# Patent

## Title
Process for the culturing of cells

## Abstract

The invention relates to a process for the culturing of cells, preferably E1-immortalized HER cells, more preferably PER.C6 cells in a reactor in suspension in a cell culture medium, wherein the cells produce a biological substance, preferably an antibody, wherein at least one cell culture medium component is fed to the cell culture and wherein the cells culture comprising the cells, the biological substance and the cell culture medium is circulated over a separation system and wherein the separation system separates the biological substance from substances having a lower molecular weight than the biological substance and wherein the biological substance is retained in or fed back into the reactor. Preferably part of the substances of lower molecular weight is continuously removed from the cell culture.

## Background
This application is a continuation application which claims priority to U.S. patent application Ser. No. 13/707115, filed Dec. 6, 2012 (U.S. Pat. No. 8,685,731, issued Apr. 1, 2014), which is a continuation of U.S. patent application Ser. No. 13/372069, filed Feb. 13, 2012 (U.S. Pat. No. 8,222,001, issued Jul. 17, 2012), which is a continuation of U.S. patent application Ser. No. 12/307995, filed Sep. 4, 2009 (U.S. Pat. No. 8,119,368, issued Feb. 21, 2012) which is a U.S. national phase of International Application no.: PCT/EP2007/005915, filed July 4, which designated the U.S. and claims priority to Europe Application No(s): 06014671.9, filed Jul. 14, 2006 and 07002571.3, filed Feb. 6, 2007.


The invention relates to a process for the culturing of cells in a reactor in suspension in a cell culture medium.
Such a process is for example known from WO04/099396. Herein it is described how the cell density of the cell culture and the yield of the desired biological material can be improved by optimizing the growth conditions in a fed-batch process.
Furthermore, WO05/095578 discloses a process for the culturing of cells by continuous perfusion culturing of a cell culture comprising cell culture medium and cells, whereto cell culture medium is added to the cell culture, the cell culture is circulated over a filter module comprising hollow fibers resulting an outflow of liquid having a lower cell density than the cell culture, and the flow within the filter module is an alternating tangential flow, wherein the cells produce a biological substance. In the example of WO05/095578 it is shown that 0.9 g/L/day product is produced, corresponding to a product concentration in the outflow of approximately 0.3 g/L.
The larger the volume of liquid containing the biological substance, the more laborious becomes the purification of the biological substance. The concentration of biological substance obtained is not that high in the processes as disclosed in WO04/099396 and WO05/095578. Therefore, downstream processing of this biological substance is cumbersome, because the biological substance needs to be concentrated before further purification steps are applied or large volumes of less concentrated biological substance need to be purified. Furthermore, the culturing of cells at lower cells densities results in lower volumetric productivity and therefore requires larger and/or more culturing vessels and thus higher investments in equipment for a given production level.
Therefore, it is the object of the invention to provide a process wherein the product is obtained from the cell culture in higher concentrations.
A further object of the present invention is to enable culturing of the cells and production of the biological material during an extended period.
These objects are achieved by a process for the culturing of cells in a reactor in suspension in a cell culture medium, wherein the cells produce a biological substance, wherein at least one cell culture medium component is fed to the cell culture and wherein the cell culture comprising the biological substance and cell culture is circulated over a separation system and wherein the separation system separates the biological substance from substances having a lower molecules weight than the biological substance and wherein the biological substance is retained in or fed back into the reactor.
For example, the invention relates to a process for the culturing of cells in a reactor in a cell culture medium, wherein the cells practice a biological substance, wherein nutrients and/or cell culture medium is/are fed to the reactor and wherein the cell culture comprising the cells and the cell culture medium is circulated over a filter having a pore size or molecular weight cut off of between 5 and 500 kD.
It has been found that by using a separation system that separates the biological substance from substances having a lower molecular weight than the biological substance, the biological substance can be accumulated in the cell culture in higher concentrations.
Hence, the present invention differs form the cell culturing described in the prior art in that it allows for accumulation of the desired biological material together with the cell mass.
In a preferred embodiment of the present invention part of the substances of lower molecular weight are continuously removed from the cell culture.
An additional advantage of the process of the present invention is that higher viable cell concentration can be reached as compared to for example batch or fed-batch processes. Furthermore, the production time—the period during which the cells produce the biological substance—can the extended compared to for example batch or fed-batch processes. Also, as compared to a batch or fed-batch process, it is possible is use a smaller reactor. Use of smaller reactors is of advantage as this reduces the equipment and facility related investments.
Also, higher concentrations of the biological substance may be obtained is shorter times.
It was found that it was possible to obtain high concentrations of biological substance within the reactor without sharply decreasing the cell viability and hence without limiting the production time. The person skilled in the art would have expected that the product inhibition, i.e. inhibition of production of the biological substance by the biological substance itself or inhibition by other macromolecules produced by the cell (such as for instance host cell proteins, enzymes or cellular debris), would occur. Furthermore, it was found that the accumulation of the desired biological material does not impair the function of the separation system.
The process of the present invention provides a considerable advantage in terms of cell density, product concentration in the cell culture and extended culturing period as compared to the processes according to WO05/095578 and WO04/099396. As a result the present process results in an improved production of the desired biological material.
Cells which can be used to produce the biological substance are in principle all cells known to the person skilled in the art, which have the ability to produce a biological product. The cells may be eukaryotic, for example, filamentous fungi, for example Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum, yeasts, for example Saccharomyces cerevisiae, Kluyveromyces lactis, Phaffia rhodozyma, yeast from the genus Pichia, for example Pichia pastoris or prokaryotic, for instance Escherichia coli, Bacilus sp. for example B. licheniformis, B. subtilis, B. amyloliquefaciens, B. alkalophilus, Streptomyces sp., Corynebacterium glutamicum, Pseudomonas sp. Examples of eukaryotic cells are for example also described in Chu, L., Robinson, D. K., (2001) Curr. Opinion Biotechn., vol. 12, p. 180-187. Preferably, the cells that are used in the process of the present invention are animal cells, in particular 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 lymphoblastold cells, E1 immortalized HER cells, mouse cells, for example NS0 cells. More preferably, E1 immortalized HER cells are used, most preferably PER C6 cells.
Primary human embryonic retina (HER) cells can be isolated from fetuses (Byrd P. Brown K W, Gallimore P H. 1982. Malignant transformation of human embryo retinoblasts by cloned adenovirus 12 DNA. Nature 298: 69-71, Byrd P J, Grand R J A, Gallimore P H. 1988. Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A+ras. Oncogene 2: 477-484). Primary cells will die upon culturing for several passages. E1-immortalized HER cells for the purpose of the present invention are derived from primary HER cells by expressing DNA encoding adenoviral E1A and E1B proteins therein, to obtain immortalized cells. Such immortalized cells can be cultured for more than 100 passages. Methods to obtain E1-immortalized HER cells have for instance been described in U.S. Pat. No. 5,994,128, in Byrd P, Brown K W, Gallimore P H. 1982. Malignant transformation of human embryo retinoblasts by cloned adenovirus 12 DNA. Nature 298: 69-71, in Byrd P J, Grand R J A, Gallimore P H. 1988. Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A+ras. Oncogene 2: 477-484, and in Gallimore, P. H., Grand, R. J. A. and Byrd, P. J. (1986). Transformation of human embryo retinoblasts with simian virus 40, adenovirus and ras oncogenes. Anticancer Res. 6, p499-508. For instance, immortalized HER cells, including PER.C1, PER.C3. PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9 cells, were general by transfection of primary HER cells using a plasmid that contained the adenovirus serotype 5 (Ad5) E1A- and E1B-coding sequences (Ad5 nucleotides 459-3510) under the control of the human phosphoglycerate kinase (“PGK”) promoter (see U.S. Pat. No. 5,994,128).
In a preferred embodiment, the cells in the process of the present invention are E1-immortalized HER cells, more preferably PER.C6 cells (see. U.S. Pat. No. 5,994,128). PER.C6 cells are exemplified by cells as deposited under ECACC No. 96022940 (see, e.g., U.S. Pat. No. 5,994,128, EP 0833934 B1).
In the process of the invention, the cells may be cultured in suspension in any form, for example as immobilized cells, single cells or in cell clusters or as a combination thereof. Preferably the cells are colored as single cells and/or as small cell clusters of not more than 100 cells, more preferably of not more than 20 cells. Cells can for example be immobilized on microcarriers such as are commercially available from for example GE Healthcare (Cytodex).
A reactor as defined herein is a system that comprises the cell culture which cell culture on its turn comprises cells and a cell culture medium. It preferably provides sterile barriers, such as air filters, to prevent other cells from contaminating the desired cells and if preferably maintains a favorable environment for the cells by providing the right culture conditions such as mixing, temperature, pH, oxygen concentration etc.
The reactor can for example be of a more permanent nature, for example the reactor can be of stainless steel or glass or can for example be of a disposable nature, for example the reactor can be a plastic flask or bag. Examples of reactors 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 (bio)reactors are used as they are favourable as they require relatively low investment costs, have great operational flexibility, short turn-around times and are easily configurable to the process. Disposable (bio)reactors are commercially available from for example Hyclone, Sartorius, Applikon or Wave.
The term ‘separation system’ is defined within the framework of the invention as a system capable of separating on basis of molecular weight. The separation system used in the process of is invention is capable of separating the biological substance from substances having a lower molecular weight than the biological substance. In other words, the molecular weight cut-off is chosen such that the molecular weight cut-off (MWCO) is smaller than, more preferably at least a factor 2, most preferably at least a factor 3 smaller than the molecular weight of biological substance. Typically, but of course depending on the molecular weight of the biological substance produced in the process of the present invention, the MWCO of the separation system is preferably at least 5, more preferably at least 10, most preferably at least 30 kDa and preferably at most 500 kDa, more preferably at most 300 kDa, most preferably at most 100 kDa. For example for an IgG with a molecular weight of 150 kDa, a separation system having a MWCO of at most 50 kDa is most preferred.
Examples of separation systems include but are not limited to filters, centrifuges and aqueous two phase extraction systems.
The term ‘liter’ 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, any filter may be used as long as the pore size or MWCO is chosen such that the biological substance is separated from substances having a lower molecular weight than the biological substance, typically this will be a pore size or MWCO of between 5 and 500 kDa. 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 filer 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 at least 0.1 mm, more preferably at least 0.5 mm, most preferably at least 0.75 mm and preferably the internal diameter of the tube is at most 10 mm, more preferably at most 6 mm, most preferably at most 1 mm. Filter modules comprising hollow fibers are commercially available from for example General Electric (GE, formerly Amersham).
By circulating the cell culture comprising the biological substance, cells and the cell culture medium over a separation system, the biological substance and cells are retained in the reactor and the liquid outflow therefore has a lower concentration of biological substance and a lower cell density than the cell culture. Usually in the process of the invention, the liquid outflow does not contain or hardly contains any biological substance and cells. Usually, the liquid outflow will essentially only contain components having a molecular weight lower than that of the biological substance. Essentially all cells and essentially all biological substance are therefore usually retained in the reactor.
Preferably, the pore size or MWCO of the filter is chosen such that the size of the pores or MWCO of the filter is smaller than, more preferably at least a factor 2, most preferably at least a factor 3 smaller than the diameter or molecular weight of the product, ensuring a high retention of product. Typically, but of course depending on the size or molecular weight of the product, i.e. biological substance proved in the process of the present invention, the pore size or MWCO of the filter is preferably at least 5, more preferably at least 10, most preferably at least 30 kDa and/or the pore size or MWCO of the filter/membrane is preferably at most 50 kDa, more preferably at most 300 kDa, most preferably at most 100 kDa.
With molecular weight cut-off (MWCO) is meantime molecular weight above which at least 90% of the particles is retained by the separation system.
Circulating the cell culture over a separation system, for example a filter means that the cell culture is passed through a separation system, for example a filter resulting in a liquid outflow and a flow which contents are kept in or fed back into the reactor. The flow which contents are kept in or fed back into the reactor will usually essentially only contain components having a molecular weight of at least equal to that of the biological substance or higher and therefore said flow will comprise more biological substance than the liquid outflow.
In principle, it is not critical when the circulation of the cell culture over the separation system is started during the process of the invention. The circulation of the cell culture may for example be started directly from the start of the process or when the viable cell density of the cells has reached a certain level.
The circulator of the cell culture over a filter may be a flow substantially perpendicular with respect to the filter surface, also known as dead-end flow or a flow substantially parallel to the filter surface, also known as tangential flow, for example unidirectional tangential flow (TFF) or cross-flow. A preferred example of cross-flow is alternating tangential flow (ATF) as with ATF it was found that filter clogging does not occur (quickly) even at very high cell densities. It is common general knowledge that in depth filtration, the final small pore filter needs to be protected from clogging by course pre-filters. This practice is based on the common general knowledge that filters with smaller pores or with a smaller MWCO clog more easily, thereby limiting the production time. If ATF is used, the use of a pre-filter becomes superfluous.
The flow may be directed by moving the cell culture, by moving the filter or bolts. The filter may for example be moved by rotation (rotating filter) or vibration (vibrating filter). Alternatively if the flow is directed by moving the cell culture only, the filter is static and the cell culture may for example be moved by way of pumps or pressure.
With “alternating tangential flow” is meant that there is one flow in the same direction as (i.e. tangential to) the filter surface(s), which flow is going back and forth, and that there is another flow in a direction substantially perpendicular to said filter surface. Alternating tangential flow can be achieved according to methods known to the person skilled to the art (for example as described to U.S. Pat. No. 6,544,424).
During the culturing of the cells, at least one cell culture medium component, for example one or more nutrients and/or cell culture medium may be fed to the cells. In the process according to the invention, it is of advantage to supplement in part or preferably in whole at least one of the depleted nutrients by way of a feed of this nutrient or these, nutrients to the reactor. For example, complete cell culture medium may be fed to the reactor, which is of advantage as a separate feed need then not be prepared separately. The cell culture medium may for example also be fed to the cells in a more concentrated form; this is of advantage as smaller volumes are easier to handle. Also one or more nutrients may be fed to the reactor. For example carbohydrates, for instance glucose or fructose; amino acids, such as glutamine and/or peptides may advantageously be fed to the reactor.
In a preferred embodiment of the invention, cell culture conditions are chosen such that cell growth rate and/or specific productivity of the cells is not limited and more preferably such that the concentration of at least one of the components of the cell culture medium remains essentially constant. Examples of limiting cell culture conditions are nutrient limitations and formation of inhibiting metabolites, such as ammonia, carbon dioxide and lactate. For example, cell culture conditions such as the feed may be chosen such that cell growth rate is not limited for example by supplying enough nutrients as to compensate for the depletion and/or to avoid the production of inhibiting metabolites such as lactate or ammonia. For example, the aeration conditions may be chosen such that carbon dioxide formation is not limiting the cell growth rate. Growing the cell under non-limiting conditions is highly advantageous from a Good Manufacturing Practice (GMF) point of view as 1) this may give a constant cell culture environment that in many cases also owes constant and good product quality and 2) this may lead to high cell viability, in some cases to a cell viability of more than 98%. High cell viability reduces the release of cell related contaminants, such as host cell proteins, which facilitates product purification. Furthermore, growing the cells at unlimited cell growth rate and/or unlimited specific productivity has the commercial advantage that it is possible to produce more biological substance to an even shorter time as higher cell density will be reached earlier in the process.
“Specific productivity” of the cells is the amount of a given biological substance produced per cell per time unit and is usually expressed in pg.cell−1day−1.
The rate of addition of at least one cell culture medium component, for example nutrients and/or cell culture medium to the cell culture (the inflow rate or perfusion rate) influences the viability and the density of the cells. In the process of the invention, the cell culture medium components such as nutrients and/or cell culture medium may be fed for example in a continuous flow, semi-continuous flow, for example step-wise flow or staggered flow. Preferably, cell culture medium component(s), for example nutrients and/or cell culture medium are added in a continuous flow.
Cell culture medium component(s), such as complete cell culture medium and/or nutrients may in principle be fed to the reactor at any time during the process. Preferably, the feed is initiated before substrates, such as glutamine and glucose have reached such low levels as to cause the growth of the cells to cease or before inhibitory metabolites, for example lactate or, ammonia reach such high levels that growth would cease. From this point onward, the cell culture medium component(s), such as nutrients and/or complete cell culture medium are preferably fed to the reactor at a rate such that substrate demand is met.
In one embodiment of the invention, cell culture medium is added at a Feed Rate according to formula (1):

Feed Rate=SFR×(total cell culture volume)×(viable cell density)  (1)

wherein the feed rate is expressed in liters per day, wherein the SFR is the Specific Feed Rate, i.e. the rate in which the cell culture medium is fed to the cell culture expressed as the volume of medium added per viable cell per time unit and wherein the viable cell density is the number of viable cells per unit of volume. The number of viable cells can be determined by the person skilled in the art, for example via the trypan blue exclusion method. The specific feed rate is preferably chosen between 0.01 and 0.3 nL/cell/day, more preferably between 0.01 and 0.2 nL/cell/day.

It may be of advantage to take into account additional parameters when adjusting the feed rate, for example the amount of glucose to be fed to the culture and/or the oxygen uptake rate. For example, for PER.C6 the feed rate of the cell culture medium and/or the nutrients is preferably chosen such that glucose concentration is kept between 3 and 20 mmol/L, more preferably between 5 and 15 mmol/L. Preferably the glucose concentration is at least 3 mmol/L, more preferably at least 5 mmol/L and preferably at most 20 mmol/L, more preferably at most 15 mmol/L.
In a special embodiment of the invented cell culture (comprising cells, biological substance and cell culture medium) is removed at least once from the reactor and liquid, for example cell culture medium or a nutrient feed is added to the reactor to compensate for the cell culture removal. Cell culture removal may lead to longer process times at high cell densities in combination with high cell viabilities resulting in a higher productivity. Cell cultures may be removed continuously or step-wise.
In a preferred embodiment of the invention, cell culture (comprising cells, cell culture medium and biological substance) is removed from the reactor as soon as the desired cell density, for example a cell density of at least 10.106 viable cell/ml, preferably of at least 20.106 viable cells/ml, more preferably of at least 30.106 viable cells/ml, for example a cell density of at most 200.106 viable cells/ml, is reached and liquid, for example cell culture medium or nutrient feed is added to the reactor to compensate for the cell culture removal. Preferably, cell culture is removed at such rate that the cell density remains in the desired cell density range. This embodiment of the invention is highly advantageous as compared to a conventional batch or fed-batch process as it combines the advantages of the process of the invention with high viability that can be maintained longer, making it possible to realize an even higher overall volumetric productivity. With ‘volumetric productivity’ is meant the amount of biological substance produced per unit reactor volume per unit time and is usually expressed in g.L−.day−1. As compared to a conventional perfusion process, this embodiment of the invention is also highly advantageous as it combines the advantages of the process of the invention with a cell culture removal stream having a highly concentration of biological substance. The high concentration of biological substance in the cell culture removal stream makes it commercially interesting to harvest the biological substance there from. In a conventional perfusion process wherein cell culture is removed, the cell culture removal stream does not contain enough biological substance to make it commercially worthwhile to harvest the biological substance and the cell culture removal stream is therefore usually regarded as waste. Hence, in this embodiment of the invention, in theory all biological substance produced can be harvested in a straight forward, economically feasible and simple manner.
In a particularly preferred embodiment of the invention, cell culture conditions are chosen such that the cell growth rate and/or specific productivity of the cells is not limited and more preferably such that also the concentration of at least one of the components of the cell culture medium, such as glucose or glutamine remains constant and cell culture is removed at least once from the reactor as soon as the desired cell density is reached and liquid, for example cell culture medium is added to the reactor to compensate for the cell culture removal.
Preferably, the rate of the outflow is chosen such that it is substantially equal to the rate of the addition of the at least one cell culture medium component, for example nutrients and/or cell culture medium minus the rate of the optional cell culture removal.
Cells that produce a biological substance are for instance cells capable of expressing a gene encoding the biological substance. Cells capable of expressing a gene encoding the biological substance may for example be prepared by transaction of the cells with a plasmid containing the gene encoding the biological substance and gene encoding a suitable selection marker, for example a gene encoding a neomycine resistance (Neo marker gene). Stably transfected cells may then be selected by selection pressure, for example—in the case of a Neo marker gene—by culturing the transfected cells to the presence of G418 (genericin) and immediate screening of the cells for cells exhibiting high-level expression of the biological substance. Methods for preparing clones of E1-immortalized HER cells expressing a protein, and methods for culturing such cells to produce the protein, are well known to the skilled person, and can for instance be found in U.S. Pat. No. 6,855,544.
Biological substances, which may be produced by the cells, for example by expressing a (recombinant) gene coding therefore are for example (recombinant) proteins, in particular receptors, enzymes, fusion proteins, blood proteins such as proteins from the blood coagulation cascade, multifunctional proteins such as for instance erythropoletin, virus or bacterial proteins for instance for use to vaccines; immunoglobulins such as antibodies, for example IgG or IgM, and the like; Preferably a protein, more preferably an antibody is produced by the cells. Preferably, the biological substances such as proteins or vaccines produced by the cells can be used as an active ingredient in a pharmaceutical preparation. In the context of the present invention, the terms ‘product’ and ‘biological substance’ are interchangeable.
Within the framework of the present invention, with pharmaceutical preparation is meant any preparation, which can be used as a medicine, in particular as a medicine in humans. Such a medicine may for example be used for diagnosis, or for prophylactic purpose such as for instance a vaccine, and/or for therapeutic purpose, such as for instance an enzyme or protein for which a patient is deficient, or an antibody to kill undesired cells. A pharmaceutical preparation may further contain a pharmaceutically acceptable carrier or excipient, examples of which are well known to the person skilled in the art.
The PER.C6 cell line can be used for production of biological substances, such as E1-deleted adenovirus (see e.g. U.S. Pat. No. 6,994,128; Nichols et al, 2002, Propagation of adenoviral vectors: use of PER.C6 cells. In: Curiel D, Douglas J T, editors. Adenoviral vectors for gene therapy. San Diego: Elsevier, p 128-167), other viruses (see e.g. WO 01/38362), or recombinant proteins (see e.g. U.S. Pat. No. 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, Jörg Knäblein (Editor)).
Examples of proteins that can be used as an active ingredient in pharmaceutical preparations (with the brand name between brackets) include Tenecteplase (TN KaseTM), (recombinant) antihemophilic factor (ReFactoTM), lymphoblastoid Interferon α-n1 (WellferonTM), (recombinant) Coagulation factor (NovoSevenTM), Etanercept, (EnbrelTM), Trastuzumab (HerceptinTM), Infliximab (RemicadeTM), Palivizumab (SynagisTM), Basiliximab (SimulectTM), Daclizumab (ZenapazTM), Rituximab (RituxanTM), (recombinant) Coagulation factor IX (BenefixTM) and Interferon β-1a (AvonexTM).
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 (RotaShieldTM), rabies vaccine (RanAvertTM), influenza vaccines and inactivated hepatitis A vaccine (VAQTATM).
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 culture (see, e.g. WO 2004/099396). Preferably, for the process of the invention when using E1 immortalized HER cells, the pH is chosen between 6.6 and 7.6 and/or the temperature is chosen between 30 and 39° C. and/or the osmolarity is chosen between 260 and 400 mOsm/kg. To maintain optimal process conditions automation to control the process conditions is desired. In order to optimize process conditions, for instance to obtain growth arrest for increased cellular productivity, during the culture a shift in the culture conditions can be applied. This may be established by for instance a temperature shift (such as from 37 to 32° C.), a pH shift or an osmolarity shift. p The process of the present invention can in principle be performed in any type of cell culture medium suitable for the culturing of cells. Guidelines for choosing a cell culture medium and cell culture conditions are well known 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, Johns Wiley & Sons.
For example the cell culture medium may for example comprise as a cell culture medium component a carbohydrate source, salts and/or amino acids and/or vitamins and/or lipids and/or detergents and/or buffers and/or growth factors and/or hormones and/or cytokines and/or trace elements. Examples of carbohydrate sources include glucose, fructose, galactose and pyruvate. Examples of salts include magnesium salts, for example MgCl2.6H2O, MgSO4 and MgSO4.7H2O iron safe, for example FeSO4.7H2O, potassium salts, for example KH2PO4, KCl; sodium salts, for example NaH2PO4, Na2HPO4 and calcium salts, for example CaCl2.2H2O. Examples of amino acids include all known proteinogenic amino acids, for example hystidine, glutamine, threonine, serine, 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; Examples of detergents include Tween® 80 and Pluronic® F68. Example of buffers include HEPES and Na2CO3. Examples of growth factors/hormones/cytokines include IGF (insulin-like growth factor), hydrocortisone and (recombinant) insulin. Examples of trace elements are known to the person skilled in the art and include Zn, Mg and Se. The cell culture medium may for example also compose other cell culture medium components, for example soy peptone or ethanol amine.
For production of biological substances according to the invention, in particular if the biological substances are to be used as an active ingredient in pharmaceutical preparations, serum free media are preferred to media containing a serum source. The reason for this is that serum source media may be contaminated with viruses, present the risk of prionic infections, and can create a major obstacle in the downstream processing of the biopharmaceutical product (i.e. the further purification of the biological substance from the cell culture). Therefore the process of the invention is preferably performed in a cell culture medium that does not comprise serum from an animal, including human, source. Since compounds from a mammalian source also present an infection risk, preferably the cell culture medium is mammalian source free (i.e. the cell culture medium does not comprise serum or components from a mammalian source). More preferably the cell culture medium is animal source free (i.e. the cell culture medium does not comprise serum or components from an animal, including human, source. Examples of serum free media that can be used for the culturing of PER.C6 cells include commercially available media, such as the instance EX CellTM VPRO medium (SAFC), HyQ® CDM4RetinoTM (HyClone), IS ProVec CD (Irvine scientific), 293-SFM II (Invitrogen).
In preferred embodiments, the biological substance produced in the process of the present invention is harvested from the flow which contents are kept in or preferably fed back into the reactor or from the cell culture that is removed from the reactor or from both. The biological substance(s) produced in the process of the present invention can be further harvested from the cell culture in so-called downstream processing, using methods dependent on the biological substance, which methods are as such well known to the skilled person. Downstream processing usually comprises several purification steps in varying combinations and order. Examples of purification steps in the downstream processing are separation steps (e.g. by affinity chromatography and/or ion exchange chromatography and/or extraction by aqueous two-phase systems and/or precipitation by for example ammonium sulphate), steps for the concentration of the biological substance (e.g. by ultrafiltration or diafiltration), steps to exchange buffers and/or steps to remove or inactivate viruses (e.g. by virus filtration, pH shift or solvent detergent treatment).
In one aspect the invention relates to a cell culture comprising mammalian cells, preferably E1-immortalized HER cells, more preferably PER.C6 cells, having a viable cell density of at least 50.106 cells/mL, preferably at least 60.106 cells/mL, in particular at least 90.106 cells/mL and a concentration of biological substance of at least 5 g/L, more preferably at least 10 g/L, in particular at least 11 g/L. In principle the concentration of biological substance can be as high as the solubility of the biological substance allows. The concentration of viable cells is typically not more than 200.106 cells/mL and preferably within the range of 80-150.106 cells/mL.
Viable cell density can for example be determined using the tryptan blue exclusion method for example by using a cell counter as is commercially available from for example Innovatis (Cedex cell counter).
With cell culture is meant the liquid comprising cell culture medium, cells and biological substance, which liquid is the result of a process for the culturing of cells in a reactor in a cell culture medium, wherein the cells produce the biological substance.
The invention will now be elucidated by way of the following examples without however being limited thereto.



DESCRIPTION OF THE FIGURES
FIG. 1. shows the viable cell density Y (106.ml−1) plotted versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process of the invention).
FIG. 2. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process of the invention).
FIG. 3. shows the viable cell density Y (106.ml−1) plotted versus the process time X (days) for process A (batch), B (fed-batch) C2 (process of the invention).
FIG. 4. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) for process A (batch), B (fed-batch) and C2 (process of the invention).
FIG. 5. shows the viable cell density Y (106.ml−1) plotted versus the process time X (days) for process A (batch), B (fed-batch) C3 (process of the invention).
FIG. 6. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A3) versus the process time X (days) for process A, and C3.
FIG. 7. shows the cumulative yield Q (% as compared to yield in process A, per L reactor volume) plotted versus the process time X (days) for process A, B and C3.
FIG. 8. shows the cell number Y (106.ml−1) plotted versus the process time X (days) for C4 (process of the invention).
FIG. 9. shows the IgG concentration of the reactor Z (% as compared to the maximum IgG concentration reached versus the process the X (days) for process C4 (one embodiment or process of the invention)



EXAMPLES
Example 1
Comparison between a Batch Process, a Fed Batch Process and the Process According to the Invention
In this example the performance of the process according to me present invention was compared to batch and fed-batch processes.
FIG. 1 shows the viable cell density Y (106.ml−1) plotted versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process of the invention).
FIG. 2. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process of the invention).
All fermentations were performed using a Sartorius Biostat B controller to control the temperature at 36.5° C., the pH between 7.2 and 6.8 and the DO at 50% air saturation and at 200 rpm. The same IgG producing PER.C6 cell line (see WO 2004/099396) was used in all experiments.
Batch Process A
The batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3×10e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.
Fed-batch Process B
The fed-batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3×10e5 cells/mL VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose and glutamine were added to keep the concentration above respectively 15 mM and 1 mM. Amino acids and peptides were added from day 5 to replenish the consumed amino acids.
Process of the Invention C1
The process of the invention was performed in a 2 L Applikon vessel. A 100 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane obtained from General Electric (GE) operated in ATF flow mode with an ATF-2 system (Refine Technology) was used to retain the cells and the IgG product. The culture was started with 3×10e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. VPRO culture medium (SAFC) supplemented with 6 mM L-Glutamin was perfused through the suspension cell culture using a Specific Flow Rate (SFR) between 0.05 and 0.2 nL/cell/day. The highest product concentration obtained was 1.4 g/L.
The process of the invention resulted in increased viable cell densities and increased product concentrations compared to mentioned cultivation modes in less time, as can be seen from FIG. 1 and FIG. 2 below.
Example 2
Comparison between a Batch Process, a Fed Batch Process and the Process According to the Invention
In this example the process according to the present invention is again compared to batch and fed-batch processes; in process C2, the CO2 pressure is controlled and a 50 kDa separation system was used.
FIG. 3 shows the viable cell density Y (106.ml−1) plotted versus the process time X (days) for process A (batch), B (fed-batch) C2 (process of the invention).
FIG. 4 shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) for process A (batch), B (fed-batch) and C2 (process of the invention)
All fermentations were performed using a Sartorius Biostat B controller to control temperature at 36.5° C., pH between 7.2 and 6.8 and DO at 50% air saturation and at 200 rpm. The same IgG (of approximately 150 kDa) producing PER.C6 cell line (see WO 2004/099396) was used in all experiments.
Batch process A
The batch process was executed at 4 L wording volume in a Sartorius B5 vessel. Cells were inoculated at 3.105 cells.mL−1 in VPRO medium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.
Fed-batch Process B
The fed-Batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3.105 cells.mL−1 in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose and glutamine were added to keep the concentration above respectively 15 mM and 1 mM. Amino Acids and peptides were added from day 5 to replenish the consumed amino acids.
Process of the Invention C2
The process of the invention was performed in a 2 L Applikon vessel. A 50 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane (GE) operated in ATF flow mode with an ATF-2 system (Refine Technology) was used to retain the cells and the IgG product. The culture was started with 3×10e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM t-Glutamin VPRO culture medium (SAFC) supplemented with 6 mM L-Glutamin is perfused through the suspension cell culture using an SPR between 0.05 and 0.2 nL.cell−1.day−1. The CO2 pressure was controlled below 15%.
Result
As can be seen from FIG. 3 and from FIG. 4, the process according to the invention results in significantly to increased viable cell densities and increased product concentrations (2415%×Batch yield; 690%×Fed-Batch yield) in equal or less time (100% Batch time; 81% Fed-Batch time).
The overall productivity increase in g.L−1.day−1 of the process of the invention is 23.9 times the Batch productivity in g.L−1.day−1) and 8.5 times the Fed-batch productivity in g.L−1.day−1. In the process of the invention C2, 11.1 g product/L was produced. Clogging of the retention device did not occur during 17 days, even with very high cell density.
Example 3
Comparison between a Batch Process, a Fed Batch Process and the Process According to the Invention
In this example the performance of the process according to the present invention with cell culture removal and again compared to batch and fed-batch processes; in process C3 cell culture has been removed.
FIG. 5 shows the viable cell density Y (106.mL−1) plotted versus the process time X (days) for process A (batch), B (fed-batch) C3 (process of the invention).
FIG. 6 shows the IgG concentration in the reactor Z (% as compared to IgG concentrate in process A3) versus the process time X (days) for process A, and C3.
FIG. 7 shows the cumulative yield Q (% as compared to yield in process A, per L reactor volume) plotted versus the process time X (days) for process A, B and C3.
All fermentations were performed using a Sartorius Biostat B controller to control temperature at 36.5° C., pH between 7.2 and 6.8 and DO at 50% air saturation and at 200 rpm. The same IgG (of approximately 150 kDa) producing PER.C6 cell line (see WO 2004/099396) was used in all experiments.
Batch process A
The batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3.106 cells.mL−1 in VPRO medium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured for 17 days.
Fed-batch Process B
The fed-Batch process was executed at 4 L working volume in a Sartorius B5 vessel. Cells were inoculated at 3.106 cells.mL−1 in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. During the culture glucose and glutamine were added to keep the concentration above respectively 15 mM and 1 mM. Amino Acids and peptides were added from day 5 to replenish the consumed amino acids.
Process of the Invention C3
The process of the invention was performed in a 2 L Applikon vessel. A 100 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membrane (GE) operated in ATF flow mode with an ATF-2 system (Refine Technology) was used to retain the cells and the IgG product. The culture was started with 3×10e5 cells/mL in VPRO medium (SAFC) supplemented with 6 mM L-Glutamin. VPRO culture medium (SAFC) supplemented 6 mM t-Glutamin is perfused through the suspension cell culture using an SPR between 0.05 and 0.2 nL.cell−1.day−1. Cell culture is removed at 10% of the working volume per day above 10.106 cells.mL−1 and at 30% of the working volume per day when the viable cell density exceeds 30.106 cells.mL−1 and onwards.
Result
As can be the seen from FIG. 5 with the process of the invention higher viable cell densities are reached fast. Furthermore, FIG. 5 also shows that the viability of the cells can be maintained longer with the process of the invention as process C3 was maintained in operation over a period of nearly 40 days, because no dogging of the retention device occurred even with high cell densities.
FIG. 6 shows that product concentrations for the process of the present invention are much higher than the product concentration in the batch process. The product flow containing the product was harvested from process C3 at approximately 200% to 250% times the final concentration in the batch process A.
FIG. 7 shows that most product is formed by the process of the present invention and that the process of the inventions can be maintained longer than the batch process A or the fed-batch B. At day 17, the cumulative yield of process C3 is 8.1 times the cumulative yield of the batch process (A3) and 2.1 times the cumulative yield of the fed-batch process (B). Also, at day 17, the batch process ended. At day 21, the cumulative yield of process C3 is 3.0 times the cumulative yield of the fed-batch process B. At day 21, the fed-batch process ended. After 39 days, the overall cumulative yield of process C3 is 25 times the cumulative yield of the batch process A and 6 times the yield of the fed-batch process B.
It can be concluded from this experiment that the overall yield of a desired biological material in the process according to the present invention can be further improved by applying a bleed of the cell culture when the cell density exceeds a certain high level.
Example 4
Culturing of and Production with CHO Cells
In this example the process according to the present invention has been performed with an IgG producing CHO cell line and includes a temperature drop to decrease cell growth.
FIG. 8 shows the cell number Y (106.ml−1) plotted versus the process time X (days) the C4 (process of the invention).
FIG. 9 shows the IgG concentration in the reactor Z (% as compared to the maximum IgG concentration reached versus the process time X (days) for process C4 (one embodiment of process of the invention)
The fermentation was performed using a Sartorius Biostat B controller to control temperate at 36.5° C. pH between 7.1 and 6.9 and DO at 40% air saturation and at 100 mm. The temperature was dropped to 32° C. on day 5.
Process of the invention C4
The process of the invention was performed in a 2 L Applikon vessel. Cell and product retention device is a 50 kD Molecular Weight Cut-Off (MWCO) hollow fiber membrane (General Electric) operated in ATF low mode, with an ATF-2 system (Refine Technology). The culture was started with 5.106 cells mL−1 in MTCM-49 culture medium (Hyclone). The medium was perfused through the suspension cell culture using an SPR between 0.1 and 0.4 nL cell−1.day −1. The CO2 pressure was controlled below 15%.
Result
The data show that the process of the invention also works when using a protein producing CHO cell line. The achieved cell density and the product concentrations are increased compared to batch culture. The data also show that in the process according to the present invention cell growth can be arrested (e.g. by a temperature drop), whereas the product accumulation in the culture system continues.
Example 5
Process of the invention Performed with a Myeloma Cell Line
The process according to the present invention can also the applied to myeloma cell lines. To this end the fermentation is performed using a Sartorius Biostat B controller to control temperature at 36.5° C., pH between 7.2 and 6.8 and DO at 40% air saturation and at 100 rpm. The cell coloring starts with inoculating the myeloma cells at 3×10e5 cells/ml in SFM4Mab culture medium (Hyclone) in a 5 L Sartorius vessel. The cell and product retention device is a 30 kD Molecular Weight Cut-Off (MWCO) hollow fiber membrane (General Electric) operated in ATF flow mode with an ATF-4 system (Refine Technology). SFM4Mab culture medium (Hyclone) is perfused through the suspension cell culture using an SPR between 0.1 and 0.4 nL.cell-1.day-1. The CO2 pressure is controlled below 15%.
Example 6
Process of the Invention Performed with an MDCK Cell Line
The process according to the present invention can also the applied to transformed MDCK cell lines in suspension. To this end the fermentation is performed using a Sartorius Biostat B controller to control temperature at 36.5° C., pH between 7.2 and 6.8 and DO at 40% at saturation and at 100 rpm. The cell culturing starts with inoculating the transformed MDCK cells at 3×10e5 cells/ml in VF-SFM culture medium (Invitrogen) in a 5 L Sartorius vessel. The cell and product retention device is a 30 kD Molecular Weight Cut-Off (MWCO) hollow fiber membrane (General Electric) operated in ATF flow mode with an ATF-4 system (Refine Technology). VP-SFM culture medium (Invitrogen) is perfused through the suspension cell culture using an SPR between 0.1 and 0.4 nL.cell−1.day−1. The CO2 pressure is controlled below 15%.

## Claims

1. Process for the culturing of cells in a reactor in suspension in a cell culture medium, comprising:
circulating a cell culture through a separation system, wherein the cell culture comprises eukaryotic cells, a biological substance produced by the cells, and a cell culture medium,
wherein at least one cell culture medium component is fed to the cell culture;
wherein the cell culture is circulated through the separation system in a flow substantially parallel to a surface of said separation system resulting in a liquid outflow and a flow wherein cell culture contents are retained or fed back into the reactor;
wherein the separation system comprises a filter having a pore size characterized by a molecular weight cut-off smaller than the molecular weight of the biological substance to separate the biological substance from substances having a lower molecular weight than the biological substance and
wherein the liquid outflow consists essentially of components having molecular weight lower than that of the biological substance, and the flow wherein cell culture contents are retained or fed back into the reactor consists essentially of components having molecular weight equal to or higher than that of the biological substance.

2. Process according to claim 1, wherein the molecular weight cut-off is smaller by at least a factor 2 than the molecular weight of the desired biological substance.

3. Process according to claim 1, wherein the molecular weight cut-off is smaller by at least a factor 3 than the molecular weight of the desired biological substance.

4. Process according to claim 1, wherein part of the substances of lower molecular weight is continuously removed from the cell culture.

5. Process according to claim 1, wherein the eukaryotic cells are mammalian cells.

6. Process according to claim 5, wherein the mammalian cells are selected from the group consisting of Chinese Hamster Ovary (CHO) cells, hybridomas, Baby Hamster Kidney (BHK) cells, myeloma cells, human cells, and mouse cells.

7. Process according to claim 5, wherein the eukaryotic cells are E1-immortalized HER cells.

8. Process according to claim 5, wherein the eukaryotic cells are PER.C6 cells.

9. Process according to claim 1, wherein the biological substance is a recombinant protein.

10. Process according to claim 1, wherein the biological substance is an antibody.

11. Process according to claim 1, wherein the separation system is a membrane filter.

12. Process according to claim 1, wherein the separation system is a hollow fiber filter.

13. Process according to claim 1, wherein the cell culture is circulated over the filter in an alternating tangential flow.

14. Process according to claim 1, wherein the cell culture medium component is fed to the cell culture in the reactor.

15. Process for the culturing of cells in a reactor in suspension in a cell culture medium, comprising:
circulating a cell culture through a separation system, wherein the cell culture comprises eukaryotic cells, a biological substance produced by the cells, and a cell culture medium, wherein at least one cell culture medium component is fed to the cell culture;
wherein the cell culture is circulated through the separation system in a flow substantially parallel to a surface of said separation system resulting in a liquid outflow and a flow wherein cell culture contents are retained or fed back into the reactor;
wherein the separation system comprises a filter having a pore size characterized by a molecular weight cut-off smaller than the molecular weight of the biological substance to separate the biological substance from substances having a lower molecular weight than the biological substance and
wherein the liquid outflow consists essentially of components having molecular weight lower than that of the biological substance and the flow wherein cell culture contents are retained or fed back into the reactor consists essentially of components having molecular weight equal to or higher than that of the biological substance and
wherein the cell culture is removed at least once from the reactor.

16. Process according to claim 15, wherein liquid is added to the reactor to compensate for the cell culture removal.

17. Process according to claim 16, wherein the liquid is cell culture medium.

18. Process according to claim 1, wherein the biological substance is harvested from the cell culture.

19. Process according to claim 15, wherein the biological substance is harvested from the cell culture that is removed at least once from the reactor.