# US 2021/0171997 A1 · Harvest operations for recombinant proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0171997 A1 - Document ID: US20210171997A1 - Status: Published application - Kind: A1 - Application number: 17/067,059 - Filed: 2020-10-09 - Published: 2021-06-10 - Earliest claimed: 2012-03-27 - Assignees: Genentech, Inc. - Applicants: Genentech, Inc. - Inventors: Michael W. LAIRD; Richard ST. JOHN; Jane V. GUNSON; Kimberly KALEAS; Deepa NADARAJAH; Rachel L.E. ADAMS; Bradley R. SNEDECOR - Web page: https://patentgrep.com/patent/US20210171997A1 ## CPC - C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00) - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - 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)} - 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) ## Abstract The present invention contemplates a method of producing a recombinant protein comprising (a) fermenting a prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, and (b) harvesting said recombinant protein under conditions where dO2 levels are greater than 0%, and (c) purifying said recombinant protein to a filtered bulk, wherein said filtered bulk does not contain detectable DHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm. The present invention also contemplates a method of producing a recombinant protein comprising (a) fermenting a menE gene-deleted prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, (b) harvesting said recombinant protein; and (c) purifying said recombinant protein to a filtered bulk, wherein said filtered bulk does not contain detectable DHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm, and wherein the recombinant protein yield is increased by about 20% or greater. ## Claims ### Claim 1 A method of producing a recombinant protein comprising (a) fermenting a prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, and (b) harvesting said recombinant protein under conditions where dissolved oxygen (dO2) levels are greater than 0%, and (c) purifying said recombinant protein to a filtered bulk for storage (FBS), wherein said filtered bulk does not contain detectable 1,4-dihydroxy-2-naphthoate (DHNA)-recombinant protein adduct, as measured by an ion exchange chromatography (IEC) assay at 310 nm. ### Claim 2 (depends on 1) The method of claim 1, wherein said analytical assay is by HPLC, RP HPLC, HIC HPLC, NMR, mass spectrometry, or UV spectroscopy. ### Claim 3 (depends on 1) The method of claim 1, wherein said dO2 is maintained at levels greater than 0% continuously throughout the harvest operations of step (b). ### Claim 4 (depends on 3) The method of claim 3, wherein the harvest operations comprise a homogenization stage. ### Claim 5 (depends on 4) The method of claim 4, wherein said dO2 is maintained at about 30% to about 75% prior to homogenization. ### Claim 6 (depends on 4) The method of claim 4, wherein said dO2 is maintained at levels greater than 75% prior to homogenization. ### Claim 7 (depends on 4) The method of claim 4, wherein said dO2 is maintained at about 50% after homogenization. ### Claim 8 (depends on 4) The method of claim 4, wherein said dO2 is maintained at levels greater than 50% after homogenization. ### Claim 9 (depends on 5) The method of claim 5, wherein said dO2 is maintained for a period of greater than or equal to 1.5 hours. ### Claim 10 (depends on 6) The method of claim 6, wherein said dO2 is maintained for a period of greater than or equal to 2 hours. ### Claim 11 (depends on 1) The method of claim 1, wherein said dO2 is maintained with overlay or sparged air, with increased back-pressure, or with agitation rate. ### Claim 12 (depends on 11) The method of claim 11, wherein the overlay air is from about 0.4 vvm to about 0.8 vvm. ### Claim 13 (canceled) ### Claim 14 (depends on 11) The method of claim 11, wherein the increased backpressure is between about 1.0 to 30 psi. ### Claim 15 (canceled) ### Claim 16 (depends on 11) The method of claim 11, wherein the agitation rate is from about 6 Watts/L to about 8 Watts/L. ### Claim 17 (depends on 16) The method of claim 16, wherein the agitation rate is targeted at about 6 Watts/L. ### Claim 18 A method of producing a recombinant protein comprising (a) fermenting a menE gene-deleted prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, (b) harvesting said recombinant protein; and (c) purifying said recombinant protein to a FBS, wherein said filtered bulk does not contain detectable 1,4-dihydroxy-2-naphthoate (DHNA)-recombinant protein adduct, as measured by an ion exchange chromatography (IEC) assay at 310 nm. ### Claim 19 (depends on 18) The method of claim 18, wherein the recombinant protein yield is increased by about 20% or greater, by about 30% or greater, by about 40% or greater, by about 50% or greater, by about 60% or greater, as compared to the yield using a control prokaryotic host cell. ### Claim 20 (depends on 1) The method of claim 1, wherein the fermentation is scale-independent. ### Claim 21 (depends on 1) The method of claim 1, wherein said recombinant protein is a recombinant polypeptide or an isolated antibody. ### Claim 22 (depends on 1) The method of claim 1, wherein said prokaryotic host cell is Escherichia coli (E. coli), Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.