阅读说明：本技术 用于生物反应器系统的灌注装置 (Perfusion device for bioreactor system ) 是由 Y·列文森 E·亚伯拉罕 S·古普塔 于 2018-09-28 设计创作，主要内容包括：公开了一种灌注装置,用于在生物反应器内的微载体上的细胞培养物生长期间从生物反应器中提取流体介质。还公开了在包含在微载体上的生物反应器中培养细胞的方法。灌注装置包括附连到过滤构件的中空管状构件。过滤构件具有能够以相对高的流量从生物反应器中提取流体介质的孔径和容积。(A perfusion apparatus is disclosed for extracting fluid medium from a bioreactor during growth of a cell culture on a microcarrier within the bioreactor. Also disclosed are methods of culturing cells in a bioreactor comprising the microcarrier. The perfusion apparatus includes a hollow tubular member attached to a filter member. The filter member has a pore size and a volume that enable extraction of the fluid medium from the bioreactor at a relatively high flow rate.)

1. An infusion device, comprising:

a hollow tubular member for perfusion of fluid from a bioreactor, the hollow tubular member having a first end defining a first opening and a second end opposite end defining a second opening, the second opening having a cross-sectional area, an

A filter member located at and attached to a second end of the hollow tubular member, the filter member completely surrounding and closing the second opening, the filter member defining an enclosed volume and a surface area, and wherein a ratio between a cross-sectional area of the second opening and the surface area of the filter member is about 1:5 to about 1: 200.

2. The perfusion device of claim 1, wherein the filter member comprises a porous mesh having an average pore size of greater than about 60 microns.

3. The perfusion device of claim 1, wherein the filter member comprises a porous mesh having an average pore size of greater than about 80 microns.

4. The perfusion device of claim 1, wherein the filter member comprises a porous mesh having an average pore size of about 60 microns to about 150 microns.

5. The perfusion device of any one of the preceding claims, wherein a ratio between a cross-sectional area of the second opening and a surface area of the filter member is about 1:15 to about 1: 100.

6. The perfusion device of any one of the preceding claims, wherein the hollow tubular member is made of stainless steel.

7. The perfusion device of any one of claims 1-5, wherein the perfusion device comprises a single-use perfusion device.

8. The perfusion device of any one of claims 1-5, wherein the hollow tubular member is made of a thermoplastic polymer, and wherein the filter member comprises a polyamide mesh.

9. The perfusion device of any one of the preceding claims, wherein the hollow tubular member is straight from the first end to the second end.

10. The perfusion device of claims 1-8, wherein the hollow tubular member comprises a first straight section, a second straight section, and an angled section between the first and second straight sections for positioning the second opening and filtering member in a position in the bioreactor that is free of contact with the rotating impeller.

11. The perfusion device of claim 10, wherein the angled section is angled about 25 ° to about 45 ° from the first straight section and about 25 ° to about 45 ° from the second straight section.

12. The perfusion device of any one of the preceding claims, wherein the hollow tubular member has a diameter of about 2mm to about 20 mm.

13. The perfusion device of any one of the preceding claims, wherein the hollow tubular member comprises an angular member located near the second end, the hollow tubular member comprising a straight section that transitions to the angular member, the angular member being at an angle of about 50 ° to about 90 ° from the straight section.

14. The perfusion device of any one of claims 1-8, wherein the hollow tubular member and the filter member are movably enclosed in a collapsible bellows, wherein the collapsible bellows includes a sterile connection port at one end for connection to a mating sterile connection port of the bioreactor.

15. The perfusion apparatus of any one of claims 1-8, wherein the filter member comprises a mesh patch on a side wall or a bottom wall of a bioreactor, the filter member further comprising a cone connecting the mesh patch to the hollow tubular member.

16. An infusion device, comprising:

a hollow tubular member for perfusion of fluid from a bioreactor, the hollow tubular member having a first end defining a first opening and a second end defining a second opening, the second opening having a cross-sectional area; and

a filter member located at and attached to the second end of the hollow tubular member, the filter member completely surrounding and closing the second opening, the filter member comprising a porous mesh having an average pore size ranging from about 60 microns or more to about 150 microns or less.

17. The perfusion device of claim 16, wherein the filter member defines an enclosed volume and a surface area, and wherein a ratio between a cross-sectional area of the second opening and the surface area of the filter member is about 1:5 to about 1:200, such as about 1:15 to about 1: 100.

18. The perfusion apparatus of claim 16 or 17, wherein the mesh of the filter member has a uniform pore size.

19. The perfusion apparatus of any one of claims 16-18, wherein the mesh comprises a stainless steel mesh.

20. The perfusion device of any one of claims 16-19, wherein the hollow tubular member comprises a first straight section, a second straight section, and an angled section between the first and second straight sections for positioning the second opening and the filter member in a position in the bioreactor that is free of contact with the rotating impeller.

21. The perfusion device of claim 20, wherein the angled section is angled about 25 ° to about 45 ° from the first straight section and about 25 ° to about 45 ° from the second straight section.

22. The perfusion device of any one of claims 16-21, wherein the hollow tubular member comprises an angular member located near the second end, the hollow tubular member comprising a straight section that transitions to the angular member, the angular member being angled from the straight section by about 50 ° to about 90 °.

23. The perfusion device of any one of claims 16-22, wherein the hollow tubular member and the filter member are movably enclosed in a collapsible bellows, wherein the collapsible bellows comprises a sterile connection port at one end for connection to a mating sterile connection port of the bioreactor.

24. The perfusion apparatus of any one of claims 16-19, wherein the filter member comprises a mesh patch on a side wall or a bottom wall of the bioreactor, the filter member further comprising a cone connecting the mesh patch to the hollow tubular member.

25. A bioreactor system comprising a bioreactor having a bioreactor volume containing microcarriers for growing cells thereon, the bioreactor having a top and defining a port, and wherein the bioreactor system further comprises a perfusion device as defined in any one of claims 16-23 received within the port.

26. A bioreactor system comprising a bioreactor having a bioreactor volume, the bioreactor containing microcarriers for growing cell growth thereon, the bioreactor system further comprising a perfusion apparatus according to claim 24, wherein the bioreactor is in communication with the perfusion apparatus.

27. A method of culturing cell growth, comprising:

seeding biological cells into a bioreactor comprising a fluid medium for cell growth, the biological cells being attached to microcarriers contained in the bioreactor;

perfusing the fluid medium contained in the bioreactor by inserting a perfusion device into the bioreactor, the perfusion device comprising a hollow tubular member having a first end defining a first opening and a second opposite end defining a second opening, the perfusion device further comprising a filter member located at and attached to the second end of the hollow tubular member, the filter member completely surrounding and closing the second opening, and wherein the fluid medium is extracted from the bioreactor by the perfusion device, the filter member preventing extraction of the microcarriers from the bioreactor; and

replenishing the fluid medium within the bioreactor to promote cell growth.

28. A method of culturing cell growth comprising:

seeding biological cells into a bioreactor comprising a fluid medium for cell growth, the biological cells being attached to microcarriers contained in the bioreactor;

perfusing a fluid medium contained in the bioreactor, placing the bioreactor in communication with a perfusion apparatus, the perfusion apparatus comprising a hollow tubular member having a first end defining a first opening and a second opposite end defining a second opening, the perfusion apparatus further comprising a filter member formed as a mesh patch in a wall of the bioreactor, the perfusion apparatus further comprising a flexible cone connected from the mesh patch to the second opening of the hollow tubular member, and wherein the fluid medium is extracted from the bioreactor by the perfusion apparatus, the filter member preventing extraction of the microcarriers from the bioreactor; and

replenishing the fluid medium within the bioreactor to promote cell growth.

Background

Bioreactors are devices that can perform biological reactions or processes on a laboratory or industrial scale and are widely used in the biopharmaceutical industry. The bioreactor may be used for batch applications, wherein the biological material supplied to the bioreactor remains in the bioreactor until the end of the reaction time. Alternatively, the bioreactor may be used for perfusion applications, wherein the fluid medium contained in the bioreactor is periodically or continuously removed and re-supplied to the bioreactor in order to replenish the nutrients contained in the fluid medium and possibly remove harmful by-products produced in the process.

In some bioreactor systems, microcarriers are added to the bioreactor to promote cell growth. For example, cells may adhere to the surface of the microcarriers for further growth and propagation. In this way, the microcarriers provide a greater surface area for cell culture growth within the reactor. In fact, some anchorage-dependent cells, such as certain animal cells, require attachment to a surface in order to grow and divide.

Microcarriers can be made of a variety of different materials, including polymers. The microcarriers may have any suitable shape, and in some applications, comprise round beads. Microcarriers may generally have a particle size of about 200 microns to about 350 microns. In some systems, microcarriers are suspended in culture medium, which is caused by bulk agitation, which optimizes and maximizes growth conditions in the bioreactor system.

One problem encountered in the past in bioreactor systems containing microcarriers is the ability to quickly remove fluid from the bioreactor without removing microcarriers or damaging microcarriers or cells growing on the microcarriers. Attempts to increase the outflow rate of a bioreactor system containing microcarriers may result in flow blockage or blockage due to improperly sized dip tubes, clogging by microcarriers, and/or tube collapse due to a suction head created upstream of the flow-driving pump. In view of the foregoing, there is a need for an apparatus and method for rapid removal of fluid media from a bioreactor containing microcarriers.

Disclosure of Invention

In general, the present disclosure relates to a perfusion apparatus that is capable of removing fluid media from a bioreactor at relatively high flow rates without damaging the bioreactor or the cells growing in the reactor. More particularly, the present disclosure relates to a perfusion apparatus specifically designed to remove culture fluid medium from a bioreactor containing microcarriers at a relatively high flow rate. As will be described in more detail below, the perfusion device is particularly suitable for removing fluid without removing or damaging the microcarriers or cells attached to the microcarriers. The present disclosure also relates to a method for promoting cell growth in a bioreactor system, wherein a perfusion device is used to remove culture fluid medium for replenishment and for further cell growth.

For example, in one embodiment, the present disclosure relates to a perfusion apparatus comprising a hollow tubular member for perfusing fluid from a bioreactor. The hollow tubular member may have a length sufficient to be inserted into the bioreactor. For example, the hollow tubular member may have a length sufficient to extend toward the bottom of the bioreactor. The hollow tubular member may extend through a port in the top or side of the bioreactor. The hollow tubular member has a first end defining a first opening and a second opposite end defining a second opening. The second opening is used for inserting the fluid medium into the bioreactor and for extracting the fluid medium. The second opening of the hollow tubular member may have a cross-sectional area designed to enable a desired volumetric flow rate to be extracted from the bioreactor.

According to the present disclosure, the perfusion device further comprises a filter member located at and attached to the second end of the hollow tubular member. The filter member completely surrounds and closes the second opening. The filter member has a length extending beyond the second end of the hollow tubular member and defines an enclosed volume. Even when the bioreactor contains microcarriers, the closed volume is of sufficient size to meet the desired fluid flow. For example, in one embodiment, the ratio between the cross-sectional area of the second opening and the surface area of the filter member is about 1:5 to about 1:200, e.g., about 1:15 to about 1: 100. The surface area of the filter member as used herein is the total surface area of the porous portion of the filter member that allows fluid to enter the hollow tubular member.

In one embodiment, the filter member comprises a porous mesh. For example, the porous mesh may comprise a screen, such as a stainless steel screen or a polymer mesh. In one embodiment, the average pore size of the mesh is greater than about 60 microns, such as greater than about 70 microns, such as greater than about 80 microns. For example, the average pore size of the mesh may be from about 60 microns to about 150 microns. In one embodiment, the apertures in the mesh are all of a substantially uniform size. In one embodiment, the mesh has a pore size of about 60 microns to about 150 microns.

The diameter of the hollow tubular member and the second opening may typically be greater than about 2mm, such as greater than about 4mm, such as greater than about 8mm, such as greater than about 10mm, such as greater than about 12mm, such as greater than about 14mm, such as greater than about 16mm, such as greater than about 18mm, such as greater than about 20 mm. The diameter of the hollow tubular member is typically less than about 50mm, for example less than about 30mm, for example less than about 20mm, for example less than about 14 mm. The hollow tubular member may be made of a variety of different materials, such as stainless steel or polymers. In one embodiment, the hollow tubular member is straight from the first end to the second end. In an alternative embodiment, the hollow tubular structure may be shaped such that the second end does not interfere with an impeller that may rotate in the bioreactor. For example, in one embodiment, the hollow tubular member may include a first straight section, a second straight section, and an angled section between the first straight section and the second straight section. The angled section may extend at an angle of about 25 ° to about 45 ° from the first straight section. Similarly, the angled section may extend at an angle of about 25 ° to about 45 ° from the second straight section. In one embodiment, the first and second straight sections are parallel to a vertical axis extending through the bioreactor.

In one embodiment, the hollow tubular member may further comprise an angle member at the second end. The hollow tubular member may comprise a straight member that transitions to an angled member. The angle member may be angled from about 50 ° to about 90 ° from the straight section. For example, in one embodiment, the angle member forms a right angle at the end of the hollow tubular member. In this regard, the angled member may be positioned toward the bottom of the bioreactor and may be substantially parallel to the bottom surface of the bioreactor when the perfusion device is extended into the bioreactor. For example, in one embodiment, the angle member may be designed to place the filter member under an impeller contained within the bioreactor.

In one embodiment, the hollow tubular member and the filter member may be completely closed for sterile closure of the port connected to the bioreactor. A plastic flexible bellows can enclose the hollow tubular member and the filter member. A sterile connection port may be attached to one end of the bellows. The bioreactor port may have a mating sterile connector. When the bioreactor and mating sterile connectors of the bellows are connected, the bellows can be collapsed and the filter member and hollow tubular member can be inserted into the bioreactor port.

In one embodiment, the perfusion apparatus may comprise a filter member located on a side wall or a bottom wall of the bioreactor. The filter member may be a mesh patch on the side wall or bottom wall of the bioreactor. A flexible cone may connect the mesh patch to the hollow tubular member for outputting fluid from the bioreactor.

The present disclosure also relates to a method of growing a cell culture in a bioreactor. The method comprises seeding cells in a bioreactor comprising a microcarrier. Biological cells can be attached to microcarriers to continue cell growth. The bioreactor may contain a fluid medium for providing nutrients and food to the growing cell culture.

In one embodiment, for example, the perfusion apparatus may be designed to remove fluidic media at a rate of greater than about 20L/day, such as greater than about 25L/day, such as greater than about 30L/day, such as greater than about 35L/day, such as greater than about 40L/day, such as greater than about 45L/day, such as greater than about 50L/day.

When the fluid medium is removed from the bioreactor, new fluid medium is added to the bioreactor to further promote the growth of the biological cells attached to the microcarriers. After a desired amount of cells have been grown, a release agent may be added to the bioreactor to separate the cells from the microcarriers. The cells can then be harvested and used as desired.

Other features and aspects of the present disclosure are discussed in more detail below.

Drawings

A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying figures wherein:

FIG. 1 is a cross-sectional view of one embodiment of a bioreactor system according to the present disclosure;

FIG. 2 is a side view of one embodiment of a perfusion device made in accordance with the present disclosure;

FIG. 3 is a side view of another embodiment of an irrigation device made in accordance with the present disclosure;

FIG. 4A is a perspective view of one embodiment of a filter member attached to a perfusion apparatus according to the present disclosure;

FIG. 4B is a side view of the filter member shown in FIG. 4A;

FIG. 5 is a side view of another embodiment of an irrigation device made in accordance with the present disclosure;

FIG. 6 is a perspective view of another embodiment of a bioreactor system according to the present disclosure;

FIGS. 7A to 7C are perspective views of one embodiment of a bioreactor system having a sterile closed connection between the bioreactor and a perfusion apparatus;

FIG. 8A is a perspective view of another embodiment of a bioreactor system according to the present disclosure;

FIG. 8B is a side view of a perfusion apparatus that may be used with the bioreactor system shown in FIG. 8A;

FIG. 9 is a side view of another embodiment of a bioreactor system according to the present disclosure;

fig. 10A and 10B show data generated from an accuracy test of a perfusion apparatus made according to the present disclosure.

Repeat use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the present disclosure.

Detailed Description

It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present disclosure.

The present disclosure relates generally to methods and systems for culturing and propagating cells and/or cell products in a bioreactor. According to the present disclosure, biological cells, such as mammalian cells, are combined with a suitable microcarrier in a bioreactor. The cells are attached to microcarriers to promote cell growth. The bioreactor contains a fluid medium, such as a fluid growth medium. The biological cells are cultured under suitable conditions and in a suitable culture medium to promote reproduction and growth of the cells until a desired number of cells can be harvested from the bioreactor.

According to the present disclosure, in addition to containing one or more microcarriers, the bioreactor is designed to operate in perfusion mode during cell culture. In particular, the fluid medium contained in the bioreactor is continuously or at least periodically removed and replenished. In the past, problems have been encountered in removing liquid media from a bioreactor containing microcarriers at a flow rate sufficient to maintain optimal growth conditions within the reactor. In this regard, the present disclosure relates to a perfusion apparatus that is capable of rapidly removing fluidic media from a bioreactor without removing microcarriers, without damaging microcarriers, and/or without damaging cell cultures within the bioreactor.

Referring to fig. 1, one embodiment of a bioreactor system according to the present disclosure is shown. The bioreactor system includes a bioreactor 10. The bioreactor 10 comprises a hollow vessel or container comprising a bioreactor volume 12 for receiving a cell culture attached to microcarriers suspended in a fluid growth medium. As shown in fig. 1, the bioreactor system may further include a rotatable shaft 14 coupled to an agitator, such as an impeller 16.

Bioreactor 10 may be made of a variety of different materials. For example, in one embodiment, bioreactor 10 may be made of a metal, such as stainless steel. Metal bioreactors are typically designed to be reusable.

Alternatively, bioreactor 10 may comprise a single-use bioreactor made of a flexible polymer film. The membrane or shape conforming material can be liquid impermeable and can have an internal hydrophilic surface. In one embodiment, bioreactor 10 may be made of a flexible polymer film designed to be inserted into a rigid structure, such as a metal container for assuming a desired shape. Polymers that can be used to make the flexible polymeric film include polyolefin polymers such as polypropylene and polyethylene. Alternatively, the flexible polymer film may be made of polyamide. In yet another embodiment, the flexible polymeric film may be formed from multiple layers of different polymeric materials. In one embodiment, the flexible polymer film may be gamma irradiated.

Bioreactor 10 may have any suitable volume, for example, bioreactor 10 may have a volume of from 100 milliliters to about 10,000 liters or more, for example, bioreactor 10 may have a volume 12 of greater than about 0.5L, for example greater than about 1L, for example greater than about 2L, for example greater than about 3L, for example greater than about 4L, for example greater than about 5L, for example greater than about 6L, for example greater than about 7L, for example greater than about 8L, for example greater than about 10L, for example greater than about 12L, for example greater than about 15L, for example greater than about 20L, for example greater than about 25L, for example greater than about 30L, for example greater than about 35L, for example greater than about 40L, for example greater than about 45L, bioreactor 10 may have a volume of generally less than about 20,000L, for example less than about 15,000, L, for example less than about 10,000 liters, for example less than about 5 liters, such as less than about 5,000 liters, for example less than about 5 liters, such as less than about 200 liters in another alternative embodiment, such as less than about 200 liters to about 200 liters of bioreactor 10, such as less than about 200 liters

In addition to impeller 16, bioreactor 10 may include various additional equipment, such as baffles, spargers, gas supplies, ports, and the like, which allow for the cultivation and propagation of biological cells. In addition, the bioreactor system may include various probes for measuring and monitoring pressure, foam, pH, dissolved oxygen, dissolved carbon dioxide, and the like.

In one embodiment, bioreactor 10 includes a top defining a plurality of ports. The ports may allow for supply lines and feed lines to and from bioreactor 12 for the addition and removal of fluids and other materials. Further, the bioreactor system may be placed in association with a load cell for measuring the mass of the culture within the bioreactor 10.

In alternative embodiments, the plurality of ports may be located at different locations on bioreactor 10. For example, in one embodiment, the ports may be located on the sidewall of the bioreactor, as shown in fig. 6-8. In another embodiment, the port may be located at the bottom of the bioreactor, as shown in fig. 9. For example, a bioreactor made of a flexible polymer membrane may include a port located at the bottom of the vessel.

As shown in fig. 1, bioreactor 10 may include a rotatable shaft 14 attached to at least one impeller 16. The rotatable shaft 14 may be coupled to a motor for rotating the shaft 14 and impeller 16. The impeller 16 may be made of any suitable material, such as a metal or a biocompatible polymer. Examples of impellers suitable for use in the bioreactor system include hydrofoil impellers, high consistency pitch blade impellers, high consistency hydrofoil impellers, rashton (Rushton) impellers, pitch blade impellers, gentle marine blade impellers, and the like. Further, the rotatable shaft 14 may be coupled to a single impeller 16 as shown in fig. 1, or may be coupled to two or more impellers. When two or more impellers are included, each impeller may be spaced apart along the axis of rotation 14. In one embodiment, impeller 16 is rotated a sufficient amount to keep the microcarriers contained in bioreactor 10 suspended in the fluid medium without damaging the biological cells attached to the microcarriers.

In one embodiment, the bioreactor system may further comprise a controller, which may comprise one or more programmable devices or microprocessors. The controller may be used to maintain optimal conditions within bioreactor 10 that promote cell growth. For example, the controller may communicate and control the thermal cycler, load sensors, control the pump, and receive information from various sensors and probes. For example, the controller may control and/or monitor pH, dissolved oxygen tension, dissolved carbon dioxide, temperature, agitation conditions, alkaline conditions, fluid growth medium conditions, pressure, foam level, and the like. For example, based on the pH reading, the controller may be configured to adjust the pH level by adding the necessary amount of acid or base. The controller may also use the carbon dioxide gas supply to lower the pH. Similarly, the controller may receive temperature information and control the fluid to be fed into the water jacket around the bioreactor to increase or decrease the temperature.

Bioreactor 10 may also be in communication with a perfusion apparatus 20 as shown in FIG. 1, in accordance with the present disclosure. Perfusion device 20 may extend through a port in the top of bioreactor 10. As shown, the perfusion apparatus 20 may extend into the bioreactor 10 and be placed near the bottom of the bioreactor without interfering with the impeller 16. The perfusion apparatus 20 is used to continuously or periodically withdraw the liquid medium from the bioreactor 10 without extracting the microcarriers contained in the bioreactor. For example, the perfusion apparatus 20 of the present disclosure can extract fluid at relatively high flow rates without removing microcarriers or damaging cells attached to microcarriers. For example, microcarriers may comprise biocompatible beads or small particles and provide attachment sites for propagation of biological cells. For example, in one embodiment, the microcarrier may be made of a polymer, such as a polysaccharide. For example, in one particular embodiment, the microcarrier may be made of dextran. The microcarrier may have a particle size or diameter of about 150 microns to about 400 microns.

Referring to fig. 2, 4A and 4B, one embodiment of an irrigation device 20 that may be used in accordance with the present disclosure is shown. Referring to fig. 2, the irrigation device 20 includes a hollow tubular member 22. The hollow tubular member 22 may include a first end 24 defining a first opening and a second, opposite end 26 defining a second opening. The hollow tubular member 22 may be made of any suitable material that is biocompatible with cell cultures. For example, the hollow tubular member 22 may be made of a metal, such as stainless steel.

In an alternative embodiment, the hollow tubular member may be made of a polymer. For example, in one embodiment, infusion device 20 may be designed to be discarded after a single use. In this embodiment, the hollow tubular member 22 may be made of a polymeric material. For example, the hollow tubular member may be made of a polyolefin such as polypropylene or polyethylene. Alternatively, the hollow tubular member 22 may be made of polyamide. Otherwise, the hollow tubular member 22 may be made of a plastic material that can be gamma irradiated.

The hollow tubular member 22 may be flexible or rigid. The hollow tubular member 22, first opening and second opening may generally have diameters suitable for the particular application and amount of fluid that needs to be extracted from the bioreactor 10. For example, the diameter of the hollow tubular member 22 may generally be greater than about 2mm, such as greater than about 4mm, such as greater than about 6mm, such as greater than about 8mm, such as greater than about 10 mm. The diameter of the hollow tubular member 22 is typically less than about 40mm, such as less than about 30mm, such as less than about 20mm, such as less than about 15mm, such as less than about 11mm, such as less than about 10mm, such as less than about 8mm

The first end 24 of the hollow tubular member 22 may include a pipe connection for connecting the hollow tubular member 22 to a plastic pipe. The pipe connections may be any of a variety of weldable pipe types. The outer diameter of the tubing connection at first end 24 may generally have an outer diameter that is appropriate for the particular application and the amount of fluid that needs to be extracted from the bioreactor. For example, the pipe connection may typically have an outer diameter of about 3mm or more, such as about 6mm or more, such as about 13mm or more, such as about 19mm or more, such as about 26 mm. The pipe connections typically have an outer diameter of about 26mm or less.

The hollow tubular member 22 may be made from a single piece of material or may be made from multiple pieces of material that are joined together. The hollow tubular member 22 may be straight from the first end 24 to the second end 26. Alternatively, the hollow tubular member 22 may include an angle member 28 as shown in fig. 2. In the embodiment shown in fig. 2, an angle member 28 extends from bioreactor 10 for directing fluid out of the bioreactor in a desired direction. The angle member 28 as shown is generally at a right angle to the straight section 30 of the hollow tubular member 22. However, the angle member 28 may be at any suitable angle relative to the straight or vertical section 30 of the hollow tubular member 22.

When used to remove fluid from the bioreactor 10, the perfusion device should be of sufficient length such that the second end 26 of the hollow tubular member 22 is located near the bottom surface of the bioreactor 10. In this regard, the length of the straight section 30 of the perfusion apparatus 20 is generally greater than the length (or depth) of the bioreactor 10. For example, the length of straight section 30 may be greater than about 110%, such as greater than about 120%, such as greater than about 150% of the length of bioreactor 10. Typically, straight section 30 is less than about 500%, such as less than 300%, for example less than about 200% of the length of bioreactor 10.

According to the present disclosure, the infusion device 20 further includes a filter member 32 located at the second end 26 of the hollow tubular member 22. The filter member 32 is shown in more detail in fig. 4A and 4B. The filter member 32 is sufficiently porous to allow the fluid medium to pass through the perfusion apparatus 20 at relatively high flow rates without allowing the microcarriers to flow or otherwise damage the microcarriers. For example, in one embodiment, the filter member 32 may be made of a porous mesh, such as a stainless steel screen. Alternatively, the filter member 32 may be made of a polymeric material. For example, in one embodiment, the filter member 32 may be made of a polyamide screen. For example, the polymer mesh may be more flexible and less fragile than a filter element made of metal. The infusion device 20 having the polymeric hollow tubular member 22 and the filter member 32 may further include a polymeric shell (not shown) surrounding the filter member 32.

The mesh may have a desired pore size. The pore size may or may not be uniform across the web. In one embodiment, the mesh has a pore size greater than about 60 microns, such as greater than about 70 microns, such as greater than about 80 microns, such as greater than about 90 microns. The pore size is typically less than about 150 microns, such as less than about 130 microns, such as less than about 120 microns, such as less than about 110 microns. It has been found that the above-mentioned pore size optimizes the fluid flow in a non-destructive manner. For example, a smaller pore size does not allow sufficient flow and clogging problems may be encountered. However, in other embodiments, a smaller aperture may be required. For example, in other embodiments, the pore size may be less than about 50 microns. For example, a filter element made of a polymer may have a smaller pore size. For example, when made from a polymer, the pore size can be about 18 microns to about 50 microns, such as about 20 microns to about 30 microns.

Referring to fig. 4A and 4B, the filter member 32 is shown in greater detail. As shown, the filter member 32 is attached to the second end 26 of the hollow tubular member 22. For example, in the illustrated embodiment, the filter member 32 completely surrounds and closes the opening at the second end 26 of the hollow tubular member 22. The filter member 32 may be attached to the hollow tubular member 22 using any suitable method or technique. For example, the filter member 32 may be welded to the hollow tubular member 22, may be adhered to the hollow tubular member 22, or may be mechanically attached to the hollow tubular member. For example, in one particular embodiment, the filter member 32 may be a resin welded to the hollow tubular member 22.

As shown in FIG. 4B, in one embodiment, the filter member 32 has a length L that extends beyond the second end 26 of the hollow tubular member 22. in this manner, the filter member 32 defines a closed volume 34. the size of the closed volume 34 may depend on the flow requirements of the system and may be proportional to the cross-sectional area of the opening of the second end 26. for example, the size of the closed volume 34 may be sufficient to allow sufficient fluid to flow through the filter member and into the hollow tubular member 22, which may be desirable for certain applications.

For example, in one embodiment, the ratio between the cross-sectional area of the opening at the second end 26 and the surface area of the filter member 32 may be greater than about 1:5, such as greater than about 1:10, such as greater than about 1:15, such as greater than about 1:20, such as greater than about 1:25, such as greater than about 1:30, such as greater than about 1:35, such as greater than about 1: 40. the ratio between the cross-sectional area of the opening at the second end 26 and the surface area 34 of the filter member 32 may generally be less than about 1:1000, such as less than about 1:500, such as less than about 1:200, such as less than about 1:150, such as less than about 1:100, such as less than about 1: 80. for example, when the diameter of the second opening at the second end 26 is from about 2mm to about 20mm, the length L of the filter member 32 is generally greater than about 20mm, such as greater than about 30mm, such as greater than about 40mm, such as greater than about 50mm, and generally less than about 500mm, such as less.

In the embodiment shown in fig. 4A and 4B, the filter member 32 has an elongated shape that terminates in an angled end 38. However, it should be understood that the filter member 32 may have any suitable shape. For example, the shape of the filter member 32 may depend on a shape that maximizes surface area while being conveniently placed in the bioreactor 10.

In accordance with the present disclosure, the cross-sectional area of the hollow tubular member 22, the enclosed volume 34 of the filter member 32, and the pore size of the filter member 32 are all selected to optimize flow. In particular, the perfusion apparatus 20 of the present disclosure is designed to allow a relatively high flow rate out of the bioreactor 10. For example, in one embodiment, the flow rate through perfusion apparatus 20 may depend on the volume of bioreactor 10. For example, perfusion apparatus 20 may be designed to extract more than about 50% of the bioreactor volume per day (24 hours), such as more than about 60% of the bioreactor volume, such as more than about 70% of the bioreactor volume, such as more than about 80% of the bioreactor volume, such as more than about 90% of the bioreactor volume, such as more than about 100% of the bioreactor volume, such as more than about 110% of the bioreactor volume, such as more than about 120% of the bioreactor volume, such as more than about 130% of the bioreactor volume, such as more than about 140% of the bioreactor volume, such as more than about 150% of the bioreactor volume. Typically, the flow rate through perfusion apparatus 20 is typically less than about 500% of the bioreactor volume per day, such as less than about 200% of the bioreactor volume per day.

In one particular example, perfusion apparatus 20 is designed to extract greater than about 20L of fluid from bioreactor 10 per day, such as greater than about 30L of fluid per day, such as greater than about 40L of fluid per day, and typically less than about 100L of fluid per day.

The embodiment of the perfusion apparatus 20 as shown in fig. 2 comprises a straight or vertical section 30 intended to be inserted into the bioreactor 10. Once inserted into the bioreactor 10, the straight or vertical section 30 remains substantially parallel to the vertical axis of the bioreactor and/or the rotatable shaft 14. Thus, the length of the straight or vertical section 30 is at least as long as the length or depth of the bioreactor 10. However, in one embodiment, the straight or vertical section 30 may interfere with the impeller 16 contained within the bioreactor 10. Thus, in other embodiments, the shape of perfusion apparatus 20 may be altered to provide a better fit within the bioreactor.

For example, referring to FIG. 3, another embodiment of an irrigation device 120 is shown. The infusion device 120 includes a hollow tubular member 122 including a first end 124 and a second opposite end 126. A filter member 132 made in accordance with the present disclosure is attached to second end 126. The hollow tubular member 122 also includes an angle member 128 at the first end 124.

In the embodiment shown in fig. 3, irrigation device 120 includes a first straight section 140, a second straight section 142, and an angled section 144. An angled section 144 is located between the first and second straight portions 140, 142. As shown in fig. 1, an angled section 144 may be included in the hollow tubular member 22 to prevent the irrigation device 120 from interfering with the impeller 16 contained in the bioreactor 10. In particular, the angled section 144 positions the second end 126 of the hollow tubular member 122 adjacent to the wall of the bioreactor 10. In one embodiment, the angular segment 144 may form an angle with the first straight portion 140 of about 10 ° to about 80 °, such as an angle of about 25 ° to about 45 °. For example, the angle between the angled section 144 and the first straight section 140 may generally be greater than about 20 °, such as greater than about 30 °, such as greater than about 40 °, and generally less than about 60 °, such as less than about 50 °. Similarly, the angle between the angular segment 144 and the second straight segment 142 may be from about 10 ° to about 80 °, such as from about 25 ° to about 45 °.

The length of straight sections 140 and 142 and the length of angled section 144 may also vary depending on the geometry of bioreactor 10 and various other factors. In one embodiment, for example, the angle segment 144 may be greater than about 5%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, and generally less than about 50%, such as less than about 40%, such as less than about 30%, such as less than about 20% of the total length of the first straight segment 140, the second straight segment 142, and the angle segment 144.

Referring to fig. 5, yet another embodiment of a perfusion apparatus 220 made in accordance with the present disclosure is shown. The infusion device 220 includes a hollow tubular member 222 including a first end 224 and a second opposite end 226. The filter member 232 is attached to the second end 226 of the hollow tubular member 222. The hollow tubular member 222 includes a first straight section 250, a second straight section 242, and an angled section 244 between the first straight section 240 and the second straight section 242. The irrigation device 220 also includes a first angled member 228 located at the first end 224 of the hollow tubular member 222.

In the embodiment shown in fig. 5, the irrigation device 220 further comprises a second angular member 250 located at the second end 226 of the hollow tubular member 222. The second angle member 250 is used to position the filter member 232 near the bottom of the bioreactor 10. For example, the second angular member 250 may form an angle with the first straight section 240 that is generally greater than about 40 °, such as greater than about 50 °, such as greater than about 60 °, such as greater than about 70 °, such as greater than about 80 °, and generally less than about 120 °, such as less than about 100 °. For example, as shown in fig. 5, in one embodiment, the second angular member 250 forms a right angle with the first straight section 240 of the hollow tubular member 222. In this way, the perfusion apparatus 220 may be placed in the bioreactor to avoid interference with the impeller. On the other hand, the second angle member 250 allows the filter member 232 to extend along the bottom of the bioreactor toward the center of the bioreactor or toward the wall of the bioreactor depending on the particular application. Accordingly, the second angle member 250 may have a length suitable for placing the filter member 232 in a desired position. For example, in one embodiment, the length of the second angle member 250 may be generally greater than about 20mm, such as greater than about 30mm, such as greater than about 40mm, such as greater than about 50mm, such as greater than about 60mm, such as greater than about 70mm, such as greater than about 80mm, such as greater than about 90mm, such as greater than about 100mm, generally less than about 500mm, such as less than about 300mm, such as less than about 200mm, such as less than about 180mm, such as less than about 160mm, such as less than about 140 mm. However, the length of the second angle member 250 may depend on the size and volume of the bioreactor 10. Thus, the length may be greater or less than the above dimensions.

Referring to fig. 6, yet another embodiment of a bioreactor system made in accordance with the present disclosure is shown. The bioreactor system includes a bioreactor 310 having a port 318 located on a sidewall of the bioreactor. The bioreactor system further includes a perfusion apparatus 320 having a hollow tubular member 322 and a filter member 332. An infusion set 320 may be inserted into port 318. The filter member 332 is similar to the filter member shown in more detail in fig. 4A and 4B. Perfusion device 320 may minimize the amount of space occupied in the bioreactor, which may allow filter member 332 to include a longer mesh with a larger surface area in some embodiments. As shown in fig. 6, allowing perfusion apparatus 320 to enter bioreactor 310 at the bottom sidewall reduces the total amount of material that protrudes into bioreactor 310 compared to embodiments of perfusion apparatuses inserted through ports at the top of the bioreactor, such as shown in fig. 1.

Referring now to fig. 7A through 7C, another embodiment of a bioreactor system made in accordance with the present disclosure is shown. The bioreactor system includes a bioreactor 410 having a port 418 located on a lower sidewall of the bioreactor. The embodiment of fig. 7A-7C also includes an infusion device 420 having a hollow tubular member 422 and a filter member 432.

In the embodiment shown in fig. 7A-7C, the infusion device 420 further includes a collapsible bellows structure 440 for fully closed, sterile access. The bellows 420 may be plastic. The hollow tubular structure 422 and the filter member 432 are completely encased in the bellows 440. Bellows 440 forms an enclosed environment that can be sterilized to contain hollow tubular structure 422 and filter member 432. The infusion device 420 also includes a rigid tunnel 446 within the bellows 440 leading to the sterile connection port 442. Sterile connection port 442 can be any commercially available sterile connection port compatible with bioreactor 410. For example, the sterile connection port may be Kleenpak manufactured by Pall Biotech^TMSterile connector, manufactured by SartoriusA sterile connector, a ready ReadyMate disposable connector manufactured by GE Healthcare L ife Sciences, or other commercially available sterile connector bioreactor 410 includes a mating sterile connector 444 in port 418 on the wall of the bioreactor.

As shown in fig. 7B, sterile connections 442 and 444 of perfusion apparatus 420 and bioreactor 410 are first connected to each other. A seal is formed between sterile connectors 442 and 444. Then, as shown in fig. 7C, an opening is formed between sterile connectors 442 and 444. Bellows 440 may then be collapsed and hollow tubular member 422 may be pushed through bioreactor 410, extending filter member 432 into bioreactor 410. As hollow tubular member 422 and filter member 432 are pushed into the bioreactor, bellows 440 collapse.

Referring to fig. 8A and 8B, yet another embodiment of a bioreactor system made in accordance with the present disclosure is shown. The bioreactor system includes a bioreactor 510 having a conical perfusion device 520. The filter member 532 of the perfusion apparatus 520 is formed as a mesh patch on the wall 511 of the bioreactor 510. Mesh patches may be located on the sidewalls 511 of the bioreactor 510 as shown in fig. 8B. The perfusion device 520 has an enclosed volume 534 formed by a cone 536 leading from the filter member 532 to the outlet hollow tubular member 522. In some embodiments, taper 536 may be flexible.

Referring to fig. 9, another embodiment of a bioreactor system made in accordance with the present disclosure is shown. The bioreactor system includes a bioreactor 610, the bioreactor 610 having a conical filling device 620, the filling device 620 being operable as a filter drain for the bioreactor 610. The filter member 632 of the perfusion apparatus 620 is formed as a mesh patch on the bottom wall of the bioreactor 610. The infusion device 620 has an enclosed volume 634 formed by a cone 636, the cone 636 leading from the filter member 632 to the outlet hollow tubular member 622. In some embodiments, taper 636 may be flexible. The design of the embodiment shown in fig. 9 allows the maximum amount of liquid to be drained from the bioreactor leaving only the microcarriers in the bioreactor. An agitator, such as impeller 16 shown in fig. 1, may be included in bioreactor 610 to prevent microcarriers from depositing on the mesh patch and clogging filter member 632.

Examples of the invention

In each test, a bioreactor system is established including a perfusion apparatus inserted into the bioreactor, the bioreactor including fluidic media, microcarriers, and mesenchymal stem cells, the perfusion apparatus is inserted into the bioreactor through a port within a sterile enclosure.

FIG. 10A shows the results of a test conducted with the perfusion apparatus shown in FIG. 2 and a bioreactor having a bioreactor volume of 3L, the test was run seven times, FIG. 10A shows the individual results of each test run, it can be seen from FIG. 10A that the perfusion apparatus was delivered within about 10% of the set point in each test run.

FIG. 10B shows the results of a test conducted with the perfusion apparatus shown in FIG. 3 and a bioreactor having a bioreactor volume of 50L the test was conducted three times FIG. 10B shows the individual results for each test run it can be seen from FIG. 10B that the perfusion apparatus was delivered within 10% of the set point (50L) in each test run.

The apparatus, apparatus and methods described herein are applicable to the culture of any desired cell line, including prokaryotic and/or eukaryotic cell lines. Furthermore, in embodiments, the apparatus, facility and method are suitable for culturing suspension cells or anchorage-dependent (adherent) cells, and for production operations configured for the production of pharmaceutical and biopharmaceutical products, such as polypeptide products, nucleic acid products (e.g. DNA or RNA), or cells and/or viruses, such as cells and/or viruses for cell and/or virus therapy.

In embodiments, the cell expresses or produces a product, such as a recombinant therapeutic or diagnostic product. As described in more detail below, examples of products produced by cells include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that specifically bind to antigens but are not related to antibody structure, such as DARPins, affibodies, adnectins or IgNARs), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutic agents (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cell therapeutic agents (e.g., pluripotent stem cells, mesenchymal stem cells and adult stem cells), vaccines or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA) or DNA (e.g., plasmid DNA), antibiotics or amino acids. In embodiments, the apparatus, facilities and methods may be used to produce biosimilar drugs.

As mentioned above, in embodiments, the apparatus, facilities and methods allow the production of eukaryotic cells, such as mammalian cells or lower eukaryotic cells, such as yeast cells or filamentous fungal cells, or prokaryotic cells, such as gram-positive or gram-negative cells, and/or products of eukaryotic or prokaryotic cells, such as proteins, peptides, antibiotics, amino acids, nucleic acids (such as DNA or RNA), to be synthesized by eukaryotic cells in a large scale manner. Unless otherwise specified herein, equipment, facilities and methods may include any desired volume or production capacity, including but not limited to laboratory scale, pilot scale and full production scale capacities.

Further, unless otherwise specified herein, the apparatus, facilities, and methods may include any suitable reactor(s), including but not limited to stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and/or spouted bed bioreactors. As used herein, "reactor" may include a fermentor or a fermentation unit, or any other reaction vessel, and the term "reactor" may be used interchangeably with "fermentor". For example, in some aspects, an example bioreactor unit may perform one or more or all of the following, supply of nutrients and/or carbon sources, injection of suitable gases (e.g., oxygen), inlet and outlet flow of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, oxygen and CO₂Maintenance of levels, maintenance of pH levels, agitation (e.g., stirring), and/or cleaning/sterilization. Example reactor units, such as fermentation units, may contain multiple reactors within a unit, for example the unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and/or a facility may contain multiple units with a single or more reactors within a facility. In various embodiments, the bioreactor may be adapted for batch, semi-fed batch, fed-batch, perfusion, and/or continuous fermentation processes. Any suitable reactor diameter may be used. In an embodiment of the present invention,non-limiting examples include 100 ml, 250 ml, 500 ml, 750 ml, 1 l, 2 l, 3 l, 4 l, 5 l, 6 l, 7 l, 8 l, 9 l, 10 l, 15 l, 20 l, 25 l, 30 l, 40 l, 50 l, 60 l, 70 l, 80 l, 90 l, 100 l, 150 l, 200 l, 250 l, 300 l, 350 l, 400 l, 450 l, 500 l, 550 l, 600 l, 650 l, 700 l, 750 l, 800 l, 850 l, 900 l, 950 l, 1000 l, 1500 l, 2000 l, 2500 l, 3000 l, 3500 l, 4000 l, 4500 l, 5000 l, 6000 l, 7000 l, 8000 l, 9000 l, 10,000 l, 15,000 l, 20,000 l and/or 50,000 l, furthermore, suitable reactors may be formed of any suitable single use of glass, stainless steel or non-compatible steel, such as stainless steel, and/or non-compatible alloys, such as stainless steel, including any other suitable metals, including stainless steel, glass, stainless steel, and/or non-compatible alloys.

In embodiments, unless otherwise indicated herein, the apparatuses, facilities, and methods described herein may further include any suitable unit operations and/or equipment not otherwise mentioned, such as operations and/or equipment for separating, purifying, and isolating these products. Any suitable facility and environment may be used, such as a conventional stick-build facility, modular, mobile, and temporary facility, or any other suitable configuration, facility, and/or layout. For example, in some embodiments, a modular clean room may be used. Further, unless otherwise specified, the devices, systems, and methods described herein may be housed and/or executed in a single location or facility, or alternatively, housed and/or executed in separate or more locations and/or facilities.

By way of non-limiting example and not limitation, U.S. publication numbers 2013/0280797; 2012/0077429, respectively; 2011/0280797, respectively; 2009/0305626, respectively; and U.S. patent No. 8,298,054; 7,629,167, respectively; and 5,656,491 describe example facilities, equipment, and/or systems that may be suitable, all of which are incorporated herein by reference in their entirety.

In embodiments, the cell is a eukaryotic cell, e.g.Mammalian cells may be, for example, human or rodent or bovine cell lines or cell strains examples of such cells, cell lines or cell strains are, for example, mouse myeloma (NSO) cell lines, Chinese Hamster Ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby Kidney cells), VERO, SP2/0, YB2/0, Y0, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK-293, VERO, PER. C6, He L A, 1, EB2, EB3, oncolytic or hybridoma cell linesThe eukaryotic cell can also be an avian cell, cell line or cell strain, e.g., CHOK1SV (L onza Biologics, Inc.)Cell, EB14, EB24, EB26, EB66 or EBvl 3.

In one embodiment, the eukaryotic cell is a stem cell. The stem cells can be, for example, pluripotent stem cells including Embryonic Stem Cells (ESCs), adult stem cells, induced pluripotent stem cells (ipscs), tissue specific stem cells (e.g., hematopoietic stem cells), and Mesenchymal Stem Cells (MSCs).

In one embodiment, the cell is a differentiated form of any of the cells described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.

In embodiments, the cell is a hepatocyte, e.g., a human hepatocyte, an animal hepatocyte, or a nonparenchymal cell. For example, the cell can be a plateable (plateable) metabolically competent human hepatocyte, a plateable induced competent human hepatocyte, a plateable quick Transporter Certified^TMHuman hepatocytes, suspension-qualified human hepatocytes (including 10 donor and 20 donor pooled hepatocytes), human hepatokupffer cells, human hepatic stellate cells, dog hepatocytes (including single and pooled Beagle (Beagle) hepatocytes), mouse hepatocytes (including CD-1 and C57BI/6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus monkey (Cynomolgus monkey) hepatocytes or Rhesus monkey (Rhesus monkey (Rhesus monkey) hepatocytes), cat hepatocytes (including brachyptic cat hepatocytes) and rabbit hepatocytes (including new zealand white rabbit hepatocytes). examples of hepatocytes are commercially available from trianglle Research L abs, LL C,6Davis driven Research Park, North Carolina Park, USA, 27709.

In one embodiment, the eukaryotic cell is a lower eukaryotic cell, such as a yeast cell, e.g., a Pichia (Pichia) (e.g., Pichia pastoris), Pichia methanolica (Pichia pastoris), Pichia kluyveri (Pichia kluyveri), and Pichia angusta (Pichia angusta)), a Komagataella (e.g., Komagataella pastoris, Komagataella parapeupatoria, or Komagataella phaffii), a Saccharomyces (Saccharomyces) (e.g., Saccharomyces cerevisiae), Saccharomyces kluyveri (Saccharomyces kluyveri), Saccharomyces uvarum (Saccharomyces uvarum), Saccharomyces cerevisiae (Saccharomyces uvarum), Candida (Candida), Candida albicans (Candida), Candida strain (Candida), Candida utilis (Candida), Candida strain (Candida), Candida utilis (Candida), Candida utilis (Candida), Candida utilis (Candida), Candida utilis (, hansenula polymorpha (Hansenula polymorpha), yarrowia lipolytica (yarrowia lipolytica), or Schizosaccharomyces pombe (Schizosaccharomyces pombe). Pichia pastoris (Pichia pastoris) is preferred. Examples of Pichia pastoris (Pichia pastoris) strains are X33, GS115, KM71, KM 71H; and CBS 7435.

In one embodiment, the eukaryotic cell is a fungal cell, e.g. Aspergillus (Aspergillus) (such as Aspergillus niger (a. niger), Aspergillus fumigatus (a. fumigus), Aspergillus oryzae (a. oryzae), Aspergillus nidulans (a. nidulans)), Acremonium (such as Acremonium thermophilum), Chaetomium (such as Chaetomium thermophilum), Chrysosporium (such as Chaetomium thermophilum), Cordyceps (Cordyceps) (such as Cordyceps militaris), corynebacterium (corynebacterium), corynebacterium (Chrysosporium), Fusarium (Fusarium) (such as Fusarium oxysporum), morbid (such as Myceliophthora), Myceliophthora (such as Myceliophthora carotovora), Myceliophthora (Myceliophthora), Myceliophthora (such as Myceliophthora carotovorax). Neurospora (Neurospora) (such as Neurospora crassa (n. crassa)), Penicillium (Penicillium), sporothrix (Sporotrichum) (such as sporothrix thermophilus (s. thermophile), rhizopus (Thielavia) (such as rhizopus terrestris (t. terrestris), rhizopus (t. heterothylica)), Trichoderma (Trichoderma) (such as Trichoderma reesei (t. reesei)), or Verticillium (such as Verticillium dahliae (v. dahliae)).

In one embodiment, the eukaryotic cell is an insect cell (e.g., Sf9, Mimic)^TMSf9，Sf21，High Five^TM(BT1-TN-5B 1-4) or BT1-Ea88 cells), algal cells (for example, of the genus Geotrichum (genus Amphora), the genus Diatom (genus Bacillariophyceae), the genus Dunaliella (Dunaliella), Chlorella (Chlorella), the genus Chlamydomonas (Chlamydomonas), the phylum Cyanophyta (cyanobacteria) (cyanobacteria)), the genus Nannochloropsis (Nannochloropsis), the genus Spirulina (Spirulina) or the genus Coccinum (Ochromas)), or plant cells (for example, cells from monocotyledonous plants (such as maize, rice, wheat or green bristlegrass), or from dicotyledonous plants (such as cassava, potato, soybean, tomato, tobacco, alfalfa, Phytophyta (Phytophyllum patens) or Arabidopsis (Arabidopsis)).

In one embodiment, the cell is a bacterium or a prokaryotic cell.

In an embodiment, the prokaryotic cell is a gram-positive cell, such as Bacillus (Bacillus), Streptomyces (Streptomyces) Streptococcus (Streptococcus), Staphylococcus (Staphylococcus) or lactobacillus (L Bacillus) can be used, such as Bacillus subtilis (b.subtilis), Bacillus amyloliquefaciens (b.amyloliquefaciens), Bacillus licheniformis (b.licheniformis), Bacillus natto (b.natto) or Bacillus megaterium (b.megaterium) in an embodiment, the cell is Bacillus subtilis (b.subtilis), such as Bacillus subtilis (b.subtilis)3NA and Bacillus subtilis (b.subtilis) 168. Bacillus (Bacillus) 1214 can be obtained from e.g. Bacillus genetics, Bacillus science 556,484, Bacillus subtilis 12, Bacillus subtilis).

In one embodiment, the prokaryotic cells are gram-negative cells, such as Salmonella (Salmonella spp.) or Escherichia coli (Escherichia coli), such as TGI, TG2, W3110, DH1, DHB4, DH5a, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, X L1-Blue, and Origami, and those derived from Escherichia coli (E.coli) B-strains, such as B L-21 or B L21 (DE3), all of which are commercially available.

Suitable host cells are commercially available, for example from culture collections, such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

In embodiments, the cultured cells are used to produce proteins, e.g., antibodies, e.g., monoclonal antibodies and/or recombinant proteins, for therapeutic use. In embodiments, the cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in an embodiment, molecules having a molecular weight of about 4000 daltons to greater than about 140,000 daltons may be prepared. In embodiments, these molecules may have a range of complexities and may include post-translational modifications, including glycosylation.

In embodiments, the protein is, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other botulinum neurotoxin serotypes), alfcosidase alpha, daptomycin (daptomycin), YH-16, chorionic gonadotropin alpha, filgrastim (filgrastim), cetrorelix (cetrorelix), interleukin-2, aldesleukin (aldesleukin), teskin (teceuleukin), denenkin-toxin linker (deniileukin diftox), interferon alpha-n 3 (injection), interferon alpha-nl, DL-8234, interferon, trinexalide (gamma-1 a), interferon gamma, thymosin alpha 1, tasomine, Digium, ViaaTAb, Fab Echitab, Crokatab, nesiritide, apremide, axatetra, ritodrel (rebitf), tetromin, calcitonin alpha (alfa), injectable osteoporotrichin (osteoporosis), osteoporosis), etanercept, polyglutamaldehyde hemoglobin 250 (bovine), curbenxol alpha, collagenase, capecitabine, recombinant human epidermal growth factor (topical gel, wound healing), DWP401, dabbepotein alpha, epoetin omega, epoetin beta, epoetin alpha, desipramine, lepirudin, bivalirudin, nona prothrombin alpha (nonacog alpha), coagulation factor IX powder injection (Mononine), etactin alpha (eptacog alpha) (activated), recombinant factor VIII + VWF, concentrated recombinant antihemophilic factor (Recommatee), recombinant factor VIII, factor VIII (recombinant), Alphnemate, octocog alpha (octocogag), factor VIII, palifermin, indikininase, tenecteplase, alteplase, pamiteplase, reteplase, neterpenase, monteplase, mentepenase, pFLC, PFFSH, FSH, finasterin, retionin, dessertraline, and further peganin, glucagon, ezetimibe, pramlintide, iniglucerase, thiolase, Leucotropin, molgramostim, triptorelin acetate, histrelin (subcutaneous implant, Hydron), deslorelin, histrelin, nafarelin, leuprorelin sustained release long acting drug (depot) (Atrigel), leuprorelin implant (DUROS), goserelin, Eutropin, KP-102 program, growth hormone, mecamylamine (growth disorder), enfavirtide, Org-33408, insulin glargine, insulin glulisine, insulin (inhalation), insulin lisine, insulin detemir, insulin (oral, RapidMist), mecamylamine, anakinra, simethionin, simon interleukin, 99mTc (99 mTc-apcitic), interferon beta-beta, interferon, bevacetin, bevacein, bevacerin, bevacizine, leupeptin, bevacizine, bellfurf (Bilive), insulin (recombinant), recombinant human insulin, insulin aspart, mecasenin, Roferon (Roferon) -A, interferon- α 2, Alfaferone, Complex α -1(interferon alfacon-1), recombinant human luteinizing hormone of interferon α, Avonex, streptokinase α, trafmine, ziconotide, talirelin, dibotramine α, atosiban, Bekaplan, eptifibatide, Zeima, CTC-111, Shanvac-B, Sc vaccine (tetravalent HPV), octreotide, lanreotide, ansetron, argasidase β, argasidase α, Raganise, copper acetate (topical gel), Labralizase, ranibizumab, interferon γ -1B (Actimmune), PEG-intron, Geranib (Tricomin), recombinant house dust mite, recombinant allergy injection, recombinant human hormone desensitization (PTH 1-84), osteoporosis), epoetin delta, transgenic antithrombin III, allicin, hyaluronidase (Vitrase), recombinant insulin, interferon-alpha (oral lozenges), GEM-21S, vapreotide (vapreotide), idum sulfatase (idursufase), omapatrilat (omapatrilat), recombinant serum albumin, certolizumab pegol (certolizumab pegol), carboxypeptidase (glucarpiase), human recombinant C1 esterase inhibitor (angioedema), lantipilase, recombinant human growth hormone, enfuvirtide (injection, Biojector 2000), VGV-1, interferon (alpha), russetitan, aviptadil (inhalation, pulmonary disease), eptifibatide, ecteinkala, oxcana, aureganan, Aurograb, cetomagnen acetate, ADI-PEG-20, LDI-200, degarelix, cindelsubex, farradex 1379, islad-247, MDX-247, teriparatide (osteoporosis), tifacogin, AA4500, T4N5 liposome lotion, rituximab, DWP413, ART-123, Chrysalin, desmoprase, Andropsin, follitropin alpha (corifollitropin alpha), TH-9507, teduglutide, Diamylad, DWP-412, growth hormone (slow release injection), recombinant G-CSF, insulin (inhalation, AIR), insulin (inhalation, Techno spheres), insulin (inhalation, AERx), RGN-303, DiaPep277, interferon beta (hepatitis C virus infection (HCV)), interferon alpha-N3 (oral), Beraslacian, transdermal insulin patch, AMG-531, P-8, Xerecpt, Obecap (Opebaceae), SVPabacacan, GV-1001, Lymphoman, leopard (ceronan), Lipolan-25, Lipolan-P36829, melanophore vaccine (Lipocalin), Lipocalin-P-8, Xenopian, Lipocalin-P-52, Lipocalin, CTP-37, Insegia, vitespen, human thrombin (frozen, surgical hemorrhage), thrombin, TransMID, snake venom plasmin (alfimeprase), Prykexi (Puricase), terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinant FGF-I (injectable, vascular disease), BDM-E, gap junction regulator (rotigotide), ETC-216, P113, MBI-AN, duramycin (inhalation, cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman Birk inhibitor concentrate, XMP-629,99 mTc-Hynic-annexin V, kahalalide F, CTCE-9908, teverelix (extended release), ozarelix, romidepsin (BAY-504798, interleukin 4, PRrnX-321, peninsulin, Pecanteddy, Petacidin, Pentakino-015, IL-21, ATN-161, cilengitide, albumin interferon (Albuferon), Biphasix, IRX-2, omega-interferon, PCK-3145, CAP-232, pasireotide, huN901-DMI, ovarian cancer immunotherapeutic vaccines, SB-249553, Oncovax-CL, Oncovax-P, BLP-25, Cervax-16, multi-epitope peptide melanoma vaccines (MART-1, gp100, tyrosinase), Nanofilide, AT (inhalation), rAAT (skin disease), CGRP (inhalation, asthma), pegsunecept, thymosin beta 4, prilin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (oral, eligen), calcitonin (oral, osteoporosis), elsamirrilin, capromorelin, Carvela, Cardevava, 131-AFII, KK-601, t-10, Urapidine, dilacta, hematide, Chrysalin (topical), rNAPc2, recombinant factor V111 (PEGylated liposomes), bFGF, PEGylated recombinant staphylokinase variants, V-10153, SonoLysis Prolyse, neuroVax, CZEN-002, islet cell neotherapy, rGLP-1, BIM-51077, LY-548806, Exenatide (controlled release, Medisorb), AVE-0010, GA-GCB, Aflorelin (avorelin), ACM-9604, linaclotide acetate (linac acetate), CETi-1, Hemospan, VAL (injectable), fast acting insulin (injectable, Viadynaud), insulin (inhaled), insulin (oral, insulin), recombinant human methionine leptin, pimira subcutaneous injection, intranasal (inhaled powder), eczema, dry powder (inhaled powder, dry asthma-1068, NBMM-1068, AT-001, PI-0824, Org-39141, Cpn10 (autoimmune/inflammatory), talctoferrin (topical), rEV-131 (ophthalmic), rEV-131 (respiratory disease), oral recombinant human insulin (diabetes), RPI-78M, Opplelbumin (oral), CYT-99007CTLA4-Ig, DTY-001, Vallarast (valategorast), interferon alpha-n 3 (topical), IRX-3, RDP-58, Tauferon, bile salt-stimulated lipase, Mepase, alkaline phosphatase, EP-2104R, melanosan (Melanotan) -II, Bremelanotan (bremelelanottide), ATL-104, recombinant human microfibrillar lylase, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, tenascin A, SI-03, GHR-002, GHRH-6615 LAB-66728, LAB-728, malaria vaccine (virosome, Pevipro), ALTU-135, parvovirus B19 vaccine, influenza vaccine (recombinant neuraminidase), malaria/HBV vaccine, anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine, Tat-toxoid, YSPSL, CHS-13340, PTH (l-34) liposome cream (Novasome), Ostabolin-C, PTH analogue (topical, psoriasis), MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85A vaccine (tuberculosis), FARA04, BA-210, recombinant pestis FIV vaccine, AG-702, OxSODrol, rBetV1, Der-pl/Der-p2/Der-p7 allergen targeted vaccine (dermatophagoides pteronyssinus allergen), PR1 peptide antigen (leukemia), mutant ras vaccine, HPV-16E7 lipopeptide vaccine, maze toxin (CML) vaccine, CML vaccine (CML), CML vaccine, WT 1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptine, tipermin (telbermin) (dermatology, diabetic foot ulcers), Lupingquwei, reticulose, rGRF, HA, alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin therapeutic vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828, ErbB 2 specific immunotoxin (anti-cancer), DT3SSIL-3, TST-10088, PRO-1762, Probocox, cholecystokinin-B/gastrin receptor binding peptide, 111 In-GF, AE-37, trasnizumab-1, antagonist G, IL-12 (recombinant), Comab-34, IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19 based radioimmunotherapeutic agents (cancer), Re-188-P-2045, AMG-386, DC/1540/KLH vaccines (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccines (peptide), NA17.A2 peptide, melanoma vaccines (pulsed antigen therapy), prostate cancer vaccines, CBP-501, recombinant human lactoferrin (FX-06, AP-214, WAP-8294A (injectable), ACP-HIP, N-11031, peptide YY [3-36] (obese, intranasal), FGLL, atacicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 (nasal, osteoporosis), F-18-CCR1, AT-1100 (celiac/diabetes), JPD-003, PTH (7-34) liposomal cream (Novasome), duramycin (ophthalmology, dry eye), CAB-2, CTCE-0214, glycosylated PEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, factor XIII, aminopolycine, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, teverrelix (immediate release), EP-51216, hGH (controlled release, Biosphere), OGP-1, Cifuwei peptide, TV4710, ALG-889, Org-41259, rhCCIO, F-991, thymopentin (pulmonary disease), r (m) CRP, liver-selective insulin, surelin, L19-IL-2 fusion protein, elastase inhibitor (elafin), NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonists (thrombocytopenia), AL-108, AL-208, nerve growth factor antagonists (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide (eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion vaccine (Therapore), EP-1043, pediatric vaccine for Streptococcus pneumoniae, malaria vaccine, Neisseria meningitidis (Neisseria meningitidis) group B vaccine, Streptococcus neonate group B vaccine, anthrax vaccine, HCV vaccine (gpEl + gpE2+ MF-59), otitis media therapy, HCV vaccine (core antigen + ISCATRIX), hPTH (1-34) (transdermal, ViaDerm), 768974, SYN-101, N-0052, PGisucumine, BIM-23190, tuberculosis vaccine, polytyrosine epitope (Tp-tyrosine), cancer vaccine, enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular targeting TNF (solid tumor), desmopressin (controlled release oral), onercept and TP-9201.

In some embodiments, the polypeptide is adalimusMonoclonal antibody (HUMIRA), infliximab (REMICADE)^TM) Rituximab ((RITUXAN)^TM/MAB THERA^TM) Etanercept (ENBRE L)^TM) Bevacizumab (AVASTIN)^TM) Trastuzumab (HERCEPTIN)^TM) Pegelagliptin (NEU L ASTA)^TM) Or any other suitable polypeptide, including biosimilars and bioremodelers.

Other suitable polypeptides are those listed below and in table 1 of us patent 2016/0097074:

watch 1

In embodiments, the polypeptide is a hormone, hemagglutination/coagulation factor, cytokine/growth factor, antibody molecule, fusion protein, protein vaccine or peptide as shown in table 2.

Table 2 exemplary products

In embodiments, the protein is a multispecific protein, such as a bispecific antibody as shown in table 3.

TABLE 3 bispecific format

These and other modifications and variations to the present disclosure may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present disclosure, which is more particularly set forth in the appended claims. Further, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the disclosure so further described in such appended claims.