JP6227562B2 - Perfusion bioreactor system and operating method thereof - Google Patents

Description

Related Applications This application is filed on January 18, 2012 with the title “PERFUSION BIOREACTOR SYSTEMS AND METHODS OF OPERATING THE SAME” (Attorney Docket No. BH-001 / L), US Provisional Patent Application No. 61 / 587,940. Insist on the interests of the issue and its priority.

  Conventional perfusion bioreactor systems and processes include bioreactors that have the function of culturing cells in a liquid medium, such as tissue culture fluid (TCF). Cultured cells and TCF are transferred from the bioreactor, eg, by pumping, and the cells are separated from TCF by conventional cell retention units. The recovered liquid from the cell holding unit, which still contains some cells, small pieces and debris, is then further processed. The recovered material used herein includes TCF that is further processed to obtain the desired product (eg, clotting factor). Filtration techniques such as dead-end depth filtration, membrane filtration, microfiltration and / or centrifugation can be used to further concentrate and / or purify the harvest from the cell retention unit.

  Other TCF effluents exiting the cell holding unit, including relatively high concentrations of cells, return directly to the bioreactor (eg, reuse or recirculate). In such a continuous perfusion bioreactor process, the collected and recirculated effluent is substantially continuous during a culture period that can be 10 days or longer. However, using this type of conventional perfusion setting can create an environment where the cell density in the bioreactor can be controlled relatively poorly.

  Accordingly, there is a need for perfusion bioreactor systems and methods that more efficiently regulate bioreactor cell density.

  In a first aspect, a perfusion bioreactor system is provided. The perfusion bioreactor system comprises (1) a bioreactor designed to contain a tissue culture medium and cells to be cultured; (2) accepts a tissue culture medium containing cells from the bioreactor and separates the cells from the tissue culture medium. A cell retention unit designed to supply tissue culture fluid and cell recycle, and to supply tissue culture fluid and cell recycle waste; and (3) accepts tissue culture fluid and cell recycle waste A cell aggregate trap that separates cell aggregates from tissue culture fluid and cell recycle effluent and returns the remaining tissue culture fluid and cells to the bioreactor.

In other embodiments, a cell aggregate trap is provided. The cell aggregates trap sedimentation chamber; at least some recycle effluent (3) tissue culture fluid containing cells; (2) tissue culture fluid and the inlet of the recycle effluent Accept trap designed so that the cells including waste trap outlet coupled to sedimentation chamber was designed to discharge and (4) cell aggregates; side flow chamber was designed to return to the bioreactor.

  In another system embodiment, a perfusion bioreactor system is provided. The perfusion bioreactor system comprises: (1) a bioreactor designed to contain tissue culture medium and cells to be cultured; (2) a cell holding unit designed to separate the cells from the tissue culture medium and supply the harvest; And (3) a cell aggregate trap designed to separate cell aggregates from tissue culture fluid and cells and to provide effluent with relatively low amounts of cell aggregates.

  In a method aspect, a method of operating a perfusion bioreactor system is provided. The method includes (1) supplying a tissue culture solution containing cells from a bioreactor to a cell holding unit, (2) separating the cells from the tissue culture solution in the cell holding unit, and collecting the tissue culture solution and the cells. And recirculating the cell culture and cell recycle effluent; (3) separating cell aggregates from the tissue culture medium and cell recirculation effluent in a cell aggregate trap. Tissue culture medium and cells may be returned to the bioreactor with relatively few cell aggregates.

  In another method aspect, a method of operating a perfusion bioreactor system is provided. The method comprises (1) supplying tissue cell fluid and cell flow from a bioreactor; (2) separating cells from tissue culture fluid in a cell holding unit and supplying a recovery; (3) cell aggregates Separating the cell aggregates from the tissue culture medium and cells in the trap, producing a tissue culture medium with a relatively reduced amount of cell aggregates. Tissue culture medium and cells may be returned to the bioreactor with relatively low amounts of cell aggregates.

  These and other features of the invention are described herein.

  It will be appreciated by those skilled in the art that the following figures are for illustrative purposes only. Such figures are not intended to limit the scope of the invention in any way.

FIG. 4 shows a block diagram of one embodiment of a perfusion bioreactor system including a cell aggregate trap according to this embodiment. FIG. 2 shows a side view of a cross section of a cell aggregate trap according to this embodiment. FIG. 2B shows an end view of the upward cross section of the cell aggregate trap along the 2B-2B cross sectional line of FIG. 2A. 2 shows a flowchart representing a method of operating a perfusion bioreactor system according to this embodiment. Fig. 6 shows another flow chart representing another method of operation of a perfusion bioreactor system according to this embodiment.

  Culture of cells (including animal, plant or microbial cells) can be used to produce biologically effective substances and pharmaceutically effective products. However, in certain cell cultures, cells can adhere to each other to some extent to form relatively large cell aggregates, cell aggregates or aggregates (hereinafter “cell aggregates”). Called). The presence of cell aggregates can cause certain process problems in the perfusion bioreactor process. Specifically, the presence of cell aggregates can cause the cell density in the bioreactor to be relatively unstable; that is, properly maintain, maintain or regulate within a desired range of cell density settings. Becomes difficult. In the presence of cell aggregates, it is also difficult to accurately measure the cell concentration. As a result, it is necessary to dispose of TCF and cells from time to time by regularly operating the waste pump of a conventional perfusion bioreactor system. Furthermore, it is also difficult to measure the amount of discarded TCF and cells. Of course, this discard also discards valuable TCF containing the desired product. Furthermore, cells in bioreactor culture, in particular animal cells or plant cells, are generally very sensitive to being subjected to mechanical shear forces. Thus, it is desirable not only to minimize the amount of material to be discarded, but also to minimize the exposure of cells to damaging shear forces. It is also desirable to be able to accurately adjust the cell concentration in the bioreactor.

Thus, according to aspects of the present invention, an improved perfusion bioreactor system is provided. The improved perfusion bioreactor system includes a cell aggregate trap that is provided, designed and / or tuned to operate in conjunction with a cell retention unit. The cell aggregates trap is functionally is based on sedimentation, wherein the cell aggregates are precipitated, it may remove from the recycle flow. By using a cell aggregate trap in conjunction with a cell retention unit in a perfusion bioreactor, a relatively large majority of cells are returned to the bioreactor, and cell aggregates that may be detrimental to the perfusion process are removed and discarded. obtain.

  According to another aspect, the perfusion bioreactor system is a bioreactor, a cell holding unit connected to the bioreactor, accepting TCF and cells from the bioreactor, separating cells from TCF, and obtaining a recovered material. Cell retention units designed to accept TCF and cell recycle effluent from the cell retention units, separate cell aggregates from TCF and cells, and return the remaining TCF and cells to the bioreactor Includes traps.

  In another aspect, a method of operating a perfusion bioreactor system is provided. The method includes providing TCF and cells, receiving TCF and cells in a cell aggregate trap, and separating the cell aggregates from TCF and cells in the cell aggregate trap. The remaining TCF and cells may be returned to the bioreactor with relatively low amounts of cell aggregates.

  In another aspect, a method of operating a perfusion bioreactor system is provided. The method feeds TCF containing cells from a bioreactor to a cell retention unit, separates the cells from TCF and feeds them, receives the remaining recirculated TCF and cells in a cell aggregate trap, and Separating cell aggregates from TCF and cells in a collection trap. The remaining TCF and cells may be returned to the bioreactor with a relatively low amount of cell aggregates. The methods, perfusion bioreactor systems and cell aggregate traps described herein may be applied to clotting factor production and / or other suitable methods for producing biological agents or factors.

  These and other aspects of the perfusion bioreactor system including the cell aggregate trap, the cell aggregate trap and the method of operation of the perfusion bioreactor system are described in FIGS. 1-4 below. FIG. 1 shows a block diagram of one embodiment of a perfusion bioreactor system (100). The perfusion bioreactor system (100) includes a bioreactor (102) that includes a bioreactor inlet (104) and a bioreactor outlet (106). The bioreactor (102) includes a culture chamber (105) designed to maintain tissue culture fluid (TCF) (108) and cells to be cultured (109). The perfusion bioreactor system (100) can be used for the production of biologics such as clotting factors. For example, the perfusion bioreactor system (100) and the method may be used for the production of a coagulation factor or other suitable factor or substance, such as Factor VII, Factor VIII or Factor IX.

An example of a method for producing Factor VIII is described in US Pat. No. 6,338,964, entitled “Process and Medium for Mammalian Cell Culture Under Low Dissolved Carbon Dioxide Concentration”, the entire contents of which are incorporated herein by reference. Is included. For example, the cell culture process can include culturing the cells in TCF with a high concentration of complexing agent and a buffer with a low concentration of added NaHCO ₃ . The cell culture process may be performed in a culture chamber, such as the culture chamber (105) of FIG. 1, which in some embodiments may be an agitated fermentor that includes an agitated impeller. The fermentor may be equipped with a microsparger at the bottom of the culture chamber or as a membrane as an oxygenation system. The TCF is a commercial DMEM / F12 formulation manufactured by JRH (Lenexa, Kansas) or Life Technologies (Grand Island, NY) supplemented with other adjuvants such as iron, Pluronic F-68 or insulin. Based media composition and essentially free of other proteins. The complexing agents histidine (his) and iminodiacetic acid (IDA) may be used, organic buffers such as MOPS (3- [N-morpholino] propanesulfonic acid), TES (N-tris [hydroxymethyl] Methyl-2-aminoethanesulfonic acid), BES (N, N-bis [2-hydroxyethyl] -2-aminoethanesulfonic acid) and TRIZMA (tris [hydroxymethyl] aminoethane) may be used; All are available, for example, from Sigma (Sigma, St. Louis, Mo.). In some embodiments, the TCF may be supplemented with known concentrations of these complexing agents and organic buffers, either individually or in combination. The TCF may contain EDTA as an iron chelator, for example at 50 μM. Other compositions, formulations, adjuvants, complexing agents and / or buffers may be used.

Cell culture may be initiated by seeding the cells from a previously grown culture. Typical bioreactor parameters include stable conditions such as temperatures of about 35 ° C. to 37 ° C., pH of about 6.8 to 7.0, dissolved oxygen (DO) of about 30% to 70% of air saturation, about It can be maintained (e.g. automatically) under conditions such as a stirring speed of 30 rpm to 80 rpm and a substantially constant liquid volume. Other bioreactor parameters may be used. DO and pH can be measured online using commercially available probes. The bioreactor process may begin with an initial doubling of cell concentration for about 1-2 days in batch mode. This can be followed by a perfusion phase where TCF is continuously pumped into the bioreactor and TCF containing cells (and may contain cell aggregates) is pumped out. The TCF flow rate is regulated and can increase in proportion to cell concentration. The cell concentration reached a high level to target in the bioreactor (e.g., about 10 × 10 ⁶ cells / ml~20x10 ⁶ cells / mL), when that may be adjusted with this concentration can achieve a steady state or stable perfusion process . In this respect, the flow rate can be kept constant. The cell density may be 4,000,000 to 40,000,000 cells / mL in the perfusion bioreactor system. Other biologics, clotting factors, cell concentrations or cell densities, etc. can also be used.

With reference to FIG. 1, cell (109) may be a eukaryotic cell or a prokaryotic cell, such as an animal, plant or microbial cell. For example, the cell (109) may be a baby hamster kidney cell (BHK cell), a hybrid of kidney cell and B cell (HKB cell), or a human fetal kidney cell (also referred to as HEK cell-HEK293 or 293 cell). . The TCF (109) may be introduced into the culture chamber (105) via the TCF inlet (105A) or elsewhere in the perfusion bioreactor system (100). Cells (109) in TCF (108) can sometimes form cell aggregates (109A) as shown in the enlarged view, depending on their nature and treatment. As used herein, the term “cell aggregate” refers to a cell aggregate, a cell aggregate, or an aggregate of cells that are linked together and adhere to form a group of cells. A “cell aggregate” that can be removed using one or more aspects of the present invention can be about 10 or more cells, about 20 or more cells, or about 40 or more cells. According to one or more aspects of the present invention, cell aggregates can be removed in the range of about 10 to about 50,000 cells, or about 40 to about 300 cells. More generally, cell aggregates (109A) that can be removed using one or more of the various aspects of the invention are such that at least some of the internal cells in the aggregates are sufficient during the perfusion process. Includes cell aggregates of a size and shape that tend to die from lack of oxygen and / or nutrients. In general, cell aggregates are very large. For example, cell aggregates (109A) with a minimum (cell aggregate width) of about 60 microns or larger, or about 100 microns or larger in size (109A) can be separated and removed by using various embodiments of the present invention. . One or more aspects of the present invention may remove cell aggregates having a minimum dimension ranging from about 60 microns to about 3000 microns, or from about 100 microns to about 500 microns. Smaller agglomerates may be further separated or removed as an alternative. The presence of cell aggregates (109A) in the bioreactor (102) is usually not desirable, and the perfusion bioreactor system (100) and method (300, 400) of the present invention has at least some and many In this case, most cell aggregates (109A) can be removed therefrom. The illustrated perfusion bioreactor system (100) includes cells (109) at a first cell concentration (C1) in fluid communication with the bioreactor (102), including TCF (108) (including cell aggregates (109). A cell holding unit (110) designed to receive from the bioreactor (102). The first cell concentration (C1) can range, for example, from about 4 × 10 ⁶ cells / mL to about 40 × 10 ⁶ cells / mL. Other ranges of cell concentrations may be used. In the embodiment shown, TCF (108) containing cells (109) at a first concentration (C1) is discharged from the outlet (106) of the bioreactor and passes through the first conduit (113) of the cell holding unit. Accepted at the entrance (112). The conduit (113) may be connected to any heat exchanger that functions to cool the TCF (108) containing cells (109) discharged from the bioreactor outlet (106). The cell holding unit (110) separates most of the cells (109) from the TCF (108) and at the outlet (114) of the first holding unit a very small amount of cells (second cell concentration (C2) ( It is designed to supply a TCF (108) recovery containing only 109) and is operable and therefore functions as such. Thus, the second cell concentration is lower than the first cell concentration (C1), ie C2 <C1, specifically C2 << C1). The second cell concentration (C2) can range, for example, from about 0.1 × 10 ⁶ cells / mL to about 2 × 10 ⁶ cells / mL. Other cell concentrations may be used. The so-called recovered material passes from the outlet (114) of the first cell holding unit through the second conduit (115) by the pumping action of the recovery pump (117) connected to the second conduit (115), for example. The harvest can be further isolated and / or purified in a downstream isolation and purification process (118). These additional isolation and purification processes (118) may be performed continuously or batchwise. For example, these downstream isolation and purification processes (118) can be performed as described in U.S. Patent Publication No. 2008/0269468, entitled `` Devices And Methods For Integrated Continuous Manufacturing Of Biological Molecules '', the disclosure of which Are hereby incorporated by reference in their entirety. For example, in batch mode, once a certain volume of collected material has been collected, typically in 1 to 4 days or more, one or more collection vessels may be disconnected from a sterile fermentation vessel and the collected material may be combined into one batch. . The next step is to remove cells, debris, and small pieces. On an industrial scale, this can be done by centrifugation and subsequent dead end membrane filtration or by dead end depth filtration followed by dead end membrane filtration. Other techniques such as tangent flow (or “cross flow”) microfiltration or any other suitable filtration technique may also be used. In any case, the product of the debris removal process is a batch of purified tissue culture fluid (cTCF). The cTCF can be purified (concentrated) by any suitable method, such as cross-flow ultrafiltration or packed bed chromatography.

  In continuous mode, a fixed volume of harvest can be purified by a continuous purification system that can be maintained under sterile conditions integrated with a perfusion bioreactor system. As used herein, the term “continuous” refers to an uninterrupted time, sequence and / or operation over an extended period of time. For example, the cell retention unit (110) may perform the initial cell retention and production of the recovered TCF recovery (108) at the outlet (114) of the first retention unit. The isolation and purification process (118) may further comprise filtering (isolating) the recovered material from the second conduit (115) through a suitable filtration system, and in some embodiments, the filtration system. CTCF is provided by a final filtration rate of about 3 microns or less, 0.45 microns or less, or 0.2 microns or less. Other filtration rates may be used.

The filtration process may be followed by a purification process including, for example, a continuous ultrafiltration separation process. In some embodiments, ultrafiltration may be performed at a specific specific flow below the transition point of the molecule of interest in the pressure dependent region of the flux versus TMP curve, where the specific specific flow is It is substantially constant throughout the continuous ultrafiltration. By using a continuous isolation and purification process (118), relatively high yields can be achieved. In some embodiments, the cTCF is approximately equivalent to the volumetric flow rate (l / hour) of cTCF, has an area (m ² ) that is about 0.1 to ² times greater, or has a volumetric flow rate of cTCF (l / hour). ) Or an ultrafiltration membrane having an area (m ² ) of about 0.3 to 1 times. Other membrane areas may be used.

After separation in the cell holding unit (110), the recirculated effluent of TCF (108) and a relatively high concentration of cells (109) is transferred to the third cell at the outlet (119) of the second holding unit. Obtained in concentration (C3). Such a third cell concentration (C3) is generally relatively higher than the first concentration (C1), ie C3> C1, which means that a small amount of cells (109) are lost in the recovery solution. Despite being seen, the volume of TCF (108) extracted at the outlet (114) of the first holding unit is larger. The third cell concentration (C3) can range, for example, from about 6 × 10 ⁶ cells / mL to about 60 × 10 ⁶ cells / mL. Other ranges of cell concentrations may be used. The cell retention unit (110) can be any known cell separation technique such as a disk filter, spin filter, flat sheet filter, microporous hollow fiber filter, crossflow filter, vortex flow filter, continuous centrifugation, centrifugal bioreactor. May be based on gravity settlers, ultrasonic devices, hydrocyclones, and the like. Any suitable type of cell retention unit that functions as it is designed and operable to divide the concentration of the first cell entering (C1) into the concentration of cells exiting (C2 and C3). 110) can be used.

  In the embodiments described herein, the perfusion bioreactor system (100) includes a cell aggregate trap (120). The cell aggregate trap (120) is configured to bring the recirculated effluent of TCF (108) and cells (109) from the cell retention unit (110) to a third cell concentration (C3) at the trap inlet (121). It is designed to accept and operate, and therefore functions as such. The cell holding unit (110) may be fluidly connected to the cell aggregate trap (120) by a third conduit (122). In some embodiments, the functions of the cell retention unit (110) and the cell aggregate trap (120) may be integrated into one unit. Thus, in such an embodiment, the conduit (122) may be omitted and the effluent of the cell holding unit (110) may be received directly into the trap inlet (121).

The cell aggregate trap (120) received the cell aggregate (109A) in the cell aggregate trap (120), the TCF (108) at the third cell concentration (C3), and the recirculating excretion of the cells (109). It works when separating from things. In one implementation, separation of cell aggregates (109A) is continuous; that is, during operation, the flow from the cell holding unit (110) is continuous. Generally, if present in the flow stream, at least some of the cell aggregates (109A), typically a fairly high percentage (%), are removed by the cell aggregate trap (120), and the fourth cell Residual TCF (108) and cells (109) at a concentration (C4) are returned to the inlet (104) of the bioreactor (102). The fourth cell concentration (C4) can range, for example, from about 5 × 10 ⁶ cells / mL to about 50 × 10 ⁶ cells / mL. Other ranges of cell concentrations may be used. In some embodiments, the method comprising the perfusion bioreactor system (100) and the cell aggregate trap (120) can remove about 20% to about 80% of the cell aggregates, but other percentages (% ) But can also remove cell aggregates.

  TCF (108) and cells (109) may exit the trap outlet (123) and enter the bioreactor inlet (104) through the fourth conduit (124). One or more recirculation pumps (125) may be provided and may be manipulated to cause TCF (108) and cell (109) flow. Such one or more recirculation pumps (125) may be in any convenient location, such as conduit 113, 122 or 124, or other suitable location. In the embodiments described herein, the pump (125) is connected to the fourth conduit (124).

In addition, the cell aggregate trap (120) is configured and operable to allow small amounts of TCF (108) and some cell aggregate (109A) to be removed from the cell aggregate trap (120). And therefore may include any suitable trap waste port (126) that functions as such. The fifth cell concentration (C5) is obtained at the trap waste port (126). The fifth cell concentration (C5) may range, for example, from about 12 × 10 ⁶ cells / mL to about 90 × 10 ⁶ cells / mL. Other ranges of cell concentrations may be used. Because some cell aggregates (109A) are removed from the process flow stream by the cell aggregate trap (120), the cell concentration (C4) is generally lower than the cell concentration (C3), ie C4 <C3 . Cell aggregates (109A) and a small amount of TCF (109) flow from the trap waste port (126) and can be discarded. The waste pump (127) is operated continuously or periodically to allow cell aggregates (109A) and a small amount of TCF (108) to pass through the waste conduit (128) to a waste container, such as a flexible bag, or other May be poured into a mold waste container.

The structure and operation of the cell aggregate trap (120) is described below for FIGS. 2A and 2B. The cell aggregate trap (120) includes a trap body (130), which may be made of a rigid material such as stainless steel, glass or plastic. Other materials may be used. The TCF (108) and cells (109) (which may contain some cell aggregates (109A)) are received at the trap inlet (121), for example at the top of the trap body. As shown, TCF (108) and cells (109) and optionally cell aggregates (109A) can be formed during the operation at an expansion zone (132) that can be formed immediately adjacent to the trap inlet (121) and the it may flow into directly sedimentation chamber of the cell aggregates trap (120). The expansion zone (132), the cross-sectional area of the sedimentation chamber (134) is gradually increased along the length of the sedimentation chamber (134) may consist wall oblique or curved. In the embodiment shown herein, the expansion zone (132) is shown as a frustoconical region. However, the transverse area of the trap entrance (121), may be used any of generally smooth transition to cross-sectional area of the sedimentation chamber (134). In general, the rate of transition of the area increase from the inlet (121) may be less than about 8.4 cm ² / cm to minimize the shear forces experienced by the cells (109), and in some embodiments May be less than about 4.2 cm ² / cm. Other transition rates may be used. However, in some embodiments, the expansion zone (132) may not exist.

The cell aggregate trap (120) may include a side flow chamber (136). The side flow chamber (136) is connected to the sedimentation chamber (134), the TCF (108) and cells (109), out of the cell aggregates trap through the trap outlet (123) (120) , cell aggregates (109A) is at during sedimentation chamber (134), configured to settle under gravity, it is designed, but operable. In the embodiment shown, the side flow chamber (136) is generally cylindrical, it is common to have extended horizontally from the side of the sedimentation chamber (134) (134S). For example, the side flow chamber (136) is generally from sedimentation chamber (134), it may expand vertically. However, other shapes, designs and orientations other than the vertical direction may be used.

Like the sedimentation expansion zone of the chamber (134) (132), cell aggregates trap (120) is in adjacent positions directly to the trap outlet (123) from the side flow chamber (136), shrink zone (138) Can be included. In some embodiments, the shrinkage zone (138) exhibits an area shrinkage transition rate of about 8.4 cm ² / cm or less, and in some embodiments, about 4.2 cm ² / cm or less. Larger or smaller transition rates may be used. In some embodiments, the D3 / D4 ratio may be greater than about 2, for example. Similarly, the sedimentation chamber bottom portion of the trap disposal port (134) (126), there may be discarded shrinkage zone (140). The trap waste port (126) may have a maximum transverse dimension D5 (eg, inner diameter). In one or more embodiments, the waste shrinkage zone (140) may exhibit an area shrinkage transition rate of about 8.4 cm ² / cm or less, and in some embodiments, about 4.2 cm ² / cm or less. . Larger or smaller transition rates may be used. In some embodiments, the D1 / D5 ratio may be greater than about 2, for example.

More particularly, sedimentation chamber (134), in some embodiments, from about 1.9cm~ about 6.4 cm, in some embodiments about 2.5cm~ about 5.1cm transverse dimension of (D1) (e.g. inside diameter) It can have a circular cross section. Maximum cross-sectional area of the sedimentation chamber (134) may be about 2.9 cm ² ~ about 32cm ^2, in some embodiments, for example, from about 5.1 cm ² ~ about 20 cm ^2. The trap entrance (121) may have a circular cross section with a transverse dimension (D2) (eg, an inner diameter) of about 0.48 cm to about 1.6 cm, and in some embodiments, for example, about 0.64 cm to about 1.3 cm. In some embodiments, the D1 / D2 ratio may be greater than about 2, for example, greater than about 4. However, other suitable cross-sectional configurations and sizes may be used.

The appropriate size of the cell aggregate trap (120) may depend on the capacity of the perfusion bioreactor system (100) (eg, its processing capacity), which may be larger or smaller based on the flow capacity. obtain. The size of the cell aggregate trap (120) may also depend on other factors such as fluid density or viscosity. Maximum cross-sectional area of the sedimentation chamber (134) may be a maximum cross-sectional area equal to or greater than that of the trap entrance (121). Maximum cross-sectional area of the embodiment shown herein, sedimentation chamber (134) is greater than the maximum cross-sectional area of the trap entrance (121). Specifically, in some embodiments, the maximum cross-sectional area of the sedimentation chamber (134) is about 4 times the maximum cross-sectional area of the trap entrance (121), about 10-fold or more, about 30 times or more, or It may be about 60 times or more.

The sedimentation chamber (134) includes an upper region (134U) and a lower region (134L). The upper region (134U) is located above the center line (142) of the side flow chamber (136), and the lower region (134L) is below the center line (142) of the side flow chamber (136). Located in. In the embodiment shown herein, the sedimentation chamber (134), from the upper end of the expansion zone (132), the total length of up to the lower end of the contraction zone (140) (Lt) may be about 9Cm～37cm, In some embodiments, it may be about 14 cm to 28 cm. The length (Lu) of the upper region (134U) from the upper end of the contraction zone (132) to the centerline (142) of the side flow chamber (136) may be about 5 cm to 18 cm, and in some embodiments It may be about 7 cm to 14 cm. The length (Ll) of the lower region (134L) from the lower end of the contraction zone (140) to the centerline (142) of the side flow chamber (136) may be about 5 cm to 18 cm, and in some embodiments May be about 7 cm to 14 cm. Other sizes may be used. In general, the ratio of Ll / Lu is desirably> 0.5, and in some embodiments, for example, Ll / Lu> 4. Other ratios may be used.

During operation, the volumetric flow rate through the cell aggregate trap (120) is generally about 0.0025 m ³ / min to about 0.0068 m ³ / min, and in some embodiments about 0.0030 m ³ / min. ~ About 0.0045m ³ / min. Other volume flow rates (eg, volume) may be used. However, in some embodiments, the liquid flow rate, in sedimentation chamber (134), it is desirable to keep the number range of the layer Reireruzu. Sedimentation chamber (134) Reynolds number in the mixing to minimize, to facilitate settling and separation of appropriate cell aggregates (109A) is about 2300 or less, about 1000 or less, or some In some embodiments, it may be less than about 500, where the Reynolds number is approximately defined by Equation 1 below:
Re = ρQ / μ Equation 1
[Where:
Q is the volume flow rate of the liquid (m ³ / s)
μ is the dynamic viscosity of the liquid (kg / (m
ρ is the density of the liquid (kg / m ³ ). ].

However, TCF (108) and cells (109), as shown resistance to that answer cell aggregates trap (120) from the trap outlet (123), to settle in sedimentation chamber (134) , in order to properly hold the cells (109) in the flow stream, the flow in the sedimentation chamber (134), for example, may have only a Reynolds number avoids the settling of cells (109).

The side flow chamber (136) may have a circular cross section that exhibits a maximum transverse dimension (D3) (eg, inner diameter) of about 1.9 cm to about 6.4 cm, and in some embodiments about 2.5 cm to about 5.1 cm. The maximum cross-sectional area of the side flow chamber (136) may be from about 2.9 cm ² to about 32 cm ² , and in some embodiments, for example, from about 5.1 cm ² to about 20 cm ² . For example, other suitable crossing shapes and sizes may be used. The total length (Ls) of the side flow chamber (136) from the inlet to the outlet of the contraction zone (138) of the side flow chamber (136) may be about 4 cm to 15 cm, and in some embodiments, It may be about 5 cm to 11 cm. The maximum size (D4) (e.g. inner diameter) of the outlet (123) of the side flow chamber (136) may be about 0.48 cm to 1.6 cm, and in some embodiments about 0.64 cm to 1.3 cm. is there. Other sizes may be used. The flow in the side flow chamber (136) may exhibit a Reynolds number greater than about 2300, or for example greater than about 4000. Other Reynolds number ranges may be used. A Reynolds number that minimizes sedimentation of the cells (109) in the side flow chamber (136) may be selected.

In some embodiments, the maximum cross-sectional area of the sedimentation chamber (134) (Asc), the maximum equivalent or greater than the cross-sectional area (Asfc) of the side flow chamber (134), that is, Asc ≧ Asfc. Specifically, the maximum cross-sectional area of the sedimentation chamber (134) (Asc) may be larger maximum cross-sectional area equal to or more than five times the side flow chamber (136). Other Asc / Asfc ratios may be used. Difference in cross-sectional area is generally, it may function to improve the precipitation Funo. D1-D5, the representative sizes described herein, are specific examples of a perfusion bioreactor system (100) in which the flow in the second conduit (115) has a volume of about 2000-3000 l per day. (Fig. 1). A perfusion bioreactor system (100) having a smaller or larger volume is an embodiment of the present invention, such as a perfusion bioreactor system where the flow in the second conduit (115) is about 100-200 l per day. 100) can benefit.

During operation, the cell aggregates trap (120), for sedimentation chamber (134) and side flow chamber (136), by a suitable dimensioning and volume flow rate as described herein, about 10 Cell aggregate (109A) larger than or equivalent to aggregated cells, greater than or equivalent to about 20 aggregated cells (109), or greater than or equivalent to about 40 aggregated cells (109) It is designed to remove and is operable and applied as such. In some embodiments, a smaller number of aggregated cells may be removed. Cells (109) and (TCF) can exit the side flow chamber (136). Thus, undesirable cell aggregates (109A) are removed by the operations of the various aspects of the invention. In some embodiments, unwanted cell aggregates (109A) removed by the cell aggregate trap (120) have a minimum lateral direction of greater than about 60 microns, greater than about 100 microns, or even greater. (D6) (see FIG. 2A). Smaller cell aggregates can also be removed. One advantage of using a cell aggregate trap (120) is that the rate of disposal of TCF (108) from the perfusion bioreactor system (100) can be reduced. Specifically, the waste cell concentration (C5) from the cell aggregate trap (120) is equal to or about 3 times higher than the first cell concentration (C1) (where C5 ≧ 3C1), or The discard rate of the TCF (108) can be slowed down so that it is equivalent or about 5 times higher than C1 (where C5 ≧ 5C1).

  In the embodiment shown, the cell aggregate trap (120) is shown installed at the outlet of the cell holding unit (110). However, it should be understood that the cell aggregate trap (120) may be placed anywhere else in the perfusion bioreactor system (100). For example, cell aggregate traps such as cell aggregate traps (120) may be located at the location of the first conduit (113) (eg, next to or next to the bioreactor outlet (106) or the cell retention unit inlet (112)). Otherwise, it may be connected to the first conduit (113)). In this embodiment, the TCF (108), which may contain cell aggregates (109A), and the recirculated effluent of cells (109) pass through the cell aggregate trap and reach the cell retention unit (110). Thus, cell aggregates (109A) can be removed from the flow stream before entering the retention unit (110). The cell aggregate trap may also be integrated into the bioreactor (102), for example at or near the bioreactor inlet (104).

Methods of operation of various embodiments of the perfusion bioreactor system (100) are described with respect to FIG. 3 below. One method (300) of operating a perfusion bioreactor system (100) is from a bioreactor (eg, bioreactor (102)) to a cell (eg, cell (109) and possibly some cell aggregates (109A). )) And supplying a cell culture unit (eg, TCF (108)) to a cell retention unit (eg, cell retention unit (110)) (302). The tissue culture medium containing cells can be at a first cell concentration (C1). Further, the method (300) further comprises separating cells from the tissue culture medium in the cell holding unit to obtain a tissue culture medium and cell harvest (eg, in the second conduit (115)) and tissue culture. Including supplying fluid and cell recycle effluent (304). The harvest may be at a second cell concentration (C2). The tissue culture fluid and cell recycle effluent may be at a third cell concentration (C3). Tissue culture fluid and cell recycle effluent is obtained in the third conduit (122). In 306, cell aggregates (eg, 109A) are separated from tissue culture fluid and cell recycle effluent in a cell aggregate trap (eg, cell aggregate trap (120)). Finally, at 308, the tissue culture and cells can be returned to the bioreactor with a relatively low amount of cell aggregates (the relatively low amount described above refers to the cell aggregate trap ( Comparison with tissue culture medium and cells that would return to bioreactor (102) if no). The returning tissue culture medium and cells can be at a fourth cell concentration (C4). The during the separation process, sedimentation chamber (134) in a cell (109) is separated from the cell aggregates (109A) is periodically or continuously, drop-off precipitated on the bottom of the sedimentation chamber (134), From the cell aggregate (for example, the cell aggregate trap (120)), for example, it may go out of the trap disposal port (126) and be discarded.

  Another specific method of operating the perfusion bioreactor system (100) is described with respect to FIG. The method (400) involves the flow of tissue culture fluid (eg, TCF (108)) and cells (eg, cells (109) and optionally cell aggregates (109A)) into a bioreactor (eg, bioreactor (102)). ) From (402). Further, the method (400) separates cells from tissue culture fluid in a cell retention unit (e.g., cell retention unit (110)) and collects the recovered material (e.g., effluent in conduit (115)). Including supplying (404). At 406, cell aggregates are separated from tissue culture fluid and cells in a cell aggregate trap (eg, cell aggregate trap (120)). At 408, tissue culture fluid and cells can be returned to the bioreactor with relatively low amounts of cell aggregates (if the above-mentioned relatively low amounts are present when there is no cell aggregate trap (120)) Comparison with tissue culture medium and cells that would return to the bioreactor (102). As can be understood from the above description, the cell aggregate trap (for example, cell aggregate trap (120)) is formed before or after the cell retention unit (for example, cell retention unit (110)) or the cell aggregate (109A). ) May be installed anywhere else in the perfusion bioreactor system (100) that can be efficiently removed from the recirculation flow stream. In addition, one or more cell aggregate traps may be placed in the perfusion bioreactor system.

  The method of this embodiment comprises about 10 or more, or about 20 or more (or about 40 or more) aggregated cells that can adhere to each other as clumps or cell populations. It is useful to remove cell aggregates that have (eg, 109A) (although in some embodiments, smaller cell aggregates may also be removed). For example, cell aggregates (eg, 109A) in the above range can be removed. As such, during operation of the perfusion process, the density in the bioreactor (102) can be controlled more closely. Yet another advantage is that the waste volume of TCF (108) can be reduced. This is therefore a significant advantage in that the loss of product volume can be minimized. This advantage is also significant in the perfusion bioreactor system (100), where only relatively small amounts of cell aggregates (109A) are formed.

  The above description only describes exemplary embodiments of cell aggregate traps, perfusion bioreactor systems including cell aggregate traps, and methods of operation of the perfusion bioreactor systems. It is not intended that the present invention be limited to such embodiments. On the contrary, the present invention encompasses various alternatives, modifications and equivalents that can be understood by those skilled in the art. For example, relatively small cell aggregates may be removed using other embodiments of cell aggregate traps if their presence is undesirable for the operation of a perfusion bioreactor system. The section headings used herein are for structural purposes only and are not to be construed as limiting the particular subject matter of the invention.

Claims (22)

A bioreactor (102) comprising a bioreactor inlet (104), a bioreactor outlet (106) and a culture chamber (105) for containing cells (109) to be cultured with tissue culture fluid (108);
An inlet (112) for receiving a tissue culture solution (108) containing cells (109) from the bioreactor (102), a cell separation for separating a part of the cells (109) from the tissue culture solution (108) A member , a first outlet (114) for supplying a collection of tissue culture fluid (108) and cells (109) and a recirculated discharge of tissue culture fluid (108) and cells (109) A cell holding unit (110) comprising a second outlet (119) and fluidly connected to the outlet (106) of the bioreactor; and the resuscitation of tissue culture medium (108) and cells (109) from the cell holding unit (110) Trap inlet (121) for receiving circulating effluent, cell aggregate (109A) is separated from tissue culture fluid (108) and recirculating effluent of cells (109) A side flow chamber (136) comprising a sedimentation chamber (134) for trapping, the remaining tissue culture fluid (108) and a trap outlet (123) for returning cells (109) to the bioreactor, A cell aggregate trap (120) fluidly coupled to the outlet (119);
A perfusion bioreactor system (100) comprising: A bioreactor (102) comprising a bioreactor inlet (104), a bioreactor outlet (106) and a culture chamber (105) for containing cells (109) to be cultured with tissue culture fluid (108);
Trap inlet (121) for receiving tissue culture solution (108) containing cells (109) from the bioreactor (102), cell aggregate (109A), tissue culture solution (108) containing cells (109) A side flow chamber (136) comprising a sedimentation chamber (134) for separation from the cell, and a trap outlet (123) for supplying the effluent of tissue culture fluid (108) comprising cells (109), A cell aggregate trap (120) fluidly connected to an outlet (106) of the reactor, and an inlet (112) for receiving tissue culture fluid (108) containing cells (109) from the cell aggregate trap (120), the tissue A cell separation member for separating a part of the cells (109) from the culture solution (108), the tissue culture solution (108) and the cells (109) ) And a second outlet for supplying the tissue culture fluid (108) and recirculated effluent of cells (109) to the bioreactor (102). 119), the cell holding unit (110) fluidly connected to the outlet (123) of the trap;
A perfusion bioreactor system (100) comprising:   The cell aggregate trap (120) includes a trap body (130) that houses a sedimentation chamber (134), the trap inlet (121) being located at the top of the trap body (130), The perfusion according to claim 1 or 2, wherein an expansion zone (132) is formed at the inlet (121), the cross-sectional area of the expansion zone (132) gradually increasing along the length of the sedimentation chamber (134). Bioreactor system (100).   The cell aggregate trap (120) includes a contraction zone (138) at the trap outlet (123) from the side flow chamber (136), the cross-sectional area of the contraction zone (138) being the trap outlet (123). The perfusion bioreactor system (100) according to claim 1 or 2, which gradually decreases towards The settling chamber (134) has a maximum cross-sectional area, the side flow chamber (136) has a maximum cross-sectional area, and the maximum cross-sectional area of the settling chamber (134) is equal to the maximum cross-sectional area of the side flow chamber (136). 3. A perfusion bioreactor system (100) according to claim 1 or 2, which is or larger.   The perfusion bioreactor system (100) of claim 5, wherein the maximum cross-sectional area of the sedimentation chamber (134) is at least five times greater than the maximum cross-sectional area of the side flow chamber (136).   The perfusion bioreactor system (100) of claim 1 or 2, wherein the side flow chamber (136) extends horizontally from the sedimentation chamber (134).   The perfusion bioreactor system (100) of claim 1 or 2, comprising a continuous flow of tissue culture fluid (108) and cells (109) through the cell aggregate trap (120). A sedimentation chamber (134) for separating cell aggregates (109A) from tissue culture fluid (108) comprising cells (109);
Trap inlet (121) for receiving the recirculated effluent of tissue culture fluid (108) and cells (109);
A side flow chamber (136) comprising a trap outlet (123) for supplying tissue culture fluid (108) containing cells (109) and operable in connection with a sedimentation chamber (134); A trap waste outlet (126) connected to a sedimentation chamber (134), designed to discharge the mass (109A);
A cell aggregate trap (120) comprising:   A trap body (130) for storing a sedimentation chamber (134), wherein the trap inlet (121) is located on top of the trap body (130), and the trap inlet (121) has an expansion zone (132) The cell aggregate trap (120) of claim 9, wherein a cross-sectional area of the expansion zone (132) is gradually increased along the length of the sedimentation chamber (134).   The trap outlet (123) of the side flow chamber (136) includes a contraction zone (138), the cross-sectional area of the contraction zone (138) gradually decreasing towards the trap outlet (123). 9. The cell aggregate trap (120) according to 9.   The settling chamber (134) has a maximum cross-sectional area, the side flow chamber (136) has a maximum cross-sectional area, and the maximum cross-sectional area of the settling chamber (134) is equal to the maximum cross-sectional area of the side flow chamber (136). The cell aggregate trap (120) of claim 9, wherein the cell aggregate trap (120) is larger or larger.   The cell aggregate trap (120) of claim 12, wherein the maximum cross-sectional area of the sedimentation chamber (134) is at least five times the maximum cross-sectional area of the side flow chamber (136). The cell aggregate trap (120) of claim 9, wherein the side flow chamber (136) extends horizontally from the sedimentation chamber (134 ) . A method for operating a perfusion bioreactor system comprising:
Supplying tissue culture fluid (108) containing cells (109) from the bioreactor (102) to the cell holding unit (110);
In the cell holding unit (110), a part of the cell (109) is separated from the tissue culture solution (108), and the tissue culture solution (108) and the recovered cell (109), and the tissue culture solution (108) and the cell A recirculated effluent of (109), wherein the cell retention unit from an inlet (112) for receiving a tissue culture fluid (108) containing cells (109), from the tissue culture fluid (108) A cell separation member for separating a portion of the cells (109), a tissue culture fluid (108) and a first outlet (114) for supplying a collection of cells (109); and a tissue culture fluid (108) and A second outlet (119) for supplying a recirculated effluent of cells (109);
Separating cell aggregates (109A) from tissue culture fluid (108) and recirculated effluent of cells (109) in a cell aggregate trap (120), wherein the cell aggregate trap comprises Trap inlet (121) for receiving tissue culture fluid (108) and recirculated effluent of cells (109), cell aggregate (109A) recirculated effluent of tissue culture fluid (108) and cells (109) A method of operating the perfusion bioreactor system comprising a sedimentation chamber (134) for separation from the side flow chamber (136) comprising a trap outlet (123).   16. The method of claim 15, comprising returning the tissue culture fluid (108) and the cells (109) to the bioreactor (102) with relatively low amounts of cell aggregates (109A).   The method of claim 15, comprising discarding the cell aggregate (109A) from the cell aggregate trap (120).   The concentration of cells discarded from the cell aggregate trap (120) is at least three times the initial cell concentration of the tissue culture fluid (108) containing cells (109) supplied to the cell retention unit (110). 16. The method of claim 15, comprising discarding cell aggregates (109A) and tissue culture fluid (108).   16. The method of claim 15, wherein the cell aggregate (109A) comprises 10 or more aggregated cells (109A) or 20 or more aggregated cells (109A).   16. The method of claim 15, wherein the cell aggregate (109A) has a minimum dimension greater than 60 microns.   16. The method of claim 15, wherein the cells (109) comprise mammalian cells or other cells that produce clotting factors, or the cells comprise BHK cells, HKB cells or HEK cells.   16. A method according to claim 15 comprising producing Factor VII, Factor VIII or Factor IX.

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