Integrated and continuous recombinant protein manufacturing
Integrated systems and methods for the manufacturing of recombinant proteins in mammalian cells are provided. The systems and methods include a single-use bioreactor (SUB), a tangential flow filtration system, a column chromatography skid, a tankless hold, a virus inactivation plug flow reactor, and pumps.
1 . An automated integrated single-use continuous cell culture system comprising, in the following order:
a. a single-use perfusion bioreactor (SUB) between 10 L and 2250 L,
b. a single-use tangential flow filtration system generating a permeate,
c. a single-use dual-column capture column chromatography skid configured to operate in parallel generating an effluent, wherein the capture column is Protein A;
d. a tankless hold comprising an elution stream chamber consisting of a tubing and having a volume of 1.1 to 3 times the volume of a single capture column;
e. a single-use virus inactivation plug flow reactor, wherein the single-use virus inactivation plug flow reactor is a continuous viral inactivation reactor including an inlet, an outlet, and a tubular flow path having a set of alternating turns that form a serpentine pattern between the inlet and the outlet, wherein the tubular flow path includes a diameter of from 0.6 cm to 0.7 cm, and wherein the set of alternating turns are segmented vertically into at least two stacked layers, wherein each of the at least two stacked layers include a thickness of from 0.7 cm to 1.2 cm;
f. an individual anion-exchange chromatography column;
g. a single pass tangential flow filtration system;
h. a single use mixer; and
i. a first set of pumps operating with a range of about 60 mL/min to about 5 L/min, or about 60 ml/min to about 600 mL/min and a second set of pumps operating with a range of about 125 mL/min to about 2.5 L/min;
wherein the single-use SUB is fluidically attached, without a tank and by tubing, to the single-use tangential flow filtration system;
wherein the permeate is transferred, without a tank, by ¼ inch to ⅜ inch single-use tubing to the single-use dual-column capture column chromatography skid;
wherein the effluent is transferred, without a tank, by single-use ¼ inch to ⅜ inch tubing to the tankless hold;
wherein the tankless hold is fluidically connected to the single-use virus inactivation plug flow reactor;
wherein the virus inactivation plug flow reactor is fluidically connected without a tank and by tubing to the anion exchange chromatography column,
wherein the anion exchange chromatography column is fluidically connected without a tank and by tubing to the single pass tangential flow filtration system, and
wherein the single pass tangential flow filtration system is fluidically connected without a tank and by tubing to the single use mixer, and wherein the first and second sets of pumps are fluidically connected to one or more of components a-i.
2 . The automated integrated single-use continuous cell culture system of claim 1 , wherein the system is capable of processing between 10 g and 5,000 g of recombinant protein per day.