# US 11,299,539 B2 · Methods of producing two chain proteins in bacteria > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,299,539 B2 - Document ID: US11299539B2 - Status: Granted - Kind: B2 - Application number: 16/138,180 - Filed: 2018-09-21 - Published: 2019-04-18 - Granted: 2022-04-12 - Earliest claimed: 2014-11-05 - Assignees: Genentech, Inc. - Applicants: Genentech, Inc. - Inventors: James Giulianotti; Dorothea E. Reilly - Web page: https://patentgrep.com/patent/US11299539B2 ## CPC - C07K 16/244: {Interleukins [IL]} - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies} - C12N 15/70: Vectors or expression systems specially adapted for E. coli - C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature - C07K 2317/31: multispecific - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the polynucleotide further comprises three copies of a promoter, wherein a first copy is in operable combination with the first translational unit, a second copy is in operable combination with the second translational unit, and a third copy is in operable combination with the third translational unit to drive transcription of the first chain, the second chain and the chaperone protein, respectively. ### Claim 3 (depends on 2) The method of claim 2, wherein the promoter is an inducible promoter. ### Claim 4 (depends on 3) The method of claim 3, wherein the inducible promoter is an IPTG-inducible promoter that drives transcription of the first chain, the second chain and the chaperone protein in the absence of IPTG induction. ### Claim 5 (depends on 3) The method of claim 3, wherein the inducible promoter is a Pho promoter that drives transcription of the first chain, the second chain and the chaperone protein when phosphate in the culture medium has been depleted. ### Claim 6 (depends on 1) The method of claim 1, wherein the polynucleotide further comprises a selectable marker and the culture medium comprises a selection agent consisting of a single antibiotic to cause the host cell to retain the polynucleotide. ### Claim 7 (depends on 1) The method of claim 1, wherein the first translational unit comprises a first translation initiation region (TIR) in operable combination with a coding region of the first chain, and the second translational unit comprises a second translation initiation region (TIR) in operable combination with a coding region of the second chain, wherein the relative translation strength of the first and second TIR is from about 1.0 to about 3.0. ### Claim 8 (depends on 1) The method of claim 1, wherein the at least one chaperone protein comprises a peptidyl-prolyl isomerase. ### Claim 9 (depends on 8) The method of claim 8, wherein the peptidyl-prolyl isomerase is an FkpA protein. ### Claim 10 (depends on 9) The method of claim 9, wherein the FkpA is E. coli FkpA. ### Claim 11 (depends on 8) The method of claim 8, wherein the at least one chaperone protein further comprises a protein disulfide oxidoreductase. ### Claim 12 (depends on 11) The method of claim 11, wherein the protein disulfide oxidoreductase is one or both of a DsbA protein and a DsbC protein. ### Claim 13 (depends on 12) The method of claim 12, wherein the at least one protein disulfide oxidoreductase is one or both of E. coli DsbA and E. coli DsbC. ### Claim 14 (depends on 1) The method of claim 1, wherein the prokaryotic host cell is a gram-negative bacterium. ### Claim 15 (depends on 14) The method of claim 14, wherein the gram-negative bacterium is E. coli. ### Claim 16 (depends on 15) The method of claim 15, wherein the E. coli is a strain with a degpS210A mutation. ### Claim 17 (depends on 16) The method of claim 16, wherein the E. coli is a strain with a genotype of W3110 ΔfhuA ΔphoA ilvG2096 (Valr) Δprc spr43H1 ΔdegP ΔmanA lacIQ ΔompT ΔmenE degpS210A. ### Claim 18 (depends on 1) The method of claim 1, wherein the polypeptide is a monomer of a heterodimer. ### Claim 19 (depends on 1) The method of claim 1, wherein the two chains of the polypeptide are linked to each other by at least one disulfide bond. ### Claim 20 (depends on 1) The method of claim 1, wherein the polypeptide is a monovalent antibody in which the first chain and the second chain comprise an immunoglobulin heavy chain and an immunoglobulin light chain. ### Claim 21 (depends on 20) The method of claim 20, wherein the monovalent antibody is capable of specifically binding an antigen. ### Claim 22 (depends on 1) The method of claim 1, wherein the polypeptide is a secretory protein. ### Claim 23 (depends on 22) The method of claim 22, wherein the secretory protein is recovered from the periplasm of the host cell. ### Claim 24 (depends on 1) The method of claim 1, wherein the growth temperature is in the range of about 30° C. to about 34° C. during the growth phase, and the production temperature in the range of about 25° C. to about 29° C. during the production phase. ### Claim 25 (depends on 1) The method of claim 1, wherein the growth agitation rate is from about 10% to about 40% higher than the production agitation rate. ### Claim 26 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. ### Claim 27 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. ### Claim 28 (depends on 27) The method of claim 27, wherein the half antibody comprises at least one hole-forming mutation or at least one knob-forming mutation. ### Claim 29 (depends on 27) The method of claim 27, wherein the polynucleotide further comprises three copies of a promoter, wherein a first copy is in operable combination with the first translational unit, a second copy is in operable combination with the second translational unit, and a third copy is in operable combination with the third translational unit to drive transcription of the heavy chain, the light chain and the chaperone protein, respectively. ### Claim 30 (depends on 29) The method of claim 29, wherein the promoter is an inducible promoter. ### Claim 31 (depends on 30) The method of claim 30, wherein the inducible promoter is an IPTG-inducible promoter that drives transcription of the heavy chain, the light chain and the chaperone protein in the absence of IPTG induction. ### Claim 32 (depends on 30) The method of claim 30, wherein the inducible promoter is a pho promoter that drives transcription of the heavy chain, the light chain and the chaperone protein when phosphate in the culture medium has been depleted. ### Claim 33 (depends on 27) The method of claim 27, wherein the polynucleotide further comprises a selectable marker and the culture medium comprises a selection agent consisting of a single antibiotic to cause the host cell to retain the polynucleotide. ### Claim 34 (depends on 27) The method of claim 27, wherein the first translational unit comprises a first translation initiation region (TIR) in operable combination with a coding region of the heavy chain variable domain, and the second translational unit comprises a second translation initiation region (TIR) in operable combination with a coding region of the light chain variable domain, wherein the relative translation strength of the first and second TIR is from about 1.0 to about 3.0. ### Claim 35 (depends on 27) The method of claim 27, wherein the at least one chaperone protein, or the first chaperone protein comprises a peptidyl-prolyl isomerase. ### Claim 36 (depends on 35) The method of claim 35, wherein the peptidyl-prolyl isomerase is an FkpA protein. ### Claim 37 (depends on 36) The method of claim 36, wherein the FkpA is E. coli FkpA. ### Claim 38 (depends on 35) The method of claim 35, wherein one or both of the second chaperone protein and the third chaperone protein comprises a protein disulfide oxidoreductase. ### Claim 39 (depends on 38) The method of claim 38, wherein the protein disulfide oxidoreductase is one or both of a DsbA protein and a DsbC protein. ### Claim 40 (depends on 27) The method of claim 27, wherein the at least one protein disulfide oxidoreductase is one or both of E. coli DsbA and E. coli DsbC. ### Claim 41 (depends on 27) The method of claim 27, wherein the prokaryotic host cell is a gram-negative bacterium. ### Claim 42 (depends on 41) The method of claim 41, wherein the gram-negative bacterium is E. coli. ### Claim 43 (depends on 42) The method of claim 42, wherein the E. coli is of a strain deficient in endogenous protease activity. ### Claim 44 (depends on 27) The method of claim 27, wherein the growth temperature is in the range of about 30° C. to about 34° C. during the growth phase, and the production temperature in the range of about 25° C. to about 29° C. during the production phase. ### Claim 45 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. ### Claim 46 (depends on 45) The method of claim 45, wherein the anti-IL13 half antibody comprises at least one knob-forming mutation. ### Claim 47 (depends on 45) The method of claim 45, wherein the heavy chain variable domain of the anti-IL13 half antibody comprises the amino acid sequence of SEQ ID NO:7 and the light chain variable domain of the anti-IL13 antibody comprises the amino acid sequence of SEQ ID NO:8. ### Claim 48 (depends on 45) The method of claim 45, wherein the heavy chain of the anti-IL13 half antibody comprises the amino acid sequence of SEQ ID NO:15 or SEQ ID NO:16. ### Claim 49 (depends on 45) The method of claim 45, wherein the light chain of the anti-IL13 half antibody comprises the amino acid sequence of SEQ ID NO:17. ### Claim 50 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. ### Claim 51 (depends on 50) The method of claim 50, wherein the anti-IL17 half antibody comprises at least one hole-forming mutation. ### Claim 52 (depends on 50) The method of claim 50, wherein the heavy chain variable domain of the anti-IL17 half antibody comprises the amino acid sequence of SEQ ID NO:18 and the light chain variable domain of the anti-IL17 half antibody comprises the amino acid sequence of SEQ ID NO:19. ### Claim 53 (depends on 50) The method of claim 50, wherein the heavy chain of the anti IL17 half antibody comprises the amino acid sequence of SEQ ID NO:26 or SEQ ID NO:27. ### Claim 54 (depends on 50) The method of claim 50, wherein the light chain of the anti-IL17 half antibody comprises the amino acid sequence of SEQ ID NO:28. ### Claim 55 (depends on 28) A method of producing a bi-specific antibody comprising a first half antibody capable of binding a first antigen and a second half antibody capable of binding a second antigen, the method comprising: combining in a reducing condition, the first half antibody with the second half antibody to produce a bi-specific antibody, wherein the first half antibody comprises at least one knob-forming mutation and the second half antibody comprises at least one hole-forming mutation, and wherein both the first half antibody and the second half antibody are produced by the method of claim 28. ### Claim 56 (depends on 55) The method of claim 55, wherein the first antigen and the second antigen are different antigens. ### Claim 57 (depends on 55) The method of claim 55, wherein the first half antibody is capable of binding IL-13. ### Claim 58 (depends on 55) The method of claim 55, wherein the second half antibody is capable of binding IL-17. ### Claim 59 (depends on 55) The method of claim 55, further comprising the step of adding a reducing agent to achieve the reducing condition. ### Claim 60 (depends on 59) The method of claim 59, wherein the reducing agent is glutathione. ### Claim 61 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. ### Claim 62 (depends on 61) The method of claim 61, wherein the first half antibody comprises at least one knob-forming mutation, and the second half antibody comprises at least one hole-forming mutation. ### Claim 63 (depends on 61) The method of claim 61, further comprising the step of adding a reducing agent to achieve the reducing condition. ### Claim 64 (depends on 63) The method of claim 63, wherein the reducing agent is glutathione.