# US 2014/0154747 A1 · Methods and devices for producing biomolecules > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154747 A1 - Document ID: US20140154747A1 - Status: Published application - Kind: A1 - Application number: 14/173,292 - Filed: 2014-02-05 - Published: 2014-06-05 - Earliest claimed: 2008-02-08 - Assignees: Boehringer Ingelheim Rcv GmbH & Co KG - Applicants: Boehringer Ingelheim RCV GmbH & Co. KG - Inventors: Jochen URTHALER; Christine ASCHER; Daniel BUCHELI - Web page: https://patentgrep.com/patent/US20140154747A1 ## CPC - C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides - B03D 1/082: {of the froth product, e.g. washing} - B03D 1/1468: {Discharge mechanisms for the sediments} - B03D 1/1487: {Means for cleaning or maintenance} - B03D 1/18: without air supply - C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor} - C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers} - B03D 1/028: {Control and monitoring of flotation processes; computer models therefor} - B03D 1/1462: {Discharge mechanisms for the froth} - B03D 2203/003: Biotechnological applications, e.g. separation or purification of enzymes, hormones, vitamins, viruses - 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)} - B03D: FLOTATION; DIFFERENTIAL SEDIMENTATION (sedimentation in general B01D21/00; in combination with other separation of solids B03B; sink-float separation B03B5/28; detergents, soaps C11D) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A scalable process and device for producing a bio molecule, in particular pharmaceutical grade plasmid DNA is described. The process includes the steps of alkaline lysis, neutralization and clarification and can be further extended. For separating the lysate and the precipitate an improved floatation method is disclosed. This method is based on attachment of CO2 bubbles on the precipitate floe. The CO2 is released from a carbonate salt during or after neutralization (acidification). The method of the invention is preferably carried out in an automated continuous mode applying devices for lysis and neutralization and a novel device for completely continuous clarification (separation of flocs and clarified lysate). ## Claims ### Claim 1 A method for producing a biomolecule of interest that is not secreted by the host cells, comprising the steps of a) cultivating host cells to produce the biomolecule of interest and optionally harvesting and resuspending the cells, b) disintegrating the cells by alkaline lysis, c) neutralizing the lysate obtained in step b), whereby a precipitate is formed, d) separating the cleared lysate from the precipitate obtained in step c), e) purifying the biomolecule of interest, wherein a carbonate salt is added in at least one of step a-c), whereby due to acidic conditions at and after step c) CO2 is released and wherein in step d) the precipitate and the lysate are allowed to separate in a clarification device. ### Claim 2 (depends on 1) The method of claim 1, wherein the carbonate salt is added in step a). ### Claim 3 (depends on 1) The method of claim 1, wherein the carbonate salt is added in step b). ### Claim 4 (depends on 1) The method of claim 1, wherein the carbonate salt is added in step c). ### Claim 5 (depends on 1) The method of claim 1, wherein the calculated theoretical carbonate concentration in the resulting lysate-floc mixture comprising attached CO2 bubbles is in the range of about 0.003 to about 0.35 M. ### Claim 6 (depends on 5) The method of claim 5, wherein the calculated theoretical carbonate concentration in the resulting lysate-floc mixture comprising attached CO2 bubbles is in the range of about 0.005 to about 0.05 M. ### Claim 7 (depends on 1) The method of claim 1, wherein the carbonate salt is NaHCO3. ### Claim 8 (depends on 1) A device for carrying out step d) in the method of claim 1 in a semi-continuous mode, comprising a container which is equipped with a) a retention layer in its lower part, b) an inlet at a position above the retention layer, c) an outlet underneath the retention layer, and d) one or more distribution means that reach to the surface of the retention layer and evenly and gently distribute a mixture of precipitate and lysate as obtained upon alkaline lysis and neutralization into the container. ### Claim 9 (depends on 1) A device for carrying out step d) in a method of claim 1 in a continuous mode, comprising a container, which is equipped with a) two outlets, wherein one is positioned at the top of the cylinder and the other one at the bottom of the container, and b) an inlet between the two outlets. ### Claim 10 (depends on 9) The device according to claim 9, wherein the container is connected with an additional drain and/or wash unit. ### Claim 11 (depends on 1) The method of claim 1, wherein at least a combination of two steps selected from steps b) to e) is operated in a continuous mode by connecting the two or more individual steps. ### Claim 12 (depends on 11) The method of claim 11, wherein in addition step a) is operated in a continuous mode by being connected to step b). ### Claim 13 (depends on 1) The method of claim 1, wherein a washing step of the precipitate is inserted between step d) and step e). ### Claim 14 (depends on 1) The method of claim 1, wherein a concentration and/or a conditioning step optionally including filtration is inserted between step d) and step e). ### Claim 15 (depends on 1) The method of claim 1, wherein the clear lysate of step d) contains the biomolecule of interest. ### Claim 16 (depends on 1) The method of claim 1, wherein said biomolecule of interest is a polynucleotide. ### Claim 17 (depends on 16) The method of claim 16, wherein the polynucleotide is DNA. ### Claim 18 (depends on 17) The method of claim 17, wherein the DNA is plasmid DNA. ### Claim 19 (depends on 1) The method of claim 1, wherein the cell mass obtained in step a) is cryo-pelleted. ### Claim 20 (depends on 1) The method according to claim 1 comprising providing a resuspension buffer comprising a carbonate for resuspending cells in step a). ### Claim 21 (depends on 1) The method according to claim 1 comprising providing a lysis buffer comprising a carbonate in step b). ### Claim 22 (depends on 1) The method according to claim 1 comprising adding a carbonate into a resuspended cell solution. ### Claim 23 (depends on 1) The method according to claim 1 comprising adding a carbonate into a lysed cell solution. ### Claim 24 (depends on 1) The method according to claim 1 comprising adding a carbonate into a neutralized lysed cell solution. ### Claim 25 (depends on 24) The method according to claim 24 comprising adding the carbonate into the neutralized lysed cell solution before the precipitate and the lysate are introduced to a clarification device. ### Claim 26 (depends on 24) The method according to claim 24 comprising adding the carbonate into the neutralized lysed cell solution after the precipitate and the lysate are introduced to a clarification device. ### Claim 27 (depends on 1) The method according to claim 1 wherein step d) is performed in a continuous mode and carried out in the clarification device which comprises a container equipped with a first outlet positioned at the top of the container and a second outlet positioned at the bottom of the container, the container further comprising an inlet positioned between the first and second outlets. ### Claim 28 (depends on 27) The method of claim 27, wherein the cleared lysate obtained in step d) leaves the clarification device through the second outlet.