WO2010003759A2

WO2010003759A2 - Cell culturing method

Info

Publication number: WO2010003759A2
Application number: PCT/EP2009/057281
Authority: WO
Inventors: Gerben Meile Zijlstra
Original assignee: Dsm Ip Assets B.V.
Priority date: 2008-06-17
Filing date: 2009-06-12
Publication date: 2010-01-14
Also published as: WO2010003759A3

Abstract

The present invention relates to the use of a retention device with tangential flow for the improvement of mass transfer in a bioreactor. The method according to this invention is particularly suited for growing of cells and maintaining cells at high cell density.

Description

CELL CULTURING METHOD

The present invention relates to a method for the improvement of mass transfer in a cell culturing device. A persistent problem in the culturing of cells in a bioreactor is the supply of sufficient amounts of nutrients to the content of the bioreactor. In particular, the supply of oxygen poses a problem in view of the relatively low solubility of oxygen in aqueous media and the problems in transfer from the gas phase into the aqueous phase. In order to improve the oxygen transfer in cell culturing devices use is being made of oxygenators, extensive bubbling of oxygen enriched gas through the content of the bioreactor, dividing an oxygen stream in the bioreactor into extremely fine bubbles and or extensive stirring of its content.

Each of these measures has its limits and even in combination these measures generally do not allow for enough oxygen to be supplied to fully fulfill the oxygen needs in high cell density cell cultures in bioreactors.

Furthermore, the cell culturing devices known in the art contain many components, which form an important cost factor in commercial large scale production.

An example of such a complicated cell culturing device is described in US 2003/0054544. This device consists of a bioreactor connected to a flow-through external cell retention module, as well as an external oxygenator (artificial lung) coupled to the combined set of bioreactor and cell retention module.

The need for so many different components also multiplies the costs and concerns for keeping the device free of contaminating organisms. Preferably, a cell culturing device should contain as few components as possible.

Hence, there is a need for an improved method to further increase the mass transfer (and in particular the oxygen transfer) in cell culturing devices which preferably needs more simple equipment than the methods known in the art.

Surprisingly, it has now been found that the mass transfer within a cell culturing device is considerably increased when a bioreactor is coupled to a tangential flow device.

The present invention therefore relates to a method for the improvement of mass transfer in a cell culturing device comprising contacting the content of the bioreactor with a gaseous composition containing oxygen and circulating the content of the bioreactor over a retention device wherein the flow is an (optionally alternating) tangential flow.

In a further embodiment the present invention relates to a method for the improvement of mass transfer in a cell suspension contained in a bioreactor comprising contacting the cell suspension with a gaseous composition containing oxygen and circulating the cell suspension over a retention device wherein the flow is an (optionally alternating) tangential flow.

In the context of the present invention "circulating the content of the bioreactor over a retention device" means that the content is transferred from the bioreactor towards and into the retention device and also is transferred back to the bioreactor.

The present invention also relates to the use of a retention device with tangential flow for the improvement of mass transfer in a cell culturing device.

In a further embodiment the present invention relates to the use of a retention device with tangential flow for the improvement of the mass transfer in a cell suspension contained in a bioreactor.

To this end an (alternating) tangential flow device is used for the improvement of the oxygen transfer in a cell culturing device comprising a bioreactor containing a cell suspension connected to a retention device coupled to a tangential flow device wherein the use comprises: (a) contacting the content of the bioreactor with a gaseous composition containing oxygen; and (b) circulating the content over a retention device wherein the flow is a (preferably alternating) tangential flow.

In a further embodiment the invention relates to the use of a retention device coupled to a tangential flow device for the improvement of the mass transfer in a cell culture of high cell density contained in a bioreactor.

With high cell density is meant here a cell density that leads to a wet cell content of at least 14%, preferably at least 19%, more preferably at least 24%, more preferably at least 29%, more preferably at least 33%.

For mammalian cells with an average cell diameter of about 15 μm this corresponds to a viable cell density of at least 60x10⁶ cells/ml, preferably at least 80x10⁶ cells/ml, more preferably at least 100x10⁶ cells/ml, more preferably at least 120x10⁶ cells/ml, more preferably at least 140x10⁶ cells/ml.

Typically, a suitable upper limit in the viable cell density may be about 80% wet cell volume. The calculation method to correlate wet cell volume and cell density is given in Example 2. In a preferred embodiment the tangential flow used according to the present invention may be an alternating tangential flow.

The process of the present invention is suitable for the culturing of animal cells, plant cells or yeast cells, especially for the culturing of mammalian cells. Examples of mammalian cells include: CHO (Chinese Hamster

Ovary) cells, hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, human cells, for example HEK-293 cells, human lymphoblastoid cells, PER.C6® cells, mouse cells, for example NSO cells. Examples of insect cells include SF-9 and SF-21 cells. Examples of yeast cells include Saccharomyces cerevisiae, Phaffia rhodozyma, Kluyveromyces lactis, or yeast cells from the genus Pichia. Examples of plant cells include Physcomitrella patens, duckweed (Wolffia australiana, Lemnaceae), Nicotiana tabacum cv. BY-2.

In particular the invention can be performed with mammalian cells such as CHO, NSO, PER.C6^® cells. The use according to the present invention is in particular very suitable for the culturing of animal cells, plant cells or yeast cells which have been transformed with one or more genes whereby the cells are able to produce one or more biological substances encoded by at least one of these genes.

The bioreactor can for example be of a disposable nature (for example the bioreactor can be a plastic flask or bag) or of a more permanent nature (for example made of stainless steel or glass). Examples of bioreactors suitable for use in the present invention include, but are not limited to stirred tank vessels, airlift vessels and disposable bags that can be mixed by rocking , shaking motion or stirring. Preferably disposable bioreactors are used as they are favorable as they require relatively low investment costs, have great operational flexibility, short turn-around times and are easily configurable to the process. Disposable bioreactors are commercially available from for example Hyclone, Sartorius, Applikon or Wave. The contacting of the contents of the cell culturing device with oxygen-containing gas can be done e.g. via the liquid surface, via sparged bubbles, via oxygen permeable membranes.

The term "retention device" as used herein is meant to include all devices with the ability to separate particles on basis of size or molecular weight. In principle, in the process of the present invention, as a retention device any filter may be used. Preferably, the pore size or Molecular Weight Cut-Off (MWCO) of the filter is chosen such that a desired separation between the cells and at least part of the - A -

substances in the cell culture contained in the cell culturing device can be established. Examples of filters suitable for use in the present invention include membrane filters, ceramic filters and metal filters. The filter may be used in any shape; the filter may for example be spiral wound or tubular or may be used in the form of a sheet. Preferably, in the process of the invention, the filter used is a membrane filter, preferably a hollow fiber filter.

With the term "hollow fiber" is meant a tubular membrane. The internal diameter of the tube is preferably between 0.3 and 6.0 mm, more preferably between 0.5 and 3.0 mm, most preferably between 0.5 and 2.0 mm. For regular perfusion mode, where the cells are retained in the bioreactor and the

(macro)molecules (including at least one biological substance produced by the cells) has to pass the filter, preferably, the pore size in the membrane is chosen such that the size of the pores is close to the diameter of the cells, ensuring a high retention of cells, while cell debris can pass the filter. In this instance use is made of microfiltration devices and the pore size preferable is at least 0.1 μm, more preferably at least 0.2 μm; as an upper limit the pore size is preferably not more than 30 μm, more preferably not more than 20 μm. When both the cells and the product have to be retained in the bioreactor and the small molecular weight components have to removed from the culture medium , the pore size in the membrane is chosen such that the size of the pores is close to the diameter of the product, ensuring a high retention of both cells and product. In this instance use is made of ultrafiltration devices and the MWCO is preferably at least 5 kD, more preferably at least 10 kD, most preferably at least 3OkD and/or the MWCO of the device is preferably at most 50OkDa, more preferably at most 30OkDa, most preferably at most 10OkDa. Retention devices are commercially available from for example

General Electric, Pall, Spectrum (formerly Amersham). For an overview of retention devices see e.g: "Mammalian cell retention devices for stirred perfusion bioreactors", Woodside, S. M., Bowen, B. D., Piret, J. M., Cytotechnology 1998, vol. 28, no 1-3 (237 p.) (1 p.3/4), pp. 163-175 or "Potential of cell retention techniques for large-scale high- density perfusion culture of suspended mammalian cells", D. Voisard, F. Meuwly, P. -A. Ruffieux, G. Baer, A. Kadouri, Biotechnology and Bioengineering, Volume 82, Issue 7, Date: 30 June 2003, Pages: 751-765,

With the term "tangential flow" is meant a flow substantially parallel to the filter surface, for example unidirectional tangential flow (TFF) or cross-flow. With the term "alternating tangential flow" is meant that there is one flow in the same direction as (i.e. tangential to) the membrane surfaces of the hollow fibers, which flow is going back and forth, and that there is another flow in a direction substantially perpendicular to said filter surface. Tangential flow can be achieved according to methods known to the person skilled in the art. For example, in US 6,544,424 it is described that alternating tangential flow can be achieved using one pump to circulate the cell culture over a filter module comprising hollow fibers and another pump to remove the liquid having a lower cell density than prior to the filter separation. Suitable equipment for establishing an alternating tangential flow over the filter module is the ATF device of Refine Technology.

In the process of the present invention, any type of cell culture medium suitable for the culturing of cells can in principle be used. Guidelines for choosing a cell culture medium and cell culture conditions are well known in the art and are for instance provided in Chapter 8 and 9 of Freshney, R. I. Culture of animal cells (a manual of basic techniques), 4th edition 2000, Wiley-Liss and in Doyle, A., Griffiths, J. B., Newell, D. G. Cell &Tissue culture: Laboratory Procedures 1993, John Wiley & Sons.

Generally, a cell culture medium for mammalian cells comprises salts, amino acids, vitamins, lipids, detergents, buffers, growth factors, hormones, cytokines, trace elements and carbohydrates. Examples of salts include magnesium salts, for example MgCI₂.6H₂O, MgSO₄ and MgSO₄.7H₂O iron salts, for example FeSO₄.7H₂O, potassium salts, for example KH₂PO₄, KCI; sodium salts, for example NaH₂PO₄, Na₂HPO₄ and calcium salts, for example CaCI₂.2H₂O. Examples of amino acids are all 20 known proteinogenic amino acids, for example histidine, glutamine, threonine, serine and methionine. Examples of vitamins include: ascorbate, biotin, choline. Cl, myo-inositol, D-panthothenate, riboflavin. Examples of lipids include: fatty acids, for example linoleic acid and oleic acid; soy peptone and ethanol amine. Examples of detergents include Tween 80 and Pluronic F68. An example of a buffer is HEPES. Examples of growth factors/hormones/cytokines include IGF, hydrocortisone and (recombinant) insulin. Examples of trace elements are known to the person skilled in the art and include Zn, Mg and Se. Examples of carbohydrates include glucose, fructose, galactose and pyruvate.

The pH, temperature, dissolved oxygen concentration and osmolarity of the cell culture medium are in principle not critical and depend on the type of cell chosen. Preferably, the pH, temperature, dissolved oxygen concentration and osmolarity are chosen such that it is optimal for the growth and productivity of the cells. The person skilled in the art knows how to find the optimal pH, temperature, dissolved oxygen concentration and osmolarity for the perfusion culturing. Usually, the optimal pH is between 6.6 and 7.6, the optimal temperature between 30 and 39°C, the optimal osmolarity between 250 and 500 mθsm/kg.

The improvement of mass transfer in a bioreactor results inter alia in the increase of oxygen transfer rate, but also in the improvement of carbon dioxide removal rate. The oxygen transfer rate (OTR = dC_o/dt) in the cell culture is a function of the oxygen transfer coefficient (k_L,oa) and the oxygen concentration difference between the gas phase (Co) and the culture medium (Co ) according to the formula dCo/dt = k_uoa (Co^* - C₀)

Similarly, the carbon dioxide removal rate (CTR = dC_c/dt) in the cell culture is a function of the carbon dioxide transfer coefficient (k_L,ca) and the carbon dioxide concentration difference between the gas phase (C_c) and the culture medium (Cc ) according to the formula dCc/dt = k_L,_ca (C_c ^* - C_c)

The cells propagated in the cell culturing device according to the the present invention may be used for the production of biological substances such as viruses (see e.g. WO 01/38362), or recombinant proteins (see e.g. US patent 6,855,544; Yallop et al, 2005, PER.C6 cells for the manufacture of biopharmaceutical proteins, Modern Biopharmaceuticals: Design, Development and Optimization, 4 Volumes, 779-807, Jorg Knablein (Editor)), e.g. proteins that can be used as an active ingredient in pharmaceutical preparations.

Examples of proteins that can be used as an active ingredient in pharmaceutical preparations (with the brand name between brackets) include Tenecteplase (TN Kase™), (recombinant) antihemophilic factor (ReFacto™), lymphoblastoid Interferon α-n1 (Wellferon™), (recombinant) Coagulation factor (NovoSeven™), Etanercept (Enbrel™), Trastuzumab (Herceptin™), Infliximab (Remicade™), Palivizumab (Synagis™), Basiliximab (Simulect™), Daclizumab (Zenapaz™), Rituximab (Rituxan™), (recombinant) Coagulation factor IX (Benefix™) and Interferon β-1 a (Avonex™).

Examples of vaccines that can be used as an active ingredient in pharmaceutical preparation include isolated protein antigens, examples of which include but are not limited to live, oral, tetravalent Rotavirus vaccine (RotaShield™), rabies vaccine (RanAvert™), influenza vaccines and inactivated hepatitis A vaccine (VAQTA™).

Description of the figures

Fig. 1/2 Mass Transfer Coefficient (k_La, 1/hour) as a function of the airflow rate (mL/min) at a stirrer speed of 100 rpm. In situation 1 the ATF is switched off, in situation 2 the ATF is switched on

Fig. 2/2 Mass Transfer Coefficient (k_La, 1/hour) as a function of the airflow rate (mL/min) at a stirrer speed of 245 rpm. In situation 1 the ATF is switched off, in situation 2 the ATF is switched on

EXAMPLES

Example 1 : Mass Transfer increase by the ATF in a 2 L Applikon Bioreactor.

In this example the Oxygen Transfer Coefficient (k_La) was determined in a Bioreactor with an ATF retention system connected. The k_La in the bioreactor with the ATF unit switched off was compared with the k_La in the bioreactor with the ATF unit switched on. The k_La was determined using the dynamic method as described in e.g. Basic Bioreactor Design (1991 ) K. Van't Riet and J. Tramper.

The measurements were performed in a 2 L Applikon bioreactor vessel equipped with 2 standard 45° angle marine impellers (vortex) at 1.2 L working volume and a standard micro-sparger. The ATF retention device used was an ATF-2 system (Refine Technology) equipped with a hollow fiber membrane (General Electric). When switched on, the ATF system was operated at a cross flow rate of 1 L/min.

The bioreactor parameters were controlled using a Biostat B controller (Sartorius) and the dissolved oxygen (DO) data were logged with data acquisition software (MFCS, Sartorius). A regular DO probe (Ingold) was used to measure the DO. The temperature was controlled at 36.5°C, the stirrer speed was controlled at 100 rpm or 245 rpm. The pH was not controlled. The measurements were performed in 0.9% NaCI and 1 g/L Pluronic.

From Fig. 1/2 and 2/2 it can be concluded that the use of an ATF unit can be used successfully to increase the mass transfer coefficient in the cell culturing device.

Example 2: Calculation of wet cell volume based on viable cell number and average viable cell diameter

Assumptions:

Average cell diameter = 15 (μm)

Cell number = 100 mln (cells/mL)

The cells are spherical

The volume of a sphere is 4/3 x π x r³ The theoretical packing density of spheres is 0.7405

The volume of 1 cell is:

4/3 x π x (15x10^"6/2)³ = 1.76715 x 10^"15 (m³/cell)

With 100 mln cells/mL the total cell volume per cubic meter is: 1 x 10¹⁴ (cells/m3) x 1.76715 x 10^"15 (m³/cell) = 0.1767 V/V%

With a theoretical packing efficiency of 0.7405, the wet cell weight becomes: 0.1767 / 0.7405 = 0.2386 V_CΘ|i_s/V|_iquιd.

Claims

1. Use of a tangential flow device for the improvement of the oxygen transfer in a cell culturing device comprising a bioreactor containing a cell suspension connected to a retention device coupled to a tangential flow device wherein the use comprises: a. contacting the content of the bioreactor with a gaseous composition containing oxygen; and b. circulating the content over a retention device wherein the flow is a tangential flow.

2. Use according to claim 1 wherein the flow is an alternating tangential flow.

3. Use according to claim 1 or 2 wherein the bioreactor contains a cell suspension.

4. Use according to claim 1 or 2 wherein the bioreactor contains a cell suspension with a wet cell content of at least 14%.

5. Use according to claim 1 or 2 wherein the bioreactor contains an animal cell suspension with a cell density of at least 60x10⁶ cells/ml.