High-speed clarification of liquid suspensions

Abstract

Systems and methods for clarifying a liquid suspension contained in a flexible pouch at high speed are provided. The method includes nesting at least one flexible bag containing the liquid suspension within at least one bag adapter, wherein at least one inlet line and / or outlet line extends from the flexible bag to an exterior of the bag adapter. The method further includes at least partially enclosing the at least one inlet line and / or outlet line within a crown cavity defined by the crown assembly. The method further includes centrifuging the at least one bag adapter and the at least one flexible bag containing the liquid suspension in a centrifuge to produce a supernatant and a precipitate in the at least one flexible bag.

Description

Technical Field

[0001] The present invention relates to methods and systems for separating substances suspended in a liquid suspension. In particular, the present invention relates to biological treatment methods and systems for clarifying a biological suspension having substances suspended therein. Background Art

[0002] Centrifuges use centrifugal force in the laboratory to separate a mixture of substances into its components. In many applications, the mixture of substances is a biological or microbial liquid suspension. An example is a cell suspension, which may come from, for example, a fermenter, a bioreactor or a similar container, and which is at least partially separated into its components by centrifugation.

[0003] Previous methods have used relatively low speed centrifugation to attempt to separate large sized cell debris and particulate contaminants (such as particles 1-10 microns in size) from liquid suspensions (such as culture media or other biological suspensions). However, for certain production purposes, such as products requiring the separation and / or removal of particles less than or equal to 1 micron in size, low speed centrifugation has been shown to be insufficient to render a liquid suspension substantially free of such particles.

[0004] Therefore, there is a need to further clarify the liquid suspension to remove more cellular debris and particulate contaminants suspended in the culture medium beyond the levels that can be achieved by low-speed centrifugation, and to develop suitable equipment for achieving such clarification. Summary of the invention

[0005] According to the present invention, a method and system for clarifying a culture medium are provided.

[0006] In particular, in one embodiment, a bag adapter is provided, the bag adapter being configured to enclose a flexible bag containing a liquid suspension and having at least one inlet line and / or outlet line fluidly coupled to the flexible bag, wherein the at least one inlet line and / or outlet line extends from the flexible bag to an exterior of the bag adapter. The bag adapter includes a lower bag receiver portion configured to receive a lower portion of the flexible bag. The lower bag receiver portion includes: a bottom wall and a circumferential lower side wall, the circumferential lower side wall extending upwardly from the bottom wall and defining a lower bag cavity together with the bottom wall, the lower bag cavity having an opening positioned opposite the bottom wall and defined by an edge of the circumferential lower side wall, the lower bag receiver portion at least partially enclosing the lower portion of the flexible bag when the lower portion of the flexible bag is received in the lower bag receiver portion through the opening of the lower bag cavity. The bag adapter also includes an upper closure portion configured to engage the lower bag receptacle portion and receive the upper portion of the flexible bag. The upper closure portion includes a top wall and a circumferential upper side wall depending from the top wall and defining an upper bag cavity with the top wall, the upper bag cavity having an opening positioned opposite the top wall and defined by an edge of the upper side wall, the upper closure portion at least partially enclosing the upper portion of the flexible bag when the upper portion of the flexible bag is received through the opening of the upper bag cavity. The bag adapter also includes at least one hole formed in the upper closure portion, providing at least one passage extending between the upper bag cavity and an exterior of the upper closure portion. The at least one passage is configured such that the at least one inlet line and / or outlet line can extend from the upper bag cavity through the at least one passage to the exterior of the upper closure portion. The bag adapter closes the flexible bag when the lower bag receptacle portion and the upper closure portion are joined together.

[0007] In a related embodiment, the bag adapter includes at least one pair of apertures.

[0008] In a related embodiment, the lower bag receptacle portion and the upper closure portion define an overlapping joint at a junction of the lower bag receptacle portion and the upper closure portion.

[0009] In a related embodiment, the bottom wall of the lower bag receiver portion includes a rounded shoulder that joins the peripheral lower side wall of the lower bag receiver portion.

[0010] In a related embodiment, the top wall of the upper closure portion includes a rounded shoulder that engages the circumferential upper side wall of the upper closure portion.

[0011] In a related embodiment, the at least one hole is formed in the circular shoulder of the upper closure portion.

[0012] In a related embodiment, a pair of holes are formed in the circular shoulder of the upper closure portion.

[0013] In a related embodiment, the peripheral lower side wall of the lower bag receptacle portion is circular in cross-section.

[0014] In a related embodiment, the circumferential upper side wall of the upper closure portion is circular in cross-section.

[0015] In a related embodiment, the upper closure portion is releasably engaged to the lower bag receptacle portion.

[0016] In addition, a crown assembly is provided for use with a rotor to at least partially close at least one inlet line and / or outlet line during centrifugation of at least one flexible bag in the rotor, the at least one inlet line and / or outlet line being fluidly coupled to the at least one flexible bag, the at least one flexible bag containing a liquid suspension. The crown assembly includes an annular crown body defining a crown cavity. The crown cavity includes: a bottom wall, a circumferential side wall spaced upwardly from the bottom wall, and a circumferential sector side wall extending between the bottom wall and the circumferential side wall. The circumferential sector side wall includes a plurality of radially inwardly directed arcuate walls circumferentially arranged around the circumferential sector side wall. The annular crown body also includes at least one hole formed in each of the plurality of radially inwardly directed arcuate walls, providing at least one channel extending between the exterior of the annular crown body and the crown cavity. The at least one channel is configured such that the at least one inlet line and / or outlet line can extend from the exterior of the annular crown body through the at least one channel to the crown cavity. The annular crown body also includes an annular mounting member extending upward from the bottom wall and centrally disposed within the annular crown body. The crown assembly also includes an elastic element supported by the annular mounting member. The crown assembly also includes a crown assembly plunger that operably engages the elastic element and is capable of vertically moving between a first position and a second position relative to the annular mounting member. The elastic element biases the crown assembly plunger to the first position, and the crown assembly plunger is capable of moving to the second position in response to the elastic element being compressed by the cover of the rotor. The crown assembly also includes an annular crown ring that is removably engaged to the annular crown body and is configured with the annular crown body to at least partially close the at least one inlet line and / or outlet line during the centrifugation of the at least one flexible bag in the rotor, and the at least one inlet line and / or outlet line is fluidly connected to the flexible bag in the crown cavity.

[0017] In a related embodiment, the annular crown includes an annular sidewall and an annular lip extending radially inwardly from the annular sidewall of the annular crown.

[0018] In a related embodiment, the annular lip extends radially inwardly and downwardly from the annular sidewall of the annular crown.

[0019] In a related embodiment, the annular lip is at least partially translucent.

[0020] In a related embodiment, an annular crown ring is installed in the crown cavity.

[0021] In a related embodiment, the crown assembly further includes at least one O-ring supported by the annular sidewall of the annular crown ring and configured to engage the circumferential sidewall of the annular crown body when the annular crown ring is installed in the crown cavity.

[0022] In a related embodiment, the crown assembly may further include a carbon fiber reinforcement disposed about the annular sidewall of the annular crown ring.

[0023] In a related embodiment, the at least one aperture comprises a pair of apertures.

[0024] In a related embodiment, the annular mount includes: an inner annular wall extending upward from the bottom wall; and an outer annular wall extending upward from the bottom wall and concentrically disposed around the inner annular wall to define a cavity between the inner annular wall and the outer annular wall of the annular mount. The cavity of the annular mount is configured to at least partially support the elastic element within the cavity of the annular mount.

[0025] In a related embodiment, the crown assembly plunger includes: an annular body defining an annular shoulder; an inner annular wall depending from the annular shoulder; and an outer annular wall depending from the annular shoulder and concentrically disposed about the inner annular wall to define a cavity between the inner annular wall and the outer annular wall of the crown assembly plunger. The cavity of the crown assembly plunger is configured to at least partially support the resilient element within the cavity of the crown assembly plunger.

[0026] In a related embodiment, the crown assembly plunger is mounted for movement at least partially within the annular mount.

[0027] In a related embodiment, the plurality of radially inwardly directed arcuate walls includes four arcuate walls.

[0028] In a related embodiment, the plurality of radially inwardly directed arcuate walls includes at least six arcuate walls.

[0029] In addition, a kit is provided. The kit includes at least one bag adapter, the at least one bag adapter being configured to enclose a flexible bag, the flexible bag containing a liquid suspension and having at least one inlet line and / or outlet line fluidly coupled to the flexible bag, wherein the at least one inlet line and / or outlet line extends from the flexible bag to an exterior of the bag adapter. The at least one bag adapter includes a lower bag receiver portion configured to receive a lower portion of the flexible bag. The lower bag receiver portion includes a bottom wall and a circumferential lower side wall, the circumferential lower side wall extending upward from the bottom wall and defining a lower bag cavity together with the bottom wall, the lower bag cavity having an opening positioned opposite the bottom wall and defined by an edge of the lower side wall, the lower bag receiver portion at least partially enclosing the lower portion of the flexible bag when the lower portion of the flexible bag is received in the lower bag receiver portion through the opening of the lower bag cavity. The bag adapter also includes an upper closure portion configured to engage the lower bag receptacle portion and receive an upper portion of the flexible bag. The upper closure portion includes a top wall, a circumferential upper side wall depending from and defined with the top wall. The upper closure portion also includes an upper bag cavity having an opening positioned opposite the top wall and defined by an edge of the upper side wall, the upper closure portion at least partially enclosing the upper portion of the flexible bag when the upper portion of the flexible bag is received through the opening of the upper bag cavity. The upper closure portion also includes at least one aperture formed in the upper closure portion, providing at least one passage extending between the upper bag cavity and an exterior of the upper closure portion. The at least one passage is configured such that the at least one inlet line and / or outlet line extends from the upper bag cavity through the at least one passage to the exterior of the upper closure portion, wherein the bag adapter closes the flexible bag when the lower bag receiver portion and the upper closure portion are engaged together. The kit also includes the aforementioned crown assembly.

[0030] In a related embodiment, the kit further includes at least one flexible bag configured to contain the liquid suspension and having at least one inlet line and / or outlet line fluidly coupled to the flexible bag.

[0031] In addition, a method of clarifying a liquid suspension contained in a flexible bag is provided. The method includes nesting at least one flexible bag containing the liquid suspension within at least one bag adapter, wherein at least one inlet line and / or outlet line extends from the flexible bag to the exterior of the bag adapter. The method also includes at least partially enclosing the at least one inlet line and / or outlet line within a crown cavity defined by a crown assembly. The method also includes centrifuging the at least one bag adapter and the at least one flexible bag containing the liquid suspension in a centrifuge to produce a supernatant and a precipitate in the at least one flexible bag.

[0032] In a related embodiment, the method includes the step of extracting the supernatant from the at least one flexible bag via at least one outlet line fluidly coupled to the at least one flexible bag.

[0033] In a related embodiment, the method includes removing the at least one flexible bag from the at least one bag adapter prior to the extracting step.

[0034] In a related embodiment, the method further comprises receiving the liquid suspension in the at least one flexible bag via at least one inlet line.

[0035] In a related embodiment, the method includes positioning the at least one flexible bag at least partially exterior to the at least one bag adapter when the liquid suspension is contained in the at least one flexible bag.

[0036] In a related embodiment, the method includes the liquid suspension being received in the at least one flexible bag via the at least one inlet line, the at least one inlet line being fluidly coupled to a source of liquid suspension.

[0037] In a related embodiment, the method further includes an inlet manifold fluidly coupled to the liquid suspension source and fluidly coupled to the at least one inlet line, the at least one inlet line fluidly coupled to the at least one flexible bag.

[0038] In a related embodiment, the bag adapter used in the method includes: a lower bag receiver portion and an upper closure portion, wherein the lower bag receiver portion is configured to close the lower portion of the flexible bag, and the upper closure portion is configured to close the upper portion of the flexible bag, and the bag adapter is configured to withstand greater gravity than the at least one flexible bag.

[0039] In a related embodiment, the method is performed in a closed system.

[0040] In a related embodiment, the method includes extracting the supernatant into an outlet manifold via the at least one outlet line, the at least one outlet line being fluidly coupled to the flexible bag containing the liquid suspension.

[0041] In related embodiments, the method includes centrifuging the bag adapter at a rate greater than or equal to 5,000 revolutions per minute.

[0042] In a related embodiment, the method includes applying a force greater than 7,000 g to the bag adapter.

[0043] In related embodiments, the method includes extracting the supernatant into a storage container.

[0044] In related embodiments, the method includes extracting the supernatant into an outlet manifold.

[0045] In a related embodiment, the method includes receiving the liquid suspension in the at least one flexible bag via at least one inlet line when a bottom portion of the flexible bag is enclosed in a lower bag receptacle portion of the bag adapter. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Exemplary embodiments of the invention and their advantages are explained in more detail below in an exemplary sense and with reference to the accompanying drawings, in which:

[0047] Figure 1 is an exploded view of an exemplary system for clarifying a liquid suspension;

[0048] Figure 2A is a cross-sectional view of an assembled system for clarifying a liquid suspension with the crown plunger in a first position;

[0049] Figure 2B is a cross-sectional view of an assembled system for clarifying a liquid suspension with the crown plunger in a second position;

[0050] Figure 3 is a perspective view of the crown assembly;

[0051] Figure 4 is another perspective view of the crown assembly;

[0052] Figure 5 is an exploded view of the crown assembly;

[0053] Figure 6 is a cross-sectional view of the crown assembly;

[0054] Figure 7 is a perspective view of an assembled bag adapter including a flexible bag enclosed therein;

[0055] Figure 8 is an exploded view of a bag adapter including a flexible bag to be enclosed therein;

[0056] Fig. 9is a cross-sectional view of a bag adapter including a flexible bag enclosed therein;

[0057] Fig. 10A is a flow chart of a method for clarifying a liquid suspension;

[0058] Fig. 10B is a flow chart of a method for clarifying a liquid suspension;

[0059] Fig. 10C is a flow chart of a method for clarifying a liquid suspension;

[0060] Fig.11A is a system view of a flexible bag having a liquid suspension partially contained therein during a containing step of the method;

[0061] Fig. 11B is a system view of a flexible bag having a liquid suspension received therein during a receiving step of the method;

[0062] Fig. 11C is a view of a system for weighing a flexible bag;

[0063] Fig.11D is a view of a flexible bag containing a liquid suspension;

[0064] Fig.11E is a view of cutting the inlet line from the flexible bag using a welding machine;

[0065] Fig.11F is a view of a cap placed on the folded end of a severed inlet line;

[0066] Fig.11G is a view of an assembled bag adapter having a flexible bag filled with a liquid suspension therein;

[0067] Fig.11H is an exploded view of a system for centrifuging a liquid suspension, the system comprising a centrifuge rotor, a crown assembly, and a bag adapter enclosing a flexible bag filled with the liquid suspension;

[0068] Fig.11I yes Fig.11H An assembled view of a system for centrifuging a liquid suspension is shown;

[0069] Fig.11J yes Fig.11I The system is shown in a partial cross-sectional view after centrifugation of the liquid suspension to form a precipitate and a supernatant;

[0070] Figure 11K is a system view of flexible bags that have been subjected to centrifugation, the bags including supernatant to be removed from the flexible bags; and

[0071] Fig.11Lis a system view of flexible bags that have been subjected to centrifugation, from which some of the supernatant has been removed and stored in a storage container. DETAILED DESCRIPTION

[0072] It should be understood that all parameter ranges described herein include the endpoints of the given ranges. In addition, all sub-ranges of each given range are contemplated as being suitable embodiments of the invention described herein.

[0073] A system and method have been developed to clarify and / or pelletize the supernatant obtained from a cell collection process and remove cell debris or particulate contaminants suspended in a liquid suspension. In some examples, the cell collection process includes a collection step, a lysis step, a cell generation step, or any combination of these steps. These steps are explained in more detail below. In some embodiments, the system 100 can include a centrifuge rotor 400, a crown assembly 200, at least one flexible bag 350, and at least one bag adapter 300.

[0074] refer to Figure 1 , showing a method for making a liquid suspension 700 (e.g. Fig.11A and Fig. 11B 1 . The system 100 includes at least a centrifuge rotor 400 (including a rotor body 424 and a cover 450), a crown assembly 200, a plurality of bag adapters 300, and a plurality of flexible bags 350 (in Figure 2A and Figure 2B It is shown in Figure 1 Not shown, but housed within bag adapter 300).

[0075] A centrifuge is a device that is configured to rotate a liquid suspension, such as liquid suspension 700, equilibrated therein at a high rotational speed, thereby causing separation of the substances suspended in liquid suspension 700. In some cases, centrifugation of liquid suspension 700 can granulate solids suspended in liquid suspension 700 into precipitate 760 (in Figure 11J-11L760) and a supernatant 770 separated from the precipitate 760 and substantially free of solids previously suspended in the liquid suspension 700. In one embodiment of the invention, the cell debris and particulate contaminants are suspended in the liquid suspension 700 prior to centrifugation. The centrifuge rotor 400 positioned in the body of a centrifuge (not shown) can be a fixed angle rotor and can be configured to rotate the liquid suspension 700 placed in a receptacle or cavity 410 positioned on the centrifuge rotor 400. The crown locator 404 is positioned at the center of the centrifuge rotor 400 and, in some embodiments, is configured to connect to a motor (not shown) of the centrifuge to rotate the centrifuge rotor 400. In one embodiment, the rotor body 422 has a top surface 424 that is radially outwardly and upwardly inclined from the crown locator 404. A plurality of radially spaced receptacles or cavities 410 are recessed from the top surface 424 into the rotor body 422. Each receptacle 410 is sized to receive a flexible bag 350, and more specifically a bag adapter 300, and may be generally cylindrical in shape, or any other shape that conforms the shape of the receptacle 410 to the shape of the bag adapter 300. That is, each receptacle 410 is configured to receive a separate bag adapter 300, which may include a flexible bag 350 filled with a liquid suspension, in a complementary fit to reduce stress on the flexible bag 350 during centrifugation.

[0076] In order to prevent the liquid suspension 700 from moving freely within the centrifuge rotor 400 during operation, and to ensure a sterile environment that prevents contaminants from interacting with the liquid suspension 700, the liquid suspension 700 is placed in a plurality of flexible bags 350 (in Figure 2A and Figure 2B 4. As described above, the centrifuge rotor 400 includes any number of receptacles 410 for the flexible bag 350 (particularly when contained within the bag adapter 300) to be placed therein. In one embodiment, the centrifuge rotor 400 includes 2 to 10 receptacles 410. In a preferred embodiment, the centrifuge rotor 400 includes 4 to 6 receptacles 410. In another embodiment, the centrifuge rotor 400 includes at least 4 receptacles 410. In another embodiment, the centrifuge rotor 400 includes at least 6 receptacles 410. Examples of particularly suitable centrifuges configured to receive the centrifuge rotor 400 are commercially available from Fiberlite Centrifuge LLC. Of Santa Clara, CA and include Model No. 6000 or 4000. Examples of suitable centrifuge rotors 400 for use with the present invention include models F9-6x1000 LEX and / or F10-4x1000 LEX, which are commercially available from Thermo Fisher Scientific Inc. of Waltham, MA.

[0077] During centrifugation, during operation of the centrifuge rotor 400, the flexible bag 350 may not adequately or may not maintain its integrity due to the high g-forces typically exerted on the flexible bag 350. To help the flexible bag 350 withstand the g-forces exerted thereon during operation of the centrifuge rotor 400 during centrifugation, the flexible bag 350 may be placed in a bag adapter 300. The bag adapter 300 is sized to be received in a receptacle 410 of a centrifuge rotor 400. As described herein, the centrifuge rotor 400 may be operated at a rotational speed of 500 to 10,500 revolutions per minute (rpm). In a preferred embodiment, and to facilitate clarification of relatively small cell debris and particulate contaminants, such as cell debris and particulate contaminants less than or equal to 1 micron in size, from the liquid suspension 700, the centrifuge rotor 400 can be operated at a rotation speed greater than or equal to 5,000 rpm, such as 5,000 rpm to 10,500 rpm, preferably 6,000 rpm to 10,500 rpm and more preferably 8,000 rpm to 10,500 rpm. In addition, these high rotation speeds may result in high g-forces exerted on the liquid suspension 700, the flexible bag 350 and / or the bag adapter 300. These g-forces may amount to greater than or equal to 7,000 times g-force, such as 7,000 times to 21,000 times g-force. In a preferred embodiment, the g-forces amount to 9,000 times to 21,000 times g-force, more preferably 15,000 times to 20,584 times g-force. At such high rotation speeds and the application of gravity, clarification can be achieved, however, at some high speeds, a bag adapter 300 can be included to accommodate the flexible bag 350 to ensure that the flexible bag 350 does not fail. Due at least to the high operating speed of the centrifuge rotor 400, contaminants such as cell debris or cells can be pelleted from the liquid suspension 700 to form a precipitate 760.

[0078] In some embodiments, a collection step 801C (shown in FIG. 10 ) occurs prior to centrifuging the liquid suspension 700. As described below, the liquid phase used in the collection step 801C may contain contaminants therein and may be granulated in the collection step 801C. The size of the contaminants contained in the liquid suspension 700 may be different from the size of the contaminants granulated in the collection step 801C. Alternatively or in addition, the collection step 801C may not be sufficient to granulate some contaminants, regardless of their size. In this case, the contaminants granulated during the clarification process may have a similar size to the contaminants granulated in the collection step 801C. The collection step 801C is described in more detail below.

[0079] Figure 1 Six bag adapters 300 and corresponding receptacles 410 are shown in the centrifuge rotor 400, however, any number of bag adapters 300 may be used, up to the maximum number of receptacles 410 included in the centrifuge rotor 400. The receptacles 410 of the centrifuge rotor 400 may be sized to receive the flexible bag 350. Alternatively or additionally, the receptacles 410 of the centrifuge rotor 400 may be sized to receive a bag adapter 300 including a flexible bag 350 therein. Figure 1 The centrifuge rotor 400 shown in is a fixed angle centrifuge rotor. That is, the receptacle 410 of the centrifuge rotor 400 is arranged with the same orientation during the rotation of the centrifuge rotor 400. Each receptacle 410 may include a central axis 412 oriented at various fixed angles relative to a horizontal plane 1000 (shown in FIG. 2 ), for example at an angle θ of 30° to 60°, or at an angle θ of 30° to 45°. Other angles θ may also be used. Therefore, the precipitate formed by centrifuging the liquid suspension 700 is usually formed off-center from the bottom of the flexible bag 350. The cover 450 is connected to the crown locator 404 and the rotor body 422, and is used to cover the receptacle 410. Therefore, the bag adapter 300 located in the receptacle 410 is also covered during the rotation of the centrifuge rotor 400, as described in more detail below.

[0080] Continue to refer Figure 1 , showing the crown assembly 200 and the bag adapter 300. Figure 3-Figure 6 When the crown assembly 200 and in reference Figure 7-Figure 9 The bag adapter 300 will be described in more detail.

[0081] Figure 2A A cross-sectional view of an assembled centrifuge rotor 400 containing a liquid suspension 700 to be centrifuged is shown. Figure 2A The specific components shown in the drawings are not shown in the drawings and are clearly shown in other drawings. Figure 2AThe cover 450 is shown positioned adjacent to the centrifuge rotor 400. The locking mechanism 452 protrudes from the cover 450 into the crown assembly 200 and further into the centrifuge rotor 400. At least when the cover 450 is secured to the centrifuge rotor 400 (shown in FIG. Figure 2B ), a central portion of the cap 450 meets the crown assembly plunger 260. In some embodiments, the crown assembly plunger 260 includes an annular body 262 defining an annular shoulder 264. The crown assembly plunger 260 may also include an inner annular wall 266 and an outer annular wall 268 depending from the annular shoulder 264, the outer annular wall depending from the annular shoulder 264 and concentrically disposed around the inner annular wall 266 to define a cavity 244 between the inner annular wall 266 and the outer annular wall 268 of the crown assembly plunger 260, wherein the cavity 203 of the crown assembly plunger 260 is configured to at least partially support the resilient element 202 within the cavity 203 of the crown assembly plunger 260.

[0082] The crown assembly plunger 260 is capable of moving from a first position (shown in FIG. Figure 2A ) moves to the second position (shown in Figure 2B 450) wherein the first position of the crown assembly plunger 260 is further from the bottom wall 214 and the second position is closer to the bottom wall 214 than the first position. In addition, the crown assembly plunger 260 can be mounted to move at least partially within the annular mount 241. The resilient element 202 is positioned within the crown assembly and biases the crown assembly plunger 260 toward the first position (i.e., away from the bottom wall 214 and the junction of the crown assembly plunger 260 and the cap 450). The junction of the crown assembly plunger 260 and the cap 450 forms the seal 206 with the assistance of the annular retaining ring 204. Figure 2A The second position (shown in Figure 2B ) the crown assembly plunger 260 is in a first position further away from the bottom wall 214, and the resilient element 202 extends, thereby biasing the crown assembly plunger 260 away from the bottom wall 214. Conversely, Figure 2BThe crown assembly plunger 260 is shown in a second position, closer to the bottom wall 214 than the crown assembly plunger 260 in the first position, and the resilient element 202 is compressed, thereby biasing the crown assembly plunger 260 toward the cover 450. The crown assembly plunger 260 is operably engaged with the resilient element 202 and is vertically movable relative to the annular mount 241 between the first position and the second position. The resilient element 202 biases the crown assembly plunger 260 to the first position. The crown assembly plunger 260 is movable to the second position in response to the resilient element 202 being compressed by the cover 450 of the rotor 400. The annular mounting member 241 includes an inner annular wall 240 extending upward from the bottom wall 214 of the crown assembly 200 and an outer annular wall 242, the outer annular wall extending upward from the bottom wall 214 and concentrically disposed around the inner annular wall 240 to at least partially define a cavity 244 between the inner annular wall 240 and the outer annular wall 242 of the annular mounting member 241, wherein the cavity 244 of the annular mounting member 241 is configured to at least partially support the elastic element 202 within the cavity 244 of the annular mounting member 241. The annular mounting member 241 extends upward from the bottom wall 214 and is centrally disposed within the annular crown body 210.

[0083] Figure 3 and Figure 4 A perspective view of an embodiment of a crown assembly is shown. In some embodiments, the crown assembly 200 is made of metal (particularly aluminum or steel, in the example spring steel) or plastic. The crown assembly 200 includes an annular crown body 210 having an upper end face or rim 212 and a lower end face or bottom wall 214, the bottom wall 214 being opposite the rim 212, and the annular crown body 210 defining an internal crown space or crown cavity 216. A removable annular lip or annular crown ring 220 extends radially inward from the periphery of the rim 212. The annular crown ring 220 can be removably coupled to the annular crown body 210 and is configured together with the annular crown body 210 to at least partially close at least one inlet line 330 and / or outlet line 340 (shown in FIG. 1 ) fluidically connected to the flexible bag 350 during centrifugation of the at least one flexible bag 350 in the rotor 400. Fig.11I 216). The annular crown ring 220 may include an annular sidewall 224 and an annular lip 222 extending radially inward from the annular sidewall 224 of the annular crown ring 220. The annular lip 222 may be aligned with a plane including the rim 212 or disposed at an angle relative to the plane. In particular, in some examples, the annular lip 222 may be inclined downward from a plane including the rim 212 at an angle of 5 degrees or more. Alternatively or in addition, the annular lip 222 may be inclined downward from a plane including the rim 212 at an angle of 10 degrees or more.

[0084] In some embodiments, the annular crown ring 220 further includes an annular sidewall 224 that engages the annular lip 222 at an angle. The crown assembly 200 may also include at least one O-ring 219 supported by the annular sidewall 224 of the annular crown ring 220. The O-ring 219 may be configured to engage the circumferential sidewall 233 of the annular crown body 210 when the annular crown ring 220 is installed in the crown cavity 216. For example, the annular sidewall 224 may include an annular sidewall outer surface 228 having an O-ring receptacle 226. The O-ring receptacle 226 is sized to receive and retain the O-ring 219. As described below, during centrifugation, the annular crown ring 220 may retain the inlet line 330 and the outlet line 340 within the crown cavity 216 of the annular crown body 210. In some examples, the annular crown 220 is a separate component from the annular crown 210 and can be coupled to the annular crown 210 via frictional engagement of the O-ring 219 and / or the sidewall 224 with the circumferential sidewall 223 of the annular crown 210. Such coupling of the crown 220 to the annular crown 210 may result in the annular crown 220 being fixedly or removably coupled to the annular crown 210. In some embodiments, the annular crown 220 is at least translucent to allow a user to view through the annular crown 220 without removing the annular crown 220 when inspecting the contents of the centrifuge rotor 400. Alternatively or in addition, the annular crown 220 is transparent for similar reasons. Advantageously, the annular crown 220 can include 3-D printed materials in whole or in part. Therefore, it is conceivable that the annular crown 220 can be formed by a 3-D printing method such as SLA (stereolithography).

[0085] The annular crown 210 also includes a circumferential sidewall 233 that is spaced upward from the bottom wall 214. The annular crown 210 also includes a circumferential sector-shaped sidewall 234 extending between the bottom wall 214 and the circumferential sidewall 233. The circumferential sector-shaped sidewall 234 includes a plurality of radially inwardly directed arcuate walls 230 circumferentially arranged around the circumferential sector-shaped sidewall 234. In addition, the annular crown 210 may include one or more holes 232 formed in each of the plurality of radially inwardly directed arcuate walls 230. The holes 232 provide at least one channel extending between the outside of the annular crown 210 and the crown cavity 216. The at least one channel is configured such that at least one inlet line 330 and / or outlet line 340 (shown in Fig.11I233 can extend from the exterior of the annular crown 210 through at least one passage to the crown cavity 216. In one embodiment, the hole 232 included in each arcuate wall 230 is a pair of holes 232. The arcuate wall 230 can be shaped to conform to the shape of at least a portion of the bag adapter 300 (shown in FIG. 2), and the hole 232 is sized to receive the inlet line 330 and the outlet line 340, which are connected to and extend from the flexible bag 350, which can be placed in the bag adapter 300. During operation of the centrifuge rotor 400, the inlet line 330 and the outlet line 340 are positioned within the holes 232 and further extend into the crown cavity 216 of the annular crown 210. At least because the annular crown ring 210 and the circumferential sidewall 233 at least partially define the crown cavity 216 of the annular crown 210, the inlet line 330 and the outlet line 340 do not swing freely during operation of the centrifuge rotor 400. In some examples, cap 710 (shown in Fig.11F and Fig.11G in) can be any combination (shown in Fig.11F 2, 3 and 4. The cap 710 can prevent the inlet line 330 or the outlet line 340 from unfolding during centrifugation, which could undesirably allow the liquid suspension 700 to escape from the flexible bag 350. When the bag adapter 300 is loaded into the receptacle 410 of the centrifuge rotor 400, the annular crown 210 is positioned so that the arcuate wall 230, for example, is at the outer bag adapter surface (as shown in FIG. 2, Fig.11H and Fig.11I ) contacts the bag adapter 300 and prevents or inhibits accidental movement of the bag adapter 300 or the flexible bag 350 during centrifugation.

[0086] Figure 5 is an exploded view of crown assembly 200. The exploded view shows annular crown body 210, resilient element 202, crown assembly plunger 260, annular retaining ring 204, and annular crown ring 220. Together, these components form crown assembly 220 as described herein.

[0087] refer to Figure 6 , shows a cross-section of the crown assembly 200. Figure 6 Particularly visible in FIG. 2 is a central hole 238 passing through the annular crown body 210. The central hole 238 is a passage extending from the bottom wall 214 into the crown cavity 216 of the annular crown body 210. The central hole 238 is defined by the inner annular wall 240 of the annular mounting member 241. Figure 6, annular retaining ring 204 is an annular structure positioned adjacent to crown assembly plunger 260 and may include an outer periphery 205 extending beyond outer periphery 261 of crown assembly plunger 260. Annular retaining ring 204 may be a resilient metal ring. When resilient element 202 applies a bias to crown assembly plunger 260, retaining ring 204 is engaged, which limits further vertical travel or extension of crown assembly plunger 260 relative to annular mount 241. When the centrifuge is ready for operation (e.g., Figure 2B As shown), the annular retaining ring 204 presses against the locking mechanism to form a seal 206.

[0088] Continue to refer Figure 6 , shows a carbon fiber reinforcement 250 disposed around the circumferential sidewall 233 around the outer circumference of the annular crown 210. Alternatively or in addition, the carbon fiber reinforcement can be disposed around the annular sidewall 224 of the annular crown ring 220. The carbon fiber reinforcement 250 can be, for example, carbon fiber filaments. The filaments can be a composite material of carbon fiber and a resin that can be cured so as to bond the filaments to the annular crown 210. Alternatively, various other high tensile, high modulus materials can be used, such as glass fibers, such as para-aramid fibers (i.e., ), thermoplastic filaments, metal wires, or other materials suitable for reinforcing the annular crown 210 instead of carbon fibers. The carbon fiber reinforcement 250 can be chemically bonded to the annular crown 210, such as by a cured resin, or physically bonded to the annular crown, such as if there is sufficient tension in the carbon fiber reinforcement 250 so that the normal force exerted on the carbon fiber reinforcement 250 by the annular crown 210 substantially eliminates sliding of the carbon fiber reinforcement 250 relative to the outer surface of the annular crown 210.

[0089] refer to Figure 7 , the bag adapter 300 is shown in an assembled view with the flexible bag 350 nested therein. The assembled bag adapter 300 includes a lower bag receiver portion 310 and an upper closure portion 320. Figure 7 Also shown are inlet and outlet lines 330, 340, which are coupled to a flexible bag 350 (shown in FIG. Figure 8 Although the inlet line 330 is Figure 7 The pipeline in the foreground is identified in FIG. 1 and the outlet pipeline 340 is identified in FIG. Figure 7 330 or the outlet line 340 may be operated as a line for receiving the liquid suspension 700 in the flexible bag 350 or as a line for extracting the supernatant 770 from the flexible bag 350 after centrifugation.

[0090] refer to Figure 8, an exploded view of the bag adapter 300 and the flexible bag 350 is shown. The lower bag receiver portion 310 includes a bottom wall 311 and a circumferential lower side wall 314 extending upward from the bottom wall 311 and defining, together with the bottom wall 311, a lower bag cavity 315 having an opening 315a located opposite the bottom wall 311. The opening 315a is defined by an edge 312 of the circumferential lower side wall 314. When the lower portion 352 of the flexible bag 350 is received in the lower bag receiver portion 310 through the opening 315a of the lower bag cavity 315, the lower bag receiver portion 310 at least partially encloses the lower portion 352 of the flexible bag 350. In one embodiment, the bottom wall 311 of the lower bag receiver portion 310 includes a rounded shoulder 316 that engages the circumferential lower side wall 314 of the lower bag receiver portion 310. In one embodiment, the cross-section of the lower bag receiver portion 310 from the periphery to the lower side wall 314 is circular.

[0091] The upper closure portion 320 is configured to be joined to the lower bag receptacle portion 310 and receive an upper portion 354 of the flexible bag 350. The upper closure portion 320 includes a top wall 321 and a circumferential upper side wall 324 depending from the top wall 321 and defining, together with the top wall 321, an upper bag cavity 325 having an opening 325a positioned opposite the top wall 321 and defined by an edge 322 of the upper side wall 324, the upper closure portion 320 at least partially enclosing the upper portion 354 of the flexible bag 350 when the upper portion 354 of the flexible bag 350 is received through the opening 325a of the upper bag cavity 325. The upper closure portion 320 also includes at least one aperture 319 formed in the upper closure portion 320, providing at least one passage extending between the upper bag cavity 325 and the exterior of the upper closure portion 320. In one embodiment, the at least one hole 319 is a pair of holes 319. The at least one passage is configured such that the at least one inlet line 330 and / or outlet line 340 extends from the upper bag cavity 325 through the at least one passage to the exterior of the upper closure portion 320. When the lower bag receiver portion 310 and the upper closure portion 320 are joined together, the bag adapter 300 encloses the flexible bag 350. In one embodiment, when the lower bag receiver portion 310 and the upper closure portion 320 are joined together, the lower bag receiver portion 310 and the upper closure portion 320 define an overlapping joint 318 at the junction of the lower bag receiver portion 310 and the upper closure portion 320. In one embodiment, the top wall 321 of the upper closure portion 320 includes a circular shoulder 326 that joins the circumferential upper side wall 324 of the upper closure portion 320. In one embodiment, Figure 7 and Figure 8At least one hole 319 or a pair of holes 319 as shown in the figure are formed in the circular shoulder 326 of the upper closure portion 320. In one embodiment, the cross-section of the circumferential upper side wall 324 of the upper closure portion 320 is circular. In one embodiment, the upper closure portion 320 is releasably engaged to the lower bag receiver portion 310.

[0092] The upper closure portion 320 includes an outer surface that conforms to the arcuate wall 230 of the annular crown 210. During operation of the centrifuge, the arcuate wall 230 of the annular crown 210 is adjacent to the outer upper closure portion 320 surface such that each of these components prevents accidental movement of the other.

[0093] Continue to refer Figure 8 , the exploded bag adapter 300 shows a lower bag receptacle portion 310 including a rim 312 defining an opening 315a. The opening 315a has an inner sidewall surface that forms a lower bag cavity 315, which is sized to enclose at least a lower portion 352 of the flexible bag 350. In addition, the exploded view of the bag adapter 300 also shows an upper closure portion 320, which has a rim 322 having an inner edge that defines an opening 325a. The opening 325a has an inner sidewall surface that forms an upper bag cavity 325, which is sized to enclose at least an upper portion 354 of the flexible bag 350. The rim 322 has an outer edge that defines the annular perimeter of the rim 322. The upper closure portion 320 also includes an outer sidewall surface that extends from the outer edge to a flange 370, which extends radially relative to the rim 322. The upper closure portion 320 also includes at least one hole 319 (in Figure 8 The at least one hole provides at least one passage between the interior of the upper closure portion 320 and the exterior of the upper closure portion 320. The hole 319 is sized to receive the inlet line 330 or the outlet line 340, or both (if at least two holes are provided) therethrough. The rim 312 of the lower bag receiver portion 310 is configured to connect the lower bag receiver portion 310 with the upper closure portion 320 (shown in FIG. Figure 7 When the overlapping joint 318 is formed, it meets the flange 370.

[0094] As mentioned above, Figure 8 Two holes 319 are shown included in the upper closure portion 320. However, in some embodiments, there may be more than two holes 319 or less than two holes 319 in the upper closure portion 320. Figure 8 As shown, the flange 370 is shown as extending radially outward relative to the rim 322. However, it is contemplated that the flange 370 may extend radially inward relative to the rim 322, as an alternative or in addition to extending radially outward relative to the rim 322.

[0095] In some embodiments, the bag adapter 300 is intended to be a single-use adapter, and therefore, the flexible bag 350 is enclosed within both the lower bag receiver portion 310 and the upper closure portion 320, and the lower bag receiver portion 310 and the upper closure portion 320 are configured to be irreversibly engaged together once the flexible bag 350 is enclosed within the lower bag cavity 315 and the upper bag cavity 325 of the lower bag receiver portion 310 and the upper closure portion 320, respectively. Alternatively, the bag adapter 300 may be intended to be a multiple-use adapter, and therefore, the flexible bag 350 is enclosed within both the lower bag receiver portion 310 and the upper closure portion 320, and the lower bag receiver portion 310 and the upper closure portion 320 are configured to be removably engaged together once the flexible bag 350 is enclosed within the lower bag cavity 315 and the upper bag cavity 325 of the lower bag receiver portion 310 and the upper closure portion 320, respectively. During operation of the centrifuge, the lower bag receiver portion 310 will be coupled to the upper closure portion 320, but the lower bag receiver portion 310 may be removed from the upper closure portion 320 either before or after centrifugation.

[0096] As explained above, the lower bag receiver portion 310 and the upper closure portion 320 are two parts of the bag adapter 300. The lower bag receiver portion 310 or the upper closure portion 320 can be made of metal, plastic or polymer alone or in combination. A specific example of a suitable material for the lower bag receiver portion 310, the upper closure portion 320 or both is polycarbonate.

[0097] refer to Fig. 9 , shows a cross-section of an assembled bag adapter 300 including a flexible bag 350 enclosed therein. The flexible bag 350 includes a port 332 for coupling with an inlet line 330 or an outlet line 340. The port 332 is configured to attach to the inlet line 330 and / or the outlet line 340. The attachment of the port 332 to the inlet line 330 and / or the outlet line 340 forms a seal so that substances outside the inlet line 330, the outlet line 340, or inside the flexible bag 350 cannot interact with substances in these areas.

[0098] Continue to refer Fig. 9, the upper closure portion 320 and the lower bag receiver portion 310 are shown coupled together and nesting the flexible bag 350. At least before centrifuging the bag adapter 300, a gap 317 can exist between the circumferential upper sidewall 324 and the circumferential lower sidewall 314. During centrifugation, the contents of the flexible bag 350 can exert pressure on the inside of the circumferential upper sidewall 324, which reduces the gap 317 between the circumferential upper sidewall 324 and the circumferential lower sidewall 314, so that the gap 317 distance becomes close to or exactly zero meters. In addition, the lip edge 313 is radially outwardly open and includes a rim 322. The lip edge 313 is configured to slide over the circumferential lower sidewall 314 when the bag adapter 300 is assembled. When the bag adapter 300 is assembled, the lip 313 frictionally engages the circumferential lower sidewall 314 , and in some embodiments, the frictional engagement of the lower circumferential sidewall 314 with the lip 313 can inhibit axial movement and / or separation of the upper closure portion 320 and the lower bag receiver portion 310 .

[0099] The centrifuge rotor 400 according to the present invention and the crown assembly 200 used therein enable the bag adapter 300 having the flexible bag 350 enclosed therein to be centrifuged. In addition, the inlet line 330 and / or the outlet line 340 protruding outward from the flexible bag 350 facilitate the centrifugation of the liquid suspension 700 without having to take special measures, such as disconnecting the inlet line 330 or the outlet line 340. In some examples, the flexible bag 350 that can be centrifuged with the help of the centrifuge rotor 400 according to the present invention includes, but is not limited to, the following: a film bag, wherein one end of the film bag is sealed, in particular glued or welded; an arrangement of a plurality of film bags, in each of the plurality of film bags, one end of at least one film bag is sealed, in particular glued or welded, wherein the other ends of the film bags are connected to each other. In particular, the flexible bag system can be centrifugal, which is described in International Patent Application Publication No. WO2019 / 166998A1. These two patent applications are hereby expressly referred to, and their contents are hereby incorporated by reference into this application. The flexible bag 350 described can be filled with a tube assembly that can be branched, in series, or some combination thereof. According to the present invention, the flexible bags 350 can be placed individually or in groups into the bag adapter 300, which in turn are placed into the sample receptacle 410 of the centrifuge rotor 400. The inlet line 330 and the outlet line 340 protrude upward from the flexible bag 350 and can still be connected to each other when they are brought to the crown assembly 200 and, as described above, are fixed there by pressure from the crown assembly 200. This pressure contact of the crown assembly 200 with the bag adapter 300 occurs from the arcuate wall 230 of the annular crown 210 to the upper closed portion 320 of the bag adapter 300. After the centrifugation process is completed, the flexible bag 350 can be removed from the bag adapter 300 and sent for further processing.

[0100] Figure 10A-10C Flowcharts of embodiments of methods 800a-800c of clarifying a liquid suspension 700 are shown. In one embodiment, methods 800a-800c include nesting 804 a flexible bag 350 in a bag adapter 300, such as in a lower bag cavity 315 of a lower bag receptacle portion 310. The flexible bag 350 may or may not be filled, or even partially filled, with the fluid suspension 700 before, during, or after any nesting 804 step occurs. That is, when the liquid suspension 700 is received 806 by the flexible bag 350, the upper portion 354, the lower portion 352, both the upper portion 354 and the lower portion 352 of the flexible bag 350 may be, respectively, or neither the upper portion 354 or the lower portion 352 may be, respectively, enclosed by the upper closure portion 320 or the lower bag receptacle portion 310. The methods 800a-800c also include receiving 806 the liquid suspension 700 comprising the first material and the second material suspended therein in at least one flexible bag 350 via the inlet line 330. The methods 800a-800c also include centrifuging 808 the bag adapter 300 comprising the at least one flexible bag 350 and the liquid suspension 700 in a centrifuge to form a supernatant 770 (e.g., as Figure 11K ) and precipitate 760 (e.g., as Figure 11K ), wherein the supernatant 770 mainly includes the first material and the precipitate 760 mainly includes the second material. Methods 800a-800c also include extracting 810 the supernatant 770 from at least one flexible bag 350 via the outlet line 340. In some examples, methods 800a-800c can be performed in a completely closed system. Therefore, methods 800a-800c can be performed without exposing any of the liquid suspension 700, the supernatant 770, or the precipitate 760 to any contaminants outside the system. Each of the steps of methods 800a-800c will be described in more detail below.

[0101] The methods 800a-800c of the present invention are particularly useful in the development of bioprocessing methods for producing recombinant protein products using eukaryotic cells (such as CHO cells) at a development scale (such as a 10, 50 or 200 liter scale). These methods can also be used for quality control or other purposes of large-scale (e.g., 500, 1000 or 2000 liter) bioprocessing. Fig. 10B , a flow chart of an embodiment of a method 800b of clarifying a liquid suspension 700 is shown. Fig. 10BThe collection step 801B is included before the liquid suspension 700 is received 806 by the flexible bag 350. In some embodiments, the collection step 801B includes culturing or receiving cells suspended in a liquid phase or a culture medium with a product of interest in a liquid phase, and is directed to a first set of bags or other containers through a manifold, each bag or other container having a size of, for example, 1.0, 1.6 or 2 liters. The bag or other container is configured to contain a liquid phase and can be centrifuged. The collection step 801B can also include, for example, centrifuging a bag or other container including a liquid phase using a bucket rotor at a speed of 500 to 5000 rpm to separate intact cells from the first supernatant, which form a precipitate during centrifugation. The precipitate in the collection step 801B mainly includes intact cells, and the first supernatant mainly includes cell debris. The collection step 801B can also include recovering the first supernatant from the bag or other container, effectively separating the first supernatant from the precipitate. The first supernatant from this first group of bags can be collected in a common bag (using, for example, the method described in International Application Publication No. WO2019 / 166998A1) and then received 806 by the flexible bag 350 as the liquid suspension 700 described above in the receiving step 806, and the other steps of the clarification method 800b process described herein then occur.

[0102] With respect to the collection step 801B, a common bag for collecting the first supernatant from the first set of bags can be manufactured with tubing attached at its outlet to the inlet of the manifold to supply the second set of bags. This combination of the common bag (with associated inlet and outlet tubing), the manifold, the second bags, and the associated tubing can be subjected to radiation sterilization after assembly in at least one outer bag or container for storing the combination and transporting it to the site of intended use.

[0103] refer to Fig. 10C , a flow chart showing another embodiment of a method 800c for clarifying a liquid suspension is shown. Fig. 10C At least including Fig. 10B The same steps as the above embodiment are shown. However, Fig. 10CAlso included is a lysis step 801C, which occurs after the collection step 801B and before the liquid suspension is received 806 by the flexible bag 350. The lysis step 801C allows for the collection of desired products that may have been granulated inadvertently or otherwise during the collection step 801B. For example, some cells that granulated during the collection step 801B may retain the desired product within the cells. In order to extract the product that may be within the cells, the lysis step 801C is performed. For the method 800c in which the lysis step 801C is performed, after the first supernatant is removed from the bag or other container, the precipitate produced during the collection step 801C is exposed to a chemical compound or lysis buffer, such as that provided by a lysis buffer located at 601 Science Drive, Arkansas, USA or Madison, Wisconsin, USA 53711. company( CytoBuster, manufactured by Inc. TM Protein extraction reagent. Chemical compounds are configured to decompose the contents in the precipitate into a lysis liquid phase. Alternatively or in addition, the precipitate is exposed to high pressure to decompose cells. Chemical compounds and / or high pressure exposure are configured to destroy, pierce or otherwise penetrate the cell membrane or cell wall included in the cells constituting the precipitate. The lysis process may be particularly effective when decomposing cells (such as prokaryotic cells) with cell walls. Therefore, the lysis liquid phase includes more incomplete and / or structurally more unstable cell fragments or particles than the cells previously included in the precipitate immediately after the collection step 801B, at least because the cell membrane and / or cell wall of these cells have been damaged. In addition to the first supernatant from the collection step 801B, the lysis liquid phase is then received 806 in the flexible bag 350 as the liquid suspension 700 described above in the receiving step 806, and the other steps of the clarification method 801C described herein occur subsequently.

[0104] refer to Figures 11A-11K , Fig.11A and Fig. 11B A flexible bag system is shown, which in the example shown has three flexible bags 350 (shown as film bags), however any number or type of flexible bags 350 as described herein may be used in the flexible bag system. Each of the flexible bags 350 has been nested within the lower bag receiver portion 310 of the bag adapter 300 and connected to the manifold 750. The nesting step 804 includes placing at least one flexible bag 350 in the lower bag receiver portion 310 of the bag adapter 300. The nesting step 804 may occur before or after the liquid suspension 700 has been received in the flexible bag 350 in the receiving step 806 ( Fig.11AThe flexible bag 350 is shown already nested in the lower bag receptacle portion 310 of the bag adapter 300 prior to receiving 806 the liquid suspension 700, but this is merely an example). It may be desirable to nest the flexible bag 350 within the lower bag receptacle portion 310 of the bag adapter 300 prior to the flexible bag 350 receiving the liquid suspension 700 to facilitate handling of the flexible bag 350.

[0105] The clarification methods 800a-800c may include at least a collection step 801B or a lysis step 801C prior to the receiving step 806. As described above, the collection step 801B includes, for example, in a closed system, the reaction or growth of cells in a collection step liquid suspension, wherein cell debris, particulates or other materials are suspended in the collection step liquid suspension. In some examples, the collection step liquid suspension is the first material mentioned above with respect to the liquid suspension 700, and the cell debris, particulates or other materials are the second material mentioned above with respect to the liquid suspension 700. At least one of the objectives of the methods described herein is to clarify the liquid suspension 700 so that the first material and the second material are substantially, if not completely, separated. The collection step 801B may include centrifuging the liquid suspension 700 in a closed system before the liquid suspension 700 is received in the flexible bag 350 described herein. The centrifugation process included in the collection step 801B occurs at a much lower rpm / gravity than the centrifugation step 808 described below that occurs during the clarification of the liquid suspension 700. For example, the collection step 801B can include centrifuging the collection liquid suspension at a rate of 500 rpm to 10,500 rpm, which can exert up to, for example, 7,000 times the gravity on the collection liquid suspension. During the collection step 801B, the centrifuge can be operated for 20 minutes to 40 minutes, wherein in a preferred embodiment, it is operated for 30 minutes, or until relatively large fragments have been pelletized from the collection liquid suspension to the satisfaction of the operator. In some embodiments, the centrifugation during the collection step 801B is performed at a temperature of 4° C. to 20° C. At least because during the collection step 801B, the centrifuge is operated at a much lower rpm and gravity than the centrifuge during the clarification method, only relatively large fragments suspended in the collection liquid suspension are pelletized. Therefore, the collection liquid suspension is separated into a collection precipitate and a collection supernatant, and the collection supernatant can ultimately be supplied to the liquid suspension container 730 as the liquid suspension 700 used in the clarification method. It is advantageous to perform the clarification method described herein after the initial collection step 801B to remove large fragments from the liquid suspension.

[0106] Continue to refer Fig.11A and Fig. 11B as well as Figure 10A-10C, methods 800a-800c include receiving 806 a liquid suspension 700 including a first material and a second material. The first material and the second material are different materials having different densities and are configured to be separated by centrifugation, particularly by high rpm / high gravity centrifugation, referred to herein as clarification. In some examples, the liquid suspension 700 is a product of a reaction and / or growth from cells that are from a previous growth / reaction step. For example, the liquid suspension 700 may contain a cell medium that includes whole cells, cell fragments, RNA, DNA, proteins, and / or combinations thereof. The first material may include cell fragments or whole cells, and the second material may include RNA, DNA, proteins (biological products secreted by host cells), and combinations thereof, or any two suitable materials that are configured to be separated from each other due to clarification.

[0107] Continue to refer Fig.11A and Fig. 11B Each of the flexible bags 350 has two connections, namely, an inlet line 330 and an outlet line 340. Fig.11A and Fig. 11B The inlet line 330 is configured to allow the flexible bag 350 to be filled with the liquid suspension 700, for example during the receiving step 806, and in the case of Fig.11A and Fig. 11B During the illustrated receiving step 806, each is in bulk communication with the manifold 750. The manifold 750 can be connected to a vacuum pump 740, which can help pump the liquid suspension 700 (e.g., from a previous lysis step 801C) or as described herein and in Figure 11K and Fig.11L The supernatant 770 is removed from the flexible bag 350 after the centrifugation step 808 shown in FIG. As an alternative or in addition to the vacuum pump 740, other pumps 740 may be used to help add or remove the liquid suspension 700 or supernatant 770 from the flexible bag 350, respectively. One or more pumps 740 may be connected in series with the manifold 750 (e.g., Fig.11A and Fig. 11B ), and / or one or more pumps 740 may be peristaltic pumps. In a preferred embodiment, a peristaltic pump is used to direct the liquid suspension 700 into the flexible bag 350 to further ensure that the methods 800a-800c remain performed in a closed system. Alternatively, the flexible bag 350 may be filled without the use of a pump 740 at all, but gravity may act on the liquid suspension 700 contained in the liquid suspension container 730 and distribute the liquid suspension 700 to each flexible bag 350 connected thereto through the manifold 750.

[0108] Other orientations of the connector are possible. Fig.11A and Fig. 11B A series connection of the flexible bag 350 to the manifold 750 is shown, but the flexible bags 350 may alternatively be arranged in parallel (not shown), or arranged in a branched arrangement (not shown). For a more detailed description of the filling process and further details about the flexible bag 350 system and individual flexible bags 350, reference is again made to International Patent Application Publication No. WO2019166998A1.

[0109] Further references Fig.11A and Fig. 11B , a clamp 754 may be included on the inlet line 330 to control when each individual flexible bag 350 is filled. The clamp 754 may have an open position and a closed position. Fig.11A , all clamps located on the inlet line 330 are in an open position, thereby allowing the liquid suspension 700 to travel through the inlet line 330. Fig.11A As shown, each flexible bag 350 has not yet received any amount of liquid suspension 770. When the receiving step 806 occurs, the liquid suspension 700 is pumped or otherwise directed through the manifold 750, and the inlet line 350 with the clamp 754 in the open position will be filled with the liquid suspension 700. By moving the clamp 754 to the closed position, the inlet line 330 is clamped closed, thereby preventing more liquid suspension 700 from entering the flexible bag 350. For example, Fig. 11B It is shown that both clamps on the inlet line 330 have been closed and the liquid suspension 700 is being transferred from the liquid suspension container 730 into the only flexible bag 350 having the clamp 754 in the open position on the inlet line 330. Thus, the dispensing of the liquid suspension 700 from the liquid suspension container 730 can be controlled during the receiving step 806.

[0110] In some examples, the flexible bag 350 is weighed as the flexible bag 350 receives the liquid suspension 700. In one example, the flexible bag 350 is placed on a rack 900, which is placed on a scale 910 to determine when the flexible bag 350 is properly filled. The flexible bag 350 nested within the lower bag receptacle portion 310 of the bag adapter 300 can be placed on the rack 900 and weighed before the liquid suspension 700 is received by the flexible bag 350, and continues to be weighed throughout the receiving step 806. Fig. 11C Several flexible bags 350 are shown nested within the lower bag receptacle portion 310 of the bag adapter 300, each of which is weighed simultaneously on the scale 910 before the flexible bags 350 receive any liquid suspension 700. Any or all of the flexible bags 350 nested within the lower bag receptacle portion 310 of the bag adapter 300 may be placed on the support 900 and weighed during this process. Fig.11D As shown, each flexible bag 350 nested within the lower bag receptacle portion 310 of the bag adapter 300 can be continuously weighed as liquid suspension 700 is added thereto from the liquid suspension container 730. Any liquid suspension 700 desired to enter any individual flexible bag 350 can do so if the clamp 754 positioned on the inlet line 330 is placed in the open position, and additional liquid suspension 700 is directed into any individual flexible bag 350 as desired. When the flexible bag 350 is sufficiently full, the clamp 754 can be closed, thereby preventing more liquid suspension 700 from entering the flexible bag 350. In some embodiments, the flexible bags 350 are filled until each flexible bag 350 contains between 0.5 kg and 1.5 kg of liquid suspension 700. In a preferred embodiment, the flexible bags 350 are filled until each flexible bag 350 contains approximately or exactly 0.9 kg of liquid suspension 700. At the end of the receiving step 806, each flexible bag 350 will contain approximately the same amount of liquid suspension 700, regardless of the exact mass of the received liquid suspension 700. This is to ensure that the centrifuge rotor 400 remains balanced during the centrifugation step 808. In some embodiments, the weight of the flexible bags 350 with liquid suspension 700 therein may be within 50 grams of each other in order to ensure that the centrifuge rotor 400 is balanced when the flexible bags 350 are ultimately loaded therein.

[0111] In order to separate the first material from the second material in the liquid suspension 700, the flexible bag 350 containing the liquid suspension 700 therein can be centrifuged. The flexible bag 350 is first separated from the liquid suspension container 730. The separation of the flexible bag 350 from the liquid suspension container 730 occurs by cutting off the inlet line 330 from the manifold 750 and occurs after the flexible bag 350 has been filled. Fig.11E and Fig.11F , for example, by cutting the inlet line 330 from the manifold 750 using a welder 920. In some examples, the welder 920 is iiB aseptic tubing welding machine, but other welding machines 920 are also suitable. Any welding machine 920 capable of cutting off the connection between the inlet tube 330 and the manifold 750 while maintaining the closed system within the flexible bag 350 is suitable. Fig.11E The inlet tube 330 is shown inserted into the welding machine 920. In some examples, such as Fig.11F As shown in FIG, the inlet tube 330, after having been severed from the manifold 750, can be folded upon itself and covered with a cap 710 to maintain a closed system within the flexible bag 350. The cap 710 can be placed and secured over the folded end of the inlet tube 330 to prevent the inlet tube 330 end from unfolding. Fig.11G, the upper closure portion 320 of the bag adapter 300 is placed over the flexible bag 350 and engages with the lower bag receiver portion 310, thereby enclosing the flexible bag 350 within the bag adapter 300. When the upper closure portion 320 is placed over the lower bag receiver portion 310, the inlet line 330 and the outlet line 340 are directed through the aperture 319 included through the upper closure portion 320.

[0112] refer to Fig.11H , during the centrifugation step 808, the bag adapters 300 with the flexible bags 350 enclosed therein are placed in separate receptacles 410 of the centrifuge rotor 400. The bag adapters 300 enclosing the flexible bags 350 are individually weighed before they are placed in the centrifuge, as described above. Each of the bag adapters 300 enclosing the flexible bags 350 is placed in a receptacle 410 in the centrifuge rotor 400 opposite a bag adapter 300 of similar weight in order to balance the centrifuge rotor 400 and assist in the centrifugation step 808. As described above, the crown assembly 200 is then placed over the bag adapters 300, wherein each upper closure portion 320 of the bag adapter 300 has a surface that conforms to and meets the arcuate wall 230 of the annular crown body 210. The inlet line 330 and the outlet line 340 lead through the aperture 232 into the crown cavity 216. Fig.11I The crown assembly 200 is shown properly placed with the inlet line 330 and the outlet line 340 disposed within the crown cavity 216 of the annular crown body 210. The crown ring 220 is then placed over the inlet line 330 and the outlet line 340, with a portion of the crown ring 220 extending radially inward from the periphery of the rim 212 of the annular crown body 210. The crown ring 220 includes an annular sidewall 224 having an O-ring receiver 226 thereon. When the O-ring 219 positioned on and protruding from the circumferential sidewall 233 of the annular crown body 210 is positioned within the O-ring receiver 226 of the annular sidewall 224 of the crown ring 220, the crown ring 220 is fixed in place and at least partially supported by the circumferential sidewall 233 of the annular crown body 210. The O-ring receiver 226 prevents the crown ring 220 from moving by physical interaction with the O-ring 219, and thus, the crown ring 220 is coupled to the annular crown 210. The annular lip 222 of the crown ring 220 secures the inlet line 330 and the outlet line 340 within the crown cavity 216 of the annular crown 210.

[0113] The centrifuge is then closed with its lid 450, and then the centrifugation step 808 is performed. During the centrifugation step 808, the centrifuge can be operated at a speed of 500rpm to 10,500rpm. In a preferred embodiment, and in order to promote the clarification of relatively small cell debris and particle contaminants (such as cell debris and / or particles with a size less than or equal to 1 micron) contained in the liquid suspension 700, the centrifuge can be operated at a speed greater than or equal to 5,000rpm, such as 5,000rpm to 10,500rpm, preferably 6,000rpm to 10,500rpm and more preferably 8,000rpm to 10,500rpm. In addition, these high rotation speeds can result in high gravitational forces applied to the liquid suspension 700, the flexible bag 350 and / or the bag adapter 300. These gravitational forces can total greater than or equal to 7,000 times of gravity, such as 7,000 times to 21,000 times of gravity. In a preferred embodiment, the gravity amount is 9,000 to 21,000 times gravity, more preferably 15,000 to 20,584 times gravity. In some examples, the centrifugation step 808 can be performed for 5 minutes to 15 minutes, preferably 8 minutes to 12 minutes and most preferably 10 minutes. The temperature of the centrifuge step 808 inside the centrifuge is maintained at 15°C to 25°C, preferably 17°C to 23°C and most preferably 20°C. At the end of the centrifugation step 808, the small fragments and particles suspended in the liquid suspension 700 will have been pelletized into a precipitate 760, and the liquid suspension 700 will be separated into a precipitate 760 and a supernatant 770 inside the flexible bag 350. Fig.11J The flexible bag 350 is shown within the centrifuge rotor 400 at the end of the centrifuge step 808 .

[0114] As used herein, "high rpm" is intended to refer to centrifugation performed at greater than or equal to 5,000 revolutions per minute. "High gravity centrifugation" is intended to refer to centrifugation performed at greater than or equal to 7,000 g (7,000 times Earth's gravity).

[0115] refer to Figure 11K, in the extraction step 810, after the centrifugation step 808, the bag adapter 300 is removed from the centrifuge. The contents of the flexible bag 350 are checked without opening the flexible bag 350 to see if a precipitate 760 and a supernatant 770 have been formed. If a precipitate 760 and a supernatant 770 have been formed, the flexible bag 350 is coupled to another manifold 750, similar to the manifold 750 used in the receiving step 806. The manifold 750 used in the extraction step 810 may be the same as or different from the manifold used in the receiving step 806, however, in order to maintain the purity of the supernatant 770, and to avoid reintroducing the liquid suspension 700 into the supernatant 770, it is preferred to use a new manifold 750 rather than reusing the manifold 750 from the receiving step 806. The outlet line 340 is coupled to the extraction manifold 750, which may be in communication with one or more pumps 740. The pump 740 may be in series with the manifold 750 and include, for example, a vacuum pump 740 or other pump system. Alternatively or additionally, one or more pumps 740 may be peristaltic pumps and direct supernatant 770 from the flexible bag 350 without being directly in series with the manifold 750 .

[0116] Further references Figure 11K , a clamp 754 may be included on the outlet line 340 to control when each individual flexible bag 350 has supernatant extracted therefrom. The clamp 754 may have an open position and a closed position. Figure 11K , all clamps 754 positioned on the outlet line 340 are in an open position, thereby allowing the liquid suspension 770 to travel through the outlet line 340. Figure 11K As shown, each flexible bag 350 has not yet had any supernatant 770 extracted therefrom. In the extraction step 810, the supernatant 770 is pumped or otherwise directed through the outlet line 340 having the clamp 754 in the open position, into the manifold 750, and ultimately into the storage container 720. By moving the clamp 754 to the closed position, the outlet line 340 is clamped closed, thereby preventing more supernatant 770 from leaving the flexible bag 350. For example, Figure 11K It is shown that the two clamps 754 on the outlet line 340 have been closed and the supernatant 770 is transferred from the flexible bag 350 with the clamps 754 on the outlet line 340 in the open position into the storage container 720. Thus, the supernatant can be controlled 770 to be extracted from the flexible bag 350 during the extraction step 810.

[0117] Alternatively, from Figure 11K The supernatant 770 can be squeezed out of the flexible bag 350 via the squeezer 930 as shown in the pump 740. Figure 11KAs shown, after the flexible bag 350 has been connected and is in bulk communication with the manifold 750, the flexible bag 350 can be placed in a squeezer 930 that presses the flexible bag 350, thereby forcing the supernatant 770 into the manifold 750 and ultimately to the storage container 720. Once the supernatant 770 is removed from the flexible bag 350, the supernatant 770 can be sent for further processing or can be stored in the storage container 720 until needed.

[0118] The specific illustrative embodiments of the present invention shown in the figures and discussed above should not be interpreted as limiting. Those skilled in the art can modify and modify these embodiments in many obvious ways within the scope of the appended claims. Therefore, the present invention is not limited to the embodiments described above.

Claims

1. A bag adapter configured to enclose a flexible bag containing a liquid suspension and having at least one inlet line and / or outlet line fluidly coupled to the flexible bag, wherein the at least one inlet line and / or outlet line extends from the flexible bag to an exterior of the bag adapter, the bag adapter comprising: a lower bag receiver portion configured to receive a lower portion of the flexible bag, the lower bag receiver portion comprising: a bottom wall; and a circumferential lower side wall extending upwardly from the bottom wall and defining, together with the bottom wall, a lower pocket cavity having an opening positioned opposite the bottom wall and defined by an edge of the circumferential lower side wall, the lower pocket receiver portion at least partially enclosing the lower portion of the flexible bag when the lower portion of the flexible bag is received in the lower pocket receiver portion through the opening of the lower pocket cavity; and an upper closure portion configured to engage the lower bag receptacle portion and receive an upper portion of the flexible bag, the upper closure portion comprising: a top wall; a circumferential upper side wall depending from the top wall and defining an upper pocket cavity together with the top wall, the upper pocket cavity having an opening positioned opposite the top wall and defined by an edge of the upper side wall, the upper closure portion at least partially enclosing the upper portion of the flexible bag when the upper portion of the flexible bag is received through the opening of the upper pocket cavity; and at least one aperture formed in the upper closure portion providing at least one passage extending between the upper bag cavity and an exterior of the upper closure portion, the at least one passage being configured such that the at least one inlet line and / or outlet line extends from the upper bag cavity through the at least one passage to the exterior of the upper closure portion, wherein the bag adapter encloses the flexible bag when the lower bag receptacle portion and the upper closure portion are engaged together.

2. The bag adapter of claim 1, wherein the at least one aperture comprises a pair of apertures.

3. The bag adapter of claim 1, wherein the lower bag receiver portion and the upper closure portion define an overlapping joint at a junction of the lower bag receiver portion and the upper closure portion.

4. The bag adapter of claim 1 wherein the bottom wall of the lower bag receiver portion includes a rounded shoulder that engages the circumferential lower side wall of the lower bag receiver portion.

5. The bag adapter of claim 1, wherein the top wall of the upper closure portion includes a rounded shoulder that engages the circumferential upper side wall of the upper closure portion.

6. The bag adapter of claim 5, wherein the at least one aperture is formed in the circular shoulder of the upper closure portion.

7. The bag adapter of claim 5, wherein a pair of holes are formed in the circular shoulder of the upper closure portion.

8. The bag adapter of claim 1, wherein the circumferential lower sidewall of the lower bag receptacle portion is circular in cross-section.

9. The bag adapter of claim 1, wherein the circumferential upper side wall of the upper closure portion is circular in cross-section.

10. The bag adapter of claim 1, wherein the upper closure portion is releasably engaged to the lower bag receiver portion.

11. A crown assembly for use with a rotor to at least partially close at least one inlet line and / or outlet line during centrifugation of at least one flexible bag in the rotor, the at least one inlet line and / or outlet line being fluidly coupled to the at least one flexible bag, the at least one flexible bag containing a liquid suspension, the crown assembly comprising: An annular crown body, the annular crown body defines a crown cavity and includes: a bottom wall; a circumferential side wall, the circumferential side wall being upwardly spaced apart from the bottom wall; a circumferential sector-shaped sidewall extending between the bottom wall and the circumferential sector-shaped sidewall, the circumferential sector-shaped sidewall comprising a plurality of radially inwardly directed arcuate walls, the arcuate walls being circumferentially arranged around the circumferential sector-shaped sidewall; at least one hole formed in each of the plurality of radially inwardly directed arcuate walls, providing at least one passage extending between the exterior of the annular crown body and the crown cavity, the at least one passage being configured such that the at least one inlet line and / or outlet line can extend from the exterior of the annular crown body through the at least one passage to the crown cavity; and an annular mounting member extending upward from the bottom wall and disposed centrally within the annular crown; an elastic element, the elastic element being supported by the annular mounting member; a crown assembly plunger operably engaged with the resilient element and vertically movable relative to the annular mount between a first position and a second position, the resilient element biasing the crown assembly plunger to the first position, the crown assembly plunger movable to the second position in response to the resilient element being compressed by a cover of the rotor; and an annular crown ring removably coupled to the annular crown body and configured together with the annular crown body to at least partially close the at least one inlet line and / or outlet line fluidly connected to the flexible bag within the crown cavity during centrifugation of the at least one flexible bag in the rotor.

12. The crown assembly of claim 11, wherein the annular crown ring includes an annular sidewall and an annular lip extending radially inwardly from the annular sidewall of the annular crown ring.

13. The crown assembly of claim 12, wherein said annular lip extends radially inwardly and downwardly from said annular sidewall of said annular crown ring.

14. The crown assembly of claim 12, wherein said annular lip is at least partially translucent.

15. The crown assembly of claim 12, wherein the annular crown ring is mounted in the crown cavity.

16. The crown assembly of claim 15, further comprising at least one O-ring supported by the annular sidewall of the annular crown ring and configured to engage the circumferential sidewall of the annular crown body when the annular crown ring is installed in the crown cavity.

17. The crown assembly of claim 12, further comprising a carbon fiber reinforcement disposed about the annular sidewall of the annular crown ring.

18. The crown assembly of claim 11, wherein the at least one aperture comprises a pair of apertures.

19. The crown assembly of claim 11, wherein the annular mounting member comprises: an inner annular wall extending upward from the bottom wall; as well as an outer annular wall extending upwardly from the bottom wall and concentrically disposed about the inner annular wall to define a cavity between the inner and outer annular walls of the annular mount, wherein the cavity of the annular mount is configured to at least partially support the resilient element within the cavity of the annular mount.

20. The crown assembly of claim 19, wherein the crown assembly plunger comprises: an annular body defining an annular shoulder; an inner annular wall depending from the annular shoulder; as well as an outer annular wall depending from the annular shoulder and disposed concentrically about the inner annular wall to define a cavity between the inner and outer annular walls of the crown assembly plunger, wherein the cavity of the crown assembly plunger is configured to at least partially support the resilient element within the cavity of the crown assembly plunger.

21. The crown assembly of claim 11, wherein said crown assembly plunger is mounted for movement at least partially within said annular mount.

22. The crown assembly of claim 11, wherein said plurality of radially inwardly directed arcuate walls comprises four arcuate walls.

23. The crown assembly of claim 11, wherein said plurality of radially inwardly directed arcuate walls comprises at least six arcuate walls.

24. A kit, comprising: At least one bag adapter configured to enclose a flexible bag containing a liquid suspension and having at least one inlet line and / or outlet line fluidly coupled to the flexible bag, wherein the at least one inlet line and / or outlet line extends from the flexible bag to an exterior of the bag adapter, the at least one bag adapter comprising: a lower bag receiver portion configured to receive a lower portion of the flexible bag, the lower bag receiver portion comprising: a bottom wall; and a circumferential lower side wall extending upwardly from the bottom wall and defining, together with the bottom wall, a lower pocket cavity having an opening positioned opposite the bottom wall and defined by an edge of the lower side wall, the lower pocket receiver portion at least partially enclosing the lower portion of the flexible bag when the lower portion of the flexible bag is received in the lower pocket receiver portion through the opening of the lower pocket cavity; and an upper closure portion configured to engage the lower bag receptacle portion and receive an upper portion of the flexible bag, the upper closure portion comprising: a top wall; a circumferential upper side wall depending from the top wall and defining an upper pocket cavity together with the top wall, the upper pocket cavity having an opening positioned opposite the top wall and defined by an edge of the upper side wall, the upper closure portion at least partially enclosing the upper portion of the flexible bag when the upper portion of the flexible bag is received through the opening of the upper pocket cavity; and at least one aperture formed in the upper closure portion providing at least one passage extending between the upper bag cavity and an exterior of the upper closure portion, the at least one passage being configured to enable the at least one inlet line and / or outlet line to extend from the upper bag cavity through the at least one passage to the exterior of the upper closure portion, wherein the bag adapter encloses the flexible bag when the lower bag receptacle portion and the upper closure portion are engaged together; The crown assembly of claim 11.

25. The kit of claim 24, further comprising: At least one flexible bag configured to contain a liquid suspension and having at least one inlet line and / or outlet line fluidly coupled to the flexible bag.

26. A method of clarifying a liquid suspension contained in at least one flexible bag having at least one inlet line and / or outlet line fluidly coupled to the flexible bag, the method comprising: nesting the at least one flexible bag containing the liquid suspension within at least one bag adapter, wherein the at least one inlet line and / or outlet line extends from the flexible bag to the exterior of the bag adapter; at least partially enclosing the at least one inlet line and / or outlet line within a crown cavity defined by the crown assembly; as well as The at least one bag adapter and the at least one flexible bag containing the liquid suspension are centrifuged in a centrifuge to produce a supernatant and a precipitate in the at least one flexible bag.

27. The method according to claim 26, further comprising the steps of: The supernatant is extracted from the at least one flexible bag via at least one outlet line fluidly coupled to the at least one flexible bag.

28. The method according to claim 27, wherein said at least one flexible bag is removed from said at least one bag adapter prior to said extracting step.

29. The method according to claim 26, further comprising: The liquid suspension is received in the at least one flexible bag via at least one inlet line.

30. The method according to claim 29, Wherein when the liquid suspension is contained in the at least one flexible bag, the at least one flexible bag is at least partially positioned outside of the at least one bag adapter.

31. The method according to claim 30, Wherein the liquid suspension is received in the at least one flexible bag via the at least one inlet line, the at least one inlet line being fluidly coupled to a source of liquid suspension.

32. The method according to claim 31, further comprising: An inlet manifold is fluidly coupled to the liquid suspension source and to the at least one inlet line, the at least one inlet line being fluidly coupled to the at least one flexible bag.

33. The method of claim 26, wherein the bag adapter comprises: a lower bag receiver portion configured to enclose a lower portion of the flexible bag and an upper closure portion configured to enclose an upper portion of the flexible bag, the bag adapter being configured to withstand greater gravity than the at least one flexible bag.

34. The method of claim 26, wherein the method is performed in a closed system.

35. The method according to claim 27, Wherein the supernatant is extracted into an outlet manifold via the at least one outlet line, the at least one outlet line being fluidly coupled to the flexible bag containing the liquid suspension.

36. The method of claim 26, wherein centrifuging the bag adapter comprises centrifuging the bag adapter at a rate greater than or equal to 5,000 revolutions per minute.

37. The method of claim 26, wherein centrifuging the bag adapter comprises applying a force greater than 7,000 g to the bag adapter.

38. The method of claim 27, wherein extracting the supernatant from the at least one flexible bag comprises extracting the supernatant into a storage container.

39. The method of claim 35, wherein extracting the supernatant from the at least one flexible bag comprises extracting the supernatant into an outlet manifold.

40. The method of claim 26, further comprising: The liquid suspension is received in the at least one flexible bag via at least one inlet line when a bottom portion of the flexible bag is enclosed in the lower bag receptacle portion of the bag adapter.

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