U.S. patent application number 09/923017 was filed with the patent office on 2001-12-27 for ultrafilteration device and method of forming same.
Invention is credited to Bowers, William F., Towle, Timothy, Yankopoulos, Basil.
Application Number | 20010054584 09/923017 |
Document ID | / |
Family ID | 26808610 |
Filed Date | 2001-12-27 |
United States Patent
Application |
20010054584 |
Kind Code |
A1 |
Bowers, William F. ; et
al. |
December 27, 2001 |
Ultrafilteration device and method of forming same
Abstract
An ultrafiltration device has a filter membrane sealed inside a
reservoir body, such as a tube. The tube has one or more ports and
a closed portion distal to the port(s), and the filter membrane is
sealed to the body along a closed contour widely surrounding the
port(s) to provide a large area filtered outflow path. The method
is effective to rapidly isolate a predetermined amount of a desired
retentate in the distal portion of the tube. The method and device
are also useful for quantitative transfer of smaller molecules and
for multi-step processing of sample arrays. A frusto-conical
peripheral filter provides high ratio of effective filter area, and
may be configured for hydrostatic deadstopping and little or no
wicking, greatly enhancing recovery time and efficiency. Linear
array strips of such chambers may be formed by bonding together
mating halves with filter areas over the chamber ports. The vessel
may include a rib to guide and orient filter during assembly,
and/or a ledge or recess to engage and align the filter, assuring
that the filter is precisely positioned and does not wander during
manufacture and bonding. In one embodiment a deflectable lip or
other integral feature of the vessel geometry seals the vessel in a
capped receiving tube, and opens under pressure to pass pressure
between the tube and vessel during centrifugation, to permit
overfilling of the vessel in a tilted rotor assembly without
spillage or leakage, and increasing the concentration ratio. The
vessels have a high filter area to volume ratio, maintain open
filter surfaces and high rates of filtration throughout the spin,
and are fully compatible with robotic loading, multistage operation
and in situ multiwell plate filtrate and/or retentate assay or
transfer. Attachment of the filter may be effected by heat welding.
Preferably the vessel and filter are positioned between a press
member and a heat sink and a superheated tool contacts the press
member to selectively deliver a defined bolus of heat to the weld
areas.
Inventors: |
Bowers, William F.;
(Topsfield, MA) ; Yankopoulos, Basil; (Peabody,
MA) ; Towle, Timothy; (Lee, NH) |
Correspondence
Address: |
NUTTER MCCLENNEN & FISH LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
26808610 |
Appl. No.: |
09/923017 |
Filed: |
August 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09923017 |
Aug 6, 2001 |
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09454391 |
Dec 3, 1999 |
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6269957 |
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60111068 |
Dec 4, 1998 |
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60116890 |
Jan 22, 1999 |
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Current U.S.
Class: |
210/455 ;
156/272.2; 210/451; 210/452 |
Current CPC
Class: |
B01L 2300/042 20130101;
B01L 2400/0409 20130101; B01L 3/5021 20130101; G01N 1/4077
20130101; B01L 2300/0681 20130101; B01D 65/003 20130101; B01D 63/16
20130101; B01D 61/18 20130101; B01L 2200/12 20130101 |
Class at
Publication: |
210/455 ;
210/451; 210/452; 156/272.2 |
International
Class: |
B01D 029/085 |
Goverment Interests
[0002] A portion of the present invention was supported by NIH
Grant# 1 R43 RR12066-01.
Claims
What is claimed is:
1. A method of forming an ultrafiltration device, comprising
providing a reservoir body having a sloped bounding wall region
including a port and a closed portion distal to the port providing
a filter membrane supported by said bounding wall region and
entirely covering the port, and sealing the filter membrane to the
sloped bounding wall region such that solvent and lower molecular
weight material pass through the filter membrane and out the port,
to effectively isolate a predetermined amount of retentate in the
closed portion distal to the port.
2. The method of claim 1, wherein the filter membrane has an active
filter area greater than about 0.5 cm.sup.2 and is bonded to said
bounding wall at plural discrete spots to prevent separation due to
gravitational peeling forces arising during centrifuging.
3. A method of forming an ultrafiltration device, comprising
providing a hollow reservoir body formed as a single integral shell
wall having a length, a proximal inlet at an inlet end, a closed
distal end, and a port located in an intermediate region between
said inlet end and said closed distal end, and sealing a filter
membrane to said shell wall around the interior of said
intermediate region and over said port, such that when centrifuged
under predetermined conditions, fluid and solutes of a
predetermined molecular weight range pass through the filter
membrane and exits said port, while a retentate having a greater
predetermined molecular weight accumulates in said closed distal
end.
4. The method of claim 3, wherein i) said closed distal end has a
receiving volume less than two percent of the volume of said
reservoir body ii) ratio of area of said filter membrane to volume
of said reservoir body is greater than 0.75/cm, and iii) volume of
said reservoir body is between about one-half and two hundred cubic
centimeters.
5. The method of claim 3, wherein said closed distal end forms a
deadstop having a volume under about 1.0% of the volume of said
body.
6. The method of claim 3, wherein said filter forms a truncated
cone-shaped active filter area when sealed.
7. The method of claim 6, wherein said filter is sealed along an
axial-release direction along narrow band segments that allow
filtration through a preponderance of its surface area to said port
while being supported by the wall of the reservoir body.
8. An ultrafiltration device, comprising a hollow, smooth,
continuous convex reservoir body having a length, a proximal inlet
end, and a closed distal end with a port located in a sloping wall
lying in an intermediate region between said inlet end and said
closed distal end, and a filter membrane sealed around the interior
of said intermediate region and over said port, such that when
centrifuged under predetermined conditions, fluid and solutes of a
predetermined molecular weight range pass through the filter
membrane and exits said port, and a retentate having a greater
predetermined molecular weight accumulates in said closed distal
end.
9. A method of forming an ultrafiltration device, comprising
providing a reservoir body having a port and a distal body portion
distal to the port providing a filter, and sealing the filter to
the body around an axial-release direction so as to entirely cover
said port, such that the device is effective to isolate a
predetermined amount of a desired retentate in the distal body
portion.
10. The method of claim 9, wherein the filter has an area of about
0.5 to about 120 cm.sup.2, more than 70% of which is active filter
area.
11. A method of forming an ultrafiltration device, comprising
providing a hollow reservoir body having a length, a proximal
inlet, and a closed distal end with a port extending through a wall
of the body in an intermediate region located between said inlet
end and said closed distal end, and sealing a filter around the
interior of said intermediate region and over said port, such that
when centrifuged under predetermined conditions, material below a
predetermined molecular weight passes through a broad region of the
filter and flows along the wall to exit said port, and solute
having a molecular weight greater than the predetermined molecular
weight is retained by the filter in said tube and accumulates in
said closed end.
12. The method of claim 11, wherein said closed distal end forms a
deadstop having a volume between about 0.3% and about 1.0% of the
volume of said body.
13. The method of claim 11, wherein said filter forms a truncated
cone-shaped active filter area when sealed.
14. The method of claim 11, wherein said closed distal end forms a
deadstop having a volume between about 0.04% and 0.3% of the volume
of said body.
15. The method of claim 11, wherein the step of sealing is
performed in stages to first drive moisture from a sealing region
of the filter and then fuse the sealing region and vessel.
16. An ultrafiltration device, comprising at least one hollow
reservoir body, each said body having a length, a proximal inlet, a
closed distal end, and a port intermediate its inlet and its distal
end, and filter sealed about its perimeter to a wall of said
intermediate region and over said port to define between said wall
and the filter an interstitial space communicating with the port,
such that when centrifuged under predetermined conditions, solvent
and solutes having a molecular weight substantially smaller than a
predetermined molecular weight pass through the filter and exit the
port via said interstitial space, and may optionally be collected
in a mating receiving well, while solutes having a molecular weight
greater than the predetermined molecular weight accumulate in the
closed distal end.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is related to, and claims the
benefit of U.S. Provisional Patent Applications No. 60/111,068
filed Dec. 4, 1998 and No. 60/116,890 filed Jan. 22, 1999, each of
which is hereby incorporated by reference herein in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Various devices are known for isolating a retentate
containing a high molecular weight material, such as DNA or
protein, through centrifugal ultrafiltration. The yields and
amounts of retentate achieved using these techniques vary greatly
due to the size, shape and position of filter membrane, the
positions of outlets and/or the presence of ledges, comers or
compartments in the devices.
[0004] Often these devices have associated limitations or
drawbacks. For example, a device may be ineffective to prevent
filtration of retentate to near dryness, or may have a design that
hinders access to, or prevents complete pipette recovery of, the
retentate due to chamber geometry, surface tension spreading, or
the like. Also, a device may attain only a low yield or poor
separation, or may require excessive centrifuge times.
Additionally, a device may be poorly adapted for, or entirely
incapable of, being prepared by or being used with robotic or other
automated devices. Further, a technique or device may be uneconomic
due, for example, to inefficient utilization of filter membrane
area, and/or to manufacturing cost, and/or to requiring a long
centrifuge time.
[0005] Therefore, a need exists for a centrifugal ultrafiltration
device that can be dependably manufactured and used.
[0006] There is also a need for a separation technique that is
rapid, effective and amenable to automated implementation.
[0007] There is also a need for improved processes for the
manufacture or assembly of filtration or concentration vessels.
SUMMARY OF THE INVENTION
[0008] One or more of the foregoing ends are achieved in accordance
with the present invention by providing a separation vessel having
a conical region extending to a closed tip and a port in the wall
of the conical region covered by a filter. The filter has a pore
size and structure such that when centrifuged, fluid material such
as solvent and solutes with a molecular weight below a threshold
level passes through the filter and out the port. The conical
region has a cone angle that causes retentate accumulating on the
inner surface of the filter to slough down into the closed tip.
Advantageously, the filter covers an area substantially larger than
the port and is supported by the underlying wall so that a large
filter area is actively used, and also resists clogging, thus
allowing fast filtration. Moreover, the conical shape, which may
extend from a cylindrical proximal or upper body portion, subtends
a large reservoir of material in the vessel while allowing the
receiving end to be sized for retaining a relatively small or minor
fraction, e.g., below two percent, or even below a twentieth of one
percent, of the total volume as retentate. Preferably, the filter
is welded or fastened around its edges to the wall of the vessel by
a process such as heat fusing or solvent welding, and covers a
region extending from above the port at least down to the port. The
vessel may have an upper flange allowing it to drop into a standard
concentration tube so that the filtrate leaving the vessel is
retained in the concentration tube and may itself be further
processed, analyzed or transferred. In one embodiment, a
deflectable member or portion of the vessel body operates as a
pressure vent between the interior of the concentration tube and
the interior of the separation vessel, allowing the vessel to be
centrifuged while tightly capped. This feature also adapts a vessel
to be overfilled and safely processed in a common tilted rotor
assembly without spillage or blowout, thereby increasing the
achievable single batch concentration ratio.
[0009] The separation vessel may be assembled by positioning a
shaped filter sheet within the cone area of the vessel using a tool
having one or more heated areas shaped to define bonding segments
in peripheral regions of the filter at which the filter is to be
fused with the body of the vessel. Preferably, the filter sheet has
a trapezoidal shape which curls, when inserted into the vessel, to
entirely cover the interior surface of a truncated-cone region of
the vessel wall. The vessel may be formed with an alignment rib or
other feature along its interior surface positioned to engage an
edge of the filter sheet and thus to orient and align the sheet as
it is curled against the curved inner wall of the vessel during
insertion. A ledge may further be provided to catch the curled,
aligned filter in position when fully inserted. Bonding of the
filter to the wall is advantageously carried out by supporting the
vessel in a heat sink while pressing a hot iron against the inner
surface to fuse the filter backing membrane to the vessel wall.
[0010] The invention in another aspect provides a centrifugal
concentrator having an alignment structure, such as a rib, which
extends in a plane through the concentrator tube axis and serves to
align a wedge-shaped membrane squarely along the axis during
insertion of the membrane and assures that the filter edges are
located away from ports of the vessel. The filter may extend
substantially the full circumference, so that the well-aligned
edges abut and seal precisely when pressed in along the axial
direction with a tack welder, such as a conical tip and/or slotted
insertion/heat sealing tool. The tool may also melt the vessel rib
over the seated butt edges. A seating ledge provided in the vessel
wall to engage the top edge of the filter further aids in orienting
or positioning the truncated cone filter membrane, and stabilizes
filter position during handling or assembly.
[0011] In yet another aspect the concentrator tube is configured to
fit into and be supported by a filtrate collection tube, and the
concentrator tube has a top sealing surface with a deflectable
sealing lip that seals against the cap of the filtrate collection
tube. The lip deflects in response to outside pressure within the
capped collection tube, opening during centrifugation to allow
venting via a bypass channel so pressure may vent from the
collection tube to the concentrator tube, without leaking or
blowing aerosols out to the centrifuge drum. This allows the
concentrator tube to be overfilled, i.e., to be loaded into a fixed
angle carrier at a higher fill level such that the fluid contents
wet the cap, and yet to be processed without spillover or leakage,
thus increasing the attainable concentration ratio and enhancing
the speed and yield of the concentration process.
[0012] The invention also contemplates a separation vessel
manufactured with a clamshell construction as part of a vessel
array having the form of a strip or row of two or more vessels. In
accordance with this aspect of the invention, a sheet of suitable
polymer material is formed with a number n of identically-shaped
troughs, each trough corresponding to one-half of the desired
chamber shape, and including one or more ports formed in a
conically sloping region thereof. A sheet of filter material is
then placed over the multi-trough polymer sheet, and may optionally
be pressed into the troughs and sealingly attached to cover the
ports. Attachment is done by advancing one or more tools, such as a
press mold or a hot wire die, which advantageously may be advanced
in a direction perpendicular to the plane of the sheet, avoiding
shear movement at the surface of the filter. A second symmetrically
shaped filter-bearing polymer sheet is then laid on top to complete
each of the vessel chambers, and the two polymer sheets are bonded
together, for example by heat fusion, solvent or ultrasonic
welding, or the like, to form a strip of n vessels. The geometry of
the strip preferably conforms to the lattice spacing of a standard
microtiter plate or multiwell receiving tray, e.g., forms a row of
n or m vessels spaced to fit a standard n.times.m array or rack
into which the strip itself is to be loaded for centrifuging. The
basic row may also be manufactured to fit a into a portion of a
single row or column of the matrix array, allowing multiple
different sets of vessel strips to be loaded in the same array,
which may, for example have dimensions kn.times.jm, where kj are
integers.
[0013] Advantageously, since each separation vessel or chamber of
the assembled array is centered around the tip of the conical
region, the retentate resides in a defined and regular lattice
position, and is accessible by a direct and unobstructed axial
motion, thus making the vessel array adapted to robotic processing,
pipette transferor assay, or operations with mechanized handling
equipment.
[0014] The vessels of the present invention advantageously present
a large surface area relative to the effective volume of the
vessel, and operate at high rotational speeds while maintaining an
open surface filter surface during operation. The filter is broadly
supported against the adjacent vessel wall, providing a large flow
area via interstitial space for filtrate passing out the ports, and
free of internal structural encumbrances that might catch fluid and
diminish its efficiency.
[0015] Assembly of the vessel and filter membrane is accomplished
in a preferred aspect of the invention by insertion of a shaped
heating tool to heat peripheral regions of the filter membrane as
it lies positioned against the wall of the vessel. The filter may
be a regenerated cellulose material on a porous polyethylene
backing, such that the tool may contact the cellulosic material
without sticking, and melt the backing to the vessel wall. The
vessel and filter may be placed between a heat sink, and a press
plate, with the heated member contacting the press plate to
transfer a defined dosage of thermal energy with controlled thermal
characteristics to the weld areas. A superheated rod and thimble
embodiment employs a two-step heater advance to preheat and then
weld the filter in place. The vessel or press plate may be provided
with protrusions or partial ribs to automatically center the heat
transfer tooling in the vessel and assure complete welding of the
intended weld lines in areas to seal the filter over the ports and
prevent ballooning of its central region.
BRIEF DESCRIPTION OF DRAWINGS
[0016] These and other features of the invention will be understood
from the discussion below and illustration of representative
embodiments in the drawings, wherein:
[0017] FIG. 1 shows a shaped sheet of filter membrane suitable for
the vessel of the present invention;
[0018] FIG. 2 shows an alternate shape for the filter membrane used
in the invention;
[0019] FIGS. 2A and 2B illustrate cutting patterns for obtaining
the membranes of FIGS. 1 and 2 respectively from large continuous
sheets;
[0020] FIG. 3 illustrates a first embodiment of a separation vessel
in accordance with the invention;
[0021] FIG. 4 illustrates assembly of a filter into the vessel of
FIG. 3;
[0022] FIG. 5 illustrates another step of assembly of the vessel of
FIG. 3;
[0023] FIG. 6 illustrates one embodiment of a heat welding inserter
tool for carrying out the step of FIG. 5;
[0024] FIG. 7 illustrates a completed separation vessel having a
square flange;
[0025] FIG. 8 illustrates a strip or cartridge array of separation
vessels in accordance with another embodiment;
[0026] FIG. 9 illustrates another embodiment of a vessel of the
invention wherein a filter membrane extends distally of the outlet
port;
[0027] FIGS. 10A-10D illustrate steps of manufacturing and use of a
strip array embodiment like that of FIG. 8;
[0028] FIGS. 11A-11D illustrate another embodiment of a
concentrator vessel and filter of the invention;
[0029] FIGS. 12A-12C illustrate venting operation of the embodiment
of FIGS. 11A-11D;
[0030] FIGS. 13A-13F illustrate tools for another manufacturing
method and its implementation; and
[0031] FIG. 14 illustrates steps of the method.
DETAILED DESCRIPTION
[0032] In general, applicant's invention contemplates a separation
vessel forming a generally elongated or tubular chamber with a
conical region in which an ultrafiltration filter membrane allows
passage of solvent and lower molecular weight material to an exit
port while directing retained higher molecular weight material to a
retentate sump. The construction offers a high ratio of filter area
to reservoir volume, high filtrate throughput per unit time, and
high efficiency of separation of the retained material. By locating
the exit port or ports near the apex of a cone, the assembly may
maintain a high filtration rate during all or a major portion of
the centrifuging separation cycle, and yet isolate a retained
faction comprising less than one or two percent of the initial
fluid volume. The shape of each filter allows utilization of filter
membrane to be highly efficient, with tessellated patterns cut from
a large continuous sheet or roll so as to have minimal or no
wastage. Further, by mounting the filter to the surrounding wall by
perimeter weld along line segments, seventy percent or more of the
filter area may be actively used. This will be appreciated from a
brief consideration of illustrative embodiment and representative
filter shapes applied to conical regions of a ported separation
vessel.
[0033] Referring to FIG. 1, an exemplary ultrafiltration filter
membrane 10 is shown. The filter membrane 10 is of a substantially
wedge-like shape, with a proximal end 12 and a distal end 14 and
two sides 16, 18. The proximal and distal ends 12, 14 are generally
curved or arcuate, while the sides 16, 18 are each substantially
straight. The filter membrane 10 has an active area 20 and an
inactive area 22. The active area 20 of the filter membrane 10
corresponds to the portion of the filter membrane that, once the
filter membrane is placed in a centrifuge tube and sealed, will be
capable of ultrafiltration. That is, the active area 20 is that
area through which, when the filter membrane is positioned in a
separation tube or vessel, the smaller fluid components permeate as
the filter membrane is centrifuged. The membrane utilization
efficiency of a device design may be calculated by dividing its
active area by its total active area, inactive area, and cutting
waste area. In the embodiment of FIG. 1, the membrane has an active
area of approximately 1.0 cm.sup.2 and an inactive area of
approximately 0.22 cm.sup.2, with no cutting waste area. Thus, the
manufacturing efficiency is approximately 0.82 or 82%. This level
of efficiency for the wedge-like filter membrane is much higher
than the utilization efficiencies of disk-shaped membrane designs
currently used in the art. An alternate embodiment of the filter
membrane 10 of FIG. 1 is shown in FIG. 2. The filter membrane 10'
depicted in FIG. 2 is also of a substantially wedge like design,
however, the filter membrane of FIG. 2 has proximal and distal ends
12', 14' that are formed from two substantially straight edges. The
sides 16', 18' which connect the ends 12', 14', however, are
generally identical to the sides 16, 18 of the filter membrane of
FIG. 1. The wedge-like design of the filter membrane 10' of FIG. 2,
and its substantially similar dimensions to the filter membrane of
FIG. 1 are such that its utilization efficiency is also
approximately 82%.
[0034] As depicted in FIGS. 2A and 2B, the filter membranes of FIG.
1 or FIG. 2, respectively, may each be laid out like tiles along a
strip, facing in alternating directions and be cut without cutting
wastage from filter membrane strips which have previously been slit
by rolling dies to form the shapes outlined above. One of ordinary
skill in the art will appreciate that the filter membrane shapes of
FIG. 1 or FIG. 2 may be varied, and also that other filter membrane
shapes may be used with the present invention, while still enjoying
its attendant advantages. Generally, however, the membrane 10 or
10', regardless of its exact shape, should have a thickness and
pore size such that it is able to retain globular solutes having a
molecular weight above a threshold, e.g., of at least about 10,000
Daltons. For DNA purification or concentration, the membrane 10 or
10' preferably should have a pore structure rated to retain
globular solutes at least above about 30,000 Daltons, to above
about 150,000 Daltons.
[0035] Referring now to FIG. 3, a reservoir body 30 or
concentration tube of the present invention in which a filter
membrane 10 or 10' of FIGS. 1 or 2 is placed. The reservoir body 30
is generally a tube which has a proximal portion 32 and a distal
portion 34 and a longitudinal axis 36. The proximal portion 32 of
the tube 30 is cylindrical, e.g., with a substantially constant
diameter, while the distal portion is substantially conical and
tapers to a closed tip 37. The distal portion 34 of the tube 30
includes at least one port area 38. Preferably, the tube 30
includes two to four port areas 38.
[0036] An exemplary tube 30 for use with the present invention
resembles a conventional microcentrifuge tube made of
polypropylene, which holds about 0.6 milliliters of material and
can, in turn, be accommodated in a larger microcentrifuge tube of
between about 1.5 and about 2.0 milliliters capacity. The tube 30
may be centrifuged at a wide range of angles and forces. The tube
is preferably formed to withstand up to 20,000 G of force. The tube
may be used in a 45.degree. fixed angle rotor, or may be placed in
an aperture of a fixture in a rotating platform device. The tube 30
may have a longitudinal length of between about 1.0 and about 5.0
centimeters, of which all but about 0.5 centimeter lies above the
port areas 38.
[0037] The port areas 38, however many are included, are generally
identical in their contour and may be located at the same or at
varying height along the longitudinal length of the tube 30 they
affect. As described more fully below in connection with FIGS. 4
and 5, the filter membrane 10 mounts in the tube 30 with a large
area filter oriented so as to be both substantially aligned to the
centrifugal force vector, and to cover the port areas 38. Although
not herein illustrated, the present invention also contemplates the
utilization of differently-shaped tubes including, but not limited
to, substantially cylindrical tubes, or tubes which are entirely
conical, lacking any proximal portion 32, and which have
essentially the entire wetted wall area other than tip 37 covered
by a filter membrane.
[0038] The distal portion 34 of the tube 30 includes a closed end
retentate area 40 in which desired retentate is isolated with high
efficiency and may be retrieved without filtering to dryness. As
shown in FIG. 3, the retentate area is located partially or
entirely distal to the port areas 38, depending upon the angle at
which the axis 36 is aligned to the centrifugal force vector. The
retentate area 40 should be shaped and placed such that a
predetermined amount of desired retentate will remain, and may be
removed as is generally known in the art, such as by pipetting or
through a robotic or otherwise automated device. For example, in a
vessel of 0.6 mL capacity, the ports may be positioned at a height
to define a retentate volume 40 of two to twenty microliters.
[0039] Referring now to FIG. 4, the filter membrane 10 of FIG. 1 is
shown during assembly being placed in the centrifuge tube 30 of
FIG. 3. The edges of the filter membrane 10 are curled over so as
to fit within the tube 30. Because of its wedge-like design, the
membrane is substantially self-guiding. Specifically, once the
narrow tip of the membrane 10 is introduced into the tube, and as
it is moved through the entry toward the distal, conical portion 34
of the tube 30, the membrane will curl over into a well aligned
frustoconical shell which conforms to the shape of the distal,
conical portion of the tube, such that its sides 16, 18 come
together and its ends 12, 14 each form a complete circumferential
edge.
[0040] The membrane 10 may be introduced into, and moved distally
throughout, the distal portion 34 of the tube with any suitable or
appropriately shaped rod, mandrel, fork or the like. The membrane
may be introduced into the distal portion 34 of the tube 30 with
the same instrument that is to seal the filter membrane as
discussed below. Although not specifically shown in FIG. 4, the
membrane 10' of FIG. 2 may be similarly introduced into, and moved
distally throughout, the distal portion 34 of the tube 30.
[0041] Once it has been moved distally into a predetermined
location in the distal portion 34 of the tube 30, the membrane 10,
which may optionally be securely maintained in position by applying
a vacuum to the outer surface of tube 30 to draw it snugly against
ports 38, (or by applying such a vacuum internally of the tip of an
insertion mandrel), is then sealed to the tube around its
circumference. As shown in FIG. 5, generally there are three bands
of sealing of the membrane filter: a proximal seal portion 50, a
distal seal portion 52, and at least one vertical edge sealing
portion 54. The filter membrane 10 may be sealed in a number of
ways, for example with adhesive or a heat melt polymer in or along
the inactive area 22, or by heat fusing with the vessel body in
that area. Regardless of the sealing technique, however, the filter
membrane 10 should be sealed to the tube 30 around the inactive
area 22 such that the filter membrane entirely covers the port
area(s) 3 8, and allows material to exit the tube only through the
filter and then through the port(s). Further, the extreme distal
end of the filter membrane 10 may extend to and be sealed
immediately distal to the port area(s) 38, or it may extend
further, into the retentate area 40.
[0042] The filter membrane 10 may be sealed by a heat-welding
inserter tool 60 as shown in FIG. 6. As noted above, the inserter
60 preferably has an elongate handle 62 and a conical tip 64 shaped
such that the inserter is capable of introducing the filter
membrane 10 to the distal portion 34 of the tube 30 and pressing
the filter membrane outward against a wall of the distal portion of
the tube prior to sealing the filter membrane thereat. Further, the
inserter 60 should be shaped, and the filter membrane 10 should be
made of a material, such that the filter membrane may be sealed by
the inserter 60 without cracking or scratching the membrane and
without sticking to the inserter.
[0043] In an exemplary embodiment, the inserter 60 has a distal end
66 that has substantially the same shape or contour as the distal
portion 34 of the tube 30. The distal end 66 of the inserter 60
also includes, at its surface, a plurality of heater wires or
ribbons 68 that also are energized by conductors contained within
the handle 62 of the inserter. These wires or ribbons 68 may be
Nichrome wire or ribbons, or ceramic ribbon bands which are
electrically powered to heat a defined region in order to
effectuate a desired seal to the filter membrane 10 in a short
time, e.g., approximately two seconds. The surface of the inserter
60 may have features such as a waffle texture, or vacuum passages
as well as registration tabs for improved gripping and positioning
of the filter membrane 10. Preferably the inserter has a plurality
of vacuum passages at the tip, to which vacuum is applied to pick
up a pre-cut piece of filter membrane 10. The filter is then
inserted to the vessel, and released from the inserter 60 as vacuum
is applied externally of the vessel ports to draw the filter
securely into position against the vessel wall, where it is then
welded, as described above. One of ordinary skill in the art,
however, will appreciate that heat may be provided to the inserter
60 using RF or other means, and that its gripping ability may be
enhanced in other ways.
[0044] FIG. 7 shows a perspective view of a tube 30 with a sealed
filter membrane 10. The tube 30 is adapted for placement within a
well of a multiwell tray for centrifuging. The proximal region 32
of the centrifuge tube 30 includes a flange 70 which may be any
shape suitable to allow the tube to be inserted into an individual
well or a multiple well container for centrifuging. In the
embodiment shown in FIG. 7, the flange 70 is substantially
square.
[0045] The tube 30 of the present invention may be used in any
conventional individual or multiple well centrifuge, with such
multiple well centrifuges including, but not limited to, rotating
platform devices. A plurality of centrifuge tubes 30 (one, two, or
as many as needed) of the type shown in FIG. 7 may be separately
placed in a multiple well carrier. This carrier may then optionally
be placed above a receiver multiwell tray 85 (FIG. 10D) used to
quantitatively collect filtrate which drips from the bottom point
of tip 37 of each tube resting inside the mating receiver well
below it. More generally, the tubes 30 may be manufactured as
strips or cartridges 80 of eight (see FIG. 8) or twelve tubes, so
each strip fills a row or column of a conventional 96-well
plateholder. One of ordinary skill in the art, however, will
appreciate, that tubes 30 of the present invention may be used in
any multiple well centrifuge, regardless of the number of tubes or
the matrix orientation of the multiple wells. Furthermore, special
adapter plates may be formed, for example, to place four or more
such tubes 30 in a larger single well so as to adapt the filtration
cell to different existing vessels or centrifuges, or to
accommodate a convenient batch size.
[0046] Also within the scope of the present invention is an array
embodiment, wherein a strip 80 containing a plurality of chambers
is made by welding or otherwise bonding a sheet or individual
wedges of membrane 10 to two molded halves along the axial plane 75
as shown in FIGS. 10A-10D, treating or cutting away excess membrane
at "e" between the tubes if needed to assure dependable joining or
sealing, and then welding together the halves to form an integral
strip in which each well half has two vertical sealed edges at 54
formed at or just next to the center plane. In this embodiment, the
filter is substantially coextensive with the entire wall of the
vessel, so, as shown by the meniscus line 76, essentially the
entire chamber surface area participates in filtration.
[0047] The tube or tubes 30, 80 of the present invention are shaped
so that their placement into an individual well centrifuge, or into
the strips or cartridges of a multi-well centrifuge, may be
performed by a robotic or another automated device. Similarly, the
unobstructed axial position of the retentate well permits addition
of sample material and removal of the retentate from the retentate
area to be performed by a robotic or other automated device.
Optionally, retentate may be analyzed directly in the retentate
area using multiwell fluorimetry, spectrophotometry, or
luminometry. Moreover, the present invention may be adapted to
perform diafiltration.
[0048] In this regard, the configuration of a filter membrane which
is sealed about its ends to a ported tube with a conical end
advantageously places the desired retentate in a small, central,
distal region, which is both on-axis, and is displaced from the
filter membrane. Thus, a pipette may accurately reach the retentate
without touching the filter membrane. The cone itself preferably
has an apex angle of about 10.degree. to about 35.degree., and most
preferably of about 10.degree. to about 20.degree., such that the
surface of the filter membrane in this region lies at a steep angle
of only about 5.degree. to about 10.degree. with respect to the
centrifugal force vector. This promotes a continuous sloughing of
the denser gel-like, higher molecular weight retentate material
that builds up on the filter surface, to efficiently channel that
retentate centrifugally down to the apex. Furthermore, by
minimizing accumulation of polarized retained macrosolutes, a
higher flux rate and minimal retention of smaller molecules is
achieved throughout the centrifuging cycle.
[0049] FIG. 9 illustrates an alternate embodiment 30' of the tube
30 of FIG. 3, in which a filter membrane 10' extends somewhat
distally to the port area(s) 38. This advantageously increases the
area 20 of the portion of the filter membrane 10' which remains
active toward the end of the centrifuging cycle when approaching
the desired retentate volume. Further, this extension beyond the
port produces hydrostatic deadstopping, which, in turn, results in
a faster approach to the desired volume, by a mechanism such as
described more fully in U.S. Pat. No. 4,632,761 of Bowers et al.
Alternatively, the distal seal portion 52 may be situated
essentially at the level of the sill of the distal-most port 38. In
this case, deadstopping is provided solely by sequestering, i.e.,
by the isolation of the retentate in the small, cup-shaped apex of
the tube 40.
[0050] When the described embodiment is made with a 0.6 milliliter
microcentrifuge tube and is spun on a rotating platform device, the
final retentate volume is approximately 0.006 milliliters. When
spun in a fixed angle 45.degree. rotor, the retentate volume is
about 0.002 milliliters. As can be seen from the approximate scale
of FIG. 9, approximately 75 percent of the 0.6 milliliter volume of
the tube lies above the proximal seal portion 50 of the filter
membrane 10', so that the 1.0 cm.sup.2 area of this design, which
is twofold larger than prior art devices of this volume range, thus
results in an exceptionally high ratio of filter membrane active
area to fluid volume throughout the course of volume reduction.
This is expected to enhance the speed of protein ultrafiltration by
a factor of at least two over that of known devices. Further
improvement in protein filtration rate, particularly at
transmembrane pressures in excess of 150 psi which are obtained
when the device of FIG. 9 is centrifuged above 12,000 rcf, will
result if the inner wall of tube 30 in the region adjacent the
active membrane area 20 is molded with a rough textured pattern
which provides microchannels for filtrate to flow laterally, along
the interstitial space between the membrane and the wall, to the
port or ports. The device shown in FIG. 10 advantageously has three
times the filter area of the embodiment of FIG. 9 and a two-thirds
greater sample volume capacity, thus further increasing both the
throughput and the speed of DNA diafiltration. Further, the
cellulosic membrane covers all portions of the plastic chamber
walls except the tip, effectively preventing adsorptive loss of DNA
to the surface of the polymer vessel wall. In addition, as
described above in regard to FIG. 9, the filter may be positioned
for hydrostatic deadstopping to more quickly reach an endpoint.
With this enhanced cycle speed, it becomes efficient to reach a
desired degree of purity, for example, to effectively remove PCR
primers, by simply performing successive diafiltration cycles. The
device thus provides a new and effective method for use in DNA
amplification to remove unused primers and harvest the PCR product
after amplification.
[0051] In this regard, it has been reported (Amicon Publication
304) that optimal retention of PCR DNA product larger than about
500 bp and clearance of smaller oligonucleotide primers using
YM-100 regenerated cellulose membranes in the Centricon.RTM. 100
device requires filtration velocities of no more than one
millimeter per minute. The present device of FIG. 9 has threefold
greater active surface area than any currently available
microcentrifuge device that employs the regenerated cellulosic
membranes needed for high recovery of DNA, and thus may be expected
to result in as much as a threefold reduction in the time required
to diafilter DNA at one millimeter per minute.
[0052] In one preferred embodiment of the present invention, the
filter membrane 10 is formed of a two-layer filter medium including
an inner regenerated cellulose ultrafilter cast on an outer porous
film of ultra-high molecular weight microporous polyethylene
(UHMWPE). One suitable material is a material sold by Millipore as
their PLCCC, PLGCD, PLCGC, PLCTK, or PLCHK filter sheet material
which is used in purification systems and has been found to retain
globular solutes having an average molecular weight over 5,000,
10,000, 30,000, 70,000 or 200,000 Daltons, respectively. Another
suitable material is a regenerated cellulose on Freudenberg or
Tyvek backing, such as the material available from the Kalle
company in Germany, or available from the Amicon Division of
Millipore as their YM line of membranes.
[0053] The regenerated cellulosic membrane does not melt, while the
polyolefin backing material of the membrane has a higher than or
similar melting point to conventional polypropylene microcentrifuge
tubes, and compatibly self-welds thereto. This property permits
simple and clean device fabrication with a heated conical inserter
60 as described above. Advantageously, after heat-welding the
filter membrane 10 against the tapered conical vessel wall surface,
the tapered inserter 60 itself, when withdrawn, pulls away from,
rather than along, the non-adherent, inner (e.g., cellulose)
surface of the filter membrane. Thus, axially press-welding the
membrane with a heated tool in this fashion produces no surface
abrasion, and the delicate filter membrane skin surface is not
damaged. That is, the geometry of contacting along a conical
surface results in the tool retracting along a surface release
direction, avoiding shear or tearing of the delicate filter
material. Prototype testing of this technique showed remarkably
high filter integrity, demonstrating manufacturing feasibility.
[0054] The advantageous filter rate and efficiency properties noted
above are also achieved with the filter membrane 10 also positioned
in or extending to the cylindrical portion of the separation tube
30. In such a case, the conical tip 37 of the tube 30 may be made
shorter, while the cone angle may be larger, or the tube may
otherwise be configured to hold the desired volume of retentate. In
each case, the filter membrane extends over and is supported by the
peripheral wall of the vessel. This construction using a filter
over the perimeter wall may also be applied to cylindrical
centrifuge tubes or to purely conical tubes as mentioned above. In
each case, a relatively large filter is primarily supported by
solid wall, providing a high ratio of active filter area to
reservoir volume, while small ports provide escape for the
filtrate.
[0055] In providing a concentration vessel wherein the filter is
coextensive with a region of the peripheral wall and the retentate
is captured in a conical tip, applicant's vessel may be seen to
both increase the available area and openness of the filter while
allowing effective recovery of extremely small volumes with high
efficiency. The ratio of sample volume to retentate volume may be
controlled by the relative height of the permeate ports and the
provision of increased sample volume in the proximal cylindrical
portion of the reservoir. Furthermore, selection of the effective
membrane pore size allows a high degree of control over the
ultimate percentage recovery and required spin down time.
[0056] The vessel of FIG. 9 may be implemented in standard sizes
identical to those of existing concentration or sedimentation
tubes. For example, that device can be configured using a
commercial 0.6 mL microcentrifuge tube to form the retentate
reservoir. This size tube can be accommodated in a one-and-a-half
to two mL filtrate tube for use with small numbers of samples in a
conventional 45.degree. fixed-angle microcentrifuge. Alternatively,
the same basic device may be arrayed in 8.times.12 racks above a 96
well microtiter tray used with a swinging platform rotor. As noted
above, the open conical retentate well is suited to robotic sample
addition and retentate harvesting using conventional laboratory
robotic equipment. Furthermore, with a 0.6 mL sample volume and a
five microliter retentate sump, the concentrator achieves
concentration by a factor of over one hundred.
[0057] However, scaling up the vessel size encounters several
significant limitations if the larger vessels are to be compatible
with existing centrifuge equipment. Thus, for example, if a similar
tube is to be used with a standard 15 mL sample or sedimentation
tube, the reception of filtrate below the retentate sump imposes
limits on the size of the microcentrifuge vessel and its contents,
whereas if one were to use a 50 mL tube, the lower rotational
speeds of the required large capacity centrifuge would
significantly limit separation speed of devices using smaller pore
size filter membrane to retain molecules in the range of 5,000 to
20,000 Daltons.
[0058] Applicant addresses these limitations in a further
embodiment of the invention illustrated in FIGS. 12A-12C, to
provide a larger effective batch size while achieving a an even
higher concentration ratio, over 2000:1, that is suitable for
concentrating extremely dilute samples or for improved
diafiltration.
[0059] FIGS. 12A and 12B illustrate a 7 mL centrifuge tube 130,
constructed in accordance with this embodiment of the invention,
and positioned within a 15 mL sample tube 140 of conventional type
and having a closure cap or lid 141. As shown, when the assembled
pair of vessels reside in a 28.degree. rotor, 6.06 ml. is the
maximal volume of filtrate that may pass into the receiver vessel
140 before reaching the same height as that of the hydrostatic dead
stop in the retentate area below the port of vessel 130, when that
port is positioned to define a three microliter retentate sump.
Furthermore, as shown in FIG. 12A at the same angle, when the
microcentrifuge vessel of seven milliliters total capacity is
filled to 5.2 mL or more, the meniscus in an angled rotor will be
at or above the top of the inner vessel 130 (the right-hand side as
shown). Thus, the volume of sample accommodated in the
microcentrifuge tube as well as the volume of filtrate which must
be accommodated in the receiving vessel below the tube both clearly
impose limits on the amount of material which may be processed in
the vessel without spillage of sample or decanting of filtrate, and
these limits are decreased when using a fixed angle rotor. For the
illustrated three microliter retentate volume, the concentration
range achieved by the vessel is 666:1 from a 2 mL sample, and rises
to 2333:1 from a 7 ML sample. Thus, the limitation of 5.2 mL
imposed by the spill line (FIG. 12A) of the centrifuge tube limits
the achievable concentration to an intermediate value of about
1733:1.
[0060] Applicant addresses this limitation in the further
embodiment of the invention by providing a separation vessel for
fitting within a larger receiving tube having a cap, and wherein
the separation vessel is configured with a check seal operating as
an internal relief vent to both retain overfilled sample under
resting oblique orientation and the high pressure conditions during
initial centrifuge operation, and to cycle air pressure from the
filtrate chamber to the retentate chamber under the negative
pressure conditions that arise as the sample level subsides in the
separation vessel and pressure rises in the receiving tube during
centrifuging.
[0061] FIG. 12C shows an enlarged sectional view of a preferred
implementation of this releasing seal construction in the
separation vessel 130 of FIG. 12B. As shown, the outer wall 131 of
vessel 130 extends upward to a flange 132 that rests upon the body
of the receiving vessel 140. The receiving vessel 140 is closed by
a cap 141 having a seal gasket 142, and the flange 132 of the
separation vessel 130 extends to a top surface 134 which bears
against the gasket material 142. This may be a compressible
urethane foam gasket material in the cap 141. As shown in the
detailed enlarged view of FIG. 12C, the upper surface 134 of the
separation tube comprises a check seal lip 134a extending upwardly
at an inwardly-directed angle against the gasket to form a
fluid-tight band closing the top of the separation tube 130 against
the gasket. The lip 134a is sufficiently thin and is disposed at an
angle so that, as pressurized air is forced up between the outer
wall 131 of the separation vessel and the inner wall of the
surrounding receiving tube 140, the increasing pressure deflects
the lip 134a downwardly, thus allowing air pressure to pass from
the vessel 140 into the separation tube 130. For this operation, a
molded bypass passage, best seen as passage 212 in FIGS. 11B and
11C communicates between the surrounding vessel and the space
surrounding the lip 134a. As shown in FIG. 12A, this seal
arrangement allows the separation tube 130 to be overfilled, that
is, to be filled to such a high level that when the tube 130 is
placed in a tilted rotor it wets a substantial portion of the
gasket 142 lying above it, and the lip seals against the relatively
high outwardly-directed pressure that initially develops in that
area during rotation at high speed before the fluid level drops,
without leaking out of the vessel 130 into the receiving tube 140,
or leaking outside the cap into the centrifuge drum. The separation
tube may therefore be loaded to a 7 mL capacity rather than the
lower, angled overflow level of 5.2 mL, thereby achieving 34%
greater effective reservoir capacity and a correspondingly
increased concentration ratio of 2333:1. For this purpose, tube 130
is used in cylindrical receiving tube of larger capacity than the
existing commercial 15 mL tube of FIG. 12A, B. For example, a
receiving tube such as tube 240 modified as illustrated in phantom
in FIG. 11C having a geometry effective to hold 7 mL of filtrate
below the port of the vessel 130 at the rotor tilt angle allows the
enhanced capacity of the vented filter vessel 130 to be fully
exploited in a fixed angle rotor. Such a receiving vessel may be
accommodated with simple modifications to existing rotors or
fixtures. The cap gasket may be formed of a higher modulus material
than the polymer conventionally used in centrifuge tube cap gaskets
to achieve, in conjunction with the deflectable lip, a suitable
positive pressure sealing and negative pressure release
characteristic.
[0062] As further seen in FIG. 12C, the outer portion 134b of the
rim of vessel 130 seats firmly against the cap gasket 142, while
the bottom circumferential edge 134c of the flange 132 seals
against the top of the collection tube 140, thus providing a double
seal to prevent leakage out of the collection tube 140. The
illustrated seven milliliter vessel may have a filter with an area
of 5.5 cm.sup.2, allowing fast spin-down times to be achieved with
the enhanced batch size.
[0063] It bears emphasis that the separation vessels and methods of
the present invention involve operation at very high rotational
speeds, and it is therefore necessary that the filter be well
attached to the underlying vessel wall so as to avoid any potential
leakage paths, and also be well supported by the wall to prevent
sagging and membrane rupture. Preferably, the inner wall of the
separation vessel is formed with a rough surface, so that while it
supports the filter over its full area, it also permits filtrate to
flow or percolate between the outside of the filter and the inside
wall of the vessel. Effectively, retentate sloughs off the inner
wall of the filter to the sump, and filtrate seeps along the outer
wall to the ports, thus keeping both the filter and the filtrate
flow paths open.
[0064] For installation of the filter membrane against a conical
surface of the vessel, applicant further contemplates that inner
surface of the separation vessel be provided with one or more
alignment structures, as illustrated in FIGS. 11A-11C. These
figures illustrate the bare vessel (FIG. 11A), its assembly with a
filter membrane (FIG. 11B) and the completed separation vessel
mounted in a receiving tube (FIG. 11C). Each figure includes a
downward facing top plan view, illustrating orientation or relative
position of the components of the vessel such as ribs, port and
sealing lip, and also includes a vertical section along the plane
identified in the corresponding top view.
[0065] FIG. 11A illustrates a top view (upper panel) and vertical
section side view of a separation vessel 230 such as the vessel of
FIG. 12 illustratively having a seven milliliter capacity. As best
seen in the top views, the vessel has four ports 238 equispaced
about its circumference, and further includes an alignment guide
having the form of a rib or blade 231 that projects radially inward
from the vessel wall along a diametral plane. Rib 231 is shown
extending from near the mouth (top) of the vessel 230 to a position
slightly above the bottom of the ports 238. Further the rib 231,
which may, for example, be approximately one-half to two
millimeters wide, is positioned in a sector between the ports and
extends radially to form an elevated wall that catches and aligns
the edges 210a, 210b of the filter membrane 210 (FIG. 11D) as it is
inserted in the vessel. The membrane 210 preferably is sized or
subtends an angle such that the filter edges 210a, 210b butt
against the rib on each side of the rib, and the filter 210 bows
outwardly in alignment against the vessel wall. As illustrated, the
membrane is adhered to the vessel wall along edges 210a, 210b by
sealing bands 215a, 215b, respectively, and is further attached at
the top and bottom edges 210c, 210d by perimeter sealing bands
215c, 215d. Preferably, the vessel 230 also is formed with a
circumferential ledge 234 formed by an indentation of the vessel
wall at a height to capture, align and retain the membrane as it is
initially inserted into the vessel. That is, the upper edge snaps
into position below the ledge 234 after the filter has been
inserted down to a level that covers the ports 238. This fully
stabilizes and positions the filter, allowing adhesive (if used) to
set, or allowing a fusing or welding tool (if used) to be inserted
and moved to join the filter to the vessel wall without risk of
dislodging or misaligning the filter.
[0066] Advantageously the sealing bands 215a-215d occupy relatively
little of the filter area; preliminary tests indicate that a band
0.5-0.75 mm wide, and having a net surface area of under one square
centimeter will dependably seal a filter of five times that area
against the wall of the large vessel of FIGS. 12A-12C discussed
above. Additional sealing bands a, b, c as illustrated in phantom
in FIG. 11D may also be provided to secure the central region of
the filter to the vessel wall and prevent ballooning caused by the
weight of the filter as the filter area becomes uncovered in
approaching the final deadstop volume.
[0067] As noted above, the installation of filter membrane in the
vessel 130 requires sealing of the filter to the vessel wall around
a peripheral area without damaging the filter itself. It is
desirable that this operation be highly automated for the
manufacture of the concentration vessels of the invention. For this
purpose, impulse welding with a shaped mandrel having heat actuated
wire or ribbon element as described above may be used to
selectively apply heat while quickly cooling down after the fusing
operation. Another approach is to use a shaped press iron inserted
against the filter. In both cases, the vessel may be supported by
an external support to prevent deformation of the vessel.
[0068] One suitable set of tools for so heat-welding the filter to
the vessel and the steps of this operation are illustrated in FIGS.
13A-13F. FIG. 13A illustrates a separation vessel 230, which may be
identical or similar to any of the above-described vessels, having
a filter membrane 210 positioned therein and covering its port 238.
Fastening of the filter 210 in vessel 230 proceeds as follows. The
vessel 230 is placed against a receiving heat sink 240 as shown in
FIG. 13B. Heat sink 240 may be a heat conductive block of material
such as copper and having a recess shaped to receive the vessel 230
and conforming to the region of the vessel 230 at which the filter
is to be bonded, e.g., the conical tip. An entry bore permits
insertion of the vessel 230, and the entry bore of block 240 is
somewhat oversized, providing a small clearance or circumferential
relief in the upper access region 241 surrounding the vessel.
[0069] A thimble-like or elongated hollow heat transfer tool 250 is
then inserted into the vessel along the axis of the vessel down to
the tip thereof. The heat transfer tool 250 has a generally
elongated shaft portion 251 and a tip portion 252. The tip portion
has a conical inner taper, and a thick wall with outer surface
protuberances described more fully below, to function as a somewhat
thickened hollow thimble element to function as a heat receiving
and transfer member, and to press against the filter selectively in
the areas to be welded. The outer contour of a welding tip portion
252 is not radially symmetric, but includes vertically running
protruding ridge 252a, 25b corresponding to the butt weld of the
filter along lines 215a, 215b (FIG. 11D), and may have ridges or
bumps in positions corresponding to the tack lines a, b, c of FIG.
11D, as well as a circumferential ridge 252c corresponding to the
upper edge perimeter weld 215c of FIG. 11D, and a lower enlarged
tip portion 252d which protrudes radially to contact and press the
filter against the inner wall of the vessel 230 at the filter's
lower edge (region 215d at edge 210d of FIG. 11D). Each of the
ridges has steep edges and a well defined upper surface, forming a
narrow strip of contact area protruding about thirty mils outwardly
of the non-contact portion of the thimble surface. Thus, outside
the intended weld lines, the thimble surface has a relief effective
to avoid heating the non-weld areas of the filter. The ridges 252a,
252b straddle the centering rib 231 (FIG. 11A), and may for example
constitute a single ridge having a narrow slot to accommodate the
rib without contact. When the tool 250 is dropped into the vessel,
this slot may orient the tool in the vessel. The various ridges,
and bumps at tack line positions, further serve to center the heat
transfer tool when it is dropped into the vessel, assuring that
when a high level of heat is later applied the intended welds will
be uniform and the vessel itself will not rack.
[0070] The heat transfer tool 250 has a central bore 255 into which
a heat applicator rod 260 is then inserted and advanced down to
contact the inner wall of the thimble region 252 of the transfer
element 250 and transfer heat into the transfer tool wall along its
contact region 253. As noted above, this thimble area of the
transfer tool has a thin wall, giving it a minimal thermal mass and
rapid heating and cooling characteristics. Heat is then transferred
to the surrounding filter. The heating rod 260 contacts the thimble
intimately, to uniformly and quickly elevate its temperature, but
heating of the filter preferentially occurs at the locations of the
protruding ridges or bumps on the external surface of the transfer
member 250, which are close to, or bear against, the vessel wall.
The tip of heating rod 260 fits precisely in the surrounding
transfer tool, and each of the tools 250, 260 is independently held
at its upper end to allow precise insertion and removal without
binding of the two elements of the heating assembly, and to allow
removal of the heating rod 260 without upsetting the bonded filter
as it cools and sets.
[0071] The transfer tool 250 or its tip region 252 may be preheated
or warmed a moderate amount before insertion in the vessel 230.
However, in accordance with a principal aspect of this assembly
method, the heater rod 260 forms the primary heat source and is
superheated, i.e., heated to a temperature which is quite high,
e.g., hundreds of degrees higher than the melting point of the
vessel material or the filter backing, so that it provides a fast
and controlled impulse of heat to bring the transfer element up to
a temperature above fusing. By way of example, the heater rod 260
may be maintained at a temperature of about 400-475.degree. C.
(750-875.degree. F.) for quickly and effectively applying thermal
energy to the transfer member 250 when inserted therein. The
transfer member 250 may initially start at a low temperature,
between ambient and 80.degree. C.
[0072] In general, applicant contemplates that the heat welding of
the filter in position in this manner is carried out as shown in
FIG. 14. First the filter is positioned in the vessel, and the
filter/vessel is placed with the transfer tool on one side of the
filter while the heat sink supports the vessel on the other side.
Welding is then preferably effected in two stages by a preheating
stage which may be accompanied by a partial compression of the
filter in the areas contacted by the ridges of the transfer tool,
followed by a compression and heating stage in which the heat
transfer tool is advanced to bear more strongly against the filter
to assure continuous fusing along the intended bond lines. The
second-stage increased pressure may be applied by advancing the
heater rod 260 five or ten mils further and/or applying greater
pressure via the heater rod 260, or, equivalently, raising the sink
240 by a small distance. The heating member 260 is then withdrawn
and the assembly is allowed to set and cool before removal of the
heat transfer tool from the vessel, and removal of the vessel from
the heat sink block.
[0073] These steps are illustrated in greater detail in FIGS.
13C-13F. As shown in FIG. 13C during the preheating stage, heating
rod 260 is advanced into the transfer tool 250 with moderate or
slight pressure or compression so that its conical tip lies against
the inner wall of the heat transfer thimble 250 and the various
protruding ridges or dots 252a, 252b. . . come into contact with
the surrounding filter. By way of example, with a filter membrane
approximately 10 mils thick, the protruding ridges may compress the
filter partially under the edge weld line positions 283 and at
circumferential band positions 281, 282, for example by 2 to 5
mils, and the thimble surface otherwise resides above the filter
elsewhere as seen at non-bonding region 284 without transferring
any appreciable heat thereto. At this stage the non-protruding
portions of the heat transfer thimble exterior surface reside ten
to fifty thousandths of an inch above the filter surface, so only
minor heat is transferred, inefficiently, by radiation or
convective means without direct contact or thermal conduction. As a
result, the filter is preferentially heated in the bonding areas,
so water in the filter material weld regions may evolve as steam
and has ample space for escaping in the gap between the transfer
tool 250 and the inner wall of the vessel 230 and filter. By way of
illustration, the heater rod 260, initially at 740-900.degree. F.,
may bring the transfer member to about 250-300.degree. F. during
the preheating stage for attaching a regenerated cellulose membrane
to a polypropylene vessel, and preheating occurs in bonding strips
ten to eighty mils wide.
[0074] After maintaining the preheating position for several
seconds to allow escape of the steam, further pressure or movement
is then applied to heater rod 260 or heat sink 240, and the
transfer tool and rod advance further into the vessel 260, firmly
establishing thermally conductive contact and sealing the filter
along the perimeter and other intended weld bands against the wall
of the vessel. This welding at full compression is illustrated in
FIG. 13D. At this point, the transfer tool temperature may
illustratively lie in the range of 350-400.degree. F., while the
temperature of the heater rod 260 may have fallen appreciably,
e.g., to about 480-600.degree. F. Following closure of clamps 270
to hold the transfer tool, the heater rod 260 is then withdrawn,
allowing the temperature of the vessel to fall by heat conduction
into the sink 240 and up the shaft of the transfer member 250 into
clamp 270, until the bond has set. In this manner a controlled
bolus of heat is preferentially transferred into regions of the
delicate filter to weld without abrading or injuring the filter
itself. The heat transfer tool is then removed and the finished
vessel 230 is withdrawn from the heat sink. As seen in FIG. 13F,
the bond lines 215a, 215c, 215d, a, b are clearly visible in the
completed assembly due to fusing together of the vessel wall and
backing material in those areas.
[0075] This manufacturing method has great advantages in that the
thermal mass of the transfer member 250 and the heating rod 260 are
precisely determined, and their starting temperatures and the
residence time of the heating rod in the heat transfer member 250
may both be set so that precisely controlled amounts of heat are
applied to the filter, both for preheating and fusing, while no
actual movement of the filter or shearing motions occur when the
assembly is at elevated temperature. The heat sink 240 establishes
a sharp thermal gradient through the wall of the vessel, allowing
the body of the vessel 230 to remain intact and assuring a fast
setting time, while high levels of pressure may be applied to
assure complete fusing along the narrow bonding lines and tack-down
regions of the filter.
[0076] The heating rod 260 may be maintained at the desired
temperature by securely mounting it in a larger heated block, for
example a copper block maintained at 900.degree. Fahrenheit, and
may be instrumented, for example with an internal thermocouple, to
conveniently monitor tip temperature and control its reheating or
residence cycles. Optionally an internal heating element may be
incorporated into the heating rod to shorten thermal recovery time
between welding cycles. Moreover, by selectively driving off water
from the membrane material to be welded, the process prevents
bubble defects from arising in the fused areas, or buckling of the
filter, and dries the relevant filter area without impairing the
activity of the cellulosic material of the filter media, which
remains hydrated without loss of function.
[0077] The filter attachment method of FIGS. 13 and 14 may be
applied more generally to other geometric configurations, such as
flat window figure configurations, or the open curved sheet or clam
shell construction of FIGS. 10A-10C. In this case when applied to
the multiple half-vessel shells of FIGS. 10A-10C, the transfer
member may be a sheet-like member having protruding contour
conforming to the general shape of the vessel wall with
press-protrusions for effecting the desired welds. The heat
applying member, rather than a conical tipped rod, may then be
shaped correspondingly, with contact areas conforming to the back
surface of the filter pressing areas of the transfer member. In
each case, the use of a transfer plate to hold the vessel and
filter assembly with minimal movement against the filter surface,
and to modulate the heat transfer characteristics of a super heated
heating assembly that is temporarily moved into position to
initiate press-welding, serves to insure the integrity of the
filter while forming uniform and dependable bond lines between the
filter and the vessel wall.
[0078] One skilled in the art will appreciate further features and
advantages of the invention based on the above-described
embodiments. Accordingly, the invention is not to be limited by
what has been particularly shown and described, but will be seen to
include further variations, modifications and adaptations within
its scope, as defined by the claims appended hereto and equivalents
thereof. All publications and references cited herein are expressly
incorporated herein by reference in their entirety.
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