U.S. patent application number 09/324527 was filed with the patent office on 2001-11-22 for static separation method using non-porous cellulose beads.
Invention is credited to GARNER, WILLIAM DARWIN, STIPANOVIC, BOZIDAR.
Application Number | 20010042718 09/324527 |
Document ID | / |
Family ID | 23263986 |
Filed Date | 2001-11-22 |
United States Patent
Application |
20010042718 |
Kind Code |
A1 |
STIPANOVIC, BOZIDAR ; et
al. |
November 22, 2001 |
STATIC SEPARATION METHOD USING NON-POROUS CELLULOSE BEADS
Abstract
An affinity separation method and system comprising an affinity
separation media with low porosity and low non-specific binding,
and a fluid containing a target compound to be isolated which is
capable of binding onto the affinity separation media in a fluid
mixing loop in a static filtration apparatus. The static filtration
apparatus comprises an intermixing-chamber containing a filtration
medium wherein a tangential flow is created for intermixing the
affinity separation media and target compound in the fluid. The
fluid is capable of passing through the filtration medium while the
affinity separation media are substantially incapable of passing
through the filtration medium. The affinity separation media are
separated from the fluid by opening the filtrate outlet so as to
allow the fluid to pass through the filtration medium of the static
filtration apparatus. The filtrate can be thereby rendering
substantially free of the target compound.
Inventors: |
STIPANOVIC, BOZIDAR; (LAKE
FOREST, IL) ; GARNER, WILLIAM DARWIN; (CHICAGO,
IL) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Family ID: |
23263986 |
Appl. No.: |
09/324527 |
Filed: |
June 2, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60087902 |
Jun 3, 1998 |
|
|
|
Current U.S.
Class: |
210/656 ;
210/198.2; 436/161 |
Current CPC
Class: |
B01J 20/3278 20130101;
B01J 20/3274 20130101; B01J 20/3204 20130101; B01D 15/1871
20130101; B01D 15/3804 20130101; B01J 20/3212 20130101; B01D
15/1807 20130101; B01J 20/3219 20130101; B01J 20/28004 20130101;
B01J 20/321 20130101; C07K 1/22 20130101; B01J 20/3265
20130101 |
Class at
Publication: |
210/656 ;
210/198.2; 436/161 |
International
Class: |
B01D 015/08 |
Claims
We claim:
1. A method of separation for isolating a target compound from a
fluid comprising: (a) forming a suspension comprising i) a fluid
containing a target compound to be isolated, and ii) a plurality of
low porosity affinity particles capable of binding the target
compound, (b) maintaining said low porosity affinity particles and
said fluid in contact for a sufficient period of time for binding
of said target compound onto said low porosity affinity particles
to be effected, (c) introducing said suspension into a static
filtration apparatus having a filtration medium, (d) creating a
tangential flow in said static apparatus thereby aiding in
separation between said fluid and said low porosity affinity
particles with the target compound attached to the surface thereof,
(e) passing said fluid devoid of the target compound through said
filtration medium of said static filtration apparatus, (f) washing
said low porosity affinity particles, and (g) eluting said target
compound from said low porosity affinity particles.
2. The method of claim 1, wherein said low porosity affinity
particles are nonporous.
3. The method of claim 2, wherein said low porosity affinity
particles are generally spherical.
4. The method of claim 3, wherein said low porosity affinity
particles are generally identical in diameter.
5. The method of claim 4, wherein said low porosity affinity
particles have an average diameter of between about 0.5 and about
25 microns.
6. The method of claim 4, wherein said low porosity affinity
particles have an average diameter between about 1 and about 3
microns.
7. The method of claim 4, wherein said low porosity affinity
particles have an average diameter at least about two-fold larger
than the average pore size of said filtration medium.
8. The method of claim 7, wherein said fluid contains particulate
and said filtration medium has an average pore size at least about
two-fold larger than the size of the largest particulate in said
fluid.
9. The method of claim 4, wherein said low porosity affinity
particles have an average diameter at least about five-fold larger
than the average pore size of said filtration medium.
10. The method of claim 7, wherein said fluid contains particulate
and said filtration medium has an average pore size at least about
five-fold larger than the size of the largest particulate in said
fluid.
11. The method of claim 1, wherein said target compound is eluted
from said low porosity affinity parties by transferring said
affinity particles having said target compound thereto from said
static filtration to a tank, passing an eluent through said tank to
remove said target compound from said low porosity affinity
particles, thereby providing eluded low porosity affinity particles
from which said target compound has been removed, and collecting
the resulting eluent containing said target compound.
12. The method of claim 10, wherein said eluted low porosity
affinity particles are then transferred back to said static
filtration apparatus and the method is repeated.
13. The method of claim 1, wherein said low porosity affinity
particles have an average diameter between about 0.5 and about 120
um.
14. The method of claim 1, wherein said low porosity affinity
particles have an average diameter range of about 60 um or
less.
15. The method of claim 12, wherein said low porosity affinity
particles have an average diameter of about 20 um or less.
16. The method of claim 13, wherein said filtration medium has an
average pore rating of less than abut 5 um.
17. The method of claim 1, wherein said low porosity affinity
particles have an average diameter of at least about 0.5 um.
18. The method of claim 15, wherein said filtration medium is a
microporous membrane having an average pore rating of less than
about 1 um.
19. A system for affinity separation of a target compound from a
fluid comprising: (a) a static filtration apparatus comprising a
housing having a feed port, a concentrate port, a filtrate port,
and a filtration medium disposed within the housing; (b) the feed
port and concentrate port being in fluid communication with the
upstream side of the filtration medium, while the filtrate port is
in fluid communication with the downstream side of the filtration
medium; and (c) a suspension comprising a plurality of low porosity
affinity particles and a fluid containing a target compound to be
isolated fed into the filtration apparatus via the feed port for
inter-mixing.
20. The system of claim 19, wherein said low porosity affinity
particles are nonporous.
21. The system of claim 19, wherein said low porosity affinity
particles are generally spherical.
22. The system of claim 19, wherein said low porosity affinity
particles are generally identical in diameter.
23. The system of claim 20, wherein said low porosity affinity
particles have an average diameter least about two-fold larger than
the average pore size of said filtration medium.
24. The system of claim 21, wherein said fluid contains
particulates and said filtration medium has an average pore size at
least about two-fold larger than the size of the largest
particulate in said fluid.
25. The system of claim 20, wherein said low porosity affinity
particles have an average diameter at least about five-fold larger
than the average pore size of said filtration medium.
26. The system of claim 23, wherein said fluid contains
particulates and said filtration medium has an average pore size at
least about five-fold larger than the size of the largest
particulate in said fluid.
27. The system of claim 19, wherein said low porosity affinity
particles have an average diameter of about 120 um or less.
28. The system of claim 19, wherein said low porosity affinity
particles have an average diameter about 60 um or less.
29. The system of claim 19, wherein said low porosity affinity
particles have an average diameter or about 20 um or less.
30. The system of claim 27, wherein said filtration medium has an
average pore rating of less than about 5 um.
31. The system of claim 19, wherein said low porosity affinity
particles have an average diameter of between about 1 and about 3
um.
32. The system of claim 19, wherein said filtration medium is a
microporous membrane having an average pore rating of less than 1
um.
33. The system of claim 19 wherein said low porosity affinity
particles are ungrafted for separation of a fusion protein
comprising a cellulose binding protein segment fused with a target
protein or peptide.
34. The system of claim 33 wherein said low porosity affinity
particles comprise a bead having a surface, said surface of the
bead as a whole representing an affinity site for attaching a
cellulose binding fusion protein segment.
35. The system of claim 19 wherein said low porosity affinity
particles have a plurality of chemical linkers attached
thereto.
36. The system of claim 19 wherein said low porosity affinity
particles are linker-coated polystyrene affinity particles having
generally less than 40% porosity.
37. The system of claim 20 wherein said low porosity affinity
particles are reconstituted cellulose affinity particles.
38. The system of claim 19 wherein said system is connected to
other said systems forming a multi-system purification method
wherein at least a product may be purified using said low porosity
affinity particles capable of binding said products in each system
for the purification of each product separate from the other using
a single source of raw material having a mixture of each product.
Description
RELATED APPLICATION
[0001] The present application claims the benefit of the filing
dates under 35 U.S.C. .sctn. 119(e) to provisional U.S. patent
application Ser. No. 60/087,902 filed on Jun. 2, 1999, which is
hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Purification systems using glass or metal tubes which
contain a packed column of separation medium, for example, beads or
particles, are known. These tubes are known as column boxes.
Because the separation medium is compacted within the column boxes,
the flow rates are slow and the column boxes have a limited
capacity. Therefore, prior art purification technology has focused
on increasing the porosity of the separation medium to increase the
flow rates and capacity within the column box. The object of these
known systems is to purify the largest amount of material within
the shortest amount of time while keeping the amount of
contaminants low and the product yields high. One problem with the
old purification technology is that increasing porosity of the
separation medium achieved faster flow rates and capacity, but
reduced product yields and quality.
[0003] A past method, which does not use a column box, utilized a
dynamic (i.e., mechanical forces at the site of filtration)
filtration system with a variety of separation medium. This old
method and apparatus also failed in part because the sheering
forces of the dynamic system damaged the separation medium and the
bio-compounds being purified.
[0004] Prior methods of affinity separation involving dynamic
filtration of cellulose and non-cellulose beads have the
disadvantage that the beads degrade due to sheering forces inherent
in dynamic filtration. These sheering forces irreversibly damage
bead supports and the sheer-sensitive biological molecules grafted
or bound to them. Generally, many porous beads will fragment due to
the agitation and sheering forces contained in the dynamic affinity
separation apparatus. The present invention overcomes the
disadvantages of the prior art by utilizing a static affinity
separation method which is gentle on beads and biological
molecules, but causes intermixing of the target compound to be
purified with the sheer-sensitive beads and biological molecules
which flow through the static tangential flow filters.
[0005] The prior art discloses the use of polystyrene beads in
dynamic filtration apparatuses for affinity separation of
biological compounds. Certain disadvantages are involved in the use
of polystyrene beads, such as high non-specific adsorbing of
biological molecules on their hydrophobic surfaces. Further,
polystyrene beads have open pores on the surface of the bead which
entrap contaminants which will co-purify with wanted products and
decrease yield and purity of a target compound. Polystyrene has
been found to exhibit a high degree of agglomerate and to adhere to
the filters used in dynamic affinity separation methods. This
agglomeration of the polystyrene beads allows for debris to become
trapped and spoils the filtration affinity system by clogging the
filter. A preferred embodiment of the present invention overcomes
the disadvantages of the prior art by adding a multitude of linkers
to the bead surface to increase bead coating. Bead coating reduces
unwanted agglomeration and filter clogging. This multitude of
linkers reduces agglomeration and non-specific binding, resulting
in increased stability and reduced entrapment of unwanted
contaminants, while enabling the attachment of the target compounds
desired to be separated.
[0006] The prior art discloses the use of cellulose in dynamic
filtration apparatuses for affinity separation of biological
compounds. The prior art cellulose particles available are highly
porous particles which exhibit entrapment of target and
contaminants. Moreover, these highly porous prior art cellulose
particles can swell from 25% to 400% of their original size in
aqueous medium. Additionally, the prior art cellulose particles are
highly sensitive to sheering forces, thus resulting in
fragmentation in a dynamic filter. The present invention uses
non-porous cellulose beads in a static tangential flow system to
prevent bead fragmentation.
BRIEF SUMMARY OF THE INVENTION
[0007] A preferred embodiment of the present invention provides an
improved affinity separation method and system comprising an
affinity separation media with low porosity and low non-specific
binding, and a fluid containing a target compound to be isolated
which is capable of binding onto the affinity separation media in a
fluid mixing loop in a static filtration apparatus. The static
filtration apparatus comprises an intermixing-chamber containing a
filtration medium having upstream and downstream sides, an inlet in
fluid communication with the upstream side of the filtration
medium, and a filtrate outlet in fluid communication with the
downstream side of the filtration medium, wherein a tangential flow
is created for intermixing the affinity separation media and target
compound in the fluid. The fluid is capable of passing through the
filtration medium while the affinity separation media are
substantially incapable of passing through the filtration medium.
The affinity separation media are separated from the fluid by
opening the filtrate outlet so as to allow the fluid to pass
through the filtration medium of the static filtration apparatus.
The filtrate can be thereby rendered substantially free of the
target compound. If the recovery of the target compound is desired
and/or if the affinity separation media are to be reused, the
affinity separation media are then thoroughly washed, and the
target compound is eluted from the affinity separation media,
filtered, and ultimately the target compound is recovered from the
filtrate and the affinity separation media may be reused.
[0008] The preferred invention apparatus and system ideally
utilizes small, reconstituted cellulose particles having no pores
and low non-specific binding properties. The cellulose particles of
the preferred embodiment of the present invention consist of small,
substantially spherical bodies with a near complete absence of
irregularities, holes, cracks and the like. The cellulose bodies
are made from viscose. This improvements results in uncross-linked,
high density, spherical cellulose separation support beads without
substantial holes, voids or craters on their surface. In certain
circumstances, as where ligands are attached to the cellulose
particles, chromatographic separation may be optimized when
substrate/sorbent interactions take place exclusively on the
outside surface of the particle. In such cases, any presence of
holes of a size that may accommodate a substrate molecule cannot be
tolerated; otherwise diffusion based interferences may adversely
effect resolution of pure compounds.
[0009] The cellulose beads are essentially non-crystalline.
Electron micrograph sections of the particles mounted in an epoxy
matrix display a structure whereby the cellulose particles show a
dense non-porous outer shell with an approximate thickness of 1,000
to 2,000 angstroms and a more porous interior of the closed-cell
type. The shape of the particles is essentially spherical. The
cellulose particles are essentially non-swellable and stable in pH
range between about 1 and 13.
[0010] The current invention relates to a unique and novel method
which eliminates the column box and utilizes low porosity. The
present method for the affinity separation of bio-compounds uses a
static filtration system instead of the column box to achieve flow
rates of liters per minute instead of milliliters per minute, which
is typical of high porosity separation media. Therefore, a low
porosity, low non-specific binding separation medium, generally the
lowest being reconstituted cellulose affinity particles, is
preferred.
[0011] An advantage of the present inventive method is the
provision of the exceptionally efficient separation through an
affinity separation procedure of a compound from a dilute solution.
A preferred embodiment of the present invention provides a means
for lessening the number of processing steps required to perform an
affinity separation as compared to known affinity separation
methods, thereby increasing the overall yield of the separation
method.
[0012] A further advantage of the preferred embodiment of the
invention is that the present inventive method is able to be
conducted in a relatively lesser amount of time as compared to
known affinity separation processes.
[0013] Moreover, a further advantage of the invention is that since
the present inventive method preferably utilizes nonporous affinity
particles, the present invention avoids those problems attendant
the use of highly porous affinity particles, e.g., fragility during
mechanical agitation, affinity particle fouling, susceptibility to
crushing, and swelling.
[0014] In addition, it is yet another advantage of the invention
that the present inventive method generally avoids problems
associated with channeling and filtration medium fouling associated
with conventional chromatographic affinity separation methods using
column boxes.
[0015] An embodiment of the present invention overcomes the
disadvantages of the prior art by adding a multitude of linkers to
the bead surface to coat the bead. Thereby, the bead pores are
covered, thus reducing unwanted porosity and non-specific binding
which may allow for entrapment and adsorption of unwanted
contaminants. The linkers may be added to the beads by covalent
bonding or in other manners known to those skilled in the art. This
linker coating enables the attachment of special chemical "hooks"
which specifically bind the desired target compounds to be
separated.
[0016] A further advantage of a preferred embodiment of the present
invention over the prior art is that the use of reconstituted
cellulose particles prevents the entrapment of target molecules and
contaminants and the swelling associated with the use of prior art
cellulose beads. In comparison, a reconstituted cellulose particle
swells no more than 15% of its original size.
[0017] A further advantage of a preferred embodiment of the present
invention over the prior art is that static tangential affinity
systems prevent the accumulation of debris and beads that usually
connect and clog tangential flow systems that may get trapped in
systems such as the prior art dynamic systems.
DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic depiction of the significant elements
used in a preferred embodiment of the present invention.
[0019] FIG. 2 is a schematic depiction of a preferred embodiment of
the apparatus of the present invention for use with the low
porosity, low nonspecific binding separation media, the preferred
separation media being reconstituted cellulose particles.
[0020] FIG. 3 is a schematic depiction of an embodiment of the
present invention for sequential purification of biomaterials.
DETAILED DESCRIPTION OF THE DRAWINGS AND PREFERRED EMBODIMENTS OF
THE INVENTION
[0021] The present invention provides an improved affinity
separation method. It has been found that the use of a static
filtration apparatus having a tangential flow can be used to
efficiently effect separation between a fluid without plugging the
filter and suspended therein affinity particles. The static
filtration apparatus can thereafter be used to separate the fluid
from the nonporous cellulose affinity particles having the bound or
complexed target thereon. Such a process enables the use of
nonporous cellulose affinity particles having small diameters and
relatively large surface areas per weight of the cellulose.
[0022] The preferred inventive method is an improved affinity
separation method comprising:
[0023] (a) introducing reconstituted cellulose affinity particles
into a fluid containing a target compound to be isolated which is
capable of specifically binding onto the cellulose affinity
particles by means of specific "hooks" which specifically bind the
target compound,
[0024] (b) filtering the resulting suspension in a static
filtration apparatus, comprising a filtration medium having
upstream and downstream sides, an inlet in fluid communication with
the upstream side of the filtration medium, and a filtrate outlet
in fluid communication with the downstream side of the filtration
medium, wherein the apparatus has a tangential flow and the fluid
is capable of passing through the filtration medium and the
nonporous cellulose affinity particles are substantially incapable
of passing through the filtration medium, and
[0025] (c) separating the cellulose affinity particles from the
fluid by opening the filtrate outlet so as to allow the fluid to
pass through the filtration medium of the static filtration
apparatus.
[0026] As a consequence of affinity of the target compound for the
bead surface, which has been rendered attractive to the target
compound, the target concentration in the fluid can be brought down
to sufficiently low levels, allowing such process to be used as a
practical industrial method of separation of individual compounds
from a mixture of a multitude of compounds in a solution.
[0027] The relative sizes of the largest debris in the fluid to be
treated, the pores of the filtration medium, and the diameter of
the reconstituted affinity particles are important in the practical
utilization of the present inventive affinity separation method.
Ideally, the filtration medium will allow all of the fluid
(including the largest debris) to pass therethrough, while
preventing any of the reconstituted cellulose affinity particles
from passing therethrough. The filtration medium, therefore, will
preferably have an average pore size at least a factor of two, and
preferably a factor of five or ten, larger than the largest debris
in the fluid to be treated, and the reconstituted cellulose
affinity particles will preferably be at least a factor of two, and
preferably a factor of five or ten, larger than the pore size of
the filtration medium. Thus, for example, if the largest debris in
the fluid to be treated is in on the order of about 0.1-0.2 micron,
the average pore size of the filtration medium will preferably be
on the order of about 1-3 microns, while the reconstituted
cellulose affinity particles will preferably be on the order of
about 10-20 microns.
[0028] In general, the smallest acceptable reconstituted cellulose
affinity particles, e.g., reconstituted cellulose affinity
particles of 60 microns diameter or less, particularly
reconstituted cellulose affinity particles of 20 microns diameter
or less, are preferably used to improve the recovery of the target
compound by increasing the available surface area per unit of
weight of the reconstituted cellulose affinity particles for
interaction with the fluid being treated. The range of size for the
reconstituted cellulose affinity particles can range from nano-size
to 120 microns. Accordingly, while pretreatment of the fluid to be
treated may not be the most desirable inasmuch as it adversely
impacts on the recovery of the target compound by introducing an
additional processing step, there may be instances in which the
recovery loss resulting from pretreatment, particularly
prefiltering and/or homogenization to reduce the sizes of the
largest debris in the fluid, will be more than offset by the
resulting ability to use smaller affinity particles having a higher
area per unit of weight of the affinity particles.
[0029] Although several separation media may be modified to yield
affinity beads which possess low porosity and reduced nonspecific
binding, the preferred embodiment includes nonporous cellulose
beads with reduced nonspecific binding. The reconstituted cellulose
affinity particles comprise a spacer and a ligand on the surface
thereof which is capable of binding to the target compound in a
fluid so as to enable the separation of that compound from the
remainder of the fluid. Then, the target compound is capable of
being removed from the reconstituted cellulose affinity particles
by changing the conditions of the solution, for example, pH, salts,
and others. The affinity ligands used to separate particular
compounds from a fluid will vary. The proper selection of the
ligand will ensure that the target compound selectively and
reversibly binds, e.g., complexes with or adsorbs onto, the
reconstituted cellulose affinity particle.
[0030] In a preferred embodiment, low porosity, low
non-specific-binding separation media includes low porosity linker
coated polystyrene carbohydrate, glass, porcelain, ceramics, and
other low porosity beads known to one skilled in the art. Examples
of beads which may be used in the present invention are disclosed
in U.S. Pat. No. 5,567,615 to Degan et al. the entire disclosure of
which is incorporated herein by reference. In yet another preferred
embodiment, the separation medium includes reconstituted, nonporous
cellulose particles. In this embodiment, reconstituted cellulose
particles exhibit pore-free surfaces with linkage and functional
groups in the bulk of the solution free to form attachments to the
target compounds that are desired to be separated. The
reconstituted cellulose particles have no pores which open onto the
particle surface into which may be trapped a target compound to be
separated, thereby lowering yield. Further, cellulose particles can
be constructed to be of consistent, small size which provides for
increase in surface area of contact and a uniform flow in the
affinity separation apparatus.
[0031] Fusion proteins, where a target protein or a peptide is
fused with segments such as glutathion transferase or
polyhystidine, require grafted affinity ligands, glutathion, or
chelated metals, such as Nickel or Zinc, respectively. In an
embodiment of the invention, affinity separation of fusion proteins
uses ungrafted cellulose beads of the present invention, without
spacers or ligands, for separation of fusion proteins comprising a
cellulose binding protein segment fused with a target protein or
peptide. In this particular case, the cellulose surface of the
beads as a whole represent an affinity site for attaching said
cellulose binding protein segment.
[0032] The reconstituted cellulose affinity particles preferably
have a surface which is smooth and nonporous and are nearly
identical.
[0033] The reconstituted cellulose affinity particles are
preferably substantially spherical shaped. Moreover, the
reconstituted cellulose affinity particles are preferably of narrow
distribution in diameter. Such reconstituted cellulose affinity
particles generally will have the lowest probability of creating
flow problems and will allow an easy selection of membrane porosity
for fast and clean separation.
[0034] The reconstituted cellulose affinity particles may be of any
suitable size. The average diameter of the reconstituted cellulose
affinity particles will typically be less than about 120 microns,
and more usually less than about 60 microns. Preferably, the
affinity particles will have an average diameter of less than about
20 microns, and more preferably less than about 10 microns. For
many uses, the reconstituted cellulose affinity particles will
advantageously have a diameter of about 1 to about 5 microns. The
reconstituted cellulose affinity particles preferably have a narrow
diameter distribution.
[0035] The preferred particles of the present invention are of
about 1-3 microns in diameter. Such small particles have relatively
high surface area per unit volume and are easily filtered in a
static filter apparatus. The efficiency of the overall affinity
separation method of the present invention is high because the near
complete recovery of the reconstituted cellulose affinity particles
with the bound target compound is achieved.
[0036] The surface of the preferred cellulose beads has low or no
affinity for the target compound in the fluid being treated. Such a
surface ensures that the target compound will reversibly complex or
otherwise attach exclusively to the ligand on the surface of the
particles rather than the surface of the particles itself, where
other components or compounds as well might get nonspecifically
bound, thereby causing impurities to be present with the target
compound. Moreover, the surface of the substrate is preferably
smooth to minimize adherence of material other than the target
compound to the attached ligand on the surface of the reconstituted
cellulose affinity particles and to provide for easy cleaning of
the reconstituted cellulose affinity particles. The beads are
preferably capable of being reused many times and are chemically
and mechanically stable such that the beads do not decompose or
otherwise pose contamination problems.
[0037] There are potentially endless numbers of ligands for
affinity separation of a given target compound. To mention a few
generally applicable examples, antibodies of the monoclonal and
polyclonal kind, active site analogs, chelated metals, particularly
Nickel, Zinc, and Copper are used in the present art as affinity
ligands.
[0038] The filtration medium used in conjunction with the static
filtration apparatus can be any suitable filtration medium and will
typically be a porous membrane, preferably a microporous polymeric
membrane. While the filtration medium may have any suitable pore
rating, e.g., on average of about 20 microns or less, on average of
about 10 microns or less, on average of about 5 microns or less, or
even about on average of 1 micron or less, the filtration medium
will preferably allow for all, or at least substantially all, of
the fluid being treated to pass through the filtration medium while
retraining all or at least substantially all, of the reconstituted
cellulose affinity particles. Thus, the pore rating of the
filtration medium is largely dependent on the size of the
reconstituted cellulose affinity particles and the size of the
largest debris (i.e., nontarget compound and/or particulate) in the
fluid being treated.
[0039] FIG. 2 schematically depicts a preferred static filtration
apparatus for use with the present inventive affinity separation
method. The static filtration apparatus comprises a housing 20
having a feed port 21, a concentrate port 22, and a filtrate port
23. A filtration medium or membrane 25 is disposed within the
housing 20. The feed port 21 and concentrate port 22 are in fluid
communication with the upstream side of the filtration medium 25,
while the filtrate port 23 is in fluid communication with the
downstream side of the filtration medium 25. Fluid is capable of
passing through the filtration medium 25, while the reconstituted
cellulose affinity particles are substantially incapable of passing
through the filtration medium 25.
[0040] In use, a mixture of the fluid containing the target
compound and the reconstituted cellulose affinity particles are fed
into the housing 20 of the static filtration apparatus via feed
port 21 in such a manner as to produce a tangential flow.
[0041] The mixture is then filtered through filtration medium 25 by
opening filtrate port 23 to allow the fluid to be removed from
within the static filtration apparatus. Washing buffer may be
introduced into the static filtration apparatus via feed port 21 to
remove any unbound material within the static filtration apparatus,
and the washing buffer may then be allowed to pass through the
filtrate port 23. Multiple washing steps, if desire, can be carried
out in the same manner.
[0042] Eluent is then introduced, via feed port 21, to detach the
target compounds from the reconstituted cellulose affinity
particles. After the target compound has been removed from the
reconstituted cellulose affinity particles and recovered, the
reconstituted cellulose affinity particles can then be reused after
washing. The washing buffer is removed by way of port 23.
[0043] FIG. 2 is merely a schematic illustration of a preferred
embodiment of the apparatus useful in the carrying out of the
present inventive method, and particular aspects of an actual
apparatus for use with the present invention may vary considerably.
EXAMPLE 1
[0044] The present invention may be further understood with
reference to the accompanying drawings. FIG. 1 schematically
depicts the significant elements used in a preferred embodiment of
the present invention. Upon commencement of the present inventive
methods, the fluid to be treated, which contains the target
compound to be separated from the remainder of the fluid, resides
in holding tank 1, while the affinity particles reside in holding
tank 2. Both the fluid and the affinity particles are transferred
to a buffer tank 3 where they are combined to form a mixture. The
mixture is then transferred to the static filtration apparatus 4
via valve 14 and inlet 5, although the fluid and reconstituted
cellulose affinity particles could be directly transferred to the
static filtration apparatus 4 without passing through the buffer
tank 3. The mixture is maintained in the static filtration
apparatus 4 without any of the fluid passing through the filtration
medium of the static filtration apparatus 4, by, for example, the
filtrate valve 7 being in the closed position. Intermixing of the
fluid and the reconstituted cellulose affinity particles is
provided by an optional agitator 12 or by a tangential fluid in
tank 3.
[0045] Thereafter, some or all of the mixture is transferred via
concentrate valve 8 in a batch or continuous (in-line) process to a
detection tank 9, wherein the concentration of the target compound
in the fluid which remains unbound to the reconstituted cellulose
affinity particles is determined. In a permanent manufacturing
process wherein similar batches of fluid are being repeatably
treated, there may be no need for any detection means after
determining an appropriate quantity of fluid, reconstituted
cellulose affinity particles, and residence time in the static
filtration apparatus inasmuch as the present inventive method is
quite consistent and reproducible as regards the recovery of the
target compound.
[0046] When it is determined, by whatever means, that a sufficient
amount of target compound has been adsorbed onto the reconstituted
cellulose affinity particles, then the filtrate valve 7 is opened
so that the fluid passes through the filtration medium of the
static filtration apparatus 4 into waste tank 6, thereby leaving
the reconstituted cellulose affinity particles in the static
filtration apparatus 4. The filtration valve 7 is then typically
closed, although, alternatively, additional fluid can be passed
into the static filtration apparatus 4 for contacting with the
reconstituted cellulose affinity particles, particularly if the
reconstituted cellulose affinity particles are not saturated with
the target compound. Such additional fluid can be passed into the
static filtration apparatus 4 in a continuous or semi-continuous
manner while some of the fluid in the static filtration apparatus 4
continues to pass through the filtration medium of the static
filtration apparatus 4.
[0047] After the addition of fluid into the static filtration
apparatus 4 is complete and the fluid within the static filtration
apparatus 4 has passed through the filtration medium, the washing
buffer valve 11 is opened to allow for washing buffer from washing
buffer tank 10 to enter the static filtration apparatus 4. The
washing buffer is allowed to intermingle with the reconstituted
cellulose affinity particles for a suitable period of time, and
then the filtrate valve 7 is again opened to allow for the washing
buffer to pass to the waste tank 6. Typically, there will be
several such wash cycles to ensure that the fluid being treated,
except for the target compound bound to the reconstituted cellulose
affinity particles, has been removed from the static filtration
apparatus 4.
[0048] The reconstituted cellulose affinity particles are then
transferred to the elution receptacle 13 via the concentrate valve
8. Such a transfer may be accomplished by using washing buffer from
the washing buffer tank 10 to transport the reconstituted cellulose
affinity particles to elution receptacle 13. Eluent from eluent
tank 15 then contacts the affinity particles in the receptacle 13
to detach the target compound from the reconstituted cellulose
affinity particles and passes the target compound, together with
beads, back into the static filter 4 via valve 14 and inlet 5. The
effluent stream exits the static filter via valve 7 and is diverted
into tank 7. An optional effluent detection means which monitors
the level of a target compound in the effluent stream may also be
installed, similar to detection tank 9.
[0049] The reconstituted cellulose affinity particles, which no
longer have the target compound bound thereto, can consequently be
washed and then reused. After washing with buffer from tank 10,
regenerated beads are transferred back to tank 2 via concentrate
valve 8.
[0050] FIG. 1 is merely a schematic illustration of a preferred
embodiment of the present invention, and the actual equipment and
its placement may be varied considerably for the embodiments of the
present invention.
EXAMPLE 2
[0051] As shown in FIG. 3, Example 2 comprises a series of
purification loops, each of which is a self contained purification
system for a particular target, e.g., a biomaterial. The
purification loops are connected, enabling the fluid, or permeate
of the previous system to be the "feed stock" or fluid to be
treated, for the next loop. Each loop may contain affinity
particles possessing different hooks or the same hooks as the
previous purification loop in the connected configuration. The
loops begin with a feed tank 1' which may be similar to the holding
tank 1 of FIG. 1. Each loop of affinity particle may purify a
specific product from a single source of fluid to be treated which
comprises a mixture of products. For example, commonly human
immunoglobulin, human albumen, and human clofting factors are
purified from raw human plasma by separate methods. Some of these
methods in some cases damage the raw human plasma, thus preventing
additional product extraction. The current invention describes a
method which enables extraction of two or more valuable
bio-products from human plasma without damaging the biomaterials
within the used raw human plasma. FIG. 3 shows the sequential
tangential flow systems where the first system purifies biomaterial
x, the second biomaterial y, the third biomaterial z, and so on. X,
y and z may represent the same or different biomaterials to be
purified. A solution containing all biomaterials passes through
each system containing bead loops which circulate low porosity
particles having chemical hooks which specifically bind either x,
y, or z, and so on. After the solution has passed through all of
the beads loops and x, y, and z biomaterials have been removed
within each of the specific bead loops, the bead loops are
disconnected from each other and each loop system is separately
processed to yield purified biomaterials x, y, and z.
[0052] It should be appreciated that the method and system of the
present invention is capable of being incorporated in the form of a
variety of embodiments, only a few of which have been illustrated
and described above. The invention may be embodied in other forms
without departing from its spirit or essential characteristics. The
described embodiments are to be considered in all respects only as
illustrative and not restrictive and the scope of the invention is,
therefore, indicated by the appended claims rather than by the
foregoing description. All changes that come within the meaning and
range of equivalency of the claims are to be embraced within their
scope.
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