# US 10,479,973 B2 · Small volume bioreactors with substantially constant working volumes and associated systems and methods > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,479,973 B2 - Document ID: US10479973B2 - Status: Granted - Kind: B2 - Application number: 14/913,734 - Filed: 2014-08-22 - Published: 2016-07-28 - Granted: 2019-11-19 - Earliest claimed: 2013-08-23 - Assignees: Massachuesetts Institute of Technology; Sanofi - Applicants: Massachusetts Institute of Technology; Sanofi - Inventors: Shireen Goh; Rajeev Jagga Ram; Michelangelo Canzoneri; Horst Blum - Web page: https://patentgrep.com/patent/US10479973B2 ## CPC - C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)} - C12M 23/58: {Reaction vessels connected in series or in parallel (combinations of bioreactors with other apparatus, C12M43/00)} - C12M 41/32: {of substances in solution} - C12M 41/34: {of gas} - C12M 41/44: {of volume or liquid level} - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} ## Abstract Control of volume in bioreactors and associated systems is generally described. Feeding and/or sampling strategies can be employed, in some embodiments, such that the working volume within the bioreactor remains substantially constant. ## Claims ### Claim 1 A method of operating a bioreactor, comprising: performing for a period of time, within the bioreactor, a biochemical reaction in which at least one eukaryotic cell is grown within a liquid medium having a volume of less than about 50 milliliters, wherein the bioreactor comprises an online sensor that is in contact with the liquid medium for monitoring viability of the at least one eukaryotic cell, a gas inlet conduit and a gas outlet conduit connected to the bioreactor, and a gas bypass conduit that connects the gas inlet conduit and the gas outlet conduit, wherein the gas bypass conduit is external to the bioreactor, and wherein, during the period of time, a pressure gradient is applied to an inlet of the bioreactor using a pump and/or to an outlet of the bioreactor using a syringe; adding a first volume of liquid to the liquid medium in the bioreactor during the period of time; removing a second volume of liquid from the liquid medium in the bioreactor during the period of time; and monitoring the volume of the liquid within the bioreactor during the period of time; wherein: during at least about 80% of the period of time, the total volume of liquid within the bioreactor does not fluctuate by more than about 20% from an average of the volume of liquid within the bioreactor; and over the entire period of time, the osmolarity of liquid within the bioreactor is maintained within a range of from about 200 osmoles per kilogram of the liquid to about 600 osmoles per kilogram of the liquid. ### Claim 2 (depends on 1) The method of claim 1, wherein the adding step is performed over a first period during the period of time, the removing step is performed over a second period during the period of time, and the first and second periods do not substantially overlap with each other. ### Claim 3 A method of operating a bioreactor, comprising: performing for a period of time, within the bioreactor, a biochemical reaction in which at least one eukaryotic cell is grown within a liquid medium having a volume of less than about 50 milliliters, wherein the bioreactor comprises at least one online sensor that is in contact with the liquid medium for monitoring viability of the at least one eukaryotic cell, a gas inlet conduit and a gas outlet conduit connected to the bioreactor, and a gas bypass conduit that connects the gas inlet conduit and the gas outlet conduit, wherein the gas bypass conduit is external to the bioreactor, and wherein, during the period of time, a pressure gradient is applied to an inlet of the bioreactor using a pump and/or to an outlet of the bioreactor using a syringe; adding a first volume of liquid to the liquid medium in the bioreactor during a first period during the period of time; removing a second volume of liquid having a volume that is within 10% of the first volume of liquid from the liquid medium in the bioreactor during a second period during the period of time, wherein the first period and the second period do no overlap; repeating the adding and removing steps at least one time during the period of time; and monitoring the volume of liquid within the bioreactor during period of time; wherein: the adding step and the removing step are performed such that, between the adding step and the removing step, substantially no liquid is removed from the bioreactor via a nonevaporative pathway, and substantially no liquid is added to the bioreactor during the period of time; and over the entire period of time, the osmolarity of liquid within the bioreactor is maintained within a range of from about 200 osmoles per kilogram of the liquid to about 600 osmoles per kilogram of the liquid. ### Claim 4 (depends on 3) The method of claim 3, wherein the adding step and the removing step are performed such that, between the adding step and the removing step, substantially no liquid is removed from the bioreactor. ### Claim 5 (depends on 3) The method of claim 3, wherein the second volume of liquid has a volume that is within 5% of the volume of the first volume of liquid. ### Claim 6 (depends on 1) The method of claim 1, wherein the biochemical reaction is performed within a liquid medium having a volume of less than about 10 milliliters. ### Claim 7 (depends on 1) The method of claim 1, wherein adding the first volume of liquid to the liquid medium comprises transporting the first volume of liquid into the bioreactor via a liquid inlet. ### Claim 8 (depends on 7) The method of claim 7, wherein the liquid inlet comprises a channel. ### Claim 9 (depends on 8) The method of claim 8, wherein the liquid inlet channel comprises a microfluidic channel. ### Claim 10 (depends on 1) The method of claim 1, wherein removing the second volume of liquid from the liquid medium in the bioreactor comprises transporting the second volume of liquid out of the bioreactor via a liquid outlet. ### Claim 11 (depends on 10) The method of claim 10, wherein the liquid outlet comprises a channel. ### Claim 12 (depends on 11) The method of claim 11, wherein the liquid outlet channel comprises a microfluidic channel. ### Claim 13 (depends on 1) The method of claim 1, wherein the liquid medium comprises a cell growth medium. ### Claim 14 (depends on 1) The method of claim 1, wherein at least one eukaryotic cell is grown during the biochemical reaction. ### Claim 15 (depends on 14) The method of claim 14, wherein the eukaryotic cell is a Chinese hamster ovary (CHO) cell. ### Claim 16 (depends on 1) The method of claim 1, wherein the first volume of liquid added to the liquid medium comprises at least one biochemical reactant. ### Claim 17 (depends on 1) The method of claim 1, wherein the second volume of liquid removed from the liquid medium comprises a product of the biochemical reaction. ### Claim 18 (depends on 1) The method of claim 1, wherein the second volume of liquid removed from the liquid medium comprises a eukaryotic cell.