System and method for processing, incubating, and/or selecting biological cells

• US 10,251,990 B2
• Filed: 04/27/2017
• Issued: 04/09/2019
• Est. Priority Date: 04/29/2016
• Status: Active Grant

• Current Assignee: Fenwal Incorporated (Fresenius SE & Co. KGaA)
• Original Assignee: Fenwal Incorporated (Fresenius SE & Co. KGaA)
• Inventors: Wegener, Christopher J., Olson, Bret M., Schlinker, Alaina, Binninger, Steven, Kadakia, Avnie A.
• Primary Examiner(s): Kim, John
• Application Number:
  US15/498,881
• Publication Number:
  US 20170313968A1
• Time in Patent Office: 712 Days
• CPC Class Codes: A61M 1/265 inducing Taylor vortices
• CPC Class Codes: A61M 1/3496 Plasmapheresis; Leucopheresis; Lymphopheresis A61M1/3472 takes precedence
• CPC Class Codes: A61M 1/362 changing physical properties of target cells by binding them to added particles to facilitate their subsequent separation from other cells, e.g. immunoaffinity
• CPC Class Codes: C12M 23/28 disposable or single use
• CPC Class Codes: C12M 25/02 Membranes; Filters filters or filtration in general B01D24/00-B01D41/00
• CPC Class Codes: C12M 27/02 Stirrer or mobile mixing elements
• CPC Class Codes: C12M 29/00 Means for introduction, extraction or recirculation of materials, e.g. pumps pumps per se F04B
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 33/14 with filters, sieves or membranes
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: G01N 2035/00465 Separating and mixing arrangements
• CPC Class Codes: G01N 35/1016 Control of the volume dispensed or introduced

Abstract

A method for automated processing of a cellular product comprising target substrate cells, the method comprising providing a separation apparatus configured to associate with a disposable sterile circuit comprising a separator in communication with the cellular product. The apparatus and disposable sterile circuit are configured to remove platelets from the cellular product to form a platelet-depleted cellular product, resuspend the platelet-depleted cellular product in media to form a resuspended platelet-depleted cellular product, receive an agent having an association with the target substrate cells of the resuspended platelet-depleted cellular product, incubate the agent with the target substrate cells over a period sufficient for the agent to bind with and/or enter the target substrate cells to form a first mixture comprising agent-target substrate cell complexes, unbound/unassociated agent, and non-target substrate cells, and remove unbound/unassociated agent to form a second mixture comprising the agent-target substrate cell complexes and non-target substrate cells.

Claims

1. A system for automated processing of a cell product comprising surface antigen expressing cells, in preparation for surface antigen expressing cell selection, the system comprising:

a reusable separation apparatus controlled by a microprocessing controller unit driven by software, wherein the apparatus and microprocessing controller unit are configurable with a plurality of settings;

a disposable sterile circuit configured to associate with the reusable separation apparatus, the disposable sterile circuit comprising a spinning membrane separator comprising a porous membrane, wherein the separator comprises an inlet and first and second outlets;

a cell product in communication with the inlet of the separator, wherein the separation apparatus is configured in a first cycle by the microprocessing controller unit to flow the cell product into the inlet of the separator, separate platelets and supernatant from the first outlet into a filtrate container, and separate remaining cellular components from the second outlet into an in-process container;

wash media within a media container in communication with the in-process container, wherein the separator is configured by the separation apparatus in a second cycle to flow the wash media into the in-process container containing the remaining cellular components to form resuspended platelet-depleted cell product;

an external injection port located on the in-process container configured to receive an agent having an association with the surface antigen expressing cells of the resuspended platelet-depleted cell product, wherein the in-process container is configured to incubate the agent with the surface antigen expressing cells over an incubation period sufficient for the agent to bind and/or associate with the surface antigen expressing cells to form a first mixture, comprising antigen-agent complexes, unbound/unassociated agent, and cells not presenting the antigen;

wherein the separation apparatus is configured in a third cycle to separate the first mixture into unbound/unassociated agent, directed from the first outlet into the filtrate container, and a second mixture, comprising antigen-agent complexes and cells not expressing the antigen, directed from the second outlet to a retentate container, wherein the second mixture is ready for target cell selection.

2. The system of claim 1, wherein the plurality of settings further comprises:

a spinner inlet flow rate of 80 mL/min, a reduction spinner revolution rate of 4000 rpm, a desired spinner inlet packed cell volume (PCV)of 6%, and a reduction retentate pump rate of 8 mL/min for the first cycle and the second cycle; and

a spinner inlet flow rate of 148 mL/min, a reduction spinner revolution rate of 2750 rpm, a desired spinner inlet packed cell volume (PCV)of 7%, and reduction retentate pump rate of 16 mL/min for the third cycle.

3. The system of claim 1, wherein the wash media comprises at least one of Phosphate-Buffered Saline (PBS), Ethylenediaminetetraacetic Acid (EDTA), Human Serum Albumin (HAS), and saline.

4. The system of claim 1, wherein the porous membrane comprises pore sizes greater than the size of platelets and the unbound/unassociated agent but less than the size of the cellular components.

5. The system of claim 4, wherein the pore sizes are within the range of 4.0 μ

m to 10.0 μ

m.

6. The system of claim 1, wherein the reusable separation apparatus controlled by a microprocessing controller unit is configured to automatically pause between the first cycle and the second cycle, and/or a beginning of the incubation period and the third cycle, to allow an operator may manually mix the in-process container.

7. The system of claim 1, wherein the incubation period further comprises an automated incubation cycle, wherein the separation apparatus controlled by the microprocessing controller unit is configured to cycle the agent and the resuspended platelet-depleted cell product from the in-process container through the separator and back to the in-process container in a cyclical pathway.

8. The system of claim 1, wherein during the incubation period, the separation apparatus controlled by the microprocessing controller unit is configured to add additional volume of fluid and/or substrate gradually and/or intermittently, via a second media container in communication with the in-process container, to the resuspended platelet-depleted cell product.

9. The system of claim 1, wherein the in-process container is configured to receive the agent within an enclosed, ventilated laboratory workspace, wherein the agent is incubated on a shaker with the surface antigen expressing cells over the incubation period to form the first mixture.

10. The system of claim 1, wherein the plurality of settings comprises:

a spinner inlet flow rate of 150 mL/min, a reduction spinner revolution rate of 3750 rpm, a desired spinner inlet packed cell volume (PCV) of 3%, and a reduction retentate pump rate of 15 mL/min for the first, second, and third cycles;

or

a spinner inlet flow rate of 80 mL/min, a reduction spinner revolution rate of 4000 rpm, a desired spinner inlet packed cell volume (PCV)of 6%, and reduction retentate pump rate of 8 mL/min for the first, second, and third cycles.