WO2012076442A1

WO2012076442A1 - Adapter for the non-invasive cultivation of cells and uses thereof

Info

Publication number: WO2012076442A1
Application number: PCT/EP2011/071697
Authority: WO
Inventors: Sylvia Baumann; Jens Hoffmann; Alexander Jockwer; Christian Klinger; Thomas Troebs
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2010-12-07
Filing date: 2011-12-05
Publication date: 2012-06-14

Abstract

Herein is reported an adapter comprising a body in form of a hollow cylinder which is made of an sterilizable material and comprises three sections wherein the upper section (1) has the form of a right circular hollow cylinder comprising an external screw thread (2) with an external diameter d, the middle section (3) has the form of a right circular hollow cylinder or a truncated hollow cone comprising at least one connector (4), and the lower section (5) has the form of a right circular hollow cylinder with a internal screw thread (6) suitable for screwing in a screw thread with an external diameter d.

Description

Adapter for the non-invasive cultivation of cells and uses thereof

Herein is reported an adapter to be placed on the neck of a shaker flask comprising connectors suitable for the addition of feed solutions or gaseous substances as well as for the withdrawal of gaseous or liquid samples. This adapter can be used for the non-invasive cultivation of cells in shaker flasks. Background of the Invention

During the development of biotechnological processes for recombinant polypeptide production employing mammalian cells a plurality of parameters have to be adjusted and optimized. Due to the large number of required individual experiments this optimization is done in shaker flasks. The cultivation of cells in shaker flasks is generally associated with a large number of steps required to be performed on each of the flasks during the cultivating. These are, for example, the addition of feed solutions or the withdrawal of samples for monitoring the cultivation progress. The cultivation in shaker flasks is normally performed in an incubator under controlled environmental conditions, such as temperature or carbon dioxide content, which should vary as little as possible during the cultivation. Every time, e.g., when a sample has to be withdrawn or a feed solution has to be added the flasks have to be removed from the incubator, the top cover of the flasks has to be removed from the neck of the flaks and the respective action has to be performed with the flasks. Thus, shaker flask cultivations actually cannot be performed at constant environmental conditions as e.g. the removal from the incubator and the opening of the top cover perturbs these conditions.

The fluctuation in the cultivation conditions makes a comparison even between flasks cultivated at the same time in the same incubator very difficult if not impossible as e.g. the time courses of the pH value, the p0₂ value, the pC0₂ value, the power input, or the temperature show abrupt changes from which each system recovers only slowly and individually. This results in hardly predictable and also reproducible metabolic changes of the cultivated cells which in turn result in different overall product yields or product quality. In US 5,672,505 an insert for a tissue culture vessel is reported. A disposable bioreactor, kit for the same and method for its production are reported in EP 2 251 407. In JP 2009-034078 a culture container flask is reported. A connector device for sealing and dispensing freeze-dried preparations is reported in US 2004/0134562. In US 2010/0038303 a disposable polymer-structured filtering kit is reported. Summary of the Invention

It has been found that by using an adapter as reported herein it is possible to cultivate cells in a shaker flask as a fed-batch or continuous cultivation without the need to remove the flasks during the cultivation from the incubator for feeding processes. Additionally the adapter as reported herein is made in a way to minimize the additional gas phase volume in the shaker flask to which it is attached. This allows the cultivation to be carried our without erratic changes e.g. in the pH value or the p0₂ value in the cultivation medium in the shaker flask as the removal from the controlled cultivation environment is no longer required. At the same time with the adapter as reported herein no impact on the gass exchange can be observed. One aspect as reported herein is an adapter comprising a body in form of a hollow cylinder which is made of a sterilizable material and comprises three sections wherein

a) the upper section (1) has the form of a right circular hollow cylinder comprising an external screw thread (2) with an external diameter d, b) the middle section (3) has the form of a right circular hollow cylinder or truncated hollow cone comprising at least one connector (4), and c) the lower section (5) has the form of a right circular hollow cylinder with an internal screw thread (6) with an internal diameter I suitable for screwing in a screw thread with an external diameter d. In one embodiment the adapter comprises one to one hundred connectors (4), or one to ten connectors (4), or two to six connectors (4), or three to four connectors (4). In one embodiment the connectors (4) are evenly distributed around the middle section and have the same distance to the upper end of the middle section (7) as to the lower end of the middle section (8). In another embodiment the angle (9) between the outer surfaces of the lower section and the middle section is of from 180° to 240°. In a further embodiment the angle is of from 181° to 225°. In another embodiment the angle is about 195°. In still an embodiment the connectors are independently of each other selected from a barbed-end-type connector, a plain- type connector, a weldable tubing, and/or a Luer-type connector. In one embodiment the external diameter d is about 25 mm, or about 40 mm, or about 42 mm, or about 45 mm, or about 52 mm. In one embodiment the internal screw thread (6) has an internal diameter I. In a further embodiment the internal diameter I is about 23 mm, or about 37.5 mm, or about 39 mm, or about 42.5 mm, or about 49.5 mm. In also an embodiment the outer diameter d is 25 mm and the inner diameter I is 23 mm, or the outer diameter d is 40 mm and the inner diameter I is 37.5 mm, or the outer diameter d is 42 mm and the inner diameter I is 39 mm, or the outer diameter d is 45 mm and the inner diameter I is 42.5 mm, or the outer diameter d is 52 mm and the inner diameter I is 49.5 mm. In another embodiment the sections have the same height. In a further embodiment the upper and lower sections have a height of about 15 to 60 mm, especially 30 to 45 mm, and the middle section has a height of about 15 mm. In also an embodiment one connector is connected on the inside of the adapter to a standpipe (10). In still another embodiment the adapter comprises three or four connectors and two of these connectors are connected on the inside to a feed mixing device (11). In a further embodiment the feed mixing device comprises a chamber for mixing a first solution and a second solution prior to their addition to the cell cultivation medium. In still another embodiment the chamber is separated from the cultivation vessel and comprises an outlet to the inside of the cultivation vessel. In another embodiment the chamber is outside of the cultivation vessel or inside the cultivation vessel. In a further embodiment the chamber has a volume of from 0.1 ml to 50,000 ml. In an embodiment the chamber has a volume of from 0.25 ml to 30,000 ml. In still a further embodiment the chamber has a volume of from 0.5 ml to 10 ml. In one embodiment the mixing chamber comprises an inlet with individual connectors for each of the solutions. In another embodiment the adapter is sterilizable.

Another aspect as reported herein is a device comprising an adapter as reported herein and a shaker flask. In one embodiment the device further comprises a lid.

Also aspects as reported herein are the use of an adapter as reported herein for the selection of cell clones in a shaker flask cultivation, or for the evaluation of media and feeds in a shaker flaks cultivation, or for the evaluation of cultivation conditions for a shaker flask cultivation.

Another aspect as reported herein is a method for cultivating a cell in a shaker flask comprising the following steps: a) cultivating a cell comprising a nucleic acid encoding a polypeptide of interest in a device as reported herein,

b) adding a feed solution to the shaker flask.

In one embodiment the adding is continuously or intermittently (as bolus feed). In one embodiment the cell is a mammalian cell. In another embodiment the mammalian cell is selected from the group of mammalian cells comprising CHO cells (e.g. CHO Kl, CHO DG44), NSO cells, SP2/0 cells, and HEK 293 cells. In a further embodiment the polypeptide of interest is a biologically active polypeptide. In also an embodiment the biologically active polypeptide is a complete antibody, or an antibody conjugate, or an antibody fragment.

Detailed Description of the Invention

Shake flasks are a common small scale format for cultivation of mammalian suspension cell cultures. Incubators providing a suitable environment by carbon dioxide (C0₂) supply and humidity control are used to ensure sufficient oxygen transfer and homogenization of cell suspension by appropriate mixing. In order to match scale-up parameters power inputs are adjustable either by adaptation of the shaking frequency, eccentricity of the shaking movement or working volumes in shake flasks.

However, removal of shake flasks from the incubator for feeding or sampling procedures during development of fed batch cultivations may lead to a sudden loss of C0₂ in the headspace of the shake flasks altering pH of the cell suspension.

Moreover, temperature drops, cell settling, local pH gradients by addition of feed solutions, and oxygen limitations may occur. After removal of shake flasks from the incubator requiring opening the door only a few seconds, recovery of the C0₂ content in the incubator may take up to ten minutes. This effect is even more significant if shake flasks are removed and put back into the incubator consecutively.

To avoid the necessity of removing the shake flasks from the incubator for feeding and sampling, a sterilizable shake flask adapter was desgined which enables addition and removal of liquids via three ports. The adapter provides an internal screw thread for the shake flask itself and an external screw thread for the vented cap. A modification of the shake flask itself is not necessary. In contrast to attached sterile filters, oxygen transfer into the shake flask was shown not to be limited by this device. Continuous feeding, e.g., is very easy to perform via continuous pumps or syringe based dosing systems. Moreover, monitoring and control of e.g. dissolved oxygen and pH is possible using in-line analytics from PreSens^®, and incubator settings for C0₂ and frequency as actuating variables. For scale-up studies, comparable and representative results can be gathered using this device since main disturbance variables can be minimized or knocked out.

Thus, with the adapter as reported herein it is possible to cultivate cells in a shaker flask as a fed-batch or continuous cultivation without the need to remove the flasks during the cultivation from the incubator due to feeding to and/or sampling from the flask.

The term„about" denotes that the thereafter following value is no exact value but is the center point of a range that is +/- 10 % of the value, or +/- 5 % of the value, or +/- 2 % of the value, or +/- 1 % of the value. If the value is a relative value given in percentages the term "about" also denotes that the thereafter following value is no exact value but is the center point of a range that is +/- 10 % of the value, or +/- 5 % of the value, or +/- 2 % of the value, or +/- 1 % of the value, whereby the upper limit of the range cannot exceed a value of 100 %.

The term "sterilizable material" as used herein denotes a material that can be made abacterial by a know method, such as autoclaving or gamma irradiation. The sterilizable material is in one embodiment for single or multiple use.

The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. The term "antibody" refers in general to a protein consisting of one or more polypeptide(s) substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the different constant region genes as well as the myriad immunoglobulin variable region genes. Antibodies may exist in a variety of formats, including, for example, Fv, Fab, and F(ab)₂ as well as single chains (scFv) or diabodies. An antibody in general comprises two so called light chain polypeptides (light chain) and two so called heavy chain polypeptides (heavy chain). Each of the heavy and light chain polypeptides contains a variable domain (variable region) (generally the amino terminal portion of the polypeptide chain) comprising binding regions that are able to interact with an antigen. Each of the heavy and light chain polypeptides comprises a constant region (generally the carboxyl terminal portion). The constant region of the heavy chain mediates the binding of the antibody i) to cells bearing a Fc gamma receptor (FcyR), such as phagocytic cells, or ii) to cells bearing the neonatal Fc receptor (FcRn) also known as Brambell receptor. It also mediates the binding to some factors including factors of the classical complement system such as component (Clq). The variable domain of an antibody's light or heavy chain in turn comprises different segments, i.e. four framework regions (FR) and three hypervariable regions (CDR).

The term„biologically active polypeptide" denotes an organic molecule, e.g. a biological macromolecule such as a peptide, protein, glycoprotein, nucleoprotein, mucoprotein, lipoprotein, synthetic polypeptide or protein, that causes a biological effect when administered in or to artificial biological systems, such as bioassays using cell lines and viruses, or in vivo to an animal, including but not limited to birds or mammals, including humans. This biological effect can be but is not limited to enzyme inhibition or activation, binding to a receptor or a ligand, either at the binding site or circumferential, signal triggering or signal modulation. Biologically active molecules are without limitation for example immunoglobulins, or hormones, or cytokines, or growth factors, or receptor ligands, or agonists or antagonists, or cytotoxic agents, or antiviral agents, or imaging agents, or enzyme inhibitors, enzyme activators or enzyme activity modulators such as allosteric substances. In one embodiment the biologically active polypeptide is a complete antibody, an antibody conjugate, or an antibody fragment.

The term "complete antibody" denotes a protein which comprises two so called light immunoglobulin chain polypeptides (light chain) and two so called heavy immunoglobulin chain polypeptides (heavy chain). Each of the heavy and light chain polypeptides of a complete antibody contains a variable domain (variable region) (generally the amino terminal portion of the polypeptide chain) comprising binding regions that are able to interact with an antigen. Each of the heavy and light chain polypeptides of a complete antibody comprises a constant region (generally the carboxyl terminal portion). The constant region of the heavy chain mediates the binding of the antibody i) to cells bearing a Fc gamma receptor (FcyR), such as phagocytic cells, or ii) to cells bearing the neonatal Fc receptor (FcRn) also known as Brambell receptor. It also mediates the binding to some factors including factors of the classical complement system such as component (Clq). The variable domain of an antibody's light or heavy chain in turn comprises different segments, i.e. four framework regions (FR) and three hypervariable regions (CDR). The term "antibody fragment" denotes a molecule other than a complete antibody that comprises a portion of a complete that binds the antigen to which the complete antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')₂, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and multispecific antibodies formed from antibody fragments.

The term "antibody conjugate" denotes a polypeptide comprising at least one domain of an antibody heavy or light chain conjugated via a peptide bond to a further polypeptide. The further polypeptide is a non-immunoglobulin peptide, such as a hormone, or growth receptor, or antifusogenic peptide, or complement factor, or cytotoxic agent or the like.

The term "cell" denotes a cell into which a nucleic acid, e.g. encoding a polypeptide of interest has been introduced/transfected. The term„cell" denotes both prokaryotic cells, which are used for propagation of plasmids, and eukaryotic cells, which are used for the expression of a nucleic acid. In one embodiment the eukaryotic cells are mammalian cells. In a further embodiment the mammalian cell is selected from the group of mammalian cells comprising CHO cells (e.g. CHO Kl, CHO DG44), BHK cells, NSO cells, SP2/0 cells, HEK 293 cells, HEK 293 EBNA cells, PER.C6^® cells (immortalized human cell line), AGE^® cells (Muscovy duck cell line), and COS cells. As used herein, the expression "cell" includes the subject cell and its progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same biological activity as screened for in the originally transformed cell are included. The adapter as reported herein comprises in one embodiment up to one hundred connectors, or in another embodiment one to ten connectors, or in another embodiment two to six connectors, or in another embodiment three to four connectors, which can be of any type as long as these connector are sterilizable, e.g. by autoclaving or irradiation, air tight and liquid tight. An exemplary connector is a Luer-lock connector or a cannula. To such a connector either directly or via a flexible tube or pipe e.g. a syringe, an automatic pump, or a dosing unit can be connected. Therewith it is possible to add solutions, e.g. feed solutions or correction fluids, to a flask without the need to remove the flask from its controlled environment and likewise without interfering with the linearity and reproducibility of the cultivation conditions. Therewith it is additionally possible to withdraw samples from a flask without the need to remove the flask from its controlled environment and likewise without interfering with the linearity and reproducibility of the cultivation conditions.

To the kind or way of addition and/or withdrawal no limitation is set. It can be independently of each other for example a bolus addition or withdrawal or a continuous addition or withdrawal.

An exemplary embodiment of the adapter as reported herein is shown in Figure 1. The adapter has to be made of a sterilizable material. This includes heat sterilization, sterilization by irradiation, and also chemical sterilization. Exemplary materials for the adapter are glassware, Teflon^®, stainless steel, polycarbonate, polyvinyl chloride, polypropylene, polyethylene terephthalate, ethylene tetra fluoro ethylene, perfluoro alkoxy, poly tetra fluoro ethylene, ethylene chloro tri fluoro ethylene, poly methylpenten, and polyvinylidene fluoride. The middle section (3) is in one embodiment made from a different material as the upper section and the lower section. In one embodiment the middle section (3) is made of rubber, PTFE, silicone, Viton^®, or combinations thereof. Thus, the adapter can either be sterilized prior to the connection to the external addition and withdrawal adapters, or together with the flexible tubes connected to the connectors of the adapter.

It is possible with the adapter as reported herein to add a feed solution or a correction fluid (acid, base, antifoam solution, etc.) to a shaker flask within the incubator batch wise or continuously without the need to remove the flask from the incubator. Also the withdrawal of samples from the cultivation medium inside the flask can be batch wise or continuously but also without the requirement to remove the flask from the incubator. This allows for a more flexible mode of cultivation using shaker flasks, such as a chemostat or perfusion cultivation. In one embodiment the addition of the feed and/or the withdrawal of the medium is continuously.

In one embodiment a continuous cultivation with or without cell retention is performed. Therefore the withdrawn cultivation medium is reapplied to the flask after the separation of cells or the application to a dialysis module. The continuous addition and/or withdrawal can be automated, e.g. by using a dispenser, automatic pump etc. In another embodiment a fed-batch cultivation with bolus addition of a feed solution is performed. The adapter as reported herein is placed between the lid of the shaker flask and the neck of the shaker flask after the inoculation of the shaker flask with cells.

The adapter as reported herein comprises three sections:

- an upper section with an outer thread for taking up the lid of the shaker flask that has the same outer diameter as the neck of the shaker flask,

- a middle section in the form of a hollow cylinder or a truncated cone that comprises the connectors, and

- a lower section with an inner thread for taking up the neck of the shaker flask that has the same inner diameter as the lid of the shaker flask. If it is intended to withdraw liquid samples from the cultivation medium during the cultivation the connector to which the sampling adapter will be connected is connected to an internal standpipe that has a length that places the lower end of the standpipe closely above the bottom of the shaker flask ensuring that the pipe's end is below the surface of the cultivation medium during the shaker flask cultivation even if the flaks is shaken (see Figure 2).

By withdrawing samples from the cultivation medium the total volume of the cultivation medium inside the shaker flask is changed, i.e. reduced. Therefore, the sample volume and/or the feed and/or the agitation rate and/or the oxygen transfer rate (k_La value) would have to be adjusted accordingly. Thus, a process with a sophisticated control adapter would be needed therefore. Alternatively, as in one embodiment, the cultivation is performed with the addition of a feed solution using a adapter as reported herein but without the removal of samples from the cultivation medium in the flasks during the cultivation. In this embodiment the volume of the cultivation medium inside the flasks increases steadily. Such a mode of operation is especially useful if the feed solution has a different osmolality than the cultivation medium used at the start of the cultivation. If a sample is withdrawn and also a feed solution with different osmolality is added, the change of the osmolality during the cultivation is more pronounced compared to a cultivation without the withdrawal of samples. This holds true also for other parameters differing between initial culture medium and added feed solutions and correction fluids (e.g. pH value, amino acid concentrations, lipid concentrations, trace element concentrations, sugar concentrations, detergent concentrations, anti foam agent concentrations, shear protectant concentrations, etc.) which can have an influence on the nutritional and/or fluidynamic properties of the medium and/or the cells. Alternatively the adapter can also be used to withdraw samples from the gas phase above the cultivation medium. In this case no standpipe is required.

The adapter as reported herein can be used together with any standardized cultivation flaks, such as Erlenmeyer-type flasks, Corning flasks, spinner flasks, or tubespins^®.

With the adapter as reported herein the gas exchange between the gaseous phase inside the shaken flask and the surrounding atmosphere in the incubator is easily possible compared to the use of e.g. sterile filters. It can be shown that oxygen limitations do not occur when an adapter as reported herein is used. The use of the adapter as reported herein can further be improved by combining it with a feed mixing device. The feed mixing device is a device for adding at least two solutions with non-physiological pH value, e.g. at least one acidic solution and at least one alkaline solution, or at least two alkaline solutions, to a cell cultivation vessel that comprises a chamber for mixing the at least two solutions with non-physiological pH value prior to their addition to the cell cultivation vessel. The term "non-physiological pH value" denotes a pH value outside the pH value range of pH 6.5 to pH 7.5. Thus, a solution with a non-physiological pH value has a pH value of below pH 6.5, i.e. pH 0 to pH 6.49, or above pH 7.5, i.e. pH 7.51 to pH 14. The chamber for mixing can be separated from the cultivation flask. It comprises an outlet to the inside of the cultivation flask. The chamber for mixing can be outside of the cultivation flask or inside of cultivation flask. The chamber for mixing can have a volume of from 0.01 ml to 5 ml. The mixing chamber comprises an inlet with individual connectors for each of the solutions to be mixed. The feed mixing device can be sterilizable. The 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. With a feed mixing device the cultivating can be performed with at least one additional degrees of freedom and, thereby, with more flexibility. With the device it is possible to use feed solutions that have a non-physiological pH value and/or a high compound concentration. The non-physiologically 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.

By the gained variability of the pH value of the feed solutions it is possible to feed solutions with any pH value or combination of pH values, i.e. alkaline, neutral or acidic solutions, and with any concentration of the contained compounds. 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 strategy not using the feed mixing device. Thus, even 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-physiologically, pH values.

The pH value of the mixed feed solution leaving the feed mixing device and being added to the cultivation medium is depending 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. By using a feed mixing device for the mixing of feed solutions the viability of the cultivated cells can be maintained for a longer time period 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.

One aspect as reported herein is the use of an adapter as reported herein for the selection of cell clones, or for the evaluation of media and feeds, or for the evaluation of cultivation conditions and course of temperature, p0₂, pC0₂, power input, or pH.

Another aspect as reported herein is the use of a shaker flask cultivation with the adapter as reported herein with a continuous addition or/and withdrawal of cultivation medium as well as cell retention as seed train cultivation.

It has been found that by using a adapter as reported herein difference in the obtainable cell density and productivity of a shaker flask cultivation compared to a shaker flask cultivation without the adapter as reported herein can be observed, especially if it is a fed-batch or continuous cultivation. In the following Table the viable cell density, viability, final volume, total cell count, osmolality after 14 days of cultivation of a shaker flask cultivation according to Example 1 are shown. Table 1.

It can be seen that by using an adapter as reported herein the cell density as well as the final volume are higher compared to those cultivations not using the adapter as reported herein. By using the adapter as reported herein the total cell count is increased by at least 70 % compared to that in the daily sampled shaker flasks, the viable cell density is increased by at least 40 % compared to that in the daily sampled shaker flasks, the viability is increased compared to that in the daily sampled shaker flasks, and the osmolality is reduced compared to that in the daily sampled shaker flasks.

As the feed volume is held constant with respect to the starting volume of the cultivation the relative intake and therewith the increase by the fed volume is more pronounced with increasing sample volume. Thus, the osmolality increases more the more sample volume is withdrawn. It has been found that by using an adapter as reported herein the daily sampling can be omitted overcoming the negative influence of an increasing osmolality in the cultivation medium in the shaker flasks. The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Description of the Figures

Figure 1 Schematic drawing of the position of the adapter as reported herein for the non-invasive addition or withdrawal from shaker flask cultivations when placed between the neck of a shaker flask and the lid of a shaker flask.

Figure 2 Schematic drawing of the adapter as reported herein comprising a standpipe for the withdrawal of liquid samples from the cultivation medium of a shaker flask.

Figure 3 Schematic drawing of the adapter as reported herein comprising in addition a feed mixing adapter.

Figure 4 Schematic drawings of different embodiments of the feed mixing adapter.

Figure 5 Course of pH and p0₂ during a shaker flask cultivation using the adapter as reported herein.

Figure 6 Exemplary barbed and plain connectors.

Figure 7 Comparison of time required for oxygen saturation from 10 % to

70 % in shaker flasks with different gas-exchange devices.

Example 1

Effect of bolus feeding on shaker flask cultivations

In this example a CHO cell line expressing an anti-IL17 antibody as reported in WO 2010/034443 (incorporated herein by reference) has been used.

The cultivation was performed for 14 days in two sets of shaker flasks. The first set of flasks was cultivation with an adapter as reported herein and the feed was added with a syringe attached via a flexible pipe to the adapter as reported herein. The second set of flasks was cultivated without the adapter as reported herein requiring the removal of the flasks from the incubator and the shaker for daily sampling and feeding.

For all flasks the starting cultivation volume was 50 ml and the inoculation cell density was 3.5 x 10⁵ cells/ml. The flasks were shaken initially at a frequency of 120 rpm which was increased during the cultivation to 140 rpm. All flasks were cultivated with the same shaking frequency profile. Feeding was started after a cultivation time of 72 hours with a first feed solution. After 120 hours of cultivation time the first feed solution was replaced by a second feed solution. The feed was added on a daily basis as bolus feed to the shaker flasks. The added feed volume is constant with respect to the starting cultivation volume. Feed solutions 1 and 2 had compared to the starting cultivation medium a higher osmolality.

The feeding of the flaks comprising a adapter as reported herein was performed with a syringe attached via a flexible pipe to the adapter whereby the content of the feed solution remaining in the pipe after the syringe plunge was completely pressed into the syringe was transferred with incubator air into the flasks ensuring the transfer of the entire feed volume.

The pH and the p0₂ value inside the shaker flasks during the cultivation were determined using PreSens sensors.

Table 2. flask 1+2 flask 3+4 flask 5+6 flask 7+8 flask flask

9+10 11+12 use of an

adapter

as yes yes no no no no reported

herein

feed la

(started

yes no yes no yes no after 72

hours)

feed lb

(started

no yes no yes no yes after 72

hours)

feed 2

(started

yes yes yes no yes yes after 120

hours)

sample

0 ml 0 ml 32.5 ml 32.5 ml 11 ml 11 ml volume The total cell count at day 14 was calculated from the viable cell density and the theoretical final volume in the shaker flask. The theoretical final volume increases with decreasing sample volume. Thus, the total cell number in the flask increases with increasing volume and same cell density.

Table 1.

Example 2

Continuously fed shaker flask cultivations

The cultivation was performed for 14 days in four shaker flasks all comprising the adapter as reported herein. The flasks were continuously fed during the cultivation by using Watson Marlow pumps. Concomitantly the pH value and the p0₂ value were monitored in the shaker flasks.

The pH value and the p0₂ value in the flasks were controlled by changing the C0₂ content of the atmosphere inside the incubator and the shaking frequency, respectively. The pH value and the p0₂ value were recorded with a PreSens Shake Flask Reader. The course of the pH value and the p0₂ value is shown in Figure 5.

To the four flasks different feed solutions were added continuously during the cultivation. Depending on the feed solution different cell densities could be obtained after 14 days of cultivation.

Example 3

Oxygen saturation

Four 500 ml shake flasks (PreSense) comprising pH and oxygen sensors were filled with 250 ml of a sodium chloride solution (8.77 g/1, about 300 mOsmol/kg). The filled flaks were placed over night in an incubator for equilibration of the sensor spots. The next day 250 μΐ of a 1 M Co(II)-chloride solution were added to each flask. Thereafter the flasks are positioned on the shaker in the incubator but left open. The pH and oxygen determination was started. The shaker was operated at 150 rpm with an excentricity of 5 cm. The incubator was operated at 0 % C02, 37 °C and 85 % relative humidity.

Once a stable readout of the sensor spots has been obtained 1 ml sodium sulfite (0.4 mol/1) was added sequentially to each flask. Directly afterwards the flaks are closed with the different lids:

1 : flaks lid as provided by corning with an integrated membrane for gas exchange (vented lid)

2: same lid as in 1 but with an adaptor as reported herein inserted between the lid and the nek of the flasks; all connectors of the adaptor were sealed gas-tight

3 : lid comprising an external filter poppet for gas exchange

4: standard lid. The sodium sulfite solution was added to the next flask only after in the previous flaks a stable oxygen value was established in order to prevent interferneces due to the opening of the incubator.

As can be seen from Figure 7 a difference with respect to the time required for oxygen saturation from 10 % to 70 % of the different lids was detected. Lid 2 comprising the adaptor as reported herein shows the same characteristics as the standard lid 1 provided by the flask manufacturer. Thus, the adaptor as reported herein does not interfere with gas exchange of the flaks but at the same time offers the possibilities to add solutions or withdraw samples without the need to remove the flask from the incubator. Whereas with the adaptor as reported herein oxygen saturations above 90 % can be obtained with lid 3 at most 75 % can be achieved.

Claims

Patent Claims

1. An adapter comprising a body in form of a hollow cylinder which is made of a sterilizable material and comprises three sections wherein a) the upper section (1) has the form of a right circular hollow cylinder comprising an external screw thread (2) with an external diameter d, b) the middle section (3) has the form of a right circular hollow cylinder or a truncated hollow cone comprising at least one connector (4), and c) the lower section (5) has the form of a right circular hollow cylinder with an internal screw thread (6) suitable for screwing in a screw thread with an external diameter d.

2. The adapter according to claim 1, characterized in that the adapter comprises two to six connectors (4), optionally evenly distributed around the middle section and optionally having the same distance to the upper end of the middle section (7) as well as to the lower end of the middle section (8).

3. The adapter according to any one of the preceding claims, characterized in that the angle (9) between the outer surfaces of the lower section and the outer surface of the middle section is of from 180° to 240°.

4. The adapter according to any one of the preceding claims, characterized in that the connectors are independently of each other selected from a barbed-end-type connector, a plain-type connector, a weldable tubing, a cannula, and/or a Luer-type connector.

5. The adapter according to any one of the preceding claims, characterized in that the external diameter d is about 25 mm, or about 40 mm, or about 42 mm, or about 45 mm, or about 52 mm.

6. The adapter according to any one of the preceding claims, characterized in that the sections have the same height.

7. The adapter according to any one of claims 1 to 5, characterized in that the upper and lower section have a height of about 30 mm, and the middle section has a height of about 15 mm.

8. The adapter according to any one of the preceding claims, characterized in that one or more connector is connected on the inside of the adapter to a standpipe (10).

9. The adapter according to any one of the preceding claims, characterized in that the adapter comprises three to four connectors and optionally that two or more of the connectors are connected on the inside to a feed mixing device (11).

10. A device comprising an adapter according to any one of claims 1 to 9 and a shaker flask.

11. The device according to claim 8 further comprising a lid.

12. Use of an adapter according to any one of claims 1 to 9 for the selection of cell clones in shaker flask cultivation.

13. Use of an adapter according to any one of claims 1 to 9 for the evaluation of media and added solutions in a shaker flask cultivation.

14. Use of an adapter according to any one of claims 1 to 9 for the evaluation of cultivation conditions for shaker flask cultivation.

15. A method for cultivating a cell in a shaker flask comprising the following steps: a) cultivating a cell comprising a nucleic acid encoding a polypeptide of interest in a device according to any one of claims 10 to 11, b) adding a solution to the shaker flask.