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US 2014/0154726 A1Published application
Perfusion Culturing Methods and Uses Thereof
OwnerJianguo Yang, Agata Villiger-Oberbek +1inventors
InventorsJianguo Yang, Agata Villiger-Oberbek, Yang Yang
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Abstract
Provided herein are improved methods of culturing a mammalian cell in a conical container, and methods that utilize these culturing methods.
Claims62
A method of culturing a mammalian cell, the method comprising:
- providing a conical container containing a mammalian cell suspended in a first liquid culture medium that occupies about 4% to about 80% of the volume of the container;
- incubating the container for a period of time at about 31° C. to about 40° C. at a reactor angle of about 5 degrees to about 85 degrees from horizontal and with a rotary agitation of about 20 revolutions per minute (RPM) to about 1000 RPM;
- and
- continuously or periodically, during the period of time, removing a first volume of the first liquid culture medium and adding to the first liquid culture medium a second volume of a second liquid culture medium, wherein the first and second volumes are about equal.
1 more element
A method of culturing a mammalian cell, the method comprising:
- culturing in a gradient perfusion process a mammalian cell suspended in a liquid culture medium under conditions that generate in the medium a fluid sheer force and dissolved oxygen (O2) concentration that is essentially the same as that achieved in a medium occupying 4% to 40% of the volume of a gas-permeable conical container when the container is positioned at a reactor angle of about 5 degrees to about 85 degrees from horizontal, incubated at a temperature of about 31° C. to about 40° C., and agitated at a frequency of about 20 revolutions per minute (RPM) to about 1000 RPM.
A method of producing a recombinant protein, the method comprising:
- providing a conical container containing a mammalian cell containing a nucleic acid that encodes a recombinant protein, wherein the cell is suspended in a first liquid culture medium that occupys about 4% to about 80% of the volume of the container;
- incubating the container for a period of time at about 31° C. to about 40° C. at a reactor angle of about 5 degrees to about 85 degrees from horizontal and with a rotary agitation of about 20 revolutions per minute (RPM) to about 1000 RPM;
- continuously or periodically, during the period of time, removing a first volume of the first liquid culture medium and adding to the first liquid culture medium a second volume of a second liquid culture medium, wherein the first and second volumes are about equal;
- and
- recovering the recombinant protein from the mammalian cell or from the first or second culture medium.
2 more elements
A method of producing a recombinant protein, the method comprising:
- culturing in a gradient perfusion process a mammalian cell containing a nucleic acid that encodes a recombinant protein, wherein the cell is suspended in a liquid culture medium under conditions that generate in the medium a fluid sheer force and dissolved oxygen (O2) concentration that is essentially the same as that achieved in a volume of liquid culture medium occupying 4% to 40% of the volume of a gas-permeable conical container when the container is positioned at a reactor angle of about 5 degrees to about 85 degrees from horizontal, incubated at a temperature of about 31° C. to about 40° C., and agitated at a frequency of about 20 revolutions per minute (RPM) to about 1000 RPM, and
- recovering the recombinant protein from the mammalian cell or the liquid culture medium.
A method for testing a manufacturing process for making a recombinant protein, the method comprising:
- providing a conical container containing a mammalian cell suspended in a first liquid culture medium occupying about 4% to about 80% of the volume of the container;
- incubating the container for a period of time at about 31° C. to about 40° C. at a reactor angle of about 5 degrees to about 85 degrees from horizontal and with an agitation of about 20 revolutions per minute (RPM) to about 1000 RPM;
- continuously or periodically, during the period of time, removing a first volume of the first liquid culture medium and adding to the first liquid culture medium a second volume of a second liquid culture medium, wherein the first and second volumes are about equal;
- detecting the recombinant protein in the cell or in the first or second culture medium;
- and
- comparing the amount of recombinant protein present in the cell or in the first or second culture medium to a reference level of recombinant protein.
3 more elements
Dependencies are inferred from the claim text.