Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/10—Perfusion
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
  • C12M33/22—Settling tanks; Sedimentation by gravity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/10—Separation or concentration of fermentation products

Definitions

• TCF tissue culture fluid
• 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 deadend 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 .
• 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.
• the harvest output stream and the recirculation output stream are substantially continuous during the cultivation period, which can be ten days or more.
• using this type of conventional perfusion configuration can lead to conditions where cell density within the bioreactor may be relatively inadequately controlled.
• a perfusion bioreactor system is provided.
• the perfusion bioreactor system is provided.
• a bioreactor configured to contain a tissue culture fluid and cells to be cultured
• 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
• 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.
• a 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.
• 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 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.
• a method of operating a perfusion bioreactor system is provided. The method
• tissue culture fluid containing cells comprises (1) providing tissue culture fluid containing cells to a cell retention unit from a bioreactor (2)
• tissue culture fluid 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
• tissue culture fluid and cells can be returned to the bioreactor having relatively lower amount of cell aggregates.
• 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.
• FIG. 1 shows a block diagram of an embodiment of a perfusion bioreactor system including a cell aggregate trap according to the embodiments.
• FIG. 2A shows a cross-sectioned side view of a cell aggregate trap according to the embodiments.
• 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.
• FIG. 3 shows a flowchart illustrating a method of operating a perfusion bioreactor system according to the embodiments .
• FIG. 4 shows another flowchart illustrating another method of operating a perfusion bioreactor system according to the embodiments.
• Culturing of cells can be used to produce biologically-active substances and pharmaceutically-active products.
• 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") .
• cell aggregates When such cell aggregates are present, they can cause certain processing problems in the perfusion bioreactor process.
• 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.
• TCF and cells need to be
• bioreactor culture particularly animal or plant cells are generally very sensitive to imparted mechanical shear
• an improved perfusion bioreactor system is
• the improved perfusion bioreactor system comprises 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
• the perfusion bioreactor system comprises a bioreactor, a cell retention unit coupled to the bioreactor, a cell retention unit
• TCF and cells from the bioreactor 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
• a method of operating a perfusion bioreactor system is provided. The method
• 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.
• a method of operating a perfusion bioreactor system is provided. The method
• TCF retention unit from a bioreactor, separating some cells from the TCF to provide a harvest output, with the remaining re- 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 .
• bioreactor systems comprising cell aggregate traps, cell aggregate traps, and methods of operating perfusion
• FIG. 1 illustrates a block diagram of an
• 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
• the perfusion bioreactor system 100 can be used for the production of biologies such as coagulation factors.
• 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.
• 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 aHC03 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
• 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, Pluronic F-68, or insulin, and can be essentially free of other proteins.
• ⁇ , ⁇ -bis [2-Hydroxyethyl] -2-aminoethanesulfonic acid ⁇ , ⁇ -bis [2-Hydroxyethyl] -2-aminoethanesulfonic acid
• TRIZMA tris [Hydroxymethyl ] aminoethane
• the TCF can be any organic buffers such as MOPS (3-[N- Morpholinolpropanesulfonic acid), TES (N- tris [Hydroxymethyl]methyl-2-aminoethanesulfonic acid), BES ( ⁇ , ⁇ -bis [2-Hydroxyethyl] -2-aminoethanesulfonic acid) and TRIZMA (tris [Hydroxymethyl ] aminoethane) can be used; all of which can be obtained from Sigma (Sigma, St. Louis, Mo.), for example.
• the TCF can be any organic buffers such as MOPS (3-[N- Morp
• the TCF can contain EDTA, e.g., 50 ⁇ , as an iron chelating agent.
• EDTA e.g., 50 ⁇
• Other compositions, formulations, supplements, complexing agents and/or buffers can be used.
• 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 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 TCF is pumped continuously into the bioreactor and the TCF containing cells (and possibly some cell aggregates) are pumped out.
• a flow rate of TCF can be controlled and increased
• a steady state or stable perfusion process can be attained when the cell concentration reaches a target high level (e.g., about 10 *10 6 eelIs/mil to 20 x10 6 cells/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 biologies, coagulation factors, cell concentrations, cell densities or the like can be employed.
• the cells 109 can be eukaryotic or prokaryotic such as animal, plant, or
• 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 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.
• 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.
• cell aggregates are quite large.
• 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.
• cell aggregates 109A can be separated and removed.
• bioreactor 102 is generally undesirable, and the present perfusion bioreactor system 100 and methods 300, 400
• the depicted perfusion bioreactor system 100 comprises a cell retention unit 110 fluidly coupled to the bioreactor 102 and
• TCF 108 containing cells 109 in a first cell concentration (CI) (including possibly some cell aggregates 109A) from the bioreactor 102.
• Example first cell concentrations (CI) 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 (CI) are expelled from the bioreactor outlet 106 and received at a cell retention unit inlet 112 by passing through a first conduit 113.
• the conduit 113 can couple to an optional heat
• 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 (C2) at a first retention unit outlet 114. Accordingly, the second cell concentration (C2) is less than the first concentration (CI), that is C2 ⁇ CI, and in
• Example second cell concentrations can range from about 0.1 x 10 ⁇ 6 cells/mL to about 2 x 10 A 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
• 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 this can be done using centrifugation followed by dead-end membrane filtration, or by dead-end depth-filtration
• 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 chromotography.
• 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
• 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.
• the filtering process can be followed by a
• the ultrafiltration can occur at a specific flow rate below the transition point of the molecule of interest in the
• the cTCF 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 cTCF in liters/hour, or even approximately equal to between 0.3 to 1 times the volumetric flow rate of the cTCF in liters/hour. Other membrane areas can be used.
• 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
• 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
• 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 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.
• 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
• the output of the cell retention unit 110 can be directly received by the trap input 121.
• 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.
• C3 third cell concentration
• 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
• Example fourth cell concentrations (C4) can range from about 5 x 10 ⁇ 6 cells/mL to about 50 x 10 ⁇ 6
• 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.
• the TCF 108 and cells 109 can exit the trap outlet
• One or more recirculation pumps 125 can be
• the one or more pumps 125 can be located at any 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.
• 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 (C5) is provided in the trap discard outlet 126.
• Example fifth cell concentration (C5) 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
• 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.
• 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 used.
• the TCF 108 and cells 109 are received at the trap inlet 121, such as at a top of the trap body 130, for example.
• 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 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
• the expansion zone 132 is shown as a frustoconical region.
• 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.
• a transitional rate of increase in area exiting from the inlet 121 can be less than about 8.4 cm 2 /cm, and in some embodiments less than about 4.2 cm 2 /cm, in an attempt to minimize shear forces imparted to the cells 109.
• Other transitional rates can be used.
• an expansion zone 132 may not be present.
• 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
• the side flow chamber 136 is generally cylindrical and extends horizontally from a side 134S of the sedimentation chamber 134.
• the side flow chamber 136 can extend generally perpendicular from the sedimentation chamber 134.
• other shapes
• the cell aggregate trap 120 can include a contraction zone 138 at a location directly adjacent to the trap outlet 123 from the side flow chamber 136.
• the contraction zone 138 can have a transitional rate of area contraction no greater than about 8.4 cm 2 /cm, and in some embodiments, of about 4.2 cm 2 /cm or less. Larger or smaller transition rates can be used.
• a D3/D4 ratio can be provided that can be greater than about 2, for example.
• 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
• the discard contraction zone 140 can have a transitional rate of area contraction no greater than about 8.4 cm 2 /cm, and in some embodiments, of about 4.2 cm 2 /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.
• the sedimentation chamber 134 can, in some embodiments, have a circular cross section having transverse dimension (Dl) (e.g., an inside diameter) of between about 1.9 cm and about 6.4 cm, and in some
• a maximum cross-sectional area of the sedimentation chamber 134 can be between about 2.9 cm 2 and about 32 cm 2 , and in some
• 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.
• D2 transverse dimension
• 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.
• 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 centerline 142 of the side flow chamber 136.
• 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 (LI) 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 between about 5 cm and 18 cm, and in some embodiments
• a volumetric flow rate through the cell aggregate trap 120 is generally held at between about 0.0025 m 3 /min and about 0.0068 m 3 /min, and in some embodiments between about 0.0030 m 3 /min and about 0.0045 m 3 /min.
• Other volumetric flow rates e.g., capacities
• 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
• Q is the volumetric flow rate of the fluid (m 3 /s)
• ⁇ is the dynamic viscosity of the fluid (kg/ (m-s)
• p is the density of the fluid (kg/m 3 ) .
• 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.
• D3 maximum transverse dimension
• a maximum cross-sectional area of the side flow chamber 136 can between about 2.9 cm 2 and about 32 cm 2 , and in some embodiments between about 5.1 cm 2 and about 20 cm 2 , for example.
• D3 maximum transverse dimension
• a maximum cross-sectional area of the side flow chamber 136 can between about 2.9 cm 2 and about 32 cm 2 , and in some embodiments between about 5.1 cm 2 and about 20 cm 2 , for example.
• 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
• 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.
• 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.
• 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 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
• a perfusion bioreactor system 100 having a capacity of about 100 to 200 liters per day of flow second conduit 115.
• the cell aggregate trap 120 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.
• 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.
• D6 minimum cross-wise dimension
• 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 (C5) from the cell aggregate trap 120 is greater than or equal to about 3 times the first cell concentration (CI) (wherein C5 ⁇ 3C1), or even greater than or equal to about 5 times
• 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
• the 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) .
• 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.
• cell aggregates 109A can be removed from the flow stream prior to entry into the retention unit 110.
• a cell aggregate trap can be integrated into the bioreactor 102, such as at or near the bioreactor inlet 104.
• perfusion bioreactor system 100 comprises, in 302, providing to a cell retention unit (e.g., cell retention unit 110) from a bioreactor (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 (CI) .
• 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) .
• C2 second cell concentration
• recirculation output of tissue culture fluid and cells can be in a third cell concentration (C3) .
• Recirculation output of tissue culture fluid and cells can be provided in third conduit 122.
• separating, in a cell aggregate trap e.g., cell aggregate trap 120
• cell aggregates e.g., cell aggregates
• tissue culture fluid and cells from the recirculation output of tissue culture fluid and cells takes place.
• 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
• the returning tissue culture fluid and cells can have a fourth cell concentration (C4).
• C4 fourth cell concentration
• 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.
• 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
• cell aggregate trap 120 cell aggregate trap 120
• 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 cell aggregate trap e.g., cell aggregate trap 120
• the cell retention unit e.g., cell
• 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
• 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.
• 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.
• section headings used herein are for organizational purposed only and are not to be construed as limiting the subject matter described in any way .

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