Perfusion bioreactor and related methods of use

Patent metadata

Field	Value
Publication number	US-11193103-B2
Application number	US-201816160465-A
Country	US
Kind code	B2
Filing date	Oct 15, 2018
Priority date	Oct 16, 2017
Publication date	Dec 7, 2021
Grant date	Dec 7, 2021

Abstract

A method of controlling a bioreactor system includes providing a cell culture in a bioreactor, wherein conditions in the bioreactor enable the cell culture to produce a protein of interest (POI), measuring process parameters (PPs) of the culture within the bioreactor by RAMAN, wherein the process parameters are selected from the group consisting of nutrient concentration, viable cell concentration, and protein attributes, measuring a predetermined weight of the bioreactor with the cell culture, removing cell-free spent media from the cell culture using a first output conduit at a first specified rate, removing cells from the cell culture using a second output conduit at a second specified rate, and introducing one or both of fresh media or nutrients into the cell culture using an input conduit at a third specified rate.

First claim

What is claimed is:

1. A method of controlling a bioreactor system, comprising:
providing a cell culture in a perfusion bioreactor, wherein conditions in the perfusion bioreactor enable the cell culture to produce a protein of interest (POI);
measuring one or more process parameters (PPs) of the culture within the perfusion bioreactor by a RAMAN probe, wherein the process parameters are selected from the group consisting of nutrient concentration, viable cell concentration, and protein attributes;
measuring a weight of the perfusion bioreactor with cell culture contents;
removing cell-free spent media from the cell culture using a first output conduit at a first specified rate;
removing cells from the cell culture using a second output conduit at a second specified rate;
introducing one or both of fresh media or nutrients into the cell culture using an input conduit at a third specified rate; and
adjusting at least one of the specified rates in response to a corresponding change in another one of the specified rates to maintain a perfusion rate of the perfusion bioreactor at a constant perfusion rate setpoint, wherein the perfusion rate defines a rate of total flow through the perfusion bioreactor such that the rate of total flow remains constant when the at least one specified rate is adjusted;
wherein the input and output conduits are adjusted based on the RAMAN probe measurements and weight measurement of the perfusion bioreactor to maintain (i) one or more of the process parameters within predetermined ranges, (ii) the weight of the perfusion bioreactor with the cell culture within a predetermined range, (iii) the third specified rate of the input conduit and the first and second specified rates of each of the output conduits within their respective predetermined ranges, and (iv) the perfusion rate of the perfusion bioreactor at the constant perfusion rate setpoint.

2. The method of claim 1 , wherein prior to adjusting the at least one of the specified rates, the method comprises:
determining a feedback loop, based on the one or more process parameters measured by the RAMAN probe, that increases or decreases the at least one specified rate in response to an opposing decrease or increase of the other specified rate, respectively;
wherein measuring the one or more process parameters of the culture within the perfusion bioreactor by RAMAN occurs at least once per hour.

3. The method of claim 1 , wherein the method is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per mL for 30 days at steady state.

4. The method of claim 1 , wherein the perfusion bioreactor has a volume of at least 10 L, and the method is configured to maintain the weight of the perfusion bioreactor and cell culture within a 20 g range.

5. The method of claim 1 , wherein the perfusion bioreactor has a volume of at least 10 L, and the method is configured to maintain the weight of the perfusion bioreactor with the cell culture within 0.1 percent of an initial weight of the perfusion bioreactor with the cell culture.

6. The method of claim 1 , wherein, when a process parameter deviates from a set point value within a respective desired range, one or more of removing cell-free media, removing cells, or introducing one or both of fresh media or nutrients, is adjusted to reduce the deviation.

7. The method of claim 1 , wherein at least two bioreactor volumes of spent media are removed through the first output conduit per day, and the constant perfusion rate setpoint is selected from one to three bioreactor volumes per day.

8. The method of claim 1 , wherein the process parameters include temperature of the cell culture and pH of the cell culture, and the temperature is maintained between 35 and 36 degrees C., and the pH is maintained between 6.85 and 7.15.

9. The method of claim 1 , wherein the process parameters include cell specific productivity, and the method is configured to maintain cells within the cell culture at a cell specific productivity of at least 15 pg/cell/day for at least 25 days.

10. The method of claim 1 , wherein the process parameters include glucose concentration, and the method is configured to maintain a glucose concentration between about 5 mM to about 85 mM, or about 1 g/L to about 15.5 g/L.

11. The method of claim 1 , wherein the process parameters include lactate concentration, and the method is configured to maintain a lactate concentration less than about 60 mM, or less than about 6 g/L.

12. The method of claim 1 , wherein the process parameters include ammonia concentration, and the method is configured to maintain an ammonia concentration less than about 15 mM.

13. The method of claim 1 , wherein each of removing cell-free spent media, removing cells, and introducing one or both of fresh media or nutrients, is controlled by a respective pump.

14. The method of claim 1 , wherein the perfusion bioreactor includes a filter configured to retain cells and allow fluid to pass through.

15. A method of controlling a bioreactor system, comprising:
providing a cell culture in a perfusion bioreactor;
measuring one or more process parameters of the cell culture within the perfusion bioreactor by a RAMAN probe;
removing cell-free spent media from the cell culture using a first output conduit at a first specified rate;
removing cells from the cell culture using a second output conduit at a second specified rate;
introducing one or both of fresh media or nutrients into the cell culture using an input conduit at a third specified rate; and
determining a first change to one or more of the first specified rate, the second specified rate, or the third specified rate based on the RAMAN probe measurements;
applying a second change to another one of the first specified rate, the second specified rate, or the third specified rate in response to the first change to maintain a perfusion rate through the perfusion bioreactor at a constant perfusion rate setpoint such that a total input and a total output of the perfusion bioreactor remains constant during the first change and the second change;
wherein the input and output conduits are automatically adjusted, by a controller receiving the RAMAN probe measurements of the perfusion bioreactor to maintain (i) one or more of the process parameters within predetermined ranges, (ii) the first specified rate and the second specified rate of each of the output conduits within respective predetermined ranges, (iii) the third specified rate of the input conduit within a predetermined range; and (iv) the perfusion rate of the perfusion bioreactor based on the constant perfusion rate setpoint.

16. The method of claim 1 , wherein:
the perfusion bioreactor has a volume of at least 10 L and includes a filter configured to retain cells and allow fluid to pass through;
each of removing cell-free spent media, removing cells, and introducing one or both of fresh media or nutrients, is controlled by a respective pump; and
the method is configured to maintain the weight of the perfusion bioreactor with the cell culture within 0.1 percent of an initial weight of the perfusion bioreactor with the cell culture, and the constant perfusion rate setpoint is selected from one to three bioreactor volumes per day.

17. The method of claim 16 , wherein:
the method is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per mL for 30 days at steady state; and
when a process parameter deviates from a set point value within a respective desired range, one or

Assignees

• Regeneron Pharma
• Regeneran Pharmaceuticals Inc

Inventors

• Angelini Matthew
• Witmer Ashley
• Debiase Anthony

Classifications

• G01N21/65

  Raman scattering · CPC title

• C12M41/48

  Automatic or computerized control (automatic analysis G01N35/00) · CPC title

• C12M41/14

  Incubators; Climatic chambers (per se B01L1/00) · CPC title

• C12M41/26

  of pH · CPC title

• C12M41/38

  of metabolites or enzymes in the cells · CPC title