U.S. patent application number 09/863485 was filed with the patent office on 2001-11-01 for recirculation container.
This patent application is currently assigned to Nexell Therapeutics Inc.. Invention is credited to Johnson, Craig L..
Application Number | 20010035377 09/863485 |
Document ID | / |
Family ID | 21710308 |
Filed Date | 2001-11-01 |
United States Patent
Application |
20010035377 |
Kind Code |
A1 |
Johnson, Craig L. |
November 1, 2001 |
Recirculation container
Abstract
A bag or reservoir for recirculation washing of blood cells
having a top outlet port and bottom inlet port. A method of
recirculation washing of blood cells using the bag in conjunction
with a spinning membrane filter. The method can be used in an
instrument for magnetic cell selection or a stand-alone cell
washing apparatus.
Inventors: |
Johnson, Craig L.; (Mission
Viejo, CA) |
Correspondence
Address: |
OPPENHEIMER WOLFF & DONNELLY LLP
840 NEWPORT CENTER DRIVE
SUITE 700
NEWPORT BEACH
CA
92660
US
|
Assignee: |
Nexell Therapeutics Inc.
|
Family ID: |
21710308 |
Appl. No.: |
09/863485 |
Filed: |
May 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09863485 |
May 23, 2001 |
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09004344 |
Jan 8, 1998 |
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6251295 |
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Current U.S.
Class: |
210/645 ;
210/195.2; 210/321.68; 210/436; 210/805 |
Current CPC
Class: |
A61M 1/262 20140204;
A61M 1/3692 20140204; A61M 1/3621 20130101; A61M 1/3603 20140204;
A61M 1/0218 20140204; A61M 1/3618 20140204 |
Class at
Publication: |
210/645 ;
210/805; 210/195.2; 210/321.68; 210/436 |
International
Class: |
B01D 061/14; B01D
063/16 |
Claims
1. A method of recirculation washing of blood cells which utilizes
a flexible plastic recirculation wash bag or reservoir having a top
port and a bottom port in conjunction with a spinning membrane
filter having an inlet port for a diluted suspension of blood cells
in buffer solution, a first outlet port for filtrate and a second
outlet port for a concentrated suspension of blood cells in buffer
solution, which comprises withdrawing a suspension of blood cells
in buffer solution from the recirculation wash bag through the top
port, mixing the suspension with additional buffer solution to form
a diluted suspension of blood cells in buffer solution, feeding the
diluted suspension into the spinning membrane filter through the
inlet port, withdrawing filtrate comprising buffer solution from
the spinning membrane filter through the first outlet port,
withdrawing a concentrated suspension of blood cells in buffer
solution from the spinning membrane filter through the second
outlet port, feeding the concentrated suspension into the bag
through the bottom port, and continuing the recirculation washing
until the desired amount of washing has been achieved.
2. The method of claim 1 wherein the suspension of blood cells
withdrawn through the top port of the recirculation wash bag is
mixed with unwashed blood cells as well as buffer solution before
feeding the diluted suspension into the spinning membrane
filter.
3. The method of claim 2 wherein the unwashed blood cells include
platelets, the filtrate comprises a suspension of platelets in
buffer solution, and the recirculation washing is continued until
the platelet content of the concentrated suspension of cells has
been reduced to the desired level.
4. The method of claim 1 wherein the recirculation wash bag at the
beginning of the recirculation wash procedure contains, in addition
to blood cells, an antibody which specifically binds an antigen on
certain of the blood cells which, the filtrate comprises a
suspension of the antibody in the buffer solution, and the
recirculation washing continues until the concentrated suspension
of cells contains a desired level of free of excess, unbound
antibody.
5. The method of claim 1 wherein washing is continued until the
fraction of starting residual has reached a predetermined value as
determined by the equation:
FSR.sub.i=FSR.sub.i-l-(F.sub.i/(B.sub.i+C.sub.i).times.(C.s-
ub.i/V.sub.i).times.FSR.sub.i-1.times.TA where i=the discrete time
interval FSR.sub.i=Fraction of Starting Residual at time t.sub.i
FSR.sub.i-1=Fraction of Starting Residual at time t.sub.i-1
F.sub.i=Filtrate volume moved at rate f measured at time interval
i-1 to i in units of ml B.sub.i=Buffer volume moved at rate b
measured at time interval i-1 to i in units of ml C.sub.i=Cell
source moved at rate c measured at time interval i in units of ml,
including the rate from the IsoFlow.TM. bag 5, as well as the rate
of addition of unwashed cells, if any, in same units V.sub.i=cell
product volume at time interval i in ml TA=Target Admittance, and
residual is the component which the cell washing is targeted to
reduce.
6. A flexible plastic bag or reservoir for recirculation washing of
blood cells which has a top port and a bottom port and an integral
coarse filter comprising a tube of semi-rigid plastic mesh
extending from the top port into the bag.
7. A flexible plastic bag or reservoir for recirculation washing of
blood cells which has a top port and a bottom port and a bubble
trap at the top which comprises plastic tubing extending into the
bag from the top port.
8. A flexible plastic bag for recirculation washing of blood cells
which has a top port and a bottom port, an integral coarse filter
comprising a tube of semi-rigid plastic mesh extending from the top
port into the bag and having a closed bottom end and a bubble trap
at the top which comprises plastic tubing extending from the top
port into the bag inside the mesh tube.
9. Bag of claim 8 wherein the mesh tube is sufficiently rigid that,
when vacuum is pulled on the bag, causing it to collapse, the mesh
tube holds an open path in the bag, so that blood cells in a buffer
solution entering the bottom port can move up to the top port.
10. A disposable set for recirculation washing of blood cells
comprising a recirculation wash bag which has a top port and a
bottom port, a spinning membrane filter which has an inlet port for
a diluted suspension of blood cells in buffer solution, a first
outlet port for filtrate and a second outlet port for a
concentrated suspension of blood cells in buffer solution, and a
filtrate bag, plus associated tubing, including tubing for a buffer
solution bag, wherein plastic tubing connects the top port of the
recirculation wash bag to a mixing zone, plastic tubing with a
buffer bag spike coupler at one end is connected to the same mixing
zone, the mixing zone is connected by plastic tubing to the inlet
port of the spinning membrane filter, the first outlet port of the
spinning membrane filter is connected by plastic tubing to the
inlet port of the filtrate bag, and the second outlet port of the
spinning membrane filter is connected by plastic tubing to the
bottom port of the recirculation wash bag.
11. A disposable set of claim 10 wherein the recirculation wash bag
has an integral coarse filter comprising a tube of semi-rigid
plastic mesh extending from the top port into the bag.
12. A disposable set of claim 10 wherein the recirculation wash bag
has a bubble trap at the top which comprises plastic tubing
extending into the bag from the top port.
13. A disposable set of claim 10 wherein the recirculation wash bag
has an integral coarse filter comprising a tube of semi-rigid
plastic mesh extending from the top port into the bag and having a
closed bottom end and a bubble trap at the top which comprises
plastic tubing extending from the top port into the bag inside the
mesh tube.
14. The disposable set of claim 10 which also includes other bags
and associated tubing for use in a magnetic cell selection
instrument, including a bag for antibody suspension in buffer, a
bag for peptide release agent solution in buffer, a bag for a
suspension of selected cells in buffer solution, and a bag for a
suspension of non-selected cells in buffer solution.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to recirculation washing of blood
cells using a spinning membrane filter, and in particular to
recirculation washing of blood cells in a magnetic cell selection
apparatus.
[0002] Fischel U.S. Pat. No. 5,034,135, issued Jul. 23, 1991, and
Schoendorfer U.S. Pat. No. 5,035,121, issued Oct. 1, 1991 disclose
spinning membrane filters comprising a cylindrical housing and
concentric grooved cylindrical rotor. The rotor is covered with a
membrane the membrane is spaced from the inner wall of the housing.
Blood is introduced into the gap between the membrane and housing.
Filtrate passes through the membrane, into the grooves of the
rotor, into tubes which communicate with the grooves, and out the
bottom center of the spinning membrane filter. Concentrated cells
are removed from the gap. FIGS. 7 and 8 in the Fischel patent
illustrate a cell washing modification in which a porous wall is
interposed between the membrane and the inner wall of the housing.
Blood is introduced into the gap between the membrane and the
porous wall and an isotonic wash solution is introduced into the
gap between the porous wall and the inner wall of the housing. FIG.
6 in the Schoendorfer patent illustratse introduction of a rinse
solution with the blood. Schoendorfer et al. U.S. Pat. No.
5,035,121, issued Oct. 1, 1991, discloses use of two spinning
membrane filters in series or parallel. A washing solution is
introduced into at least one of the spinning membrane filters.
[0003] Duff U.S. Pat. No. 5,234,608, issued Aug. 10, 1993,
discloses a spinning membrane filter of the type which is preferred
for use in conjunction with this invention. According to the
disclosure, cell-rich concentrate is removed from the upper portion
of the gap between the membrane and the inner wall of the housing,
cell-poor plasma filtrate is removed from the bottom center of the
spinning membrane filter. Source cell suspension is mixed with
cell-rich concentrate and introduced to the lower portion of the
gap area.
[0004] Schoendorfer et al. U.S. Pat. Nos. 4,675,106, issued Jun.
23, 1987, U.S. Pat. No. 4,753,729, issued Jun. 28, 1988, and U.S.
Pat. No. 4,816,151, issued Mar. 28, 1989, disclose drive mechanisms
for spinning membrane filters.
[0005] Moubayed et al. U.S. Pat. No. 5,536,475 discloses a
semi-automated instrument for selection of blood cells using
paramagnetic beads which are coated with a binding agent such as an
antibody which binds specifically to the cells to be selected. The
instrument comprises a primary magnet associated with a primary
container and a secondary magnet associated with a secondary
container. Blood cells, liquid and beads are agitated in the
primary container to form a conjugate between the beads and the
selected cells. The primary magnet is then moved into a position
adjacent the primary container to magnetically capture the
bead/cell conjugate and the non-selected cells and liquid are
removed. The primary magnet is then moved into a position away from
the primary container to release the bead/cell conjugate. Wash
solution is added and the contents of the primary container are
agitated, then the primary magnet is moved into the position
adjacent the primary container to again capture the bead/cell
conjugate and the wash solution is removed. The primary magnet is
again moved into a position away from the primary container to
release the bead/cell conjugate. Liquid containing a reagent which
releases the selected cells from the beads is added and the
contents are again agitated. The primary magnet is again moved into
the position adjacent the primary container to capture the beads.
The released cells and liquid are introduced to the secondary
container. The secondary container is positioned adjacent to the
secondary magnet to capture any beads which may have escaped the
primary magnet. The instrument is used with a disposable set
comprising plastic bags for wash liquid, cell suspension and bead
suspension, interconnected with plastic tubing.
[0006] The semi-automated instrument disclosed in the Moubayed et
al. patent is sold by Baxter Healthcare Corporation. under the
trademark Isolex.RTM. 300 SA. A modified version of the instrument
is sold by the Baxter Healthcare Corporation under the trademark
Isolex.RTM. 300i. The 300i differs from the 300 SA in that it is
fully automated and it includes a spinning membrane filter for
washing the selected cells and also for removing platelets from the
source cells prior to selection.
[0007] Chapman et al. International Publication WO 95/13837,
published May 26, 1995, discloses a peristaltic pumping assembly of
a type which is used to move fluids in the Isolex.RTM. 300 SA and
Isolex.RTM. 300i instruments. Deniega et al. International
Publication WO 95/14142, published May 26, 1995, discloses an
organizer frame of a type which is used with the peristaltic
pumping assembly in the Isolex.RTM. 300 SA and Isolex.RTM. 300i
instruments. The organizer frame is also used on a machine for
separation of platelets from whole blood. Deniaga discloses a
tubing set which includes a spinning membrane filter and a
reservoir for platelet-poor packed blood cells. The reservoir has a
top and bottom port. Packed cells from the outlet of the spinning
membrane filter enter through the top inlet port of the reservoir.
Whole blood from a patient enters through the bottom inlet
port.
[0008] Recirculation washing of selected blood cells is performed
in the Isolex.RTM. 300i utilizing the spinning membrane filter in
conjunction with a recirculation wash bag which has both inlet and
outlet ports at the bottom and no port at the top. The bag is a 600
ml bag with the inlet and outlet ports separated by about 2 inches.
The bag has been able to concentrate cell suspensions that normally
start at about 400 ml. This bag performed better when it was
occasionally massaged. This is the only way to process more than
about 5.times.10.sup.10 cells in the bag.
[0009] The above-cited U.S. patents and International Publications
are each incorporated herein by reference.
SUMMARY OF THE INVENTION
[0010] This invention includes a method, a bag and a disposable set
for recirculation washing of blood cells. The invention can be used
for washing of blood cells in a magnetic cell selection instrument,
but can also be used for washing whole blood or other blood cell
products.
[0011] The recirculation wash bag is a flexible plastic bag which
has a top port and a bottom port. In one embodiment, an integral
coarse filter comprising a tube of semi-rigid plastic mesh extends
from the top port into the bag. This filter provides mild
resistance to larger cell aggregates. In another embodiment, the
bag includes a bubble trap at the top comprising tubing extending
into the bag from the top port. In the preferred embodiment, the
bag includes both the semi-rigid integral filter and the bubble
trap; the tubing for the bubble trap fits inside the plastic mesh
tube to provide a space to accumulate air around the tubing. When a
system incorporating the bag is primed with buffer solution, vacuum
is pulled on the bag. Because the filter is semirigid, it holds
open a path through the otherwise collapsed bag for the cells to
move up to the top port.
[0012] The method of the invention utilizes a flexible plastic
recirculation wash bag and a spinning membrane filter. The spinning
membrane filter has an inlet port for a diluted suspension of blood
cells in buffer solution, a first outlet port for filtrate, and a
second outlet port for a concentrated suspension of blood cells in
buffer solution. The recirculation wash bag has a top outlet port
and a bottom inlet port. Preferably, the recirculation wash bag
includes the integral coarse filter and bubble trap described
above.
[0013] The method comprises withdrawing a suspension of blood cells
in buffer solution from the recirculation wash bag through the top
port, mixing the suspension with additional buffer solution to form
a diluted suspension of blood cells in buffer solution, feeding the
diluted suspension into the spinning membrane filter through the
inlet port, withdrawing filtrate comprising buffer solution from
the spinning membrane filter through the first outlet port,
withdrawing a concentrated suspension of blood cells in buffer
solution from the spinning membrane filter through the second
outlet port, feeding the concentrated suspension into the bag
through the bottom port, and continuing the recirculation washing
until the desired amount of washing has been achieved. A method for
determining when the desired amount of washing has been achieved,
based on an estimate of "residual," is described below. The
residual represents the target component for reduction (e.g.,
platelets, antibody, etc., as described below).
[0014] In one embodiment of the method, the suspension of blood
cells withdrawn through the top port of the recirculation wash bag
is mixed with unwashed blood cells as well as buffer solution
before feeding the diluted suspension into the spinning membrane
filter. In one aspect of this embodiment, the unwashed blood cells
include platelets, the filtrate comprises a suspension of platelets
in buffer solution, and the recirculation washing is continued
until the platelet content of the concentrated suspension of cells
has been reduced to the desired level.
[0015] In another embodiment of the method, the recirculation wash
bag at the beginning of the recirculation wash procedure contains,
in addition to blood cells, an antibody which specifically binds an
antigen on certain of the blood cells, the filtrate comprises a
suspension of the antibody in the buffer solution, and the
recirculation washing continues until the concentrated suspension
of cells contains the desired amount of excess, unbound
antibody.
[0016] In another embodiment of the method, the recirculation wash
bag at the beginning of the recirculation wash procedure contains
blood cells which have been selected in a magnetic cell selection
procedure and a peptide release agent which was used to release the
selected cells from a cell/magnetic bead conjugate, the filtrate
comprises a solution of the peptide release agent in buffer
solution, and the recirculation washing is continued until the
peptide release content of the concentrated suspension of cells has
been reduced to the desired level.
[0017] The disposable set of the invention comprises the
recirculation wash bag and the spinning membrane filter having
ports as described above, and a filtrate bag, plus associated
tubing, including tubing for a buffer solution bag. Plastic tubing
connects the top port of the recirculation wash bag to a mixing
zone. Plastic tubing with a buffer bag spike coupler at one end is
connected to the same mixing zone. The mixing zone is connected by
plastic tubing to the inlet port of the spinning membrane filter.
The first outlet port of the spinning membrane filter is connected
by plastic tubing to the inlet port of the filtrate bag. The second
outlet port of the spinning membrane filter is connected by plastic
tubing to the bottom port of the recirculation wash bag.
[0018] The disposable set may also include other bags and
associated tubing for use in a magnetic cell selection instrument,
such as a bag for antibody suspension in buffer solution, a bag for
peptide release agent solution in buffer solution, a bag for a
suspension of the non-selected cells in buffer solution, and an end
product bag for washed cells. A bag for unwashed cells (also
referred to as a cell source bag) and/or a bag for buffer solution
may be included in the set, but in the preferred embodiment these
items are supplied separately.
[0019] Use of a flexible recirculation wash bag with ports at the
top and bottom and flow from bottom to top provides several
advantages as compared to a bag with inlet and outlet ports at the
bottom, as currently used on the Isolex.RTM. 300i. First, using a
flexible bag allows the volume to be varied depending on the number
of cells. Exiting from the top has the advantage of removing the
less dense supernatant preferentially. This aids in making the
concentration ratio high. (The importance of high concentration
ratio is discussed below). For large volumes or slow flow rates,
some sedimentation of the larger cells also aids in reducing the
cell concentration at the outlet port. The system has the advantage
of having the most washed and most concentrated cells at the bottom
with the least washed and least concentrated cells at the top.
Additional advantages include the following: (1) allows accurate
residual estimates which in turn allow optimal residual levels
instead of just reduction; (2) provides more uniform processing of
cells which leads to a more uniform product for the selection
process; (3) manual massaging of the bag during the wash is not
required, permitting hands-free operation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 illustrates the preferred embodiment of the
recirculation wash bag of this invention. In the description which
follows the recirculation wash bag having the configuration shown
in FIG. 1 is referred to as the IsoFlow.TM. bag.
[0021] FIG. 2 illustrates a disposable set of this invention which
is adapted for use on a magnetic cell selection device such as the
Isolex.RTM. 300i.
[0022] FIG. 3 illustrates a disposable cell wash set of the
invention which is adapted for use on a stand-alone cell washing
apparatus.
DETAILED DESCRIPTION OF THE INVENTION
[0023] IsoFlow.TM. Recirculation Wash Bag
[0024] Referring to FIG. 1, the IsoFlow.TM. bag is indicated
generally by the numeral 5. The bag is made of a flexible plastic
such as and includes bottom port 1 and top port 2. An integral
coarse filter comprising a tube of semi-rigid plastic mesh 3
extends from the top port into the bag to within about 1/2 to 3
inches, preferably about 1 inch, from the bottom of the bag. The
mesh tube is about 1/2 to about 1.5 inches in diameter, preferably
about 1 inch in diameter, and is preferably closed at its lower
end. The tube's mesh (opening) size is in the range of about 80-400
microns, preferably about 230 microns. The bag includes a bubble
trap at the top which is created by inserting tubing 4 into the top
port about 1/2 to 3 inches, preferably about 1.5 inches. Suitable
materials of construction include polyvinyl chloride (PVC) for the
bag, polyester (e.g. Cleartuf.RTM., shell) for the mesh tube
filter, and PVC for the tubing. Volume of the bag can vary, but
will generally be between 100 and 1500 ml. As presently designed
for use on the Isolex.RTM. 300i, the bag holds a volume of 400 ml.
The mesh could be replaced by some other semi-rigid, rigid or
combination structure that facilitates flow from bottom to top.
[0025] Isolex.RTM. 300i Cell Washing System
[0026] Referring to FIG. 2, the disposable set of this invention
comprises the IsoFlow.TM. bag 5 and spinning membrane filter 6 and
associated tubing, including tubing for connecting a bag containing
buffer solution. Spinning membrane filter 6 (sometimes referred to
simply as "spinning membrane" or "spinner") has the construction
shown in FIG. 2 of Duff U.S. Pat. No. 5,234,608. The membrane is a
nominal 4 micron polycarbonate membrane. The buffer solution bag is
not shown, but is indicated at 7; it is a standard flexible plastic
bag with a bottom outlet port, and is supplied separately. The top
port 2 of IsoFlow.TM. bag 5 is connected by tubing 8 having a
sampling device 8a to the bottom right channel 9b (indicated by
dotted lines) of clamp manifold 9. Channel 9b is a mixing zone for
mixing cells from IsoFlow.TM. bag 5 with buffer solution from bag 7
and (in the platelet separation step described below) with unwashed
cells from bag 44. Channel 9b of clamp manifold 9 is connected by
tubing 10 to the inlet port 11 of spinning membrane filter 6. The
bottom port 1 of IsoFlow.TM. bag 5 is connected by tubing 12 to the
bottom left channel of clamp manifold 9 and tubing 13 connects the
bottom left channel of clamp manifold 9 to the outlet port 14 of
spinning membrane filter 6. Tubing 15 connects the outlet port of
buffer solution bag 7 to the top right channel of clamp manifold
16; tubing 17 connects the top right channel of clamp manifold 16
to the bottom left channel of clamp manifold 18; tubing 19 connects
the bottom left channel of clamp manifold 18 to the bottom right
channel of clamp manifold 18 and tubing 20 connects the bottom
right channel of clamp manifold 18 to the bottom right channel 9b
of clamp manifold 9. Tubing 15 is connected to a buffer bag spike
coupler 21 and a sterilizing filter 22. Tubing 23 connects filtrate
outlet port 24 of spinning membrane filter 6 with the top right
channel of clamp manifold 25. Tubing 26 connects the top right
channel of clamp manifold 25 with Y-connector 27. Tubing 28
connects Y-connector 27 to the inlet port 29 of filtrate (waste)
bag 30. On tubing 28 is a clamp 31. Tubing 32 connects Y-connector
27 to Y-connector 33. Tubing 32 carries a clamp 40. Tubing 34
connects Y-connector 33 to inlet port 35 of waste bag 36. Tubing 37
connectes Y-connector 33 to inlet port 38 of waste bag 39. Tubing
41 connects the top right channel of clamp manifold 25 to pressure
transducer protector 42.
[0027] There are three configurations of clamp manifolds shown in
FIG. 2. All configurations have clamps capable of obstructing the
tubing that runs through them on a flat platen (not shown) in the
center of the manifolds. The dotted lines in the upper and/or lower
portions of the clamp manifolds indicate the locations of channels
within the manifolds. The dotted lines in clamp manifold 45 show
that the bottom channel connects all 4 tubes. The dotted lines in
clamp manifolds 9 and 18 show that there are two bottom
channels--the left channel connects the two left tubes and the
right bottom connects the two right tubes. The dotted lines in
clamp manifolds 16 and 25 show that the bottom left channel
connects the tubes on the left and the top right channel connects
the tubes on the right.
[0028] In the preferred embodiment illustrated in FIG. 2, the
disposable set of the invention also includes other bags and
containers and associated tubing adapted for use on a magnetic cell
separation instrument such as the the Isolex.RTM. 300i. Tubing 43
connects a cell source bag (not shown, but indicated at 44) with
the bottom channel of clamp manifold 45. Tubing 46 connects the
bottom channel of clamp manifold 45 with the bottom left channel
18a of clamp manifold 18. Channel 18a is a mixing zone for buffer
from bag 7 and unwashed cells from bag 44. Tubing 43 is connected
to a starting cells spike coupler 47.
[0029] Bag 48 is a bag for antibody which reacts specifically with
cells to be selected on the Isolex.RTM. 300i. For example, where
CD34+ cells are to be selected, bag 48 will contain anti-CD34
antibody. The bag has an injection site 49 for injection of the
antibody solution and an outlet port 50 connected to a sterilizing
filter 51. Tubing 52 connects sterilizing filter 51 to the bottom
channel of clamp manifold 45.
[0030] Bag 53 is a bag for a peptide release agent which displaces
the antibody from the cells after the cells have been magnetically
selected. Bag 53 has an injection site 54a for injection of a
solution of the peptide and an outlet port 54 connected to a
sterilizing filter 55. Tubing 56 connects sterilizing filter 55 to
the bottom channel of clamp manifold 45.
[0031] Cylinder 57 is the primary magnet separation chamber. It has
a vent filter 59 and an injection site 58 for injection of
paramagnetic microbeads coated with an antibody which binds
specifically to the antibody in bag 48. It has a bottom port 60
which serves as both inlet and outlet for cell suspensions. In use
it is mounted on a rocker mechanism as described in Moubayed et al.
U.S. Pat. No. 5,536,475. Port 60 is connected by tubing 61 to the
bottom left channel of clamp manifold 16. That channel is connected
by tubing 62 to the right top channel of clamp manifold 16. The top
right channel of manifold 16 is connected by tubing 72 to the top
right chamber of clamp manifold 25. The bottom left channel of
clamp manifold 16 is also connected by tubing 63 to Y-connector 64
and the latter is connected by tubing 65 to the bottom channel of
clamp manifold 45. Y-connector 64 is also connected by tubing 66 to
a pressure transducer protector 67.
[0032] Bag 68 is the secondary magnet separation bag described in
Moubayed et al. U.S. Pat. No. 5,536,475. It has inlet port 69 and
outlet port 70. Inlet port 69 is connected by tubing 71 to the
bottom left channel of clamp manifold 18. Outlet port 70 is
connected by tubing 73 to the bottom right channel of clamp
manifold 18.
[0033] Bag 74 is a selected cell wash bag. It has two bottom ports.
Inlet port 75 is connected by tubing 77 which has a sampling device
77a to the bottom right channel 9b of clamp manifold 9. Outlet port
76 is connected by tubing 78 to the bottom left channel of clamp
manifold 9. If desired, an IsoFlow.TM. bag can be substituted for
the selected cell wash bag.
[0034] Bag 79 is an end product bag. It has an injection site 80
and an inlet port 81. Tubing 82 carrying sampling device 82a and
clamp 83 connects inlet port 81 with the bottom channel of clamp
manifold 45.
[0035] Frame 84 is an organizer frame as described in Denieaga et
al. International Publication WO 95/14142 for use with a
peristaltic pump assembly (not shown) as described in Chapman et
al. International Publication WO 95/13837. Tubing 13, 15, 26 and 46
each passes through one of the four pumping modules of the
peristaltic pump assembly.
[0036] The volume of bags can vary, depending upon the volume of
cells to be processed. In the the commercial Isolex.RTM. 300i
instrument, each of bags 30, 36 and 39 has a volume of 2000 ml,
each of bags 48, 53 and 79 has a volume of 150 ml, and bag 74 has a
volume of 600 ml. For use in this system, the IsoFlow.TM. bag 5 has
a volume of 400 ml.
[0037] At the beginning of a cell selection procedure, the
disposable set of FIG. 2 is placed on the Isolex 300i. Bag 7
containing buffer and bag 44 containing source cells are attached.
The source cells are typically a leukapheresis product from a cell
separation device such as a Fenwall 3000 CS. The buffer bag has a
capacity of 4000 ml and a starting volume of at least 3500 ml. The
cell source bag has a capacity of 1000 ml and a starting volume of
about 500 ml. By appropriate operation of clamps in the clamp
manifolds and the pumps on tubing 13, 15, and 46, buffer solution
is added to the following elemments and connecting tubing to prime
the system: Isoflow.TM. bag 5, secondary magnet pouch 68, spinning
membrane filter 6, filtrate bag 30, selected cell wash bag 74,
release agent bag 53, antibody bag 48, cell source bag 44. During
the prime, fluid is added to the Isoflow.TM. bag, the air is
removed from the top part of the bag, more fluid is added through
the bottom part, and excess air is released through tubing 8 to
waste bag 30.
[0038] At this point the system is ready for removal of platelets
from the leukapheresis product in cell source bag 44, using the
method of this invention. For purpose of the following description:
clamps in clamp manifold 45 are designated clamps C1, C2, C3, C4;
clamps in clamp manifold 9 are designated C5, C6,C7, C8; clamps in
clamp manifold 16 are designated C9, C10, C11, C12; clamps in clamp
manifold 18 are designated C13, C14, C15, C16; clamps in clamp
manifold 25 are designated C17, C18, C19, C20; the pump on tubing
46 is designated P1, the cell source pump; the pump on tubing 15 is
designated P2, the buffer pump; the pump on tubing 13 is designated
P3, the recirculation pump; the pump on line 26 is designated P4,
the filtrate pump; and the rotor of spinning membrane filter 6 is
designated as pump PS.
[0039] Prior to beginning cell wash, clamps C6, C8, C10, C11, C12,
C14, C16 and C20 are opened, pumps P2, P3, P4 and P5 are moving.
This circulates buffer solution from bag 7, into the inlet port 11
and out of outlet ports 14 and 24 of spinning membrane filter 6,
into bottom port 1 and out of top port 2 of IsoFlow.TM. bag 5, and
into filtrate bag 30.
[0040] To conduct recirculation washing of the blood cells for
platelet removal, clamps C1, C6, C8, C12, C14, C16 and C20 are
open, pumps P1, P2, P3, P4, and PS are moving. A suspension of
unwashed blood cells is withdrawn from cell source bag 44 through
tubing 43 to the bottom channel of clamp manifold 45, then out
through tubing 46 to the bottom left channel 18a of clamp manifold
18 where it is mixed with buffer solution. The buffer solution is
withdrawn from buffer bag 7 through tubing 15 to the top right
channel of clamp manifold 16, then out through tubing 17 to the
bottom left channel 18a of clamp manifold 18. The diluted
suspension of blood cells in buffer solution flows out of the
bottom left channel 18a through tubing 19 into the bottom right
channel of clamp manifold 18, then out through tubing 20 to the
bottom right channel 9b of clamp manifold 9, where it is mixed with
additional buffer solution from top port 2 of Isoflow.TM. bag 5.
The diluted suspension of blood cells in buffer solution flows from
channel 9b through tubing 10 to the inle port 11 of spinning
membrane filter 6. Platelets, a few red cells, and buffer flow
through the membrane and out through outlet port 24 through tubing
23 to the top right channel of clamp manifold 25, then out through
tubing 26 and 28 to filtrate bag 30 (clamp 31 open, clamp 40
closed). (The nominal 4 miron membrane used removes about 95% of
platelets from a leukapheresis product, while about 50% of red
cells are also removed.) A concentrated suspension of blood cells
in buffer flows from the exit port 14 of spinning membrane filter 6
through tubing 13 to the bottom left channel of clamp manifold 9,
then out through tubing 12 through the bottom port 1 into
Isoflow.TM. bag 5. As the process continues, a suspension of blood
cells in buffer solution flows out of the top of the Isoflow.TM.
bag 5. These cells are mixed in mixing zone 9b with unwashed cells
from source bag 44 and are recirculated through the spinning
membrane filter 6. Recirculation washing is continued until the
desired level of platelet removal has been achieved.
[0041] After platelet removal, antibody in buffer solution is
transferred to the concentrated suspension of blood cells in buffer
solution in the Isoflow.TM. bag 5. For transfer of antibody
solution from bag 48 to Isoflow.TM. bag 5, clamps C3, C6, C8, C14,
C16 and C20 are open and pumps P1, P3 and P5 are moving. The
antibody and cells are mixed in mixing zone 9b. Then the antibody
tubing is rinsed with buffer solution while the antibody/cell
suspension circulates through the Isoflow.TM. bag 5 and spinning
membrane filter 6. This occurs with clamps C6, C8, C10, C11, C14,
C16 and C20 open,and with pumps P1, P2, P3 and P5 moving. Next the
antibody/cell suspension is circulated through the IsoflowTm bag 5
and spinning membrane filter 6 to sensitize the cells by binding
with the antibody. This is accomplished with clamps C6, C8 and C20
open, and with pumps P3 and P5 moving.
[0042] After the cells have been sensitized by binding with
antibody, they are washed to remove excess unbound antibody using
the method of this invention. With clamps C6, C8, C12, C14, C16 and
C20 open and with pumps P2, P3, P4 and P5 moving, a suspension of
blood cells in buffer solution and containing excess unbound
antibody is withdrawn from Isoflow.TM. bag 5 through top port 2 and
flows through tubing 8 to the mixing zone 9b in clamp manifold 9.
Buffer solution is withdrawn from buffer bag 7 through tubing 15,
clamp manifold 16, tubing 17, clamp manifold 18 (left channel),
tubing 19, clamp manifold 18 (right channel) and tubing 20, as
previously described, to mixing zone 9b, where it is mixed with the
suspension of blood cells from Isoflow.TM. bag 5 to form a diluted
suspension of blood cells containing excess unbound antibody. This
diluted suspension flows through tubing 10 to inlet port 11 of the
spinning membrane filter 6. Filtrate comprising antibody in buffer
solution flows out of outlet port 24, through tubing 23, clamp
manifold 25, tubing 26, tubing 28, and port 29 into filtrate bag
30. A concentrated suspension of blood cells in buffer solution
flows from the outlet port 14 of the spinning membrane filter 6,
through tubing 13, clamp manifold 9 (bottom left channel), tubing
12 and bottom port 1 into Isoflow.TM. bag 5. The recirculation
washing is continued until the cell suspension contains the desired
level of unbound antibody.
[0043] After antibody sensitization and removal of excess unbound
antibody, the cells are transferred to primary magnet separation
chamber 57. Antibody-coated paramagnetic microbeads are mixed with
the cells to form a conjugate between the microbeads and the
sensitized cells, the conjugate is magnetically separated from the
non-sensitized cells, the non-sensitized cells are transferred to
waste bag 36, peptide release agent from bag 53 is added to the
chamber 57 to release the selected cells, the selected cells are
transferred to the secondary magnet separation bag where any
remining microbeads are separated magnetically, and the selected
cells are transferred to selected cell wash bag 74. The selected
cells are then recirculation washed to remove excess peptide
release agent using spinning membrane filter 6, all in conventional
manner. If desired, selected cell wash bag can be an Isoflow.TM.
bag, and the recirculation wash to remove peptide release agent can
be conducted using the method of this invention. After removal of
peptide release agent, the selected cells are transferred to end
product bag 79.
[0044] Stand-Alone Cell Washing System
[0045] FIG. 3 illustrates a disposable set of the invention which
is adapted for use on a standalone cell washing apparatus, i.e., an
apparatus which does not include a cell selection function such as
the magnetic cell selection of the Isolex.RTM. 300i instrument.
[0046] The disposable set includes Isoflow.TM. bag 5 having top
port 2 and bottom port 1, spinning membrane filter 6 having inlet
port 11 for a diluted suspension of blood cells, outlet port 14 for
a concentrated suspension of blood cells, and outlet port 24 for
filtrate, and filtrate bag 30 having inlet port 29. It may also
include one or more of washed cell bag 79 having outlet port 81,
unwashed cell bag 44 having outlet port 47, and buffer solution bag
7 having outlet port 21. Top port 2 of Isoflow.TM. bag 5 is
connected by tubing 8 to connector 89. Port 21 of buffer bag 7 is
connected by tubing 15 to Y-connector 95 and the latter is
connected by tubing 20 carrying clamp C1 to connector 89. Port 47
of unwashed cell bag is connected by tubing 43 carrying clamp C3 to
Y-connector 93 and then by tubing 91 to connector 89. Connector 89
serves as a mixing zone for unwashed cells in buffer solution from
bag 44, recirculating cells in buffer solution from bag 5 and
buffer solution from bag 7. Connector 89 is connected by tubing 10
to inlet port 11 of spinning membrane filter 6. Filtrate outlet
port 24 of spinner 6 is connected by tubing 23 to Y-connector 94
and by tubing 26 to the inlet port 29 of filtrate bag 30. Connector
95 is connected by tubing 92 carrying clamp C2 to connector 94.
Connector 94 is connected by tubing 41 to pressure transducer 90.
Oulet port 14 of spinner 6 is connected by tubing 13 to the bottom
port 1 of Isoflow.TM. bag 5. Y-connector 93 is connected by tubing
82 carrying clamp C4 to inlet port 81 of washed cell bag 79.
[0047] During recirculation washing, a suspension of blood cells in
buffer solution is withdrawn from the Isoflow.TM. bag 5 through the
top port 2 and flows through tubing 8 to mixing zone 89. Unwashed
cells in buffer solution are withdrawn from bag 44 through port 47
and (with clamp C3 open and clamp C4 closed) through tubing 43 to
Y-connector 93 and then through tubing 91 to mixing zone 89 by the
transfer pump P2. Buffer solution is withdrawn from bag 7 through
port 21 and tubing 15 to connector 95 by the buffer pump P2. With
clamp C1 open, buffer flows through tubing 20 to mixing zone 89. A
diluted suspension of blood cells in buffer solution flows from
mixing zone 89 through tubing 10 to inlet port 11 of spinner 6. A
concentrated suspension of blood cells in buffer solution flows
through outlet port 14 of spinner 6 through tubing 13 and inlet
port 1 into Isoflow.TM. bag 5 by recirculation pump P3. Filtrate
flows through outlet port 24 in spinner 6 and tubing 23 to
connector 94 and, with clamp C2 closed, through tubing 26 and inlet
port 29 into filtrate bag 30 by pump P4. Recirculation washing is
continued until the desired amount of target component has been
removed from the blood cells. Clamps C1, C2 and C3 are then closed,
clamp C4 is opened, and the direction of pump P1 is reversed, so
that the suspension of washed cells flows from bag 5 through tubing
8, 91 and 82 and port 81 into washed cell bag 79. The lines, bag
and spinner are then rinsed by closing clamps C1 and C3, opening
clamps C4 and C2, and pumping buffer with pump P2 in series with
pumps P1 and P3 to rinse the spinner, Isoflow.TM. bag and
tubing.
[0048] System Controls
[0049] In carrying out the recirculation washing method of this
invention, the filtrate rate (f) is typically fixed at about 70
ml/min. During the transfer of cells into the wash circuit, the
recirculation rate (r) provides the primary pressure regulation
(using the concentration ratio CR described below) and varies from
14 to 70 ml/min. During the recirculation phase the recirculation
rate ranges from about 24 to 70 ml/min. The buffer solution rate
(b) ranges from 0 to 70 ml/min. to maintain a minimum scale volume
and as a secondary pressure regulation mechanism. The rotor of the
spinning membrane filter operates at a maximum of 3700 RPM and a
minimum of about 2340 RPM during normal processing.
[0050] The Isolex.RTM. 300i system is automatically controlled
using microprocessors. These microprocessors in-turn control 5
banks of 4 clamps each (clamps C1-C20), 1 bank of pumps (pumps P1
-P4), 1 spinner motor drive P5 (drive for the rotor of spinning
membrane filter 6), and 1 rocker assembly for container 57 with an
integral magnet carriage to facilitate separation of magnetic beads
(not shown, but described in Moubayed et al. U.S. Pat. No.
5,536,475). The system uses feedback from 6 weight scales (not
shown), 2 pressure transducers (not shown, but attached to line 66
at 67 and to line 41 at 42, and 3 sets of fluid and tubing
detectors (not shown but attached to lines 61, 66 and 41). During
the Isolex.RTM. 300i procedure the bags 44, 53, 48 and 79 are hung
on weight scales 1, 2, 3 and 4, respectively. Bags 74 and 5 are
hung together on weight scale 5. Buffer bag 7 is hung on weight
scale 5. Buffer bag 7 is hung on weight scale 6. Bags 36, 39 and 30
are not hung on a scale. Weight scale 5 is used to determine the
cell product volume in the wash circuit by substracting out the
reference weight when the Isoflow.TM. bag is empty. The weight
scales are in the tower of the Isolex.RTM. 300i instrument.
[0051] The stand-alone cell washing system will also run
automatically using microprocessors. These microprocessors in turn
control 1 bank of 4 clamps each, 1 bank of 4 pumps and 1 spinner
motor drive. The system will require feedback from 4 weight scales,
2 pressure transducers, and 3 sets of fluid and tubing
detectors.
[0052] The size of the cell mass is minimized by increasing the
concentration ratio (CR) as far as possible. CR is the ratio of the
rate of unwashed undiluted cell volume coming into the spinning
membrane filter to the rate of washed cell volume exiting the
spinning membrane filter. In the wash circuit, there are four
variables to control CR, the recirculation rate (r), the buffer
solution rate (b), the cell source rate (c), and the filtrate rate
(f). The relationship is c+b=r+f, and CR =c/r=1+(f-b)/r.
[0053] For both the Isolex.RTM. 300i and the Stand-alone system,
the cells are concentrated and washed automatically. We have found
that by concentrating, diluting, and concentrating again multiple
times, the volume can be more consistently controlled. Thus,
between every other cell product cycle through the spinner (i.e.,
spinning membrane filter) the cell volume is diluted and
reconcentrated. If the number of cycles left is predicted to be
less than 2.5 cycles, the dilutions stop. During dilutions, the
filtrate pump P4 is stopped, the buffer pump P2 runs at a fixed
rate and the recirculation pump P3 runs at about 110% of the buffer
rate. This allows the membrane to be rinsed and dilutes the cell
concentrate through the port with the more concentrated cells.
[0054] The transmembrane pressure is regulated by controlling the
concentration ratio CR using the recirculation pump P3. The
concentration ratio CR is contolled to a target pressure by a PID
(Proportional/Integrative/Derivative) control through the pressure
measurements. The pressure measurements are taken from the pressure
transducer connected to the filtrate line and are adjusted for the
centrifugal effects on the fluid to yield a trans-membrane
pressure. If the bag volume drops below the target volume, CR is no
longer the controlling parameter. Instead, the scale weight is
controlled by the buffer pump P2 and CR is calculated as: CR=c/r.
Given CR, the recirculation rate is calculated as r=70/CR-1 where
CR is limited to >=2.
[0055] Filtrate rate (f) is set to its maximum in order to minimize
the time to process the cells. Filtration pressure is an indicator
of the concentration of blood cells along the membrane of the
spinning membrane filter. However, if either the spinner 6, buffer
pump P2 or recirculation pump P3 are not up to speed, the filtrate
rate is reduced. The ratio of the measured spinner 6, buffer pump,
or recirculation pump rate to the respective commanded rate is
calculated. The filtrate rate is then calculated as
f.sub.1=3/4*MRR*TFR+1/4*TFR, where f.sub.1 is the minimum ratio
adjusted rate to be commanded in ml/min, MRR is the minimum rate
ratios described above, TFR is the target filtrate rate (70
ml/min). The filtrate rate is further reduced when the pressure
error (E.sub.p) described above is less than -5 mmHg. When this
condition is true the filtrate rate is set to
f.sub.2=f.sub.1+E.sub.p+5, where f.sub.2 is the final command
filtrate rate and f.sub.1 is the minimum ratio adjusted filtrate
rate described above. During dilutions, the filtrate rate is set to
0.
[0056] Recirculation rate (r) is the primary regulating variable.
The buffer solution rate (b) is used to regulate the concentration
ratio CR between values of 1 and 2. The buffer pump P2 provides the
primary regulation to the scale weight management control. When the
Isoflow.TM. bag 5 fluid volume weight drops below the target (20-35
ml), the buffer is commanded to about 78 ml/min. This is
approximately 8 ml/min faster than the filtrate pump P4. This
causes the bag weight to rise. Once the weight rises about 5 ml,
the buffer once again becomes secondary to the concentration ratio
control, the buffer pump P2 is regulated according to the equation
b=(70+f)/2-r*(CR-1).
[0057] Because the blood cells can be damaged by stress, the
contoller automatically adjusts the rotor spin rate of the spinning
membrane filter. As the recirculation rate (r) is decreased the
exposure time of the cells in the spinning membrane filter
increases as follows: t=v/(r+f), where t and v are time and volume,
respectively, in the spinning membrane. When r slows, stress on the
cells increases. The controller counteracts this by decreasing the
spin rate linearly when r is reduced.
[0058] The amount of washing is based on an estimate of "residual".
The residual represents the target component for reduction (e.g.,
platelets, antibody). This estimate is made possible by the mixing
properties of the IsoFlow.TM. bag. The estimate is calculated
similar to how serial dilutions would calculate the residual.
However, it is recalculated several times a second. The equation
is
FSR.sub.i=FSR.sub.i-1-(F.sub.i/(B.sub.i+C.sub.i).times.(C.sub.i/V.sub.i).t-
imes.FSR.sub.i-1.times.TA
[0059] where i=the discrete time interval
[0060] FSR.sub.i=Fraction of Starting Residual at time t.sub.i
[0061] FSR.sub.1=Fraction of Starting Residual at time
t.sub.i-1
[0062] F.sub.i=Filtrate volume moved at rate f measured at time
interval i-1 to i in units of ml
[0063] B.sub.i=Buffer volume moved at rate b measured at time
interval i-1 to i in units of ml
[0064] C.sub.i=Cell source moved at rate c measured at time
interval i in units of ml, including the rate from the IsoFlow.TM.
bag 5, as well as the rate of addition of unwashed cells, if any,
in same units
[0065] V.sub.i=cell product volume at time interval i in ml
[0066] TA=Target Admittance
[0067] The Target Admittance is the unitless constant that
represents the ease with which a given substance passes through the
membrane (the inverse of membrane impedance). For platelet wash the
Target Admittance has been found to be between 0.5 and 1.0 with a
preferred setting of 0.7. For antibody and release agent wash the
Target Admittance has been found to be between 0.7 and 1.2 with a
preferred setting at 1. The optimal level for the antibody used for
CD34.sup.+ selection on the Isolex.RTM. 300i has been found to be
in the range of 50-150 micrograms.
[0068] An estimate of the average number of times a cell has been
through the spinning membrane acts as a backup for determining when
to end a wash. Cell cycles are estimated based on the following
equation:
Cell
cycles.sub.i=.intg.(R.sub.j+F.sub.j-B.sub.j)/V.sub.j=.intg.C.sub.j/V.-
sub.j
[0069] where
[0070] R.sub.j=Recirculation volume moved at rate r measured at
time interval j in units of ml, and Cell cycles.sub.i=Number of
cycles through the spinning membrane device that the cell product
has experienced at time interval i.
* * * * *