Perfusion cell culture apparatus

Abstract

There is described a perfusion cell culture apparatus comprising a bioreactor, a spent-media separation member; and a pressure source. The bioreactor, separation member and pressure source are fluidly connected so that the pressure source is operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow. The separation member is operable to retain cells and separate spent- media from the cell suspension. The pressure source is also operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow. Also described is a method of cell culture perfusion using the perfusion cell culture apparatus and a kit of parts comprising the spent-media separation member and pressure source.

Description

[0001] PERFUSION CELL CULTURE APPARATUS

[0002] FIELD

[0003]

[0001] The present disclosure relates to a perfusion cell culture apparatus and methods of perfusion cell culture. The present disclosure also extends to use of the perfusion cell culture apparatus and methods in the production of cell biomass for cultivated meat.

[0004] BACKGROUND

[0005]

[0002] The world population is set to increase to almost 10 billion people within the next 50 years. As a result, there will be nearly two billion additional people to feed by 2050. This rising population will lead to an increase in global demand for meat by approximately 73% by the year 2050. The agricultural industry will have to scale, potentially doubling in size, to meet this demand. Of the earth’s habitable land, 39% is currently used to produce feed to rear livestock for the meat industry. It takes three years to rear a single cow for slaughter, or 6-12 months for pigs and poultry. Therefore, a large area of arable land is required to feed these animals to term. Currently, 80 billion animals are slaughtered each year for meat with 1 .2 billion slaughtered in the UK alone.

[0006]

[0003] Cultivated meat has the potential to address the substantial global problems associated with livestock farming and the environmental impact of meat production along with animal welfare, food security and human health. Cultivated meat is a meat produced by in vitro cell cultures of animal cells. It is a form of cellular agriculture, with such agricultural methods being explored in the context of increased consumer demand for protein. Cellular agriculture relates to the production of animal-sourced foods from cell culture.

[0007]

[0004] Perfusion processes are used in multiple different systems that involve cell culture to provide a high level of processes intensification, but this comes at the cost of increasing media volumes used for the culture, a great deal of capital expenditure for the equipment, and a ceiling and floor in the bioreactor size to which the process can be scaled.

[0008]

[0005] Perfusion processes overcome the main limitation regarding cell culture in bioreactors of the build-up of metabolic waste, which hinders cell proliferation. These processes achieve a reduction in this build-up by the removal of spent media and addition of fresh media throughout the culture. This makes process intensification to achieve high cell densities more viable.

[0009]

[0006] Some of the current perfusion bioreactors are designed around being perfusion capable. The noticeable drawback of these bioreactors can be that they are specialised equipment, and can only perfuse cultures within this format, rather than being ancillary equipment that could be attached to other bioreactors, other bioreactors being a more scalable format such as an airlift reactor or stirred tank bioreactor (or fermentor).

[0010]

[0007] External equipment that can be attached to standard bioreactors are also known. However, these still have a common theme that highly specialised parts and equipment limit the size at which these devices can be produced.

[0008] It is therefore an object of aspects of the present disclosure to address one or more of the above mentioned or other problems.

[0011] SUMMARY

[0012]

[0009] According to a first aspect of the present disclosure, there is provided perfusion cell culture apparatus comprising: i. a bioreactor; ii. a spent-media separation member; and

[0013] Hi. a pressure source, wherein the bioreactor, separation member and pressure source are fluidly connected so that the pressure source is operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow, wherein the separation member is operable to retain cells and separate spent-media from the cell suspension, and wherein the apparatus comprises a pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow.

[0014]

[0010] According to a further aspect of the present invention, there is provided a method of cell culture perfusion, comprising arranging a perfusion cell culture apparatus comprising: i. a bioreactor; ii. a spent-media separation member; and

[0015] Hi. a pressure source, wherein the bioreactor, separation member and pressure source are fluidly connected so that the pressure source is operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow, wherein the separation member is operable to retain cells and separate spent-media from the cell suspension, and wherein the apparatus comprises a pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow, and performing cell culture perfusion with the apparatus.

[0016]

[0011] According to a further aspect of the present invention, there is provided a kit of parts for a perfusion cell culture apparatus, the kit of parts comprising: i. optionally a bioreactor; ii. a spent-media separation member; and

[0017] Hi. a pressure source, wherein the separation member and pressure source are operable to be fluidly connected with each other and with a bioreactor so that the pressure source is operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow, wherein the separation member is operable to retain cells and separate spent- media from the cell suspension, and wherein the kit of parts comprises a pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow, wherein the kit of parts optionally further comprises i. optionally, a spent-media collection member; ii. optionally, a level sensor;

[0018] Hi. optionally, a culture air supply; and / or iv. optionally, an air filter.

[0019]

[0012] According to the present disclosure, there is further provided apparatus for perfusion cell culture comprising: i. a spent-media separation member; and ii. a pressure source, wherein the separation member and pressure source are operable to be fluidly connected with a bioreactor so that the pressure source is operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow, wherein the separation member is operable to retain cells and separate spent-media from the cell suspension, and wherein the apparatus comprises a pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021]

[0013] The apparatus of the present is operable to provide perfusion cell culture using pressure differentials between parts of the apparatus to cause reversable cell suspension flow. The pressure differentials are produced by changes in air flow provided by pressure sources that are not required to contact the cell suspension or culture media.

[0022]

[0014] The commonality introducing the complexity in known external equipment that can be attached to standard bioreactors is a moving part that must contact the sterile media to move the culture around a system of a bioreactor and cell retention device. This complexity is introduced by keeping the culture sterile and providing this movement without inducing levels of shear stress that would damage the mammalian cells in the culture.

[0023]

[0015] A main challenge that this invention addresses is improving scalability by providing a way to move culture liquid through the cell retention device without the need for moving parts in contact with the culture liquid, whereas traditional pumps involve the use of moving rotors or diaphragms to drive the flow. Reducing moving parts that contact the sterile culture may allow both scale up and down by removing the need for specialised parts. As a further effect, the simplicity of a pump with fewer moving parts, the need to produce highly specialised components that are required in other pumps for moving cell culture suspension will greatly reduce the cost of manufacture of the pump and CAPEX for a perfusion pump system.

[0024]

[0016] Advantageously, the perfusion cell culture apparatus of the present invention provides a simplified device that is more suitable for scale-up compared to prior art apparatus.

[0025]

[0017] The apparatus of the present disclosure may allow for fewer moving parts to be in contact with the cell suspension. In particular, the apparatus of the present invention may help to address the complexity of prior art perfusion systems by reducing the requirement for moving parts of the pump to contact the cell suspension and culture media.

[0026]

[0018] Using pressure to drive the liquid motion may allow for a reduction of moving parts inside the sterile boundary of the apparatus meaning that standard process vessels can be used for the pumping system. With such moving parts outside of the sterile boundary, items such as a compressor can be of more standard types.

[0027]

[0019] Another important advantage of the apparatus of the present disclosure is the scalability of the system. The apparatus may provide for improved ease of scale-up compared to prior art systems.

[0028]

[0020] The bioreactor may be any suitable type of bioreactor operable to have a cell suspension culture moved into and out of the bioreactor by changes in air flow affecting the relative pressures of the bioreactor and fluidly connected spent-media separation member.

[0029]

[0021] The bioreactor may comprise a stirred tank bioreactor, a hollow-fibre bioreactor, a wave motion bioreactor, and / or an airlift bioreactor.

[0030]

[0022] The spent-media separation member is operable to retain cells and separate spent-media from the cell suspension. The spent-media separation member may be operable to be, or may be, fluidly connected (directly or indirectly, for example via a spent-media collection member) to a pressure source operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow and / or a pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow.

[0031]

[0023] The separation member may comprise any cell retention / spent-media separation component that allows for cells to be moved from a bioreactor through the separation member and then returned back to the bioreactor absent a portion of spent-media.

[0032]

[0024] The separation member may be autoclavable, such as operable to function after at least 10 autoclave cycles at a temperature of at least 100°C, such as at least 120°C, for at least 15 minutes on each cycle.

[0025] The separation member may be operable to function after cleaning with aggressive substances, such as concentrated acid or caustic.

[0033]

[0026] The separation member may comprise a filter, such as a hollow-fibre filter; a hydrocyclone, an acoustic separator, a metal filter (such as stainless steel), and / or a sediment tank.

[0034]

[0027] The diameter of the hollow-fibres of the separation member may be at least 0.5 mm. The diameter of the hollow-fibres of the separation member may be at least 4 mm, such as at least 7 mm. The diameter of the hollow-fibres of the separation member may be up to 15 mm, such as up to 12 mm or up to 10 mm. The diameter of the hollow-fibres of the separation member may be from 0.5 mm to 15 mm, such as from 4 to 12 mm or from 7 to 10 mm. Advantageously, a diameter within this range may provide improve fluid flow.

[0035]

[0028] The spent-media filter may be operable to retain cells from the cell suspension at a feed flow side of the filter and permit media from the cell suspension to pass through the filter into a permeate side of the filter. As such, the spent-media filter may be operable to be, or may be, fluidly connected at a feed flow inlet to a bioreactor. The spent-media filter may be operable to be, or may be, fluidly connected at permeate flow outlet to a spent-media collection member. The spent-media filter may be operable to be, or may be, fluidly connected (directly or indirectly, for example via a spent-media collection member) at the feed flow side and / or permeate flow side of the filter to a pressure source operable to actuate movement of a cell suspension into contact with the filter from the bioreactor by effecting a change in air flow and / or a pressure source operable to actuate return of the cell suspension from contact with filter to contact with the bioreactor by effecting a change in air flow.

[0036]

[0029] The apparatus may be operable to produce tangential flow filtration (TFF) using pressure driven flow of the cell suspension. The spent-media separation member may comprise a tangential flow filter.

[0037]

[0030] The separation filter may comprise any suitable membrane. The membrane may comprise a tubular membrane. The membrane may comprise a hollow-fibre membrane.

[0038]

[0031] The membrane of the separation filter may comprise metal, such as steel, for example stainless steel. The membrane of the separation filter may comprise a stainless steel tangential flow membrane,

[0039]

[0032] The separation membrane may comprise a coating, such as a coating comprising an inorganic component, for example a coating comprising titanium dioxide. The coating may comprise a sintered inorganic coating. The membrane of the separation filter may comprise a stainless steel tangential flow membrane that comprises a coating, such as a sintered coating, comprising titanium dioxide.

[0040]

[0033] The membrane may comprise a nominal pore size of at least 0.05 pm and / or a nominal pore size of up to 0.5 pm, such as up to 0.3 pm, or up to 0.2 pm, or up to 0.1 pm.

[0034] The separation member may have an internal diameter of from 20 mm to 1 .5 m, and / or an internal length of from 0.5 m to 6 m.

[0041]

[0035] According to a further aspect of the present disclosure, there is provided apparatus for perfusion cell culture, the apparatus comprising a tangential flow filtration separation member operable to separate spent-media from cells in a cell suspension (such as a separation member as otherwise described herein), wherein the separation member comprises a metal membrane, such as a stainless steel membrane, that comprises a coating comprising an inorganic coating (such as a sintered inorganic coating, for example comprising titanium dioxide). The metal membrane may comprise a hollow-fibre membrane, such as a hollow-fibre membrane comprising a diameter of at least 4 mm, such as at least 7 mm.

[0042]

[0036] According to a further aspect of the present disclosure, there is provided use of a membrane for perfusion cell culture, the membrane comprising a tangential flow filtration membrane operable to separate spent-media from cells in a cell suspension (such as a separation member as otherwise described herein), wherein the membrane comprises a metal membrane, such as a stainless steel membrane, that comprises a coating comprising an inorganic coating (such as a sintered inorganic coating, for example comprising titanium dioxide). The metal membrane may comprise a hollow-fibre membrane, such as a hollow-fibre membrane comprising a diameter of at least 4 mm, such as at least 7 mm.

[0043]

[0037] According to a further aspect of the present disclosure, there is provided a method of perfusion cell culture, such as a method according to the aspects of the present disclosure provided herein, wherein the method comprises use of a tangential flow filtration membrane operable to separate spent-media from cells in a cell suspension (such as a separation member as otherwise described herein), wherein the membrane comprises a metal membrane, such as a stainless steel membrane, that comprises a coating comprising an inorganic coating (such as a sintered inorganic coating, for example comprising titanium dioxide) in perfusion cell culture. The metal membrane may comprise a hollow-fibre membrane, such as a hollow-fibre membrane comprising a diameter of at least 4 mm, such as at least 7 mm.

[0044]

[0038] Advantageously, use of such a coated metal membrane may provide for a scalable separation member for perfusion culture in bioreactors. Such a membrane may provide for a high number of autoclave cycles and be able to work at high pressures. In addition, such membranes may allow for use of aggressive cleaning substances such as concentrated acid or caustic.

[0045]

[0039] The apparatus of the present disclosure comprises a pressure source (or pump) operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow. The change in air flow may cause the pressure of the spent-media separation member to fall below the pressure of the bioreactor to thereby actuate movement of the cell suspension from the bioreactor into contact with the separation member.

[0040] As used herein, ‘air’, whether in terms of ‘airflow’ or otherwise, may refer to any suitable gas. The gas may comprise air, such as air extracted from the environment external to the apparatus. Such gas / air may be substantially sterile / sterilised / cell culture suitable as detailed below.

[0046]

[0041] The pressure source operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor may be operable to draw air from the separation member so as to cause a pressure drop in the separation member.

[0047]

[0042] The apparatus of the present disclosure comprises a pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow. The change in air flow may cause the pressure of the spent-media separation member to rise above the pressure of the bioreactor to thereby actuate movement of the cell suspension from the separation member into the bioreactor.

[0048]

[0043] The pressure source operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor may be operable to push air into the separation member so as to cause a pressure rise in the separation member.

[0049]

[0044] The pressure source operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor (the ‘bioreactor-separation member pressure source) and the pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor (the ‘separation member-bioreactor pressure source’) may comprise the same or separate pressure sources.

[0050]

[0045] The apparatus may be free of moving pressure source (or pump) parts that are operable to contact the cell suspension or culture media.

[0051]

[0046] The pressure source(s) may be any suitable type of air pressure source operable to produce the described pressure changes in the apparatus.

[0052]

[0047] The apparatus may comprise any suitable control circuit to alternate between the bioreactor being at a higher or lower pressure than the separation member.

[0053]

[0048] For bioreactors that operate at ambient pressures, the apparatus may comprise a pressure source operable to produce a vacuum as a low-pressure source. The apparatus may also comprise a pressure source (the same pressure source or different) operable to produce a high- pressure source at a pressure that is at a suitable level above ambient to achieve the required return flow rate. For large bioreactors that operate with a positive pressure, the low pressure can be a pressure that is lower than the bioreactor, but still above ambient pressure so that the bioreactor integrity is not compromised, yet there is still a differential that drives liquid out of the bioreactor. The high pressure may be set to be suitably above the bioreactor operating pressure to drive the flow back into the bioreactor. With the two pressure settings, the apparatus can advantageously be configured to deal with bioreactors that operate at ambient or elevated pressures.

[0054]

[0049] Where a pressure source of the apparatus is operable to introduce air into the bioreactor / separation member, the air may be air of a suitable quality for cell culture and / or the air may be passed through a filter to produce cell culture suitable air prior to contact with the cell suspension.

[0055]

[0050] The apparatus may comprise a spent-media collection member operable to receive spent- media separated from the cell suspension by the separation member. The collection member may be fluidly connected to the spent-media separation member, such as to a permeate outlet of the separation member.

[0056]

[0051] The collection member may comprise a collection vessel operable to receive and hold at least a portion of the spent media.

[0057]

[0052] The collection member may be operable to function at a pressure of >100 kPa, such as >500 kPa, such as >1000 kPa. The collection member may comprise a pressure vessel, such as a pressure rated glass vessel. The collection member may comprise a plastic vessel, such as an autoclavable plastic vessel. The collection member may comprise a pressure rated stainless steel vessel.

[0058]

[0053] The apparatus of the present disclosure may comprise a sensor operable to detect a desired point at which the cell culture suspension flow is to be switched. The sensor may be operable to communicate with a control circuit to indicate when the desired point has been reached so as to actuate a switch in the flow direction.

[0059]

[0054] The sensor may comprise a sensor operable to detect the level or amount of spent-media in the spent-media collection member.

[0060]

[0055] When the separation member is at a lower pressure than the bioreactor, the cell suspension may flow out of the bioreactor, through the cell retention device to a collection member. A sensor may monitor the volume that flows into the collection member and maintain a pressure differential until sufficient volume has flowed to flush through the cell retention device. When the required volume has flowed, the apparatus may comprise a microcontroller that may shut the low-pressure source and open a high-pressure source. This reverse of pressure gradient may return culture media and cell suspension from the collection member back through the cell retention device to the bioreactor. While this cycle of liquid flow occurs, a standard pump can be used to draw off liquid across the cell retention device so that a cell-free permeate can be removed from the bioreactor system.

[0061]

[0056] The sensor may comprise a load sensing balance. The load sensing balance may be arranged so as to be operable to measure the load of liquid in the collection member. The load balance may measure the volume of spent-media in the collection member and combined with a microcontroller, may be used to actuate whether the collection vessel is pressured by a low pressure source or a higher pressure source (such as a vacuum supply source or a high pressure process air supply source). A load balance may be arranged under the collection member to provide the data on whether to draw of return media from the bioreactor. Advantageously, use of a balance sensor may provide a particularly simple and low-cost sensor.

[0062]

[0057] Other types of sensors may be used in the apparatus of the present disclosure, such as ultrasonic level switches, for example at the high and / or low mark; and / or a direct contact probe.

[0063]

[0058] The apparatus may comprise a culture air supply operable to provide air suitable for cell culture to the bioreactor.

[0064]

[0059] The apparatus may comprise an air filter operable to provide permeate air that is suitable for cell culture. The permeate air may be sterile air. The permeate air will contact the culture to supply oxygen (or other gasses) to the metabolising cells. Using such permeate / sterilised air to move culture liquid through a separation member advantageously retains a low contaminant risk to the cell culture system while allowing for a reduction in the number of specialised parts required to pump cell cultures between processing equipment.

[0065]

[0060] The apparatus may comprise an air filter arranged between culture air supply and the bioreactor.

[0066]

[0061] The apparatus may comprise an air filter arranged between the collection member and the pressure source(s).

[0067]

[0062] The apparatus may comprise an air filter arranged between the separation member and the pressure source(s).

[0068]

[0063] The surface area of the air filter may be large enough to allow a gas flow rate that achieves the desired rate of fluid flow. The filter may have pore sizes small enough to sterilise the flow of the permanent gas. The air filter may have an average pore diameter of about 0.22pm.

[0069]

[0064] The apparatus may be operable to comprise single direction flow of the cell suspension, such as single direction flow between a bioreactor and a spent-media separation member. The apparatus may comprise non-return values to provide single direction flow of the cell suspension.

[0070]

[0065] The apparatus may comprise more than one bioreactor, such as at least 2 or at least 3 bioreactors. The apparatus may comprise more than one bioreactor in sequence and / or in parallel. The apparatus may comprise a first bioreactor that is fluidly connected to a further bioreactor, such as so that contents (such as cell suspensions and media) from the first bioreactor and the further bioreactor may be contacted together, such as at a point that is prior to contact of the cell suspension with the spent-media separation member.

[0071]

[0066] The apparatus may comprise multiple bioreactors and further comprise single flow direction, such as single flow direction into and out of the spent-media collector and / or single flow direction between bioreactors. An apparatus comprising multiple bioreactors may comprise single direction flow to a convergence point at which content from at least two bioreactors are operable to contact, and the convergence point may be prior to contact with the spent-media separation member. The apparatus may comprise single direction flow from the convergence point (or points) to the spent-media separation member and from the spend-media separation member to a bioreactor, such as back to a flow originating bioreactor.

[0072]

[0067] The apparatus may comprise any suitable means to provide single direction flow, such as a peristaltic, positive displacement, centrifugal, lobe and / or sinusoidal pump.

[0073]

[0068] The apparatus, such as an apparatus comprising single direction flow, may comprise a flow control system. The flow control system may comprise a level indicating controller (LIC), flow transmitter (FT), flow indicating controller (FIC), pressure indicating controller (PIC) and / or a pressure transmitter (PT).

[0074]

[0069] The apparatus of the present disclosure may be for the production of cell biomass to be used in the production of cultivated meat. As such, the method of the present disclosure may be a method for the production of cultivated meat comprising using the perfusion cell culture apparatus of the present disclosure in the production of cultivated meat.

[0075]

[0070] As used herein, unless otherwise expressly specified, all numbers such as those expressing values, ranges, amounts or percentages may be read as if prefaced by the word “about”, even if the term does not expressly appear. The term “about” when used herein means + / - 10% of the stated value. Also, the recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within that range (e.g. 1 to 5 can include 1 , 2, 3, 4 when referring to, for example, a number of elements, and can also include 1 .5, 2, 2.75 and 3.80, when referring to, for example, measurements). The recitation of end points also includes the end point values themselves (e.g. from 1.0 to 5.0 includes both 1.0 and 5.0). Also, any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[0076]

[0071] Singular encompasses plural and vice versa. For example, although reference is made herein to “a filter”, “a” pressure source, “a” bioreactor, and the like, one or more of each of these and any other components can be used. “Including”, “for example” and like terms means including for example but not limited to. The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Additionally, although the present disclosure has been described in terms of “comprising”, the processes, materials, and coating compositions detailed herein may also be described as “consisting essentially of’ or “consisting of’.

[0077]

[0072] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and / or C, the list can comprise A alone; B alone; C alone; A and B in combination; A and C in combination, B and C in combination; or A, B, and C in combination.

[0078]

[0073] Where ranges are provided in relation to a genus, each range may also apply additionally and independently to any one or more of the listed species of that genus.

[0079]

[0074] All of the features contained herein may be combined with any of the above aspects in any combination.

[0080]

[0075] For a better understanding of the present disclosure, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the following experimental data and figures.

[0081] BRIEF DESCRIPTION OF DRAWINGS

[0082]

[0076] Figure 1 shows a schematic plan of a cell culture apparatus according to the present disclosure.

[0083]

[0077] Figure 2 is a chart showing capacitance against day of culture for a perfusion system in a 20L bioreactor using an established cell culture filtration system.

[0084]

[0078] Figure 3 is a chart showing total cells and viability for a perfusion system in a 20L bioreactor using an established cell culture filtration system.

[0085]

[0079] Figure 4 is a chart showing capacitance against day of culture for a perfusion system in a 20L bioreactor using an apparatus according to the present disclosure.

[0086]

[0080] Figure 5 is a chart showing total cells and viability for a perfusion system in a 20L bioreactor using an apparatus according to the present disclosure.

[0087]

[0081] Figure 6 is a chart showing comparison of total cell densities for three different replicates of perfusion systems in a 20L bioreactors using an apparatus according to the present invention.

[0088]

[0082] Figure 7 shows a further schematic plan of a cell culture apparatus according to a second embodiment of the present disclosure.

[0089]

[0083] Figure 8 shows a further schematic plan of a cell culture apparatus according to a third embodiment of the present disclosure.

[0090]

[0084] Figure 9 is a chart showing the cell densities obtained with the apparatus as shown in Figure 8

[0091] EXAMPLES

[0092]

[0085] Different experiments were performed to demonstrate the successful application of the apparatus of the present disclosure for cell culture.

[0093]

[0086] A schematic representation of the cell culture apparatus 100 according to the present disclosure used in the examples is shown in Figure 1. Apparatus 100 contains ceramic coated stainless steel tangential flow filter 102, which functions as a cell retention device to separate spent culture media from the cell suspension. Filter 102 comprises fibres with a diameter of 9.6 mm and a nominal pore size of 0.05 to 0.1 pm, with the ceramic coating comprising titanium dioxide sintered to the steel. The cell suspension is provided through two-way fluid flow connection 104a from stirred ambient pressure bioreactor 104. Apparatus 100 further contains process vacuum supple pump 106x (low pressure pump) and process air supply pump 106y (high pressure pump), which are each fluidly connected to spent media collection vessel 108 through air filter 116 via two-way flow connection 106a. Collection vessel 108 is fluidly connected to the permeate flow side of filter 102 via two-way flow connection 108a. Culture air supply is provided into bioreactor 104 via pump 110 through air filter 112. Low pressure pump 106x is operable to draw a cell suspension out of bioreactor 104 into filter 102, which retains cells in the feed flow side of the filter while allowing spent media to pass into the permeate side of filter 102 and then on into collection vessel 108. Level sensing balance 114 is configured to be able to detect when the amount of spent media in collection vessel 108 is at the desired level and once that level is reached a microcontroller (no shown) is provided to deactivate pump 106x and activate high pressure pump 106y which causes the cell suspension to flow back into bioreactor 104 via connection 104a. In such a manner, apparatus 100 produces a tangential flow of fluid through filter 102, allowing continuous perfusion culture through variation in pressure.

[0094]

[0087] Figures 2 and 3 show the results of a recirculating perfusion experiment in a 20L bioreactor using an established hollow-fibre cell culture filtration system, while Figures 4 and 5 represent results from experiments carried out in comparable conditions but using the apparatus according to the present disclosure. Both systems were able to reach comparable cell densities, showing the use of the apparatus of the present disclosure for cell culture applications.

[0095]

[0088] Figure 6 shows three different replicates for a full perfusion experiment using the apparatus according to the present invention, showing the possibility of reaching high cell densities (around 10 million cells / ml) and the reproducibility of the results.

[0096]

[0089] Figure 7 shows a schematic representation of the cell culture apparatus 200 according to the present disclosure. Apparatus 200 is configured in a similar manner to apparatus 100, except that is additionally contains two further bioreactors and is configured for single direction flow by using non-return values. Apparatus 200 contains a bioreactor 202 and tangential flow filter 204 as described for apparatus 100, and in addition contains further bioreactors 206a and 206b, as well as single direction hollow fibre filters 208a-d. In use, cell suspension and media flows in a single direction from an outlet of bioreactor 202, through one-way filters 208a or 208c to contact with flow from bioreactor 206a or 206b before the combined flow then passes through respective one-way filter 208b or 208d before contacting tangential flow filter 204. The retained cell suspension is then returned to bioreactor 202 through further one-way flow. The apparatus of Figure 7 demonstrates how the apparatus of the present invention may be duplicated, parallelised and / or combined with non-return valves to provide a flow with a single direction. This configuration of the apparatus may be combined with any of the above cell retention devices such as hollow fibre filters, stainless steel filter modules, hydro cyclones and the like.

[0097]

[0090] Figure 8 shows a schematic representation of the cell culture apparatus 300 according to the present disclosure. Apparatus 300 is configured in a similar manner to apparatus 100 with the exception that the apparatus is configured for single direction flow. To effect single direction flow, apparatus 300 uses a centrifugal pump 302, positive displacement pump 304 and peristaltic pump 306 in combination, or alternatively may use one of the types of pump. Apparatus 300 also contains a flow control system formed of level indicating controller (LIC), flow transmitter (FT), flow indicating controller (FIC), pressure indicating controller (PIC) and a series of pressure transmitters (PT). In use, cell suspension is operable to flow from bioreactor 308 via pumps 304 and 306 in single direction flow to tangential flow filter 310. The retained cell suspension is then returned to bioreactor 308 through further one-way flow.

[0098]

[0091] Figure 9 shows a cell culture run using apparatus 300.

[0099]

[0092] Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0100]

[0093] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0101]

[0094] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0102]

[0095] The present disclosure is not restricted to the details of the foregoing embodiment(s). The present disclosure extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

CLAIMS1 . A perfusion cell culture apparatus comprising: i. a bioreactor; ii. a spent-media separation member; andHi. a pressure source, wherein the bioreactor, separation member and pressure source are fluidly connected so that the pressure source is operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow, wherein the separation member is operable to retain cells and separate spent-media from the cell suspension, and wherein the apparatus comprises a pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow.

2. A perfusion cell culture apparatus according to claim 1 , wherein the bioreactor comprises a stirred tank bioreactor, a hollow-fibre bioreactor, a wave motion bioreactor, and / or an airlift bioreactor.

3. A perfusion cell culture apparatus according to claim 1 or 2, wherein the separation member is autoclavable, such as operable to function after at least 10 autoclave cycles at a temperature of at least 100°C, such as at least 120°C for at least 15 minutes on each cycle.

4. A perfusion cell culture apparatus according to any preceding claim, wherein the separation member is operable to function after cleaning with aggressive substances, such as concentrated acid or caustic.

5. A perfusion cell culture apparatus according to any preceding claim, wherein the separation member has an internal diameter of from 20 mm to 1 .5 m, and / or an internal length of from 0.5 m to 6 m.

6. A perfusion cell culture apparatus according to any preceding claim, wherein the separation member comprises a filter, such as a hollow-fibre filter; a hydrocyclone, an acoustic separator, a sediment tank and / or a tangential flow filter.

7. A perfusion cell culture apparatus according to claim 6, wherein the filter comprises a membrane, such as a tubular membrane and / or a hollow-fibre membrane, optionallywherein the diameter of the hollow-fibres is from at least 0.5 mm up to 15 mm such as from 4 to 12 mm or from 7 to 10 mm.

8. A perfusion cell culture apparatus according to claim 7, wherein the membrane comprises metal, such as steel, for example stainless steel, and / or comprises a nominal pore size of at least 0.05 pm and / or a nominal pore size of up to 0.5 pm, such as up to 0.3 pm, or up to 0.2 pm, or up to 0.1 pm.

9. A perfusion cell culture apparatus according to claims 7 or 8, wherein the membrane comprises a coating, such as a coating comprising an inorganic component, such as a sintered inorganic coating, for example a coating comprising titanium dioxide.

10. A perfusion cell culture apparatus according to claim 6 to 8, wherein the spent-media separation member is fluidly connected at a feed flow inlet to the bioreactor such that the spent-media separation member is operable to retain cells from the cell suspension at a feed flow side of the separation member and permit media from the cell suspension to pass through the separation member into a permeate side of the separation member.11 . A perfusion cell culture apparatus according to any preceding claim, wherein the pressure source operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor is operable to draw air from the separation member so as to cause a pressure drop in the separation member and / or is operable to push air into the separation member so as to cause a pressure rise in the separation member.

12. A perfusion cell culture apparatus according to any preceding claim, wherein the apparatus is free of moving pressure source parts that are operable to contact the cell suspension or culture media.

13. A perfusion cell culture apparatus according to any preceding claim, wherein the air flow comprises air of a suitable quality for cell culture and / or wherein the air flow is passed through a filter to produce cell culture suitable air prior to contact with the cell suspension.

14. A perfusion cell culture apparatus according to any preceding claim, further comprising a spent-media collection member operable to receive spent-media separated from the cell suspension by the separation member, optionally wherein the collection member comprises a collection vessel operable to receive and hold at least a portion of the spent media.

15. A perfusion cell culture apparatus according to claim 15, wherein the spent-media collection member is fluidly connected to the spent-media separation member, such as to a permeate outlet of the separation member and / or, wherein the collection member is operable to function at a pressure of >100 kPa, such as >500 kPa, such as >1000 kPa.

16. A perfusion cell culture apparatus according to any preceding claim, further comprising a sensor, such as a load sensing balance, ultrasonic level switch and / or a direct contact probe, operable to detect the level or amount of spent-media in the spent-media collection member.

17. A perfusion cell culture apparatus according to claim 17, wherein the sensor is operable to communicate with a control circuit to indicate when a desired point has been reached so as to actuate a switch in the flow direction of the culture media and cell suspension.

18. A perfusion cell culture apparatus according to any preceding claim, further comprising a culture air supply operable to provide air suitable for cell culture to the bioreactor.

19. A perfusion cell culture apparatus according to any preceding claim, further comprising an air filter operable to provide permeate air that is suitable for cell culture, such as sterile air.

20. A perfusion cell culture apparatus according to claim 19, wherein the air filter has an average pore diameter of about 0.22 pm and / or wherein the permeate air is operable to contact the culture to supply gasses to the metabolising cells.

21. A perfusion cell culture apparatus comprising a tangential flow filtration separation member operable to separate spent-media from cells in a cell suspension, wherein the separation member comprises a metal membrane, such as a stainless steel membrane, that comprises a coating comprising an inorganic coating (such as a sintered inorganic coating, for example comprising titanium dioxide).

22. A perfusion cell culture apparatus according to any preceding claim, wherein the apparatus is operable to comprise single direction flow of the cell suspension.

23. A perfusion cell culture apparatus according to any preceding claim, wherein the apparatus comprises at least two bioreactors in sequence and / or in parallel.

24. A method of cell culture perfusion, comprising arranging a perfusion cell culture apparatus according to any of claims 1 to 23, and performing cell culture perfusion with the apparatus.

25. Use of perfusion cell culture apparatus, the apparatus according to any one of claims 1 to 23.

26. A kit of parts for a perfusion cell culture apparatus, such as an apparatus according to any one of claims 1 to 23, the kit of parts comprising: i. optionally a bioreactor; ii. a spent-media separation member; andHi. a pressure source, wherein the separation member and pressure source are operable to be fluidly connected with a bioreactor so that the pressure source is operable to actuate movement of a cell suspension into contact with the separation member from the bioreactor by effecting a change in air flow, wherein the separation member is operable to retain cells and separate spent-media from the cell suspension, and wherein the kit of parts comprises a pressure source operable to actuate return of the cell suspension from contact with the separation member to contact with the bioreactor by effecting a change in air flow, wherein the kit of parts optionally further comprises i. optionally, a spent-media collection member; ii. optionally, a level sensor;Hi. optionally, a culture air supply; and / or iv. optionally, an air filter.

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