Note: Descriptions are shown in the official language in which they were submitted.
81781023 PERFUSION BIOREACTOR SYSTEMS COMPRISING A CELL AGGREGATE TRAP AND METHODS OF OPERATING THE SAME RELATED APPLICATIONS [0001] The present application claims the benefit of and priority to U.S. Provisional Patent Application No. 61/587,940 filed January 18, 2012, and entitled "PERFUSION BIOREACTOR SYSTEMS AND METHODS OF OPERATING THE SAME" (Attorney Docket No. BH-001/L). BACKGROUND [0002] Conventional perfusion bioreactor systems and processes include a bioreactor that functions to culture cells in a fluid medium such as a tissue culture fluid (TCF). The cultured cells and TCF are removed from the bioreactor, such as by pumping, and the cells are separated from the TCF by a conventional cell retention unit. A harvest output stream from the cell retention unit still containing some cells, particles, and debris is then further processed. Harvest output as used herein contains the TCF that is further processed to obtain the desired product (e.g., coagulation factor). Filtration technologies, such as dead- end depth filtration, membrane filtration, microfiltration, and/or centrifugation can be used to further concentrate and/or purify the harvest output from the cell retention unit. [0003] Another output stream of TCF exiting the cell retention unit having a relatively high concentration of cells is directly returned (e.g., recycled or re-circulated) to the bioreactor. In such a continuous perfusion bioreactor process, the harvest output stream and the recirculation 1 Date Recue/Date Received 2020-04-30 CA 02861270 2014-07-15 WO 2013/109520 PCT/US2013/021533 output stream are substantially continuous during the cultivation period, which can be ten days or more. However, using this type of conventional perfusion configuration can lead to conditions where cell density within the bioreactor may be relatively inadequately controlled. [0004] Accordingly, there is a need for perfusion bioreactor systems and methods that more effectively control bioreactor cell density. 2 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 SUMMARY [0005] In a first embodiment, a perfusion bioreactor system is provided. The perfusion bioreactor system comprises (1) a bioreactor configured to contain a tissue culture fluid and cells to be cultured; (2) a cell retention unit configured to receive tissue culture fluid containing cells from the bioreactor, separate some cells from the tissue culture fluid and provide harvest output of tissue culture fluid and cells, and provide a recirculation output of tissue culture fluid and cells; and (3) a cell aggregate trap configured to receive the recirculation output of tissue culture fluid and cells, separate cell aggregates from the recirculation output of tissue culture fluid and cells, and return the remaining tissue culture fluid and cells to the bioreactor. [0006] In another embodiment, a cell aggregate trap is provided. The cell aggregate trap comprises (1) a sedimentation chamber; (2) a trap inlet configured to receive a recirculation output of tissue culture fluid and cells; (3) a side flow chamber configured to return at least some of the recirculation output of tissue culture fluid containing cells to a bioreactor; and (4) a discard trap outlet coupled to the sedimentation chamber configured to output cell aggregates. [0007] In another system embodiment, a perfusion bioreactor system is provided. The perfusion bioreactor system comprises (1) a bioreactor configured to contain a tissue culture fluid and cells to be cultured; (2) a cell retention unit configured to separate some cells from the tissue culture fluid and provide harvest output; and (3) a 3 81781023 cell aggregate trap configured to separate cell aggregates from the tissue culture fluid and cells and provide an output having relatively lower amount of cell aggregates. [0008] In a method embodiment, a method of operating a perfusion bioreactor system is provided. The method comprises (1) providing tissue culture fluid containing cells to a cell retention unit from a bioreactor (2) separating in the cell retention unit some cells from the tissue culture fluid to provide a harvest output of tissue culture fluid and cells and a recirculation output of tissue culture fluid and cells; and (3) separating in a cell aggregate trap, cell aggregates from the recirculation output of tissue culture fluid and cells. The tissue culture fluid and cells can be returned to the bioreactor having relatively lower amount of cell aggregates. [0009] In another method embodiment, a method of operating a perfusion bioreactor system is provided. The method comprises (1) providing a flow of tissue culture fluid and cells from a bioreactor; (2) separating in a cell retention unit some cells from the tissue culture fluid to provide a harvest output; and (3) separating in a cell aggregate trap, cell aggregates from the tissue culture fluid and cells, so as to produce tissue culture fluid having relatively lower amounts of cell aggregates. The tissue culture fluid and cells can be returned to the bioreactor having relatively lower amount of cell aggregates. [0009a] The present application as claimed relates to: - a cell aggregate trap, comprising: a trap inlet for receiving a recirculation output of tissue culture fluid and cells; a sedimentation chamber to separate cell aggregates from the 4 Date Recue/Date Received 2020-04-30 81781023 tissue culture fluid containing cells; a side flow chamber having a trap outlet to provide an output of tissue culture fluid containing cells being in fluid communication with the sedimentation chamber; and a discard trap outlet coupled to the sedimentation chamber configured to output cell aggregates, the sedimentation chamber comprising an upper region and a lower region, the upper region being positioned above a centerline of the side flow chamber, while the lower region being positioned below the centerline of the side flow chamber; - a perfusion bioreactor system, comprising: a bioreactor having a bioreactor inlet, a bioreactor outlet and a culture chamber to contain a tissue culture fluid and cells to be cultured; a cell retention unit fluidly coupled to the bioreactor outlet and having an inlet for receiving tissue culture fluid containing cells from the bioreactor, a cell separation technology to separate some cells from the tissue culture fluid, the cell separation technology being disc filters, spin filters, flat sheet filters, micro-porous hollow fiber filters, cross-flow filters, vortex-flow filters, continuous centrifuges, centrifugal bioreactors, gravity settlers, ultrasonic wave devices, or hydrocyclones, a first outlet to provide harvest output of tissue culture fluid and cells, and a second outlet to provide a recirculation output of tissue culture fluid and cells; and a cell aggregate trap as described herein fluidly coupled to the second outlet; - a perfusion bioreactor system, comprising: a bioreactor having a bioreactor inlet, a bioreactor outlet and a culture chamber to contain a tissue culture fluid and cells to be cultured; a cell aggregate trap as described herein fluidly coupled to the bioreactor outlet; and a cell retention unit 4a Date Recue/Date Received 2020-04-30 81781023 fluidly coupled to the trap outlet and having an inlet for receiving tissue culture fluid containing cells from cell aggregate trap, a cell separation technology to separate some cells from the tissue culture fluid, the cell separation technology being disc filters, spin filters, flat sheet filters, micro-porous hollow fiber filters, cross-flow filters, vortex-flow filters, continuous centrifuges, centrifugal bioreactors, gravity settlers, ultrasonic wave devices, or hydrocyclones, a first outlet to provide harvest output of tissue culture fluid and cells, and a second outlet to provide a recirculation output of tissue culture fluid and cells to the bioreactor; and - a method of operating a perfusion bioreactor system as described herein, comprising: providing tissue culture fluid containing cells to the cell retention unit from the bioreactor; separating in the cell retention unit some cells from the tissue culture fluid to provide a harvest output of tissue culture fluid and cells, and a recirculation output of tissue culture fluid and cells; and separating in the cell aggregate trap, cell aggregates from the recirculation output of tissue culture fluid and cells. [0010] These and other features of the present teachings are set forth herein. 4b Date Recue/Date Received 2020-04-30 CA 02861270 2014-07-15 WO 2013/109520 PCT[US2013/021533 BRIEF DESCRIPTION OF THE DRAWINGS [00].1] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. [0012] FIG. 1 shows a block diagram of an embodiment of a perfusion bioreactor system including a cell aggregate trap according to the embodiments. [0013] FIG. 2A shows a cross-sectioned side view of a cell aggregate trap according to the embodiments. [0014] FIG. 2B shows an upwardly looking cross-sectioned end view of an embodiment of a cell aggregate trap taken along section line 2B-2B of FIG. 2A. [0015] FIG. 3 shows a flowchart illustrating a method of operating a perfusion bioreactor system according to the embodiments. [0016] FIG. 4 shows another flowchart illustrating another method of operating a perfusion bioreactor system according to the embodiments. GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 DESCRIPTION OF VARIOUS EMBODIMENTS [0017] Culturing of cells (including animal, plant, or microbial cells) can be used to produce biologically-active substances and pharmaceutically-active products. However, in certain cell cultures, the cells can, to some extent, adhere to one another and form relatively large cell agglomerates, cell clumps, or aggregations (hereinafter referred to as "cell aggregates"). When such cell aggregates are present, they can cause certain processing problems in the perfusion hioreactor process. In particular, the presence of cell aggregates can cause a cell density within the bioreactor to be relatively unstable, i.e., it is difficult to adequately maintain, hold, or control within a desired cell density set point range. It is also difficult to accurately measure the cell concentration in the presence of cell aggregates. As a result, from time-to-time, TCF and cells need to be discarded by periodically operating a discard pump of the conventional perfusion bioreactor system. Furthermore, it is difficult to determine the amount of discarded TCF and cells. Of course, this discard also discards valuable TCF containing the desired product. Moreover, the cells in bioreactor culture, particularly animal or plant cells are generally very sensitive to imparted mechanical shear forces. Accordingly, it is not only desired to minimize discarded material, but it is desirable to minimize exposure of the cells to possibly damaging shear forces. Furthermore, it is desired to be able to precisely control cell density within the bioreactor. [0018] Therefore, according to embodiments of the present invention, an improved perfusion bioreactor system is provided. The improved perfusion bioreactor system comprises 6 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 a cell aggregate trap that is provided, configured and/or adapted to operate in conjunction with a cell retention unit. The cell aggregate trap is functionally based upon sedimentation wherein cell aggregates settle out and can be removed from a re-circulating flow stream. By the use of a cell aggregate trap in conjunction with the cell retention unit in the perfusion bioreactor system, a relatively high majority of the cells can be returned to the bioreactor and cell aggregates, that can be detrimental to the perfusion process, can be removed and discarded. [0019] According to other embodiments, the perfusion bioreactor system comprises a bioreactor, a cell retention unit coupled to the bioreactor, a cell retention unit configured to receive TCF and cells from the bioreactor, separate some cells from the TCF and provide a harvest output, and a cell aggregate trap configured to receive a recirculation output of TCF and cells from the cell retention unit, separate cell aggregates from the TCF and cells, and return the remaining TCF and cells to the bioreactor. [0020] In another embodiment, a method of operating a perfusion bioreactor system is provided. The method comprises providing TCF and some cells, received the TCF and cells in a cell aggregate trap, and separating in the cell aggregate trap cell aggregates from the TCF and cells. The remaining TCF and cells can be returned to the bioreactor having relatively low amount of cell aggregates. [0021] In another embodiment, a method of operating a perfusion bioreactor system is provided. The method comprises providing a TCF containing cells to a cell retention unit from a bioreactor, separating some cells from the TCF to provide a harvest output, with the remaining re- 7 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 circulated TCF and cells being received by a cell aggregate trap, and separating in the cell aggregate trap, cell aggregates from the TCF and cells. Remaining TCF and cells can be returned to the bioreactor having relatively lower amount of cell aggregates. The methods, perfusion bioreactor systems, and cell aggregate traps described herein can be adapted for coagulation factor production and/or other suitable processes for producing biological agents or factors. [0022] These and other embodiments of perfusion bioreactor systems comprising cell aggregate traps, cell aggregate traps, and methods of operating perfusion bioreactor systems are described below with reference to FIGs. 1-4. FIG. 1 illustrates a block diagram of an embodiment of a perfusion bioreactor system 100. The perfusion bioreactor system 100 comprises a bioreactor 102 having a bioreactor inlet 104 and a bioreactor outlet 106. The bioreactor 102 comprises a culture chamber 105 configured to hold a tissue culture fluid (TCF) 108 and cells 139 to be cultured. The perfusion bioreactor system 100 can be used for the production of biologics such as coagulation factors. For example, the perfusion bioreactor system 100 and methods can be used to manufacture coagulation factors such as Factor VII, VIII, or Factor IX, or other suitable factors or substances. [0023] Example methods for production of Factor VIII are described in US 6,338,964 entitled "Process and Medium For Mammalian Cell Culture Under Low Dissolved Carbon Dioxide Concentration," the disclosure of which is hereby incorporated by reference in its entirety herein. For example, a cell culture process can comprise culturing cells in a TCF which contains a high concentration of a complexing agent and a buffer which is low in added NaHCO3 8 GA 02861270 2014-07-15 WO 2013/109520 PCT/US2013/021533 concentration. The cell culture process can be carried out in a culture chamber, such as culture chamber 105 in FIG. 1, which can be a stirred tank fermenter with stirring impellers in some embodiments. The fermenter can be provided with a microsparger at a bottom of the culture chamber or a membrane as an oxygenation system. The TCF can be a medium composition based on a commercially available DMEM/F12 formulation manufactured by JRH (Lenexa, Kansas) or Life Technologies (Grand Island, N.Y.) supplied with other supplements such as iron, Piuronic F-68, or insulin, and can be essentially free of other proteins. Compiexing agents histidine (his) and iminodiacetic acid (IDA) can be used, and organic buffers such as MOPS (3-[N- Morpholino]propanesuifonic acid), TES (N- tris[Hydroxymethyi]methy1-2-aminoethanesulfonic acid), BES (N,N-bisL2-Hydroxyethyij-2-aminoethanesulfonic acid) and TRIZMA (tris[Hydroxvmethvl]aminoethane) can be used; all of which can be obtained from Sigma (Sigma, St. Louis, Mo.), for example. In some embodiments, the TCF can be supplemented with known concentrations of these complexing agents and organic buffers individually or in combination. The TCF can contain EDTA, e.g., 50 uM, as an iron chelating agent. Other compositions, formulations, supplements, complexing agents and/or buffers can be used. [0024] Cell cultivation can be started by inoculating with cells from previously-grown culture. Typical bioreactor parameters can be maintained (e.g., automatically) under stable conditions such as temperature at about 35 C to 37 C, pH at about 6.8 to 7.0, dissolved. oxygen. (DO) at about 30% to 70% of air saturation, stirring speed at about 30 rpm to 80 rpm, and approximately constant liquid volume. Other bioreactor parameters can be used. DO and pH can be measured on-line using commercially-available probes. The bioreactor 9 GA 02001270 2014-07-15 WO 2013/109520 PCT/US2013/021533 process can be started in batch mode for about. 1-2 days, allowing the initial cell concentration to double. This can be followed by a perfusion stage wherein the TOT is pumped continuously into the bioreactor and the TOE' containing cells (and possibly some cell aggregates) are pumped out. A flow rate of TOE' can be controlled and increased proportionally with the cell concentration. A steady state or stable perfusion process can be attained when the cell concentration reaches a target high level (e.g., about. 10 x106celis/mL to 20 x106ceils/mL) in the bioreactor and can be controlled at this concentration. At this point, the flow rate can be held constant. The cell density can be held. between about 4 million to about 40 million cells per milliliter in the perfusion bioreactor system. Other biologics, coagulation factors, cell concentrations, cell densities or the like can be employed. [0025] Referring again to FIG. 1, the cells 109 can be eukaryotic or prokaryotic such as animal, plant, or microbial cells. For example, the cells 109 can be baby hamster kidney cells (BHK cells), hybrid of kidney and B cells (HKB cells), human embryonic kidney cells (HEK cells - also referred to as HEK 293 or 293 cells), or the like. The TCF 109 can be introduced into the culture chamber 105 through TCF inlet 105A, or elsewhere in the perfusion bioreactor system 100. The cells 109 in the TCF 108 can, due to their properties and processing, at times form cell aggregates 109A, as shown in the enlarged view. "Cell aggregates" as used herein means a cell agglomerate, cell clump, or aggregation of cells that are connected and adhered to each other to form a grouping of cells. "Cell aggregates" that can be removed by using one or more of the present embodiments can number about 10 or more cells, about 20 or more cells, or even about 40 or more cells. One or GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 more of the present embodiments can remove cell aggregates in a range of from about 10 cells to about 50,000 cells, or even in a range of from about 40 cells to about 300 cells. More generally, cell aggregates 109A that can be removed by using one or more of the various embodiments can include cell agglomerates of a size and shape where at least some internal cells in the agglomerate will tend to die off due to lack of adequate oxygen and/or nutrients during the perfusion process. Generally, cell aggregates are quite large. For example, cell aggregates 109A having a minimum dimension (across the cell aggregate) of about 60 microns or more, or even 100 microns or more can be separated and removed by using various embodiments of the invention. One or more of the present embodiments can remove cell aggregates having a minimum dimension in a range of about 60 microns to about 3,000 microns, or even in a range of about 100 microns to about 500 microns. Additionally or alternatively, smaller cell aggregates can be separated and removed. The presence of cell aggregates 109A in the bioreactor 102 is generally undesirable, and the present perfusion bioreactor system 100 and methods 300, 400 described herein can remove at least some, and in many cases, most of the cell aggregates 109A therefrom. The depicted perfusion bioreactor system 100 comprises a cell retention unit 110 fluidly coupled to the bioreactor 102 and configured to receive TCF 108 containing cells 109 in a first cell concentration (Cl) (including possibly some cell aggregates 109A) from the bioreactor 102. Example first cell concentrations (Cl) can range from about 4 x 10"6 cells/mL to about 40 x 10^6 cells/mL. Other cell concentration ranges can be used. The TCF 108 containing cells 109 in the first concentration (Cl), in the depicted embodiment, are expelled from the bioreactor outlet 106 and received at a cell 11 GA 02001270 2014-07-15 WO 2013/109520 PCT/US2013/021533 retention unit inlet 112 by passing through a first conduit 113. The conduit 113 can couple to an optional heat exchanger 113H that functions to cool the TCF 108 containing cells 109 that are expelled from the bioreactor outlet 106. The cell retention unit 110 is configured and operational, and therefore functions to separate most of the cells 109 from the TCF 108 and provide a harvest output of TCF 108 containing only a small amount of cells 109 having a second cell concentration (02) at a first retention unit outlet 114. Accordingly, the second cell concentration (02) is less than the first concentration (Cl), that is 02 < Cl, and in particular, 02 << C1). Example second cell concentrations (02) can range from about 0.1 x 10^6 cells/mL to about 2 x 10^6 cells/mL. Other cell concentration ranges can be used. The so-called harvest output passes from the first retention unit outlet 114, through second conduit 115, such as by a pumping action of a harvest pump 117 coupled to the second conduit 115. The harvest output can be further isolated and/or purified in downstream isolation and purification processes 118. These additional isolation and purification processes 118 can be carried out in a continuous or batch fashion. For example, these downstream isolation and purification processes 118 can be carried out as described in US Pub. No. 2008/0269468 entitled 'Devices And Methods For Integrated Continuous Manufacturing Of Biological Molecules," the disclosure of which is hereby incorporated by reference herein in its entirely. In a batch mode, for instance, once a specified volume of harvest has been collected, which is typically after 1-4 days or more, one or more harvest collection vessels can be disconnected from a sterile fermentation vessel and the collected material can be designated as one harvest batch. The next step is to remove cells, debris, and particles. In industrial scale 12 GA 02861270 2014-07-15 WO 2013/109520 PCT/US2013/021533 this can be done usina centrifugation followed by dead-end. membrane filtration, or by dead-end depth-filtration followed by dead-end membrane filtration. Another technique such as tangential flow (or "crossflow") microfiltration or any other suitable filtration technique can be also used- In any case, the product of the particle removal process is a batch of clarified tissue culture fluid (cTCF). This cTCF can be purified (concentrated) by any suitable process such as crossflow ultrafiltration or by packed. bed chromatography. [0026] In a continuous mode, a volume of the harvest output can be purified by a continuous purification system integrated, with the perfusion bioreactor system, which can be maintained under sterile conditions. "Continuous" as used herein means uninterrupted in time, sequence, and/or operation for prolonged periods. For example, the cell retention unit 110 can carry out initial cell retention and produce a harvest output of clarified TCF 108 at first retention unit outlet 114. The isolation and purification process 118 can comprise further filtering (isolation) of the harvest output provided by the second conduit. 115 by a suitable filter system having, in some embodiments, a final filter rating of about 3 microns or smaller, 0.45 microns or smaller, or even 0.2 microns or smaller to provide cTCF. Other filter ratings can be used [0027] The filtering process can be followed by a purification process comprising a continuous ultrafiltration separation process, for example. in some embodiments, the ultrafiltration can occur at a specific flow rate below the transition point of the molecule of interest in the pressure-dependent region of the flux versus TMP curve, wherein the specific flow rate is maintained substantially constant throughout the continuous ultrafiltration. Relatively higher yields can be achievable using a 13 GA 02001270 2014-07-15 WO 2013/109520 PCT/US2013/021533 continuous isolation and purification. process 118. In some embodiments, the cTOF is passed through an ultrafiltration membrane having an area in square meters approximately equal to between 0.1 to 2 times the volumetric flow rate of the cTOF in liters/hour, or even approximately equal to between 0.3 to I times the volumetric flow rate of the cTOF in liters/hour. Other membrane areas can be used. [0028] Following separation within the cell retention unit 1101 a recirculation output of TCF 108 and a relatively higher concentration of cells 109 are provided in a third cell concentration (C3) at a second retention unit outlet 119. The third cell concentration (C3) is generally relatively higher than the first concentration (Cl), that is C3 > Cl because although some small volume of cells 109 will be lost in the harvest output stream, the volume of TCF 108 extracted at the first retention unit outlet 114 is greater. Example third cell concentrations (C3) can range from about 6 x 10^6 cells/mL to about 60 x 10^6 cells/mL. Other cell concentration ranges can be used. The cell retention unit 110 can be based upon any known cell separation technology, such as disc filters, spin filters, flat sheet filters, micro-porous hollow fiber filters, cross-flow filters, vortex-flow filters, continuous centrifuges, centrifugal bioreactors, gravity settlers, ultrasonic wave devices, hydrocyclones, and the like. Any suitable type of cell retention unit 110 can be used that is configured and operational, and therefore functional to separate an incoming first cell concentration (Cl) into outgoing cell concentrations (02 and C3). [0029] In the depicted embodiment, the perfusion bioreactor system 100 comprises a cell aggregate trap 120. The cell aggregate trap 120 is configured and operational, and therefore functional to receive the recirculation output 14 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 of TCF 108 and cells 109 at the third cell concentration (C3) at a trap inlet 121 from the cell retention unit 110. Cell retention unit 110 can be fluidly coupled to the cell aggregate trap 120 by a third conduit 122. In some embodiments, the functions of cell retention unit 110 and the cell aggregate trap 120 can be integrated into one single unit. Accordingly, in such an embodiment, the conduit 122 can be eliminated and the output of the cell retention unit 110 can be directly received by the trap input 121. [0030] The cell aggregate trap 120 functions to separate cell aggregates 109A from the recirculation output of TCF 108 and cells 109 at the third cell concentration (C3) received at the cell aggregate trap 120. In one implementation, the separation of cell aggregates 109A is carried out continuously; that is the flow is continuous from the cell retention unit 110 during operation. Generally, when present in the flow stream, at least some, and generally a relatively high percentage of the cell aggregates 109A are removed by the cell aggregate trap 120 and a remaining TCF 108 and cells 109 in a fourth cell concentration (C4) are returned to the inlet 104 of the bioreactor 102. Example fourth cell concentrations (C4) can range from about 5 x 10^6 cells/mL to about 50 x 10^6 cells/mL. Other cell concentration ranges can be used. In some embodiments, the perfusion bioreactor system 100 and methods including the cell aggregate trap 120 can remove about 20 percent to about 80 percent of cell aggregates, although other percentages of cell aggregates can be removed. [0031] The TCF 108 and cells 109 can exit the trap outlet 123 and pass through fourth conduit 124 to the bioreactor inlet 104. One or more recirculation pumps 125 can be provided and operated to cause flow of the TCF 108 and cells 109. The one or more pumps 125 can be located at any GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 convenient location, such as in conduits 113, 122, or 124 or other suitable locations. In the depicted embodiment, the pump 125 is coupled to the fourth conduit 124. [0032] Additionally, the cell aggregate trap 120 can comprise any suitable trap discard outlet 126 that is configured and operational, and therefore functional to allow a small amount of TCF 108 and some cell aggregates 109A to be removed from the cell aggregate trap 120. A fifth cell concentration (05) is provided in the trap discard outlet 126. Example fifth cell concentration (05) can range from about 12 x 10'6 cells/mL to about 90 x 10'6 cells/mL. Other cell concentration ranges can be used. Because some cell aggregates 109A have been removed from the process flow stream by the cell aggregate trap 120, the cell concentration (C4) is generally less than the cell concentration (03), that is 04 < 03. The cell aggregates 109A and small volumes of TCF 109 can flow from the trap discard outlet 126 to be discarded. A discard pump 127 can be continuously or periodically operated to flow cell aggregates 109A and a small amount of TCF 108 through the discard conduit 128 to a discard, such as a flexible bag, or other type of discard container. [0033] The structure and operation of the cell aggregate trap 120 will now be described with reference to FIGs. 2A and 2B. The cell aggregate trap 120 comprises a trap body 130 that can be made out of a rigid material, such as stainless steel, glass, or plastic. Other materials can be uscd. The TCF 108 and colic 109 (possibly including some cell aggregates 109A) are received at the trap inlet 121, such as at a top of the trap body 130, for example. As depicted, the TCF 108 and cells 109 and possibly cell aggregates 109A can, during operation, flow directly into an expansion zone 132 that can be formed at a location directly 16 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 adjacent to the trap inlet 121 and into a sedimentation chamber 134 of the cell aggregate trap 120. The expansion zone 132 can be made up of angled or curved walls that gradually increase a cross-sectional area of the sedimentation chamber 134 along a length of the sedimentation chamber 134. In the depicted embodiment, the expansion zone 132 is shown as a frustoconical region. However, any generally smooth transition between the cross- sectional area of the trap inlet 121 to the cross sectional area of the sedimentation chamber 134 can be used. In general, a transitional rate of increase in area exiting from the inlet 121 can be less than about 8.4 cm2/cm, and in some embodiments less than about 4.2 cm2/cm, in an attempt to minimize shear forces imparted to the cells 109. Other transitional rates can be used. However, in some embodiments, an expansion zone 132 may not be present. [0034] The cell aggregate trap 120 can comprise a side flow chamber 136. The side flow chamber 136 is constructed, configured, and operational in conjunction with the sedimentation chamber 134 to allow TCF 108 and cells 109 to exit the cell aggregate trap 120 through trap outlet 123, while allowing the cell aggregates 109A to settle out under the force of gravity within the sedimentation chamber 134. In the depicted embodiment, the side flow chamber 136 is generally cylindrical and extends horizontally from a side 134S of the sedimentation chamber 134. For example, the side flow chamber 136 can extend generally perpendicular from the sedimentation chamber 134. However, other shapes, configurations and orientations other than perpendicular can be used. [0035] Similar to the expansion zone 132 of the sedimentation chamber 134, the cell aggregate trap 120 can include a contraction zone 138 at a location directly 17 CA 02861270 2014-07-15 WO 2013/109520 PCT/US2013/021533 adjacent to the trap outlet 123 from the side flow chamber 136. In some embodiments, the contraction zone 138 can have a transitional rate of area contraction no greater than about 8.4 cm2/cm, and in some embodiments, of about 4.2 cm2/cm or less. Larger or smaller transition rates can be used. In some embodiments, a D3/D4 ratio can be provided that can be greater than about 2, for example. Similarly, a discard contraction zone 140 can be provided at the trap discard outlet 126 at a bottom of the sedimentation chamber 134. The trap discard outlet 126 can have a maximum transverse dimension D5 (e.g., an inside diameter). In one or more embodiments, the discard contraction zone 140 can have a transitional rate of area contraction no greater than about 8.4 cm2/cm, and in some embodiments, of about 4.2 cm2/cm or less. Larger or smaller transition rates can be used. In some embodiments, a D1/D5 ratio can be greater than about 2, for example. [0036] In more detail, the sedimentation chamber 134 can, in some embodiments, have a circular cross section having transverse dimension (D1) (e.g., an inside diameter) of between about 1.9 cm and about 6.4 cm, and in some embodiments between about 2.5 cm and about 5.1 cm. A maximum cross-sectional area of the sedimentation chamber 134 can be between about 2.9 cm2 and about 32 cm2, and in some embodiments between about 5.1 cm2 and about 20 cm2, for example. The trap inlet 121 can have a circular cross section having transverse dimension (D2) (e.g., an inside diameter) of between about 0.48 cm and about 1.6 cm, and in some embodiments between about 0.64 cm and about 1.3 cm, for example. In some embodiments, a D1/D2 ratio can be greater than about 2, or even greater than about 4, for example. However, other suitable cross-sectional shapes and sizes can be used. 18 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 [0037] Suitable dimensions of the cell aggregate trap 120 can be dependent on a capacity of the perfusion bioreactor system 100 (e.g., a volumetric throughput thereof), and the dimensions thereof can be enlarged or decreased based upon the flow capacity. The dimensions of the cell aggregate trap 120 can also depend on other factors such as fluid density or viscosity, or the like. The maximum cross-sectional area of sedimentation chamber 134 can be equal to or larger than the maximum cross-sectional area of trap inlet 121. In the depicted embodiment, the maximum cross-sectional area of sedimentation chamber 134 is larger than the maximum cross- sectional area of trap inlet 121. In particular, in some embodiments, the maximum cross-sectional area of sedimentation chamber 134 can be about 4 times or larger, about 10 times or larger, about 30 times or larger, or even about 60 times or larger than the maximum cross-sectional area of trap inlet 121. [0038] The sedimentation chamber 134 comprises an upper region 134U and a lower region 134L. The upper region 134U is positioned above a centerline 142 of the side flow chamber 136, while the lower region 134L Is positioned below the dente/line 142 of the side flow chamber 136. In the depicted embodiment, a total length (Lt) of the sedimentation chamber 134 from an upper end of the expansion zone 132 to a lower end of the contraction zone 140 can be between about 9 cm and 37 cm, and in some embodiments between about 14 cm and 28 cm. A length (Lu) of the upper region 134U from an upper end of the contraction zone 132 to the centerline 142 of the side flow chamber 136 can be between about 5 cm and 18 cm, and in some embodiments between about 7 cm and 14 cm. A length (L1) of the lower region 134L from a lower end of the contraction zone 140 to the centerline 142 of the side flow chamber 136 can be 19 CA 02861270 2014-07-15 WO 2013/109520 PCT/US2013/021533 between about 5 cm and 18 cm, and in some embodiments between about 7 cm and 14 cm. Other dimensions can be used. Generally, it is desired that a ratio of Ll/Lu > 0.5, and in some embodiments Ll/Lu > 4, for example, can be employed. Other ratios can be used. [0039] During operation, a volumetric flow rate through the cell aggregate trap 120 is generally held at between about 0.0025 m3/min and about 0.0068 m3/min, and in some embodiments between about 0.0030 m3/min and about 0.0045 m3/min. Other volumetric flow rates (e.g., capacities) can be used. However, in some embodiments, it is desirable to keep the liquid flow generally within a laminar Reynolds number range within the sedimentation chamber 134. Reynolds numbers within the sedimentation chamber 134 can be less than about 2300, less than about 1000, or even less than about 500 in some embodiments, in order to minimize mixing and promote adequate settling and separation of the cell aggregates 109A, wherein the Reynolds Number is approximately defined by Equation 1. Re = pQ/p Equation 1. where: Q is the volumetric flow rate of the fluid (m3/s), u is the dynamic viscosity of the fluid (kg/(ms), and p is the density of the fluid (kg/m3). [0040] However, to promote adequate retention of the cells 109 in Lhe flow stream such LliaL Lhe TCF 108 and cells 109 can exit the cell aggregate trap 120 from the trap outlet 123, and generally resist setting out in the sedimentation chamber 134, the flow within the sedimentation chamber 134 can have Reynolds numbers sufficient to avoid settling of the cells 109, for example. CA 02861270 2014-07-15 WO 2013/109520 PCT/US2013/021533 [0041] The side flow chamber 136 can have a circular cross section having maximum transverse dimension (D3) (e.g., an inner diameter) of between about 1.9 cm and about 6.4 cm, and in some embodiments between about 2.5 cm and about 5.1 cm. A maximum cross-sectional area of the side flow chamber 136 can between about 2.9 cm2 and about 32 cm2, and in some embodiments between about 5.1 cm2 and about 20 cm2, for example. However, other suitable cross-sectional shapes and sizes can be used. A total length (Ls) of the side flow chamber 136 from an entry into the side flow chamber 136 to an exit end of the contraction zone 138 can be between about 4 cm and 15 cm, and in some embodiments between about 5 cm and 11 cm. The maximum outlet dimension (D4) (e.g., an inner diameter) of the outlet 123 from the side flow chamber 136 can be between about 0.48 cm and 1.6 cm, and in some embodiments between about 0.64 cm and 1.3 cm. Other dimensions can be used. Flow in the side flow chamber 136 can have a Reynolds number of greater than about 2300, or even greater than about 4000, for example. Other Reynolds number ranges can be used. The Reynolds numbers can be selected to minimize setting of cells 109 in the side flow chamber 136. [0042] In some embodiments, a maximum cross-sectional area (Asc) of the sedimentation chamber 134 is equal to or larger than a maximum cross-sectional area (Asfc) of the side flow chamber 134, that is Asc Asfc. In particular, the maximum cross-sectional area (Asc) of the sedimentation chamber 134 can be the same, or even 5 times or more larger than a maximum cross-sectional area of the side flow chamber 136. Other Asc/Asfc ratios can be used. The difference in cross-sectional areas can generally function to improve sedimentation capacity. Representative dimensions D1-D5 described herein are directed towards an example embodiment 21 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 of a perfusion bioreactor system 100 having a capacity of about 2000 to 3000 liters per day of flow in second conduit 115 (FIG. 1). Perfusion bioreactor systems 100 having smaller or larger capacities can benefit by using an embodiment of the invention, such as a perfusion bioreactor system 100 having a capacity of about 100 to 200 liters per day of flow second conduit 115. [0043] In operation, the cell aggregate trap 120, through appropriate dimensioning and volumetric flow rates provided in the sedimentation chamber 134 and side flow chamber 136, as recited herein, is configured and operational, and, thus adapted to remove cell aggregates 109A of greater than or equal to about 10 aggregated cells, greater than or equal to about 20 aggregated cells 109, or even greater than or equal to about 40 aggregated cells 109. In some embodiments, a smaller number of aggregated cells can be removed. Cells 109 and TCF 108 are allowed to exit the side flow chamber 136. Thus, undesirable cell aggregates 109A are removed by operation of various embodiments of the invention. In some embodiments, the undesirable cell aggregates 109A removed by the cell aggregate trap 120 can have a minimum cross-wise dimension (D6) (See FIG. 2A) of greater than about 60 microns, greater than about 100 microns, or even larger. Smaller size cell aggregates can be removed. One advantage of the use of the cell aggregate trap 120 is that a rate of discard of TCF 108 from the perfusion bioreactor system 100 can be reduced. In particular, a rate of discard of TCF 108 can be slowed such that a discard cell concentration (05) from the cell aggregate trap 120 is greater than or equal to about 3 times the first cell concentration (Cl) (wherein C5 3C1), or even greater than or equal to about 5 times (Cl) (wherein 05 5C1). 22 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 [0044] The cell aggregate trap 120 in the depicted embodiment is shown installed at the exit from the cell retention unit 110. However, it should be understood that the cell aggregate trap 120 can be placed elsewhere in the perfusion bioreactor system 100. For example, a cell aggregate trap like the cell aggregate trap 120 can be provided in the location of the first conduit 113 (e.g., adjacent to the bioreactor outlet 106 or cell retention unit inlet 112, or otherwise coupled to the first conduit 113). In this embodiment, the recirculation output of TCF 108 and cells 109 including possibly cell aggregates 109A passes through the cell aggregate trap and then to a cell retention unit 110. Thus, cell aggregates 109A can be removed from the flow stream prior to entry into the retention unit 110. Optionally, a cell aggregate trap can be integrated into the bioreactor 102, such as at or near the bioreactor inlet 104. [0045] Methods of operating various embodiments of the perfusion bioreactor system 100 will now be described with reference to FIG. 3. One method 300 of operating the perfusion bioreactor system 100 comprises, in 302, providing to a cell retention unit (e.g., cell retention unit 110) from d Lioieactor (e.g., bioreactor 102), a tissue culture fluid (e.g., TCF 108) containing cells (e.g., cells 109 and possibly some cell aggregates 109A). The tissue culture fluid containing cells can be in a first concentration (Cl) Furthermore, the method 300 comprises, in 304, separating in the cell retention unit some cells from the tissue culture fluid to provide a harvest output (e.g., in second conduit 115) of tissue culture fluid and cells and a recirculation output of tissue culture fluid and cells. The harvest output can be in a second cell concentration (C2). The recirculation output of tissue culture fluid and cells can be in a third cell concentration (C3). Recirculation output 23 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 of tissue culture fluid and cells can be provided in third conduit 122. In 306, separating, in a cell aggregate trap (e.g., cell aggregate trap 120), cell aggregates (e.g., 109A) from the recirculation output of tissue culture fluid and cells takes place. Finally, in 308, returning the tissue culture fluid and cells to the bioreactor having relatively lower amount of cell aggregates can be accomplished (the relatively lower amount is in comparison to the tissue culture fluid and cells that would be returned to the bioreactor 102 without the cell aggregate trap 120). The returning tissue culture fluid and cells can have a fourth cell concentration (C4). Periodically or continuously during the separation process, cell aggregates 109A separated from the cells 109 within the sedimentation chamber 134 can settle to the bottom of the sedimentation chamber 134 and can exit and be discarded from the cell aggregate trap (e.g., cell aggregate trap 120), such as from trap discard outlet 126. [0046] Another example method of operating the perfusion bioreactor system 100 will now be described with reference to FIG. 4. The method 400 comprises, In 402, providing a flow of tissue culture fluid (e.g., TCF 108) and cells (e.g., cells 109 and possibly some cell aggregates 109A) from a bioreactor (e.g., bioreactor 102). Furthermore, the method 400 comprises, in 404, separating in a cell retention unit (e.g., cell retention unit 110) some cells from the tissue culture fluid to provide a harvest output (e.g., output in conduit 115). In 406, separating, in a cell aggregate trap (e.g., cell aggregate trap 120), cell aggregates from the tissue culture fluid and cells takes place. In 408, returning the tissue culture fluid and cells to the bioreactor having relatively lower amount of cell aggregates can be accomplished (the relatively lower amount is in 24 GA 02861270 2()14-07-15 WO 2013/109520 PCT/US2013/021533 comparison to the tissue culture fluid and cells that would be returned to the bioreactor 102 without the cell aggregate trap 120). As should be recognized from the above, the cell aggregate trap (e.g., cell aggregate trap 120) can be placed after or before the cell retention unit (e.g., cell retention unit 110), or elsewhere in the perfusion bioreactor system 100 where cell aggregates 109A can be effectively removed from a recirculation flow stream thereof. Furthermore, more than one cell aggregate trap can be provided in the perfusion bioreactor system. [0047] The methods according to embodiments are useful for removing cell aggregates (e.g., 109A) having greater than or equal to about 10 aggregated cells, greater than or equal to about 20 aggregated cells (or even greater than or equal to about 40 aggregated cells) that can be adhered together as a clump or mass (although smaller cell aggregates can be removed in some embodiments). Ranges of cell aggregates (e.g., 109A) as disclosed above can be removed, for example. As such, density within the bioreactor 102 during the operation of the perfusion process can be relatively more tightly controlled. Furthermore, In another advantage, discard volume of the TCF 108 can be reduced. Accordingly, this has a significant benefit in that product volume loss can be minimized. This advantage is significant even in perfusion bioreactor systems 100 where only a relatively small amount of cell aggregates 109A are formed. [0048] The foregoing description discloses only example embodiments of cell aggregate traps, perfusion biorcactor systems including a cell aggregate trap, and methods of operating the perfusion bioreactor systems. It is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and CA 02861270 2014-07-15 WO 2013/109520 PCT/US2013/021533 equivalents, as will be appreciated by those of skill in the art. For example, other embodiments of the cell aggregate trap can be used to remove relatively small cell aggregates should their presence be undesirable to the performance of the perfusion bioreactor system. The section headings used herein are for organizational purposed only and are not to be construed as limiting the subject matter described in any way. 26
