for BB-FCF
and 516 nm for Azorubine. FIG. 9 shows the selective nature of the
separation.
[0130] To improve the separation of BB-FCF from Azorubine a
different cartridge configuration was utilised as follows:
[0131] 5 kDa polyacrylamide restriction membrane
[0132] Support Grid
[0133] 200 kDa polyacrylamide separation membrane
[0134] Support Grid
[0135] 1 kDa ultrafiltration restriction membrane
[0136] 5 kDa polyacrylamide restriction membrane
[0137] This separation allowed both molecules to quickly transfer
to the product stream from the sample stream. Then over time the
Azorubine passed into the buffer stream, whilst the BB-FCF was
retained. The fact that the Azorubine would transfer through the 1
kDa Omega ultrafiltration membrane enabled separation of Azorubine
from BB-FCF more effectively than previously. FIG. 10 shows that
after a three hour separation close to 74% of the BB-FCF was still
present in the downstream whilst only 15% of the Azorubine
remained. This separation shows the highly selective nature of the
separation, which opens many possibilities for its use with a
number of different molecular separations.
[0138] In new electrophoresis system it has been found that
micromolecules under 5 kDa can be separated with this technology.
When the polyacrylamide membranes are used in combination with
certain commercially available membranes, molecules as small as
.about.200 Da can be separated and purified.
[0139] There were several problems encountered in the separation of
micromolecules using an unmodified electrophoresis system.
Difficulty retaining micromolecules in the system has been overcome
with the addition of combinations of membranes. However, these
membranes themselves posed problems in that they are not designed
to retain liquids and can produce large levels of
electro-endo-osmosis. The liquid retention problem has been solved
by backing the membranes with the hydrogel polyacrylamide
membranes, which also helped to reduce the electro-endo-osmosis
levels.
[0140] Several examples demonstrating the capability of the present
invention in separating macromolecules have been shown though many
other possible micromolecules of commercial interest do exist. For
example this technology could be used in the separation and
purification of cytokines and growth factors for use in the
pharmaceutical and research industries. Currently cytokines and
growth factors account for over 50% of the biotechnology based
pharmaceutical product sales. Other areas where the use of the
present technology could improve current separation strategies and
be of substantial commercial benefit include purification of
pharmaceutical drugs, food additives, agro-chemicals and fine
chemicals.
[0141] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
* * * * *