METHODS OF MAKING RECOMBINANT IL-12 ALBUMIN BINDING DOMAIN FUSION PROTEINS
Provided herein are methods for making recombinant IL-12 albumin binding domain (ABD) fusion proteins. The subject methods advantageously include a continuous downstream purification step that leads to high yield production of purified cytokine-based proteins.
1 . A method of making a protein comprising:
a) culturing a plurality of mammalian host cells that each comprise a polynucleotide encoding for the protein in a bioreactor comprising a liquid culture medium,
wherein the host cells are cultured under conditions wherein the protein is produced and secreted by the host cells into the liquid culture medium,
b) removing liquid culture medium comprising the secreted protein from the bioreactor;
c) purifying the protein by passing the liquid culture medium removed from the bioreactor directly over one or more chromatography columns,
wherein the one or more chromatography columns are operably and continuously linked to the perfusion bioreactor; and
d) collecting the purified protein from the liquid culture medium,
wherein the protein is an IL-12 albumin binding domain (ABD) fusion protein.
2 . The method of claim 1 , wherein the bioreactor is a perfusion mode bioreactor.
3 . The method of claim 2 , wherein the liquid culture medium comprising the secreted protein is removed from the perfusion mode bioreactor and replaced with fresh liquid culture medium at set time intervals.
4 . The method of claim 3 , wherein the set time interval is 12 hours, 24 hours, 48 hours, or 72 hours.
5 . The method of claim 2 , wherein the liquid culture medium comprising the secreted protein is continuously removed from the perfusion mode bioreactor and continuously replaced with fresh liquid culture medium.
6 . The method of claim 3 , wherein the liquid culture medium is removed from the bioreactor starting at least about 96 hours after beginning the culturing step a).
7 . The method of claim 3 , wherein the liquid culture medium comprising the secreted protein is removed from the perfusion mode bioreactor and replaced with an equal volume of fresh liquid culture medium.
8 . The method of claim 3 , wherein a partial volume of the liquid culture medium comprising the secreted protein is partially removed from the perfusion mode bioreactor and replaced with an equal volume of fresh liquid culture medium.
9 . The method of claim 1 , wherein the host cells are prevented from leaving the perfusion bioreactor during the removing step b) using a cell separation system.
10 . The method of claim 1 , wherein the host cells are cultured in the presence of nutrients that are periodically replenished in the liquid culture medium.
11 . The method of claim 1 , wherein the plurality of mammalian host cells are cultured at a density of 20-100×10 6 cells/mL of liquid culture medium in the bioreactor.
12 . The method of claim 1 , wherein the purifying c) separates one or more proteolytic enzymes from the protein.
13 . The method of claim 1 , wherein the albumin binding domain of the IL-12 ABD fusion protein comprises a variable heavy domain comprising SEQ ID NO:1 or a variant thereof, and a variable light domain comprising SEQ ID NO:5 or a variant thereof.
14 . The method of claim 13 , wherein the albumin binding domain is an scFv that comprises the amino acid sequence of SEQ ID NO:9 or a variant thereof.
15 . The method of claim 1 , wherein the IL-12 of the IL-12 fusion protein is a single chain IL-12 comprising a p35 subunit covalently attached to a p40 subunit.
16 . The method of claim 15 , wherein the p40 subunit has the amino acid sequence of SEQ ID NO:10 or a variant thereof.
17 . The method of claim 15 , wherein the p35 subunit has the amino acid sequence of SEQ ID NO: 11 or a variant thereof.
18 . The method of claim 15 , wherein the single chain IL-12 has the amino acid sequence of SEQ ID NO:12.
19 . The method of claim 1 , wherein the IL-12 ABD fusion protein has the amino acid sequence of SEQ ID NO:13.
20 . The method of claim 1 , wherein the one or more chromatography columns comprise an ion exchange chromatography column, a hydrophobic interaction column, an affinity column, a pseudo-affinity column or a size exclusion chromatography column.
21 . The method of claim 20 , wherein the one or more chromatography columns comprises an affinity column.
22 . The method of claim 21 , wherein the pseudo-affinity column comprises a Cibacron Blue ligand.
23 . The method of claim 22 , wherein the pseudo-affinity column comprises an affinity chromatography media depicted in FIG. 14 .