JP7634938B2 - Systems and methods for therapeutic cell expansion and differentiation in bioreactors - Patents.com - Google Patents

Description

COPYRIGHT AND TRADE DRESS NOTICE A portion of the disclosure of this patent document contains material that is subject to copyright protection. This patent document may display and/or describe matter that is or may be the trade dress of the owner. The copyright and trade dress owner has no objection to anyone reproducing by facsimile the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright and trade dress rights.

RELATED APPLICATION INFORMATION This application is a continuation of U.S. application Ser. No. 16/282,129, filed Feb. 21, 2019, and claims priority from U.S. application Ser. No. 62/634,077, filed Feb. 22, 2018, entitled BIOREACTOR SYSTEMS AND METHODS FOR DIFFERENTIATION OF CELLS, which are expressly incorporated herein by reference.

FIELD OF THEINVENTION Systems and methods for complete and efficient media exchange, especially at large volume scale, for the expansion and directed differentiation of therapeutic cells, including cell aggregates in bioreactors, microcarriers with attached cells, or single cells.

Bioprocesses involving cells grown in suspension culture with mixing in bioreactors have been developed for a wide range of cell and gene therapy applications. Depending on their type and properties, these therapeutic cells grow clumped together as aggregates, attached to the surface of microcarriers (MCs), or suspended as individual cells. As part of the upstream process, the liquid medium in which the cells are suspended needs to be exchanged; that is, the spent medium is removed and fresh medium is added. This is necessary to replenish nutrients or supply specific growth hormones, as well as to remove metabolic waste products and other undesirable by-products. Upstream processes requiring medium exchange are cell expansion (increasing the total number of cells) and directed differentiation (directing pluripotent cells to turn into specific cell types).

There are various techniques for performing media exchange in bioreactors. One common method is to temporarily suspend agitation and allow all cell aggregates, MCs with cells growing on their surface, or suspended single cells to settle by gravity to the bottom of the bioreactor. Once a bed of settled cell aggregates, MCs with attached cells, or single cells is formed, the supernatant of spent media is removed, fresh media is added, and agitation is resumed to resuspend the cell aggregates, MCs with attached cells, or single cells. This method has two potential problems, which become worse as the working volume of the bioreactor increases. First, the temporary cessation of mixing can result in cell damage through undesired flocculation, nutrient depletion, and deviations in critical process parameters such as temperature, pH, and dissolved oxygen levels. Second, it is difficult to completely remove all spent media, since withdrawing the supernatant too close to the settled cell aggregates, MCs with attached cells, or bed of single cells can result in cell loss, while using a filtered retention device can result in flocculation, blockage, and cell damage. Certain processes, such as multi-step directed differentiation of pluripotent stem cell (PSC) aggregates, can suffer from reduced efficiency and yield if growth factors remaining in the previously used residual media are not completely removed between each differentiation step. A process that can achieve complete media exchange in large-scale bioreactors while minimizing potential damage to the cells would greatly improve the yield and efficiency of the process for cell expansion and differentiation, and would therefore be an invaluable tool for the commercial manufacturing of emerging cell and gene therapies.

PSCs can be derived from human embryos or by inducing pluripotency in adult somatic cells. The distinguishing property of PSCs is their ability to differentiate into virtually any cell type in the human body, making them a promising cell therapy tool to potentially treat a wide variety of different disease indications. Furthermore, PSCs can be grown indefinitely as cell aggregates in culture, which is crucial to meet dosage needs that can range from millions to even billions of cells per person. Attempting to produce huge amounts of cells at a commercial scale using traditional 2D manufacturing platforms would be extremely costly and therefore unfeasible. Instead, 3D suspension culture in bioreactors represents the best option for the development and scale-up of PSC bioprocesses.

One prerequisite for PSC production is a directed differentiation step performed in vitro that induces cells to transform into the target cell type. First, a cell expansion phase occurs in a bioreactor. The differentiation phase requires multiple media exchange steps in situ in the bioreactor once the expansion phase is complete. While complete media exchange can be achieved relatively easily and completely on a small scale in an R&D setting, achieving it on a large scale for commercial production presents a major challenge.

To achieve successful commercial manufacturing of cell therapy products, rapid, efficient, and scalable media exchange techniques are required for bioprocesses involving therapeutic cells.

This application discloses a methodology for complete media exchange with minimal damage to therapeutic cells growing as cell aggregates, on the surface of microcarriers, or as suspended single cells, using at least two bioreactors and one external device designed for therapeutic cell separation and retention. Instead of therapeutic cells remaining in the bioreactor during media exchange, they are instead concentrated in the spent media and removed to an external separation and retention device that thoroughly washes them with fresh media before returning them to a different bioreactor prepared with the same media used for washing.

Various types of separation and retention devices can be used for this process. Their methods of operation can include centrifugation (conventional or continuous centrifugal force), acoustic sedimentation, or simple filtration. The main purpose of these devices is to isolate, concentrate, and wash therapeutic cells such as cell aggregates, MCs with attached cells, or single cells in suspension.

This novel method for complete media exchange eliminates the problems associated with gravity-based settling and enables large-scale production of therapeutic cells. The technique is particularly useful for directed differentiation of pluripotent stem cells (PSCs), which grow as cell aggregates.

Envisioned embodiments utilize at least two bioreactors and one external device designed for the separation and retention of therapeutic cells. After the expansion or differentiation process step is completed in the first bioreactor, the spent media containing the therapeutic cells is transferred to the separation and retention device, where the therapeutic cells are collected and concentrated. They are then washed with fresh media required for the next process step, and then immediately transferred to the second bioreactor, already pre-filled with the same media and pre-conditioned for the required parameters such as temperature, pH, and dissolved oxygen.

Since a prolonged delay phase during cell growth can be detrimental to the overall expansion efficiency, it is desirable to have a preconditioned bioreactor ready for the therapeutic cells to return to immediately after they leave the separation and retention device. The total time to prefill and precondition the bioreactor is likely to take longer than the concentration and washing steps of the separation and retention device, especially if the bioreactor volume is larger. Thus, while the expansion step is underway in the first bioreactor, prefilling and preconditioning of the second bioreactor can begin. After all the therapeutic cells have left the first bioreactor, it can in turn be prefilled and preconditioned as the third bioreactor. The "third" (previously first) bioreactor here should be ready to receive the therapeutic cells after they have moved from the second bioreactor to the separation and retention device and back from the device. Alternatively, if the first bioreactor cannot be prepared in time, an entirely new, third bioreactor can be pre-filled and pre-conditioned instead; any number of bioreactors can be used as dictated by the time required to pre-fill and pre-condition the bioreactors. This time will only increase as the cell production process and bioreactor volumes scale up. By using separation and retention devices as a bridge to cycle between bioreactors, multiple complete media exchange steps can be accomplished efficiently and quickly, even at large scale.

Another embodiment utilizes only one bioreactor and one external separation device. Depending on the time required for concentrating and washing the therapeutic cells in the external device, a single bioreactor can be used for a complete media exchange. After all the spent media is removed from the bioreactor, it must be refilled and conditioned before the therapeutic cells are concentrated and washed in the external device and ready to be returned. Otherwise, the same problems associated with the sedimentation method, where the cells sit idle without proper mixed culture conditions, can occur. However, as the bioreactor volume increases, the time required to fill and precondition with media also increases excessively. Therefore, it may be more difficult to avoid negative impacts on the therapeutic cells with this method compared to methods with two or more bioreactors.

FIG. 1 shows the multi-step directional differentiation process for the generation of insulin-producing pancreatic β-cells in vitro; FIG. 2 shows the system of the present application with two vertical wheel bioreactors and one general example of an external device designed for the separation and retention of therapeutic cells such as cell aggregates, microcarriers (MCs) with cells attached to their surface, or suspended single cells; FIG. 3 shows the system of the present application with multiple vertical wheel bioreactors and separation and retention devices designed for therapeutic cell separation and retention.

The present application provides systems and methods for the expansion and differentiation of cells grown as aggregates, on the surface of an MC, or as single suspension cells in a bioreactor with complete media exchange. In a typical embodiment, the operation of two bioreactors alternates, with an external separation and retention device acting as a bridge between them. However, the concepts described herein can also facilitate the use of more than two bioreactors, as well as multiple separation and retention devices.

Furthermore, the medium exchange technique described herein is particularly useful for the large-scale differentiation of pluripotent stem cells (PSCs), but can also be utilized for the expansion of other anchorage-dependent cells, such as mesenchymal stem cells (MSCs) or human primary cells or cell aggregates grown on microcarriers.

Furthermore, various types of bioreactors can be used for cell expansion and differentiation. In a preferred embodiment, a vertical wheel bioreactor is used due to its relatively low shear rate, which minimizes potential damage to anchorage-dependent cells. However, other bioreactor configurations with alternative agitation methods, such as stirred impellers, can be used.

As previously mentioned, a unique requirement for PSC production is a directed differentiation step that induces cells to transform into the target cell type. For example, Figure 1 shows a four-step sequence in which various growth factors are used to induce pluripotent cells through various steps until insulin-producing pancreatic β cells are produced.

Typically, the differentiation phase requires multiple media exchange steps in situ in the same bioreactor where the expansion phase was completed. Between each differentiation step, PSC aggregates settle to the bottom of the vessel, the spent media is removed, and new media with specific growth factors is added for the next differentiation step. As already mentioned, there are two challenges. First, settling of cell aggregates to the bottom of the bioreactor can lead to cell damage. Second, it is difficult to completely remove the spent media, whether from the supernatant above the level of the settled cell aggregates or via a filtered cell retention device. Any carryover media from the previous differentiation step can reduce the efficiency of the subsequent differentiation stage, using a completely different media. Ideally, the no longer desired growth factors and any remaining by-products should be completely removed before adding another medium with new growth factors for the next differentiation step.

To solve the problems inherent in large-scale differentiation of cell aggregates, directed differentiation, which requires complete media exchange, can be performed in a vertical wheel bioreactor system in conjunction with an external separation and retention device. Instead of allowing the cell aggregates to settle, the culture medium is removed to a device connected to the outside of the vertical wheel bioreactor. Within the separation and retention device, the cell aggregates can be concentrated and washed with buffer and fresh media for the next differentiation step, which completely removes the spent media, stale growth factors, and by-products. The concentrated and washed cell aggregates are then immediately returned to a different vertical wheel bioreactor, pre-filled with the same differentiation media used for washing and ready for the next differentiation step. In this way, PSC aggregates can be rapidly and efficiently differentiated in large volumes without damaging the cells. This new large-scale differentiation process will improve the overall yield of target cell production as well as the manufacturability of differentiated cells on a commercial scale.

Various separation and retention devices such as centrifugation (conventional or continuous anti-centrifugal force), acoustic sedimentation, or simple filtration can be used for this process. Any of these devices can be easily coupled with the vertical wheel bioreactor to allow complete media exchange, thus improving the yield and efficiency of cell expansion or differentiation even at large scale. The entire cell manufacturing process, including cell expansion and directed differentiation aspects, can be performed and optimized at large scale in a single-use vertical wheel bioreactor.

Figure 2 shows the system of the present application with a general example of a separation and retention device 20 and two bioreactors 30, 32. The first bioreactor 30, which in the illustrated embodiment is a vertical wheel bioreactor, is used, for example, to grow PSC aggregates in an expansion medium. The low shear humid environment of the vertical wheel bioreactor allows these aggregates to reach high cell densities at large scale (e.g., 50 L working volume) during the expansion phase.

At the transition from the expansion phase to the differentiation phase, the PSC aggregates can be harvested from the first bioreactor 30 and transferred to a separation and retention device 20. Various types of separation and retention devices can be used for this process. Their methods of operation can include centrifugation (conventional or continuous anti-centrifugal force), acoustic sedimentation, or simple filtration. The main purpose of these devices is to isolate, concentrate, and wash cell aggregates, MCs with attached cells, or single cells in suspension. One example of a separation and retention device is the kSep Scalable Single-Use Automated Centrifuge System available from Sartorius Stedim Plastics GmbH, Goettingen, Germany.

As the spent expansion medium flows into the separation and retention device 20, the PSC aggregates will ideally be concentrated in a small volume of spent medium without being compacted. Once the aggregates are concentrated, the spent medium fluid flow is replaced with the first differentiation medium. In this way, the cell aggregates are washed, completely removing the old expansion medium and replacing it with differentiation medium.

Finally, the PSC aggregates are discharged from the separation and retention device 20 into the second bioreactor 32, which is already pre-filled and pre-conditioned with the first differentiation medium. Pre-conditioning means correcting the bioreactor for important parameters such as temperature, pH, and dissolved oxygen required for the next process step. Since pre-conditioning usually takes longer than the time required to concentrate and wash in the separation and retention device, the second bioreactor can start the pre-filling and pre-conditioning process before the PSC aggregates leave the first bioreactor. Thus, the pre-filled and pre-conditioned second bioreactor 32 will be ready to be inoculated with the washed PSC aggregates immediately after leaving the separation and retention device 20.

Once the differentiation steps are completed in the second bioreactor 32, the PSC aggregates (now consisting of different cell types) can be concentrated and washed in the same manner using the separation and retention device 20 and returned to the first bioreactor 30 pre-filled and pre-conditioned with yet another differentiation medium. By alternating back and forth between the two bioreactors via the separation and retention device 20, complete medium exchange can be achieved continuously throughout the multi-step differentiation process without adversely affecting cell viability, quality, pluripotency, or yield. This back and forth process can be performed as many times as necessary to execute the sequence of FIG. 1.

If the time to precondition the bioreactor is shorter than the time required for the concentration and washing steps, a single bioreactor can be used for that particular expansion and/or differentiation step. Depending on the preconditioning time requirements for the different scale steps of the bioprocess, any number of bioreactors and external equipment can be used in different combinations as needed, especially for large scale production.

One potential process variation could involve following the differentiation process described above until cell aggregates of a particular intermediate cell type are produced. At this point, only a portion of the cell aggregates are exposed to a particular growth factor, while the remaining portion is exposed to a different growth factor. This would result in two differentiation pathways that terminate in different final target cell types.

For example, FIG. 3 shows a system of the present application having multiple separation and retention devices and bioreactors. In this case, each parallel path would require at least two bioreactors and one separation and retention device for the alternating medium exchange process described with reference to FIG. 2.

More specifically, the process begins by growing a batch of PSC aggregates using a first bioreactor 50. The aggregates are transferred to a first general example of a separation and retention apparatus 52. The batch of aggregates is washed with a first differentiation medium A and then a portion of it is transferred to a second bioreactor 60 that is pre-filled with medium A. Thereafter, the remaining aggregates in the separation and retention apparatus 52 are washed with a second differentiation medium B and transferred to a third bioreactor 62 that is pre-filled with medium B. The second bioreactor 60 and the third bioreactor 62 are then operated in parallel to carry out their respective differentiation steps.

The PSC aggregates in the second and third bioreactors 60, 62 are then transferred to separation and retention devices 70, 72, respectively. It is possible for the first device 52 to function as device 70, 72, or both. That is, a single separation and retention device can be used for multiple differentiation pathways, provided that the enrichment and washing steps are staggered in timing. However, to maintain clarity, the separation and retention devices 70, 72 in the two parallel pathways are uniquely identified in FIG. 3.

The PSC aggregates are transferred from the second bioreactor 60 to the separation and retention device 70 for concentration and washing, and then transferred to the fourth bioreactor 80 for differentiation. The PSC aggregates are transferred from the third bioreactor 62 to the separation and retention device 72, and then transferred to the fifth bioreactor 82. Either the fourth or fifth bioreactor 80, 82 may be the first bioreactor 50 used to start the process, provided that the bioreactor 50 is sufficiently pre-filled and pre-conditioned beforehand. Similarly, instead of transferring the aggregates to the fourth and fifth bioreactors 80, 82, the output from the control device 70, 72 can be returned to the second and third bioreactors 60, 62. The reader will appreciate the various variations available, limited only by the time required to pre-fill and pre-condition each bioreactor, or by the number of bioreactors available. Multiple differentiation pathways are also possible, branching from multiple intermediate progenitor cell types, each requiring its own unique growth factors, bioreactors, separation and retention devices, and terminating in a different final target cell.

One factor to consider is the method of media transfer between the bioreactor and the separation and retention device. Peristaltic pumps are commonly used to move liquid media through flexible plastic tubing. However, the physical squeezing action of the pump can break up cell aggregates larger than 400 micrometers in diameter or cause grinding of MCs against each other, damaging surface-bound cells. The viability of suspended single cells is unlikely to be significantly affected because individual cells are smaller than both the vortex size of the fluid turbulence and the gaps between the insides of the squeezed tubing.

Instead of using pumps, media transfer can be achieved by creating an internal pressure difference between the bioreactor and the separation and retention device. Liquid transfer by pressure difference can be achieved relatively easily if the container is constructed of a material such as stainless steel to withstand the internal pressure. Single-use bioreactors with a rigid frame to seal the flexible plastic film container can also be successfully pressurized without risk of operational hazards. In preparation for media transfer, the harvest valve of the bioreactor is aseptically connected to the separation and retention device via fixed plastic tubing. Gas is then pumped into the headspace of the bioreactor to pressurize it. When the clamp is opened, the liquid containing the cell aggregates or MCs with attached cells will flow to the low pressure external device. The transfer rate can be controlled by adjusting the gas pressure applied to the bioreactor. The second tubing connecting the separation and retention device to the second bioreactor remains fixed during this first pressure transfer step.

Another method for media transfer involves a system of lifts that can be used to vary the height of the bioreactors relative to each other. Thus, if one bioreactor is raised high enough relative to the other, gravity will cause the therapeutic cells to fall through the tubing from the raised bioreactor to the bioreactor below.

Once the cell aggregates or MCs with attached cells have been washed, the separation and retention device can be pressurized and then the second clamp opened to allow flow out of the device. By utilizing this technique, the aggregates and MCs can avoid the potentially damaging effects of physical feeding.

As used herein, "plurality" means two or more. As used herein, a "set" of items may include one or more of such items. Terms such as "comprising," "including," "carrying," "having," "containing," "involving," and the like, as used herein in the written description or claims, should be understood to mean open-ended, i.e., including but not limited to. With respect to the claims, only the transitional phrases "consisting of" and "consisting essentially of," respectively, are closed or semi-closed transitional phrases. In the claims, the use of ordinal terms such as "first," "second," "third," etc., does not indicate a priority, precedence, or order of one claim element relative to other elements or a chronological order in which the method acts are performed, but is used only as a marker to distinguish a particular claim element's name from another element having the same name (but using ordinal terms) to distinguish the claim element. As used herein, "and/or" means that the listed items are alternatives, but the alternatives also include any combination of the listed items.

Claims (17)

1. A method of cell expansion of therapeutic cells grown as cell aggregates, attached to the surface of microcarriers, or grown as single cells in suspension, comprising:
a. suspending therapeutic cells, including cell aggregates, microcarriers with attached cells, or single cells, in a first medium in a first bioreactor having a first working volume;
b. Expanding or differentiating the therapeutic cells in the first bioreactor;
c. pre-filling a second bioreactor having a second working volume with a second medium;
d. pre-adjusting process parameters of the second bioreactor containing the second medium;
e. transferring all of the first medium containing the therapeutic cells from the first bioreactor to an external device for cell concentration and medium exchange;
f. concentrating the therapeutic cells within the external device and washing the therapeutic cells with the second medium to remove the first medium;
g. transferring the concentrated and washed therapeutic cells from the external device to the pre-loaded and pre-conditioned second bioreactor;
h) expanding or differentiating said concentrated, washed and transferred therapeutic cells in said second bioreactor until a desired expansion of said therapeutic cells is completed. i. pre-filling a third bioreactor having a third working volume with a third medium;
j. pre-adjusting process parameters of the third bioreactor containing the third medium;
k. transferring all of the second medium containing the therapeutic cells from the second bioreactor to an external device;
l. concentrating and washing the therapeutic cells within the external device using the third medium;
m. transferring the concentrated and washed therapeutic cells from the external device to the pre-loaded and pre-conditioned third bioreactor;
2. The method of claim 1, further comprising: expanding or differentiating the concentrated and washed therapeutic cells in the third bioreactor until a desired expansion of the therapeutic cells is complete. The method of claim 2, wherein the third bioreactor is the first bioreactor. 2. The method of claim 1, further comprising repeating steps c. through h. one or more times with one or more subsequent bioreactors and subsequent media in place of the second bioreactor and the second media. The method of claim 2 , wherein the first, second and third working volumes are the same. The method of claim 2 , wherein the second working volume is greater than the first working volume and the third working volume is greater than the second working volume. The method of claim 4, wherein step e. is performed using only a single external device. The method of claim 4, wherein each time step e. is performed, a different external device is used. The method of claim 1, wherein the transferring step is performed using a pump or a fluid pressure differential. 1. A method for directing differentiation of therapeutic cells growing as cell aggregates, on the surface of microcarriers, or as single cells in suspension, comprising:
a. suspending therapeutic cells, including cell aggregates, microcarriers with attached cells, or single cells, in a first differentiation medium containing a first growth factor in a first bioreactor having a first working volume;
b. pre-filling a second bioreactor with a second differentiation medium and adding a second growth factor to be used in the next differentiation step;
c. pre-adjusting process parameters of the second bioreactor;
d. transferring all of the first differentiation media, growth factors, and therapeutic cells from the first bioreactor to an external device for cell concentration and media exchange;
e. concentrating the therapeutic cells within the external device and washing the therapeutic cells with the second differentiation medium to remove previous growth factors;
f) transferring said concentrated and washed therapeutic cells from said external device to said preconditioned second bioreactor. g. pre-filling a third bioreactor having a third working volume with a third medium;
h. pre-adjusting process parameters of the third bioreactor containing the third medium;
i. transferring all of the second differentiation medium containing the therapeutic cells from the second bioreactor to an external device;
j. concentrating and washing the therapeutic cells within the external device using the third medium;
11. The method of claim 10, further comprising the step of: k. transferring the concentrated and washed therapeutic cells from the external device to the pre-loaded and pre-conditioned third bioreactor. The method of claim 11, wherein the third bioreactor is the first bioreactor. 11. The method of claim 10, further comprising repeating steps b. through f. one or more times using one or more subsequent bioreactors and subsequent media in place of the second bioreactor and the second differentiation medium. The method of claim 13, wherein all of the bioreactors used have the same working volume. The method of claim 13, wherein step d. is performed using only a single external device. The method of claim 13, wherein each time step d. is performed, a different external device is used. The method of claim 10, wherein the transferring step is performed using a pump or a fluid pressure differential.

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