WO2022197465A1

WO2022197465A1 - Methods and devices for adding reagents to bioreactors

Info

Publication number: WO2022197465A1
Application number: PCT/US2022/018888
Authority: WO
Inventors: Michael Brown; Ranjeet Patil
Original assignee: Emd Millipore Corporation
Priority date: 2021-03-17
Filing date: 2022-03-04
Publication date: 2022-09-22

Abstract

A method and device for processing biological fluids that includes providing biological fluids within a cell culture media to a bioreactor and growing biological cells therein; delivering the biological fluid wherein a biological fluid is delivered to the bioreactor via an external re-circulating loop or, for example, a port in a manifold; optionally sampling the biological fluid through sample ports; optionally sensing a condition of the biological fluid; optionally delivering the biological fluid to a second manifold which comprises a weldable sample port; adding a reagent to the biological fluid via the sample port; and returning the biological fluid to the bioreactor.

Description

Title: METHODS AND DEVICES FOR ADDING REAGENTS TO BIOREACTORS BACKGROUND Related Applications [0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63/162,206, filed on March 17, 2021, the entire contents of which is incorporated by reference herein in its entirety. Field of the Disclosure [0002] Embodiments of the present disclosure relate to the processing of biological fluids. More particularly, embodiments relate to methods and systems for processing biological materials, wherein reagent(s) are added to a bioreactor comprising a fluid loop for processing biological materials, pharmaceuticals, and therapeutics. Description of Related Art [0003] Methods for cell cultivation within biological fluids, e.g., cell culture media and various processing agents, is conducted using a single use bioreactor or a fermentor container. Conditions within the bioreactor or fermentor, such as gas flow rates (e.g., air, oxygen, nitrogen, etc.), temperature, pH and dissolved oxygen levels, turbidity, and agitation speed/circulation rate, are monitored by sensors and controlled to continuously optimize these conditions within the bioreactor, so that cell cultivation is efficiently achieved. [0004] Current single-use bioreactors include ports for drawing samples for testing for quality control purposes. And, ports are used to introduce various gases and liquids into the bioreactor. Ports may also be used to measure various process parameters including but not limited to the temperature or the pH within the bioreactor. The monitoring and controlling of, for e.g., dissolved oxygen, pH, proteins, etc., and other parameters during bioprocessing is useful to optimize/control various bioprocesses. [0005] Also, agents may be added during bioprocessing. These reagent(s) are added to the bioprocess container, such as a bioreactor, to induce a biologic response, e.g., a transfecting reagent, used to transfect the cells. A plasmid, for a product of interest for cultivating cells to produce during bioprocessing, is a common addition to the bioreactor. Adding a reagent to the bioreactor, typically via a port on the top of a bioreactor, tends to be an inefficient process as the reagent may become trapped in the foam layer that is present due to the normal aeration gases added or formed within the bioreactor, which reduces the efficacy of the reagent and particularly so during high shear mixing. Moreover, the high-volume of biological fluids, e.g., 50 to 3000 liters within bioreactors and a relatively low concentration of cells during addition to the bioreactor, also makes transfection inefficient. [0006] Also, the addition of a relatively low volume reagent to a fluid within a relatively high-volume bioreactor creates a non-concentrated condition which results in less interaction of the reagent with the target molecule or cell. Furthermore, adding the reagent to a high volume increases the mix time in order to gain a homogenous solution, further limiting the effectiveness of the reagent. These limitations of prior art inventions, separately and in tandem are less productive and/or require larger amounts of reagent in the manufacturing process. [0007] Prior attempts to add reagent to bioreactors efficiently have not been successful. Furthermore, current methods of adding reagents, such as transfecting reagents and/or activating agents, in low volumes have not been efficient. Adding these reagents via a re-circulating external loop, which overcome the aforementioned limitations, optionally disposed within manifolds, connected therewith for bioreactors and methods for adding reagents, without increasing the complexity of the bioreactors, therefore, represents an advance in the art. It is to be understood that the efficacy of many reagents, for many differing purposes, not just specific to a transfection reagent, can be enhanced using embodiments of the devices and methods described herein. SUMMARY OF SOME EMBODIMENTS [0008] External loops for adding reagents to a bioreactor, substantially as shown in and/or described in connection with at least one of the figures, as set forth more completely in the claims, are disclosed. It is to be understood that embodiments of the bioreactors and systems discussed herein may be single-use components or multi-use components, i.e., manifolds or bioreactors. The disclosure herein describes some embodiments of systems for the addition of reagents to a bioreactor, such as a single- use bioreactor or biocontainer, for cell culturing and methods of using the same. In some embodiments, an external loop is used to add raw materials and reagents to the bioprocess. In some embodiments, the external loop is a recirculating loop that is in fluid communication with a bioreactor. In some embodiments, the external loop used to add raw materials and reagents to the bioprocess is a port capable of housing a sensor or probe and an external loop. In some embodiments, the external loop provides a smaller volume and/or a smaller cross-sectional area in which to add a reagent, resulting in a more concentrated delivery of the reagent, wherein the interaction between the reagent and the cells is increased and therefore increases the effectiveness of the reagent on either the target product or cell producing the product. In some embodiments, the external loop comprises a first tube connected to an outlet of the bioreactor on a first end and a manifold on a second end and a second tube connected to a second end of the manifold that is connected to an inlet of the bioreactor, wherein a biological fluid can be recirculated out of and into the bioreactor. Embodiments of the devices and the methods relate to any bioprocess and/or biopharma manufacturing process wherein adding a reagent promotes a more concentrated addition to increase effectiveness, including but not limited to bioreactor processes. [0009] Various novel and inventive features of the present disclosure, as well as details of exemplary embodiments thereof, will be more fully understood from the following description and drawings. BRIEF DESCRIPTION OF THE FIGURES [0010] FIG.1 depicts a bioreactor manifold connector body having connector ports and having sterile connector ends, and an inner bore, according to embodiments of the disclosure; [0011] FIG.2 depicts a second bioreactor manifold connector body having connector ports, and having a sterile connector ends, and an inner bore, according to embodiments of the disclosure; [0012] FIG.3 depicts the second bioreactor manifold connector body having connector ports, and a third connector, according to embodiments of the disclosure; [0013] FIG.4 depicts a manifold system, according to some embodiments of the disclosure; [0014] FIG.5 depicts a bioreactor manifold system comprising a bioreactor manifold connector body having connector ports, an optional sensor that is a single use sensor in some embodiments, a recirculation pump, a bioreactor containing medium, such as a cell media, connectors, which can be sterile connectors, and a sampler for removing and/or adding medium to an inner volume of the bioreactor, according to some embodiments of the disclosure; [0015] FIG.6 depicts a second bioreactor manifold system comprising a bioreactor manifold connector body, wherein the bioreactor manifold connector body can be any of the bioreactor manifold connector body having connector ports, an optional sensor that is a single use sensor in some embodiments, a recirculation pump, a bioreactor containing medium, such as a cell media, connectors, which can be sterile connectors, and a sample ports for removing and/or adding medium to an inner volume of the bioreactor, according to some embodiments of the disclosure; [0016] FIG.7 is a flow chart that depicts at least one method of a biological process, according to embodiments of the disclosure; and [0017] FIG.8 is a flow chart that depicts a second method of a biological process, according to embodiments of the disclosure. DETAILED DESCRIPTION OF SOME EMBODIMENTS [0018] So the manner in which the features disclosed herein can be understood in detail, a more particular description of the embodiments of the disclosure, briefly summarized above, may be had by reference to the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this disclosure and are therefore not to be considered limiting of its scope, for the embodiments described and shown may admit to other equally effective embodiments. It is also to be understood that elements and features of one embodiment may be found in other embodiments without further recitation and that identical reference numerals are sometimes used to indicate comparable elements that are common to the figures. [0019] The terms “bioreactor,” “bag,” and “container” are generally used interchangeably within this disclosure. A flexible bioreactor, bag, or container connotes a flexible vessel that can be folded, collapsed, and expanded and/or the like, capable of containing, for example, a biological fluid. A single use bioreactor, bag, or container, typically also flexible, is a vessel that is used once and discarded. A multi- use bioreactor is generally made of stainless steel and can be washed, sterilized and re- used. [0020] The term “sterile” is defined as a condition of being free from contaminants and, particularly within the bioprocessing industry, free from undesirable viruses, bacteria, germs, and other microorganisms and/or adventitious agents. [0021] The term “upstream” is defined as first step processes in the processing of biological materials, such as microbes/cells, mAbs, proteins, including therapeutic proteins, viral vectors, etc., are grown or inoculated in bioreactors within cell culture media, under controlled conditions, to manufacture certain types of biological products. [0022] The term “downstream” indicates those processes in which biological products are harvested, tested, purified, concentrated and packaged. [0023] The term “culture media” indicates a solution to provide nutrients to maintain and support the growth of living cells in a biological fluid. [0024] The term “transient transfection” indicates, for e.g., nucleic acid/genetic material entering the cell that will be expressed temporarily and is not integrated into the cellular genome. [0025] Turning to the figures, FIGS. 1-6 depict some embodiments of the bioreactor manifold systems (10, 70) according to some embodiments of the disclosure. FIG. 1 depicts a bioreactor manifold connector body (20) having connector ports (30a, 30b), and having sterile connector ends (23), and an inner bore (21), according to embodiments of the disclosure. Connector port (30a) may further comprise a septum (32) for the introduction of a reagent. Connector port (30b) may further comprise internal threads so that another component, such as sensor, may be connected therewith. It is further contemplated that the connector port (30b) itself be screwed into internal threads of the bioreactor manifold connector body (20) or have a plug (not shown). [0026] FIG. 2 depicts a second bioreactor manifold connector body (25) having connector ports (30a, 30b), and having a sterile connector ends (23), and an inner bore (21), according to embodiments of the disclosure. As shown, both connector ports (30a, 30b), which are hollow stems, comprise internal threads. The second bioreactor manifold body (25) further comprises a weldable tubing line (32) and a probe port (32c) that comprises a fitting. The fitting replaces a probe that would permit the addition of reagents or removal of cell culture. [0027] FIG. 3 depicts the second bioreactor manifold connector body (25) having connector ports (30a, 30b), and a third connector (30b), according to embodiments of the disclosure. The second bioreactor manifold connector body (25) further comprises tubing (36a, 36b) at opposing ends of the second bioreactor manifold connector body (25). As will be explained below, cells can enter the tubing (36a) from a bioreactor (as described below), become transfected injection into the weldable tubing (32) or modified probe port (32c) and exit the second bioreactor manifold connector body (25) at the tubing (36b). [0028] FIG. 4 depicts a manifold system (60), according to some embodiments of the disclosure. The manifold system (60) comprises the second bioreactor manifold connector body (25) of FIG.3 and further comprises barbs (29) at opposing ends. Each barb (29) has a tubing (36a, 36b) connected therewith and a sterile connector (24) at each end. The manifold system (60) further comprises two sample ports (35) disposed between the tubing (36a) and the two connector ports (30b). A vent (37) is downstream of the connector ports (30b). A weldable tubing (32), adjacent the barb (29) and tubing (36b), is downstream of the vent (37). As will be described further below, the manifold system (60) is an external loop. In other words, at least some embodiments of the external loop comprise wherein the manifold system (60) is part of a recirculating loop located outside of the bioreactor. And, the manifold system (60) provides a way to introduce an agent into the fluid in a concentrated manner before being re-introduced into the bioreactor. It is to be understood that the connector ports (30b), wherein the connector ports have threads, are capable of having an additional component joined therewith. For example, the additional component nay be a barb fitting to connect tubing therewith, a sampling device, a vent, a connector, a sterile connector, a probe port to connect a sensor, such as a turbidity sensor, a pH sensor, a dissolved oxygen sensor, and the like. In some embodiments, a sensor is directly fitted, e.g., screwed into the connector port (30b). [0029] FIG. 5 depicts a bioreactor manifold system (10) comprising a bioreactor manifold connector body (20) having connector ports (30), an optional sensor (38) that is a single use sensor in some embodiments, a recirculation pump (50), a bioreactor (40) containing medium (42), the recirculation pump (50) being in fluid communication with the bioreactor (40) via a first tube (36a), such as a cell media, connectors (22, 24) connected to an inlet and an outlet of the bioreactor (40), which can be sterile connectors, and a sampler (28) for removing and/or adding medium to an inner volume of the bioreactor (40), according to some embodiments of the disclosure. As depicted, there are eight connector ports (30), although the bioreactor manifold connector body may have from two to twelve connector ports (30). It should be noted that the bioreactor manifold connector body (20) may be coupled to any location on the bioreactor vessel (40), so long as that location is accessible by a bioreactor port. The medium (42) can be removed from the bioreactor (40) through the connector (22), pumped into the bioreactor manifold connector body (20) and returned to the bioreactor (40) through the connector (24). [0030] FIG. 6 depicts a second bioreactor manifold system (70) comprising a bioreactor manifold connector body, wherein the bioreactor manifold connector body can be any of the bioreactor manifold connector body (20, 25, 60) having connector ports (30b), an optional sensor (38) that is a single use sensor in some embodiments, a recirculation pump (50), a bioreactor (40) containing medium (42), such as a cell media, connectors (22, 24, 26), which can be sterile connectors, and a sample ports (35) for removing and/or adding medium to an inner volume of the bioreactor (40), according to some embodiments of the disclosure. As depicted, the second bioreactor manifold system (70) comprises the manifold system (60) of FIG. 4 and the second bioreactor manifold connector body (25) in fluid communication therewith tubing (72). The medium (42) can be removed from the bioreactor (40) through the connector (22) and pumped into the bioreactor manifold connector body (60), where the media (42) may be sampled via sample ports (35) and/or sensed by sensor (38). The medium (42) then travels out of the bioreactor manifold connector body (60) into tubing (72), through connector (24) and into the second bioreactor manifold connector body (25). At this point, a reagent, such as a critical reagent, such as a transfecting agent or an activating agent, can be added via the weldable sample line (32) and returned to the bioreactor (40) through the connector (26). As described herein, the transfecting agent or activating agent only acts on a small volume of the media (42) when added at the weldable sample line (32) and is therefore more efficient. In other words, the low cross- sectional area of the external manifold connector body (25) permits the transfecting reagent added at that area to act on more cells than the transfecting agent would act on if merely added to the bioreactor (40). The recirculation pump (50) is in fluid communication with the bioreactor (40) via a first tube (36a), such as a cell media, connectors (22, 24) connected to an inlet and an outlet of the bioreactor (40) and the first manifold connector body (60) and the second manifold connector body (25) similarly as with respect to Figure 5. [0031] FIG. 7 is a flow chart that depicts at least one method 100 of a biological process, according to embodiments of the disclosure. The method 100 starts and proceeds to step 102, wherein a biological fluid is delivered from a bioreactor to a manifold. In some embodiments, the manifold is a MOBIUS® Dual SENSORREADY or a MOBIUS® Quad SENSORREADY that comprises sample ports and/or sensor(s), as marketed by the EMD Millipore Corporation, Burlington, Mass, USA. At step 104, which is optional, wherein the biological fluid is sampled through sample ports. The method 100 proceeds to step 106, which is optional, wherein a condition of the biological fluid is sensed by a sensor, such as a pH, turbidity, or dissolved oxygen sensor or other sensors for biological processes as are known to those in the art. At step 108, the biological fluid optionally enters a second manifold, such as a MOBIUS® Dual SENSORREADY, which comprises a weldable sample port. At step 110, a reagent, such as a critical reagent, such as a transfecting agent, e.g., transfection plasmids, is added to the biological fluid via the weldable sample port. The transfecting agent may be particularly efficacious in transfecting cells because of the ratio of the volume of the biological fluid to the volume of the transfecting agent. At step 112, a decision is made whether to add an additional reagent, which can be the same reagent added at step 110 or a different reagent. If the answer is yes, the method returns to step 102. If the answer is no, the method 100 ends. [0032] FIG.8 is a flow chart that depicts a second method 200 of a biological process, according to embodiments of the disclosure. The second method 200 starts and proceeds to step 202, at which point a biological fluid is delivered from a biocontainer or bioreactor to a manifold having sample ports, as discussed herein. At step 204, which is optional, the biological fluid is sampled through sample ports. The method 200 proceeds to step 206, which is optional, wherein a condition of the biological fluid is sensed by a sensor, such as a pH, turbidity, or dissolved oxygen sensor or other sensors for biological processes as are known to those in the art. At step 208, a reagent(s) is added to a fluid stream of the biological fluid via one or more ports. As described herein, the one or more ports has a smaller cross-section so that the reagent(s) can act on a small volume of the biological fluid. At step 210, a decision is made whether to add another reagent, such as a critical reagent, such as a transfecting agent, e.g., transfection plasmids, via a port on the manifold. The transfecting agent may be particularly efficacious in transfecting cells because of the ratio of the volume of the biological fluid to the volume of the transfecting agent. If the answer is yes, the method returns to step 202. If the answer is no, the method proceeds to step 212, wherein the reagent(s)-treated biological fluid is returned to the bioreactor or biocontainer. Then the method 200 ends. [0033] At least one effect of embodiments of this disclosure is the provision of methods and devices for the robust, optimized addition of reagents, wherein by adding the reagent(s) to an external recirculating loop that is connected to a volume within a bioreactor, a smaller volume is created in which to add the reagent, resulting in a more concentrated delivery of the reagent. A more concentrated delivery increases interaction with, for e.g., cells, and thus increasing the effectiveness of the reagent on either the target product or cell producing a product. Addition of a reagent through an external loop also decreases the mix time of the reagent, resulting in a more homogenous solution that synergistically increases the effectiveness of the reagent. An increase in effectiveness results in higher productivity in, for e.g., transient transfections and a reduction in the amount of reagent required for processing. Embodiments of the disclosure permit higher transfection efficiency of cell lines for transfection of a biological product used for medicinal therapy applications and achieve higher productivity. Embodiments of the methods and/or systems of the disclosure also deliver reagents in a direct way for the reagents to interact with the target molecule within the bioreactor and achieve synergistic reaction efficiencies, resulting in less reagent used within the production bioprocesses. Furthermore, the addition of the reagent in this manner eliminates the risk of the reagent becoming localized within the foam layer at the surface of the liquid within the bioreactor, which would result in an increase of the concentration of the reagent in the biocontainer/bioreactor required to interact with the target molecule or cell. In some embodiments, an equal or an approximately equal volume of reagent, added to a process, results in a more efficient transfection. [0034] Reference throughout this specification to “one embodiment,” “certain embodiments,” “one or more embodiments,” “some embodiments,” or “an embodiment” indicates that a feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Therefore, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in one embodiment,” “some embodiments,” or “in an embodiment” throughout this specification are not necessarily referring to the same embodiment. [0035] Although some embodiments have been discussed above, other implementations and applications are also within the scope of the following claims. Although the specification describes, with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be further understood that numerous modifications may be made to the illustrative embodiments and that other arrangements and patterns may be devised without departing from the spirit and scope of the embodiments according to the disclosure. Furthermore, particular features, structures, materials, or characteristics may be combined in any suitable manner in any one or more of the embodiments. [0036] Publications of patent applications and patents and other non-patent references, cited in this specification are herein incorporated by reference in their entirety in the entire portion cited as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.

Claims

CLAIMS What is claimed is: 1. A method for processing biological fluids, comprising: providing biological fluids within a cell culture media to a bioreactor and growing biological cells therein; delivering the biological fluid from the bioreactor to a manifold; optionally sampling the biological fluid through one or more sample ports; optionally sensing a condition of the biological fluid; adding a reagent to the biological fluid via an external loop wherein the efficacy of the reagent/reaction is enhanced; and returning the biological fluid to the bioreactor. 2. The method of claim 1, wherein an additional reagent is added to the biological fluid. 3. The method of claim 2, wherein the additional reagent comprises the same reagent added or a different reagent. 4. The method of claim 1, wherein the reagent is a transfecting plasmid capable of transfecting the biological cells. 5. The method of claim 1, wherein the sample/addition port is weldable to the manifold. 6. The method of claim 1, further comprising delivering the biological fluid to a second manifold having a sample/addition port. 7. The method of claim 1, wherein the external loop comprises a first tube connected to an outlet of the bioreactor and to a first end of the manifold body and a second tube connected to a second end of the manifold body and an inlet of the bioreactor. 8. The method of claim 1, wherein the bioreactor manifold connector body comprises between two and twelve connector ports. 9. The method of claim 8, wherein the transfecting agent is delivered via at least one of the two to twelve connector ports. 10. A bioreactor manifold system, comprising: a bioreactor manifold connector body having connector ports; a sensor, optionally; a recirculation pump; a bioreactor having an inner volume capable of containing media; at least two connectors; and a sampler, wherein the sampler is capable of removing and/or adding media to the inner volume of the bioreactor. 11. The bioreactor manifold system of claim 10, wherein the sensor is a single use sensor. 12. The bioreactor manifold system of claim 10, wherein the media is a cell media. 13. The bioreactor manifold system of claim 10, wherein at least one of the at least two connectors is a sterile connector. 14. The bioreactor manifold system of claim 10, wherein the bioreactor manifold connector body comprises between two and twelve connector ports. 15. The bioreactor manifold system of claim 10, wherein the bioreactor manifold connector body comprises an external loop having a smaller cross- sectional area than the bioreactor, resulting in a more concentrated delivery of a reagent, wherein the interaction between the reagent and cells within the external loop is increased and therefore increases the effectiveness of the reagent on either a target product or a cell producing the target product.