Patent Publication Number: US-2021189454-A1

Title: System and method for clarifying a cell culture harvest solution

Description:
This application claims the benefit of U.S. Provisional Patent Application Ser. Nos. 62/670,220 and 62/827,009, the disclosures of which are incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     This document relates generally to the field of purification, particularly, cell culturing and biologic manufacturing and, more particularly, to a system and method for clarifying a cell culture harvest solution. 
     BACKGROUND 
     In the biologics manufacturing field, there is a need for equipment which has a limited footprint so that it can form part of a “microfacility.” Such a microfacility would permit speedy and efficient substitution of components operated under isolators between batch operations. Single-use technology would allow such substitution and prevent the need for costly cleaning and revalidation as well. Thus, the microfacility would offer high yield at higher speed of setup and functionality at substantially lower cost. 
     Past proposals for clarifying cell cultures as part of the biologics manufacturing process exist, including the disclosure in International Application No. PCT/EP2018/058366, the disclosure of which is incorporated herein by reference. This approach represents an improvement over past approaches by providing a system and method for clarifying a cell culture harvest in an easy, reliable, and inexpensive manner. However, creating and using a “horizontal” cake filter positioned upon an associated filter support at the bottom of the container presents a challenge if any “regeneration” of the filter cake is desired in an easy and efficient manner. This is because the direction of fluid flow through the filter surface is aligned with the direction of gravity. 
     Because the cake is also not easily removed from the filtration vessel, the size of that vessel must be large enough to accommodate a finite amount of the material (e.g., diatomaceous earth) forming the filter cake. When the vessel is filled beyond a certain point, further introduction of solution is not permitted and the filtration vessel must be emptied or cleaned in order to reuse it. This can impact processing time and cost, since the ratio of the area of filtration to the volume of production of filtrate is low. 
     It is also known to filter a chemical solution mixed with a dynamic filtration media using a system having a large, rigid vessel with a so-called “candle” filter. In a typical arrangement, the solution is filtered through the candle filter such that a filter cake forms on the surface of the candle filter. The filter cake may be discharged once the operation is complete, and the vessel must be cleaned and revalidated before it can be reused. Thus, the vessel is usually costly and does not permit easy use and operation in a limited space. 
     Accordingly, a need is identified for a system and method for clarifying a cell culture harvest solution that provides further efficiencies in certain manufacturing environments. Toward this end, the system and method in some embodiments would include one or more small volume vessels, including a disposable filtration vessel that includes one or more candle filters. The system could thus be readily applied as part of a “microfacility” for clarifying the cell culture harvest solution, and then simply disposed of once processing is complete. In one particularly advantageous example, the clarifying system and method would also use the filtrate as a source of a backflush fluid to discharge the filter cake from the candle filter, thereby increasing efficiency and avoiding the need for the introduction of a separate backflush fluid into a sterile environment and eliminating the corresponding costs/challenges. 
     SUMMARY 
     According to a first aspect of the disclosure, a system for clarifying a cell culture harvest solution, including target molecules and dynamic filter media is provided. The system comprises a filtration vessel comprising a flexible liner, the filtration vessel including at least one filter having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter adapted, during filtration operation, to permit a filtrate including target molecules to pass therethrough and said cake, during filtration operation, adapted to prevent unwanted solid materials from passing therethrough; and a backflush source including a backflush fluid and fluidly connected to the filtration vessel via the at least one filter, said backflush source, during backflush operation, adapted to supply backflush fluid back through the at least one filter for removing the cake formed on the filter. 
     In some embodiments, the backflush source is a backflush vessel adapted for receiving a portion of the filtrate from the filtration vessel. In some embodiments, the system further includes a bioreactor vessel or intermediate vessel within which the cell culture harvest solution and dynamic filter media is mixed and capable of supplying the cell culture harvest solution to the filtration vessel. In some embodiments, the system further includes a source of dynamic filter media for being combined with the cell culture harvest solution after delivery from a bioreactor or intermediate vessel. In some embodiments, a bioreactor vessel is provided for supplying the cell culture harvest solution to the filtration vessel and an auxiliary vessel is provided for supplying the dynamic filtration media. 
     In some embodiments, an actuator is provided for causing the flexible liner to collapse and cause liquid therein to pass through the at least one filter. The actuator may comprise a source of pressurized fluid. In some embodiments, the at least one filter is suspended within the filtration vessel. In some embodiments, a waste collector in communication with the filtration vessel is provided for receiving waste therefrom. In some embodiments, a source of buffer is provided in communication with the filtration vessel. In some embodiments, a pump is provided for pumping liquid to the filtration vessel. In some embodiments, a plurality of (candle) filters are in the filtration vessel. 
     In some embodiments, the backflush source includes a flexible liner, and the system further includes an actuator for causing the flexible liner to collapse and cause fluid therein to pass through the at least one filter and into the filtration vessel. In some embodiments, the filtration vessel further includes a valve for selectively allowing for the draining of fluid therefrom. In some embodiments, the valve is located adjacent to a bottom portion of the vessel. 
     A further aspect of the disclosure pertains to a method for clarifying a cell culture harvest solution including target molecules and a dynamic filter media into a filtrate including the target molecules. The method comprises delivering the cell culture harvest solution to a filtration vessel including at least one filter adapted for allowing a filtrate but not the dynamic filter media to pass therethrough, the filter having a surface with a surface area, and driving a first liquid flow through the filter to allow a cake to form on the filter and the filtrate to result from passing through the cake; and backflushing the at least one filter. 
     In some embodiments, the backflushing step comprises passing a liquid through the filter to discharge the cake from the filter. In some embodiments, the method comprises compressing the filtration vessel prior to the backflushing step. In some embodiments, the method includes the step of delivering filtrate to a collector after the backflushing step. In some embodiments, the method comprises the step of compressing the filtration vessel after the backflushing step. In some embodiments, the method comprises the step of introducing a buffer to the filtration vessel after the backflushing step. In some embodiments, the method further includes the step of discharging waste from the filtration vessel after the backflushing step. In some embodiments, the method further includes the step of driving a second liquid flow through the at least one filter to allow another cake to form on the surface of the filter. In some embodiments, the method further includes the step of opening a valve to drain the filtration vessel. In some embodiments, the method further includes the step of combining the cell culture harvest solution after discharge from a bioreactor or intermediate vessel with the dynamic filter media. 
     According to a further aspect of the disclosure, a method for clarifying a cell culture harvest solution including target molecules and a dynamic filter media into a filtrate including the target molecules but excluding certain impurities is provided. The method comprises delivering a cell culture harvest solution to a filtration vessel including a compressible liner and at least one filter adapted for allowing a filtrate but not the dynamic filter media to pass therethrough, the filter having a surface with a surface area, such that a cake of the dynamic filter media forms on the filter; and compressing the liner to cause liquid within the liner to flow through the filter to create a filtrate. 
     In some embodiments, the method includes the step of backflushing the at least one filter with the liquid. In some embodiments, the backflushing step comprises backflushing the filter with the filtrate. The step of backflushing the at least one filter with the liquid may comprise backflushing the filter with liquid from a backflush vessel in communication with the filtration vessel. 
     The method may further comprise passing liquid from the filtration vessel through the filter after the backflushing step. In some embodiments, the compressing step is completed after the delivering step, but before the backflushing step. In some embodiments, the compressing step is completed after the backflushing step. This disclosure also pertains to an apparatus for clarifying a cell culture harvest solution, including target molecules and dynamic filter media. The apparatus comprises a filtration vessel including at least one candle filter having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter adapted, during normal operation, to permit a filtrate including target molecules to pass therethrough and said cake, during normal operation, adapted to prevent unwanted solid materials from passing therethrough, the filtration vessel including a flexible liner for receiving the cell culture harvest solution and in fluid communication with the at least one candle filter. 
     In some embodiments, the apparatus comprises an actuator for collapsing the flexible liner. In some embodiments, the filtration vessel comprises a rigid or semi-rigid outer container for receiving the flexible liner. In some embodiments, the flexible liner includes a drain associated with a valve. In some embodiments, the flexible liner includes an agitator, or the at least one candle filter is suspended within the flexible liner. In some embodiments, the filtration vessel includes a vent in fluid communication with an interior compartment of the flexible liner. 
     This disclosure also pertains to an apparatus used to form a system in combination with a collection vessel for receiving the filtrate, and a backflush source including a backflush fluid and fluidly connected to the filtration vessel via the at least one filter, said backflush source, during backflush operation, adapted to supply backflush fluid back through the at least one filter for removing the cake formed on the filter. The backflush source may include a flexible liner, and the actuator may be adapted for collapsing the flexible liner of the backflush source. 
     According to still a further aspect of the disclosure, a system for clarifying a cell culture harvest solution, including target molecules and dynamic filter media is provided. The system comprises a filtration vessel including at least one filter, such as a candle filter, having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter adapted, during normal operation, to permit a filtrate including target molecules to pass therethrough and said cake, during normal operation, adapted to prevent unwanted solid materials from passing therethrough; and a backflush vessel including a flexible liner for containing a backflush fluid and fluidly connected to the filtration vessel via the at least one filter, said backflush vessel, during backflush operation, adapted to supply backflush fluid back through the at least one filter for removing the cake formed on the filter, the backflush vessel including a flexible liner. 
     Yet another aspect of the disclosure relates to a system for clarifying a cell culture harvest solution, including target molecules and dynamic filter media. The system comprises a filtration vessel including at least one filter, such as a candle filter, having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter adapted to permit a filtrate including target molecules to pass therethrough and said cake adapted to prevent unwanted solid materials from passing therethrough; and a backflush source fluidly connected to the filtration vessel via the at least one filter, said backflush source adapted to receive filtrate from the filtration vessel and supply the filtrate back through the at least one filter for removing the cake formed on the filter. 
     Still a further aspect of the disclosure relates to a system for clarifying a cell culture harvest solution, including target molecules and dynamic filter media. The system comprises a filtration vessel including a first disposable liner and at least one filter, such as a candle filter, having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter adapted to permit a filtrate including target molecules to pass therethrough and said cake adapted to prevent unwanted solid materials from passing therethrough, and a backflush source fluidly connected to the filtration vessel via the at least one filter, the backflush source comprising a second disposable liner. 
     Yet a further aspect of the disclosure relates to a system for clarifying a cell culture harvest solution, including target molecules and dynamic filter media. The system comprises a compressible filtration vessel including at least one filter, such as a candle filter, having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter adapted to permit a filtrate including target molecules to pass therethrough and said cake adapted to prevent unwanted solid materials from passing therethrough, and a backflush source fluidly connected to the compressible filtration vessel via the at least one filter. In some embodiments, the backflush source comprises a compressible backflush vessel. 
     A further aspect of the disclosure relates to a system for clarifying a cell culture harvest solution, including target molecules and dynamic filter media. The system comprises a filtration vessel including at least one filter, such as a candle filter, having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter adapted to permit a filtrate including target molecules to pass therethrough and said cake adapted to prevent unwanted solid materials from passing therethrough; and a compressible backflush vessel fluidly connected to the compressible filtration vessel via the at least one filter. In some embodiments, the filtration vessel comprises a compressible filtration vessel. 
     In any disclosed embodiment, the system may form part of a microfacility. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING FIGURES 
         FIG. 1  is schematic view of one embodiment of the clarification system according to one embodiment; 
         FIG. 1A  illustrates one embodiment of a disposable filtration vessel; 
         FIG. 1B  illustrates one embodiment of a disposable backflush vessel; 
         FIGS. 2A, 2B, and 2C  are schematic views of one embodiment of the system according to the disclosure; 
         FIGS. 3-13  illustrate a clarification cycle according to an embodiment of the disclosure; and 
         FIG. 14  is a flow chart, which includes the sequence of events depicted in  FIGS. 3-13 . 
     
    
    
     DETAILED DESCRIPTION 
     In one embodiment, a system disclosed herein comprises a filtration vessel comprising a flexible liner, disposed within a rigid or semi-rigid container and at least one filter disposed within the flexible liner having a surface adapted for separating a solid material from a liquid material, the filtration vessel being adapted for applying a pressure difference between the rigid or semi-rigid container and the flexible liner and/or the at least one filter and a downstream collection or backflush vessel, the filtration vessel being further adapted for removing solid waste from the flexible liner. In some embodiments the filtration vessel and the flexible liner comprise a port. The liner and vessel may be sealed together using a common component, such as for example a lid or cover. In some embodiments the port comprises a valve. In some embodiments the filtration vessel is adapted for pressure dispense operation. In some embodiments the system is housed in a microfacility. 
     In another embodiment, a method disclosed herein comprises adding a mixture comprising a liquid and a solid to the filtration vessel, applying a pressure difference between the interior of the rigid or semi-rigid container and the flexible liner to produce solids retained on the filter and a filtrate and backflushing the solids from the filter with a rinsing liquid or the filtrate and removing the solids from the flexible liner through a port in the liner. In some embodiments the port comprises a valve. In some embodiments the filter is regenerated. In some embodiments the filter is reused. In some embodiments the method further comprises an upstream process. In some embodiments the method further comprises a downstream process. In some embodiments, the method is performed in a microfacility. 
     In another embodiment, a system disclosed herein comprises a manifold adapted to connect with a source of fluid in need of filtration, a dynamic filter media, and a filtration vessel. The filtration vessel comprises a flexible liner, disposed within a rigid or semi-rigid container and at least one filter disposed within the flexible liner having a surface adapted for separating a solid material from a liquid material, the filtration vessel being adapted for applying a pressure difference between the between the rigid or semi-rigid container and the flexible liner and/or the at least one filter and a downstream collection or backflush vessel, the filtration vessel being further adapted for removing solid waste from the flexible liner. In some embodiments the filtration vessel and the flexible liner comprise a port. The liner and vessel may be sealed together using a common component, such as for example a lid or cover. In some embodiments the port comprises a valve. In some embodiments the filtration vessel is adapted for pressure dispense operation. In some embodiments the manifold is housed in a microfacility. 
     In another embodiment one or more steps in the aforementioned method are repeated. In some embodiments the system or one or more of its components are disposable. Preferably, the flexible liner is disposable. 
       FIG. 1  illustrates the concept of a clarifying system  10  of the present disclosure in an illustrative embodiment. In the embodiment, a cell culture harvest solution or “feed” source  12  is provided. As disclosed in PCT/EP2018/058366, feed source  12  may be a bioreactor (such as, for example, a high cell density fixed bed or suspension bioreactor) or it may be a different vessel downstream from a bioreactor. In any case, feed source  12  is arranged to hold (in the case of it being a vessel) and act as a source of a cell culture harvest solution comprised of a cell culture harvest that requires subsequent clarification, and may further comprise filtration, to recover a product of interest. Feed source  12  (of which there may be more than one, and feeding may be done in a continuous, semi-continuous or batch mode) is thus connected by feed conduit  15  to a filtration vessel  14  via an inlet  14   a . The filtration vessel  14  is thus downstream of the feed source  12 . The feed conduit  15  may also communicate with one or more sources  13  for providing source materials used in processing the cell culture harvest. Source  13  may comprise a buffer for a washing/chasing the solution. The buffer source  13  may simply be a source of water (such as water for injection). In one or more embodiments, the cell culture harvest may comprise one or more compounds allowing the formation of floccules, including but not limited to fatty acids having 7 to 10 carbon atoms and derivatives thereof, ureides and electropositive compounds. 
     The filtration vessel  14  may also include a filtrate outlet  14   b  at any location through which the clarified feed or “filtrate” from the filtration vessel  14  flows. This filtrate includes target molecules or cells of interest for subsequent downstream processing and/or collection (such as if the filtrate is the product of interest). The filtration vessel includes one or more protruding finger-like or “candle” filters, which may be in any suitable form to reach into the inner volume of the filtration vessel and form a filter surface area. One form of candle filter (as shown in  FIG. 1 ) is a vertically oriented (extending in a direction from top toward bottom of vessel), filter. While a vertical orientation suspended from a top wall of the vessel  14  (or an associated structure) is shown, the candle filter  16  may project from any wall of the vessel  14  (including a sidewall or a bottom wall). 
     The surface area of the candle filter  16  for achieving filtration is provided along the exterior surface of the sides thereof. The candle filter  16  can be made of a polymer material (e.g., polyethylene) with a porosity that allows retention of filter aid (or dynamic filter media) particles, while allowing permeation of the liquid phase (i.e., the filtrate). In  FIG. 1 , the candle filter  16  is suspended from the top within the filtration vessel  14  and thus is fixed to and depends from an upper portion thereof. In alternative embodiments, the candle filter  16  could also be located along other portions of the vessel (e.g., the side or bottom wall). Locating the filter in a position farthest from the bottom of the vessel has advantages that will be described below. Accordingly, the filter  16  is considered static, in the sense that it is not free or unrestrained to travel about the vessel  14 . Although shown in  FIG. 1  to include a single candle filter  16 , more than one filter may be provided, as outlined further in the description that follows. 
     The filtration vessel  14  may comprise a rigid or semi-rigid container C, but could be flexible as well (e.g., a bag-in-bag arrangement). It may be made of plastic or metal or any other suitable material known to one skilled in the art. In order to make a disposable arrangement that does not require cleaning or validation, the filtration vessel  14  may include an inner, flexible or collapsible liner, which may take the form of a bag  18  as indicated in the broken lines (e.g., a single-use, two or three-dimensional flexible polymeric bag in any suitable configuration comprising chemically resistant materials). The bag  18  may be disposed within the rigid or semi-rigid container C, and thus form the filtration vessel as an assembly (that is, an inner disposable vessel for receiving the cell culture harvest solution from the upstream feed source  12 , and also an outer (possibly reuseable) vessel that may receive a fluid (air) for compressing or squeezing the bag  18  to reduce its volume and cause liquid therein to flow through the candle filter(s)  16  (which as discussed below may include an accumulated cake of a dynamic filter media). 
       FIG. 1A  illustrates one particular example of a candle filter  16  positioned in a flexible bag  18  for forming the feed receiving portion of the filtration vessel  14 . The rigid outer container within which the bag  18  is disposed is not shown. In this example, the candle filter  16  comprises two spaced candle filters, each of which comprises a filtration media  16   a  (e.g., polyethylene having a selected porosity, such as for example 0.7 microns). The filtration media  16   a  may be elongated and/or slender, forming a finger-like structure along the exterior surface of the candle filter  16 , and could also be located within the candle filter  16 . The end of the candle filter (distal from the top of the vessel  16 ) may be tapered and may be formed by simply bonding the ends of the material together. This allows for the candle filter  16  to be positioned in the vessel  14  so as to project from the top (or side) thereof into the vessel volume. Thus, the media  16   a  is designed and located to be subject to having a cake filter formed on the surface thereof. The filtration media  16   a  may extend over a support (not shown), which may comprise a mesh, grid, or like porous material to provide rigidity and prevent the filter media from collapsing. The media may attach to the support using an adhesive or other suitable means for attachment or binding. The bag  18  may be welded or otherwise attached to lid  14   d  (such as along a depending extension of it) for sealing and enclosing the candle filter  16  against unwanted ingress. The candle filter  16  (at the end proximal to the vessel top) is fluidly connected to outlet  14   b . The lid or top portion  14   d  may also include the inlet  14   a , the outlet  14   b , and vent  14   e  (which may be aseptic/sterile or non-aseptic) for the filtration vessel  14 , as shown, and also serves as a closure for the filtration vessel  14  (and bag  18  in particular, which may be peripherally bonded to the body of the lid  14   d  to create a fluid-tight seal). 
     The bag  18  may also include an outlet or drain for discharging waste, which may be associated with a valve for allowing for the selective discharge (see, e.g.,  FIGS. 3-13 ). The filtration vessel  14  can be sized quite small (e.g., in one example 1.5 to 1.8 L) but could also be used with a larger feed source (e.g. up to 200-500 L bioreactor). The size of the filtration vessel is scalable to the size of the upstream process. In some embodiments the system comprises a bioreactor having a volume from 1.5 L to 1000 L, preferably from 10 L to 750 L and more preferably from 200 L to 500 L. 
     The outlet  14   b  may communicate, through filtrate conduit  17 , with a filtrate collector, such as a collection vessel  20 . This collection vessel  20 , in operation, receives the filtrate travelling from the filtration vessel  14  via the one or more filters  16 . A backflush source may also be provided (along with suitable valving) via the filtrate conduit  17 . The backflush source may be a backflush vessel  22  containing backflush liquid (e.g., water (including ultrapure water, USP water, EFI) or equilibration buffer (e.g., PBS or HBS)). 
     The backflush vessel  22  is used (via conduit  17 ) to introduce the backflush fluid (meaning liquid or gas (e.g., compressed air) back through the candle filter  16  in reverse direction (that is, from the outlet  14   b  for delivering the filtrate to the collection vessel  20 , which could alternatively be used as a backflush source instead of vessel  22 ). This permits the reusability or regeneration of the candle filter(s)  16  (before the media buildup begins to negatively affect flow rate through the filter  16 ) so that more feed can be supplied to the filtration vessel  14  and fed through filter  16 . As discussed below, the backflush fluid may be sourced from the filtrate of the feed source  12  or from the buffer source  13  or some other source. One embodiment includes some portion of the filtrate to be transmitted to the backflush vessel to be used for the backflush fluid. Importantly, this effectively simplifies the process (as opposed to using a buffer or other liquid for backflushing) as no adjustments need to be made to the process or solution due to the common backflush fluid introduction. 
     As indicated in  FIG. 1B , the backflush vessel  22  may comprise an inner, flexible or collapsible liner, which may take the form of a bag  22   a . The bag  22   a  may be connected to a rigid lid or top portion  22   b  that may also include a port  22   c  serving as a fluid inlet or outlet, a first outlet or vent  22   d  in communication with the interior compartment of the bag  22   a , and a second outlet or vent  22   e  for communicating with a space external to the bag  22   a . The lid  22   b  thus serves as a closure for the backflush vessel  22  (and bag  22   a  in particular, which may be peripherally bonded to the body of the lid  22   b  to create a fluid-tight seal). The bag  22   a  may be provided in a rigid or semi-rigid container D (see  FIG. 1 ), thus forming the backflush vessel  22  as an assembly (that is, an inner disposable vessel for receiving a backflush fluid, which as noted above may be a portion of the filtrate, and also an outer vessel, which as noted below may receive a fluid (air) for compressing or squeezing the inner vessel to reduce its volume and cause liquid therein to flow through the candle filter(s)  16 ) in reverse). The size of the backflush vessel  22  can be quite small (e.g. in one particular example 1.0 to 1.2 L) to hold only enough fluid (in this case, filtrate) to accomplish the backflush function during backflush operation, but is easily scalable to meet the requirements of a particular arrangement in which it is used. The impetus for forcing the backflush liquid to flow into the filtration vessel  14  may be an actuator. In the illustrated embodiment the actuator takes the form of a gas source  24 , such as a vessel containing gas under pressure (e.g., compressed air), which is delivered to the backflush source  22  via conduit  25 , but could also be mechanical in nature (e.g., a piston or pump). As shown in  FIG. 1 , this gas source  24  may also be connected via a conduit  27  to the filtration vessel  14  to introduce a fluid (gas) between the bag  18  and the inner wall of the filtration vessel  14  to cause the former to collapse and thus force liquid to flow through the candle filter  16  to the outlet  14   b  to the collection vessel  20  (which, as noted below, may be repeated during the overall clarification process). 
     The filtration vessel  14  may also include a waste outlet or drain  14   c , which may be connected to or associated with a waste collector or vessel  26 . However, the waste could also be ejected or recovered from the filtration vessel  14  in other ways (such as, for example, by applying suction to a dip tube or the like projecting into the filtration vessel). The filtration vessel  14  may optionally include an agitator for use in agitating the contents, perhaps to maintain homogeneity of the solution, which agitator may operate in a non-contact manner (e.g., a magnetic impeller) or via a dynamic seal capable of maintaining sterile conditions). When flexible liner  18  is present agitation can be provided using a pressure differential. 
     In use, and with reference to  FIGS. 2A-2C , the feed solution (which may contain the cell culture harvest and other materials including a filter aid or suitable dynamic filter media  30 ) may be delivered from the feed source  12  to the filtration vessel  14 . The dynamic filter media  30  may include diatomaceous earth (DE), which is shown in suspension in  FIG. 2A , and may be delivered from a separate source  11 , as indicated in  FIG. 1 ). The dynamic filter media  30  is introduced for the purpose of filtering certain undesirable impurities from the harvest solution such as cells, cell debris, or other waste products from the feed solution and passing downstream a clarified filtrate of the cell culture harvest solution, including the target molecule for further processing. Alternatively, the dynamic filter media  30  may be added to the filtration vessel  14  separate from the feed solution, such as by providing it in the form of a slurry from an auxiliary vessel  12   a  via a separate conduit  29  (as shown in  FIG. 2A ) or via shared conduit  15 . 
     When extraction of the filtrate is desired, a pressure differential may be created, such as by using a pump (see, e.g.,  FIG. 3 ), As indicated in  FIGS. 2A and 2B , this will generate a flow generally perpendicular to a lateral side surface of the candle filter  16  and also generally perpendicular to the direction of gravity G. In other embodiments where filters  16  are positioned within the vessel but arranged differently with respect to the vessel bottom, the flow can be generated in a direction otherwise not aligned with the direction of gravity, as would be the case with a bottom positioned filter with a purely horizontal surface). This flow will cause at least a portion of the dynamic filter media  30  to accumulate on the outer surfaces (lateral sides, but also possibly bottom if present) of candle filter  16  (see action arrows A for indicating the approximate direction of the flow transverse or opposite to the direction of gravity due to pressure differential). As a result, a cake K is formed that includes tortuous paths or tunnels which may act to block or filter the impurities or waste within the filtration vessel  14  as the filtrate is collected outside, but does not substantially block the pores of the underlying filtration media  16   a  of the candle filter  16 . 
     Extraction of filtrate to the collection vessel  20  (or alternatively the backflush source  22 , if provided with filtrate) may continue until the accumulated dynamic filter media  30  creates a layer on the candle filter  16  that eventually impedes flow to an undesirable level. This limit may be determined using a sensor, such as a flowmeter (see  FIGS. 3-13 ), in the associated conduit  17 . Using suitable valving and the source of pressurized gas  24 , the backflush liquid may be caused to flow through the outlet  14   b  and via candle filter  16  into filtration vessel  14 . As shown in  FIG. 2C , this backflush action causes the dynamic filter media  30  to be dispersed back into suspension within the solution of the filtration vessel  14  (note action arrows B) with some of it eventually settling at the bottom of the vessel  14 . Additional feed solution may be added from feed source  12 , and the process can be repeated as often as desired or necessary to optimally clarify the harvest solution and collect the filtrate. An optional added step may be to introduce buffer from source  13  into the filtration vessel  14  to wash the dynamic filter media  30  that settles to the bottom of the vessel  14 , which may be done to capture any target molecules hidden or trapped therein when the buffer is withdrawn through candle filter  16  using the same sequence. A buffer can be used to later wash the spent dynamic filter media  30  from the filtration vessel  14 , as outlined further in the following description. 
     In situations where the above process is completed (or, at any time at which the level of dynamic filter media in the vessel is such that the efficiency of the candle filter(s)  16  might be hampered, such as if the level of dynamic filter media reaches the lower extent of the filter in the illustrated embodiment), any remaining waste product or dynamic filter media  30  may be discharged. This may involve introducing liquid (buffer) from the source  13  to promote flowability, which forms a slurry similar to wet sand (which, as discussed below, may optionally be further compacted or squeezed to cause liquid to flow through conduit  17  to collection vessel  20 , and thereby enhance recovery of the target molecules of interest). The slurry may be discharged from the filtration vessel  14 , such as via the waste outlet or drain  14   c , and the clarifying process may be repeated by introducing a new batch of feed/cell culture harvest solution from the feed source  12 . 
     As can be appreciated, this arrangement allows the dynamic filter media  30  and/or candle filter(s)  16  to be repeatedly regenerated for further use, as desired, in a simple and efficient manner until the waste on the bottom of the vessel grows in height to a point where it impedes the functioning of the candle filter(s)  16 . In light of the ability to continuously reuse the candle filter  16  with the regenerated media  30 , the ratio of filter area to the volume of production can increase considerably, as compared to a horizontal filter fixed at the bottom of a vessel. Also, the dynamic filter media  30  may be discharged from the filtration vessel  14  when desired via outlet or drain  14   c . This permits the filtration vessel  14  to be designed and manufactured to a considerably smaller form factor (since being overwhelmed by the settled dynamic filter media  30  during a campaign is delayed). This smaller vessel will be much less expensive to produce and will facilitate a reduction of manufacturing footprint. Thus, the system  10  is readily adapted for use as an integral component of a limited space or “microfacility” for performing bioprocessing. 
     In the case where the filtration vessel  14  includes the inner liner or bag  18  in a rigid or semi-rigid outer container, a step may also be performed of causing the bag to collapse within the filtration vessel  14 . This may be achieved using gas from the gas source  24  to enter the area in the space between the bag  18  and the rigid or semi-rigid container C of the filtration vessel  14  via conduit  27 . This compression reduces the volume of the inner compartment of the bag  18 , and thus forces feed solution or buffer within the bag  18  to exit via the outlet  14   b , and also may compress or squeeze any dynamic filter media  30  present and not caked on the candle filter(s)  16 . This step may be implemented when at least some of the dynamic filter media  30  is accumulated on the candle filter  16 , as shown in  FIG. 2B , such as during the initial filtering step, or after the backflush step is completed, as outlined in the following description. 
       FIGS. 3-13  schematically illustrate the above-described system  10  and process in further detail, and  FIG. 14  is a corresponding flow chart. Noteworthy from  FIG. 3  is the presence of two candle filters  16  in the filtration vessel  14 . As noted above, only one filter can be used and even more than two filters can be utilized when more filtration capability is required or sought. 
       FIG. 4  illustrates a first step of filling the filtration vessel  14 , which in the illustrated embodiment includes the flexible liner or bag  18 . This may be achieved using a pump P to deliver the solution including a cell culture harvest and dynamic filter media from the upstream feed source  12  (10 L suspension bioreactor) by opening and closing corresponding valves V to establish fluid communication. A vent  14   e  associated with the filtration vessel  14  may be opened to allow for the liquid inflow. Pressure sensors S and flow meters F may be provided throughout the system  10  to regulate and monitor the flow of liquid throughout. 
     Once the filtration vessel  18  is provided with fluid (as shown in  FIG. 4 ), the next step in the illustrated embodiment may be to fill the backflush vessel  22 , if not already filled with liquid. The backflush vessel  22  may be partially or completely filled. This backflush vessel  22  may also include the liner or flexible bag  22   a , as shown in  FIG. 1B  and may be quite small compared to the filtration vessel  14 . Vent  14   e  may be closed during this step. 
     In one possible embodiment, this filling of the backflush vessel  22  may involve withdrawing liquid from the filtration vessel  14  itself, as indicated in  FIG. 5  (by using a pump, or using pressure, or any combination thereof), and may involve opening the vent  22   d  associated with the backflush vessel  22  (and bag  22   a  in particular). Thus, the backflush vessel  22  contains filtrate in this situation, which may include the target molecules, but not the dynamic filter media  30 . This embodiment provides the advantages of maintaining the same chemical environment rather than introducing a liquid that could affect the environment and require further adjustment. However, as noted below, buffer (e.g., from a prior cycle) may be provided to backflush vessel  22  instead of or in addition to the filtrate. After the backflush vessel  22  is sufficiently full from filtrate passing through the candle filters  16 , additional filtrate making its way through the candle filters  16  may then be withdrawn from the filtration vessel  14  and redirected through conduit  17  to the collection vessel  20 , as indicated in  FIG. 6 . In the course of this process, the cake (not shown) is thus formed on a surface of the filters  16 . 
     The bag  18  associated with the filtration vessel  14 , which includes the dynamic filter media  30 , may then be at least partially collapsed ( FIG. 7 ). This may be done using compressed air provided to conduit  27  via source (not shown). This squeezing of the bag  18  is done to force remaining filtrate to the collection vessel  20 . 
     When the evacuation of bag  18  is completed (which may be determined using flow meter F or visually), the backflush liquid may be delivered from vessel  22  ( FIG. 8 ) to the filtration vessel  14 , with vent  14   e  again opened to facilitate the inflow. The vessel  22  may be pressurized externally (e.g., squeezed by the application of external pressure in the case of a bag  22   a ), possibly using the same source of gas for causing the bag  18  to collapse (that is, provided via conduit  25  to the space between the vessel  22  and the liner or bag  22   a , which may be in communication with vent  22   e ), but alternatively a different source can be used, as could a pump instead to withdraw the liquid. This compression of the liner or bag  22   a  delivers the backflush liquid in reverse through the filters  16  into the filtration vessel  14 . This liquid flow serves to dislodge accumulated dynamic filter media  30  and waste from the filter surface, which is placed in solution again or permitted to settle at the bottom of the filtration vessel  14 . This process regenerates the candle filter and provides for reuse of the dynamic filter media. New feed with or without additional dynamic filter media may also be added to the bag  18 . 
     The bag  18  forming part of the filtration vessel  14  may then be caused to collapse again, as indicated in  FIG. 9 , which may be achieved by supplying fluid (air) via conduit  27  to the interior of vessel  14 , with vent  14   e  closed. This squeezes and compacts the bag  18 , and also any dynamic filter media  30  released from the filter surface (during the present cycle or a prior cycle), which at this stage is settled at the bottom with and has the consistency of wet sand. The resulting compaction and reduction in volume of the bag  18  forces the remaining “heel” of the vessel fluid through the filters  16  to be delivered to the collection vessel  20 , and thus enhances the recovery of the target molecule or product of interest. 
     The above-described steps create a single cycle, which may of course be repeated as necessary or desired (see  FIGS. 10-13 ). Specifically,  FIG. 11  illustrates the delivery of buffer from source  13  to the filtration vessel  14 , which may be done to wash the dynamic filter media  30  and then filter the liquid using the above-described squeezing technique, as indicated in  FIG. 12 . Buffer may also be delivered to the backflush vessel  22  for use during a subsequent cycle. Once the clarifying is completed, waste may be discharged from the bag  18  via the drain  14   c  (such as by opening a valve V), which may lead to a waste vessel  26  or drum, as indicated in  FIG. 13 . 
       FIG. 14  is a flow chart illustrating the above steps (some optional, such as heel filtration, second heel filtration, or buffer chase) for a method  100  of achieving clarification, but references an initial step  102  of providing a cell culture harvest solution. This may be done using an upstream bioreactor  12  as the feed source (or an intermediate vessel downstream of the bioreactor). The delivery rate may be arranged so as to correspond to the emptying of filtrate from the filtration vessel  14  and the release of the cake from the filter  16 . The steps as noted above may be performed, including: (1) step  104 , delivering feed to the filtration vessel  14 ; (2) step  106 , filtration into the backflush vessel  22 ; (3) step  108 , filtration from the filtration vessel  14  to the collection vessel  20 ; (4) step  110 , “heel” filtration (squeezing bag  18 ); (4) step  112 , backflushing to release the cake from candle filter(s)  16 ; and (5) step  114 , a second “heel” filtration. This cycle may then be repeated, as necessary or until the filtration vessel  14  accumulates sufficient dynamic media such that a buffer “chase,” step  116 , is introduced, and the “cake” (dynamic filter media) is discharged as waste, step  118 . This chart also indicates that the clarified filtrate of collection vessel  20  at step  108  may be delivered to a further downstream filtering process, step  120 , including further filtration and polishing, which may be used to recover the target molecules of interest from the filtrate. As can be understood, the above-described efficient and economical clarification process may reduce the need for further downstream filtration steps, and thus enhance the overall process from generation of the cell culture harvest solution to recovery of a final product of interest. 
     The system  10  and method described may be provided with one or more single use components. For instance, the filtration vessel  14  may be made disposable (or, alternatively, just the liner or bag  18 ), and the same can be done for the backflush vessel  22  (including for bag  22   a ). As can be appreciated, the use of disposable components, and bags  18 ,  22   a  in particular, reduces the operating costs, and avoids the need for cleaning and associated validation. The vessels  14  and  22  may also be made to operate under sterile conditions, and the ability to regenerate the dynamic filter media  30  through multiple cycles allows for a continuous process to be realized that maintains sterility (as compared to the need to change or clean a filter during each clarification cycle for a typical cell harvest solution). 
     Example 
     As one example of a filtration vessel  14 :
         Internal diameter 11 cm;   Outside diameter 12 cm;   Working volume height: 19 cm;   Overall height including the support: 24 cm;       

     As one example of a back-flush vessel  22 :
         ID=11 cm;   OD=12 cm;   Working volume height=15 cm;   Overall height=16 cm.       

     This arrangement is sufficient to clarify 8 to 12 L of harvest without performing the cake discharge. 
     With cake discharge the same filtration vessel  14  would allow to clarify 100 L with 10 cycles. This system integrates intensification technologies, thereby drastically reducing the size of each compartment and hence creating a low footprint production and purification system. The production and purification of the biomolecule can be performed as a continuous and automated process based on this system: from cell culture to final product purification minimizing human intervention. The process intensification and integration enable the containment of all compartments into an isolator ensuring the safety of process operators and the environment. The system has a small footprint. In some embodiments, the footprint of the system is less than about 50 m2, 40 m2, 30 m2, 20 m2, 10 m2, 5 m2, or less. In some embodiments, the footprint of the system is from about 5 m2 to 10 m2, 5 m2 to 20 m2, 5 to 30 m2, 5 to 40 m2 5 to 50 m2. In an example, the footprint is less than 10 m2. 
     As used herein, the following terms have the following meanings: 
     “A”, “an”, and “the” as used herein refers to both singular and plural unless the context clearly dictates otherwise. By way of example, “a compartment” refers to one or more than one compartment. 
     “About” as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/−20% or less, preferably +/−10% or less, more preferably +/−5% or less, even more preferably +/−1% or less, and still more preferably +/−0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier “about” refers is itself also specifically disclosed. 
     “Comprise,” “comprising,” and “comprises” and “comprised of” as used herein are synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open-ended terms that specifies the presence of what follows, e.g. component, and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. 
     The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. 
     The terms “Cell culture harvest”, “culture harvest” and “harvest” are used herein as synonyms and refer to the unclarified cell culture obtained at the end of culturing cells in a bioreactor. The cultured cells or the grown cells also are referred to as host cells. 
     The term “bioreactor” as used herein refers to any device or system that supports a biologically active environment, for example for cultivation of cells or organisms for production of a biological product. This would include cell stacks, roller bottles, shakes, flasks, stirred tank suspension bioreactors, high cell density fixed bed perfusion bioreactors, etc. 
     The diatomaceous earth used in the method or system according to the disclosure can be of various grades, wherein the grade gives an indication of the size of the pores present in the diatomaceous earth. The grade of diatomaceous earth used in a method or system according to the disclosure depends on the morphology, particularly when used in cell culture purification, the size and the shape, of the cells from which a clarified cell culture is to be obtained. For example, for CHO cell cultures, Celpure 300® grade or Celpure 100® grade can be used. For CHO cell cultures grown in an adherent environment such as with a high cell density fixed bed bioreactor, Cellpure 100® grade or Celpure 65® grade can be used. The smaller the floccules, the finer the grade of DE needed, in general. 
     The term “filtration” or “separation” refers to the removal of the aqueous phase, containing the soluble molecules of interest, from insoluble particles. 
     The term “target molecule” refers to an organic molecule in a living organism, having characteristics typical of molecules found in or secreted by living organisms including individual cells and that may be naturally occurring or may be artificial (not found in nature and not identical to a molecule found in nature). Example target biomolecules include but are not limited to proteins, peptides, amino acids, glycoproteins, nucleic acids, nucleotides, nucleosides, oligonucleotides, sugars, oligosaccharides, lipids, hormones, proteoglycans, carbohydrates, polypeptides, polynucleotides, polysaccharides. 
     The foregoing has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the embodiments to the precise form disclosed. Obvious modifications and variations are possible in light of the above teachings. All such modifications and variations are within the scope of the appended claims when interpreted in accordance with the breadth to which they are fairly, legally and equitably entitled.