PROCESS FOR THE PRODUCTION OF RECOMBINANT PROTEINS
The invention relates to the field of recombinant production of proteins in host cells. In particular, the invention relates to processes for manufacturing recombinant proteins in host cells which processes reduce the misincorporation of norleucine in place of methionine.
1 - 20 . (canceled)
21 . A process for producing a recombinant protein comprising the steps of:
a) providing host cells capable of producing a recombinant protein,
b) providing an amount of liquid medium containing between 0 and 1 g methionine per kg of liquid medium,
c) culturing said host cells in the liquid medium,
d) inducing production of the recombinant protein in the liquid medium, and
e) at the time or after induction adding an amount of methionine to the liquid medium while culturing said host cells to produce the recombinant protein,
wherein the amount of methionine per kg of liquid medium added in step e) is higher than the amount of methionine per kg of liquid medium contained in the liquid medium in step b).
22 . A process for reducing norleucine misincorporation during the production a recombinant protein comprising the steps of:
a) providing host cells capable of producing a recombinant protein,
b) providing an amount of liquid medium containing between 0 and 1 g methionine per kg of liquid medium,
c) culturing said host cells in the liquid medium,
d) inducing production of the recombinant protein in the liquid medium, and
e) at the time or after induction adding an amount of methionine to the liquid medium while culturing said host cells to produce the recombinant protein,
wherein the amount of methionine per kg of liquid medium added in step e) is higher than the amount of methionine per kg of liquid medium contained in the liquid medium in step b).
23 . The process according to claim 21 , wherein the host cells are harvested after step e) and the amount of methionine added in step e) is such that the concentration of methionine in the liquid medium at or immediately prior to harvest is between 0.25 g/kg and 1.5 g/kg.
24 . The process according to claim 21 , wherein the amount of methionine added in step e) is between 0.25 g and 2.0 g per kg of liquid medium provided in step b).
25 . The process according to claim 21 , wherein the amount of methionine contained in the liquid medium in step b) and/or step c) is less than 0.5 g/kg.
26 . The process according to claim 25 , wherein no methionine is contained in the liquid medium in step b) and/or step c).
27 . The process according to claim 21 , wherein step c) comprises growing the culture to an OD600 of at least 50.
28 . The process according to claim 21 , wherein step c) and/or step e) comprise a step of culturing said host cells in fed-batch culture.
29 . The process according to claim 21 , wherein feed containing a carbon source is added during step c) and step e) and the amount of carbon source added per unit time to the liquid medium in step e), is lower than in step c).
30 . The process according to claim 21 , wherein step d) is initiated when a 50% increase in dissolved oxygen occurs in the liquid medium or when a predefined OD600 is reached.
31 . The process according to claim 21 , wherein said host cells are bacterial cells.
32 . The process according to claim 21 , wherein no leucine and/or isoleucine is contained in the liquid medium in step b) and/or step c) and/or step e).
33 . The process according to claim 21 , wherein said host cells produce recombinant protein upon induction with IPTG and optionally wherein step d) comprises addition of IPTG.
34 . The process according to claim 21 , wherein the duration of step e) is between 12 and 96 hours or the duration of step e) is between about 12 and about 96 hours.
35 . The process according to claim 21 , wherein more than 75% of the carbon source consists of glycerol.
36 . The process according to claim 21 , wherein the recombinant protein is an antibody or a Fab′ fragment.
37 . The process according to claim 21 , wherein at least step e) is carried out in a bioreactor.
38 . The process according to claim 21 , wherein the process comprises the step of recovering the recombinant protein, a further step of purifying the recombinant protein and, optionally, a further step of formulating the recombinant protein.
39 . The process according to claim 38 , wherein the process comprises lyophilizing the recombinant protein.
40 . A recombinant protein preparation obtained by the process according to claim 21 .