Method for continuous centrifugal bioprocessing

• US 4,939,087 A
• Filed: 05/12/1987
• Issued: 07/03/1990
• Est. Priority Date: 05/12/1987
• Status: Expired due to Fees

• Current Assignee: Washington State University
• Original Assignee: Washington State University
• Inventors: Elliott, Michael L., Brouns, Thomas M., Van Wie, Bernard J.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): McMahon, Timothy M.
• Application Number:
  US07/048,718
• Time in Patent Office: 1,148 Days
• Field of Search: 435/240.25, 435/240.46, 435/240.47, 435/243, 435/251.1, 435/255, 435/256, 435/284, 435/286, 435/312, 435/813
• US Class Current: 435/394
• CPC Class Codes: B04B 2005/0471 with additional elutriation separation of different particles
• CPC Class Codes: B04B 5/0442 with means for adding or withdrawing liquid substances during the centrifugation, e.g. continuous centrifugation
• CPC Class Codes: C12M 27/10 Rotating vessel
• CPC Class Codes: C12M 41/00 Means for regulation, monitoring, measurement or control, e.g. flow regulation controlling or regulating chemical, physical or physicochemical processes B01J19/0006; heating or cooling apparatus for laboratory use B01L7/00; electro optical investigation of individual particles, flow cytometers G01N15/14; automatic analysis G01N35/00; controlling or regulating in general G06N
• CPC Class Codes: C12M 41/40 of pressure
• CPC Class Codes: Y10S 435/813 Continuous fermentation

Abstract

Bioprocessing apparatus and method using centrifugal acceleration of cells to maintain a culture within a reaction zone. The reactor has an inflow at a relatively distance inlet and an outflow at an inwardly spaced point toward the rotational axis. The reaction chamber has an inwardly diverging zone with a velocity gradient which captures cells of varying sizes. A recycle system is preferably used to recycle medium containing nutrients for the culture microbes. Byproducts of the cells are removed by the circulated medium and can be extracted using a desired extraction process.

Claims

1. A method for long term culturing of cells or other microorganisms forming a microorganism culture, comprising:

installing a microorganism within a reaction chamber of a bioreactor having a rotatable rotor which mounts a reactor which defines at least portions of said reaction chamber;

said bioreactor being capable of centrifugally rotating the reactor and reaction chamber;

rotating the microorganism culture contained within the reaction chamber to thereby create centrifugal forces acting upon the microorganisms forming the microorganism culture;

supplying a flow of fluid culture medium to the rotating rotor and reaction chamber;

controllably forcing fluid culture medium through the microorganism culture held within the reaction chamber in a manner which creates fluid dynamic forces on the microorganisms which are in opposition to the centrifugal forces applied to the microorganisms;

forming a velocity gradient within the fluid culture medium as the culture medium flows within the reaction chamber;

said velocity gradient varying from a relatively faster velocity to slower velocities countervailing to the centrifugal forces acting on the microorganism;

fluidizing the microorganism culture by controlling the flow of fluid culture medium through the reaction chamber to maintain microorganisms of the microorganism culture in a fluidized mass within the reaction chamber;

suspending the microorganisms of the microorganism culture within the reaction chamber substantially balanced by countervailing fluid drag forces and centrifugal forces;

removing fluid culture medium from the rotating rotor and reaction chamber;

recirculating fluid culture medium removed from the rotating rotor and reaction chamber back through the reaction chamber;

feeding the microorganisms of the microorganism culture which are suspended in the reaction chamber by supplying suitable nutrients for the microorganisms in the fluid culture medium as the fluid culture medium is forced through the reaction chamber.

2. A method according to claim 1 wherein said suspending of the microorganisms of the microorganism culture within the reaction chamber is performed for at least one day.

3. A method according to claim 1 and further comprising extracting a desired product of the microorganism culture from the recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor.

4. A method according to claim 1 and further comprising extracting a desired product of the microorganism culture from the recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor;

said extracting including removing said desired product from recirculating fluid culture medium using immunoaffinity binding.

5. A method according to claim 1 and further comprising treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture.

6. A method according to claim 1 and further comprising:

sensing pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture.

7. A method according to claim 6 wherein said treating is accomplished by passing carbon dioxide into the recirculating fluid culture medium.

8. A method according to claim 1 and further comprising:

sensing pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture;

extracting a desired product of the microorganism culture from the recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor.

9. A method according to claim 1 and further comprising controlling temperature of recirculating fluid culture medium.

10. A method according to claim 1 and further comprising:

sensing pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture;

controlling the temperature of recirculating fluid culture medium.

11. A method according to claim 1 and further comprising:

sensing the pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture;

controlling the temperature of recirculating fluid culture medium;

extracting a desired product of the microorganism culture from the recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor.

12. A method for long term culturing of cells or other microorganisms forming at least one microorganism culture, comprising:

installing at least one microorganism within at least one reaction chamber of a bioreactor having at least one rotatable rotor which mounts at least one reactor which defines at least portions of said at least one reaction chamber;

said bioreactor being capable of centrifugally rotating the at least one reactor and reaction chamber;

rotating the at least one microorganism culture contained within the at least one reaction chamber to thereby create centrifugal forces acting upon microorganisms forming the microorganism culture;

supplying at least one flow of at least one fluid culture medium to the rotating rotor and reaction chamber;

circulating at least one flow of at least one fluid culture medium to the rotating rotor, through the reaction chamber, and from the rotating rotor;

controllably forcing at least one fluid culture medium through the microorganism culture held within the at least one reaction chamber in opposition to the centrifugal forces applied to the microorganisms;

fluidizing the at least one microorganism culture by controlling the flow of the at least one fluid culture medium through the at least one reaction chamber to maintain microorganisms of the at least one microorganism culture in at least one fluidized mass within the at least one reaction chamber;

suspending the microorganisms of the at least one microorganism culture within the at least one reaction chamber substantially balanced by countervailing fluid drag forces and centrifugal forces;

said microorganisms being suspended for periods of at least one day;

removing fluid culture medium from the rotating rotor and at least one reaction chamber;

feeding the microorganisms of the at least one microorganism culture suspended in the at least one reaction chamber by passing suitable nutrients for the microorganisms in the at least one fluid culture medium as the fluid culture medium is forced through the at least one reaction chamber.

13. A method according to claim 12 and further comprising extracting at least one desired product of at least one microorganism culture from recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor.

14. A method according to claim 12 and further comprising recirculating fluid culture medium removed from at least one reaction chamber back through the at least one reaction chamber.

15. A method according to claim 12 and further comprising:

recirculating fluid culture medium removed from the at least one reaction chamber back through the at least one reaction chamber;

extracting at least one desired product of at least one microorganism culture from recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor.

16. A method according to claim 12 and further comprising:

recirculating fluid culture medium removed from the at least one reaction chamber back through the at least one reaction chamber;

extracting at least one desired product of at least one microorganism culture from recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor;

said extracting including removing said at least one desired product from recirculating fluid culture medium using immunoaffinity binding.

17. A method according to claim 12 and further comprising:

recirculating at least one fluid culture medium removed from the rotating rotor and at least one reaction chamber back through at least one reaction chamber;

sensing pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture.

18. A method according to claim 12 and further comprising:

recirculating fluid culture medium removed from the at least one reaction chamber back through the at least one reaction chamber;

sensing pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture;

extracting at least one desired product of at least one microorganism culture from recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor.

19. A method according to claim 12 and futher comprising:

recirculating fluid culture medium removed from the rotating rotor and at least one reaction chamber back through the at least one reaction chamber;

sensing pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture;

extracting at least one desired product of at least one microorganism culture from recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor;

said extracting including removing said desired product from recirculating fluid culture medium using immunoaffinity binding.

20. A method according to claim 12 and further comprising:

recirculating fluid culture medium removed from the rotating rotor and at least one reaction chamber back through the at least one reaction chamber;

sensing pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture;

extracting at least one desired product of the microorganism culture from recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor;

controlling temperature of recirculating fluid culture medium.

21. A method according to claim 12 and further comprising extracting at least one desired product of said at least one microorganism culture from fluid culture medium removed from the rotating rotor at least one reaction chamber.

22. A method for long term culturing of cells or other microorganisms forming at least one microorganism culture, comprising:

installing at least one microorganism within at least one reaction chamber of a bioreactor having at least one rotatable rotor which mounts at least one reactor which defines at least portions of said at least one reaction chamber;

said bioreactor being capable of centrifugally rotating the at least one reactor and reaction chamber;

rotating the at least one microorganism culture contained within the at least one reaction chamber to thereby create centrifugal forces acting upon the microorganisms forming the microorganism culture;

supplying at least one flow of at least one fluid culture medium to the rotating rotor and reaction chamber;

controllably forcing at least one fluid culture medium through the microorganism culture held within the at least one reaction chamber in opposition to the centrifugal forces applied to the microorganisms;

forming a velocity gradient within the fluid culture medium as the culture medium flows within the reaction chamber;

said velocity gradient varying from a relatively faster velocity to slower velocities countervailing to the centrifugal forces acting on the microorganism;

fluidizing the at least one microorganism culture by controlling the flow of the at least one fluid culture medium through the at least one reaction chamber to maintain microorganisms of the at least one microorganism culture in at least one fluidized mass within the at least one reaction chamber;

suspending the microorganisms of the at least one microorganism culture within the at least one reaction chamber substantially balanced by countervailing fluid drag forces and centrifugal forces;

said microorganisms being suspended for periods of at least one day;

removing fluid culture medium from the rotating rotor and at least one reaction chamber;

recirculating fluid culture medium removed from the at least one reaction chamber back through at least one reaction chamber;

feeding the microorganisms of the at least one microorganism culture suspended in the at least one reaction chamber by passing suitable nutrients for the microorganisms in the at least one fluid culture medium as the fluid culture medium is forced through the at least one reaction chamber;

sensing pH of recirculating fluid culture medium;

treating recirculating fluid culture medium to adjust the pH of the recirculating fluid culture medium to maintain desired pH conditions for the microorganism culture;

extracting at least one desired product of the microorganism culture from recirculating fluid culture medium while maintaining the microorganism culture suspended in the bioreactor;

controlling temperature of recirculating fluid culture medium.