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US 11,299,539 B2Granted
Methods of producing two chain proteins in bacteria
AssigneeGenentech, Inc.
InventorsJames Giulianotti, Dorothea E. Reilly
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Abstract
Provided herein are methods of producing a polypeptide containing two chains, such as an antibody including a light chain and a heavy chain. In particular, methods are provided for producing heterologous secretory proteins in bacteria through utilization of optimized expression vectors and culture processes.
Claims64
A method of producing a polypeptide comprising two chains in a prokaryotic host cell, the method comprising:
- (a) culturing the host cell to express the two chains of the polypeptide in a culture medium under conditions comprising:
- a growth phase comprising a growth temperature and a growth agitation rate, and
- a production phase comprising a production temperature and a production agitation rate,
- whereby upon expression the two chains fold and assemble to form a biologically active polypeptide in the host cell;
- wherein the host cell comprises a polynucleotide comprising
- (1) a first translational unit encoding a first chain of the polypeptide;
- (2) a second translational unit encoding a second chain of the polypeptide;
- and
- (3) a third translational unit encoding at least one chaperone protein selected from the group consisting of peptidyl-prolyl isomerases, protein disulfide oxidoreductases, and combinations thereof;
- wherein the growth temperature is from 2 to 10° C. above the production temperature, and the growth agitation rate is from 50 to 250 rpm above the production agitation rate;
- wherein:
- (i) the growth agitation rate is sufficient to achieve an oxygen uptake rate in the host cell during the growth phase of from 0.5 to 2.5 mmol/L/min above a peak oxygen uptake rate in the host cell during the production phase, or (ii) the peak oxygen uptake rate of the host cell during the growth phase is in the range of 3.5 to 4.5 mmol/L/min, and the oxygen uptake rate of the host cell during the production phase is in the range of 1.0 to 3.0 mmol/L/min;
- and
- (b) recovering the biologically active polypeptide from the host cell.
11 more elements
A method of producing a polypeptide comprising two chains in a prokaryotic host cell, the method comprising:
- (a) culturing the host cell to express the two chains of the polypeptide in a culture medium under conditions comprising:
- a growth phase comprising a growth temperature and a growth agitation rate, and
- a production phase comprising a production temperature and a production agitation rate,
- whereby upon expression the two chains fold and assemble to form a biologically active polypeptide in the host cell;
- wherein the host cell comprises a polynucleotide comprising:
- (1) a first translational unit encoding a first chain of the polypeptide;
- (2) a second translational unit encoding a second chain of the polypeptide;
- (3) a third translational unit encoding a first chaperone protein;
- (4) a fourth translational unit encoding a second chaperone protein;
- and
- (5) a fifth translational unit encoding a third chaperone protein,
- wherein the first, second and third chaperone proteins are selected from the group consisting of peptidyl-prolyl isomerases, protein disulfide oxidoreductases, and combinations thereof;
- wherein the growth temperature is from 2 to 10° C. above the production temperature, and the growth agitation rate is from 50 to 250 rpm above the production agitation rate;
- wherein:
- (i) the growth agitation rate is sufficient to achieve an oxygen uptake rate in the host cell during the growth phase of from 0.5 to 2.5 mmol/L/min above a peak oxygen uptake rate in the host cell during the production phase, or (ii) the peak oxygen uptake rate of the host cell during the growth phase is in the range of 3.5 to 4.5 mmol/L/min, and the oxygen uptake rate of the host cell during the production phase is in the range of 1.0 to 3.0 mmol/L/min;
- and
- (b) recovering the biologically active polypeptide from the host cell.
14 more elements
A method of producing a half antibody comprising a heavy chain and a light chain in a prokaryotic host cell, the method comprising:
- (a) culturing the host cell to express the heavy chain and the light chain in a culture medium under conditions comprising:
- a growth phase comprising a growth temperature and a growth agitation rate, and
- a production phase comprising a production temperature and a production agitation rate,
- whereby upon expression the heavy chain and the light chain assemble to form a half antibody in the host cell;
- wherein the host cell comprises a polynucleotide comprising:
- (1) a first translational unit encoding the heavy chain of the half antibody;
- (2) a second translational unit encoding the light chain of the half antibody;
- (3) a third translational unit encoding a first chaperone protein;
- (4) a fourth translational unit encoding a second chaperone protein;
- and
- (5) a fifth translational unit encoding a third chaperone protein,
- wherein the first, second and third chaperone proteins are selected from the group consisting of peptidyl-prolyl isomerases, protein disulfide oxidoreductases, and combinations thereof;
- wherein the growth temperature is from 2 to 10° C. above the production temperature, and the growth agitation rate is from 50 to 250 rpm above the production agitation rate;
- wherein:
- (i) the growth agitation rate is sufficient to achieve an oxygen uptake rate in the host cell during the growth phase of from 0.5 to 2.5 mmol/L/min above a peak oxygen uptake rate in the host cell during the production phase, or (ii) the peak oxygen uptake rate of the host cell during the growth phase is in the range of 3.5 to 4.5 mmol/L/min, and the oxygen uptake rate of the host cell during the production phase is in the range of 1.0 to 3.0 mmol/L/min;
- and
- (b) recovering the half antibody from the host cell.
14 more elements
A method of producing an anti-IL13 half antibody comprising a heavy chain and a light chain in a prokaryotic host cell, the method comprising:
- (a) culturing the host cell to express the heavy chain and the light chain in a culture medium under conditions comprising:
- a growth phase comprising a growth temperature and a growth agitation rate, and
- a production phase comprising a production temperature and a production agitation rate, wherein
- (i) the heavy chain comprises a heavy chain variable domain comprising an HVR-H1 of SEQ ID NO:9, an HVR-H2 of SEQ ID NO:10, and an HVR-H3 of SEQ ID NO:11;
- and
- (ii) the light chain comprises a light chain variable domain comprising an HVR-L1 of SEQ ID NO:12, an HVR-L2 of SEQ ID NO:13, and an HVR-L3 of SEQ ID NO:14, whereby upon expression the heavy chain and light chain assemble to form an anti-IL13 half antibody in the host cell;
- wherein the host cell comprises a polynucleotide comprising:
- (1) a first translational unit encoding the heavy chain of the half antibody;
- (2) a second translational unit encoding the light chain of the half antibody;
- (3) a third translational unit encoding a first chaperone protein;
- (4) a fourth translational unit encoding a second chaperone protein;
- and
- (5) a fifth translational unit encoding a third chaperone protein,
- wherein the first, second and third chaperone proteins are selected from the group consisting of peptidyl-prolyl isomerases, protein disulfide oxidoreductases, and combinations thereof;
- wherein the growth temperature is from 2 to 10° C. above the production temperature, and the growth agitation rate is from 50 to 250 rpm above the production agitation rate;
- wherein:
- (i) the growth agitation rate is sufficient to achieve an oxygen uptake rate in the host cell during the growth phase of from 0.5 to 2.5 mmol/L/min above a peak oxygen uptake rate in the host cell during the production phase, or (ii) the peak oxygen uptake rate of the host cell during the growth phase is in the range of 3.5 to 4.5 mmol/L/min, and the oxygen uptake rate of the host cell during the production phase is in the range of 1.0 to 3.0 mmol/L/min;
- and
- (b) recovering the anti-IL13 half antibody from the host cell.
16 more elements
A method of producing an anti-IL17 half antibody comprising a heavy chain and a light chain in a prokaryotic host cell, the method comprising:
- (a) culturing the host cell to express the heavy chain and the light chain in a culture medium under conditions comprising:
- a growth phase comprising a growth temperature and a growth agitation rate, and
- a production phase comprising a production temperature and a production agitation rate,
- wherein (i) the heavy chain comprises a heavy chain variable domain comprising an HVR-H1 of SEQ ID NO:20, an HVR-H2 of SEQ ID NO:21, and an HVR-H3 of SEQ ID NO:22;
- and (ii) the light chain comprises a light chain variable domain comprising an HVR-L1 of SEQ ID NO:23, an HVR-L2 of SEQ ID NO:24, and an HVR-L3 of SEQ ID NO:25, whereby upon expression the heavy chain and the light chain assemble to form an anti-IL17 half antibody in the host cell;
- wherein the host cell comprises a polynucleotide comprising:
- (1) a first translational unit encoding the heavy chain of the half antibody;
- (2) a second translational unit encoding the light chain of the half antibody;
- (3) a third translational unit encoding a first chaperone protein;
- (4) a fourth translational unit encoding a second chaperone protein;
- and
- (5) a fifth translational unit encoding a third chaperone protein,
- wherein the first, second and third chaperone proteins are selected from the group consisting of peptidyl-prolyl isomerases, protein disulfide oxidoreductases, and combinations thereof;
- wherein the growth temperature is from 2 to 10° C. above the production temperature, and the growth agitation rate is from 50 to 250 rpm above the production agitation rate;
- wherein:
- (i) the growth agitation rate is sufficient to achieve an oxygen uptake rate in the host cell during the growth phase of from 0.5 to 2.5 mmol/L/min above a peak oxygen uptake rate in the host cell during the production phase, or (ii) the peak oxygen uptake rate of the host cell during the growth phase is in the range of 3.5 to 4.5 mmol/L/min, and the oxygen uptake rate of the host cell during the production phase is in the range of 1.0 to 3.0 mmol/L/min;
- and
- (b) recovering the anti-IL17 half antibody from the host cell.
15 more elements
A method of producing a bispecific antibody comprising a first half antibody capable of binding IL13 and a second half antibody capable of binding IL17, the method comprising:
- (a) culturing a first prokaryotic host cell to express a first heavy chain and a first light chain of the first half antibody, wherein (i) the first heavy chain comprises a first heavy chain variable domain comprising an HVR-H1 of SEQ ID NO:9, an HVR-H2 of SEQ ID NO:10, and an HVR-H3 of SEQ ID NO:11;
- and (ii) the first light chain comprises a first light chain variable domain comprising an HVR-L1 of SEQ ID NO:12, an HVR-L2 of SEQ ID NO:13, and an HVR-L3 of SEQ ID NO:14, whereby upon expression the first heavy chain and the first light chain assemble to form the first half antibody in the host cell;
- and
- (a′) culturing a second prokaryotic host cell to express a second heavy chain and a second light chain of the second half antibody, wherein (i) the second heavy chain comprises a second heavy chain variable domain comprising an HVR-H1 of SEQ ID NO:20, an HVR-H2 of SEQ ID NO:21, and an HVR-H3 of SEQ ID NO:22;
- and (ii) the second light chain comprises a second light chain variable domain comprising an HVR-L1 of SEQ ID NO:23, an HVR-L2 of SEQ ID NO:24, and an HVR-L3 of SEQ ID NO:25, whereby upon expression the second heavy chain and the second light chain assemble to form the second half antibody in the host cell;
- wherein the first host cell comprises a first polynucleotide comprising:
- (1) a first translational unit encoding the first heavy chain;
- (2) a second translational unit encoding the first light chain;
- and
- the second host cell comprises a second polynucleotide comprising:
- (1′) a third translational unit encoding the second heavy chain;
- (2′) a fourth translational unit encoding the second light chain;
- wherein both the first polynucleotide and the second polynucleotide further comprise:
- (3) a fifth translational unit encoding a first chaperone protein;
- (4) a sixth translational unit encoding a second chaperone protein;
- and
- (5) a seventh translational unit encoding a third chaperone protein,
- wherein the first, second and third chaperone proteins are selected from the group consisting of peptidyl-prolyl isomerases, protein disulfide oxidoreductases, and combinations thereof;
- wherein both the first host cell and the second host cell are separately cultured in a culture medium under conditions comprising:
- a growth phase comprising a growth temperature and a growth agitation rate, and
- a production phase comprising a production temperature and a production agitation rate,
- wherein the growth temperature is from 2 to 10° C. above the production temperature, and the growth agitation rate is from 50 to 250 rpm above the production agitation rate;
- wherein:
- (i) the growth agitation rate is sufficient to achieve an oxygen uptake rate in the host cell during the growth phase of from 0.5 to 2.5 mmol/L/min above a peak oxygen uptake rate in the host cell during the production phase, or (ii) the peak oxygen uptake rate of the host cell during the growth phase is in the range of 3.5 to 4.5 mmol/L/min, and the oxygen uptake rate of the host cell during the production phase is in the range of 1.0 to 3.0 mmol/L/min;
- (b) recovering the first half antibody from the first host cell;
- (b′) recovering the second half antibody from the second host cell;
- and
- (c) combining the first half antibody with the second half antibody in a reducing condition to produce a bi-specific antibody capable of binding to both IL-13 and IL-17.
25 more elements
Dependencies are inferred from the claim text.