Improved process for the culturing of cells

(56) References cited:

WO-A-2004/099396 WO-A-2005/095578

US-A- 4 806 484 US-A- 5 286 646 • JAMES M. PÍRÉT ETAL: Mammalian cell and protein distributions in ultrafiltration hollowfiber bioreactors, BIOTECHNOLOGY AND BIOENGINEERING, vol. 36, no. 9, 1 November 1990 (1990-11-01), pages 902-910, XP055039117, ISSN: 0006-3592, DÓI: 10.1002/bit.260360905 • FALKENBERG FW ETAL: In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermenter, JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 179, no. 1, 13 February 1995 (1995-02-13), pages 13-29, XP004021096, ISSN: 0022-1759, DÓI:

10.1016/0022-1759(94)00266-Y

ΕΡ 2 634 242 Β1

Note: Within nine months ofthe publication ofthe mention ofthe grant ofthe European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall nőt be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).

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ΕΡ 2 634 242 Β1

Description [0001] The invention relates to a process for the culturing of cells in a reactor in suspension in a cell culture médium. [0002] 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 matéria! can be improved by optimizing the growth conditions in a fedbatch process.

[0003] Furthermore, WO05/095578 discloses a process for the culturing of cells by continuous perfusion culturing of a cell culture comprising cell culture médium and cells, wherein cell culture médium is added to the cell culture, the cell culture is circulated over a filter modulé comprising hollow fibers resulting in an outflow of liquid having a lower cell density than the cell culture, and the flow within the filter modulé is an alternating tangential flow, wherein the cells produce a biological substance. In the examples 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.

[0004] 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 nőt 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 cell 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.

[0005] Therefore, it is the object of the invention to provide a process wherein the product is obtained from the cell culture in higher concentrations.

[0006] This object is achieved by a process for the culturing of eukaryotic cells in a reactor in suspension in a cell culture médium, wherein the cells produce a biological substance, selected from proteins and vaccines, which can be used as an active ingredient in a pharmaceutical preparation, wherein at least one cell culture médium component is fed to the cell culture and wherein the cell culture comprising the cells, the desired biological substance and cell culture is circulated over a filter using a tangential flow and wherein the filter has a poré size characterized by a molecular weight cut-off smaller than the molecular weight ofthe desired biological substance to separates the desired biological substance from substances having a lower molecular weight than the desired biological substance, wherein the liquid outflow from the filter essentially only contains components having a molecular weight lower than that of the desired biological substance and wherein the desired biological substance is retained in orfed back intő the reactor.

[0007] For example, the invention relates to a process for the culturing of cells in a reactor in a cell culture médium, wherein the cells produce a biological substance, wherein nutrients and/or cell culture médium is/are fed to the reactor and wherein the cell culture comprising the cells and the cell culture médium is circulated over a filter having a poré size or molecular weight cut off of between 5 and 500kD.

[0008] 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.

[0009] Hence, the present invention differs form the cell culturing described in the prior art in that it allows for accumulation ofthe desired biological matéria! together with the cell mass.

[0010] In a preferred embodiment of the present invention part of the substances of lower molecular weight are continuously removed from the cell culture.

[0011] An additional advantage ofthe process ofthe present invention is that higherviable cell concentration can be reached as compared to for example batch orfed-batch processes. Furthermore, the production time - the period during which the cells produce the biological substance - can be extended compared to for example batch orfed-batch processes. Alsó, as compared to a batch or fed-batch process, it is possible to use a smaller reactor. Use of smaller reactors is of advantage as this reduces the equipment and facility related investments.

[0012] Alsó, higher concentrations of the biological substance may be obtained in shorter times.

[0013] Itwas found that itwas 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 ofthe biological substance by the biological substance itself or inhibition by other macromolecules produced by the cell (such as for instance hőst cell proteins, enzymes or celiuiar debris), would occur. Furthermore, itwas found that the accumulation ofthe desired biological matéria! does nőt impair the function of the separation system.

[0014] 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 resuit the present process results in an improved production ofthe desired biological matéria!. [0015] 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

ΕΡ 2 634 242 Β1 fungi, fór example Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum, yeasts, fór example Saccharomyces cerevisiae, Kluyveromyces lactis, Phaffia rhodozyma, yeast from the genus Pichia, fór example Pichia pastoris or prokaryotic, fór instance Escherichia coli, Bacillus sp, fór example B. licheniformis, B. subtilis, B. amyloliquefaciens, B. alkalophilus, Streptomyces sp., Corynebacterium glutamicum, Pseudomonas sp. Examples of eukaryotic cells are fór example alsó deseribed in Chu, L., Robinson, D. K., (2001) Curr. Opinion Biotechn., vol. 12, p. 180-187. Preferabiy, the cells that are used in the process ofthe present invention are animai cells, in particular mammalian cells. Examples of mammalian cells include CHO (Chinese Hamster Ovary) cells, hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, humán cells, fór example HEK-293 cells, humán lymphoblastoid cells, E1 immortalized HER cells, mouse cells, fór example NSO cells. More preferabiy, E1 immortalized HER cells are used, most preferabiy PER.C6 cells.

[0016] Primary humán embryonic retina (HER) cells can be isolated from fetuses (Byrd P, Brown KW, Gallimore PH. 1982. Malignant transformation of humán embryo retinoblasts by cloned adenovirus 12 DNA. Natúré 298: 69-71, Byrd PJ, Grand RJA, Gallimore PH. 1988. Differential transformation of primary humán embryo retinái cells by adenovirus E1 regions and combinations of E1A + ras. Oncogene 2: 477-484). Primary cells will die upon culturing fór several passages. E1-immortalized HER cells tor the purpose of the present invention are derived from primary HER cells by expressing DNA encoding adenoviral E1A and E1 B proteins therein, to obtain immortalized cells. Such immortalized cells can be cultured tor more than 100 passages. Methods to obtain E1-immortalized HER cells have tor instance been deseribed in US patent5,994,128, in Byrd P, Brown KW, Gallimore PH. 1982. Malignant transformation of humán embryo retinoblasts by cloned adenovirus 12 DNA. Natúré 298: 69-71, in Byrd PJ, Grand RJA, Gallimore PH. 1988. Differential transformation of primary humán embryo retinái cells by adenovirus E1 regions and combinations of Ε1A + ras. Oncogene 2: 477-484, and in Gallimore, P.H., Grand, R.J .A. and Byrd, P.J. (1986). Transformation of humán embryo retinoblasts with simian vírus 40, adenovirus and ras oncogenes. AntiCancer Rés. 6, p499-508. Fór instance, immortalized HER cells, including PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9 cells, were generated by transfection of primary HER cells using a plasmid that contained the adenovirus serotype 5 (Ad5) E1 A- and E1 B-coding sequences (Ad5 nucleotides 459-3510) under the control ofthe humán phosphoglycerate kinase (PGK) promoter (see US patent 5,994,128).

[0017] In a preferred embodiment, the cells in the process ofthe present invention are E1-immortalized HER cells, more preferabiy PER.C6 cells (see U.S. Patent 5,994,128). PER.C6 cells are exemplified by cells as deposited under ECACC No. 96022940 (see, e.g., U.S. Patent 5,994,128, EP 0833934 B1).

[0018] In the process ofthe invention, the cells may be cultured in suspension in any form, fór example as immobilized cells, single cells or in cell clusters or as a combination thereof. Preferabiy the cells are cultured as single cells and/or as small cell clusters of nőt more than 100 cells, more preferabiy of nőt more than 20 cells. Cells can fór example be immobilized on microcarriers such as are commercially available from fór example GE Healthcare (Cytodex).

[0019] 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 médium. It preferabiy provides sterilé barriers, such as air filters, to prevent other cells from contaminating the desired cells and it preferabiy maintains a favorable environment fór the cells by providing the right culture conditions such as mixing, temperature, pH, oxygen concentration etc.

[0020] The reactor can fór example be of a more permanent natúré, fór example the reactor can be of stainless steei or glass or can fór example be of a disposable natúré, fór example the reactor can be a plastic flask or bag. Examples of reactors suitable fór use in the present invention include, bút are nőt limited to stirred tank vessels, airlift vessels and disposable bags that can be mixed by rocking , shaking motion or stirring. Preferabiy 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 fór example Hyclone, Sartorius, Applikon orWave.

[0021] The term 'separation system’ is defined within theframework ofthe invention as a system capable of separating on basis of molecular weight. The separation system used in the process ofthe 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 preferabiy at least a factor 2, most preferabiy at least a factor 3 smaller than the molecular weight of the biological substance. Typically, bút of course depending on the molecular weight of the biological substance produced in the process of the present invention, the MWCO ofthe separation system is preferabiy at least 5, more preferabiy at least 10, most preferabiy at least 30kDa and preferabiy at most 500kDa, more preferabiy at most 300kDa, most preferabiy at most 100kDa. Fór example fór an IgG with a molecular weight of 150kDa, a separation system having a MWCO of at most 50kDa is most preferred.

[0022] Examples of separation systems include bút are nőt limited to filters, centrifuges and aqueous two phase extraction systems.

[0023] The term 'filter’ 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 ofthe present invention, any filter may be used as long as the poré

ΕΡ 2 634 242 Β1 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 poré size or MWCO of between 5 and 500kDa. Examples of filters suitable fór use in the present invention include membráné filters, ceramic filters and metál filters. The filter may be used in any shape; the filter may fór example be spirál 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 membráné filter, preferably a hollow fiber filter. With the term hollow fiber is meant a tubular membráné. The internál diameter of the tűbe is at least 0.1 mm, more preferably at least 0.5 mm, most preferably at least 0.75 mm and preferably the internál diameter of the tűbe 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 fór example General Electric (GE, formerly Amersham).

[0024] By circulating the cell culture comprising the biological substance, cells and the cell culture médium 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 ofthe invention, the liquid outflow does nőt 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 ofthe biological substance. Essentially all cells and essentially all biological substance are therefore usually retained in the reactor.

[0025] The poré size or MWCO of the filter is chosen such that the size of the pores or MWCO of the filter is smaller than, preferably at least a factor 2, more preferably at least a factor 3 smaller than the diameter or molecular weight of the product, ensuring a high retention of product. Typically, bút of course depending on the size or molecular weight of the product, i.e. biological substance produced in the process ofthe present invention, the poré size or MWCO ofthe filter is preferably at least 5, more preferably at least 10, most preferably at least 30kDa and/or the poré size or MWCO ofthe filter/membrane is preferably at most 500kDa, more preferably at most 300kDa, most preferably at most 100kDa. [0026] With molecular weight cut-off (MWCO) is meant the molecular weight above which at least 90% ofthe particles is retained by the separation system.

[0027] Circulating the cell culture over a separation system, fór example a filter means that the cell culture is passed through a separation system, fór example a filter resulting in a liquid outflow and a flow which contents are kept in or fed back intő the reactor. The flow which contents are kept in orfed back intő the reactor will usually essentially only contain components having a molecularweightofat least equal to that ofthe biological substance or higher and therefore said flow will comprise more biological substance than the liquid outflow.

[0028] In principle, it is nőt critical when the circulation ofthe cell culture over the separation system is started during the process of the invention. The circulation of the cell culture may fór example be started directly from the start of the process or when the viable cell density ofthe cells has reached a certain level.

[0029] The circulation of the cell culture over a filter may be a flow substantially perpendicular with respect to the filter surface, alsó known as dead-end flow or a flow substantially parallel to the filter surface, alsó known as tangential flow, fór 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 nőt occur (quickly) even at very high cell densities. It is common generál knowledge that in depth filtration, the final small poré filter needs to be protected from clogging by course pre-filters. This practice is based on the common generál 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 ofa pre-filter becomes superfluous.

[0030] The flow may be directed by moving the cell culture, by moving the filter or both. The filter may fór example be moved by rotation (rotating filter) orvibration (vibrating filter). Alternatively ifthe flow is directed by moving the cell culture only, the filter is static and the cell culture may fór example be moved by way of pumps or pressure.

[0031] With alternating tangential flow is meant that there is one flow in the same direction as (i.e. tangential to) the filtersurface(s), which flow is going back and forth, and that there is anotherflow in a direction substantially perpendicular to said filter surface. Alternating tangential flow can be achieved according to methods known to the person skilled in the art (fór example as described in US 6,544,424).

[0032] During the culturing ofthe cells, at least one cell culture médium component, fór example one or more nutrients and/or cell culture médium 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. Fór example, complete cell culture médium may be fed to the reactor, which is of advantage as a separate feed need then nőt be prepared separately. The cell culture médium may fór example alsó be fed to the cells in a more concentrated form; this is of advantage as smaller volumes are easier to handle. Alsó one or more nutrients may be fed to the reactor. Fór example carbohydrates, fór instance glucose or fructose; amino acids, such as glutamine and/or peptides may advantageously be fed to the reactor.

[0033] In a preferred embodiment ofthe invention, cell culture conditions are chosen such that cell growth rate and/or specific productivity of the cells is nőt limited and more preferably such that the concentration of at least one of the components ofthe cell culture médium remains essentially constant. Examples of limiting cell culture conditions are nutrient limitations and formation of inhibiting metabolites, such as ammónia, carbon dioxide and lactate. Fór example,

ΕΡ 2 634 242 Β1 cell culture conditions such as the feed may be chosen such that cell growth rate is nőt limited fór example by supplying enough nutrients as to compensate fór the depletion and/or to avoid the production of inhibiting metabolites such as lactate or ammónia. Fór example, the aeration conditions may be chosen such that carbon dioxide formation is nőt limiting the cell growth rate. Growing the cell under non-limiting conditions is highly advantageous from a Good Manufacturing Practice (GMP) point of view as 1) this may give a constant cell culture envíronment that in many cases alsó gives constant and good product quality and 2) this may lead to high cell viability, in somé cases to a cell viability of more than 98%. High cell viability reduces the release of cell related contaminants, such as hőst 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 in an even shorter time as higher cell density will be reached earlier in the process.

[0034] 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.celHday¹.

[0035] The rate of addition of at least one cell culture médium component, fór example nutrients and/or cell culture médium to the cell culture (the inflow rate or perfusion rate) influences the viability and the density ofthe cells. In the process ofthe invention, the cell culture médium component(s), such as nutrients and/or cell culture médium may be fed fór example in a continuous flow, semi-continuous flow, fór example step-wise flow or staggered flow. Preferably, cell culture médium component(s), fór example nutrients and/or cell culture médium are added in a continuous flow. [0036] Cell culture médium component(s), such as complete cell culture médium 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 ofthe cells to cease or before inhibitory metabolites, fór example lactate or, ammónia reach such high levels that growth would cease. From this point onward, the cell culture médium component(s), such as nutrients and/or complete cell culture médium are preferably fed to the reactor at a rate such that substrate demand is met.

[0037] In one embodiment ofthe invention, cell culture médium is added at a Feed Rate according to formula (1):

Feed Rate = SFR x (totál cell culture volume) x (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 médium is fed to the cell culture expressed as the volume of médium 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, fór 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.

[0038] It may be of advantage to take intő account additional parameters when adjusting the feed rate, fór example the amount of glucose to be fed to the culture and/or the oxygen uptake rate. Fór example, fór PER.C6 the feed rate of the cell culture médium 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.

[0039] In a special embodiment of the invention cell culture (comprising cells, biological substance and cell culture médium) is removed at least once from the reactor and liquid, fór example cell culture médium óra nutrientfeed is added to the reactor to compensate fór 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 culture may be removed continuously or step-wise.

[0040] In a preferred embodiment ofthe invention, cell culture (comprising cells, cell culture médium and biological substance) is removed from the reactor as soon as the desired cell density, fór example a cell density of at least 10.10⁶viable cells/ml, preferably ofat least 20.10⁶ viable cells/ml, more preferably ofat least 30.10⁶ viable cells/ml, fór example a cell density of at most 200. 10⁶ viable cells/ml, is reached and liquid, fór example cell culture médium or nutrient feed is added to the reactor to compensate fór the cell culture removal. Preferably, cell culture is removed at such rate that the cell density remains in the desired cell density rangé. This embodiment of the invention is highly advantageous as compared to a conventional batch orfed-batch process as it combines the advantages ofthe process ofthe invention with high viability that can be maintained longer, making it possible torealizean even higheroverall 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'íday'¹. As compared to a conventional perfusion process, this embodiment of the invention is alsó highly advantageous as it combines the advantages ofthe process ofthe invention with a cell culture removal stream having a high 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 nőt contain enough

ΕΡ 2 634 242 Β1 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.

[0041] In a particuíaríy preferred embodiment ofthe invention, cell culture conditions are chosen such that the cell growth rate and/or specific productivity of the cells is nőt limited and more preferably such that alsó the concentration of at least one of the components of the cell culture médium, 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, fór example cell culture médium is added to the reactor to compensate fór the cell culture removal.

[0042] 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 médium component, fór example nutrients and/or cell culture médium minus the rate ofthe optional cell culture removal.

[0043] Cells that produce a biological substance are fór instance cells capable of expressing a gene encoding the biological substance. Cells capable of expressing a gene encoding the biological substance may fór example be prepared by transfection ofthe cells with a plasmid containing the gene encoding the biological substance and gene encoding a suitable selection marker, fór example a gene encoding a neomycine resistance (Neo marker gene). Stably transfected cells may then be selected by selection pressure, fór example - in the case of a Neo marker gene - by culturing the transfected cells in the presence of G418 (genericin) and immediate screening of the cells fór cells exhibiting high-level expression ofthe biological substance. Methods fór preparing clones of E1-immortalized HER cells expressing a protein, and methods fór culturing such cells to produce the protein, are well known to the skilled person, and can fór instance be found in US 6,855,544.

[0044] Biological substances, which may be produced by the cells, fór example by expressing a (recombinant) gene coding therefore are fór example (recombinant) proteins, in particular receptors, enzymes, fusion proteins, blood proteins such as proteins from the blood coagulation Cascade, multifunctional proteins such asfor instance erythropoietin, vírus or bacterial proteins fór instance fór use in vaccines; immunoglobulins such as antibodies, fór 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 ofthe present invention, the terms ’product’ and 'biological substance’ are interchangeable.

[0045] Within the framework ofthe 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 fór example be used fór diagnosis, orfor prophylactic purpose such asfor instance a vaccine, and/orfortherapeutic purpose, such asfor instance an enzyme or protein fór 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 ofwhich are well known to the person skilled in the art.

[0046] The PER.C6 cell line can be used fór production of biological substances, such as E1-deleted adenovirus (see e.g. US patent 6,994,128; Nichols et al, 2002, Propagation of adenoviral vectors: use of PER.C6 cells. In: Curiel D, Douglas JT, editors. Adenoviral vectors fór gene therapy. San Diego: Elsevier. p 129-167), other 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 forthe manufacture of biopharmaceutical proteins, Modern Biopharmaceuticals: Design, Development and Optimization, 4 Volumes, 779-807, Jörg Knáblein (Editor)).

[0047] Examples of proteins that can be used as an active ingredient in pharmaceutical preparations (with the brand name between brackets) includeTenecteplase (TN Kasé™), (recombinant) antihemophilicfactor(ReFacto™), lymphoblastoid Interferon a-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-la (Avonex™).

[0048] Examples of vaccines that can be used as an active ingredient in pharmaceutical preparation include isolated protein antigens, examples ofwhich include bút are nőt limited to live, órai, tetravalent Rotavirus vaccine (RotaShield™), rabies vaccine (RanAvert™), influenza vaccines and inactivated hepatitis A vaccine (VAQTA™).

[0049] The pH, temperature, dissolved oxygen concentration and osmolarity ofthe cell culture médium are in principle nőt critical and depend on the type ofcell chosen. Preferably, the pH, temperature, dissolved oxygen concentration and osmolarity are chosen such that it is optimál fór the growth and productivity ofthe cells. The person skilled in the art knows how to find the optimál pH, temperature, dissolved oxygen concentration and osmolarity forthe culture (see. e.g. WO 2004/099396). Preferably, forthe process ofthe 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 400mOsm/kg. To maintain optimál process conditions automation to control the process conditions is desired. In order to optimize process conditions, fór instance to obtain growth árrést fór increased cellular productivity, during the culture a shift in the culture conditions can be applied. This may be established by fór instance a temperature shift (such as from 37 to 32 °C), a pH shift or an osmolarity shift.

[0050] The process of the present invention can in principle be performed in any type of cell culture médium suitable

ΕΡ 2 634 242 Β1 for the culturing of cells. Guidelines for choosing a cell culture médium 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, John Wiley & Sons.

[0051] For example the cell culture médium may for example comprise as a cell culture médium 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 MgCÍ2.6H₂O, 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 include all known proteinogenic amino acids, for example hystidine, glutamine, threonine, serine, methionine. Examples of vitamins include: ascorbate, biotin, choline.CI, 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 médium may for example alsó comprise other cell culture médium components, for example soy peptone or ethanol amine.

[0052] For production of biological substances according to the invention, in partieuiar ifthe biological substances are to be used as an active ingredient in pharmaceutical preparations, serum free média are preferred to média containing a serum source. The reason for this is that serum source média may be contaminated with viruses, present the risk of prionic infections, and can create a major obstaele in the downstream processing of the biopharmaceutical product (i.e. the further purification ofthe biological substance from the cell culture). Therefore the process ofthe invention is preferably performed in a cell culture médium that does nőt comprise serum from an animal, including humán, source. Since compounds from a mammalian source alsó present an infection risk, preferably the cell culture médium is mammalian source free (i.e. the cell culture médium does nőt comprise serum or components from a mammalian source). More preferably the cell culture médium is animal source free (i.e. the cell culture médium does nőt comprise serum or components from an animal, including humán, source. Examples of serum free média that can be used for the culturing of PER.C6 cells include commercially available média, such as for instance EX Cell™ VPRO médium (SAFC), HyQ® CDM4Retino™ (HyCIone), IS ProVec CD (Irvine scientific), 293-SFM II (invitrogen).

[0053] In preferred embodiments, the biological substance produced in the process ofthe present invention is harvested from the flow which contents are kept in or preferably fed back intő the reactor or from the cell culture that is removed from the reactororfrom both. The biological substance(s) produced in the process ofthe 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 ofthe biological substance (e.g. by ultrafiltration or diafiltration), steps to exchange buffers and/or steps to remove or inactivate viruses (e.g. by vírus filtration, pH shift or solvent detergent treatment).

[0054] 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 partieuiar at least 90.10⁶ cells/mL and a concentration of biological substance of at least 5 g/L, more preferably at least 10g/L, in partieuiar 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 nőt more than

200.10⁶ cells/mL and preferably within the rangé of 80-150.10⁶ cells/mL.

[0055] 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).

[0056] With cell culture is meantthe liquid comprising cell culture médium, cells and biological substance, which liquid is the result of a process for the culturing of cells in a reactor in a cell culture médium, wherein the cells produee the biological substance.

[0057] The invention will now be elucidated by way of the following examples without however being limited thereto.

Description ofthe figures [0058]

Fig. 1/9. shows the viable cell density Y (1O⁶.ml^_1) plotted versus the process time X (days) for process A (batch), B (fed-batch) and C1 (process ofthe invention).

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Fig. 2/9. 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 ofthe invention).

Fig. 3/9. shows the viable cell density Y (1O⁶.ml¹) plotted versus the process time X (days) fór process A (batch), B (fed-batch) C2 (process ofthe invention).

Fig. 4/9. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A) versus the process time X (days) fór process A (batch), B (fed-batch) and C2 (process ofthe invention).

Fig. 5/9. shows the viable cell density Y (1O⁶.ml^_1) plotted versus the process time X (days) fór process A (batch), B (fed-batch) C3 (process ofthe invention).

Fig. 6/9. shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A3) versus the process time X (days) fór process A, and C3.

Fig. 7/9. shows the cumulative yield Q (% as compared to yield in process A, per L reactor volume) plotted versus the process time X (days) fór process A, B and C3.

Fig. 8/9. shows the cell number Y (1O⁶.ml^_1) plotted versus the process time X (days) fór C4 (process ofthe invention).

Fig. 9/9. shows the IgG concentration in the reactor Z (% as compared to the maximum IgG concentration reached versus the process time X (days) fór process C4 (one embodiment of process of the invention)

Examples

Example 1: Comparison between a batch process, a fed batch process and the process according to the invention.

[0059] In this example the performance ofthe process according to the present invention was compared to batch and fed-batch processes.

[0060] Fig. 1/9 shows the viable cell density Y(10⁶.ml^_1) plotted versus the process time X (days) fór process A (batch), B (fed-batch) and C1 (process ofthe invention).

[0061] Fig. 2/9 shows the IgG concentration in the reactorZ (% as compared to IgG concentration in process A) versus the process time X (days)for process A (batch), B (fed-batch) and C1 (process ofthe invention).

[0062] 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 [0063] The batch process was executed at 4 L working volume in a Sartorius B5 véssél. Cells were inoculated at 3x10e5 cells/mL in VPRO médium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured fór 17 days.

Fed-Batch process B [0064] The fed-batch process was executed at 4 L working volume in a Sartorius B5 véssél. Cells were inoculated at 3x10e5cells/mL in VPRO médium (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 ofthe invention C1 [0065] The process of the invention was performed in a 2 L Applikon véssél. A 100 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membráné 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 3x10e5 cells/mL in VPRO médium (SAFC) supplemented with 6 mM L-Glutamin. VPRO culture médium (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 1.4g/L.

[0066] The process ofthe 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.

[0067] 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 50kDa separation system was used.

[0068] Fig. 3/9 shows the viable cell density Y (10⁶.ml^_1) plotted versus the process time X (days) fór process A (batch),

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Β (fed-batch) C2 (process ofthe invention).

[0069] Fig. 4/9 shows the IgG concentration in the reactorZ (% as compared to IgG concentration in process A) versus the process time X (days) fór process A (batch), B (fed-batch) and C2 (process ofthe invention) [0070] All fermentations were performed using a Sartorius Biostat B controller to controi temperature at 36.5 °C, pH between 7.2 and 6.8 and DO at50% airsaturation and at 200 rpm. The same IgG (of approximately 150kDa) producing PER.C6 cell line (see WO 2004/099396) was used in all experiments.

Batch process A [0071] The batch process was executed at 4 L working volume in a Sartorius B5 véssél. Cells were inoculated at 3.10⁵cells.mL^-1 in VPRO médium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured fór 17 days.

Fed-Batch process B [0072] The fed-Batch process was executed at 4 L working volume in a Sartorius B5 véssél. Cells were inoculated at 3.10⁵ cells.mL^-1 in VPRO médium (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 ofthe invention C2 [0073] The process of the invention was performed in a 2 L Applikon véssél. A 50 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membráné (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 3x10e5 cells/mL in VPRO médium (SAFC) supplemented with 6 mM L-Glutamin. VPRO culture médium (SAFC) supplemented with 6 mM L-Glutamin is perfused through the suspension cell culture using an SPR between 0.05 and 0.2 nL.cell¹ .day^-1. The CO₂ pressure was controlled below 15%.

Resuít [0074] As can be seen from Fig. 3/9 and from Fig 4/9 ,the process according to the invention results in significantly increased viable cell densities and increased product concentrations (2415% x Batch yield; 690% x Fed-Batch yield) in equal or less time (100% Batch time; 81% Fed-Batch time).

[0075] The overall productivity increase in g.L^-1 .day^-1 ofthe process ofthe 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 ofthe invention C2,11.1 g product/L was produced. Clogging of the retention device did nőt 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.

[0076] 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.

[0077] Fig. 5/9 shows the viable cell density Y (10⁶.ml^_1) plotted versus the process time X (days) fór process A (batch), B (fed-batch) C3 (process ofthe invention).

[0078] Fig. 6/9 shows the IgG concentration in the reactor Z (% as compared to IgG concentration in process A3) versus the process time X (days) fór process A, and C3.

[0079] Fig. 7/9 shows the cumulative yield Q (% as compared to yield in process A, per L reactor volume) plotted versus the process time X (days) fór process A, B and C3.

[0080] All fermentations were performed using a Sartorius Biostat B controller to controi temperature at 36.5 °C, pH between 7.2 and 6.8 and DO at50% airsaturation and at 200 rpm. The same IgG (of approximately 150kDa) producing PER.C6 cell line (see WO 2004/099396) was used in all experiments.

Batch process A [0081] The batch process was executed at 4 L working volume in a Sartorius B5 véssél. Cells were inoculated at 3.10⁵cells.mL^-1 in VPRO médium (SAFC) supplemented with 6 mM L-glutamin and subsequently cultured fór 17 days.

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Fed-Batch process Β [0082] The fed-Batch process was executed at 4 L working volume in a Sartorius B5 véssél. Cells were inoculated at 3.10⁵ cells.mL^-1 in VPRO médium (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 ofthe invention C3 [0083] The process of the invention was performed in a 2 L Applikon véssél. A 100 kDa Molecular Weight Cut-Off (MWCO) hollow fiber membráné (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 3x10e5 cells/mL in VPRO médium (SAFC) supplemented with 6 mM L-Glutamin. VPRO culture médium (SAFC) supplemented 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. Cell culture is removed at 10% ofthe working volume per day above 10.10⁶ cells. mL^-1 and at 30 % ofthe working volume per day when the viable cell density exceeds

30.10⁶ cells.mL^-1 and onwards.

Result [0084] As can be seen from Fig. 5/9 with the process ofthe invention higher viable cell densities are reached fást. Furthermore, Fig. 5/9 alsó shows that the viability ofthe cells can be maintained longer with the process ofthe invention as process C3 was maintained in operation over a period of nearly 40 days, because no clogging ofthe retention device occurred even with high cell densities.

[0085] Fig. 6/9 shows that product concentrations fór 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.

[0086] Fig. 7/9 shows that most product is formed by the process ofthe present invention and that the process ofthe invention 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 fedbatch process (B). Alsó, 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 fedbatch process B.

[0087] It can be concluded from this experiment that the overall yield of a desired biological matériái in the process according to the present invention can be further improved by applying a bleed ofthe cell culture when the cell density exceeds a certain high level.

Example 4: Culturing of and production with CHO cells.

[0088] 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.

[0089] Fig. 8/9 shows the cell number Y (1O⁶.ml^_1) plotted versus the process time X (days) fór C4 (process of the invention).

[0090] Fig. 9/9 shows the IgG concentration in the reactor Z (% as compared to the maximum IgG concentration reached versus the process time X (days) fór process C4 (one embodiment of process of the invention) [0091] The fermentation was performed using a Sartorius Biostat B controller to control temperature at 36.5°C, pH between 7.1 and 6.9 and DO at 40% air saturation and at 100 rpm. The temperature was dropped to 32°C on day 5.

Process ofthe invention C4 [0092] The process ofthe invention was performed in a 2 L Applikon véssél. Cell and product retention device is a 50 kD Molecular Weight Cut-Off (MWCO) hollow fiber membráné (General Electric) operated in ATF flow mode with an ATF-2 system (Refine Technology). The culture was started with 5.10⁶ cells.mL^-1 in MTCM-49 culture médium (Hyclone). The médium 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%.

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Result [0093] The data show that the process of the invention alsó 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 alsó 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 ofthe invention performed with a myeloma cell line.

[0094] The process according to the present invention can alsó be 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 culturing starts with inoculating the myeloma cells at 3x10e5 cells/ml in SFM4Mab culture médium (Hyclone) in a 5 L Sartorius véssél. The cell and product retention device is a 30 kD Molecular Weight Cut-Off (MWCO) hollow fiber membráné (General Electric) operated in ATF flow mode with an ATF-4 system (Refine Technology). SFM4Mab culture médium (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 ofthe invention performed with an MDCK cell line.

[0095] The process according to the present invention can alsó be 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% air saturation and at 100 rpm. The cell culturing starts with inoculating the transformed MDCK cells at 3x10e5 cells/ml in VP-SFM culture médium (Invitrogen) in a 5 L Sartorius véssél. The cell and product retention device is a 30 kD Molecular Weight Cut-Off (MWCO) hollow fiber membráné (General Electric) operated in ATF flow mode with an ATF-4 system (Refine Technology). VP-SFM culture médium (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%.