FEED MIXING DEVICE AND ITS USE

Abstract

Herein is reported a feed mixing device for adding feed solutions with a non-physiologically pH value to a cell cultivation vessel comprising a chamber for mixing the feed solutions prior to their addition to the cell cultivation vessel as well as its use. With the feed mixing device as reported herein feed components can be provided in solution at a pH value at which they 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 the physiological pH value. This allows performing the cultivation with more flexibility compared to a cultivation in which the pH value of the feed solution is limited to a small range around the pH value of the cultivation.

Claims

1. A device for adding at least two feed solutions each with a non-physiological pH value to a cell cultivation vessel comprising separate inlets for the feed solutions, a chamber for mixing the feed solutions, and a single outlet for adding the mixed feed solutions to the cell cultivation vessel, whereby the ratio of the volume of the chamber for mixing the feed solutions to the volume of cultivation medium in the cultivation vessel is of from 0.8 ml/L to 1.2 ml/L.

2. The device according to claim 1, characterized in that the ratio is about 1 ml/L.

3. The device according to claim 1, characterized in that the device is separated from the cultivation vessel and the outlet is to the inside of the cultivation vessel.

4. The device according to claim 1, characterized in that the feed mixing device is outside of the cultivation vessel or inside the cultivation vessel.

5. The device according to claim 1, characterized in that the chamber for mixing the feed solutions has a volume of from 0.5 ml to 1,000 ml.

6. The device according to claim 1, characterized in that the device is made of sterilizable material.

7. A cell cultivation apparatus comprising a) a cell cultivation vessel, b) a gas supply, c) at least two liquid storage units for feed solutions each with a non-physiological pH value, d) a sensor for determining the pH value in the cell cultivation vessel, and e) a device according to claim 1.

8. Use of a device according to claim or of an apparatus according to claim 7 in a fed-batch or continuous cultivation of a cell.

9. A method for the production of a polypeptide comprising the steps of: cultivating a cell comprising a nucleic acid encoding the polypeptide in a fed-batch or continuous cultivation within a cultivation vessel, wherein the cultivation vessel comprises a device for adding at least two feed solutions, wherein the device comprises separate inlets for the feed solutions, a chamber for mixing the feed solutions, and a single outlet for adding the mixed feed solution to the cell cultivation vessel; mixing at least two feed solutions, wherein at least one feed solution is an acidic feed solution and has a pH value of less than pH 6.5, and at least one feed solution is an alkaline feed solution and has a pH value of more than pH 7.5, in the chamber to produce a mixed feed solution; adding the mixed feed solution to the cultivation vessel containing the cultivation medium; further cultivating the cell comprising the nucleic acid encoding the polypeptide in the fed-batch or continuous cultivation within the cultivation vessel, wherein the ratio of the volume of the mixed feed solution in the chamber to the volume of the cultivation medium in the cultivation vessel is from 0.8-1.2 ml chamber volume per 1 liter volume of the cultivation medium in the cultivation vessel; and recovering the polypeptide from the cultivation medium or the cells.

10. The method according to claim 9, wherein any acidic feed solution has a pH value of pH 6.5 or lower, and/or any alkaline feed solution has a pH value of pH 8.0 or higher, and the mixed feed solutions have a pH value of from pH 7 to pH 7.5.

11. The method according to claim 9, wherein the mixing is immediately prior to the addition to the cultivation vessel.

12. The method according to claim 9, wherein any acidic feed solution has a pH value of pH 4.5, or lower, and/or any alkaline feed solution has a pH value of pH 10.0 or higher.

13. The method according to claim 9, wherein from two to four separate feed solutions are added to the cultivation medium whereof at least one is an acidic feed solution and at least one is an alkaline feed solution.

14. The method according to claim 9, characterized in that each of the alkaline or acidic feed solutions comprises at least a compound selected from amino acid, sugar, vitamin, trace element, lactate, and growth factor.

15. A method for obtaining a polypeptide with a reduced G(0) glycoform and/or increased G(1) glycoform comprising the steps of: cultivating a cell comprising a nucleic acid encoding the polypeptide in a fed-batch or continuous cultivation within a cultivation vessel, wherein the cultivation vessel comprises a device for adding at least two feed solutions, wherein the device comprises separate inlets for the feed solutions, a chamber for mixing the feed solutions, and a single outlet for adding the mixed feed solution to the cell cultivation vessel; mixing at least two feed solutions, wherein at least one feed solution is an acidic feed solution and has a pH value of less than pH 6.5, and at least one feed solution is an alkaline feed solution and has a pH value of more than pH 7.5, in the chamber to produce a mixed feed solution; adding the mixed feed solution to the cultivation vessel containing the cultivation medium; further cultivating the cell comprising the nucleic acid encoding the polypeptide in the fed-batch or continuous cultivation within the cultivation vessel, wherein the ratio of the volume of the mixed feed solution in the chamber to the volume of the cultivation medium in the cultivation vessel is from 0.8-1.2 ml chamber volume per 1 liter volume of the cultivation medium in the cultivation vessel 0.8 ml/L to 1.2 ml/L; and recovering the polypeptide from the cultivation medium or the cells, wherein the feed solution mixture has a pH value upon addition to the cultivation vessel of from pH 4.0 to pH 6.0.

Description

DESCRIPTION OF THE FIGURES

[0059] FIG. 1 Scheme of the general course of the pH value of a cell cultivation.

[0060] 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.

[0061] 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%.

[0062] 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).

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] FIG. 9 Acidic peak fraction: left: use of a device as reported herein; dark right: without a device as reported herein.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] FIG. 16 Dependency of the G(0) fraction on the pH value of the feed solution.

[0075] FIG. 17 Dependency of the G(1) fraction on the pH value of the feed solution.

[0076] FIGS. 18+19 Exemplary schemes of the device as reported herein.

[0077] FIG. 20 Dependency of the host cell protein content on the pH value of the feed solution.

EXAMPLES

Materials

Antibody

[0078] 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 Solutions

[0079] 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.

[0080] In this setup it is ensured that the concentration as well as the number of components as well as the volume of the added feed 1 is the same as after the combination of feed 2 and feed 3. 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 Mixing Device Dimensions

[0081] Volume of the chamber for mixing the feed solutions: 1.146 ml

[0082] Volume of the cultivation medium at the start of a cultivation with a 2l -cultivation vessel: 1.2 l

[0083] Feed flow through the feed mixing device: 36 g/d

Example 1

[0084] Four 2 l-cultivation vessels (Sartorius Biostat B-DCU Quad, Sartorius, Goettingen, Germany) have been inoculated in parallel with an inoculum solution pre-cultivated in the same shaker flask. Two cultivation vessels comprised the device as reported herein whereas the other two cultivation vessels comprised two individual conventional feeding devices with a Luer fitting but without a mixing chamber.

[0085] All cultivations were performed with a constant aeration rate, constant temperature and constant agitation speed over the entire cultivation. All feed rates are calculated based on the start working volume and are given in feed volume per fermentation starting volume per day.

[0086] 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.

[0087] 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.

[0088] Thus, the added volume as well as the added amount of all feed components is identical in all four cultivations (see Tables 1 and 2).

TABLE-US-00001 TABLE 1 parameter feed solution 1 feed solution 2 feed solution 3 use of device as no yes yes reported herein pH value alkaline alkaline acidic volume flow 100% 50% 50% concentration 1× 2× 2× feed rate 0.03 1/d 0.015 1/d 0.015 1/d total feed volume 100% 50% 50%

TABLE-US-00002 TABLE 2 cultivation 1 cultivation 2 cultivation 3 cultivation 4 feed 1 feed 1 feed 2 + feed 3 feed 2 + feed 3 +sodium +potassium +sodium +potassium chloride chloride chloride chloride feed pH feed pH mixed feed pH mixed feed pH value 9.5 value 9.5 value 7.2 value 7

[0089] 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.

[0090] As can be seen from the figures 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.

Example 2

[0091] In this example only the influence of the pH value of the feed solutions on different parameters of the cultivation, such as base consumption, lactate formation, growth kinetic or product formation is analyzed. All other parameters were kept comparable.

[0092] 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.

[0093] The pO.sub.2 value was adjusted to a value of 35% air saturation and determined with a pO.sub.2 probe (Mettler-Toledo InPro 6820). The pO.sub.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.

[0094] 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 4.0 l 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).

[0095] 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.sup.3. The same power input was used in the pre-cultivation to ensure comparability and avoid a rapid change in the conditions.

[0096] 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.

[0097] 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.

[0098] As 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.sup.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.sub.2, and a relative humidity of 85%.

[0099] After transfer of the inoculation medium to the main cultivation samples were withdrawn on a daily basis. The main cultivation was performed as a fed-batch cultivation, wherein the feeding was started approximately 72 hours after start of the main cultivation.

[0100] Five different cultivations were carried out simultaneously. The parameters thereof are given in Table 3.

TABLE-US-00003 TABLE 3 parameter cultivation 1 cultivation 2 cultivation 3 cultivation 4 cultivation 5 use of device as yes yes no yes yes reported herein feed 1 alkaline alkaline complete feed alkaline alkaline feed 2 acidic acidic — acidic acidic pH set of feed 9.5 7.0 9.5 4.5 4.5 change of pH no no no Yes, change to no set of feed pH set of 9.5 after pH value dropped to lower pH band border

Results

[0101] By using feed solution 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.

[0102] In 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.sub.2 in the cultivation medium after a cultivation time of 14 days is depicted.

[0103] As can be seen from FIG. 10 the amount of added base is dependent on the pH value of the feed solution.

[0104] As can be seen from FIG. 11 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. By using a device as reported herein the overall amount of lactate is reduced by about 30% compared to a combined feed.

[0105] As can be seen from FIG. 12 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.

[0106] As can be seen from FIG. 13 the osmolality in the cultivation medium after a cultivation time of 14 days is depending on the pH value of the feed solution.

[0107] In FIG. 14 the pCO.sub.2 value in the cultivation medium after a cultivation time of 14 days is shown. It can be seen that the pCO.sub.2 is dependent on the pH value of the feed solution, whereby the highest pCO.sub.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.sub.2 value after a cultivation time of 14 days can be dramatically reduced avoiding unphysiologically high pCO.sub.2 values in the cultivation medium.

[0108] In 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%.

[0109] In 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.

[0110] In 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.

Example 3

Behavior of the Mixed Feeds

[0111] 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.

[0112] After an incubation time of 110 min. a slight precipitate was observed for the feeds mixed and incubated at room temperature. In the feeds mixed and incubated at 4° C. a precipitate was formed already shortly after the mixing was performed.