MX2012014894A - Compartmentalized continuous micro-reactors for the production of biopharmaceuticals in ovary cells of chinese hamster. - Google Patents

Abstract

The present invention refers to a group of micro-devices that a) have a plurality of cylindrical-shaped compartments obstructed by a central structure (toroidal or doughnut-type compartments); b) have a texture in the surface of the channel that promotes the cellular adherence and the retention or proliferation of the recombinant cells of the mammal, particularly but not exclusively, ovary cells of Chinese hamster; c) are fed with a culture medium in a continuous manner by some type of pumping device. This plurality of micro-devices may be used in screening applications for culturing recombinant cells of mammals, particularly but not exclusively CHO cells, or for the production of biopharmaceuticals or other recombinant proteins.

Description

MICRO-REACTORS CONTINUOS COMPARTAMENTALIZADOS FOR THE PRODUCTION OF BIOPHARMACY IN OVARIAN CELLS OF CHINESE HAMSTER TECHNICAL FIELD The present invention corresponds to the field of biotechnology applied to the production of biopharmaceuticals for animals and humans. In particular, this invention relates to the design of continuous micro-reactors for the production of biopharmaceuticals in mammalian cells, particularly in Chinese hamster ovary cells (CHO cells).

OBJECT OF THE INVENTION An object of the present invention is a set of microreactors that share in their design the characteristics of (a) possessing a set of micro-compartments in series, each conformed by a partially cylindrical clogged space (toroidal shape); (b) having a texture on the surface (s) of the channel that promotes cell adhesion and the retention or proliferation of recombinant Chinese hamster ovary cells; (c) being fed with culture medium continuously by some pumping device.

BACKGROUND The market for biopharmaceutical products, particularly therapeutic recombinant proteins for humans (e.g., erythropoietin, monoclonal antibodies, insulin, clotting factors, etc.) amounts to billions of dollars worldwide. For example, only erythropoietin and its analogs report a total worldwide sales exceeding 13 billion dollars in 201 1. Many of these biopharmaceuticals are glycoproteins. Therefore, in order to exert a suitable biological activity, these recombinant proteins must be expressed in an organism capable of glycosylation, for example a mammalian cell or a yeast. Chinese hamster ovary cells (CHO cells) are the host par excellence for the expression of glycosylated proteins. The conventional CHO cell culture processes, both at the laboratory and commercial level, are suspension cultures. The cells are kept suspended in a culture bottle, in a flask, or in a stirred tank, by the action of some mechanism of mechanical agitation. Typically, the culture medium is fed only once at the start of the culture (batch or batch culture) or several times during the course of the culture period (batch-fed or fed-batch culture). Some commercial processes of production of biopharmaceuticals in stirred tanks use culture methods by perfusion. In perfusion systems, free or particle-adhered cells (called micro-carriers) are retained in the reactor (by some physical retention method or recovery strategy and cellular recirculation) while the fresh culture medium is continuously fed and the partially or totally exhausted medium is withdrawn to the same volumetric flow. In this way, (a) the nutrients are continuously renewed in the culture, and (b) the metabolites, products, and waste compounds of the cells are continuously removed. These two factors maintain an appropriate atmosphere in the reactor for extended times. These improved conditions make it possible to maintain the high cell density in the tank promoted by the retention mechanism. The object of this invention is a plurality of micro-devices that, due to their design and operation characteristics, make possible the anchoring and retention of CHO cells in a series of micro-compartments continuously fed with culture medium. In these perfusion systems, it is possible to achieve culture densities superior to those reported for perfusion cultures in stirred tanks. Consequently, the ability to house, retain and maintain more cells per unit volume of the reactor, translates into the observance of higher productivities (in terms of mass or molar units of biopharmaceutical per unit volume per unit of time) in the micro- continuous devices described here.

BRIEF DESCRIPTION OF THE FIGURES Figure 1-A. Illustrative scheme of the operation principle of the continuous culture micro-devices object of the invention. Recombinant CHO cells (dark gray circles), capable of expressing the biopharmaceutical Y, are exposed to a continuous flow of culture medium (recirculating medium or fed in a single pass). The cells remain attached to the surface of the microcompartments of the device and continuously release the bio-drug of interest (Y's). The surfaces of each micro-compartment have been textured by carving cavities of 5 to 25 μp in them? in diameter, preferably 15 μ. The surface of the channel of the device has been coated with some compound or material that favors cell adhesion (circles light gray) illustrative but not limiting fibronectin, adhesion factor, or collagen, among others.

Figure 1-B. Preferential configuration of micro-compartment devices in series. In general, each device must have more than one cylindrical compartment clogged (toroidal compartment).

Figure 1-C. Geometrical details of a toroidal compartment.

Figure 1-D. Image of a device built in PMMA. In this particular case, a device with a series of 8 toroidal compartments in series has been manufactured in a block of PMMA. Tygon® hoses are arranged at the entrance and exit of each micro-channel to inject and remove culture medium.

Figure 1-E. Illustrative scheme that exemplifies a strategy of feeding culture medium to the micro-channels of the device. A syringe pump (Harvard 33 twin syringe pump) from Harvard Apparatus, Inc. (Holliston, MA, USA) is used to supply culture medium continuously.

Figure 2-A. Cell density in different sections of a toroidal behavior at a time of 24 hours of static incubation after inoculation of the compartment with a cell suspension of 5X103 cel / mL. The image corresponds to the 'linear section of entrance to the toroidal space.

Figure 2-B. Cell density in different sections of a toroidal behavior at a time of 24 hours of static incubation after inoculation of the compartment with a cell suspension of 5X105 cel / mL. The image corresponds to the previous section of a toroidal space.

Figure 2-C. Cell density in different sections of a toroidal behavior at a time of 24 hours of static incubation after inoculation of the compartment with a cell suspension of 5X105 cel / mL. The image corresponds to the posterior section of a toroidal space.

Figure 3-A. Concentration of functional monoclonal antibody quantified in the output of a micro-device at 12, 13, 14, 15, 16, 17, 24, 36, 42 and 48 hours of continuous feeding for the case where the surface of the micro-channel has Textured with semi-spherical cavities for cell anchoring and treated with fibronectin (·).

BRIEF DESCRIPTION OF THE INVENTION The present invention refers to a set of devices that have as distinctive characteristics: (a) possess a series of cylindrical compartments obstructed by a central structure (compartments of toroidal geometry or donut type); (b) having a texture on the surface (s) of the channel that promotes cell adhesion and the retention or proliferation of recombinant Chinese hamster ovary cells; (c) being fed with culture medium continuously by some pumping device. This type of micro-devices can be used as continuous perfusion bioreactors for the production of glycosylated recombinant proteins. In these reactors, the productivities with respect to recombinant protein produced per unit time and per unit volume (ie mg protein mL "1 min" 1) are more than two orders of magnitude higher than those observed in suspension culture systems of CHO cells.

DETAILED DESCRIPTION OF THE INVENTION The main embodiment of the present invention consists of a family of micro-devices for the culture of recombinant CHO cells (and other recombinant mammalian cells) and the production of therapeutic recombinant proteins for human or veterinary use (biopharmaceuticals) and other recombinant proteins. The plurality of devices object of this invention possess as distinctive characteristics: (a) possess a series of cylindrical compartments obstructed by a central structure (compartments of toroidal geometry or donut type); (b) having a texture on the surface (s) of the channel that promotes cell adhesion and the retention or proliferation of recombinant Chinese hamster ovary cells; (c) being fed with culture medium continuously by some pumping device (see Figure 1-A). The combination of these distinguishing features provide the invention with a significant competitive advantage against other devices used for CHO cell culture and for the production of recombinant proteins. The general rational that supports this invention is described below. For its proper growth and metabolic functioning, mammalian cells require particular conditions of nutrients (nature and concentration), pH, temperature, concentration of salts, concentration of possible inhibitory substances, etc. Ideally, a culture system (bio-reactor) that promotes a more homogeneous and more controllable micro-environment, will provide advantages to cell culture processes (in general) and mammalian cells (in particular). In a micro-device for the cultivation of serial toroidal compartments (MD-CTS), given the surface / volume relationships, the phenomena of mass transfer, heat, and momentum can be better controlled, such that a suitable microenvironment is propitiated for growth cellular and / or the production of a recombinant protein. The use of geometric configurations with toroidal compartments in series (Figure 1 -B and Figure 1-C) is a determining factor for the proper performance of the devices that are the subject of this invention. In each compartment originate, due to their particular geometry, a diversity of zones of low flow velocity (see Figure 2-A, 2-B and 2-C), particularly in the part of the annular space posterior to the obstructive volume of the space toroidal (with respect to the flow, see figure 2-C). The preferred embodiment of this invention consists of devices containing eight toroidal compartments arranged in series, such that in each compartment there are zones of low local speed. In general, it is in these areas of lower flow velocity that the appearance of the first nuclei of cell adhesion is observed when CHO cells (or other mammalian cells) are inoculated and cultured statically in the devices. A second distinctive element of this invention is the treatment of the surface of the micro-devices to motivate cell adhesion, in particular the adhesion of CHO cells. Conventionally, CHO cells grow attached to substrates when they are cultured in the presence of fetal bovine serum. However, for commercial applications related to the production of therapeutic proteins for human use, the use of fetal bovine serum (SBF) is restricted by international regulations given the risk of viral contamination. Thus, conventionally, CHO cells used in commercial processes of biopharmaceutical production are cultured in SBF-free media. In the absence of SBF, CHO cells lose their adhesion capacity. Consequently, CHO cell lines destined for pharmaceutical use in humans are adapted to grow in suspension (by culture under conditions of progressively lower SBF concentration). With the purpose of achieve high cellular concentrations of CHO cells in micro-channels, adhered to the surface with sufficient force such that they are capable of supporting a certain continuous flow without detaching, the cell surface or alternatively the surface of the micro-channel, must be treated. In the preferred embodiment of this invention, the surface of the micro-devices is treated to promote cellular anchoring. This treatment consists of (a) texturizing the surface (s) of the micro-channels by carving semi-spherical cavities of between 5 and 20 μ? T? in diameter, preferably 15 μp? in diameter, such that these cavities can be used as sites for the individual anchoring of CHO cells; and (b) depositing on the textured surface of the micro-channels compounds that favor cell adhesion, preferably not exclusively materials such as fibronectin, cell adhesion factor, collagen, gelatin, among others (Figure 1-A). In the preferred embodiment of this invention, CHO recombinant cells, enabled by genetic manipulation to express a recombinant therapeutic protein (e.g., a biosimilar monoclonal antibody from Infliximab®), were inoculated into a device with eight toroidal compartments. The total length of the arrangement of compartments in this preferred configuration is 2.37 cm in length, 2.0 mm in width (in the widest section) and 0.1 mm in depth (see Figure 1-B and 1-C) and statically cultivated (without activating the continuous flow pump) in suitable culture medium. After an incubation period of 24 to 96 hours under static conditions (preferably 48 hours), the continuous flow of culture medium is activated. In the preferred embodiment of this invention, using the aforementioned geometry and channel dimensions, the continuous flow range that must be applied is 1 to 3 μ? / ????. For this purpose, flow pumps can be used continuous non-pulsatile, preferably more not exclusively dual reciprocal syringe pumps (Figure 1 -E). The continuous flow is a third fundamental element of this invention. The continuous supply of fresh culture medium ensures that the needs for nutrient consumption for growth and / or cellular maintenance and production of recombinant protein are satisfied. Additionally, the continuous removal of culture medium (partially or totally exhausted) also guarantees the removal of substances excreted by cells (metabolites such as organic acids, C02, etc.) that could inhibit growth, detonate cellular apoptosis, or inhibit cell growth. growth of the product of interest. Given that the cellular density that is reached in these channel devices is high (both by cellular retention strategies and by the uninterrupted supply of nutrients) the production of cell excretion compounds is presumed high. Therefore, the continuous removal of medium is particularly advantageous in these high cell density perfusion systems.

Considering the geometrical dimensions and the recommended operating conditions for the micro-devices of serial compartments that are the subject of this invention, the flux originated in the device is of a laminar nature. The laminar and stable nature of the flow (non-pulsatile) guarantees a low level of mechanical stress, this representing a significant advantage of the culture systems object of this invention. Mammalian cells in general, and CHO cells in particular, are sensitive to high levels of mechanical stress induced by agitation or by forces caused by the explosion of bubbles.

EXAMPLE 1. CELLULAR ACCESSION IN A SERIAL MICRODISPOSITION OF TOROIDAL COMPARTMENTS This example illustrates a feasible design that leads to the expression of a biosimilar monoclonal antibody from Infliximab® in CHO cells attached to the surface of a micro-device comprised of a series of 8 toroidal compartments (see Figure 1-B and 1-C) of 2.37 mm in length. In this configuration, the space through which the fluid circulates has, in all its sections, at least 0.5 mm in width and 0.1 mm in depth (see Figure 1-B). The micro-device was manufactured in a rectangular block of PMMA (6 mm thick, 30 mm wide and 45 mm long). At the beginning and end of the series of compartments, a 1 mm diameter cylindrical reservoir was arranged to facilitate the coupling of hoses to dispense or remove culture medium. The device was designed in CAD-CAM® and micro-machined in the PMMA block using a vertical machine MAKINO F3, CA, USA, with a position accuracy of 0.0015 mm. The micro-machining was performed using a 0.410 μ tip. diameter). Once the micro-microdevice was built, it was covered with a lid made of PDMS (Sylgard 184, Dow Corning Corp., Midland, MI, USA). The surfaces of the body of the micro-device (built in PMMA) and the cover (made in PDMS) were treated with a "corona-discharge" device (Electro-Technic-Products Inc., Chicago, ILL, USA, model BD20A), joined using liquid PDMS and thermally sealed.

The lower surface of the micro-device of serial toroidal compartments was treated to favor cell adhesion. This treatment consisted in (a) texturizing the lower surface of the micro-channel by carving semi-spherical cavities of 15 μp? of diameter (average equivalent diameter of a CHO cell) such that these cavities can be used as sites for individual anchoring, and (b) deposit on the textured surface of the micro-device a compound that promotes cell adhesion, fibronectin in this case particular.

In devices manufactured in this way, a suspension of CHO cells with a concentration of 5X105 cells / mL was dispensed by means of a syringe pump (Harvard 33 twin siringe pum; from Harvard Apparatus, Inc.; Holliston, MA, USA). Cells were suspended in culture medium containing CD OptiCHO ™ (Invitrogen, Carlsbad, CA, USA) and CHO CD EfficientFeed ™ B (Invitrogen, Carlsbad, CA, USA) supplemented with 200 mM L-glutamine (Invitrogen, Carlsbad, CA, USES). In particular, in this application example, recombinant CHO cells producing a biosimilar monoclonal antibody from Infliximab® were taken as a model. This cell line was derived from the CHO-S line (Invitrogen, Carlsbad, CA, USA). The inoculant suspension of recombinant CHO cells was incubated in the micro-devices of serial toroidal compartments for a period of 48 hours under static conditions (without activation of the continuous flow). This incubation period has the purpose of promoting cellular adhesion to the surface of the microdevice.

At the end of this period of static incubation, the average cellular density in the micro-channels was calculated in the range of 1X107 to 1X108 cells per mL equivalent, a significant (but not complete) cellular coverage of the micro-channel surface is observed (see Figure 2-A, 2-B and 2-C). These cell densities are equivalent to or higher than those reported for shallow tank perfusion culture systems (= 1-5X10 cel / mL). After the activation of the continuous supply of culture medium, at a volumetric flow condition of 3 μ? 7 ???, the cell density increased progressively. Figure 3 presents performance results of the reactor in question. Specifically, the concentration of the biosimilar monoclonal antibody of Iníliximab® determined at the output of the micro-device at different times of continuous operation is presented. In this example, the continuous flow of culture medium feed was activated at a volumetric ratio of 3 μL m. The concentration of the biosimilar monoclonal antibody of Iníliximab® was analyzed by a specific ELISA method for functional Iníliximab®. The concentration of monoclonal antibody increased exponentially with respect to time during the first 48 hours of uninterrupted continuous feeding. This result suggests that the cell density in the micro-device did not reach its maximum during this period and that there is still space on the surface of the micro-device to house a greater number of cells.

EXAMPLE 2. PROJECTION OF THE PRODUCTION OF A MONOCLONAL ANTIBODY IN A BATTERY OF MICRODISPOSITIVES OF SERIOUS COMPARTMENTS In this application example, performance indicators of the micro-devices of serial compartments reason for this invention are compared, during their period of batch operation (static incubation) and continuous. Additionally, they are included in said comparison, performance indicators of a conventional cell culture system CHO in suspension, specifically a fed-batch instrumented reactor (final volume of 1.5 L) operated for a period of 14 days (Table 1). The productivities in terms of the product of interest, expressed in mass antibody units functional unit per unit volume per unit of time, are superior by at least two orders of magnitude in the continuous system object of the present invention that in the conventional suspension culture system.

Table 1. Comparison of characteristics and relevant indicators of performance in a micro-device of serial toroidal compartments (MD-CTS) operated in batches (static incubation), a MD-CTS system operated continuously (Example 1) and a conventional system of cell culture in suspension operated in fed batch mode (Reactor 1 -L (susp.)).

MD-CTS (continuous) 3.20 0.39 1 1.64 191.75 X Reactor 1L (susp.) 826.00 ± 90 20160 0.059 1 X [b] agitated tank suspension culture system; variable flow of fresh medium feed without removal of culture medium.

The practical implications of these results explain the potential advantages of the devices object of this invention. Comparing against cell culture systems CHO in fed batch suspension (conventionally used in the field biopharmaceutical), a micro-channel device could produce per unit time the same amount of product in a volume 190 times smaller. Alternatively, per unit volume, a continuous microchannel system could produce the same amount of product in a 190 times shorter operating time. In equal time and volume, a continuous micro-channel device would produce 190 times more product.

Claims (6)

CLAIMS Having described my invention enough, I consider it as a novelty and therefore claim as my exclusive property, what is contained in the following clauses:

1. The set of micro-devices characterized by (a) having a series of cylindrical compartments obstructed by a central structure (compartments of toroidal geometry or donut type); (b) possess a texture on the surface (s) of the channel that promotes cell adhesion and the retention or proliferation of mammalian recombinant cells, particularly but not exclusively, Chinese hamster ovary cells; and (c) being fed with culture medium continuously by some pumping device.

2. The plurality of micro-devices described in claim 1, characterized in that they possess in particular a series of cylindrical compartments obstructed by a central structure and semi-spherical cavities with a diameter in the range of 5 to 20 μ? on the surface (s) exposed to the flow.

3. The plurality of micro-devices described in claim 1, characterized in that additionally the surface (s) of their micro-channels have been treated with a compound that promotes cell adhesion, in a nonlimiting manner, fibronectin, factor of cellular adhesion, collagen, among other substances.

4. The use of the micro-devices described in claim 1 for the purpose of screening conditions (eg, temperature, pH, culture media, etc.) for the culture of mammalian cells and particularly, but not exclusively, cells CHO recombinants.

5. The use of the micro-devices described in claim 1 for the purposes of production of therapeutic recombinant proteins for human use (biopharmaceuticals) and other glycosylated recombinant proteins in cultures of mammalian cells and particularly, but not exclusively, recombinant CHO cells.

6. The use of the micro-devices described in claim 1 for the purposes of production of therapeutic recombinant proteins for veterinary use in cultures of mammalian cells and particularly, but not exclusively, recombinant CHO cells.