# US 9,751,930 B2 · Process for purifying proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,751,930 B2 - Document ID: US9751930B2 - Status: Granted - Kind: B2 - Application number: 13/812,350 - Filed: 2011-07-27 - Published: 2013-07-11 - Granted: 2017-09-05 - Earliest claimed: 2010-07-27 - Assignees: UCB Pharma S.A. - Applicants: Gavin Barry Wild - Inventors: Gavin Barry Wild - Web page: https://patentgrep.com/patent/US9751930B2 ## CPC - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 1/30: by precipitation - C07K 16/241: {Tumor Necrosis Factors} - C12N 9/90: Isomerases (5.) - C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00) - C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature - C07K 2317/55: Fab or Fab' - 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) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} ## Abstract The present invention provides method for purifying a recombinant protein from a gram-negative bacterial host cell sample or extract thereof wherein said host cell expresses a recombinant protein and a recombinant disulphide isomerase DsbC; comprising: a. adjusting the pH of the host cell sample or extract thereof to a pH of 5 or less to precipitate the recombinant disulphide isomerase; and b. separating precipitated recombinant disulphide isomerase DsbC from the recombinant protein to produce a recombinant protein sample. ## Claims ### Claim 1 A method for purifying a recombinant protein of interest from gram-negative bacterial host cells, wherein said gram-negative bacterial host cells express the recombinant protein of interest and a recombinant disulphide isomerase, wherein the method comprises: collecting said gram-negative bacterial host cells from culture medium; releasing said recombinant protein of interest and said recombinant disulphide isomerase from said collected gram-negative bacterial host cells to form a gram-negative bacterial host cell extract; separating said gram-negative bacterial host cells from the released recombinant protein of interest in said gram-negative bacterial host cell extract to form a liquid gram-negative bacterial host cell extract, wherein the pH of the liquid gram-negative bacterial host cell extract is lower than the pI of the recombinant protein of interest; adjusting the pH of the liquid gram-negative bacterial host cell extract to a pH of 5 or less to precipitate the recombinant disulphide isomerase from the recombinant protein of interest, which remains in solution; and separating the precipitated recombinant disulphide isomerase from the recombinant protein of interest remaining in solution to produce a liquid recombinant protein sample containing a reduced quantity of recombinant disulphide isomerase compared to the quantity of recombinant disulphide isomerase prior to said separating step. ### Claim 2 (depends on 1) The method according to claim 1, wherein the pH of the liquid gram-negative bacterial host cell extract is adjusted to a pH of 4.5 or less. ### Claim 3 (depends on 2) The method according to claim 2, wherein the pH of the liquid gram-negative bacterial host cell extract is adjusted to a pH of 4.5 to 3.0. ### Claim 4 (depends on 2) The method according to claim 2, wherein the pH of the liquid gram-negative bacterial host cell extract is adjusted to a pH of 3 or less. ### Claim 5 (depends on 1) The method according to claim 1, wherein the step of separating the precipitated recombinant disulphide isomerase from the recombinant protein of interest remaining in solution comprises centrifuging said liquid gram-negative bacterial host cell extract to separate the precipitate from the recombinant protein of interest to form said liquid recombinant protein sample and purifying said liquid recombinant protein of interest from said recombinant protein sample by chromatography. ### Claim 6 (depends on 1) The method according to claim 1, wherein the disulphide isomerase comprises a histidine tag. ### Claim 7 (depends on 1) The method according to claim 1, wherein the gram-negative bacterial host cells are Escherichia coli cells. ### Claim 8 (depends on 1) The method according to claim 1, wherein the gram-negative bacterial host cells comprise FKBP-type peptidyl-prolyl cis-trans isomerase (Fkpa) and/or seventeen kilodalton protein (Skp). ### Claim 9 (depends on 1) The method according to claim 1, wherein the pH of the liquid gram-negative bacterial host cell extract is adjusted to less than 5 using glacial acetic acid. ### Claim 10 (depends on 1) The method according to claim 1, wherein the liquid gram-negative bacterial host cell extract is held at a pH of 5 or less for one hour or less. ### Claim 11 (depends on 1) The method according to claim 1, wherein separating the precipitated recombinant disulphide isomerase from the recombinant protein of interest comprises centrifugation and/or filtration. ### Claim 12 (depends on 1) The method according to claim 1, said method further comprising a purification step of subjecting the liquid recombinant protein sample to chromatography. ### Claim 13 (depends on 12) The method according to claim 12, wherein the chromatography is ion-exchange chromatography. ### Claim 14 (depends on 1) The method according to claim 1, wherein the pH of the liquid recombinant protein sample is adjusted to a pH of 5 to 7. ### Claim 15 (depends on 1) The method according to claim 1, wherein releasing said recombinant protein of interest and said recombinant disulphide isomerase from said gram-negative bacterial host cells to form a gram-negative bacterial host cell extract comprises adding an extraction buffer to the gram-negative bacterial host cell extract and heating the gram-negative bacterial host cell extract/extraction buffer composition. ### Claim 16 (depends on 1) The method according to claim 1, wherein the recombinant protein of interest has a pI of 6 to 9. ### Claim 17 (depends on 16) The method according to claim 16, wherein the recombinant protein of interest has a pI of 8 to 9. ### Claim 18 (depends on 1) The method according to claim 1, wherein the recombinant protein of interest is a recombinant antibody. ### Claim 19 (depends on 18) The method according to claim 18, wherein the recombinant antibody is a monoclonal, humanized or chimeric antibody, or a binding fragment thereof. ### Claim 20 (depends on 19) The method according to claim 19, wherein the binding fragment is a Fab or Fab′ fragment. ### Claim 21 (depends on 18) The method according to claim 18, wherein the recombinant antibody is specific for human TNFα. ### Claim 22 (depends on 21) The method according to claim 21, wherein the antibody comprises a heavy chain wherein the variable domain comprises a CDR having the sequence given in SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, and SEQ ID NO: 3 for CDRH3 and a light chain and wherein the variable domain comprises a CDR having the sequence given in SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 for CDRL2 and SEQ ID NO: 6 for CDRL3. ### Claim 23 (depends on 22) The method according to claim 22, wherein the antibody comprises the light chain variable region sequence given in SEQ ID NO: 7 and the heavy chain variable region sequence given in SEQ ID NO: 8. ### Claim 24 (depends on 23) The method according to claim 23, wherein the antibody is a Fab fragment and comprises a heavy chain having the sequence given in SEQ ID NO: 10 and a light chain having the sequence given in SEQ ID NO: 9. ### Claim 25 (depends on 1) The method of claim 1, wherein the recombinant disulphide isomerase is disulphide isomerase A (DsbA), disulphide isomerase B (DsbB), disulphide isomerase C (DsbC), disulphide isomerase D (DsbD) or disulphide isomerase G (DsbG). ### Claim 26 (depends on 25) The method of claim 25, wherein the recombinant disulphide isomerase is disulphide isomerase C (DsbC). ### Claim 27 (depends on 5) The method according to claim 5, wherein the chromatography is ion-exchange chromatography. ### Claim 28 (depends on 15) The method according to claim 15, wherein said releasing said recombinant protein of interest and said recombinant disulphide isomerase from said gram negative bacterial host cells to form a gram-negative bacterial host cell extract comprises: adding the extraction buffer to said gram-negative bacterial host cells separated from the culture medium to form the gram-negative bacterial host cell extract; heating the gram-negative bacterial host cell extract to which said extraction buffer has been added to a temperature of 50° C. to 60° C. for a period of 10 to 16 hours; centrifuging said heat treated gram-negative bacterial host cell extract/extraction buffer to separate gram-negative bacterial host cells from extracted material in the supernatant and collecting said supernatant; adjusting the pH of said supernatant to between 3 and 5 to precipitate disulphide isomerase in said supernatant; centrifuging the pH adjusted supernatant to separate precipitated disulphide isomerase from soluble proteins in said supernatant to form a recombinant protein sample; optionally adjusting the pH of said recombinant protein sample to a pH of 5 to 7; and purifying said recombinant protein of interest from said recombinant protein sample by column chromatography. ### Claim 29 (depends on 15) The method according to claim 15, wherein releasing said recombinant protein of interest and said recombinant disulphide isomerase from said gram-negative bacterial host cells to form a gram-negative bacterial host cell extract comprises separating said gram-negative bacterial host cells from the culture medium, resuspending the separated gram-negative bacterial host cells in an extraction buffer and heating the gram-negative bacterial host cell/extraction buffer composition to a temperature between 30° C. and 70° C. ### Claim 30 (depends on 29) The method according to claim 29, wherein said gram-negative bacterial host cell/extraction buffer composition is heated to a temperature between 40° C. and 65° C. ### Claim 31 (depends on 29) The method according to claim 29, wherein said gram-negative bacterial host cell/extraction buffer composition is heated to a temperature between 40° C. and 60° C. ### Claim 32 (depends on 29) The method according to claim 29, wherein said gram-negative bacterial host cell/extraction buffer composition is heated to a temperature between 50° C. and 60° C. ### Claim 33 (depends on 29) The method according to claim 29, wherein said gram-negative bacterial host cell/extraction buffer composition is heated to a temperature between 30° C. and 70° C. for a period of between one (1) and 24 hours. ### Claim 34 (depends on 29) The method according to claim 29, wherein said gram-negative bacterial host cell/extraction buffer composition is heated to a temperature between 30° C. and 70° C. for a period of between four (4) and 24 hours. ### Claim 35 (depends on 29) The method according to claim 29, wherein said gram-negative bacterial host cell/extraction buffer composition is heated to a temperature between 30° C. and 70° C. for a period of between four (4) and 18 hours. ### Claim 36 (depends on 29) The method according to claim 29, wherein said gram-negative bacterial host cell/extraction buffer composition is heated to a temperature between 30° C. and 70° C. for a period of between six (6) and 16 hours. ### Claim 37 (depends on 1) The method according to claim 1, said method comprising adjusting the pH of the liquid gram-negative bacterial host cell extract to a pH of 3 to 5. ### Claim 38 (depends on 37) The method according to claim 37, wherein the pH of the liquid gram-negative bacterial host cell extract is held at a pH of 3 to 5 for a period of less than one hour and the pH is then raised to a pH of 5 to 7. ### Claim 39 (depends on 1) The method according to claim 1, wherein the step of collecting said gram-negative bacterial host cells from the culture medium comprises centrifugation of said gram-negative bacterial host cells to form a cell pellet and removal of the supernatant from said cell pellet. ### Claim 40 (depends on 1) The method according to claim 1, wherein the step of collecting said gram-negative bacterial host cells from the culture medium comprises filtering the cell culture containing said gram-negative bacterial host cells to separate said gram-negative bacterial host cells from said culture medium and recovering the filtered cell mass. ### Claim 41 (depends on 1) The method according to claim 1, wherein the step of collecting said gram-negative bacterial host cells from the culture medium comprises concentrating said gram-negative bacterial host cells in said culture medium and recovering the concentrated cell mass.