Microfiltration of cell retention perfusate

Abstract

A microfiltration apparatus is disclosed, which includes a bioreactor capable of holding a biological fluid to be filtered; a cell retention device disposed between a perfusate pump and the bioreactor; a feed pump disposed between the cell retention device and the bioreactor, wherein the feed pump, the bioreactor, and the cell retention device are fluidly connected; a first recirculating loop for flowing from the cell retention device to the bioreactor, returning a cell retention retentate to the bioreactor; a collection vessel; and a plurality of microfiltration units having a microfiltration membrane, wherein filtration retentate flows are arranged in series, and wherein permeate flows flow to the collection vessel.

Description

Attorney Docket No.: P24-222-SEC-WO01MICROFILTRATION OF CELL RETENTION PERFUSATEBACKGROUNDRelated Applications

[0001] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 729,642, filed December 9, 2024, the entire content of which is incorporated herein in its entirety.Field

[0002] Methods and systems for the processing of biological fluids are disclosed. Embodiments described herein more particularly relate to systems and processes for the clarification and / or perfusion of biological fluids.Related Art

[0003] Processes for growing, feeding, and harvesting live cellular cultures, such as monoclonal antibodies, cells, viruses, and other biological products, are difficult to develop. Many critical attributes and properties, which impact cell growth and productivity, must be monitored and controlled. Changes or losses of control in upstream processes during cell growth can affect the concentration and therefore harvest of product from a biological fluid or culture. This is especially true of continuous and semi-continuous processes, as opposed to batch processes. For batch processes, a cell culture is maintained for a set duration, followed by harvesting the entire culture within the batch. In continuous harvesting systems, e.g., perfusion processes, spent culture is removed and replaced by fresh media, while cell culture permeate is collected during a run. Perfusion culture can achieve higher cell density, run for longer duration, and improve product quality7. Higher volumes of material, however, are generated from perfusion cell culture processes, which pose challenges in downstream processing of biological fluids, such as clarification and filtration. Upstream and downstream processes are, therefore, subject to balancing the interests of processing times, product concentrations, and quality. Moreover, many of the downstream processes are expensive to conduct and any impact is therefore commensurately significant. Accordingly, process intensification, i.e., the coupling of heretofore discrete processes, leading to greater efficiencies, is an important goal.

[0004] Prior art inventions include filtration devices, such as depth filtration and tangential flow filtration devices. Theoretically, in perfusion processes, as the pore size of the tangentialAttorney Docket No.: P24-222-SEC-WO01 flow filters increases, protein sieving and in turn, the protein recovery will increase. However, these devices with increased pore sizes may permit cell debris and other contaminants to remain in the fluid and therefore require additional clarification before the biological fluid can be subjected to the next downstream operation, e.g., affinity capture, anion and cation exchange chromatography, etc., to avoid column clogging. In contrast, when such filtration devices contain membranes with narrow pore size, capacities are low and, therefore, many filters are required to clarify the biological fluid. Low capacities are manifested by way of rapid fouling or rapid decrease in protein recovery over time. Thus, the filters with larger pores are preferred despite the need for further clarification. Many bioprocessors prefer tighter pore size membranes during clarification and purification for enhanced reliability and safety, which resultingly suffer from low filter throughput and protein retention (sieving) and therefore require secondary clarification operations.

[0005] The additional clarification requires further considerations such as need for appropriate filter devices in combination with peripherals such as pumps and tubing with accompanying dedicated hardware. Additionally, such devices, e. g. depth filters, are unfit for direct fluid connection with previous device and bioreactor due to contamination concerns. Therefore, need arises whereby additional clarification is performed with minimal addition of peripheral parts, and with devices that can be integrated seamlessly with sterile fluid path with devices that are upstream.

[0006] Single-pass tangential flow filtration (SPTFF) systems described in prior arts can achieve higher permeate conversion through a single pass, therefore avoiding recirculation loops. However, SPTFF systems have long, staged flow paths and employ membranes for the concentration of proteins, making their applications limited. The membranes for SPTFF can be configured with step-wise decreases in cross-sectional membrane areas resulting in higher concentration factors at a given feed flux compared to multi-pass TFF. but cannot be used for retention of cells or cell debris in sterile assemblies.

[0007] Methods and cell culturing apparatus for producing antibodies, therapeutic proteins, and other biological products that balance processing expense, quality and concentration while providing increased product recovery represent an advance in the art. Methods and apparatus, employing open pore size membrane(s), without the drawbacks described above, that clarify biological fluids and other perfusates, operating in sterilized, closed, continuous processes represent inventive advances in the art.Attorney Docket No.: P24-222-SEC-WO01SUMMARY

[0008] Microfiltration systems for clarification of cell culture perfusate and methods for using same in perfusion cell culture processes; substantially as shown in and / or described in connection with at least one of the figures, as set forth more completely in the claims, are disclosed. In some embodiments, systems described herein comprise two or more microfiltration units, such as TFF filters, wherein each microfiltration unit delivers permeate to a collection vessel and no other components or operations between the microfiltration units. In some embodiments, the system or assembly comprises a bioreactor in direct fluid communication with a cell retention device, and two or more microfiltration units. In some embodiments, tangential flow filtration system that enables the use of perfusion to produce biological products, such as antibodies, therapeutic proteins, blood factors and enzymes, and the like, and generates a product stream that can be delivered to downstream processing without further clarification.

[0009] Various novel and inventive features of the present disclosure, as well as details of exemplary embodiments thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE FIGURES

[0010] FIG. 1 depicts a tankless TFF system comprising serialized microfiltration apparatus for clarification of cell culture perfusate where the final retentate from the microfiltration apparatus is sent to waste, according to some embodiments of the disclosure;

[0011] FIG. 2 depicts a TFF system comprising serialized microfiltration apparatus for clarification of cell culture perfusate where the perfusate from cell retention device is collected in an intermediate vessel and final retentate from the microfiltration apparatus is sent to waste, according to some embodiments of the disclosure;

[0012] FIG. 3 depicts a tankless TFF system comprising serialized microfiltration apparatus for clarification of cell culture perfusate where the final retentate from the microfiltration apparatus is returned to the bioreactor, according to some embodiments of the disclosure;

[0013] FIG. 4 depicts a tankless TFF system comprising serialized microfiltration apparatus w here the final retentate is returned to retentate line of the cell retention device, according to some embodiments of the disclosure;Attorney Docket No.: P24-222-SEC-WO01

[0014] FIG. 5 depicts a TFF system comprising serialized microfiltration apparatus which clarifies cell culture perfusate collected in an intermediate vessel, where the final retentate is returned to the intermediate vessel, according to some embodiments of the disclosure; and

[0015] FIG. 6 depicts a tankless TFF system comprising serialized microfiltration apparatus for clarification of cell culture perfusate where the final retentate is connected to the feed line of the first microfiltration device, according to some embodiments of the disclosure.DETAILED DESCRIPTION OF SOME EMBODIMENTS

[0016] So the manner in which the features disclosed herein can be understood in detail, a more particular description of the embodiments of the disclosure, briefly summarized above, may be had by reference to the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this disclosure and are therefore not to be considered limiting of its scope, for the embodiments described and shown may admit to other equally effective embodiments. It is also to be understood that elements and features of one embodiment may be found in other embodiments without further recitation and that identical reference numerals are sometimes used to indicate comparable elements that are common to the figures.

[0017] The terms “bioreactor,” “bag,” and “container” are generally used interchangeably within this disclosure. A flexible bioreactor, bag. or container connotes a flexible vessel that can be folded, collapsed, and expanded and / or the like, capable of containing, for example, a biological fluid. A single use bioreactor, bag, or container, typically flexible, is a vessel that is used once and discarded.

[0018] The term “perfusion” refers to as a process that uses a method to keep cells in a bioreactor while continuously exchanging the cell culture media.

[0019] The term “cell retention device” (CRD) refers to a device that retains cells inside the bioreactor during a perfusion process of cell culturing, w hile fresh media is added, and products of interest, w aste products and spent (or depleted) media are continuously removed, achieving and maintaining high cell densities and viabilities typically over extended periods of time.

[0020] The term “molecular weight cut-off' or “MWCO” of a membrane refers to the lowest molecular weight of the molecules in daltons (Da) for which 90% of the molecules is retained by the membrane, or the molecular weight of the molecule that is 90% retained by the membrane.Attorney Docket No.: P24-222-SEC-WO01

[0021] The term “Pellicon® XL50” represents a family of tangential flow filtration devices manufactured and marketed by the EMD Millipore Corporation, Burlington, MA. USA. The devices may contain membranes made with polyethersulphone. a composite regenerated cellulose, or surface-modified polyvinylidene fluoride. Some membranes are specified for molecular weight cutoffs ranging from 10 kDa to 1000 kDa. Some membranes have nominal pore sizes ranging from 0. 1 pm to 0.65 pm.)

[0022] The term “tangential flow filtration” (TFF) is a defined as a mode of filtration wherein the sample flows parallel to the surface of membrane such that particles bigger than the pore size of the membrane are retained on the upstream side of the membrane while the smaller particles permeate across the membrane, allowing the biological products to be concentrated or reformulated. Tangential flow filtration membranes include, but are not limited to, ultrafiltration (UF) membranes, microfiltration (MF) membranes, reverse osmosis (RO) membranes and nanofiltration (NF) membranes.

[0023] The term alternating tangential flow filtration (ATF) is similar to TFF and uses a diaphragm pump to alternate the flow direction over a membrane surface.

[0024] The term single pass tangential flow filtration (SPTFF) is similar to TFF and uses a series of membranes to concentrate a biological fluid in a single pass without recirculating a retentate.

[0025] The terms “ultrafiltration membrane” and “UF membrane” are used herein to refer to a membrane that has pore sizes in the range of between about 1 nanometer to about 100 nanometers.

[0026] The terms “microfiltration membranes” and “MF membranes” are used herein to refer to membranes that have pore sies in the range between about 0.1 micrometers to about 10 micrometers.

[0027] The expressions "serialized processing," "processing in series," "serial operation." and "operation in series" refer to distributing a liquid in a TFF system to one filtration unit (e.g., filtration module, TFF cassette) at a time, such that the retentate flow of a preceding unit serves as the feed flow for a subsequent, adjacent unit.

[0028] The term “diverter plate” refers to a solid plate that can be stacked against TFF cassettes on both of its faces to enable serialized processing.

[0029] The term “sterile” is defined as a condition of being free from contaminants and, particularly, free from undesirable viruses, bacteria, germs, and other microorganisms.Attorney Docket No.: P24-222-SEC-WO01

[0030] The term “upstream” is defined as first step processes in the processing of biological materials, such as microbes / cells, mAbs. proteins, including therapeutic proteins, viral vectors, etc., are grown or inoculated in bioreactors within cell culture media, under controlled conditions, to manufacture certain types of biological products.

[0031] The term "downstream" indicates those processes occurring after cell culturing and clarifying, e.g., chromatography and diafiltration, in which biological products are concentrated, purified, polished, and packaged.

[0032] In general, an order of unit operations is cell culture development within a bioreactor, clarification, affinity chromatography, virus inactivation, purification chromatography, polishing chromatography, viral clearance, concentration / diafiltration, and final filling. Not all of these unit operations are necessarily present in a standard bioprocessing batch. Nonetheless, as one unit process follows another, the subsequent steps are said to be downstream of the former.

[0033] The term “clarification” is defined as a process, wherein large insoluble contaminants, usually cells and cell debris are separated from the feedstock or harvest.

[0034] The term “purification” is defined as a downstream process, wherein bulk contaminants and impurities, including host cell proteins, DNA and process residuals are removed from the product stream.

[0035] The term “polishing” is defined as a downstream process, wherein trace contaminants or impurities that resemble the product closely in physical and chemical properties are eliminated from the purified product stream.

[0036] The term “chromatography” is defined as a downstream process suitable for biological chromatographic techniques, comprising, but not limited to, protein A chromatography, affinity chromatography, hydrophobic interaction chromatography, column chromatography, and ion exchange chromatography, e.g., anion exchange chromatography, cation exchange chromatography, and mixed mode chromatography.

[0037] Turning now7to the drawings, some embodiments of the disclosure include various systems 100, 200, 300, 400, 500, and 600, which have a bioreactor 102, one or more feed pumps 108, 112 and / or perfusate pumps 118, a microfiltration train 106, and collection vessels 104. Some embodiments of systems 100, 200, 300, 400, 500, and 600 further comprise an intermediate vessel 122. Some embodiments of systems 100, 200, 300, 400, 500, and 600 comprise a three-w ay retentate port 114. It is to be understood that the fluid communicationAttorney Docket No.: P24-222-SEC-WO01 between any of the devices and apparatus described herein may be connected by tubing 132, hard plumbing, e.g., pipes, connectors, and / or valves, and / or combinations thereof. Additionally, it is to be understood that any connections to build the microfiltration train 106 may be made using hard pipes, flexible tubing, and / or specially designed diverter plates stackable between two microfiltration devices. It is also to be understood that any of the feed collected in any collection vessel 104 or intermediate vessel 122 can be the feed for another downstream process, such as a 0.22 micron (p) pore size filtration or capture step.

[0038] As described herein, a small pore size microfiltration membrane inside a microfiltration unit 106a, 106b, 106c is, for example, a 0.22 pm or > 0. 1 pm. In some embodiments, pore size of this membrane represented in molecular weight cutoff is, for example, > 500 kDa. The processed product can be fed into the next unit operation (for e.g., Protein A chromatography for monoclonal antibodies (mAbs) and / or other downstream biological processes known to those in the art without further clarification. A large pore size microfiltration membrane inside a microfiltration unit 106a, 106b, 106c is, for example, a <0.45 pm.

[0039] FIG. 1 depicts a tankless microfiltration assembly 100 for clarification of cell culture perfusate, according to some embodiments of the disclosure. The tankless microfiltration assembly 100, as depicted, comprises a serialized microfiltration membrane train 106a, 106b, 106c driven by a cell retention system perfusate pump 110 along with a cell retention device 120 and a feed pump 108 in fluid communication with a bioreactor 102. As can be seen, there are three microfiltration units 106a, 106b. 106c in the microfiltration train. In practice, there may be two, three, four, or up to any reasonable amount, e.g., ten. In operation, the bioreactor 102 cultures cells or other biological products therein. A feed pump 108 delivers the biological fluid, e.g., cell culture perfusate, to a cell retention device 120. A retentate is delivered back to the bioreactor 102 while a perfusate is delivered to the microfiltration units 106a, 106b, 106c by perfusate pump 110. The microfiltration units 106a, 106b. 106c deliver a retentate in series to the adjacent microfiltration unit 106a, 106b, 106c . . . 106n while a permeate is delivered to a collection vessel 104. The permeate in the collection vessel 104 can then, optionally, become the feed to another downstream process, e.g., filtration, capture, chromatography, etc. In this embodiment, the retentate from the final microfiltration unit 106c can be discarded as waste 134. The assembly 100 utilizes the perfusate pump 110 as part of the cell retention device 120 to drive product through the TFF assembly comprising of serialized microfiltration membranes of smaller pore size (e.g., 0.22 pm, 0.45 pm). The microfiltration assembly 100 retains cellAttorney Docket No.: P24-222-SEC-WO01 debris and impurities while the product molecule is permeated through the membrane, which may be directly fed into the next unit operation. This assembly 100 utilizes fewer pumps and eliminates the need for an intermediate storage tank.

[0040] FIG. 2 shows a microfiltration assembly 200 and method aimed at clarifying cell culture perfusate, according to some embodiments of the disclosure. The microfiltration assembly 200, as depicted, comprises a serialized microfiltration membrane train 106a, 106b, 106c driven by a cell retention system perfusate pump 110 and a second feed pump 112, wherein an intermediate vessel 122 is disposed therebetween. A cell retention device 120 and a feed pump 108 in fluid communication with a bioreactor 102 and the perfusate pump 110. As can be seen, there are three microfiltration units 106a, 106b, 106c in the microfiltration train. In practice, there may be two, three, four, or up to any reasonable amount, e.g., ten. In operation, the bioreactor 102 cultures cells or other biological products therein. A feed pump 108 delivers the biological fluid, e.g., cell culture perfusate, to a cell retention device 120. A retentate is delivered back to the bioreactor 102 while a perfusate is delivered to the intermediate tank by perfusate pump 110, and the pump 112 delivers the contents of the tank to the microfiltration units 106a, 106b, 106c. The microfiltration units 106a. 106b, 106c deliver a retentate in series to the adjacent microfiltration unit 106a, 106b, 106c . . . 106n while a permeate is delivered to a collection vessel 104. The permeate in the collection vessel 104 can then, optionally, become the feed to another downstream process, e.g., filtration, capture, chromatography, etc. In this embodiment, the retentate from the final microfiltration unit 106c, or whichever microfiltration unit 106 may be the furthest downstream from the other microfiltration units, can be discarded as waste. In this microfiltration assembly 200, a cell culture perfusate is collected in an intermediate surge vessel 122. A feed pump 108 drives the product from the intermediate vessel 122 into the three microfiltration units 106a, 106b, 106c.Attorney Docket No.: P24-222-SEC-WO01Graph 1 : Flux v. Transmembrane pressure (TMP) in PSI

[0041] Graph 1 is generated using the embodiment of FIG. 2, using three serialized 0.22 micron Pellicon® XL50 devices, flux in liters of permeate per squared meter of membrane per minute (LMM), also sometimes referred to as also sometimes denoted as liters per squared meter of membrane per hour (LMH).Graph 2: Flux v. TMP (PSI).

[0042] Graph 2 is generated using the embodiment of FIG. 2, using three serialized 0.45 micron Pellicon® XL50 devices, flux in liters of permeate per squared meter of membrane per minute (LMM), also sometimes referred to as also sometimes denoted as liters per squared meter of membrane per hour (LMH).

[0043] Monoclonal antibody (mAb) perfusate was collected and pooled from a bioreactor utilizing a 3 pm TFF cell retention system. The expressed mAb was an IgGl molecule at a concentration of 0.5 g / L, produced from a Chinese Hamster Ovary (CHO) cell line in a chemically defined cell culture media. The mAb may, optionally, be other forms of IgGAttorney Docket No.: P24-222-SEC-WO01 isoforms or biologicals. A secondary' TFF system was assembled using three 0.22 gm Durapore Pellicon® XL50 devices each with an area of 50 cm2. As shown in FIG. 2, the fdtration module comprises a feed inlet to receive the feed into the first filtration module, and a manifold segment that comprises a first manifold for receiving and carrying the feed into the second filtration module, and a second manifold for receiving and carrying retentate out of the second filtration module and into the third filtration module, a retentate outlet from the third filtration module, permeate outlets for each of the three devices, and, optionally, a second manifold segment for receiving and carrying permeate out of each of the three filtration modules, wherein the flow path through the manifold segment and the filtration modules is serial. In some embodiments, to accommodate more membrane area than that provided by an individual cassette within each filtration module, a plurality’ of TFF cassettes are stacked on one or both faces of, and are fluidly connected to, the manifold segment, wherein the liquid flow path is parallel through the cassettes. The manifold segments described herein may be soft tubing, hard tubing or diverter plates, where applicable. Manifold segments described herein may contain pressure sensors on the inlet of first filtration module, and at all or third retentate ports. Optionally, the manifold segments include several three-way valves or sampling ports for collecting samples for analytical measurements.

[0044] At variable feed fluxes, conversion of feed to permeate for the entire system as w ell as each individual filter was measured at increasing retentate pressures. Graph 1 shows an increase in conversion at lower fluxes, achieved at lower transmembrane pressures. Graph 2 demonstrates the same assembly and method using 0.45 pm Durapore® Pellicon® XL50 devices.Attorney Docket No.: P24-222-SEC-WO01Graph 3: Mass v. Flow conversion from feed to permeate through 0.22 m Pellicon® XL50 filters.

[0045] Graph 3 shows a strong correlation between flow conversion from feed to permeate flow and the conversion of protein mass from feed to permeate flow. Mass conversion here is calculated as the percent of mass permeating through the membrane as compared to the mass entering the system through the feed. This correlation suggests that mAb recovery can be achieved and optimized as conversion is optimized.Graph 4: mAb recovery v. Throughput (L / m2). Three 0.22 pm Pellicon® XL50 devices were assembled as depicted in FIG. 2 and mAb perfusate collected separately from a bioreactor was recirculated through the system at 1LMM at a TMP of 7.5 PSI up to a throughput of 6400 L / m2. The permeate and retentate lines were returned to the feed vessel, with grab samples of the permeate regularly taken to analyze flux and mAb concentration during an extended duration of operation, showing the calculated mAb recovery throughout the duration of the experiment using the permeate flux and permeate mAb concentrations.Attorney Docket No.: P24-222-SEC-WO01Turbidity0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0Throughput (L / mA2)Graph 5. Turbidity of recirculated feed and permeate grab samples from each filter.Graph 6. mAb conversion from feed to permeate for each individual filter, and the relative residence time of feed through each filter as compared to filter 1.

[0046] As shown in Graph 5, the turbidity of the product is significantly reduced after filtration by the 0.22 pm filters. This reduction in turbidity indicates some removal of cell debris and other impurities, enabling further downstream processing.

[0047] Graph 6 demonstrates the benefit gained from serializing multiple smaller area membranes as opposed to operating with a single larger mem brane. As feed flows and permeates through the first filter, the actual feed flux into the second filter is reduced, resulting in a greater residence time in the device. As seen in Graph 1 and Graph 2, greater mAb conversion is observed at lower feed fluxes. This trend continues as retentate descends the trainAttorney Docket No.: P24-222-SEC-WO01 of membranes, resulting in an increase of >20% residence time between the third and first devices.

[0048] FIG. 3 depicts a tankless TFF system 300 comprising of serialized microfiltration membranes of smaller pore size driven by a cell retention system perfusate pump where the final retentate is returned to the bioreactor 102, according to some embodiments of the disclosure. The system 300 depicts a tankless microfiltration assembly 100 for clarification of cell culture perfusate, according to some embodiments of the disclosure. The tankless microfiltration assembly 300, as depicted, comprises a serialized microfiltration train 106a, 106b, 106c driven by a cell retention system perfusate pump 110 along with a cell retention device 120 and a feed pump 108 in fluid communication with a bioreactor 102. As can be seen, there are three microfiltration units 106a, 106b. 106c in the microfiltration train. In practice, there may be two. three, four, or up to any reasonable amount, e.g.. ten. In operation, the bioreactor 102 cultures cells or other biological products therein. A feed pump 108 delivers the biological fluid, e.g., cell culture perfusate, to a cell retention device 120. A retentate is delivered back to the bioreactor 102 while a perfusate is delivered to the microfiltration units 106a. 106b, 106c by perfusate pump 110. The microfiltration units 106a. 106b, 106c deliver a retentate in series to the adjacent microfiltration unit 106a, 106b, 106c . . . 106n while a permeate is delivered to a collection vessel 104. The permeate in the collection vessel 104 can then, optionally, become the feed to another downstream process, e.g., filtration, capture, chromatography, etc. In this embodiment, the retentate from the final microfiltration unit 106c can be returned to the bioreactor 102. The assembly 100 utilizes the perfusate pump 1 10 as part of the cell retention device 120 to drive product through the TFF assembly comprising of serialized microfiltration membranes of smaller pore size (e.g., 0.22 pm, 0.45 pm). The microfiltration assembly 100 retains cell debris and impurities while the product molecule is permeated through the membrane, which may be directly fed into the next unit operation. This assembly 100 utilizes fewer pumps and eliminates the need for an intermediate storage tank. The fluid is driven by a cell retention system perfusate pump 110 where the final retentate is returned to the bioreactor 102 via a port from the last microfiltration unit 106, here 106c, i.e., the retentate from the last device in series is diverted to the bioreactor 102 to improve, e.g., mAb recovery. Otherwise, the assembly 300 is similar to the assembly 100.

[0049] FIG. 4 depicts a tankless TFF system 400 comprising of serialized microfiltration units 106a, 106b, 106c (as with other embodiments within the dislcosure, the number of serializedAttorney Docket No.: P24-222-SEC-WO01 microfiltration units can be any suitable amount, e.g., two, three, four, five, six, and more, etc.) driven by a cell retention system perfusate pump 110 (disposed between the cell retention device and the plurality of serialized microfiltration units 106a- 106c) where the final retentate (from the last microfiltration unit, as shown, microfiltration unit 106c) is returned to retentate line of the cell retention device 120 via port three-way 114, according to some embodiments of the disclosure. The bioreactor 102 supplies a biological fluid to the cell retention device 120 via feed pump 118. In another mode of the process, the retentate line from the last device in series is connected to the retentate line at a three-way retentate port 114, which is disposed between the bioreactor 102 and the output of the cell retention device 120, for improving mAb recovery. A collection vessel 104 collects the permeate from each of the microfiltration units 106a. 106, b, 106c . . . 106n.

[0050] FIG. 5 depicts a TFF system 500 comprising of serialized microfiltration membranes of smaller pore size clarifies cell culture perfusate collected in an intermediate vessel, where the final retentate is returned to this intermediate vessel and volume of this vessel is significantly smaller than the bioreactor volume, according to some embodiments of the disclosure. The microfiltration assembly 500, as depicted, comprises a bioreactor 102 in fluid communication with a serialized microfiltration units train 106a, 106b, 106c, driven by a cell retention system perfusate pump 110 (disposed between the cell retention device 120 and an intermediate vessel 122) and a second feed pump 112 (disposed between the intermediate 122 and the plurality of microfiltration units, as shown 106a- 106c), wherein a feed pump 108 is disposed between the cell retention device 120 and the bioreactor 10, wherein a collection vessel 104 collects permeate from the plurality of microfiltration units 106a, 106b, 106c. A retentate line 132 returns retentate from the final microfiltration unit (here, 106c) directly to the intermediate vessel 122.

[0051] As can be seen, there are three microfiltration units 106a, 106b, 106c in the microfiltration train. In practice, there may be two, three, four, or up to any reasonable amount, e.g., ten. In operation, the bioreactor 102 cultures cells or other biological products therein. A feed pump 108 delivers the biological fluid, e.g., cell culture perfusate, to a cell retention device 120. A retentate is delivered back to the bioreactor 102 while a perfusate is delivered to the intermediate tank by perfusate pump 110, and the pump 112 delivers the contents of the tank to the microfiltration units 106a, 106b, 106c. The microfiltration units 106a, 106b, 106c deliver a retentate in series to the adjacent microfiltration unit 106a, 106b, 106c . . . 106n while aAttorney Docket No.: P24-222-SEC-WO01 permeate is delivered to a collection vessel 104. The permeate in the collection vessel 104 can then, optionally, become the feed to another downstream process, e.g., filtration, capture, chromatography, etc., such as a subsequent filtration step, capture step or other downstream process. In another mode, the retentate from the last device in series is recycled to the surge tank, or intermediate vessel 122, upstream of the pump 112 that feeds the microfiltration units 106a, 106b, 106c.

[0052] FIG. 6 depicts a tankless TFF system 600 comprising a plurality of serialized microfiltration units 106a, 106b, 106c driven by a cell retention system perfusate pump 110 where the final retentate delivered from the microfiltration unit 106c, as depicted here, microfiltration unit 106c, is connected to the three-way retentate port 114, which is disposed just upstream of the first microfiltration device 106a. i.e., between the pump 110 and the microfiltration unit 106a, according to some embodiments of the disclosure. The final retentate from the final microfiltration unit, here 106c, is connected to the feed line of the first microfiltration unit 106a to increase the recovery of mAh and other various biologicals.

[0053] It is to be understood that for the microfiltration assemblies depicted in Figures 1-6, the first filtration unit 106a, second filtration unit 106b. and third filtration unit 106c may, optionally, all have a membrane of similar pore size or could be subsequently smaller pore size. For example, the first filtration unit 106a may have a pore size of 0.44pm, the second filtration unit 106b may have a pore size of 0.44pm, and the third filtration unit 106c may have a pore size of 0.2pm. The microfiltration units separate cell debris and other impurities from the product molecule using a small pore size microfiltration membrane (for example, a <0.2 pm). The processed product can be fed into the next unit operation (for e.g., Protein A chromatography for monoclonal antibodies (mAbs) and / or other downstream biological processes known to those in the art) without further clarification. It is also to be understood that many differently-sized devices, e.g.. may be employed. By way of example and not limitation, a first device may be 50 cm2and a 25 cm2for a final device.

[0054] It is to be understood that any of the microfiltration apparatus 100, 200, 300, 400, 500, and 600 may further comprise / comprises wherein: a final retentate line leading from a final microfiltration unit to the bioreactor; wherein the filtration retentate flows to a waste collection; a second feed pump disposed between an intermediate vessel and the microfiltration units and wherein the final retentate line flows to the intermediate vessel; wherein the microfiltration membrane has a pore size of molecular weight cut-off of greater than or equal to 500 kDa;Attorney Docket No.: P24-222-SEC-WO01 wherein the microfiltration membrane has a pore size of 0.22 microns; wherein the microfiltration membrane has a pore size of greater than 0.1 microns to 10 microns; wherein the microfiltration membrane has a pore size of 0. 1 microns; wherein microfiltration membrane has a pore size smaller than or equal to 0.45 microns; wherein three to twenty microfiltration units are arranged in series; wherein the microfiltration units are connected for tangential flow filter filtration, alternating flow filtration, or single pass tangential flow filtration; wherein the plurality of microfiltration units are connected using hard piping, flexible tubing, or by a manifold; wherein the microfiltration apparatus 100, 200, 300, 400, 500, and 600 further comprises a diverter plate arranged between the plurality of microfiltration units; wherein the microfiltration units comprising one or more microfiltration membranes have the total installed area of 50 cm2to 22 cm2; wherein the microfiltration apparatus comprises one or more microfiltration membranes have the total installed area of 25 cm2to 22 cm2; wherein a first microfiltration unit has total installed membrane area of 50 cm2to 22 m2and a second microfiltration unit has total installed membrane area of 0.1 to 0.9 times that of the first microfiltration unit; or wherein a first microfiltration unit has total installed membrane area of 25 cm2to 11 m2and a second microfiltration unit has total installed membrane area of 1.1 to 10 times that of the first microfiltration unit.

[0055] Embodiments of the disclosure also comprise methods for perfusing cells within a cell culture media. For example, some embodiments of the disclosure include a method for processing biological fluids, via a second recirculating loop to the storage tank and a second permeate is delivered to final fill container for storage as a biological product.

[0056] Reference throughout this specification to “one embodiment,” “certain embodiments,” “one or more embodiments,” “some embodiments,” or “an embodiment” indicates that a feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Therefore, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in one embodiment,” “some embodiments,” or “in an embodiment” throughout this specification are not necessarily referring to the same embodiment.

[0057] Although some embodiments have been discussed above, other implementations and applications are also within the scope of the following claims. Although the specification describes, with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure.Attorney Docket No.: P24-222-SEC-WO01It is therefore to be further understood that numerous modifications may be made to the illustrative embodiments and that other arrangements and patterns may be devised without departing from the spirit and scope of the embodiments according to the disclosure. Furthermore, particular features, structures, materials, or characteristics may be combined in any suitable manner in any one or more of the embodiments.

[0058] Publications of patent applications and patents and other non-patent references, cited in this specification are herein incorporated by reference in their entirety in the entire portion cited as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.

Claims

Attorney Docket No.: P24-222-SEC-WO01CLAIMSWhat is claimed is:

1. A microfiltration apparatus, comprising: a bioreactor capable of holding a biological fluid to be filtered; a cell retention device disposed between a perfusate pump and the bioreactor; a feed pump disposed between the cell retention device and the bioreactor, wherein the feed pump, the bioreactor, and the cell retention device are fluidly connected; a first recirculating loop for flowing from the cell retention device to the bioreactor, returning a cell retention retentate to the bioreactor; a collection vessel; and a plurality’ of microfiltration units having a microfiltration membrane, wherein filtration retentate flows are arranged in series, and wherein permeate flows flow to the collection vessel.

2. The microfiltration apparatus of claim 1 , further comprising a final retentate line leading from a final microfiltration unit to the bioreactor.

3. The microfiltration apparatus of claims 1-2, wherein the filtration retentate flows to a waste collection.

4. The microfiltration apparatus of claims 1-3, further comprising a second feed pump disposed between an intermediate vessel and the microfiltration units and wherein the final retentate line flows to the intermediate vessel.

5. The microfiltration apparatus of claims 1-4, wherein the microfiltration membrane has a pore size of molecular weight cut-off of greater than or equal to 500 kDa.

6. The microfiltration apparatus of claims 1-5, wherein the microfiltration membrane has a pore size of 0.22 microns.

7. The microfiltration apparatus of claims 1-6, wherein the microfiltration membrane has a pore size of greater than 0.1 microns to 10 microns.

8. The microfiltration apparatus of claims 1-7, wherein the microfiltration membrane has a pore size of 0. 1 microns.

9. The microfiltration apparatus of claims 1-8, wherein microfiltration membrane has a pore size smaller than or equal to 0.45 microns.

10. The microfiltration apparatus of claims 1-9, comprising three to twenty microfiltration units arranged in series.Attorney Docket No.: P24-222-SEC-WO0111. The microfiltration apparatus of claims 1-10, wherein the microfiltration units are connected for tangential flow filter filtration, alternating flow filtration, or single pass tangential flow filtration.

12. The microfiltration apparatus of claims 1 -1 1 , wherein the plurality of microfiltration units are connected using hard piping, flexible tubing, or by a manifold.

13. The microfiltration apparatus of claims 1-12, further comprising a diverter plate arranged between the plurality of microfiltration units.

14. The microfiltration apparatus of claims 1-13, wherein the microfiltration units comprising one or more microfiltration membranes have the total installed area of 50 cm2to 22 m215. The microfiltration apparatus of claims 1-14, wherein the microfiltration units comprising one or more microfiltration membranes have the total installed area of 25 cm2to 22 m216. The microfiltration apparatus of claims 1-15, wherein a first microfiltration unit has total installed membrane area of 50 cm2to 22 m2and a second microfiltration unit has total installed membrane area of 0. 1 to 0.9 times that of the first microfiltration unit=17. The microfiltration apparatus of claims 1-16, wherein a first microfiltration unit has total installed membrane area of 25 cm2to 11 m2and a second microfiltration unit has total installed membrane area of 1. 1 to 10 times that of the first microfiltration unit.

18. A microfiltration apparatus, comprising: a bioreactor capable of holding a biological fluid to be filtered; a collection vessel; a cell retention device disposed between a perfusate pump and the bioreactor; a feed pump disposed between the cell retention device and the bioreactor, wherein the feed pump, the bioreactor, and the cell retention device are fluidly connected; a plurality of microfiltration units having filtration retentate flows arranged in series, and wherein permeate flows flow to a collection vessel; and a first recirculating loop comprising a three-way port for flowing liquid from the cell retention device to the bioreactor, returning a cell retention retentate to the bioreactor and for receiving a retentate flow from the final microfiltration unit.

19. A microfiltration apparatus, comprising: a bioreactor capable of holding a biological fluid to be filtered;Attorney Docket No.: P24-222-SEC-WO01 a collection vessel; a cell retention device disposed between a perfusate pump and the bioreactor; a feed pump disposed between the cell retention device and the bioreactor, wherein the feed pump, the bioreactor, and the cell retention device are fluidly connected; a three-way retentate port disposed in a tube between the perfusate pump and the plurality of microfiltration units for receiving a filtration retentate flow; a plurality of microfiltration units having filtration retentate flows arranged in series, and wherein permeate flows flow to a collection vessel; and a first recirculating loop for flowing liquid from the cell retention device to the bioreactor, returning a cell retention retentate to the bioreactor.

20. A method for processing biological fluids, comprising; providing biological fluids within a cell culture media to a bioreactor and growing biological cells therein; delivering the biological fluid to a first stage TFF device, wherein cells are retained within the first stage TFF device and producing a permeate; perfusing the biological fluid by returning the biological fluid to the bioreactor via first recirculating loop; delivering the permeate to a storage tank; feeding the permeate from the storage tank to a second stage TFF device, wherein the permeate is clarified, wherein some of the permeate is recirculated via a second recirculating loop to the storage tank and a second permeate is delivered to final fill container for storage as a biological product.

21. A microfiltration apparatus, comprising: a bioreactor capable of holding a biological fluid to be filtered; a cell retention device disposed between a perfusate pump and the bioreactor; a feed pump disposed between the cell retention device and the bioreactor, wherein the feed pump, the bioreactor, and the cell retention device are fluidly connected; a first recirculating loop for flowing from the cell retention device to the bioreactor, returning a cell retention retentate to the bioreactor; a collection vessel; andAttorney Docket No.: P24-222-SEC-WO01 a plurality7of microfiltration units having a microfiltration membrane, wherein filtration retentate flows are arranged in series, and wherein permeate flows flow to the collection vessel.

22. The microfiltration apparatus of claim 21 , further comprising a final retentate line leading from a final microfiltration unit to the bioreactor.

23. The microfiltration apparatus of claims 21-22, wherein the filtration retentate flows to a waste collection.

24. The microfiltration apparatus of claims 21-23, further comprising a second feed pump disposed between an intermediate vessel and the microfiltration units and wherein the final retentate line flows to the intermediate vessel.

25. The microfiltration apparatus of claims 21-24, wherein the microfiltration membrane has a pore size of molecular weight cut-off of greater than or equal to 500 kDa.

26. The microfiltration apparatus of claims 21-25, wherein the microfiltration membrane has a pore size of 0.22 microns.

27. The microfiltration apparatus of claims 21-26, wherein the microfiltration membrane has a pore size of greater than 0. 1 microns to 10 microns.

28. The microfiltration apparatus of claims 21-27, wherein the microfiltration membrane has a pore size of 0. 1 microns.

29. The microfiltration apparatus of claims 21-28, wherein microfiltration membrane has a pore size smaller than or equal to 0.45 microns.

30. The microfiltration apparatus of claims 21-29, comprising three to twenty microfiltration units arranged in series.

31. The microfiltration apparatus of claims 21-30, wherein the microfiltration units are connected for tangential flow filter filtration, alternating flow filtration, or single pass tangential flow filtration.

32. The microfiltration apparatus of claims 21 -31, wherein the plurality of microfiltration units are connected using hard piping, flexible tubing, or by a manifold.

33. The microfiltration apparatus of claims 21-32, further comprising a diverter plate arranged between the plurality of microfiltration units.

34. The microfiltration apparatus of claims 21-33, wherein the microfiltration units comprising one or more microfiltration membranes have the total installed area of 50 cm2to 22 m2Attorney Docket No.: P24-222-SEC-WO0135. The microfiltration apparatus of claims 21-34, wherein the microfiltration units comprising one or more microfiltration membranes have the total installed area of 25 cm2to 22 m236. The microfiltration apparatus of claims 21 -35, wherein a first microfiltration unit has total installed membrane area of 50 cm2to 22 m2and a second microfiltration unit has total installed membrane area of 0. 1 to 0.9 times that of the first microfiltration unit=37. The microfiltration apparatus of claims 21-36, wherein a first microfiltration unit has total installed membrane area of 25 cm2to 11 m2and a second microfiltration unit has total installed membrane area of 1. 1 to 10 times that of the first microfiltration unit.

38. A microfiltration apparatus, comprising: a bioreactor capable of holding a biological fluid to be filtered; a cell retention device disposed between a perfusate pump and the bioreactor: a feed pump disposed between the cell retention device and the bioreactor, wherein the feed pump, the bioreactor, and the cell retention device are fluidly connected; a first recirculating loop for flowing from the cell retention device to the bioreactor, returning a cell retention retentate to the bioreactor; a collection vessel; and a plurality of microfiltration units having a microfiltration membrane, wherein filtration retentate flows are arranged in series, and wherein permeate flows flow to the collection vessel.

39. The microfiltration apparatus of claim 38, further comprising a final retentate line leading from a final microfiltration unit to the bioreactor.

40. The microfiltration apparatus of claims 38-39, wherein the filtration retentate flows to a waste collection.

41. The microfiltration apparatus of claims 38-40. further comprising a second feed pump disposed between an intermediate vessel and the microfiltration units and wherein the final retentate line flows to the intermediate vessel.

42. The microfiltration apparatus of claims 38-41, wherein the microfiltration membrane has a pore size of molecular weight cut-off of greater than or equal to 500 kDa.

43. The microfiltration apparatus of claims 38-42, wherein the microfiltration membrane has a pore size of 0.22 microns.Attorney Docket No.: P24-222-SEC-WO0144. The microfiltration apparatus of claims 38-43, wherein the microfiltration membrane has a pore size of greater than 0. 1 microns to 10 microns.

45. The microfiltration apparatus of claims 38-44. wherein the microfiltration membrane has a pore size of 0. 1 microns.

46. The microfiltration apparatus of claims 38-45, wherein microfiltration membrane has a pore size smaller than or equal to 0.45 microns.

47. The microfiltration apparatus of claims 38-46, comprising three to twenty microfiltration units arranged in series.

48. The microfiltration apparatus of claims 38-47, wherein the microfiltration units are connected for tangential flow filter filtration, alternating flow filtration, or single pass tangential flow filtration.

49. The microfiltration apparatus of claims 38-48. wherein the plurality of microfiltration units are connected using hard piping, flexible tubing, or by a manifold.

50. The microfiltration apparatus of claims 38-49, further comprising a diverter plate arranged between the plurality of microfiltration units.

51. The microfiltration apparatus of claims 38-50, wherein the microfiltration units comprising one or more microfiltration membranes have the total installed area of 50 cm2to 22 cm252. The microfiltration apparatus of claims 38-51, wherein the microfiltration units comprising one or more microfiltration membranes have the total installed area of 25 cm2to 22 cm253. The microfiltration apparatus of claims 38-52, wherein a first microfiltration unit has total installed membrane area of 50 cm2to 22 m2and a second microfiltration unit has total installed membrane area of 0. 1 to 0.9 times that of the first microfiltration unit=54. The microfiltration apparatus of claims 38-53. wherein a first microfiltration unit has total installed membrane area of 25 cm2to 1 1 m2and a second microfiltration unit has total installed membrane area of 1. 1 to 10 times that of the first microfiltration unit.

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