Continuous filtration by means of feed cycling

Abstract

Processes and systems for continuous filtration of a liquid feed are provided where amounts of liquid feed sample are routed from two or more sources to a cycling tank, either in series or simultaneously, prior the liquid feed sample being routed to a filtration system, until a total product volume has been processed.

Description

CONTINUOUS FILTRATION BY MEANS OF FEED CYCLINGBACKGROUND

[0001] Filtration typically involves the removal of impurities and other low molecular weight species from a bioproduction sample in which a target molecule, such as a protein, peptide, nucleic acid, or antibody, resides. Other uses for filtration include purification where the product can reside in either the retentate or the permeate (or both). While filtration can be performed at several stages throughout a bioproduction process, it is frequently performed as a preliminary step to remove impurities such as host cell proteins (HCP) and nucleic acids (NA) or as a final step to purify and sterilize a final product pool containing the target molecule.

[0002] Filtration is typically performed in “batch” mode, that being, one batch of a production run being filtered after which the filtration system is flushed or cleaned in preparation for the next batch. Filtration may also be performed as a batch recirculation operation, in which a sample volume undergoes several iterations of passing through a filtration assembly, with a retentate stream from the filtration assembly being recirculated back to a feed tank and, if necessary or desired, fresh buffer solution being introduced to the feed tank to replace any volume lost during filtration. The repeated filtration of the sample continues until final processing conditions are met, at which point the product is recovered from the system. Alternatively, a sample volume can travel through a series of single-pass filtration assemblies ( / .e., in-line filtration), with buffer solution being introduced between each stage of filtration, if necessary or desired. By performing filtration with a series of single-pass filtration stages, the inflow and outflow of the sample through the filtration system can be continuous. However, since the process is typically stopped for maintenance after each production “batch,” the entire process is a batch process.SUMMARY

[0003] Filtration methods and systems are provided in which a continuous inflow and outflow of a bioproduction sample (alternatively referred to as a“liquid feed sample,” “feed sample” or “product”) can be achieved with use of batch filtration processes and assemblies. Such methods and systems can provide for continuous production, while requiring less membrane area and / or buffer volume than that typically required for in-line filtration. Such methods also generally use a smaller footprint so valuable floor space is also saved.

[0004] In one aspect, the invention relates to a process for the continuous filtering of a first amount of a liquid feed sample from, a sample source, a continuous sample source or continuous feed stream, to a cycling tank (also may be referred to as a feed tank). The feed stream is then routed to a filter system, typically a tangential flow filtration (TFF) system. During the filtration of the first amount of liquid feed sample, one or more additional amounts of liquid feed sample are routed to the cycling tank either in series or in parallel ( / .e., simultaneously) to maintain a level of liquid feed sample in the cycling tank to ensure a continuous feed stream for the filter system. This process may continue until a total product volume is processed through the filter system.

[0005] In another aspect, the additional amounts of liquid feed sample may come from multiple sample sources, for example from several cell culture apparatus.

[0006] In another aspect, the filter system is a TFF system and, in certain instances, the filter system is a single-pass tangential flow filtration (SPTFF) system.

[0007] In certain aspects, the liquid feed sample may be concentrated or diluted as a result of the filtration process. In other aspects, the feed stream may be concentrated or diluted prior to or after the filtration process.

[0008] The feed stream is filtered as a result of the filtration process with the target molecule or molecules, e.g., monoclonal antibodies, becoming a larger percentage of the non-aqueous constituents of the filtered liquid when compared to the unfiltered liquid feed sample. In some aspects, the concentration of the target molecule or molecules is increased by at least 5%, 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 1000% or more, as compared to the unfiltered liquid feed sample. Likewise, in some aspects, theconcentration of one or more impurities, e.g., host cell proteins (HCP) and / or nucleic acids (NA), are reduced when compared to the unfiltered liquid feed sample. In some aspects, the concentration of the one or more impurities is reduced by at least 5%, 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, as compared to the unfiltered liquid feed sample. In a preferred aspect, the target molecule(s) is / are concentrated and the one or more impurities are reduced in concentration as a result of the filtration process of the present invention when compared to the unfiltered liquid feed sample.

[0009] The filtration process of the present invention is applicable to any liquid feed sample so long as the filters are compatible with the liquid feed sample. One of ordinary skill in the art can identify an appropriate filter type (e.g., size porosity, membrane material, etc.) for a specific liquid feed sample to be filtered. In one aspect, the liquid feed sample is a cell culture media from a cell culture vessel, wherein the cell culture housed in the cell culture vessel is engineered to produce a biological molecule with therapeutic qualities and / or is suitable for use in research and development in the fields of biotechnology and medicine.

[0010] Exemplary biological molecules that may be produced by the cell cultures that produce the liquid feed sample for the filtration process of the present invention include, but are not limited to, are monoclonal antibodies as well as other proteins and glycoproteins with known or potential therapeutic properties.

[0011] In one aspect, the present invention provides a process for filtering a liquid feed sample, comprising: (a) routing a first amount of a liquid feed sample from a first sample source to a cycling tank; (b) simultaneously or in series with the first amount of liquid feed sample, routing a second amount of liquid feed sample from a second sample source to the cycling tank; (c) upon collection of a suitable quantity of liquid feed sample in the cycling tank, routing a continuous feed stream of the liquid feed sample from the cycling tank to a tangential flow filtration (TFF) system to filter the continuous feed stream of the liquid feed sample; (d) during the filtration of the liquid feed sample, routing one or more additional amounts of liquid feed sample to thecycling tank to maintain a continuous feed stream of liquid feed sample to the TFF system; (e) continuing to provide liquid feed sample to the cycling tank until a total product volume is processed through the TFF system.

[0012] In another aspect, the present invention provides a process for filtering a liquid feed sample, comprising: (a) routing a first amount of a liquid feed sample from a first sample source to a cycling tank; (b) upon collection of a suitable quantity of liquid feed sample in the cycling tank, routing a continuous feed stream of the liquid feed sample from the cycling tank to a tangential flow filtration (TFF) system to filter the continuous feed stream of the liquid feed sample; (c) during the filtration of the liquid feed sample, routing one or more additional amounts of liquid feed sample to the cycling tank to maintain a continuous feed stream of liquid feed sample to the TFF system; (d) continuing to provide liquid feed sample to the cycling tank until a total product volume is processed through the TFF system.

[0013] In yet another aspect, the present invention provides that the amounts of liquid feed sample come from one or more different sample sources.

[0014] In yet another aspect, the present invention provides that the TFF system is a Single-Pass Tangential Flow Filtration (SPTFF) system.

[0015] In yet another aspect, the present invention provides that the liquid feed sample is concentrated as a result of the filtration.

[0016] In yet another aspect, the present invention provides that the liquid feed sample is concentrated prior to being delivered to the cycling tank.

[0017] In yet another aspect, the present invention provides that the liquid feed sample is a cell culture media.

[0018] In yet another aspect, the present invention provides that the liquid feed sample comprises monoclonal antibodies.

[0019] In yet another aspect, the present invention provides that the liquid feed sample is diluted prior to being delivered to the cycling tank.

[0020] In yet another aspect, the present invention provides that the filtration is diafiltration and wherein the process additionally comprises a diafiltration buffer tank being in fluid communication with the feed stream.

[0021] In yet another aspect, the present invention provides that the process further comprises a holding tank between the cycling tank and the TFF system.In yet another aspect, the present invention provides that the process further comprises flushing the TFF system after a total product volume has been processed.

[0022] In some embodiments, the continuous filtration process includes a tangential flow filtration (TFF) step. The continuous filtration process can be, for example, a constant volume filtration process or an optimum volume filtration process.

[0023] In further embodiments, the liquid feed sample can be concentrated prior to being delivered to the cycling tank(s), such as by tangential flow filtration (TFF) process or a single-pass tangential flow filtration (SPTFF) process. The process can optionally further include alternately routing the retentate from the first and second amounts of liquid feed sample to a holding tank prior to delivering the retentate to a subsequent filtration assembly. The retentate produced by filtration of each amount can alternately be filtered by the filtration assembly, such as by single-pass tangential flow filtration (SPTFF), until a total product volume is recovered. The process can further include cleaning cycling tank after the end of the process. The filtration assembly can be cleaned and / or flushed after the completion of the process.

[0024] In some embodiments, the filtration assembly comprises a tangential flow filtration (TFF) assembly. The system can further include a single-pass tangential flow filtration (SPTFF) assembly upstream of the cycling tank and / or downstream of the cycling tanks to concentrate a sample prior to and / or following filtration. The system can also include a holding tank or means for containing a surge volume upstream or downstream of the cycling tanks.

[0025] In another embodiment, the system comprises a diafiltration buffer tank. A diafiltration pump can be in fluid communication with the diafiltration buffer tank and the cycling tank(s), or with a fluid conduit configured to direct retentate to the cycling tank(s). The system can further comprise multiple diafiltration buffer tanks which can have the same or different buffer solutions.

[0026] In yet a further embodiment, the fluid contacting flow paths of the system are sterilized or sanitized to create a closed system and comprise sterilized / sanitized assemblies which have been sterilized / sanitized using gamma irradiation, ETO, NaOH or other method, with aseptic connection using tube welding or aseptic connectors, and using sterile filters on the inlet and outlet lines to the continuous diafiltration system for bioburden control.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0028] FIG 1 is a schematic diagram of a continuous ultrafiltration system of the invention that is comprised of minimal components while still achieving a continuous filtration process.

[0029] FIG. 2 is a graph showing the difference in total membrane area (m2) requirements between the system and methods of the present invention and the prior art discontinuous filtration method (batch mode).

[0030] FIG. 3 is a graph showing the difference in total membrane loading (kg / m2) requirements between the system and methods of the present invention and the prior art discontinuous filtration method(batch mode).

[0031] FIG. 4 is a graph showing the difference in total tank volume (L) requirements between the system and methods of the present invention and the prior art discontinuous filtration method (batch mode).

[0032] FIG. 5 is table showing a comparison of various parameter requirements between TFF batch mode and continuous ultrafiltration (UF / DF).

[0033] FIG. 6 is a table showing reagent consumption comparison (NaOH, WFI (water for injection) and Buffer) between TFF batch mode and continuous ultrafiltration (UF / DF).DETAILED DESCRIPTIONDefinitions

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] As used herein, the singular forms “a,” “an,” and “the” include plural unless the context clearly dictates otherwise.

[0036] “Continuous filtration process” or “CFP” is used herein to refer to a process by which feed streams from more than one source are combined in series or in parallel prior to being directed to a filter assembly. A CFP may also be referred to as a “continuous filtration system” or “filtration system.”

[0037] “Continuous filtration assembly” or “CFA” is used herein to refer to a filtration assembly configured for use in a continuous process and containing one or more filtration modules (e.g., a TFF assembly).

[0038] “Continuous diafiltration process” or “CDF process” is used herein to refer to a process by which buffer exchange of a liquid feed sample occurs by adding buffer and removing permeate with minimally uninterrupted inflow and outflow to and from a diafiltration assembly.

[0039] “Continuous diafiltration assembly” or “CDF assembly” is used herein to refer to a filtration assembly configured for use in a continuous diafiltration process and containing one or more filtration modules (e.g., a TFF assembly). A “filtration assembly” refers to an assembly comprising one or more filtration units or filtration modules, for example (including but not limited to), a cassette filter, a spiral-wound filter, a capsule filter, or a hollow-fiber filter. A filtration assembly may include one or more filtration units or filtration modules operating in series or in parallel. Series operation refers to modules where the retentate of the previous module becomes the feed for the subsequent module.

[0040] “TFF assembly” and “TFF system” are used interchangeably herein to refer to a tangential flow filtration assembly in which fluid travels tangentially along a surface of a filtration membrane in a filtration module of the system.

[0041] “SPTFF assembly” and “SPTFF system” are used interchangeably herein to refer to a single pass tangential flow filtration system that is configured for operation in a single-pass mode, where the fluid passes once through the system.

[0042] A “cassette” refers to a cartridge or flat plate filter element comprising stacked filtration (e.g., ultrafiltration or microfiltration) membrane sheet(s).

[0043] A “spiral-wound filter element” refers to a filter element comprising filtration membrane sheet(s) and, optionally, separator material wound around a central core. A spiral-wound filter element is typically housed in a capsule.

[0044] A “capsule” refers to a filtration element comprising a matrix of filtration membrane sheet(s) or filtration material within a holder-less cartridge.

[0045] A “hollow-fiber filter element” refers to a filter element comprising a bundle of filtration membrane tubes.

[0046] “Filtration membrane” refers to a selectively permeable membrane for separating a feed into a permeate stream and a retentate stream in a filtration process (e.g., a TFF process, a diafiltration process). Filtration membranes include for example, but are not limited to, ultrafiltration (UF) membranes, microfiltration (MF) membranes, reverse osmosis (RO) membranes and nanofiltration (NF) membranes.

[0047] The terms “ultrafiltration membrane” and “UF membrane” are generally defined as a membrane that has pore sizes in the range of between about 1 nanometer to about 100 nanometers, or alternately defined by the "molecular weight cut off" of the membranes, expressed in units of Daltons, and abbreviated as MWCO. In various embodiments, the present invention utilizes ultrafiltration membranes having MWCO ratings in the range from about 1 ,000 Daltons to a 1 ,000,000 Daltons.

[0048] The term “microfiltration membranes” and “MF membranes” are used herein to refer to membranes that have pore sizes in the range between about 0.1 micrometers to about 10 micrometers.

[0049] The terms “liquid feed sample,” “feed,” “feed sample,” “liquid feed stream” or “liquid stream” refer to the solution that is delivered (e.g., continuously or as a batch) to a filtration module to be filtered. The feed thatis delivered to a filtration module for filtration can be, for example, feed from a feed container (e.g., vessel, tank) external or internal to the system, or retentate flowing in a tube from a preceding filtration module upstream of the diafiltration system.

[0050] Further, the terms “liquid feed sample,” “feed” or “feed sample,” “liquid feed stream” or “liquid stream” are interchangeable and refer to a liquid feed sample or feed fluidly moving from one vessel, device, etc., to another vessel, device, etc. Such movement is usually the result of pumping (e.g., by use of a peristaltic pump or other type of pump generally known to one of skill in the art) but may be by other means such as gravity.

[0051] The term “amount” and “amounts,” as used herein, refer to a nondefined amount of a sample (for example, of a liquid feed sample) as measured, e.g., by volume or weight. The term(s) are not limited to any specific volume(s) or weight(s), etc. The terms also are not limited to any specific units of measurement. The term(s) may refer to a portion of an identified amount or an entirety of an identified amount, i.e., a portion or an entirety of a liquid feed sample.

[0052] “Product” refers to a target compound in a feed sample. Typically, a product will be a biomolecule (e.g., protein) of interest, such as a monoclonal antibody (mAb). It can reside in the retentate or permeate or both.

[0053] The term “total product volume” refers to a non-defined volume equal to one production run, wherein a “production run” is defined as a total quantity or volume processed contiguously or continuously. The total product volume is typically limited to the total volume available for a production run. It is non-defined because various product runs may have differing volumes depending on, for example, the product being produced (e.g., some products may be in higher demand and necessitate a larger production run), availability of raw materials (e.g., a limited supply of a specific media may result in a smaller production run) or the capacity of the system used (e.g., a smaller processing system may be limited in total volume that can be processed continuously or contiguously as compared to a larger system).

[0054] The term “filtration” generally refers to the act of separating the feed sample into two streams, a permeate and a retentate, using membranes.

[0055] The terms “permeate” and “filtrate” refer to that portion of the feed that has permeated or has been filtered through the membrane. In other words, the portion of the feed stream that has traversed the membrane. The terms may be used interchangeably.

[0056] The term “retentate” refers to the portion of the solution that has been retained by the membrane, and the retentate may be enriched in a retained species.

[0057] “Feed line” or “feed channel” refers to a conduit for conveying a feed from a feed source (e.g., a feed container) or “sample source” to one or more processing units in a filtration assembly. The feed stream flows through the feed line or feed channel.

[0058] “Retentate line” or “retentate channel” refers to a conduit in a filtration assembly for carrying retentate.

[0059] “Permeate line” or “permeate channel” refers to a conduit in a filtration assembly for carrying permeate.

[0060] The term “plurality,” when used herein to describe processing units, refers to two or more processing units (e.g., two or more filtration units).

[0061] “Fluidly connected” refers to two or more components of a filtration assembly that are connected by one or more conduits (e.g., a feed channel, a retentate channel, a permeate channel) such that a liquid can flow from one component to the other.

[0062] “Processing” refers to the act of filtering (e.g., by TFF) a feed containing a product of interest and subsequently recovering the product in a concentrated or purified form.

[0063] “Purified” refers to a product made to have a higher concentration of desired constituents or product of interest and a lower concentration of contaminants as compared to the unfiltered sample.

[0064] A “cycle tank” or “cycling tank” is used herein to refer to a vessel configured to store a fluid and which can be single-use or multi-use tank composed of, for example, plastic, glass, or metal. It can also be capable of receiving or including an internal mixer to mix a held fluid, including, for example, conventional batch tanks, bags, and single-use bags. The cycle tank or cycling tank can also be configured integrally into a feed line or feedchannel such that it can capture a surge volume of a feed and need not be a separate and distinct component. It is preferred for the tanks described here to demonstrate good mixing in order to achieve performance efficiencies.This can be accomplished for example, by the use of an internal mixer and / or by the design of the tank and retentate flow inlet to assist mixing. In a diafiltration process, diafiltrate may be added directly to the tank but it is preferred to add it to the retentate line before the combined fluids are introduced to the tank. This may facilitate mixing.

[0065] A “pump” is used herein to refer to a method or device configured to impart flow such as inducing gravity flow, applying a pressure differential to a fluid, including, for example a magnetic levitation pump, a peristaltic pump, or a diaphragm pump.

[0066] “Trans-membrane pressure drop” is the module average pressure drop across a filtration membrane.

[0067] “Cross flow” is the retentate flow rate between an inlet and an outlet of a filtration module. Unless otherwise stated, “cross flow” refers to an average cross flow.

[0068] “Permeate flux” refers to an area normalized flow rate of permeate in a permeate channel (e.g., Liters / m2 / hr, LMH) of a filtration module.

[0069] “Cross flow flux” refers to an area normalized average flow rate of retentate in a feed channel (e.g., Liters / m2 / min, LMM) of a filtration module.

[0070] “Valves” refers to methods or assemblies that stop or regulate fluid flow and / or redirect fluid flow through system conduits. This may include physically moving tubes between tanks in open systems, “pinching” tubes, on- off valves, control valves and multiport valves.

[0071] A description of example embodiments follows.Filtration Systems and Methods for Continuous Production

[0072] In the biopharmaceutical industry, filtration is traditionally performed as a batch process in which a sample undergoes several iterations of filtration, as with a TFF system, or one pass, as with a SPTFF system.Traditional batch filtration is an inherently discontinuous production process, as inflow and outflow of the product to and from the filtration system haltswhile the sample undergoes the several iterations of filtration or the process is stopped between batches for cleaning and / or sterilization. As the biopharmaceutical industry moves towards continuous processing for several of its unit operations (e.g., perfusion production and harvest, flow-through chromatography, single-pass concentration), there is a need for filtration methods and systems that are capable of providing continuous inflow and outflow of a product. While in-line filtration can provide for continuous inflow and outflow of a product, such systems require undesirably large membrane areas, multiple storage tanks and buffer volumes and become very difficult to operate in a cGMP environment to balance multiple flows. There is a need for filtration systems and methods that can perform filtration in a continuous production process that do not require undesirably large membrane areas, multiple storage tanks and buffer volumes and are easy-to-operate.

[0073] Conventional batch filtration is typically run for only a few hours to minimize protein degradation through multiple pump passes, manage bioburden, and balance the batch production line to time allow for pre- and post-processing operating steps. The use of cycling allows for continuous operation to the balance production line, longer utilization of membrane for reduced area requirements, and enables compact systems with sterile or sanitized components to manage bioburden, without adding significant complexity.

[0074] In methods and systems of the present invention, filtration can be performed by cycling between feed samples provided by two or more sample sources or feed sources. As such, multiple amounts of an overall volume are provided for filtration resulting in continuous inflow and outflow of a product to and from the filtration system. Such filtration systems can be placed between any process or unit operation that exists either upstream or downstream: for example, between SPTFF concentration, chromatography, clarification and / or virus filtration unit operations. Such multiple filtration systems can be run in series or in parallel and can include bound products such as chromatography beads, cell substrates, or adsorbing polymers where products are sequentially adsorbed, washed, eluted, and regenerated in a series of successive steps done using membranes to facilitate the separation and retention, if desired.

[0075] An example of a filtration system of the present invention 100 is represented in FIG. 1 . The system may comprise either a TFF system 150a, an SPFTT system 150b or both in series. An exemplary cycling tank 110 is represented. One or more sample sources (not shown) feed into the cycling tank. An optional holding tank 120 is represented. The feed is pumped from the cycling tank to the optional holding tank via pump 160a and / or from the cycling tank or holding tank to the TFF filtration system via pump 160b. A TFF filter 130 is also represented (RET = retentate; FEED = feed from either the holding tank or directly from the cycling tank; PER = permeate). In some embodiments, optionally, a second cycling tank may be employed in the event, for example, that the liquid feed sample is of large quantity or the first cycling tank needs cleaning or servicing. The operator would then switch between cycling tanks as necessary or desired.

[0076] In one embodiment, the permeate and / or retentate is collected from the TFF filter until the continuous filtration is completed. In the TFF system, at least a portion of the retentate may be recirculated into the holding tank or feed line as necessary to ensure the desired level of filtration. Continuous filtration is completed when the feed stream is exhausted ( / .e., total product volume is processed). Optionally, a buffer flush may be performed with suitable buffer coming from a buffer tank 180a. The choice of buffer will depend on the product being processed and can be determined by one of ordinary skill in the art. Non-limiting examples are; phosphate buffered saline (PBS), Dulbecco’s phosphate buffered saline (DPBS), Hank’s balanced saline solution (HBSS), HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) and other suitable buffers known to one of ordinary skill in the art. In another embodiment, diafiltration may optionally be performed with suitable buffer (DF) coming from a diafiltration buffer tank 170. The choice of diafiltration buffer will depend on the product being processed and can be determined by one of ordinary skill in the art.

[0077] In other embodiments, the filtration process may be a SPTFF filtration process. Feed may be pumped (via pump 160c) directly to the SPTFF system 150b from, for example, cycling tank 110 or pumped to a holding tank 140 prior to the SPTFF system. In another embodiment, the feedmay be the retentate from the TFF system 150a. In one scenario, the TFF system is used for diafiltration and the SPTFF system for filtration. To reiterate, feed may be pumped directly to the SPTFF system from the cycling tank 110 avoiding the TFF system or disposing of the TFF system altogether.

[0078] As with the TFF system, the permeate and / or retentate can be collected from the SPTFF system. Continuous filtration is completed when the feed stream is exhausted ( / .e., total product volume is processed). Optionally, a buffer flush may be performed with suitable buffer coming from a buffer tank 180b. The choice of buffer will depend on the product being processed and can be determined by one of ordinary skill in the art.

[0079] For clarity, the present invention may be operated as a TFF system, an SPTFF system or a TFF system followed in series by an SPTFF system. For further clarity, the system may be operated as filtration system or a diafiltration system. In another aspect, if both TFF and SPFTT systems are used in series, the TFF system may be operated as a diafiltration system and the SPFTT system as a filtration system. In yet another aspect, both the TFF and SPTFF systems, alone or together, may be operated first as a diafiltration system followed by being operated as a filtration system or operated as a filtration system followed by being operated as a diafiltration system.

[0080] Although not shown, appropriate valving may be used to control flow rates and direct flow of the feed stream, retentate, permeate and any pre- and post-operation fluids (e.g., flush buffers, cleaning solutions, etc.; see, below). Flow rates can be controlled with valving ( / .e., toggling the valve(s) fully or partially) in addition to or separately from control via the various pumps. Valving may be used to control flows from diafiltration tanks, buffer tanks, feed streams, cycling tanks, etc. Valving (and pumps) may be controlled manually or by automated systems. Suitable automated systems are known to one of ordinary skill in the art.

[0081] Further, one or more of feed flow rate, retentate flow rate or permeate flow rate may be controlled. Control of the various flow rates of part or of all of the entire system may be via manual control or may be automated. Automation may be by computer-controlled systems or other means such as mechanically controlled timers. Control of one or more of the feed, retentateand / or flow rate may be used to ensure the appropriate transmembrane pressure (TMP) is maintained for the filtration scenario being performed and is dependent upon filter type, feed material and desired process result.

[0082] Pre-filtration and pre-diafiltration preparation and post-filtration and post-diafiltration cleaning components can optionally be included in the system. For example, with regard to pre-diafiltration preparation, containers for storing water (e.g., reverse osmosis deionized (RODI) water) and regeneration solutions (e.g., detergents, enzymes, acids, bases, etc.) are in fluid communication with a first buffer / regeneration valve, which selectively directs the flow of buffer and regeneration solutions to the continuous filtration assembly. Second and third cleaning valves can also be included to direct flow of cleaning solutions to and from the continuous filtration assembly, as necessary.

[0083] In one embodiment, systems of the present invention encompass continuous filtration assemblies that are tangential flow filtration (TFF) assemblies. TFF assemblies can include cassettes, spiral-wound filter elements, capsules, or hollow-fiber filter elements. TFF provides an efficient method for performing filtration while also enabling concentration of a sample in a same unit operation, if desired. As such, TFF is advantageous for use in filtration processes.

[0084] In another embodiment, systems of the present invention include additional TFF assemblies located upstream and / or downstream of the filtration assembly to, for example, concentrate a sample prior to / following filtration or exchange buffers through diafiltration. Such TFF assemblies can be single-pass TFF assemblies (SPTFF), if desired.

[0085] In embodiments, filtration systems of the present invention include standard bioproduction equipment, such as conventional batch tanks, pumps, and control valves. The systems further include switching valves and / or multiport valves in fluid communication with the cycling tanks and filtration assembly to allow a main product flow to be cycled between filling, filtration, and product recovery operations while the filtration system (e.g., a TFF system) is operated continuously.

[0086] Systems and methods of the present invention advantageously allow smaller amounts of product to undergo filtration, while providing for continuous production. By filtering smaller amounts, process time, and hence number of pump passes, can be shorter than typical batch filtration processes. It is desirable to limit the number of pump passes of the sample as biomolecules can be damaged while travelling through pumps and filtration equipment, which can result in a lower product yield and lower product quality. Additionally, such systems and methods can be used in-line with other processing operations, while also meeting high-buffer exchange requirements, if needed, for bioprocessing operations. By breaking up a sample into multiple aliquots, smaller cycle tanks and filtration assemblies may be used, providing for smaller system footprints and smaller cycle times. Furthermore, by sequentially running multiple cycles of product through a continuously operating filtration assembly, idle time between batches, as occurs in traditional batch filtration, is eliminated. With continuous operation of a filtration system, protein loading per membrane area can be increased, such that discarding the membrane at the end of the process, and consequently eliminating cleaning time, cleaning solutions, and re-use and revalidation efforts, becomes more economically viable, particularly for large operations.

[0087] In embodiments, adequate time remains at the end of each filtration cycle to provide for buffer flushing to recover higher amounts of product. Buffer flushing can be performed by, for example, introducing a buffer solution, air or gravity drain to push additional product located at or in the membrane of the filtration assembly or in fluid conduits / piping, back to a current cycle tank (also referred to as back-flushing or flushing). The contents of the cycle tank are then provided as feed for a subsequent unit operation. Alternatively, a sample can first be pumped out of a cycle tank and then chased by a buffer solution into a separate product collection tank, the contents of which are subsequently provided as feed for the next unit operation.

[0088] In further embodiments, adequate time remains at the end of each filtration cycle to provide for a rapid regeneration cycle to be performed on themembrane, which can assist with maintaining constant filtration fluxes over extended cycling operations. Rapid regeneration cycles may not need to be performed following each filtration cycle but can be invoked periodically or on an as-needed basis.

[0089] In yet further embodiments, following all filtration cycles, the system undergoes a full or partial clean-in-place (CIP) procedure. Alternatively, or in addition, all or some of the fluid conduits and system components are replaced. For example, TFF filtration assemblies that include single-use modules may have such modules removed and discarded, or cycling tanks that include single-use bags may have such bags discarded.

[0090] Control methods for filtration can be effected by various control strategies. In one embodiment, continuous filtration processes are controlled through monitoring and adjustment of TFF membrane crossflow and transmembrane pressure (TMP). TFF membrane crossflow can be controlled by adjusting a feed flow rate, a retentate flow rate, an average crossflow rate, and / or a pressure drop setpoint for a feed pump. TMP can be controlled by a retentate pressure control valve or by an overlay pressure applied to the cycle tank. During filtration, a recirculating product volume can be controlled to either a constant set point (e.g., constant-volume filtration) or to a variable set point based on an algorithm that optimizes a concentration of the product and / or buffer species in the cycle tank (e.g., optimum filtration). Volume control can be monitored by a level probe or by load cells associated with the cycle tanks. Alternatively, flowmeters, totalizers, and / or weight scales can be used to measure a buffer addition flow rate and a permeate removal flow rate. Such measurements can be provided to a controller, which can monitor the filtration process and effect adjustments to crossflow and TMP, such as by toggling valves or adjusting pump speeds.

[0091] Endpoint control for filtration can also be effected by various endpoint control strategies. In one embodiment, a filtration endpoint can be based on a totalized volume of feed added and / or a totalized volume of permeate removed, as measured by flow totalizers or weight scales located at or near fluid conduits and tanks of the system, or time-based measurements. In another embodiment, a filtration endpoint can be triggered based upon ameasurable quality of the permeate or retentate stream that indicates product processing has occurred. For example, a filtration endpoint can be a solution adjustment such as conductivity reduction where the filtration is run to reduce the conductivity prior to a next step such as TFF or an ion exchange chromatography process. This is especially true if the filtration process is a diafiltration process. Examples of measurable qualities include pH, conductivity, refractive index, UV, turbidity, particle-size measurement, or near-real-time direct measurement of an excipient, impurity, or target product concentration (e.g., as determined by high-performance liquid chromatography (HPLC) or other analytical equipment) at-line or inline.

[0092] Product recovery can also be effected by various recovery strategies. In one embodiment, flow rate and / or pump speed setpoints are monitored for a feed pump and / or filtration pump. In a further embodiment, a tank recovery endpoint is monitored and controlled based upon measured values obtained from level, weight, and / or air sensors, a measured volume totalization, a timed drain duration, or other means of sensing that a tank is empty.

[0093] Lastly, buffer flush recovery can be controlled by various recovery strategies. In some embodiments, buffer flushing is monitored and controlled based upon a buffer pump flow rate, speed, and / or discharge pressure. A buffer flush endpoint can be triggered by a flush duration, totalized volume, recovery vessel of buffer vessel weight, an air sensor, or other measurable quality of the protein / buffer solution, such as an ultraviolet (UV) signal.

[0094] In some embodiments, the continuous filtration systems and methods of the present invention can also optionally provide for filtration of relatively low molecular-weight species from other sample components. Examples of filter-permeable components from retained components include salts from proteins, proteins from nucleic acids, protein products from cells or flocs, viruses and host cell proteins from cells, refolding reagents from proteins, alcohol from proteins, unreacted polyethylene glycol (PEG) from PEGylated proteins, unreacted toxins from antibody drug conjugates (ADCs,) and unreacted carbohydrates from conjugate vaccines.

[0095] Systems and methods of the present invention can include filtration by microfiltration, ultrafiltration, reverse osmosis, or nanofiltration, depending upon the molecules to be separated. Particular examples of suitable filtration membranes include Biomax®-30kD membranes and Ultracel®-30kD membranes (EMD Millipore, Bedford, MA) or other suitable membrane size cutoffs to retain the components of interest.

[0096] Examples of suitable buffer solutions for use in systems and methods of the present invention, when needed, include water, purified waters, phosphate buffered saline (PBS), acetates, and histidines.EXEMPLIFICATIONExample 1 : Reduction of Membrane Usage and System Size

[0097] The feasibility of continuous filtration was estimated in a study where 2 different processes and 12 different series were tested (several feed and diafiltration concentrations). Process 1 utilized the cUFDF (continuous ultrafiltration / diafiltration) process of the present invention and the second process was a control scenario using a standard batch process ( / .e., prior art process). Parameters tested were: 1) Total Membrane Area (m2); 2) Total Membrane Loading (kg / m2); and 3) Total Tank Volume (L). The series concentrations were as in Tablel .Table 1 - The 12 Series had different inlet and diafiltration concentrations: (CbDF = Concentration Before Diafiltration):No. Starting Concentration (g / L) CbDF (g / L)1 10 302 10 503 10 704 10 1005 15 306 15 507 15 708 15 1009 20 3010 20 5011 20 7012 20 100

[0098] As can be seen in Figures 2, 3 and 4, the scenarios utilizing the process system of the present invention, resulted in markedly lower total membrane needed, markedly lower total tankage needed and greatly increased total membrane loading, as compared to the standard batch procedure. Thus, the process of the present invention saves membrane size and system size for the same production. Additionally, single use apparatus usage is reduced since one unit is often used for longer process runs. Manual operations are also reduced since constant disconnecting and reconnecting are virtually eliminated.Example 2. Conservation of Production Resources

[0099] Another advantage to the processes of the present invention is the reduction in resource usage. The data shown in Figure 5 shows typical process parameters for one day of production comparing batch mode (openflow path), continuous UF / DF (open flow path) and continuous UF / DF (closed flow path.

[0100] The data shown in Figure 6 shows theoretical consumptions for the same three process scenarios.Example 3. Exemplary Continuous Filtration Process Procedure

[0101] Raw product is collected into a cycling tank (for example, the Mobius Mix, EMD Millipore, Burlington, MA).

[0102] When enough product is collected, continuous transfer to the TFF process system is initiated.

[0103] TFF process system may be one of an ultrafiltration or a diafiltration system.

[0104] Purified product is recovered to the SPTFF stage tank.

[0105] Product is concentrated thru SPTFF while TFF system continues to process raw product from the production process by collecting raw product in the cycling tank from multiple production sources.

[0106] Flushing and cleaning steps can be done between the filtration cycles, for example, once a day or as needed.

[0107] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0108] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:1 . A process for filtering a liquid feed sample, comprising:(a) routing a first amount of a liquid feed sample from a first sample source to a cycling tank;(b) simultaneously or in series with the first amount of liquid feed sample, routing a second amount of liquid feed sample from a second sample source to the cycling tank;(c) upon collection of a suitable quantity of liquid feed sample in the cycling tank, routing a continuous feed stream of the liquid feed sample from the cycling tank to a tangential flow filtration (TFF) system to filter the continuous feed stream of the liquid feed sample;(d) during the filtration of the liquid feed sample, routing one or more additional amounts of liquid feed sample to the cycling tank to maintain a continuous feed stream of liquid feed sample to the TFF system;(e) continuing to provide liquid feed sample to the cycling tank until a total product volume is processed through the TFF system.

2. A process for filtering a liquid feed sample, comprising:(a) routing a first amount of a liquid feed sample from a first sample source to a cycling tank;(b) upon collection of a suitable quantity of liquid feed sample in the cycling tank, routing a continuous feed stream of the liquid feed sample from the cycling tank to a tangential flow filtration (TFF) system to filter the continuous feed stream of the liquid feed sample;(c) during the filtration of the liquid feed sample, routing one or more additional amounts of liquid feed sample to the cycling tank to maintain a continuous feed stream of liquid feed sample to the TFF system;(d) continuing to provide liquid feed sample to the cycling tank until a total product volume is processed through the TFF system.

3. The process of Claims 1 and 2, wherein said additional amounts of liquid feed sample come from one or more different sample sources.

4. The process of any of the previous claims, wherein the TFF system is a Single-Pass Tangential Flow Filtration (SPTFF) system.

5. The process of any of the previous claims, wherein the liquid feed sample is concentrated as a result of the filtration.

6. The process of any of the preceding claims, wherein the liquid feed sample is a cell culture media.

7. The process of any of the preceding claims, wherein the liquid feed sample comprises monoclonal antibodies.

8. The process of any of the preceding claims, wherein the liquid feed sample is concentrated prior to being delivered to the cycling tank.

9. The process of any of the preceding claims, wherein the liquid feed sample is diluted prior to being delivered to the cycling tank.

10. The process of any of the preceding claims, wherein the filtration is diafiltration and wherein the process additionally comprises a buffer tank being in fluid communication with the feed stream.

11. The process of any of the preceding claims, further comprising a holding tank between the cycling tank and the TFF system.

12. The process of any of the preceding claims, further comprising flushing the TFF system after a total product volume has been processed.

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