US20140051121A1 - Feed mixing device and its use - Google Patents

Feed mixing device and its use Download PDF

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US20140051121A1
US20140051121A1 US13/909,591 US201313909591A US2014051121A1 US 20140051121 A1 US20140051121 A1 US 20140051121A1 US 201313909591 A US201313909591 A US 201313909591A US 2014051121 A1 US2014051121 A1 US 2014051121A1
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feed
cultivation
value
solutions
reported
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US13/909,591
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Sylvia Baumann
Jens Hoffmann
Alexander Jockwer
Christian Klinger
Thomas Troebs
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Hoffmann La Roche Inc
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Roche Diagnostics GmbH
Hoffmann La Roche Inc
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Assigned to F. HOFFMANN-LA ROCHE AG reassignment F. HOFFMANN-LA ROCHE AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ROCHE DIAGNOSTICS GMBH
Assigned to HOFFMANN-LA ROCHE INC. reassignment HOFFMANN-LA ROCHE INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: F. HOFFMANN-LA ROCHE AG
Priority to US15/713,268 priority Critical patent/US10837042B2/en
Priority to US17/083,048 priority patent/US20210040528A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/16—Apparatus for enzymology or microbiology containing, or adapted to contain, solid media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/26—Conditioning fluids entering or exiting the reaction vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F2215/00—Auxiliary or complementary information in relation with mixing
    • B01F2215/04—Technical information in relation with mixing
    • B01F2215/0413—Numerical information
    • B01F2215/0436—Operational information
    • B01F2215/044—Numerical composition values of components or mixtures, e.g. percentage of components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F35/00—Accessories for mixers; Auxiliary operations or auxiliary devices; Parts or details of general application
    • B01F35/80—Forming a predetermined ratio of the substances to be mixed
    • B01F35/88—Forming a predetermined ratio of the substances to be mixed by feeding the materials batchwise
    • B01F35/883—Forming a predetermined ratio of the substances to be mixed by feeding the materials batchwise using flow rate controls for feeding the substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure

Definitions

  • a feed mixing device that allows simultaneous feeding of two or more feeding solutions with non-physiological pH value to a cultivation medium whereby the pH value of the combined feed solutions is adjusted, e.g. to a physiological pH value, prior to the addition to the cultivation medium within the feed mixing device.
  • Feeding of aqueous solutions with physical properties different from the cultivation medium will affect physical parameters of the cultivation medium like osmolality or pH value. Due to poor solubility or required stabilization of different components, the pH value of feeding solutions sometimes has to be changed to non-physiological values.
  • Luan, Y. T., et al. report strategies to extend longevity of hybridomas in culture and promote yield of monoclonal antibodies (Biotechnol. Lett. 9 (1987) 691-696). Improved fermentation processes for NS0 cell lines expressing human antibodies and glutamine synthetase is reported by Dempsey, J., et al. (Biotechnol. Prog. 19 (2003) 175-178). Bibila, T. A., et al., report monoclonal antibody process development using medium concentrates (Biotechnol. Prog. 10 (1994) 87-96).
  • WO 2009/132616 a supply system is reported.
  • a method and apparatus for producing alcohol or sugar using a commercial-scale bioreactor is reported in WO 2010/045168.
  • US 2003/0092652 a protected one-vial formulation for nucleic acid molecules, methods of making the same by in-line mixing, and related products and methods are reported.
  • feed components can be provided e.g. in solutions with a pH value at which the components have good solubility and/or good stability, whereby the pH value can be/is different from the pH value of the cultivation medium, i.e. different from a physiologically acceptable pH value of pH 6.5 to pH 7.5, i.e. the solutions have independently of each other a pH value of less than pH 6.5 or more than pH 7.5.
  • This allows performing a cultivation with more flexibility compared to a cultivation in which e.g. the pH value of the feed solution is limited to a small range around the pH value of the cultivation medium.
  • One aspect as reported herein is a device for adding at least two solutions each with a non-physiological pH value to a cell cultivation vessel comprising a chamber for mixing the solutions prior to their addition to the cell cultivation vessel.
  • the ratio of the volume of the chamber for mixing the solutions to the volume of the cultivation medium in the cultivation vessel is of from 0.8 ml/l to 1.2 ml/l. In one embodiment the ratio is of from 0.9 ml/l to 1.1 ml/l. In one embodiment the ratio is about 1 ml/l. In one embodiment the ratio is 0.95 ml/l. In one embodiment the volume of the cultivation medium is the volume of liquid at the start of the cultivation in the cultivation vessel.
  • the at least two solutions each with a non-physiological pH value are at least one acidic solution and at least one alkaline solution. In one embodiment the at least two solutions each with a non-physiological pH value have a pH value independently of each other of less than pH 6.5 or more than pH 7.5. In one embodiment the at least two solutions each with a non-physiological pH value have a pH value independently of each of pH 0 to pH 6.49 or pH 7.51 to pH 14.
  • the pH value of the acidic and alkaline solution differs by at least 0.5 pH units from the pH value of the cultivation medium.
  • the acidic solution has a pH value of pH 6.5 or lower.
  • the acidic solution has a pH value of pH 4.0 or lower.
  • the alkaline solution has a pH value of pH 8.0 or higher.
  • the alkaline solution has a pH value of 10.0 or higher.
  • the device is for adding of from two to four separate solutions with non-physiological pH value, whereof optionally at least one is an acidic solution and at least one is an alkaline solution.
  • each of the solutions is a feed solution comprising at least one compound selected from amino acid, sugar, vitamin, trace element, lactate, and growth factor.
  • the chamber for mixing the solutions is separated from the cultivation vessel and comprises an outlet to the inside of the cultivation vessel. In one embodiment the chamber is outside of the cultivation vessel or inside the cultivation vessel.
  • the chamber has a volume of from 0.1 ml to 50,000 ml. In one embodiment the chamber has a volume of from 0.25 ml to 30,000 ml. In one embodiment the chamber has a volume of from 0.5 ml to 1,000 ml.
  • the chamber has a volume of about 1.15 ml, or about 8 ml, or about 80 ml, or about 200 ml, or about 400 ml, or about 800 ml, or about 1.6 l, or about 4 l, or about 8 l, or about 16 l, or about 40 l.
  • the mixing is immediately prior to the addition to the cultivation vessel.
  • the chamber for mixing the solutions comprises an inlet with individual connectors for each of the solutions.
  • the device is sterilizable.
  • Another aspect as reported herein is the use of a device as reported herein in the fed-batch or continuous cultivation of cells.
  • cultivation vessel comprising a device as reported herein.
  • the mixed feed solutions have a pH value of from pH 4.5 to pH 9.5 upon addition to the cultivation vessel. In one embodiment the mixed feed solutions have a pH value of from pH 6.5 to pH 7.5 upon addition to the cultivation medium.
  • the cultivation vessel has a volume of about 2 l, or 10 l, or 20 l, or 100 l, or 250 l, or 500 l, or 1,000 l, or 2,000 l, or 5,000 l, or 10,000 l, or 20,000 l, or 50,000 l.
  • the volume of the cultivation medium is about 1.2 l, or about 8 l, or about 16 l, or about 80 l, or about 200 l, or about 400 l, or about 800 l, or about 1,600 l, or about 4,000 l, or about 8,000 l, or about 16,000 l, or about 40,000 l.
  • the device as reported herein is operated at room temperature.
  • an aspect as reported herein is a method for obtaining a polypeptide with a reduced G(0) glycoform and/or increased G(1) glycoform comprising the following steps:
  • polypeptide is an antibody or an Fc-fusion polypeptide.
  • a widely used format for the production of therapeutic polypeptides or biomass is the fed-batch fermentation.
  • Cell densities of mammalian cell cultures often exceed 100*10 5 cells/ml in fed-batch fermentations resulting in challenges to provide sufficient amounts of the required cultivation substrates due to low solubility and/or impaired stability of certain substances or substance classes.
  • feed solutions with non-physiological pH values i.e. alkaline or acidic pH values
  • a feed mixing device as reported herein can be used, enabling continuous mixing of at least two feed solutions just before addition into the cultivation vessel.
  • feed components can be dissolved at pH values at which these have good solubility and/or good stability, whereby the pH value can be clearly different from the pH value of the cultivation medium, i.e. different from a physiological pH value. This allows more flexible pH values for a feed solution, as the pH value is now adjusted directly prior to the addition to the cultivation medium and cell suspension.
  • the device as reported herein is a feed mixing device useful in the cultivation of cells during which compounds have to be added, such as in a fed-batch cultivation or in a continuous cultivation.
  • a feed mixing device as reported herein the cultivating can be performed with at least one additional degree of freedom and, thereby, with more flexibility.
  • the device as reported herein it is possible to use feed solutions that have a non-physiological pH value and/or a high compound concentration.
  • the non-physiological pH value can be required e.g. for stabilizing pH-sensitive feed components.
  • Prior to the addition to the cultivation vessel and, therewith, to the cultivation medium any pH value can be adjusted. This allows in a discontinuous or continuous feeding process to add defined amounts of compounds. For example, by the addition of defined amounts of ions a pre-defined osmolality can be adjusted.
  • feed solutions with any pH value i.e. alkaline, neutral or acidic solutions, and with any concentration of individual components can be used. It is also possible to exert a pH gradient in the added feed, whereby also essentially the same amount of substances can be added compared to a conventional feeding strategies not using the device as reported herein.
  • feed solutions can be used in which the components due to their low solubility or impaired stability have to be provided at extremely alkaline or acidic, i.e. non-physiological, pH values.
  • the pH value of the mixed feed solution leaving the feed mixing device and being added to the cultivation medium depends on the volumetric mixing ratio of the individual feeds and on the residence time within the mixing chamber and, therewith, on the volume flow of the individual feed solutions.
  • the total volume flow into the cultivation vessel through the feed mixing device is of from 1 to 1.5 g/h/l. In one embodiment the volume flow is of from 1.15 g/h/l to 1.35 g/h/l. In one embodiment the volume flow is about 1.25 g/h/l.
  • the unit g/h/l denotes mass of feed/cultivation time/cultivation volume.
  • the cultivation volume is the volume of liquid in the cultivation vessel at the start of the cultivation.
  • the viability of the cultivated cells can be maintained for a longer period of time above a pre-defined level and, therewith, allows for a longer overall cultivation time. At the same time the lactate concentration and the glucose consumption can be reduced.
  • FIG. 1 The general course of the pH value of a cell cultivation is shown in FIG. 1 .
  • the pH value of the cultivation decreases and approaches the lower margin of the pre-defined pH range ( FIG. 1 , “2”). This is due to the formation of lactate and the accumulation of carbon dioxide in the cultivation medium.
  • An engaged pH control mechanism ensures that the pH value is maintained at the lower margin of the pre-set pH range by the addition of base.
  • the base addition is continued until the cell metabolism changes and the lactate in the cultivation medium is re-metabolized and/or the accumulated carbon dioxide is removed ( FIG. 1 , “3”).
  • the pH value increases until it reaches the upper margin of the pre-set pH range ( FIG. 1 , “4”).
  • the pH control mechanism maintains the pH value at this upper margin value by the addition of acid.
  • An alkaline feed solution can maintain the pH value of the cultivation medium above the lower margin of a pre-set pH range e.g. without engagement of the pH control mechanism.
  • the change of the pH value in the cultivation vessel can be counteracted by changing the ratio of the individual volume flow rates of the two or more feed solutions added to the cultivation medium using the feed mixing device as reported herein.
  • a pH control mechanism or at least the time of engagement thereof (and likewise the added amounts of acid and base) might be obsolete or can be reduced, respectively.
  • the pH value of the combined feed solutions can be adjusted to any target value.
  • the pH value of the combined feed solutions can be changed during the cultivation.
  • the variable pH value of the combined feed solutions can be used to support or replace other means for adjusting the cultivation pH value.
  • the feed solutions may comprise any compound.
  • compounds, such as vitamins can be stabilized at (high) non-physiological pH values.
  • the pH value is adjusted prior to the addition to the cultivation medium with the feed mixing device as reported herein to a pH value in the physiological range.
  • the time at which the fed compound is kept at a stability impairing pH value is reduced.
  • the pH value in the cultivation medium can be controlled online by adjusting the ratio and the individual flow rates of the feed solutions ( FIG. 1 , “5”).
  • the individual feeds can independently of each other be a dispersion, an emulsion, or a solution. Solutions can be transported to the feed mixing device by using conventional pumps or any other known mechanical or flow-mechanical method. If a feed is no true solution the feed can, e.g., be added by using a mechanical transportation method.
  • the scale of the feed mixing device is variable and, thus, the device can be used with any type and size of cultivation device, such as e.g. with a cultivation vessel (stirred tank), chip, or flow pipe reactor.
  • cultivation vessel stirred tank
  • chip chip
  • flow pipe reactor any type and size of cultivation device, such as e.g. with a cultivation vessel (stirred tank), chip, or flow pipe reactor.
  • the G(0) glycoform of a produced polypeptide, especially of a produced immunoglobulin can be reduced compared to a cultivation with a single alkaline feed and without employing the device as reported herein.
  • the G(1) glycoform can be increased by using a device as reported herein and adjusting the separates feeds to a mixed acidic feed compared to a cultivation with a single alkaline feed and without employing the device as reported herein.
  • the content of host cell protein in the cultivation medium prior to downstream processing can be changed. This should be possible by using specific feed solutions.
  • an adjusted feed strategy comprising pH adjusted feed solutions with a defined concentration of protons, i.e. a defined pH value
  • an identical or at least similar amount of feed components could be added and concurrently the amount of base or acid required in order to correct the pH value of the cultivation medium can be reduced and also concurrently the host cell protein content in the cultivation supernatant could be reduced.
  • the feed mixing device as reported herein is used in one embodiment for reducing the host cell protein content in a cell cultivation supernatant.
  • FIG. 1 Scheme of the general course of the pH value of a cell cultivation.
  • FIG. 2 Course of the viable cell density: open: single feed (feed 1); filled: separate feeds (feed 2 and feed 3); circle: with potassium chloride; square: with sodium chloride.
  • FIG. 3 Course of the viability: open: single feed (feed 1); filled: separate feeds (feed 2 and feed 3); circle: with potassium chloride; square: with sodium chloride. It can be seen that the viability by using the mixing device as reported herein remains longer at a value more than 90%.
  • FIG. 4 Course of the pH value: open: single feed (feed 1); filled: separate feeds (feed 2 and feed 3); circle: with potassium chloride; square: with sodium chloride. Course is comparable until the start of the feeding (after 72 hours).
  • FIG. 5 Course of glucose consumption: open: single feed (feed 1); filled: separate feeds (feed 2 and feed 3); circle: with potassium chloride; square: with sodium chloride.
  • the glucose consumption is reduced when a mixing device as reported herein is used.
  • FIG. 6 Course of lactate formation: open: single feed (feed 1); filled: separate feeds (feed 2 and feed 3); circle: with potassium chloride; square: with sodium chloride. Lactate formation and onset of re-metabolism is improved upon using a mixing device as reported herein.
  • FIG. 7 Course of glutamine consumption: open: single feed (feed 1); filled: separate feeds (feed 2 and feed 3); circle: with potassium chloride; square: with sodium chloride.
  • FIG. 8 Course of ammonia accumulation: open: single feed (feed 1); filled: separate feeds (feed 2 and feed 3); circle: with potassium chloride; square: with sodium chloride.
  • FIG. 9 Acidic peak fraction: left: use of a device as reported herein; dark right: without a device as reported herein.
  • FIG. 10 Dependency of the required amount of added base (after 14 days of cultivation) on the pH value of the feed solution. Circle: without a device as reported herein; square: with a device as reported herein.
  • FIG. 11 Dependency of lactate formation (after 14 days of cultivation) on the pH value of the feed solution. Circle: without a device as reported herein; square: with a device as reported herein.
  • FIG. 12 Dependency of the glutamine concentration (after 14 days of cultivation) in the cultivation medium on the pH value of the feed solution. Circle: without a device as reported herein; square: with a device as reported herein.
  • FIG. 13 Dependency of the osmolality in the cultivation medium (after 14 days of cultivation) on the pH value of the feed solution. Circle: without a device as reported herein; square: with a device as reported herein.
  • FIG. 14 Dependency of the dissolved carbon dioxide (after 14 days of cultivation) on the pH value of the feed solution. Circle: without a device as reported herein; square: with a device as reported herein.
  • FIG. 15 Dependency of viable cell density (after 14 days of cultivation) on the pH value of the feed solution. Circle: without a device as reported herein; square: with a device as reported herein.
  • FIG. 16 Dependency of the G(0) fraction on the pH value of the feed solution.
  • FIG. 17 Dependency of the G(1) fraction on the pH value of the feed solution.
  • FIG. 18 + 19 Exemplary schemes of the device as reported herein.
  • FIG. 20 Dependency of the host cell protein content on the pH value of the feed solution.
  • An exemplary antibody used in the method and examples as reported herein is an anti-IL17 antibody as reported in WO 2010/034443 (incorporated herein by reference).
  • Feed 1 This solution comprises all feed components (amino acids and pyruvate) at a pH value of about 9.5.
  • Feed 2 This solution comprises at a double concentration the components soluble at an acid pH value of about pH 1.5.
  • Feed 3 This solution comprises at a double concentration the components soluble at a basic pH value of about pH 10.
  • the pH value of feed 1 is about pH 9.5 and the pH value of the combined feeds 2 and 3 is about pH 7.2.
  • Feed flow through the feed mixing device 36 g/d
  • the feed in the cultivation vessels comprising the feed mixing device was started after 72 hours cultivation time with feed 2 and feed 3 as continuous feed with the same volume flow.
  • the feeds were combined in the feed mixing device and added to the cultivation medium after mixing.
  • the feed in the cultivation vessels comprising the conventional feeding device was started after 72 hours cultivation time with feed 1 at a volume flow twice that of the corresponding cultivations with the feed mixing device.
  • FIG. 2 The course during the cultivation of the viable cell density is shown in FIG. 2 , the course of the cell viability is shown in FIG. 3 , the course of the pH value in the cultivation medium is shown in FIG. 4 , and the course of the glucose consumption is shown in FIG. 5 .
  • FIG. 6 shows the course of the lactate concentration during the cultivation.
  • the course of glutamine concentration during the cultivation is shown in FIG. 7 .
  • FIG. 8 shows the course of the ammonia concentration during the cultivation.
  • the amount of the acidic peak of the produced immunoglobulin is shown in FIG. 9 .
  • the viability can be maintained above 90% for an extended period of time by using the device as reported herein.
  • the course of the pH value is comparable for the first 72 hours, i.e. prior to the start of the feeding. Thereafter the pH value of the cultivations employing the feed mixing device is below that of the other cultivations.
  • the glucose consumption is reduced in the cultivations employing the device as reported herein.
  • the maximum lactate concentration during the course of the cultivation with the feed mixing device is lower compared to the maximum lactate concentration of the cultivation without the feed mixing device.
  • the feed solution were composed in such a way that only the pH value after the mixing is different but all other parameters, such as the added amount of feed components, feed volume, or osmolality, are comparable. Therefore the feed solutions were not added at a constant feed rate but added by a gravimetric feeding controller.
  • the pO 2 value was adjusted to a value of 35% air saturation and determined with a pO 2 probe (Mettler-Toledo InPro 6820).
  • the pO 2 probe was calibrated at process conditions after 72 hours of gassing in based on the mean value determined with a certified gas analytics (GA4, Dasgip).
  • the aeration during the cultivation was kept at a constant rate of 75 ml/min of a mixture of nitrogen, air, pure oxygen and carbon dioxide.
  • the fraction of carbon dioxide in the total gas flow was constant at 7 vol % of the total gas flow and was changed solely due to increased demand of the pH control.
  • the pH value of the cultivation medium was adjusted with a 1 mol/l sodium carbonate solution as base and carbon dioxide as acid to a set point of 7.0 +/ ⁇ 0.05 pH units.
  • the required carbon dioxide was added to the total carbon dioxide flow of 75 ml/min.
  • the pH probe (Mettler Toledo 405-DPAS-SC-K8S/200) was calibrated with reference buffer solution of pH values 7.0 and 4.01 and after equilibration of the cultivation medium for at least 72 hours under process conditions as mean value of a blood gas analyzer (Bioprofile, PHOx BGA).
  • the cultivation was performed at a constant stirrer speed of about 230 rpm. A mixer a dish stirrer was used. The power input was about 80 W/m 3 . The same power input was used in the pre-cultivation to ensure comparability and avoid a rapid change in the conditions.
  • An anti-foam solution was added based on the foam formation. No anti-foam probe was employed. The anti-foam amount required by the cultivation vessel with the highest foam formation was also added to the other cultivation vessels. As anti-foam agent 1% medical grade Dow was used.
  • the cultivation medium was a chemically defined medium. For each fermentation one liter of medium was used. The inoculation volume was 200 ml. Thus, the cultivation was performed with a starting volume of 1,200 ml.
  • cell line a CHO cell transfected with a nucleic acid encoding an anti-IL17 antibody was used.
  • the cell density in the 200 ml inoculation volume was adjusted to ensure a cell density of about 3.5 ⁇ 10 5 cells/ml in the cultivation.
  • the power input in the inoculation cultivation was kept at the same value as the thereafter following main cultivation.
  • the inoculation cultivation was performed at about 36.5° C., 7% CO 2 , and a relative humidity of 85%.
  • the main cultivation was performed as a fed-batch cultivation, wherein the feeding was started approximately 72 hours after start of the main cultivation.
  • the pH value of the cultivation medium is directly affected. Also affected but only indirectly is the amount of acid and/or base that has to be added during the cultivation by the pH control mechanisms.
  • FIGS. 10 to 17 the influence of the pH value of the feed solution on the parameters amount of added base, amount of added acid, resulting pCO 2 in the cultivation medium after a cultivation time of 14 days is depicted.
  • the amount of added base is dependent on the pH value of the feed solution.
  • the amount of lactate in the cultivation medium after a cultivation of 14 days is dependent on the pH value of the feed solution whereby the use of a feed solution of a pH value of 9.5 results in the highest amount of lactate.
  • the overall amount of lactate is reduced by about 30% compared to a combined feed.
  • the amount of glutamine in the cultivation medium after a cultivation time of 14 days is dependent on the pH value of the feed solution.
  • the osmolality in the cultivation medium after a cultivation time of 14 days is depending on the pH value of the feed solution.
  • FIG. 14 the pCO 2 value in the cultivation medium after a cultivation time of 14 days is shown. It can be seen that the pCO 2 is dependent on the pH value of the feed solution, whereby the highest pCO 2 value was obtained with alkaline feed solutions, either as single feed or as mixed feed using the device as reported herein. It can be seen that by using a feed of a lower pH value the pCO 2 value after a cultivation time of 14 days can be dramatically reduced avoiding unphysiologically high pCO 2 values in the cultivation medium.
  • FIG. 15 the viable cell density after a cultivation time of 14 days is shown. It can be seen that the viable cell density of the cultivations is at or above a value of 90%.
  • FIG. 16 the amount of the G(0) glycoform depending on the pH value of the feed solution is shown. It can be seen that with a neutral and an alkaline feed solution comparable amounts of the G(0) glycoform were obtained. With an acidic feed solution the amount of the G(0) glycoform was reduced.
  • FIG. 17 the amount of the G(1) glycoform depending on the pH value of the feed solution is shown. It can be seen that with a neutral and alkaline feed solution comparable amounts of the G(1) glycoform were obtained. With an acidic feed solution the amount of the G(1) glycoform was increased.
  • An alkaline feed solution of a pH value of 11.3 and an acidic feed solution of a pH value of about 1.0 were combined to obtain a target pH value of about pH 6.5.
  • the mixing of the individual solutions was performed at room temperature and at 4° C. by combining 10 ml of each feed solution of the respective temperature.

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US5081036A (en) 1987-01-23 1992-01-14 Hoffmann-La Roche Inc. Method and apparatus for cell culture
JP2509630B2 (ja) 1987-07-29 1996-06-26 三井石油化学工業株式会社 培養方法及び培養装置
ES2053410T1 (es) 1991-06-17 1994-08-01 Life Technologies Inc Metodo para la preparacion de un medio de cultivo de celulas.
US5856179A (en) * 1994-03-10 1999-01-05 Genentech, Inc. Polypeptide production in animal cell culture
RU2102472C1 (ru) * 1994-07-19 1998-01-20 Научно-исследовательский институт микробиологии МО РФ Способ получения биомассы чумного микроба
JPH10201467A (ja) * 1997-01-20 1998-08-04 Ishikawajima Harima Heavy Ind Co Ltd 被培養体の生長判定方法および培養装置
US20060003448A1 (en) 2003-12-30 2006-01-05 Richard Fike Dry powder cells and cell culture reagents and methods of production thereof
US20030092652A1 (en) 1998-08-14 2003-05-15 Maria Bruno Protected one-vial formulation for nucleic acid molecules, methods of making the same by in-line mixing, and related products and methods
CA2340416C (en) * 1998-08-14 2009-06-02 Valentis, Inc. Co-lyophilized complex of a nucleic acid vector and a formulating agent
KR100283499B1 (ko) * 1998-12-23 2001-03-02 이금용 폐기물 매립지의 매립가스 발생량 조절방법 및 그 장치
EP2295533A3 (en) * 2000-11-06 2011-07-13 Life Technologies Corporation Dry powder cells and cell culture reagents and methods of production thereof
FR2836910B1 (fr) * 2002-03-08 2005-02-11 Amenagement Urbain & Rural Procede de degradation de la matiere organique par voie mycelienne
US6924124B1 (en) 2002-08-23 2005-08-02 Immunex Corporation Feeding strategies for cell culture
AU2003295584A1 (en) 2002-11-18 2004-06-15 Taurus Hsa Llc Method for continuous, automated blending of solutions from acids and bases
EA012617B1 (ru) 2005-03-24 2009-10-30 ДСМ АйПи АССЕТС Б.В. Способ микробиального производства ценного соединения
WO2008013809A1 (en) 2006-07-24 2008-01-31 Amgen Inc. Cell culture methods
PT2115126E (pt) 2007-03-02 2015-08-24 Wyeth Llc Utilização de cobre e glutamato na cultura de células para a produção de polipeptídeos
PT2195035T (pt) * 2007-09-28 2018-03-07 Engene Inc Composições de poliplexos de ácido nucleico de quitosano de alta concentração
DE102008021483A1 (de) 2008-04-29 2009-12-03 Forschungszentrum Jülich GmbH Zuleitungssystem
CN102617735B (zh) 2008-09-29 2015-09-09 罗氏格黎卡特股份公司 针对人il17的抗体及其应用
US8349591B2 (en) * 2008-10-16 2013-01-08 Scientek Llc Method and apparatus for producing alcohol or sugar using a commercial-scale bioreactor
RU2577972C2 (ru) * 2010-04-26 2016-03-20 Новартис Аг Способ получения рекомбинантного полипептида
SG185038A1 (en) * 2010-04-26 2012-11-29 Novartis Ag Improved cell culture medium
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JP2014500025A (ja) 2014-01-09
KR101507254B1 (ko) 2015-03-30
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CA2817874A1 (en) 2012-06-14
BR112013009934A2 (pt) 2016-07-05
US20210040528A1 (en) 2021-02-11
JP6117885B2 (ja) 2017-04-19
JP2016028596A (ja) 2016-03-03
EP2649176A1 (en) 2013-10-16
MX345885B (es) 2017-02-22
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