SEQUENTIAL PROCESSING OF BIOLOGICAL FLUIDS

Abstract

A process for the sequential processing of opaque and transparent biological fluids such as whole blood, apheresis blood, bone marrow blood, umbilical cord blood, buffy coat or cultured cells by processing steps in a hollow cylindrical centrifugal processing chamber (300) which is part of a disposable set. At least three different procedures selected from washing, incubation, transduction, separation, density gradient separation, dilution and volume adjustment are each carried out once or repeated a number of times according to a given processing profile in the processing chamber. Each procedure involves an input into the processing chamber, an operation in the processing chamber and an output from the processing chamber by displacement of a piston (310). The at least three different procedures are sequentially chained one after the other to constitute an overall sequential operation in the processing chamber and its disposable set. A first application is incubation for binding magnetic beads with human blood cells or stem cells. A second application is transduction by which foreign genetic material is inserted into human blood cells or stem cells by a virus. A third application is reconditioning biological fluids to achieve reproducible concentration and volumes of blood cells or stem cells.

Claims

1. A process for the sequential processing of opaque and transparent biological fluids such as whole blood, apheresis blood, bone marrow blood, umbilical cord blood, buffy coat or cultured cells by processing steps selected from washing, incubation, transduction, separation, density gradient separation, dilution and volume adjustment in a hollow cylindrical centrifugal processing chamber for processing or mixing the biological fluids and/or mixing of the biological fluids with reagents and/or diluents, wherein the centrifugal processing chamber contains within its generally cylindrical wall an axially movable member such as a piston which is axially movable to vary the volume of a separation space within the processing chamber to intake or to output biological fluids into or from the separation space, wherein the processing chamber is part of a disposable set containing the biological fluids and reagents and/or diluents, the disposable set constituting an enclosed sterile environment, characterized in that, using a single processing chamber as part of a single disposable set, at least three different procedures selected from washing (including pre-washing and post-washing as separate procedures), incubation, transduction, separation, density gradient separation, dilution and volume adjustment are carried out; each said procedure being carried out once or repeated a number of times according to a given processing profile in the processing chamber of the single disposable set; each said procedure involving an input into the processing chamber produced by axially moving the movable member, an operation in the processing chamber and an output from the processing chamber produced by axially moving the movable member; the at least three different procedures being sequentially chained one after the other in the processing chamber to constitute a chained sequence of the different procedures as an overall sequential operation in the processing chamber and its single disposable set, all of the biological fluids, reagents and/or diluents necessary for carrying out all of said at least three different procedures being contained in the enclosed sterile environment of the single disposable set used for the overall sequential operation.

2. A process as claimed in claim 1, wherein the chained sequence constituting an overall sequential operation in the processing chamber comprises one of the following sequences of procedures (i), (ii) and (iii): (i) pre-washing; addition of reagents and incubation; post-washing, (ii) washing; dilution; centrifugal separation; dilution; volume adjustment of the product, and (iii) preliminary thawing; washing; sampling and dilution; volume adjustment/extraction.

3. A process as claimed in claim 2, wherein the following procedures are carried out sequentially: pre-washing; addition of reagents and incubation; post-washing.

4. A process as claimed in claim 3, which is for binding magnetic beads with human blood cells or stem cells.

5. A process as claimed in claim 2, wherein the following procedures are carried out sequentially: washing; dilution; centrifugal separation; dilution; volume adjustment of the product.

6. A process as claimed in claim 5, which is for virus transduction with lymphocytes T-cells or other human cells.

7. A process as claimed in claim 2, wherein the following procedures are carried out sequentially: preliminary thawing; washing; sampling and dilution; volume adjustment/extraction.

8. A process as claimed in claim 7, which is for recondition dosing of multiple doses of cellular products.

9. A process as claimed in claim 1, wherein the position of the movable member is monitored by an infrared sensor.

10. A process as claimed in claim 1, wherein the volume of liquids treated in the processing chamber of one disposable set for one overall sequential operation is from four to sixteen times the maximum treatment volume of the processing chamber.

11. A process as claimed in claim 1, wherein the processing chamber is associated with two external pinch valves controlling tubing of the disposable set for selecting two biological additives for switching bags of the disposable set without user intervention.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0033] The invention will be further described by way of example, with reference to the accompanying drawings wherein:

[0034] FIG. 1A is a flow diagram of a first application of the invention;

[0035] FIG. 1B is a completed diagram of the first application.

[0036] FIG. 2A is a flow diagram of a second application of the invention;

[0037] FIG. 2B is a completed diagram of the second application.

[0038] FIG. 3A is a flow diagram of a third application of the invention; and

[0039] FIG. 3B is a completed diagram of the third application.

[0040] FIG. 4 is a perspective view of a cabinet for housing the processing chamber of an apparatus for carrying out the process according to the invention.

[0041] FIG. 5 is a diagram of a disposable kit or set usable in the process according to the invention for carrying out the given applications.

DETAILED DESCRIPTION

[0042] Application 1: Binding Magnetic Beads with Human Blood Cells or Stem Cells

[0043] The first application relates to the blending of magnetic beads of human blood cells or stem cells, like lymphocytes T cells, lymphocytes B cells or hematopoietic stem cells with magnetic beads.

[0044] This application illustrates the sequential carrying out of the following procedures: washing, dilution, incubation and washing.

[0045] The first stage is to precondition the cell-based product, usually coming from an apheresis procedure for collecting peripheral blood stem cells and rarely also coming from bone marrow harvesting, in order to get rid of unnecessary elements such as human platelets, aggregates, clots, cell debris but also to hydrate the cells by adding biological nutriments. Cryo-protectant solution like Dimethyl sulfoxide should also be removed via this stage if the product was previously cryopreserved.

[0046] Then, the second step relates to adding a dose of reagents, like magnetic beads, or coloration medium, or a dose of stain to the preconditioned biological products.

[0047] The third step relates to incubation of the solution. The purpose of this step is to bind magnetic beads to targeted cells, or to colorize activated cells to make them highly sensitive to photodynamic treatment, or to bind other solutions via varied mechanism with targeted cells. During this phase, incubation can be done at ambient temperature during a given time, usually starting from 10 minutes up to 2 hours with a typical time of 30 minutes, in a separation chamber while constantly mixing the solutions. It can also be done under a controlled temperature, time and constant mixing in a blood bag via a device, for example that described in WO 2014/181158 A1, and commercialised by Biosafe SA under the Trademark Smart-Max. Such devices serve for mixing biological specimens contained in flexible storage bags at controlled temperature, which flexible storage bags may serve, in addition to storage, for the collection, freezing storage or transfer of biological specimens.

[0048] Finally, the last step is another washing step for the removal of excess reagents, like unbound magnetic beads not attached to targeted cells to avoid incorrect selection or counting of targeted cells, or to stop the coloration process by medium exchange, or to remove other solutions via the supernatant followed by suspension of cells in fresh medium.

[0049] All those steps are done are done sequentially under an automated system and in a sterile environment for instance using the aforementioned Sepax technology.

[0050] This first application is illustrated in FIG. 1A and FIG. 1B. As shown in FIG. 1A, this application of the process includes a pre-wash step 10, followed by reagent addition 20 and incubation 30. The reagent addition 20 and incubation 30 are repeated n times via loop 40. Then the final incubated product is subjected to a post-wash step 50.

[0051] As shown in FIG. 1B: [0052] The pre-wash step 10 involves washing a cell-based product typically from an apheresis procedure, platelet removal, and DMSO removal if applicable. The product is re-suspended to a given volume to have cells that are not too concentrated and to prevent de-hydration of cells. [0053] The reagent addition step 20 involves adding a specific volume of magnetic beads and/or a cell staining agent or an activation reagent, [0054] In the incubation step 30, the cells are mixed with added reagent during a specific time inside or outside the centrifugal processing and separation chamber, for instance of a Sepax system. If this is done outside, place the cell bag on a device like the Smart-Max for mixing and temperature control. [0055] The post-wash step 50 serves to remove reagent (e.g. beads), as applicable
Application 2: Virus Transduction with Lymphocytes T Cells

[0056] The second application relates to the transduction of lymphocytes T cells, or other human cells such as hematopoietic stem cells, by the addition of a virus.

[0057] This application illustrates the sequential carrying out of the following procedures: washing, dilution, transduction, dilution, volume adjustment.

[0058] The first stage is to precondition the cell-based product, usually coming from an apheresis procedure for collecting peripheral blood stem cells or from expanded cells after a cell culture process, by performing a washing procedure in order to remove unwanted elements such as platelets, aggregates, clots or cell debris. Once unwanted elements are removed, cells are also rehydrated by adding biological nutriments and could be suspended in a fixed desired volume. Medium culture should also be removed if cells were previously cultured and also Dimethyl sulfoxide if the product was previously cryopreserved.

[0059] Then, the second stage consists of diluting the pre-conditioned biological product in stage 1 with a solution containing the virus later used for transduction. In this context, usually the solution containing the virus is primed inside the centrifugation chamber containing the cells.

[0060] The third stage consists of spinning at a high speed, between 1200 to 1700 g, in order to separate cells from suspension media and putting them in close contact with the virus for initiating a transduction process. By spinning at high g-forces, T lymphocytes will separate from media and will stick to the external surface of the centrifugation chamber. At the same time, virus inserted in the chamber will be spread in the fluid in the separation chamber, and close contact will initiate the transduction process of T lymphocytes cells with the virus.

[0061] At the end of the high-spin process, the solution is then diluted with a culture medium solution, or saline solution, in order to reach the desired final volume.

[0062] Finally, the entire solutions can be split in one or several solution bags if the dose needs to be used several times for later use.

[0063] This second application is illustrated in FIG. 2A and FIG. 2B. As shown in FIG. 2A, this application of the process includes a wash step 60, followed by dilution 70 and a high-spin operation 80, then dilution 90 and an optional splitting step 100 for splitting the product into n bags.

[0064] As indicated in FIG. 2B: [0065] The washing step 60 washes a cell-based product, usually coming from an apheresis procedure, or a cell culture, with a wash buffer. [0066] In the dilution step 70, a specific volume of virus is added to the cells inside the separation chamber of a Sepax system. [0067] This is then subjected to a high-spin step 80 in the Sepax centrifugal processing chamber to bring the virus into close contact with the cells, though a high g-force and a long spin duration. [0068] In the dilution step 90 culture medium is added to reach a target volume. [0069] In the splitting step 100, the total volume is split into n smaller bags. This requires manual intervention to change bags and open/close clamps.
Application 3: Recondition Dosing (after Cryo, Including Accurate Dosing for Patient Infusion)

[0070] This application illustrates the sequential carrying out of the following procedures: washing, separation, dilution, volume adjustment.

[0071] Usually, in a standard autologous transplantation set-up, apheresis procedures are performed in pre-conditioned patients. Peripheral blood, which is enriched of blood cells and hematopoietic stem cells due to the pre-conditioning of the patient, is cryopreserved during the time the patient is undergoing chemotherapies or radiotherapies. Once the patient has been treated, patient cells are thawed and reconditioned prior to being transplanted.

[0072] The third application is a repeatable automated method for obtaining multiple doses of cellular products having identical blood cells or stem cells concentration starting from a cryopreserved apheresis solution.

[0073] The first stage relates to thawing a cellular product under a controlled temperature and mixing environment for example using the device described in WO 2014/181158 A1, and commercialised by Biosafe SA under the Trademark Smart-Max. Such devices are for mixing biological specimens contained in flexible storage bags at controlled temperature, which flexible storage bags may serve, in addition to storage, for the collection, freezing storage or transfer of biological specimens,

[0074] Then, the second stage consists of performing a washing procedure that will eliminate cryo-protectant solution, aggregates formed during the cryopreservation process or even clots.

[0075] Then, the third stage consists of taking a sample for measuring cell concentration. The sample is analysed via a tierce technology, such as cell counter or flow-cytometer, necessary for cell concentration calculation. Once the actual density of cells has been calculated, a dilution factor is adjustable on an automated Sepax platform in order to reach the desired cell concentration. Those two previous steps allow a reproducible and homogenized cell concentration to be obtained independently of the collection process or the patient.

[0076] Once a targeted cell concentration is obtained, the Sepax system allows the user to select the volume that should be divided on each bag, or the number of bags that should contain the total cell solution. By doing so, a fixed number of bags or volume dose per bag, all containing an identical cell concentration can be achieved.

[0077] This third application is illustrated in FIG. 3A and FIG. 3B. As shown in FIG. 3A, this application of the process includes a thawing step 110, followed by washing 120, sampling 130 and dilution 140. A first extraction step 150 extracts x ml to a bag, repeated n times via loop 160, A second extraction step 170 extracts y ml to a Quality Control bag, this step being repeated m times via loop 180 typically only once.

[0078] As indicated in FIG. 3B: [0079] Thawing operation 110 serves to thaw cell products. [0080] In washing operation 120, cryo-protectant is removed. [0081] In sampling operation 130, washed cells are extracted to an output bag for sampling. This operation requires the manual step of sample taking. [0082] In dilution step 140, the cell concentration is adjusted for patient dosing. [0083] The first extraction step 150 extracts a given volume of cells to a transfer bag for accurate patient dosing. This requires manual intervention to change bags and open/close clamps. [0084] The second extraction step 170 prepares the last m bags for Quality Control (sterility testing). Typically, m=1.

[0085] FIG. 4 is a perspective view of a cabinet 200 for housing the processing chamber of an apparatus for carrying out the invention, which apparatus is part of a system according to EP-B-0 912 250 and EP-B1 144 026, the contents whereof are herein incorporated by way of reference. As shown the cabinet 200 has a front with an upwardly-protruding part-cylindrical housing 210 having on top a swinging closure 220 for receiving inside the generally cylindrical centrifugal processing chamber (not shown) of the system. In its bulging front, the part-cylindrical housing 210 has a window 215 for viewing an inserted cylindrical processing chamber and its contents. At the rear of the cabinet 200 is an upwardly-extending post (shown partly cut-away) for suspending bags of a disposable set. Also at the rear of cabinet 200 is a display screen 240 for displaying parameters of the processing protocol according to integrated software. The sides 250 of cabinet 200 are flat and at least one of these flat sides 250 is provided with two pinch valves 260 having slots 270 for accommodating tubing of a disposable set associated with the cabinet 200, this tubing leading to two bags of the disposable set supported on the cabinet's sides 250. Above the pinch valves 260 are protrusions 265 for guiding the tubing. These two pinch valves 260 can be arranged for automatically selecting, by the software associated with the cabinet 200, two biological additives such as virus, for priming the processing chamber and later switching with the medium solution for resuspension, enabling switching bags without user intervention.

[0086] FIG. 5 schematically shows the layout of a disposable set or kit for use in the process according to the invention to carry out all of the three described applications. The disposable set comprises a centrifugal processing chamber 300 containing a movable member or piston 310 that defines a processing space 320 of variable volume in the chamber 300. The chamber 300 is connected via a tubing line 330 to a 5-way valve 340. One outlet of the valve 340 is connected by a tubing line 350 which branches out to connect to input spikes 304 for saline solutions or other solutions and to bags 394a and 304b for saline solutions or various other products. Another outlet of valve 340 is connected by a tubing line 360 to a waste bag 303. A third outlet of valve 340 is connected via a tubing line 370 and a filter 372 to input spikes 301 for the connection of blood bags or bags of other biological fluids. A fourth outlet of valve 340 is connected via a tubing line 380 to a set of output spikes 302, and branched off to a collection bag 305. The tubing line 380 also leads to a sampling port 382 and a sampling pillow 384.

[0087] The various tubing lines are fitted with pinch valves 352 that can be manually actuated when the disposable set is being set up, or some can be automatically operated during use, see for example the magnetically-operated pinch valves 260, FIG. 4.

[0088] The described kit or disposable set forms an enclosed sterile environment, even with bags connected to the spikes 301, 302 and 304.

BeadWash

[0089] For the beadwash application, one or two apheresis blood units can be connected to the kit via the input spikes 301. Then the blood apheresis units are filtered through the input filter 372 for removing any debris, clots and any unwanted residuals.

[0090] The pre-wash step consist of washing cells, and for this a washing solution, such as a saline solution NaCl is connected either using the spikes 304 or filled in one available bag 304a. Once the pre-washing step finished, waste solution is redirected in waste bag 303 and cells remain in the chamber 300.

[0091] Then the second step consists of adding reagent including magnetic beads in the processing chamber 300, that has been filled in the second available bag 304b. Then an incubation step is done inside chamber 300.

[0092] Finally, a post-wash step is done by pumping the same saline solution as in the pre-washing step, and finally incubated cells with magnetic beads are extracted in the collection bag 305, or in any custom bag connected by a spike 302.

Transduction

[0093] With the virus transduction application, the first step consists of connecting the apheresis unit(s) with the input spike(s) 301, connecting saline solution with spikes 304, adding a solution containing the virus in a bag 304a, adding a medium solution for cell nutriment in bag 304b, and finally connecting multiple output bags, up to three bags, via spikes 302.

[0094] The first step consists of pumping apheresis unit via input bag in 1, then washing via the saline solution connected by spikes 304, then cells remain in the chamber 300 and the virus solution (from bag 304a) is added to the chamber 300, the solution is centrifuged with a high spin force, then the solution is suspended in a culture medium from bag 304b, and finally the solution is split into equivalent or different volumes in up to three bags connected to spikes 302.

Reconditioning and Dose

[0095] With this application, first a cryopreserved blood unit is thawed. Then the bag of thawed blood solution is connected to input spike 301 and the solution pumped into chamber 300. Then, the previously-connected saline solution is connected via spikes 304 or an available bag 403a is used to wash the apheresis unit in the chamber 300. Once the cells are washed, a partial or total cell solution in the chamber 300 is extracted in the collection bag 305, followed by a sample taken for measuring cell concentration via the sampling port 382. Then, the volume can be pumped inside the chamber 300 again, diluted with saline solution from a bag connected to spike 304 or an available bag 304a, and then split in several bags connected on the output spikes 302.

[0096] The kit optionally includes a cryopreparation line with a sampling pillow 384 in case a sample needs to be taken outside the laminar flow, and a DMSO resistant in the tubing path 386 in case the final solution needs to be added with cryoprotectant solution for further freezing.