U.S. patent application number 15/067694 was filed with the patent office on 2016-07-21 for preparative chromatography column.
This patent application is currently assigned to Bio-Rad Laboratories, Inc.. The applicant listed for this patent is Bio-Rad Laboratories, Inc.. Invention is credited to Maurice Agee, Sebastien Lefebvre, Lilian Portier.
Application Number | 20160206972 15/067694 |
Document ID | / |
Family ID | 49006155 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160206972 |
Kind Code |
A1 |
Lefebvre; Sebastien ; et
al. |
July 21, 2016 |
PREPARATIVE CHROMATOGRAPHY COLUMN
Abstract
A chromatography column design and a packing device, as well as
a method for obtaining a column with a very compact and uniform bed
are provided. The bed remains compact and uniform during operation
of the column and during sanitization, storage and transportation
of the column. The amount of hardware, particularly the hardware
parts in contact with product, is minimized, especially with the
implementation of an internal liner. With this internal liner, the
packed bed can be separated from the column in intact form, or even
prepared separately from, and outside of, the column.
Inventors: |
Lefebvre; Sebastien; (Saint
Beauzire, FR) ; Portier; Lilian; (Paslieres, FR)
; Agee; Maurice; (Yssac-la-Tourette, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bio-Rad Laboratories, Inc. |
Hercules |
CA |
US |
|
|
Assignee: |
Bio-Rad Laboratories, Inc.
Hercules
CA
|
Family ID: |
49006155 |
Appl. No.: |
15/067694 |
Filed: |
March 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13771371 |
Feb 20, 2013 |
9327213 |
|
|
15067694 |
|
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61601904 |
Feb 22, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B65B 1/24 20130101; B01D
15/206 20130101; G01N 2030/565 20130101; F16L 33/00 20130101; G01N
30/56 20130101; B01D 15/22 20130101; G01N 30/603 20130101; F16L
11/00 20130101; G01N 30/6021 20130101; G01N 30/6082 20130101; Y10T
29/49865 20150115 |
International
Class: |
B01D 15/22 20060101
B01D015/22; B01D 15/20 20060101 B01D015/20 |
Claims
1. A method of packing a chromatography column, said method
comprising: (a) placing a tube of flexible, water-impermeable
material in a rigid column shell, said tube being open at a first
end and closed at a second end with a bottom plate comprising a
layer of porous material over a rigid and liquid-impermeable base,
said layer of porous material facing said first end of said tube
and said rigid and liquid-impermeable bottom plate base having a
bottom plate port therein for discharge of liquid; (b) placing a
slurry of separation medium particles in said tube within said
rigid column shell and placing a piston in said tube over said
slurry, said piston having a layer of porous material facing said
slurry and a piston port for supply of liquid over said layer of
porous material; (c) exerting a downward circulation of liquid
between the said piston and the said bottom plate to settle and
compact the said slurry; and (d) lowering said piston over said
slurry to compact said particles into a packed bed.
2. The method of claim 1, wherein the piston or the rigid column
shell further comprises a releasable seal between the tube and the
piston.
3. The method of claim 2, wherein the seal is selected from the
group consisting of an O-ring, a lobe joint, and a scraper
seal.
4. The method of claim 1, wherein the flexible, water-impermeable
material is elastic.
5. The method of claim 1, wherein the chromatography column further
comprises a slurry injection port, and wherein the placing
comprises injecting the slurry of separation medium particles
through the slurry injection port.
6. The method of claim 1, comprising assisting packing of the
slurry with percussive tapping from under the rigid and
liquid-impermeable bottom plate base.
7. The method of claim 6, wherein the percussive tapping comprises
a periodic projecting of a hammer upwards against a projection
plate positioned under the rigid and liquid-impermeable bottom
plate base.
8. The method of claim 8, wherein impulses from the percussive
tapping are dampened by one or more shock absorbers.
9. The method of claim 8, wherein the one or more shock absorbers
comprises an elastic block.
10. The method of claim 8, wherein the one or more shock absorbers
comprises a spring.
11. The method of claim 6, wherein the frequency of the percussive
tapping is between 0.1 and 300 Hz.
12. The method of claim 6, wherein the amplitude of the percussive
tapping is less than 15 mm.
13. The method of claim 1 further comprising attaching a column
shell extension to said rigid column shell to extend the height of
said rigid column shell prior to placing said tube of flexible,
water-impermeable material in said rigid column shell, and wherein:
step (a) comprises placing said tube of flexible, water-impermeable
material in both said rigid column shell and said column shell
extension; step (b) comprises placing a sufficient quantity of said
slurry within said tube to extend into both said rigid column shell
and said column shell extension; step (c) comprises compacting all
of said particles into a portion of said tube within said rigid
column shell; said method further comprising: (e) removing said
column shell extension after step (c) to leave said rigid column
shell containing said packed bed.
14. The method of claim 13, wherein step (e) further comprises
compressing the tube at a position below the column shell extension
and above the rigid column shell and the piston.
15. The method of claim 14, wherein the compressing comprises the
pressing of pins against the wall of the tube.
16. The method of claim 15, wherein the pressing comprises the
piercing of the tube.
17. The method of claim 13, comprising assisting packing of the
slurry with percussive tapping from under the rigid and
liquid-impermeable bottom plate base.
18. The method of claim 17, wherein the column shell extension and
the piston are isolated from the rigid and liquid-impermeable
bottom plate base, such that column shell extension and the piston
are subjected to a reduced force associated with the percussive
tapping.
19. The method of claim 13 further comprising closing the top of
said column shell extension with a cap that supports said piston,
and for securing said first end of said tube of flexible,
water-impermeable material to said cap, prior to step (c).
20. The method of claim 13 further comprising: (f) severing said
tube of flexible, water-impermeable material above said packed bed
to leave a shortened length of said tube encircling said packed bed
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of application Ser. No.
13/771,371, filed Feb. 20, 2013, which claims the benefit of U.S.
Provisional Patent Application No. 61/601,904, filed Feb. 22, 2012,
the contents of which are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to preparative chromatography
columns, i.e., chromatography columns in which molecular species
are extracted from source solutions in high amounts for commercial
use rather than for analytical purposes.
[0004] 2. Description of the Prior Art
[0005] The chromatography columns to which the present invention is
primarily directed are preparative chromatography columns designed
for plug flow of a mobile phase through a packed bed of solid or
semi-solid stationary phase made from soft (semi-solid) or rigid
(solid) particles. The typical such column is a cylinder closed at
each end by plates, each plate equipped with a fluid port, a
distribution system, and a filter. The cylinder is uniformly filled
with a separation medium or media, packed to form a "bed." The
filters have pore sizes that are smaller than the particle size of
the media to retain the media within the cylinder, and yet large
enough to allow the process liquids (i.e., the mobile phase) to
pass through the filters and hence the column. The distribution
system ensures that the process liquid is spread across the full
width of the bed, thereby making maximal use of the bed.
[0006] The typical preparative chromatography column is large
enough in diameter that separation within the column can be
performed at a commercially useful throughput rate, i.e., one that
will produce the extracted species at an economically viable
production rate. The typical column is also small enough in depth
that the pressure drop through the column is low, thereby avoiding
the need for a high pump pressure to force the mobile phase through
the column. There must be sufficient depth however to provide the
mobile phase with a residence time that is long enough to allow
proper interaction between the mobile and stationary phases. The
typical column also contains a plunger or piston head that is
lowered to contact and compress the stationary phase to a desired
height. In operation, the mobile phase enters the column at the top
through the plunger which includes a distributor plate to spread
the mobile phase across the full width of the bed, thereby making
maximal use of the bed.
[0007] The performance of a preparative chromatography column of
the type described above is very sensitive to the degree of
uniformity of the stationary phase. Optimal operation is achieved
when the bed is homogeneous, compact, and of uniform depth such
that the mobile phase is evenly distributed across the width of the
bed. In the present state of art, the need for a stationary phase
that is adjustable yet stable during purification, sanitization,
storage and transportation of the column has led to columns with
adjustable mechanical assemblies. Sealing between parts of the
column, especially between the plunger and the column tube or
between the bottom plate and the tube, requires tight adjustment of
the parts, close alignment of the parts, and a smooth interior
surface of the column tube. In addition, particularly in columns
that are used for purifying food or drugs, the parts of the column
that are in contact with the product, including the tube itself,
the plates, and the distributors, must be made of special materials
that are stable and inert, i.e., that neither leach into the
product nor corrode upon contact with the product. As a
consequence, the number of column parts, the volume of the column,
the materials from which the parts are made, and the dimensional
precision of the parts, make the column expensive and thus not
suitable for single-use applications. The use of other forms of
media, such as membranes or monoliths, could be simpler and less
expensive but often fail to provide the same degree of
purification.
[0008] The transportation of packed columns on long distances, for
instance with the pre-packed or disposable columns applications, is
also a challenge for maintaining the performance of the column. All
the packing methods which only use downwards strengths, such as
flow circulation and axial compression for column packing, succeed
to settle the media particles in some kind of equilibrium after a
short or long period, where each particle finds support on the
lower particles. But this arrangement might not be optimal:
excessive voids can still take place due to the friction of the
tube walls, or due to the difference in size and shape of
particles. The random stacking of these particles is also not
optimal: the vibration, shocks and tilting and thermal variations
during the transportation often induce local re-arrangements of the
particles resulting with further compression of the bed, forming of
supernatant, initiation of bed cracking. This prevents the
transportation of packed columns on long distances, especially when
non compressible chromatography media is used, where the bed is not
maintained under compression.
SUMMARY OF THE INVENTION
[0009] It has now been discovered that the problems cited above can
be addressed and at least partially mitigated by a specific design
of the column, a special packing device and a specific packing
method. This design can be significantly simplified and reduced in
cost in some embodiments by the use of a liner inside the
chromatography column. The liner can be, for example, a tubular and
flexible water-impermeable film that will contain the separation
media and allow movement of the piston within the liner. This
configuration allows the media in the form of either a slurry or
dry particles to be packed with the packing method described
hereafter into a compact and uniform bed, and keeps the bed compact
and uniform during operation of the column and during
transportation. Use of the liner also promotes and enhances
sanitization, storage and transportation of the column, and uses a
minimal amount of hardware, particularly the hardware parts that
will be in contact with the product. Use of the liner also allows
the packed bed to be separated from the column. In some
embodiments, the liner is transparent, allowing a user to readily
see the contents within the liner. The rigid external shell
containing the liner can in different embodiments be transparent or
not.
[0010] Further advantages include the ability to separate
contaminated waste from non contaminated waste, in the case of
disposable columns, and the better stability of the packed bed over
a large number of cycles, thanks to the packing method
described
[0011] Also provided is a packing method and apparatus able to
obtain very high performance and reproducible packing, in terms of
bed height and particles arrangement. This objective is obtained by
the conjunction of a precise dosing of the media and the packing
method combining flow packing and rhythmic (percussive) packing.
While this aspect can be used with a column liner as described
above, the inclusion of a liner with the packing method is
optional. In some embodiments in which the percussive packing is
used, the chromatography resin (i.e., the separation medium
material) is rigid or semi-rigid, e.g., such as a ceramic apatite
(including but not limited to hydroxyapatite and fluoroapatite) or
silica.
[0012] Also provided herein is a new method of applying and
tightening a filter in the bore hole of a piston or a bore hole in
the bottom plate of the column. The method involves heating the
piston and/or column such that the area for the filter thermally
expands. Eventually the filter can be cooled to shrink its outer
diameter. The filter is then added and the piston and/or column is
allowed to cool to ambient temperature, while the filter warms up
to ambient temperature thereby tightening the area tightly around
the filter, thereby affixing the filter to cover the bore hole.
[0013] In some embodiments, a chromatography column is provided
comprising: a rigid column shell, a bottom plate secured to the
column shell and comprising a layer of porous material over a rigid
and liquid-impermeable base and the base has a port therein for
passage of liquid, a piston comprising a layer of porous material
over a rigid and liquid-impermeable base and the base having a port
therein for passage of liquid, the piston fitting within a tube
within the column shell, and the tube being of flexible,
water-impermeable material containing a packed bed of separation
medium, the tube being open at a first end, closed at a second end
with the bottom plate, and encircling the piston.
[0014] In some embodiments, the column further comprises a
releasable seal between the tube to the piston. In some
embodiments, the piston comprises the seal. In some embodiments,
the rigid column shell comprises the seal. In some embodiments, the
seal is selected from the group consisting of an O-ring, a lobe
joint, and a scraper seal.
[0015] In some embodiments, the piston fits within the column shell
with clearance between the piston and the column shell to allow
liquid to flow past the piston and thereby equalize pressure above
and below the piston when the piston is moved within the column
shell.
[0016] In some embodiments, the flexible, water-impermeable
material is elastic.
[0017] In some embodiments, the layer of porous material on the
bottom plate is planar and the base has a concave upper surface
tapering toward the port in the base, whereby the layer of porous
material contacts the base along a periphery of the base while
leaving a gap between the layer and the base inside the periphery.
In some embodiments, the layer of porous material on the piston is
planar and the piston has a concave surface facing the layer of
porous material and tapering toward the port in the piston, whereby
the layer of porous material contacts the concave surface along a
periphery of the concave surface while leaving a gap between the
layer and the concave surface inside the periphery.
[0018] In some embodiments, the layer of porous material on the
bottom plate is planar and the base has a concave upper surface
tapering toward the port in the base, whereby the layer of porous
material contacts the base along a periphery of the base while
leaving a gap between the layer and the base inside the periphery,
and the layer of porous material on the piston is planar and the
piston has a concave surface facing the layer of porous material
and tapering toward the port in the piston, whereby the layer of
porous material of the piston contacts the concave surface of the
piston along a periphery of the concave surface while leaving a gap
between the layer and the concave surface inside the periphery.
[0019] In some embodiments, the column further comprises a column
shell extension removably attachable to the column shell to extend
the height of the column shell, and wherein the tube of flexible,
water-impermeable material is of sufficient length to extend
through both the column shell and the column shell extension, the
column shell extension being of sufficient width to receive the
piston with clearance between the piston and the extension to allow
liquid to flow past the piston and thereby equalize pressure above
and below the piston when the tube of flexible, water-impermeable
material is within both the column shell and the column shell
extension and the piston is moved within the tube. In some
embodiments, the column further comprises a cap that attaches to
the column shell extension and that supports the piston, and means
for securing the first end of the tube of flexible,
water-impermeable material to the cap. In some embodiments, the
column further comprises a slurry injection port allowing for
injection of a slurry of chromatography media under the piston in
the column.
[0020] In some embodiments, the column further comprises under the
base, a percussion table capable of rhythmically tapping the base
to improve packing of particles of a separation medium in the
column.
[0021] In some embodiments, the chromatography column is a
preparative chromatography column.
[0022] Also provided are methods of packing a chromatography
column. In some embodiments, the method comprises: (a) placing a
tube of flexible, water-impermeable material in a rigid column
shell, the tube being open at a first end and closed at a second
end with a bottom plate comprising a layer of porous material over
a rigid and liquid-impermeable base, the layer of porous material
facing the first end of the tube and the base having a port therein
for discharge of liquid; (b) placing a slurry of separation medium
particles in the tube within the column shell and placing a piston
in the tube over the slurry, the piston having a layer of porous
material facing the slurry and a port for supply of liquid over the
layer of porous material; (c) exerting a downward circulation of
liquid between the piston and the bottom plate to settle and
compact the slurry; and (d) lowering the piston over the slurry to
compact the particles into a packed bed.
[0023] In some embodiments, the method further comprises assisting
packing of the slurry with percussive tapping from under the base.
In some embodiments, the percussive tapping occurs at a frequency
from 0.2 to 100 Hz with an amplitude less than 5 mm.
[0024] In some embodiments, the method further comprises attaching
a column shell extension to the column shell to extend the height
of the column shell prior to placing the tube of flexible,
water-impermeable material in the column shell, and wherein: step
(a) comprises placing the tube of flexible, water-impermeable
material in both the column shell and the column shell extension;
step (b) comprises placing a sufficient quantity of the slurry
within the tube to extend into both the column shell and the column
shell extension; step (c) comprises compacting all of the particles
into a portion of the tube within the column shell; the method
further comprising: (d) removing the column shell extension after
step (c) to leave the column shell containing the packed bed.
[0025] In some embodiments, the method further comprises closing
the top of the column shell extension with a cap that supports the
piston, and for securing the first end of the tube of flexible,
water-impermeable material to the cap, prior to step (c).
[0026] In some embodiments, the method further comprises: (e)
severing the tube of flexible, water-impermeable material above the
packed bed to leave a shortened length of the tube encircling the
packed bed.
[0027] In some embodiments, the layer of porous material on the
bottom plate is planar and the base has a concave upper surface
tapering toward the port in the base, whereby the layer of porous
material contacts the base along a periphery of the base while
leaving a gap between the layer and the base inside the
periphery.
[0028] In some embodiments, the layer of porous material on the
piston is planar and the piston has a concave surface facing the
layer of porous material and tapering toward the port in the
piston, whereby the layer of porous material contacts the concave
surface along a periphery of the concave surface while leaving a
gap between the layer and the concave surface inside the
periphery.
[0029] Also provided is a tube of flexible, water-impermeable
material open at a first end and closed at a second end with a
bottom plate comprising a layer of porous material over a rigid and
liquid-impermeable base, the layer of porous material facing the
first end of the tube and the base having a port therein for
passage of liquid. In some embodiments, the flexible,
water-impermeable material is elastic. In some embodiments, the
layer of porous material is planar and the base has a concave upper
surface tapering toward the port, whereby the layer contacts the
base along a periphery of the base while leaving a gap between the
layer and the base inside the periphery.
[0030] Also provided is a chromatography column (including or
optionally lacking a liner). In some embodiments, the column
comprises: a rigid water-impermeable column shell containing a
packed bed of separation medium, the shell forming a tube being
open at a first end, closed at a second end with a bottom plate,
and encircling a piston, the shell or piston comprising a port for
supply of liquid; the bottom plate secured to the column shell and
comprising a layer of porous material over a rigid and
liquid-impermeable base and the base having a port therein for
passage of liquid, and the piston fitting within the shell and
comprising a seal between the piston and the shell.
[0031] In some embodiments, the seal is selected from the group
consisting of an O-ring, a lobe joint, and a scraper seal. In some
embodiments, the piston comprises the seal. In some embodiments,
the rigid column shell comprises the seal.
[0032] In some embodiments, the layer of porous material on the
bottom plate is planar and the base has a concave upper surface
tapering toward the port in the base, whereby the layer of porous
material contacts the base along a periphery of the base while
leaving a gap between the layer and the base inside the
periphery.
[0033] In some embodiments, the layer of porous material on the
piston is planar and the piston has a concave surface facing the
layer of porous material and tapering toward the port in the
piston, whereby the layer of porous material contacts the concave
surface along a periphery of the concave surface while leaving a
gap between the layer and the concave surface inside the
periphery.
[0034] In some embodiments, the layer of porous material on the
bottom plate is planar and the base has a concave upper surface
tapering toward the port in the base, whereby the layer of porous
material contacts the base along a periphery of the base while
leaving a gap between the layer and the base inside the periphery,
and the layer of porous material on the piston is planar and the
piston has a concave surface facing the layer of porous material
and tapering toward the port in the piston, whereby the layer of
porous material of the piston contacts the concave surface of the
piston along a periphery of the concave surface while leaving a gap
between the layer and the concave surface inside the periphery.
[0035] In some embodiments, the column further comprises a column
water-impermeable shell extension attachable to the column shell to
water-impermeably extend the height of the column shell, and
wherein the shell extension being of sufficient width to receive
the piston with clearance between the piston and the extension to
allow liquid to flow past the piston and thereby equalize pressure
above and below the piston. In some embodiments, the column further
comprises a cap that attaches to the column shell extension and
that supports the piston. In some embodiments, the column further
comprising a slurry injection port which can be used to inject a
slurry of chromatography media under the piston in the column.
[0036] In some embodiments, the chromatography column is a
preparative chromatography column.
[0037] Also provided is a method of packing a preparative
chromatography column as described above. In some embodiments, the
method comprises: (a) placing a slurry of separation medium
particles in the column shell closed on bottom end with the bottom
plate and placing a piston in the column shell over the slurry, (b)
closing the top of the column shell extension with a cap that
supports the piston; (c) exerting a downward circulation of liquid
between the piston and the bottom plate to settle and compact the
slurry; (d) lowering the piston over the slurry to compact the
particles into a packed bed; and (e) removing the column shell
extension after step (d) to leave the column shell containing the
packed bed. In some embodiments, the column further comprises under
the base, a percussion table capable of rhythmically tapping the
base to improve packing of particles of a separation medium in the
column.
[0038] In some embodiments, the rigid column shell and the tube of
flexible, water-impermeable material are sufficiently high to
contain the media in suspension (slurry), further comprising a
piston with a gasket or protuberance to seal the tube to the piston
all along the tube.
[0039] Also provided are methods of attaching a filter to a bore
hole in a bottom plate of a chromatography column and/or a bore
hole in a piston for packing a chromatography column. In some
embodiments, the method comprises: heating the bottom plate and/or
piston such that an opening comprising the bore hole expands;
placing a filter into the heated opening; and allowing the bottom
plate and/or piston to cool, thereby contracting the opening such
that the filter is fixed in the opening.
[0040] In some embodiments, the filter placed into the heated
opening is cooled below ambient temperature such that the filter
expands upon returning to ambient temperature.
[0041] In some embodiments, the method further comprises fastening
the filter to the bottom plate and/or piston at least one position
on the filter aside from the filter edge. In some embodiments, the
fastening comprises adding one or more screw through the filter and
into the bottom plate and/or piston.
[0042] Further features, aspects, objects, and advantages of the
invention will be apparent from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 is a cross section of a tubular liner attached to a
bottom plate. This and all other figures herein are within the
scope of the present invention.
[0044] FIG. 2 is a cross section of the tubular liner and bottom
plate of FIG. 1 positioned inside a column tube.
[0045] FIG. 3 is a cross section of the components of FIG. 2 shown
with a separation medium being placed inside the liner.
[0046] FIG. 4 is the same view as FIG. 3 after addition of the
separation medium.
[0047] FIG. 5 is the same view as FIG. 4 with a piston added to
compact the separation medium.
[0048] FIG. 6 is the same view as FIG. 5, showing the direction of
movement of the piston.
[0049] FIG. 7 is the same view as FIGS. 5 and 6 after the bed of
separation medium has been fully packed.
[0050] FIG. 8 is the same view as FIG. 7 after the piston is
lowered against the bed.
[0051] FIG. 9 is the same view as FIG. 8, showing the line sealed
over the piston.
[0052] FIG. 10 is the same view as the preceding Figures, showing
removal of certain parts of the structure following the sealing of
the liner.
[0053] FIG. 11 is the same view as the preceding Figures, showing a
subsequent stage in which a cap is placed over the top of the
column.
[0054] FIG. 12 is a cross section of a cartridge incorporating the
separation medium, the liner, and an outer tube.
[0055] FIG. 13 is a cross section view of the dismantling of the
column of the preceding Figures.
[0056] FIG. 14 is a cross section view of an arrangement of stacked
columns, each column of the stack being of the construction of
those in the preceding Figures.
[0057] FIG. 15 is a schematic of the installation needed for the
packing of the column.
[0058] FIG. 16 is the same view as FIG. 5, showing the percussion
table.
[0059] FIG. 17 is the same view as FIG. 16, where the tube
extension is supported by a separate frame.
[0060] FIG. 18 is the same view as FIG. 17, with different solution
for the holding of liner, and with a slurry injection valve.
[0061] FIG. 19 shows an alternative solution for the design of the
column and packing system where the external clamp is replaced by
an O-ring on the piston.
[0062] FIG. 20 is the same view as FIG. 19, after the piston is
lowered against the bed
[0063] FIG. 21 is the same view as the FIG. 27, showing a
subsequent stage in which a cap is placed over the top of the
column.
[0064] FIG. 22 shows a cross section of a column with tubular liner
sealed via a gasket with the piston all along the column tube
[0065] FIG. 23 shows the same view as FIG. 22, but an embodiment in
which the piston and bottom directly seal against the tubular
liner.
[0066] FIG. 24 depicts positioning of the filter in an opening
comprising the bore of the piston.
DETAILED DESCRIPTION OF SELECTED EMBODIMENTS
[0067] FIGS. 1 to 11 depict one illustrative embodiment of the
invention and show the stepwise transformation of the column from
an empty condition to a packed column. These pictures focus on the
column design, without any details on the packing apparatus. The
succession of figures illustrates the different parts of the
column, methods of assembly and disassembly, and the advantages of
the invention. FIGS. 12 through 14 show variations on the
embodiment of FIGS. 1 to 11. FIGS. 15 to 18 focus on the packing
apparatus: FIG. 15 showing the principle of the installation and
FIGS. 16 to 18 showing variations of the design related to the
packing method. FIGS. 19 to 20 depict one alternative embodiment of
the embodiment of FIGS. 5 to 11 and show the stepwise
transformation for obtaining a packed column. It is understood
however that the invention as a whole is not restricted to the
constructions shown in these Figures or the description below of
their use.
[0068] FIG. 1 depicts a tubular and flexible water-impermeable film
101 of a plastic material such as low-density polyethylene or any
polymer of similar physical characteristics. The tubular film is
open at one end (i.e., the top end in the view shown in the Figure)
and closed at the other end by a bottom plate 102, in this case by
the tubular film being placed around the outer edge of a bottom
plate. The bottom plate 102 contains rigid and liquid-impermeable
base 102a which serves as a mobile phase distributor, with a layer
of porous material 102b on the upper side of the distributor. A
bottom process port 103 extends from the lower side of the
distributor, for discharge of liquid from the column. In the
embodiment shown in FIG. 1, the bottom plate 102 is surrounded with
a ring 104 to secure the film 101 around the bottom plate in a
fluid-tight manner. Tightening of the ring 104 can be achieved by
using a ring of elastomeric material, or by a clamp, a collar, a
wire, or any other device or means that will form a hermetic seal
between the bottom plate and the plastic film. The seal can also be
formed by an adhesive or by welding. Another solution is also
further described with FIG. 19. In this embodiment, the layer of
porous material 102b is planar and the base 102a has a concave
upper surface tapering toward the bottom port 103, leaving a gap
between the porous layer and the base for collection of the liquid
and avoidance of dead volume.
[0069] The inner diameter of the tube 101, without the securing
ring 104, can be equal to or slightly larger than the bottom plate
102, or smaller but sufficiently elastic so as to expand under
further hydraulic pressure. The length of the tube 101 is great
enough that the interior volume of the tube will contain the slurry
volume that will be used in forming a bed of a desired height.
[0070] FIG. 2 depicts an assembly containing the following
components:
[0071] Column Tube 105.
[0072] The column tube, also referred to herein as a column shell
is rigid and supports the pressure inside the column. The diameter
of the column tube 105 is identical to the diameter of the tubular
plastic film 101 which is thereby fully supported by the column
tube 105 and serves as a liner for the tube. If the tubular plastic
film 101 is elastic and can expand, the column tube 105 can be
slightly larger in diameter than the tubular film 101 to allow the
tubular film to expand under internal pressure until the film 101
is in full contact with the tube 105. Such an expansion will ensure
that the film 101 remains free of folds as it is being packed with
media.
[0073] Tube Extension 106.
[0074] This extension, which is also referred to herein as a column
shell extension, is mounted above and aligned with the column tube
105 and supports the pressure inside the column during the packing
operation. Unlike the column tube 105, the tube extension 106 can
be disconnected after the media packing is completed, as explained
below and illustrated in a subsequent Figure. The diameter of the
tube extension 106 can be equal to or slightly larger than that of
the tubular plastic film 101 to offer the same benefits as those of
the column tube 105.
[0075] Upper Tie 107.
[0076] At the junction between the tube extension 106 and the
column tube 105 is a clearance for an upper tie 107, which can for
example be a clamp, a collar, a wire, or any such device that can
releasably secure the tubular film to a piston (explained below and
depicted in a subsequent Figure). When engaged, the upper tie 107
has an inner diameter approximately equal to that of the tube
extension 106, and provides a smooth continuation of the inner
cylindrical surfaces of the tube extension 106 and the column tube
105. The tie 107 likewise surrounds the tubular plastic film 101
and supports the pressure inside the column during the packing
operation. This upper tie 107 can be tightened by an external tool
through an aperture between the tube extension 106 and the column
tube 101. An alternative to such an aperture is a groove in the
column tube 105, with an open slot for access of the tightening
tool. A welding machine that welds the plastic film to the piston
can be used in place of the tie 107.
[0077] Fixing Clamp 108.
[0078] This clamp mechanically secures the tube extension 106 to
the column tube 105 in a coaxial orientation with full alignment.
The fixing clamp 108 also supports the pressure inside the column
during the packing operation. The fixing clamp 108 in this
embodiment is a nut that engages a shoulder 109 on the tube
extension and is internally threaded to engage a threaded surface
110 on the column tube 105. As the fixing clamp 108 is being
screwed to column tube 105, the fixing clamp 108 tightens the
shoulder 109 of the tube extension 106 against the column tube 105.
In the example shown in FIG. 2, the shoulder 109 contains a slot
111 on one right side to allow access to the upper tie 107. Any
device that serves the function of a fixing clamp can be used,
including for example a tri-clamp, a bolted flange, and tie rods.
When packing is performed by an automated instrument, the tube
extension 106 and column tube 105 can be held together by the
components of the machine while leaving access to the upper tie 107
for the tightening tool.
[0079] Bed Height Adjustment 112.
[0080] Optionally, the bottom plate 102 can be adjusted in height
by a bed height adjustment system 112. This element allows an
operator to adjust the bed height without changing the height of
the column tube 105. In the embodiment shown, the bed height
adjustment system 112 is represented as a stack of spacers, setting
a controlled distance between the bottom plate 102 and a column
support plate 113 at the bottom of the column. In the embodiment
shown, the column support plate 113 is fixed to the column tube 105
by a three-part fixing clamp 114 similar to the fixing clamp 108
joining the tube extension 106 to the column tube 105. As in the
fixing clamp 108, alternative means include a tri-clamp, a bolted
flange, and tie rods. Still others will be apparent to those of
skill in the art.
[0081] FIG. 3 shows the start of the procedure for forming a packed
bed in the column of the preceding Figures. A media slurry 121,
i.e., a slurry of particles of the separation medium, is poured
into the tubular plastic film 101 which is supported by the tube
extension 106 attached to the column tube 105. The bottom process
port 103 is closed at this stage, for example by a valve (not
shown) on the column. The amount of media slurry that is poured is
precisely selected such that when the slurry is packed down, the
upper surface of the resulting packed bed will be just below the
upper tie 107. Although shown as a suspension, the media can also
be in the form of dry particles.
[0082] FIG. 4 shows a stage in which the media once poured has been
allowed to settle for a period of time sufficient to leave a layer
of clear supernatant 122 above the settled slurry 123. The starting
amount of slurry should be such that enough space 124 is left in
the tube extension 106 to accommodate the piston in the succeeding
step.
[0083] FIG. 5 depicts the column with a top assembly or cap 131
attached. The top assembly 131 includes a piston 132, a filter, and
a top process port 133. These are similar to the corresponding
features of the bottom plate 102, including a mobile phase
distribution system within the piston. A piston shaft 134 supports
the piston 132 and allows the piston to be lowered along the axis
of the column tube 105. In the embodiment shown, the piston shaft
134 is a tube concentric with the column tube 105 and tube
extension 106. An O-ring 135 between the piston shaft 134 and the
piston 132 provides a water-tight seal and holds the piston 132 to
the piston shaft 134 by friction, but a mechanical coupling can
also be added if friction is not sufficient to hold the piston 132
under the piston shaft 134. The piston 132 fits inside the column
tube and column tube extension as well as the tubular film in a
manner permitting lowering of the piston within these parts.
[0084] The top assembly 131 holds the pressure inside the column
during the packing operation. The top assembly 131 has a bore with
an O-ring, scraper seal or other sealing means to allow the piston
shaft 134 to slide in the bore along the column axis while ensuring
a water-tight seal between the top assembly 131 and the piston
shaft 134. The top assembly 131 also includes seal that
hermetically seals top assembly 131 to the tubular plastic film
101. In the shown embodiment, the top assembly 131 is constructed
in two parts: a bottom part 136 and a top part 137. The top part
137 has an outer thread that engages an internal thread in the bore
in the center of the bottom part 136. Thus, by rotating the bottom
part 136 relative to the top part 137, the distance between the two
parts can be varied. On their outer edges, the bottom part 136 and
top part 137 have two opposing chamfers which accommodate an O-ring
138. When the bottom part 136 and top part 137 approach each other,
the opposing chamfers do likewise and push the O-ring 138 radially
against the tubular plastic film 101 which then tightens against
the bore of the tube extension 106. The top assembly 131 can be
tightened over the tubular plastic film 101 to ensure that the
pressure inside the column can be held. If the film 101 is
sufficiently elastic, an alternative solution to assembly 131 is
possible as further depicted on FIG. 18.
[0085] The top assembly 131 is secured to the tube extension 106 in
the following manner. Once the slurry has settled, a layer of
supernatant 122 remains, and the piston is immersed in the
supernatant. If the slurry has not been allowed to settle, the
piston must be placed in the empty space 124 above the slurry.
Media should not travel to the region above the piston during this
process. Once the piston 132 is in place, the top assembly 131 is
tightened against the tubular plastic film 101. With the top
assembly 131 secured, the inner side of the tubular plastic film is
sealed against the bottom plate 102 and also against the top
assembly 131. At this stage the piston 132 is not tightly secured
to the tubular plastic film 101, and liquid can freely move between
the region below the piston and the region above the piston. This
avoids the need for a dynamic seal, i.e., a seal between a moving
part as the piston 132 and a static part as the liner 101. Shear
stress on the plastic film 101 is thus avoided, as is the need for
an expensive column tube with a calibrated and smooth bore. A
further benefit is that neither the tube extension 106 nor the
column tube 105 are required to be made of food-grade or drug-grade
material. This reduces the cost of the column even further.
[0086] FIG. 6 illustrates the start of the packing operation. As
further shown on FIGS. 15 to 18, the optimal packing performance is
obtained with a combination of percussion and flow packing. The
percussion system is not shown on FIGS. 6 and 7 which only
represents the "flow packing," which consists of circulating the
mobile phase, i.e., the packing buffer, between the top process
port 133 and the bottom process port 103 under controlled flow
speed, controlled pressure, or both.
[0087] During packing operation, as further described with FIGS. 15
to 18, the slurry bed begins to consolidate. This creates flow
resistance and thus increases both the pressure drop across the bed
and the hydraulic pressure inside the column. Since the piston 132
is not tight against the column tube 105, the pressure under the
piston and the pressure above the piston equilibrate to each other,
and as the pressure drops across the bed increases, some liquid
below the piston will move above the piston until the pressure
equalizes. Since the top chamber is sealed on the upper side,
however, most of the mobile phase circulates through the packed bed
to reach the bottom process port where it can escape the
column.
[0088] The upper chamber, i.e., the region 141 above the piston
132, serves as an air trap. If air is introduced into the column
with the mobile phase, the bubbles move to the outer side of the
piston where they can rise within the upper chamber through the
clearance between the piston 132 and the tubular film 101. This
leaves the bed free of air as the bed is being formed, a condition
that is of value towards obtaining a homogeneous and compact bed as
needed for the purification.
[0089] FIG. 7 depicts a consolidated (packed) bed 142. When the bed
is consolidated, the pressure drop across the bed is stabilized.
Since the pressure is now constant in the column and is the same
both above and below the column, the flow rate of the mobile phase
exiting the column is equal to the flow rate of the mobile phase
entering the column through the piston.
[0090] FIG. 8 depicts removal of the supernatant. Once the bed 142
is fully consolidated, the piston 132 is moved down in the
supernatant until the piston 132 meets the bed 142. As noted above,
the quantity of media originally introduced in the column is
selected to cause the height where the piston 132 meets the
consolidated bed 142 to be level with the position where tubular
film 101 can be tightened against the piston 132 by the upper tie
107.
[0091] As the piston 132 is being lowered within the column, the
internal volume between the column tube 105, the tube extension
106, the top assembly 131 and the bottom plate 102 is reduced by
the volume consumed by the length of the piston shaft 134 that is
inside the column. Since liquid is almost incompressible, the top
process port 133, bottom process port 103, or both should be left
open so that the volume consumed by the piston shaft 134 is
compensated by an equal volume of mobile phase leaving the column
by either port.
[0092] FIGS. 9 and 10 depict the sealing of the piston 132. The
upper tie 107 is first tightened to form a hermetic seal between
the tubular plastic film 101 and the piston 132, as shown in FIG.
9. Once the seal is formed, the top assembly 131, the tube
extension 106, and the supernatant 122 above the piston 132 can be
removed to achieve the condition shown in FIG. 10. Removal of the
top assembly 131, the tube extension 106, and the supernatant 122
can be achieved by first disengaging the piston shaft 134 from the
piston 132, then emptying the supernatant 122 through an aperture
inside the piston shaft. The seal between the top assembly 131 and
the tubular plastic film 101 is then broken. The top assembly 131,
piston shaft 134 and the tube extension 106 are then removed.
[0093] If the separation medium has been compressed in the column
during packing, as is typical with soft or semi-rigid media, the
medium usually exerts a force against the piston, the magnitude of
the force being related to the Young's modulus of the medium. If
the tubular plastic film 101 cannot hold this strength on its own
and tends to expand and reduce the density or uniformity of the
bed, an external device can be used to maintain the position of the
piston. The tool used to tighten the upper tie 107 is an example of
one such device.
[0094] If desired, the section of tubular plastic film extending
above the piston can be cut and removed, since it serves no
function in the further operation of the column, other than to
preserve the option of resuspending the medium in the tubular
plastic film at a later point in time. Resuspension can be
accomplished by remounting the top assembly 131 and detaching the
upper tie 107, followed by proceeding with an inverted sequence
from FIG. 9 back toward FIG. 1.
[0095] FIG. 11 depicts the configuration of the column for
purification. A cap 131 is secured to the top of the column tube
105. The cap 131 serves to hold the hydraulic pressure in the
column during the use of the column, i.e., during sanitization,
purification, etc. The cap 131 also maintains the mechanical
pressure of the bed 142 against the piston 132, as described above.
Still further, the cap 131 can be used to further compress the bed
by pushing down the piston, while either the bottom process port
103 or the top process port 133 is left open to allow removal of an
equal volume of mobile phase.
[0096] The configuration of the packed column as shown in FIG. 11
offers multiple advantages:
[0097] (1) The overall size of the column is minimal, since the
tube extension 106 used in the column preparation has been
removed.
[0098] (2) The number of parts in contact with product is minimal:
they include only the piston 132, the bottom plate 102, the process
ports 103, 133, and the inner surface of the tubular plastic film
101. Since food-grade and drug-grade materials are expensive,
minimizing their use lowers the cost of the column. This also
allows the column to be used as a "disposable" column.
[0099] (3) Most of the parts of the column, i.e., the column tube
105, the support plate 113, the tube extension 106, and the top
assembly 131, are for mechanical support only. There is no
requirement that they be constructed of materials compatible with
drugs or food, or materials compatible with the process solutions.
Since they do not come in contact with the product, they can be
recycled for other applications without risk of cross
contamination.
[0100] (4) In preparative chromatography columns in which the
piston is smaller than the column tube, the mobile phase
distribution system often fails to extend the mobile phase to the
outer parts of the column. Poor irrigation of these outer parts can
raise sanitation concerns. With the present invention, the tubular
plastic film 101 is tight around the piston 132, and the column
section inside the tubular plastic film under the piston is very
close to the piston itself, thereby mitigating concerns regarding
distribution and sanitation.
[0101] (5) The column can withstand high hydraulic pressures, and
its ability to do so is limited only by the dimensions of its
mechanical components. The tubular plastic film is supported in
every direction and serves as a liner which requires no mechanical
resistance, except at the sections where the plastic film 101 is
tightened against the bottom plate 102 or the piston 132. Sections
of the tubular plastic film that are not supported may expand in
response to high pressure inside the column, leading to a risk of
rupture, but this risk can be mitigated by filling the space
between the cap top assembly 131 and the piston 132 and the space
between the bottom plate 102 and the support plate 113 with an
external liquid. This external liquid will not be in contact with
column interior, and yet it will hold the film where it is not
supported with mechanical parts.
[0102] (6) The tubular plastic film 101 can be thin and
transparent, which facilitates temperature sensing and pressure
sensing, as well as the detection of optical, ultrasonic or other
signals through the film. The film thus avoids the need for costly
instruments designed for food and drug contact and the need for
materials that are compatible with the process solutions. The film
also eliminates concerns of cross-contamination between runs.
Instrumentation for sensing and detection can be placed in direct
contact with the film, for example by inserting such
instrumentation in the column tube, or placed in contact with
external fluid as described above.
[0103] (7) The use of a thin, transparent tubular plastic film can
also be beneficial for heat exchange, whether by electrical
resistance heating on the column tube or by circulation of liquid
outside the tubular plastic film. Fine grooves can be formed in the
column tube 105 to provide channels in which cooling or heating
liquid can circulate without direct contact with the process liquid
inside the column.
[0104] (8) The column tube can be made of an opaque material, such
as plastic, steel or stainless steel. A slot can be included in the
column wall along the length of the column to allow observation of
the bed. The optimal slot is one that is wide enough for
observation through it but narrow enough to avoid protrusion of the
tubular plastic film through the slot under hydraulic pressure. The
slot can thus serve as a low-cost sight glass.
[0105] (9) A tubular plastic film with high mechanical resistance,
such as one made of plastic film coextruded with a textile
structure, can be used without the top assembly 131 and the support
plate 113 or even without the column tube 105.
[0106] (10) The features described above are readily adapted in
preparing a chromatography cartridge as depicted in FIG. 12. This
cartridge contains a piston 132, bottom plate 102, a top process
port 133, a bottom process port 103, ties (such as rings 104, 107),
the tubular plastic film 101, a low-cost outer tube 168, and a
clamp 169 for maintaining the desired distance between the piston
132 and the bottom plate 102. The low cost tube 168 fits inside the
wider column tube 105 depicted in the preceding Figures.
[0107] Dismantling of the column of FIGS. 1 through 11 is depicted
in FIG. 13. The top assembly 131 is opened and parts including the
bottom plate 102, the tubular plastic film 101, the lower and upper
ties 104, 107, and the piston 132 are lifted from the column tube
105. As an alternative, the upper tie 107 can be loosened and the
piston 132 removed, to permit removal of the packed media from
above, and the bottom clamp 114 then dismounted to allow the bottom
plate 102 and tubular plastic film 101 to be removed. The column
tube 105, bottom clamp 114, bed height adjustment system 112, top
assembly 131, bottom clamp 114, and other parts can then be
re-used.
[0108] FIG. 15 presents the principle of a further packing method
using percussive tapping to the base of the column, thereby
assisting uniform packing. In this picture, the column is just
depicted by its top plate 170, bottom plate 102 and bed of media
172. It also shows how flow packing can be performed, by using a
pump 174 optionally followed with an air trap 179 for ensuring that
the mobile phase will be free from gas.
[0109] The flow of mobile phase figured with arrows 173 exerts
downward strength against the media particles which adds to the
gravity force. This forces the media particles to settle in stable
manner against lower particles or column walls. The higher this
downward strength, the faster the particle settles and the stronger
is the strength exerted by this particle against lower layers,
inducing some local rearrangement until equilibrium is met. Flow
packing prevents large particles from settling before small
particles. If the particles were instead allowed to settle by
gravity, the big particles would to settle first and collect at the
bottom of the column while the small particles settle more slowly,
causing them to become concentrated at the top of the column.
Faster settling also reduces the time where percussion has to be
maintained and hence reduce the risk of damage of the particles.
This downward strength has to be maintained during all the time
percussion is performed.
[0110] The percussion table 175 can contain a mobile mass 175a,
referred to here as a hammer, actuated vertically by an actuator
175b fixed on the floor. This hammer 175a is periodically projected
against a plate 175d, to which its energy is transmitted. In the
embodiment depicted in the FIGS. 15 to 18, the plate 175d is linked
to the ground by elastic blocks 175c with low stiffness at low
frequency so as to minimize the shock absorption. These elastic
blocks 175c can be replaced with springs. Shocks can also be less
absorbed by implementation of cylindric joints in place of 175c
which just guide the plate 175d without constraint on the vertical
movement. With a given period, the hammer 175a exerts shock to the
plate 175d which lifts it up slightly (configuration on the right).
Under the gravity force of the table 175d and the stiffness of the
elastic blocks, the percussion plate comes back to its lower
position (configuration on the left) resulting with another
shock.
[0111] In some embodiments, percussion can be provided with a
rotary hammer drill, with rotation inhibited.
[0112] The percussion provides energy and upward acceleration to
the particles to move from equilibrium of higher energy to
equilibrium of lower energy (ie: more stable). The crossing from
one to the other requires energy to leave the first equilibrium,
for instance, to climb over a neighbor particle or force it to move
sideward or overcome a static friction.
[0113] This percussion differs in many manners from sinusoidal
vibration, performed by the rotation of an unbalanced mass or
vertical back and forth movement of a mass with an actuator fixed
on the vibrating table. In the methods described herein, the
frequency of the percussion is low (for example, from about 0.2 to
about 100 Hz with an amplitude less than about 5 mm, while
sinusoidal vibration exerts usually above about 50 Hz. In some
embodiments, the percussion involves very high acceleration due to
the hurtling of the mass of the hammer while acceleration is
limited on sinusoidal vibration. In some embodiments, the
percussion only acts along vertical direction, in the longitudinal
axis of the column, while sinusoidal vibrator is usually multi
directional in a plane, or unidirectional if combined by an
opposing pair. In some embodiments, the percussion targets
essentially particle rearrangement in the bed, while with higher
frequency sinusoidal vibration, it often targets friction reduction
(wall effect, etc.).
[0114] The strengths of the two actions, percussion and downward
strengths, add themselves along the vertical axis. Percussion is
discontinuous, while vertical strengths (gravity+flow circulation)
are continuous. Maximum magnitude of strength of percussion is
generally higher than downwards strength. Hence the resultant
strength is always vertical, sometimes in upward direction, during
the upward percussion impact where its magnitude is higher than
downward strength, sometimes in downward direction, during the
downward percussion impact added with downward strength or when no
percussion takes place. The mean value of the resultant strength is
positive in downward direction for allowing the media packing.
[0115] The FIG. 16 depicts one embodiment of the column assembly
with the tube extension, similar to FIG. 5, mounted on the
percussion table. The percussion table is shown in two
configurations:in low position on the left when the hammer 175a is
at rest, in high position on the right when 175d is hurt by the
hammer 175a. In this embodiment the whole column assembly is
submitted to the percussion.
[0116] The FIG. 17 depicts one embodiment alternative to the one of
FIG. 16, where the top parts comprising the top assembly 131, the
tube extension 106, the piston 132 and the piston shaft 134 are
isolated from the bottom parts of the column and from the
percussion table 175. These top parts are mounted on an independent
frame 180 attached to the tube extension 106. The liner 101, made
of soft material, allows for uncoupling of the top parts from
percussion, while keeping the water tightness inside the column
between the bottom parts submitted to percussion and top parts
uncoupled from percussion. Thus, in some embodiments, the top parts
are not submitted to the mechanical strength of the percussion with
consequent material fatigue over time. This also reduces the weight
over the percussion table 175, hence its inertia. The inertia being
reduced, the acceleration is increased, considering Newton's
law.
[0117] The FIG. 18 depicts one embodiment alternative to the one on
FIG. 16 and FIG. 17 for the top assembly 131. This assumes that the
liner 101 is sufficiently soft to be tucked over a neck 184 fitted
above the tube extension 106. Once the liner 101 is tucked over,
the top part 137 is mounted and tight against the neck 184, by a
nut 181, or a clamp or a bolted flange as for 108 in FIG. 2. When
tightened, the liner 101 is pinched water tight. This solution for
sealing the liner in the column can be easily implemented on every
embodiments of this invention.
[0118] Another optional change compared to the former embodiments
also appears on FIG. 18. The top plate 137 incorporates a slurry
injection port 182. This slurry injection port 182 encloses a valve
which can close the aperture during the packing operation and is
hence designed to resist hydraulic pressure during this operation.
The hollow shape of 137 allows for positioning the piston 132 above
the aperture of the slurry port so as the slurry can be injected
under the piston 132. The dotted line 132s shows the piston 132
when positioned in the upper position above the slurry port. By
injecting the media through 182 while mobile phase is also injected
through the piston 132, the slurry is carried in the flow and
quickly settled in the bottom of the column. This configuration
ensures that media particles don't travel to the region above the
piston. It also allows adjusting the level of media in the column
if the upper surface of the packed bed does not reach the upper tie
107, for instance due to bad dosing of media. It also allows direct
"dosing" of the media in the column during bed construction. A
possible method can be to fill the column with packing buffer, with
piston 132 positioned above the slurry port aperture, and
continuously circulate packing buffer from top process port 133 to
bottom process port 103 while injecting media through 182 until the
upper surface of the resulting packed bed will be just below the
upper tie 107. To avoid that the side positioning of 182 induces an
uneven distribution of the media in the column, several slurry
injection ports 182 can be implemented, or the media can eventually
be resuspended in place, after this initial filling, by upflow
circulation of packing buffer from bottom process port 103 to top
process port 133, or by air sparging through the bottom process
port 103. The packing operation with combination of percussion and
flow packing can then be repeated with the slurry injection port
shut off.
[0119] The FIGS. 19 to 21 depict an alternative design of the
column, especially for the sealing of the liner 101 with the piston
132 and eventually also with the bottom plate 102, but the
principle of the packing method stays unchanged. In this
embodiment, the sealing between the liner and the piston 132, and
eventually of the bottom plate 102 as shown on FIG. 19, is not
obtained by external tie such as 104 and 107 in former embodiments,
but by a seal 183 that can be for example an O-ring or any other
seal including but not limited to a lobe joint or a scraper seal.
In this case, the hollow of 105 is precisely dimensioned, at least
where parts 132 and 102 have to sit, so as the seal 183 makes a
sealing between the liner 101 and the piston 132 when engaged at
the top of 105 or between the liner 101 and the bottom plate 102
when engaged in the bottom of 105.
[0120] As explained in formers section describing FIGS. 5 & 6,
the piston 132 does not fit tightly with the film 101 when
positioned in the tube extension 106. With the embodiment of FIG.
19, this can be obtained by designing the hollow of 106 with a
larger diameter than in tube 105 so that the seal 183 does not fit
tightly between the piston 132 and the liner 101 in the tube
extension 106. This supposes that the liner is soft enough to
expands to the inner diameter of 106 under hydraulic pressure. The
same packing method as described in FIGS. 5 to 7 can be applied.
When the bed is consolidated as represented on the FIG. 19, the
piston 132 is lowered and engaged in the tube 105. This generates a
chamfer (i.e. an edge), adapting the inner diameter from the inner
diameter of 106 on the upper edge of 105 to the diameter where the
seal 183 can fit tightly between the piston 132 and the liner 101.
This chamfer hence guides the piston and progressively compresses
the seal 183 until the piston 132 meets the bed and gets sealed
with 101 as shown on FIG. 20.
[0121] At this position, the piston 132 must be held in place to
allow the dismounting of the piston shaft 134, the top assembly 131
and the tube extension 106. The holding of the piston has to
withstand the strength exerted by the media, e.g., as discussed
above. FIG. 20 depicts two possible solutions with 185 and 186 that
can be optionally be combined for holding the piston. Other
solutions that can hold the piston in place are also possible. Part
186 is one or several needles guided through an aperture in 105.
When introduced in the column, this needle(s) 186 pierces the liner
and holds the piston as a stop piece to counter the upper strength
applied by the media against the piston 132. This needle(s) 186 can
be connected to a vessel and hence can remove the supernatant above
the piston 132 before the dismounting of the upper parts (106, 134,
101). If for any reason, the liner 101 must not be pierced (for
instance, if the tube extension and liner are left for allowing
multiple packing and unpacking), one or several pins 185 can be
fitted in the same way than 186 with the difference that the pin(s)
do not pierce the liner but are simply pushed inside the column as
shown on the magnified view FIG. 20a. The design of the solution
ensures that 185 and/or 186 can be maintained on 105 without
preventing the dismounting of the upper parts 106, 134 and 101.
[0122] Once these upper parts have been removed, the cap 131 can be
installed as depicted on FIG. 21. The pin(s) 185 or needle(s) 186
can be dismounted after the cap is installed, or left in position.
They can also be removed before the cap 131 is installed, if an
external force holds the piston in place between the moment the
needle(s) 185 or pin(s) 186 are removed and the moment where cap
131 is installed.
[0123] One interesting extension of the embodiment described in
FIGS. 19 and 20 is that this solution can be easily adapted to a
configuration without the liner 101. In this case, the piston 132
and the bottom plate 102 are directly in contact with the tube 105.
In this aspect, the tube extension 106 is mounted in a water tight
fashion on the top of the column tube 105 by a seal. In some
aspects, the tube extension 106 and the tube 105 can be designed as
a single assembly in one piece or made of parts welded or assembled
together. In this aspect, the parts 105 and 106, being in contact
with the product, are ideally made of food-grade or drug-grade
material.
[0124] This invention is useful in plug-flow chromatography
applications of any scale, from small diameters for laboratory
usage up to a large industrial scale. The invention can also be
used with any kind of separation media, including matrices based on
natural polymers, organic polymers, and inorganic materials such as
hydroxyapatite, silica, TiO.sub.2, and diatomaceous earth.
[0125] The invention is also readily adapted to expanded bed
applications, by using the upper assembly and the tube extension
during the period where bed is being expanded. The invention is
readily adapted to packing techniques other than that described
above. The invention can also be practiced by stacking columns as
shown in FIG. 14 while using a single tubular plastic film, by
repeating the sequence of FIGS. 2 through 9 multiple times. When
packed with the same separation medium, the stacked columns can
save floor space. With stacked columns, the mobile phase is
distributed equally between the different inlet ports of the
columns and collected from the different outlets of the column at
the same time, achieving a result equivalent to that obtainable
with a larger column. When packed with different kinds of media,
the stacked columns can be used for the different purification
steps. Since each section of the column will have its dedicated
inlet and outlet process ports, the column sections can also be
used with different buffers and different instruments, as
independent columns.
[0126] The concept of column with liner can also be used in a more
conventional chromatography column design such as the one depicted
on FIG. 22. In this embodiment, the piston 132 encloses a seal 183
that can be for example an O-ring or any other seal including but
not limited to a lobe joint or a scraper seal, as on FIG. 19. But
contrarily to the previously-described designs where the column
tube is made of 2 sections 106 and 105, in the embodiment of FIG.
22, the column tube is made of one cylindrical section 105
sufficiently tall to contain the volume of slurry. The piston 132
is tight all along the column tube 105. Being constantly submitted
to the hydraulic pressure in the column, the piston 132 is held in
place, for example, by being attached to a central screw 191 that
can be raised or lowered by turning a central nut 190 pivoting in
the cover 137. In the embodiment of FIG. 22, the liner is
positioned in the tube 105 and retained on the upper edge of 105 by
a wedge 192, or, if the liner is sufficiently soft, the liner can
be tucked over a neck as depicted in former designs such as FIG. 18
or 19. One advantage of the embodiment depicted in FIG. 22 is that,
for regulated products, the material of the column tube does not
need to be validated for contact with drug or food. Of course,
where the product is regulated, the liner will have such
validation. This minimizes the use of costly high grade
material.
[0127] If the liner is sufficiently thick and soft, the column
design can be simplified as depicted on FIG. 23. Compared to the
FIG. 22, the sealing between the bottom plate 102 and the liner, as
well as between the piston 132 and the liner 101 is obtained
directly by mechanical compression, without seal such as 183 in
FIG. 22. For minimizing the shear stress between the piston 132 (or
bottom plate 102) and the liner 101, the outer edge of the piston
and bottom plate can enclose a protuberance (for example, that is
bead-shape), which ensures a smooth installation or movement of the
piston 132 (or bottom plate 102) in the liner 101. In the
embodiment of FIG. 23, the protuberance (which provides water
tightness) is positioned at the same level as the filter. This
ensures that the column inner volume is submitted to the flow of
the mobile phase and reduces the risk of a dead spot in the
column.
[0128] The methods and concepts described herein can also be used
for preparing a compact and homogeneous bed made of particles for
purposes other than chromatography. One example of such a purpose
is filtering; another is use of the bed as a preliminary step for
chromatography. For example, aspects of the invention can be used
in fusing a bed of particles into a monolith structure by
polymerization. Aspects of the invention can also be used for
preparing chromatography media between the bottom and top plates
inside the plastic film, with other media configurations, such as
discs. Aspects of the invention is not limited to columns of
circular cross section. By eliminating the need for dynamic
sealing, the invention can easily be adapted to columns of
virtually all cross section shapes, such as polygons, ellipses,
etc. The tubular plastic film can also be an elastomeric film, such
as EPDM or silicone.
[0129] As noted above, also provided herein is a method of applying
a filter to a bore hole of a bottom plate of a chromatography
column and/or piston for packing a chromatography column. This
method can be applied in conjunction with the other methods
described herein (e.g., use of a liner and/or percussive packing)
or can be used in the absence of such methods. An embodiment is
depicted in FIG. 24, which depicts positioning of the filter (3) in
the bore of the piston. As noted elsewhere, the same action can be
applied to fix a filter to the bore of the bottom plate. This
fixing plays at least two roles: it serves as a sealing technique,
to prevent the media from circumventing the filter and entering the
distribution chamber behind the filter, and it serves as a
mechanical fixing for holding the filter (3) in place while being
submitted to the mobile flow. Indeed, when injecting a mobile phase
downward, the flow resistance of the filter (3) due to its porous
material is converted to an axial strength, which tends to push the
filter (3) away from the piston. The filter (3) can be retained by
its outer diameter, and eventually with additional fixings
(fasteners) distributed on its surface. FIG. 24 depicts an
embodiment in which filter screws (28) are sealed with filter screw
gaskets (29). The filter screw gaskets (29) assist in preventing
diffusion of liquid or contaminants in the thread of the screws.
They also prevent the mobile phase from entering in the holes of
the filter where filter screws (28) seat. As the fasteners can
create local singularities in the distribution, their size and
number can be minimized. In some embodiments, fasteners for holding
the filter are not included at all where the fixing of the filter
by shrinking is sufficient.
[0130] The shrinking technique employs the thermal expansion of
materials such as polypropylene or polyethylene used for the piston
(2) or bottom plate. The piston (2) and bottom plate enclose an
opening (27) comprising the bore, the opening having a diameter
slightly smaller than the filter at ambient temperature, and
slightly larger or equal to filter diameter at higher temperature.
For instance, polypropylene heated from 30.degree. C. to
100.degree. C. expands by around 1%. If needed, the filter can be
cooled down so as to cumulate the thermal expansion of the filter
or bottom plate bore with the thermal shrinkage of the filter
material at cool temperature.
[0131] In the claims appended hereto, the term "a" or "an" is
intended to mean "one or more." The term "comprise" and variations
thereof such as "comprises" and "comprising," when preceding the
recitation of a step or an element, are intended to mean that the
addition of further steps or elements is optional and not excluded.
All patents, patent applications, and other published reference
materials cited in this specification are hereby incorporated
herein by reference in their entirety. Any discrepancy between any
reference material cited herein or any prior art in general and an
explicit teaching of this specification is intended to be resolved
in favor of the teaching in this specification. This includes any
discrepancy between an art-understood definition of a word or
phrase and a definition explicitly provided in this specification
of the same word or phrase.
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