Harvest capture with settled resin

Abstract

The invention provides tangential flow filtration systems and methods for capture and harvest of a biologic product, and related compositions.

Description

Docket No. 1580.00231WOHARVEST CAPTURE WITH SETTLED RESINFIELD OF THE INVENTION

[0001] The invention relates to bioprocessing systems and methods for the manufacture of biologic products produced by cultured cells.BACKGROUND

[0002] In the biotechnology and pharmaceutical industries, a number of different process operations are generally used in the purification of cell-produced biologic products from bioreactor systems. These may include centrifugation, multiple filtration operations, and one or more chromatography operations. Typically, each conventional purification operation lowers product yield. Methods are needed to streamline the purification process to decrease time, attendant costs, and a volume of water in the output, while maintaining high product yield and purity.BRIEF SUMMARY

[0003] The present invention provides methods for performing a combined capture and harvest operation initiated by a direct addition of a capture resin to a cell culture fluid or lysate, without the need for prior clarification steps such as prior centrifugation and / or filtration step. The ensuing steps utilize a combination of diafiltration and concentration operations through a tangential flow filtration device to produce a biologic product at high yield and of high purity. In aspects, direct addition of resin to cell culture fluid or lysate containing a biologic product includes contacting the cell culture fluid or lysate with the resin in a secondary vessel or in a recirculating retentate fluid circuit of a tangential flow filtration module, which may also be referred to herein for conciseness as a “recirculation loop”.

[0004] In one aspect, provided is a method for isolating a biologic product from a process fluid. The method includes performing a capture operation by contacting the process fluid with an affinity resin in a process vessel for a period of time sufficient to allow binding of the biologic product to the affinity resin. The method adds a wash buffer to the process vessel and performs a wash operation by circulating the process fluid and resin mixture in a first direction through a tangential flow filtration (TFF) module with the wash buffer, thereby separating the mixture into a waste permeate stream and retentate fluid streams. The filtration module and the recirculation vessel are interconnected in a retentate fluid circuit, and the method recirculates the retentateDocket No. 1580.00231WO fluid stream through the retentate fluid circuit for a first number of diafiltration volumes (DVs) while directing the waste permeate stream to a waste container outside the fluid circuit, thereby producing a clarified retentate fluid. The method stops the circulation of the process fluid and resin mixture through the TFF module and extracts the process fluid and wash buffer through the TFF module via a permeate pump to allow the affinity resin to settle in the clarified retentate fluid. The method performs an elution operation by contacting the clarified retentate fluid with a volume of elution buffer for a period of time sufficient to allow disassociation of the biologic product from the affinity resin. The method then performs a harvest operation by extracting a second permeate fluid stream that includes the biologic product through the TFF module while the affinity resin is retained in the retentate stream without recirculating the resin through the retentate fluid circuit.

[0005] In one aspect, the method pumps the clarified retentate fluid to a fritted column and performs an elution operation by contacting the clarified retentate fluid with a volume of elution buffer for a period of time sufficient to allow disassociation of the biologic product from the affinity resin. The method then performs a harvest operation by extracting a permeate fluid stream comprising the biologic product through the TFF module while the affinity resin is retained in the retentate stream.

[0006] The method may also include where the retentate fluid enters the recirculation vessel at a top surface of the recirculation vessel such that an entrance of the retentate fluid is not submerged in the retentate fluid in the recirculation vessel.

[0007] The method may also include reversing a flow of a recirculation pump to pump the retentate fluid in a second direction opposite the first direction to force the retentate fluid into the recirculation vessel.

[0008] The method may also include where when a flow of the recirculation pump is reversed, air is pumped in the second direction to force the retentate fluid into the recirculation vessel.

[0009] The method may also include where the waste permeate stream from the wash operation comprises wash fluid, debris, and contaminants from the process fluid.

[0010] The method may also include where the retentate fluid is retained in the recirculation vessel with a frit.

[0011] The method may also include where the retentate fluid is retained in the recirculation vessel with a valve.Docket No. 1580.00231WO

[0012] The method may also include where a process line in which the retentate fluid enters the recirculation vessel at a top surface of the recirculation vessel is configured such that an entrance of the retentate fluid is submerged in the retentate fluid in the recirculation vessel, and wherein the process line comprises a vent.

[0013] The method may also include where when a flow of the recirculation pump is reversed, air is pumped in the second direction via the vent to force the retentate fluid into the recirculation vessel.

[0014] The method may also include where the TFF module utilizes a tubular / spiral flat sheet filter medium.

[0015] The method may also include where the circulation is stopped by stopping a recirculation pump and / or closing a valve below the TFF module.

[0016] The method may also include where during the wash operation, fluid lost to the permeate stream is not replaced in order to concentrate the fluid in the process vessel.

[0017] The method may also include where the permeate fluid stream comprising the biologic product is pumped by the permeate pump into a product vessel.

[0018] The method may also include pumping air or liquid into the recirculation vessel to resuspend the affinity resin in solution.

[0019] The method may also include where the biologic product is an antibody, a recombinant protein, or a virus particle.

[0020] The method may also include where the resin is functionalized with any suitable affinity ligand. In one example, Fc-binding ligands or ligands that bind to virus particles is used.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0021] Non-limiting embodiments of the present disclosure are described by way of example with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. The accompanying drawings are provided for purposes of illustration only, and the dimensions, positions, order, and relative sizes reflected in the figures in the drawings may vary. In the figures, identical or nearly identical or equivalent elements are typically represented by the same reference characters, and similar elements are typically designated with similar reference numbers, with redundant description omitted. For purposes of clarity and simplicity, not every element is labeled in every figure, nor is every element of each embodiment shownDocket No. 1580.00231WO where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure.

[0022] FIG. l is a block flow diagram depicting a capture / harvest operation in accordance with an aspect of the invention.

[0023] FIG. 2 illustrates a system that may be utilized in connection with a capture / harvest operation in accordance with an aspect of the invention.

[0024] FIG. 3 illustrates a system that may be utilized in connection with a capture / harvest operation in accordance with an aspect of the invention.

[0025] FIG. 4 illustrates a system that may be utilized in connection with a capture / harvest operation in accordance with an aspect of the invention.

[0026] FIG. 5 illustrates a fritted column in accordance with an aspect of the invention.

[0027] FIG. 6 is a graph of product concentration in the permeate outlet for the capture / harvest operation compared to a conventional harvest process in accordance with one embodiment.

[0028] FIG. 7 is a graph representing a pressure profile during a harvest capture TFF step.

[0029] FIG. 8 is a graph representing pressure profiles for primary and secondary depth filtration during conventional AAV clarification

[0030] FIG. 9 is a graph illustrating a flux profile during TFF of clarified AAV material.

[0031] FIG 10 is a graph illustrating AAV9 capsid yield for Harvest Capture.

[0032] FIG. 11 is a graph illustrating the standard multi-step clarification and purification process.DETAILED DESCRIPTION

[0033] Recovery of cell-produced biologic products, which may include antibodies and other recombinant proteins, virus particles, viral vectors, including adeno associated virus (AAV) particles or lentivirus (LV) particles or AAV vectors, or LV vectors, as well as other nucleic acid vectors including e.g., eukaryotic or bacterial plasmid vectors, and other nucleic acid based products, including antisense oligonucleotides, mRNA, siRNA, shRNA, cDNA, etc., involves the physical separation of the biologic product from producer or host cells and / or cell debris in the cell culture fluid or lysate, abbreviated herein as “CF”. Product recovery operations are typically initiated when producer cells have reached a certain predetermined cell density,Docket No. 1580.00231WO characterized as a viable cell density (VCD) or total cell density (TCD). Generally, the CF at this stage contains a high density of cells and cell debris. In operations where the cells are lysed to release the biologic product, the CF will contain high concentrations of host cell protein (HCP) and DNA (hcDNA) as well as other cell debris and / or virus particles and will typically also have a viscosity greater than 1 centipoise (cP), for example in a range of 2-30 cP, or 2-20 cP, or 2-10 cP. In either case, at the initial stage of product recovery from the CF, the CF will be characterized by high cell density or high turbidity, or both, and may also have a viscosity greater than 1 cP. Due to its high cell density and / or high turbidity, the CF is typically subjected to several clarification and concentration operations, including centrifugation, filtration, and diafiltration, to obtain a fluid of sufficient purity and concentration to be further purified by column chromatography, including affinity chromatography.

[0034] The present harvest capture technology provides a method for performing a combined affinity capture and harvest operation in bioprocessing applications. This technology utilizes a single tangential flow filtration operation and one or more parallel column operations to perform capture of the biologic product and then perform a harvest operation without the need for prior clarification steps, including e.g., prior centrifugation and / or prior filtration steps. Instead, as described in detail below, an appropriate capture resin, such as an appropriately functionalized affinity resin, is contacted directly with the unclarified CF under conditions permitting binding of the biologic product to the resin in a capture operation. In another example, the capture resin is an ion exchange resin (IEX), hydrophobic interaction chromatography resin (HIC), multimodal chromatography resin (MMC), or immobilized metal ion affinity chromatography resin (IMAC).

[0035] The capture phase may also include an optional concentration step. The capture operation, which may be performed in several modes as discussed in detail infra, is followed by a wash operation (which may be preceded by an optional concentration step), which proceeds by diafiltration through a filtration module. The filtration module comprises a filter medium, which may include a tangential flow filtration (TFF) medium, a tangential flow depth filtration (TFDF) medium, or a tubular / spiral flat sheet filter medium that may be referred to herein as a tangential flow chromatography filter or “TFCF”.

[0036] For non-woven flatsheet filter medium in the TFF module, the filter media may be constructed using a wetlaid method with base fibers of polyethylene, polypropylene, polyester or other similar base fibers. The non-woven flatsheet is then cut into continuous strips, and the strips are formed into a spiral, tubular device through ultrasonic welding on the continuous seam.Docket No. 1580.00231WOOther forms of welding or gluing may be used on the seam of the media. Alternatively, other types of manufacturing processes other than wetlaid methods may be used to form the non-woven flatsheet filter media.

[0037] When using the harvest capture filter to concentrate and wash cells and cell debris out of the system, it is necessary to operate in TFF mode to prevent filter plugging or fouling. In conventional harvest capture technologies, biologic product is then eluted from the capture resin and collected in a harvest operation that includes a series of diafiltration and concentration steps. However, as described herein, once large debris and other foulants have been removed from the system, TFF are no longer required to prevent filter fouling. This creates an opportunity to settle the resin directly in the flowpath to efficiently wash, elute, clean in place (CIP), and regenerate the resin, as described infra. The harvest operation also isolates the resin, which may then be regenerated. In an example, different fluids may be used in the CIP process to remove debris and other materials from the vessels, flow streams, resin, and other materials in the processes herein. For example, an alkaline solution may be used for CIP, such as sodium hydroxide or potassium hydroxide.

[0038] Conventional harvest capture technologies have limitations that prevent adoption in all bioprocessing applications. One limitation of conventional harvest capture is that enough liquid must be present to keep the resin from packing into a dense immovable slug. The consequence of requiring enough liquid to keep resin suspended in the flowpath is that the final product concentration and volume is dependent on the amount of liquid in the flowpath. Fluid in the flowpath that contains the product must be exchanged with a fluid that does not contain product, such as with diafiltration. The recovery of a product by diafiltration in TFF can be modeled using the equation below, where N is the number of diafiltration volumes, and R is the retention factor of the product.

[0039] % Recovery = 1 - eN(R-1)

[0040] In contrast, traditional chromatography is not dependent on the amount of liquid in the column. Instead, the volume of resin determines how much liquid is needed to displace the product. Traditional column chromatography can reach higher product concentrations and lower final volumes. The final volume becomes significant in certain applications, such as when the resin demand is high or when salt in the product must be diluted prior to a subsequent step. In conventional harvest capture methods, some bioprocessing applications would expand theDocket No. 1580.00231WO product volume to that greater than the initial feed volume, which becomes inconvenient to store and manage at manufacturing scales.

[0041] The methods and systems described here advantageously reduce process time, decrease costs, and increase efficiency as well as provide high product yield due at least in part to a reduction in the number of operational steps required. Because the process does not use TFF in the elution phase, an amount of water, buffers, or other liquids added to the system is reduced. The produced product is at a higher concentration with a lower volume of liquids. For example, compared to processes using the fritted column described herein, using a TFF process for elution at 45% solids would typically require approximately 6.5x more elution buffer to recover the biologic product. The product produced by the fritted column process accordingly has a volume of water usage that is lower by the same 6.5x factor.

[0042] In addition, the methods described herein provide a biologic product of high purity. For example, as described in detail infra, where the biologic product is recombinant virus particles, the recovered virus particles have low levels of contaminating host cell protein and nucleic acids. Alternatively, where the biologic product is recombinant protein, such as an antibody or monoclonal antibody, the recovered antibody has low levels of contaminates, including viruses.

[0043] FIG. l is a flow chart depicting a capture / harvest operation in accordance with an aspect of the invention.

[0044] In block 102, a process fluid containing a target biologic product is introduced to a recirculation vessel. The term “target biologic product” refers to a product of interest produced by cells. Exemplary biologic products include recombinant proteins, antibodies, nucleic acid vectors, including viral vectors such as AAV vectors or lentiviral (LV) vectors, virus particles, including AAV and LV particles and virus-like particles (VLPs). Producer cells may be bacterial cells, yeast cells, insect cells, or mammalian cells. The biologic product may be secreted from the cells or otherwise released from the cells into the cell culture fluid (CCF), for example by cell lysis. Where the cells are lysed to release the biologic product, the CCF may also be referred to as the “lysate”.

[0045] An important aspect of the methods described here is that it is not necessary to subject the CCF to any clarification processes prior to contacting with a capture resin. As discussed above, the CCF at this stage will be characterized by high cell density or high turbidity, or both, and may also have a viscosity greater than 1 centipoise (cP). For example, in some aspects the CCF may have for a cP of from 1.5-30 cP, or 1.5-20 cP, or 1.5-10 cP, or a cP of about 1.5, aboutDocket No. 1580.00231WO2, about 3, about 4, abut 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20. In the following discussion, the CCF is referred to as the process fluid, the product stream, or the product fluid. Accordingly, the process fluid at the start of a capture / harvest operation in accordance with the methods described here is characterized by a high cell density and / or high turbidity and optionally a viscosity greater than 1 cP, as defined in more detail in the following paragraphs.

[0046] In aspects where the process fluid is characterized by a high cell density, cell density may be measured as viable cell density or “VCD”, including VCD pre-lysis where the cells are lysed to release the cell product to be recovered, or total cell density “TCD” which includes both viable and non-viable cells. For example, the process fluid may have a VCD or TCD of from lxl0A5 (10E5) to 10E9 cells per milliliter (ml). In some aspects, the process fluid may have a VCD or TCD of from about 10E5 to 10E6 cells / ml or about 10E6 to 10E7 cells / ml, or about 10E8 to 10E9 cells / ml, for mammalian and insect cells. In other aspects, for example where the cells are bacterial cells, cell density may be measured in units of optical density (OD). For example, where the cells are E. coli cells, the process fluid may have an OD of from 1-350 at 600 or 620 nm, or from 30-300 or from 30-250.

[0047] In aspects where the process fluid is characterized by a high turbidity, turbidity may be measured in nephelometric turbidity units (NTUs). In some aspects, the process fluid may have a turbidity of from about 100-30,000 NTU. In some aspects, the process fluid may have a turbidity of from about 100-10,000 NTU, or from about 100-5,000 NTU, or from about 100- 2,500 NTU, or from about 100-1,000 NTU, or from about 100-500 NTU. In some aspects, the process fluid may have a turbidity of from about 200-1,000 NTU or about 300-1,000 NTU, or about 400-1,000 NTU. In some aspects, the process fluid may have a turbidity of about 300, about 400, about 500, about 600, about 700, about 800, or about 900 NTU. In this context, the turbidity of the process fluid refers to its turbidity prior to addition of the capture resin. In general, the resin may add from about 3,000-6,000 NTU or more to the fluid's turbidity, depending on the amount of resin added. The methods and systems described here allow for capture of the biologic product from highly turbid process fluids, without the need for prior clarification steps such as prior centrifugation or filtration operations.

[0048] In aspects where the process fluid is characterized by a viscosity greater than 1 cP, the process fluid may have a viscosity of from about 5-100 cP, or from 5-50 cP, or from 5-25 cP. In some aspects, the process fluid may have a viscosity of about 2, about 5, about 10, or about 15Docket No. 1580.00231WO cP. The process fluid may be characterized by a viscosity greater than 1, for example, where the cells producing the biologic product are lysed to release the biologic product into the process fluid prior to capture.

[0049] In some aspects, the process fluid is characterized by one or more of a high cell density, which may be a high viable cell density (VCD), a high total cell density (TCD) or high optical density (OD), a high turbidity, and / or a viscosity greater than 1, where high cell density, high turbidity, and viscosity are defined by the ranges discussed above.

[0050] With reference to FIG. 1, in capture phase 104, the process fluid containing the target biologic product is contacted with a capture resin, for example, an affinity resin, in a recirculation vessel to produce a resin mixture. In other examples, the resin is an IEX, HIC, MMC, or IMAC resin. The resin mixture is recirculated through a system that includes a TFF filter. During capture, process fluid containing the biologic product as well as cells and / or cell debris, or including cell lysate where the cells are lysed to release the biologic product, and having a high cell concentration and / or high turbidity, as described above, is contacted with a capture resin for a period of time under conditions suitable for binding of the biologic product to the resin. The period of time is selected based on the particular resin in accordance with the manufacturer's recommendations. Conditions suitable for binding may include, for example, a specified temperature, in accordance with the resin manufacturer's recommendations.

[0051] In aspects where the cells are lysed in order to release biologic product, the cells may be lysed prior to initiating the capture operation using any suitable method, such as a chemical or mechanical method.

[0052] In accordance with the methods described here, the contacting of the capture phase 104 may be performed by direct addition of capture resin to a primary process vessel, which may be a bioreactor, or by any other suitable method, for example by addition of process fluid to a secondary process vessel containing the resin, or the resin may recirculate within a filter module flow path, also referred to as a retentate flow path. Thus, in aspects, the capture operation may be performed by addition of capture resin directly to a bioreactor or directly to a secondary process vessel in fluid communication with a bioreactor. Alternatively, the resin may reside in a retentate fluid circuit of a filtration module that is fluidly connected to a bioreactor or secondary process vessel. In accordance with any of the foregoing configurations, the retentate fluid circuit may also be fluidly connected to one or more of a wash buffer tank, an elution buffer tank, and a regeneration buffer tank. In another example, the capture operation may be performed in anDocket No. 1580.00231WO external column or vessel, such as a fritted column. Examples employing a fritted column are discussed in greater detail with respect to FIG. 4 infra.

[0053] Suitable resins for use in capture phase 104 of biologic product in accordance with the methods described here are in the form of beads or other particulates having a mean particle diameter that is about 1.0 to 10 times larger than the average pore size of the filter medium. Suitable capture resins include affinity resins, ion exchange resins, hydrophobic interaction chromatography (HIC) resins, multimodal chromatography resins (MMC), and immobilized metal affinity chromatography (IMAC) resins. In some aspects of the methods described here, the resin may have a mean particle size of about 20 microns, about 30 microns, or about 50 microns. In some aspects, the resin may have a mean particle size of up to 200 microns, for example from 20-50 microns or from 50-200 microns, or about 100 microns, about 150 microns, or about 200 microns.

[0054] Suitable affinity resins include a chemistry or ligand chemistry capable of binding the biologic product to the resin with high affinity. For example, in some aspects the resin is functionalized with a ligand, such as Staphylococcus aureus Protein A or a derivative thereof, capable of binding the Fc region of an antibody or other Fc-containing protein. In some aspects, the resin is functionalized with a ligand capable of binding a virus particle, such as an AAV or LV particle. In some aspects, the ligand is capable of binding one or more AAV capsid proteins. Suitable resins that may be utilized include resins formed of discrete polymeric particles functionalized with an affinity ligand where the polymeric particles may be made from a polysaccharide such as agar, agarose, dextran, starch, cellulose, pullulan, etc., and stabilized variants and derivatives thereof; or where the particles are made from a synthetic polymer such as polystyrene, polyvinylether, polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, etc. Suitable affinity resins include regenerated resins and single use or disposable resins. Suitable affinity resins are commercially available.

[0055] For example, AVIPure® AAV affinity resins and CaptivA® Protein A affinity resins from Repligen Corp. (Waltham MA) provide capture of AAV virus particles (also sometimes referred to in the art as AAV vectors) and Fc-containing proteins, respectively. Other suitable resins include resins functionalized with Protein A or derivatives of Protein A, such as Eshmuno® A (MilliporeSigma) and MabCaptureC® (Thermo Fisher Scientific); resins functionalized with other ligands, including metal chelates for isolation of recombinant proteins, especially histidine- tagged proteins, such as Fractogel® Metal Chelate (MilliporeSigma) or Capto® ChelatingDocket No. 1580.00231WO(Cytiva); and other resins functionalized with ligands for capture of virus particles, such as POROS® CaptureSelect AAV (Thermo Fisher Scientific) and Capto® AVB (Cytiva) for isolation of AAV, or CaptureSelect Lenti VSVG (Thermo Fisher Scientific) for capture of VSV-G pseudotyped LV particles.

[0056] With reference to FIG. 1, following capture 104, the method proceeds to a wash phase 106 in which a volume of wash buffer is added in order to dislodge nonspecific binding of cellular debris, proteins, and nucleic acids from the resin particles and wash away unbound cells, cell debris, and other contaminants, including e.g., host cell nucleic acids including DNA and RNA, host cell proteins (HCP), and virus particles where the biologic product is not the virus particles, by diafiltration through a TFF module as described herein, interconnected with the process vessel in a fluid circuit. In some aspects, the flow-through or permeate stream is returned to the bioreactor or secondary process vessel. In other aspects, the flow-through or permeate stream is sent directly to a waste container. Suitable buffers for the wash operation include, for example, include neutral pH buffers such as PBS, 10-50 mM sodium phosphate, or neutral Tris buffer. Additional salt additives may also be added to the solution..

[0057] In some aspects, the wash buffer does not include a phosphate buffer. In some aspects, the wash buffer may include 0.15M sodium chloride (NaCl), neutral pH.

[0058] A TFF module for use in the systems and methods described herein comprises one or more of a plurality of filtration media or elements, encased in a filter housing. The filter housing may include any inlets, outlets, filter supports, valves, or any other suitable process equipment that supports a TFF module process. The filtration media may be any suitable membranes or other filter materials as described herein. Some example filters membranes may be generally characterized in the industry as having lumens of less than 2 mm in diameter, and “tubes” which term may be used where the lumen has a diameter larger than 2 mm, for example in the range of 2-12 mm. The filter elements used herein may refer to fiber or tube shaped filter elements that collectively encompass lumens ranging from 0.2-12 mm in diameter, which is also referred to as the internal diameter or “ID” of the filter element. In some aspects, the filter elements are constructed of non-woven fibers having a pore size in the range of 50-200 microns. In aspects, the filter medium may be in the form of a flat sheet spiral wound into a tubular form, which may also be referred to as “tubular / spiral wound” or in the context of the present invention, a tangential flow chromatography filter or “TFCF.” In aspects, the filtration medium is constructed from a nonwoven polypropylene / polyethylene polymer having a pore size of from 50-200Docket No. 1580.00231WO microns. Suitable membranes include nonwoven wetlaid membranes. In aspects, the TFCF medium is not formed by extrusion. In aspects, the filter medium may be a "tangential flow depth filtration" (TFDF) filter. TFDF refers to a filtration process that combines tangential flow filtration with depth filtration. TFDF systems employ non-laminar feed flows through a filter medium that includes a thick wall of high porosity comprising tortuous flow paths which may include settling zones and narrowing channels. In another example, the filter medium is a flat mesh rolled into a tube with an inner diameter of approximately 5 to 20 mm.

[0059] The filter housing of the TFF module includes a process fluid inlet to bring process fluid into the housing at an upstream or proximal end of the module and a retentate outlet to bring retentate fluid out of the housing from the downstream or distal end of the module. In one aspect, the TFF module is configured to flow the process fluid upwards vertically through the filter housing. In other aspects, the TFF module is configured to flow the process fluid downwards vertically through the filter housing.

[0060] The filter housing also includes at least one permeate outlet to bring permeate fluid out of the housing. The housing may include other ports, for example a vent port and a drain port. In some aspects, the filter medium is encapsulated in the filter housing to provide an integral device that may be a single-use or disposable unit. In some aspects, the single-use or disposable unit may be sterile. In some aspects, the single-use or disposable unit may be sterilized by ethylene oxide gas sterilization or by irradiation, for example X-ray irradiation, gamma irradiation or electron beam irradiation.

[0061] Each element of the filter medium is comprised of a plurality of non-woven polymer fibers characterized by a pore rating of from 10-50 microns, or from 20-50 microns, or about 30 microns, about 40 microns, or about 50 microns. In aspects, the polymer fibers are sintered. In other aspects, the polymer fibers are melt-blown.

[0062] Porosity (P) is calculated as a weight percentage based on the density (d) of the hollow fiber element(s) measured in grams per cubic centimeter (g / cc). Where the hollow fiber element(s) consist of more than one type of polymer, a “dA” term takes into account the aggregate or blended density of the material (dA) such that porosity of the aggregate material is calculated as:

[0063] P = l-(d / dA).

[0064] For example, where the filter element(s) are made of bi-component materials, dA is calculated as a sum of each polymer's density multiplied by its weight percentage in the material.Docket No. 1580.00231WOThus, for a material comprised of two polymers, Pl and P2, present in amounts of 70 / 30 weight percent, respectively, each having a density dl and d2, respectively, the aggregate density is calculated as

[0065] dA = (0.70 x dl) + (0.3 x d2)

[0066] In some aspects, the depth filtration (TFDF) elements are defined by a porous wall of from about 2-10 mm thick or about 2-6 or 4-10 mm thick, defining a lumen having an ID if from about 1-12 mm in diameter or from about 3-6 mm or about 4-5 mm in diameter, where the porosity of the wall is in the range of from about 50-80% (0.50-0.80) or from about 50-70%. In some aspects, the filter element(s) have a pore rating of from 10-50 microns, or from 20-50 microns, or from 30-50 microns. In some aspects, the hollow fiber element(s) have a pore rating of about 30 microns, about 40 microns, or about 50 microns.

[0067] The depth filter medium may be defined by its cross-sectional area and number of hollow fiber elements that comprise the medium, as well as by parameters of the hollow fiber element(s) that form the medium, such as the ID, wall thickness, porosity, and length of the hollow fiber element(s). In some aspects, the hollow fiber depth filter medium may be defined by its permeability in terms of its normalized water permeability (NWP) measured as LMH / psi. In some aspects, the NWP of the hollow fiber depth filter medium is from about 100,000 to about 150,000 LMH / psi.

[0068] In aspects where the filter medium is a tangential flow chromatography or “TFC” filtration medium, the filter elements are constructed of non-woven fibers having a pore size in the range of 50-200 microns. In aspects, the TFC filter medium may be in the form of a flat sheet spiral wound into a tubular form, referred to herein as a tangential flow chromatography filter or “TFCF”. In some aspects, the TFCF medium is constructed from a nonwoven polypropylene / polyethylene polymer having a pore size of from 50-200 microns. In aspects, the TFCF medium includes a porous wall having a thickness of from 0.1 to 0.5 mm defining a lumen having an ID of from 1-12 mm, a porosity of from about 60-90%, and a pore rating of 10, 20, 30, 40, 50, 100, 150, or 200 microns. Suitable membranes include nonwoven wetlaid membranes. In aspects, the TFCF medium is not formed by extrusion.

[0069] In some aspects, the hollow fiber elements of the TFF filtration media are constructed of a material that includes one or more of polysulfone, polyethersulfone (PES) or modified polyethersulfone (mPES). In embodiments, the polysulfone, PES or mPES has an anisotropic structure.Docket No. 1580.00231WO

[0070] The hollow fibers for use in the filter units may be formed from a variety of materials using a variety of processes. For example, hollow fibers may be formed by assembling numerous particles, filaments, or a combination of particles and filaments into a tubular shape. The pore size and distribution of hollow fibers formed from particles and / or filaments will depend on the size and distribution of the particles and / or filaments that are assembled to form the hollow fibers. The pore size and distribution of hollow fibers formed from filaments will also depend on the density of the filaments that are assembled to form the hollow fibers. For example, mean pore sizes ranging from 0.5 microns to 50 microns may be created by varying filament density.

[0071] Suitable particles and / or filaments include both inorganic and organic particles and / or filaments. In some embodiments, the particles and / or filaments may be mono-component particles and / or mono-component filaments. In some embodiments, the particles and / or filaments may be multi-component (e.g., bi-component, tri-component, etc.) particles and / or filaments. For example, bi-component particles and / or filaments having a core formed of a first component and a coating or sheath formed of a second component, may be employed, among many other possibilities.

[0072] In various embodiments, the particles and / or filaments may be made from polymers. For example, the particles and / or filaments may be polymeric mono-component particles and / or filaments formed from a single polymer, or they may be polymeric multi-component (i.e., bi- component, tri-component, etc.) particles and / or filaments formed from two, three, or more polymers. A variety of polymers may be used to form mono-component and multi-component particles and / or filaments including polyolefins such as polyethylene and polypropylene, polyesters such as polyethylene terephthalate and polybutylene terephthalate, polyamides such as nylon 6 or nylon 66, fluoropolymers such as polyvinylidene fluoride (PVDF) and polytetrafluoroethylene (PTFE), among others. Suitable polyethylene polymers include, without limitation, high-density polyethylene (HDPE) and high- or ultra-high-molecular weight polyethylene (UHMWPE).

[0073] Particles may be formed into tubular shapes by using, for example, tubular molds. Once formed in a tubular shape, particles may be bonded together using any suitable process. For instance, particles may be bonded together by heating the particles to a point where the particles partially melt and become bonded together at various contact points (a process known as sintering), optionally, while also compressing the particles. As another example, the particlesDocket No. 1580.00231WO may be bonded together by using a suitable adhesive to bond the particles to one another at various contact points, optionally, while also compressing the particles.

[0074] Filament-based fabrication techniques that can be used to form tubular shapes include, for example, simultaneous extrusion (e.g., melt-extrusion, solvent-based extrusion, etc.) from multiple extrusion dies, or electrospinning or electrospraying onto a rod-shaped substrate (which is subsequently removed), among others.

[0075] Filaments may be bonded together using any suitable process. For instance, filaments may be bonded together by heating the filaments to a point where the filaments partially melt and become bonded together at various contact points, optionally, while also compressing the filaments. As another example, filaments may be bonded together by using a suitable adhesive to bond the filaments to one another at various contact points, optionally while also compressing the filaments.

[0076] In particular embodiments, numerous fine extruded filaments may be bonded together to at various points to form a hollow fiber, for example, by forming a tubular shape from the extruded filaments and heating the filaments to bond the filaments together, among other possibilities.

[0077] In some aspects, the hollow fiber elements of the depth filter medium are formed from sintered or melt-blown polymer fibers. The terms “fibers” and “filaments” in the context of “polymer fibers” or “polymer filaments” are used interchangeably herein. Polymers that may be used include polyolefins such as polyethylene and polypropylene, polyesters such as polyethylene terephthalate and polybutylene terephthalate, polyamides such as nylon 6 or nylon 66, fluoropolymers such as polyvinylidene fluoride (PVDF) and polytetrafluoroethylene (PTFE), among others. Suitable polyethylene polymers include, high-density polyethylene (HDPE) and high- or ultra-high-molecular weight polyethylene (UHMWPE). In some aspects, the polymer is selected from polypropylene, a polyester, and mixtures thereof.

[0078] The term “sintered” in this context refers to the use of heat and optionally pressure in a bonding process. In this process, the polymer fibers are heated to a point where the filaments partially melt and become bonded together at various contact points, optionally, while also compressing the filaments. Thus, sintering bonds fibers where they touch, creating void spaces between the fibers. Numerous fine extruded filaments may be bonded together to at various points to form a hollow fiber, for example, by forming a tubular shape from the extruded filaments and heating the filaments to bond the filaments together.Docket No. 1580.00231WO

[0079] The term “melt-blown” refers to the use of a gas stream at an exit of a filament extrusion die to attenuate or thin out the filaments while they are in their molten state. Melt-blown filaments are described, for example, in US 5607766 to Berger. Mono- or bi-component filaments may be attenuated as they exit an extrusion die using known melt-blowing techniques to produce a collection of filaments. The collection of filaments may then be bonded together in the form of a hollow fiber.

[0080] In some aspects, hollow fibers for use in the filter medium of the filter module described here may be formed by combining bicomponent filaments having a sheath of first material which is bondable at a lower temperature than the melting point of the core material. For example, hollow fibers may be formed by combining bicomponent extrusion technology with melt-blown attenuation to produce a web of entangled biocomponent filaments, and then shaping and heating the web, for example in an oven or using a heated fluid such as steam or heated air, to bond the filaments at their points of contact. An example of a sheath-core melt-blown die is schematically illustrated in US 5,607,766 in which a molten sheath-forming polymer and a molten core-forming polymer are fed into the die and extruded together. The molten bicomponent sheath-core filaments are extruded into a high velocity air stream, which attenuates the filaments, enabling the production of fine bicomponent filaments. US 3,095,343 to Berger shows an apparatus for gathering and heat-treating a multi-filament web to form a continuous tubular body, such as a hollow fiber, of filaments randomly oriented primarily in a longitudinal direction, in which the body of filaments are, as a whole, longitudinally aligned and are, in the aggregate, in a parallel orientation, but which have short portions running at random in non-parallel diverging and converging directions. In this way, a web of sheath-core bicomponent filaments may be pulled into a confined area, for example by using a tapered nozzle having a central passageway forming member, where it is gathered into tubular rod shape and heated or otherwise cured to bond the filaments.The hollow fiber depth filter medium does not have a defined pore size. However, pore size

[0081] Pore sizes for the filter medium may be determined using methods known in the art, for example a “bubble point test.” The bubble point test is based on the fact that, for a given fluid and pore size, with constant wetting, the pressure required to force an air bubble through a pore is inversely proportional to the pore diameter. In practice, this means that the largest pore size of a filter can be established by wetting the filter material with a fluid and measuring the pressure at which a continuous stream of bubbles is first seen downstream of the wetted filter. The pointDocket No. 1580.00231WO at which a first stream of bubbles emerges from the filter material is a reflection of the largest pore(s) in the filter material, with the relationship between pressure and pore size being based on Poiseuille's law which can be simplified to P=K / d, where P is the gas pressure at the time of emergence of the stream of bubbles, K is an empirical constant dependent on the filter material, and d is pore diameter. In this regard, pore sizes determined experimentally may be measured using a device such as a POROLUX™ 1000 Porometer (Porometer NV, Belgium), or similar device.

[0082] Given the large pore sizes of the filter media for use in the methods described here, a passage / retention test may be used to determine pore size, rather than a bubble point test. In accordance with the methods described here, the mean pore size of the hollow fiber element or elements forming the filter medium is selected to retain the resin particles utilized for capture of the biologic product. In some aspects, the mean pore size of the material forming the porous wall of the element or elements is from 1.0 to 10 times smaller, or from about 2-5 times smaller, than the mean diameter of the resin. In some aspects, the wall of the hollow fiber element(s) is characterized by a porosity of from about 50-70% (0.50-0.70), or from about 55-70%. In some aspects, the hollow fiber element(s) have a pore rating of from 10-50 microns, or from about 20 microns, about 30 microns, about 40 microns, or about 50 microns, depending on the size of the resin particles.

[0083] In some aspects, the as-formed filter element may be further coated with a suitable coating material either on the inside or outside of the fiber, which coating process may also act to reduce the pore size of the hollow fiber.

[0084] In some aspects, the capture resin beads have a mean diameter of 20 microns, 50 microns, or 75 microns, and the filter medium comprises elements in which porosity of the wall is in the range of from about 50-90% (0.50-0.90) or about 50%, about 60%, about 70%, about 80%, or about 90%. In some aspects, the element(s) have a pore rating of from 10-50 microns, or about 10 microns, about 20 microns, about 30 microns, about 40 microns, or about 50 microns.

[0085] Returning to FIG. 1, the wash phase 106 operation includes a number of diafiltration steps sufficient to remove cells, cell debris, and related impurities from the resin using a diafiltration buffer, which may for example, phosphate buffered saline (PBS) or other suitable buffer. The extent of resin washing performed during the wash phase 106 may be measured in terms of diafiltration volumes (DVs). A single DV is the starting volume of the slurry (liquids and solids) in the process vessel following capture 102. Thus, one DV has been processed whenDocket No. 1580.00231WO the volume of permeate is equal to equal the starting volume. Diafiltration may be performed in a batch or continuous process. In accordance with some aspects of the methods described here, diafdtration is carried out using from 5-20 DVs. The diafiltration operation is preferably performed at an average process flux of about 500-4000 LMH, or about 1000-4000 LMH.

[0086] Returning to FIG. 1, in the settle phase 108, the method settles the resin and the product in the recirculation vessel. When being settled, the resin may be concentrated, for example, to about 1 to 80 percent by volume (% v / v), or from about 10-70% v / v or from about 20-60% v / v. In some aspects, the resin may be concentrated by a factor of from 10 to 100 times (10-100X).

[0087] The resin may be settled by stopping an operation of any recirculation valves and / or closing any valves in the recirculation loop that proceeds from the recirculation vessel through the filter and back to the recirculation vessel. In one example, a valve beneath the filter is closed while the pump continues operation. In this manner, the resin in the recirculation loop below the valve is transferred to the recirculation vessel. In an example, a valve above the filter is closed while the pump continues operation to transfer resin out of the filter, through the recirculation loop, and into the recirculation vessel. In another example, the recirculation pump under the filter is reversed to force the resin and the fluids backwards through the filter and into the recirculation vessel. In another example, air is pumped through the recirculation loop to force the fluids and resin out of a portion of the recirculation loop and into the recirculation vessel.

[0088] A pump on the outlet of the permeate stream that exits the filter housing may be started or engaged to pump liquids out of the closed recirculation loop. For example, any wash buffers, cells, cell debris, or other contaminants that pass through the filter are pumped away from the recirculation loop. The remaining capture resin with the product are settled into the recirculation vessel and / or the filter while the wash buffer and the contaminants are extracted through the permeate line.

[0089] In another aspect, the method settles the resin and the product in the recirculation loop. That is, the pump 212 is stopped and any appropriate valves are closed to allow the resin and the product to reside in the filter, the recirculation vessel 202, and any connection lines of the recirculation loop.

[0090] In another aspect, the resin is settled in an external column, such as a fritted column. Instead of forcing the resin back into the recirculation vessel, the resin is pumped to the fritted column by operation of the recirculation pump. When the resin is pumped into the fritted column, the wash buffer or any other remaining fluids may be extracted from the fritted column andDocket No. 1580.00231WO elution fluid may be pumped to the fritted column. Examples using a fritted column for the settle phase 108 and the elution phase 110 are illustrated and discussed in greater detail in FIG. 4 infra. An example fritted column is illustrated and discussed in FIG. 5 infra.

[0091] Returning to FIG. 1, an elution phase 110 is initiated by addition of an elution buffer, either to the recirculation vessel or a secondary vessel containing the resin-bound biologic product, or to the retentate fluid circuit containing the settled resin-bound biologic product. The elution buffer is formulated to disrupt the high affinity binding between the resin and biologic product and may be selected based on manufacturer's instructions for the particular resin utilized. The capture resin is incubated with elution buffer for a period of time, or residence time, and under conditions suitable for maximum dissociation of the biologic product from the resin. The time and conditions, which may include a specified pH and salt concentration, may also be determined in accordance with manufacturer's instructions for the resin.

[0092] Returning to FIG. 1, following the elution phase 110, a harvest operation 112 is initiated by extracting the dissociated biologic product through the filter module and collecting product from the permeate stream. For example, the permeate pump may be started to extract the biologic product through the filter while the capture resin is retained in the recirculation vessel and / or the filtration loop. If the resin is to be retained in the recirculation vessel 202, a frit, a screen, or other type of filter that will not pass the resin may be introduced in the outlet of the recirculation vessel 202. In another example, to allow the settle phase 108 to occur in the recirculation loop, the resin is allowed to break apart and drain into the filter 204 and the recirculation loop along with the dissociated biologic product and the elution fluid. Any resin in the filter 204 is unable to pass through the filter membrane allowing the biologic product to be removed from the filter.

[0093] Advantageously, because the recirculation loop is not actively pumping the resin in a diafiltration process to remove the biologic product, excess or additional fluids are not required to maintain the resin in suspension and prevent the resin from becoming a slug that blocks the filter.

[0094] In some aspects, an additional filtration step utilizing a 0.8 / 0.2 micron or other similar graded membrane filter such as 0.45 / 0.2 micron glass fiber or polymeric capsule filter may be added to the permeate coming off the filter into the harvest vessel to remove any large particles that may have come off the TFF, TFDF, or TFCF filter during the elution step.

[0095] It should also be understood that multiple harvest / capture flowpaths and modules as described herein may be connected in parallel. In an aspect, at least two flowpaths and modulesDocket No. 1580.00231WO are connected in parallel such that the resin of a first module may be regenerated while that in a second parallel flowpath / module continues to perform the capture and elution steps, thereby enabling continuous operation.

[0096] FIG. 2 illustrates a system that may be utilized in connection with a capture / harvest operation in accordance with an aspect of the invention. As illustrated in FIG. 2, the capture operation is carried out in a recirculation vessel 202A feed pump 210 may be used to add resin or process fluid to the recirculation vessel 202 from a feed addition vessel 206. For example, where the capture phase 104 is performed by direct addition of resin to a primary process vessel, such as a recirculation vessel 202, the feed pump 210 may be used to transfer resin to the primary process vessel. The feed pump 210 may later be used to transfer the biologic product feed to the recirculation vessel 202 from the feed addition vessel 206. The feed pump 210 may be used at a different time to transfer wash buffer during a wash phase 106 and / or elution buffer during an elution phase 110. In alternate embodiments, multiple vessels and / or multiple pumps may be used to perform the functions described as being performed by feed pump 210 and feed addition vessel 206.

[0097] During capture phase 104, the process fluid is incubated with resin for a period of time, or residence time, under conditions suitable for binding of the biologic product to the resin, as discussed above. During this period, the process fluid and resin may be mixed using any suitable means. In one aspect of the methods described here, the mixing is performed by operation of an impeller of the recirculation vessel 202. In accordance with this aspect, valve 222 remains closed. In another aspect, the process fluid and resin may be mixed by recirculation through the filter 204 by operation of recirculation pump 212. In accordance with this aspect, valve 216 and valve 222 are opened while valve 218 remains closed. The resin and the product recirculate through the filter housing and back into the recirculation vessel 202. Only a minimal amount of the liquid or particles exit through the filter in the permeate stream because the valve 218 is closed.

[0098] Following capture phase 104, a wash phase 106 in initiated. The wash phase 106 may include a series of concentration and diafiltration steps as necessary to remove contaminants and impurities from the product-bound resin. During wash, valve 216, valve 222, valve 218, and valve 228 are opened. Fluid circulates through the filter 204 by operation of the recirculation pump 212. The recirculation pump 212 may be, for example, a low shear centrifugal pump. The filter 204 separates the fluid into a retentate stream containing product-bound resin and aDocket No. 1580.00231WO permeate stream containing cells and / or cell lysate and other debris. Valve 228 may be opened and the permeate stream may be directed by operation of a permeate pump 214 to a waste vessel 230, container, waste stream, or in any suitable alternative not depicted in the figure. The permeate stream may be directed to a secondary container, for example to capture producer cells. The permeate pump 214 may be, for example, a peristaltic pump.

[0099] The retentate stream flows through a retentate fluid loop back into the recirculation vessel 202. Buffer, cell culture media, or optionally additional process fluid as described below, may be added to the recirculation vessel 202, for example via an inlet line, the feed pump 210, or via any other suitable process, in order to replace fluid volume lost to the permeate stream.

[0100] In examples, the recirculation loop is routed to a top surface of the recirculation vessel 202. The conduit that delivers the retentate to the top of the recirculation vessel 202 may terminate at a level above the level of liquid and resin in the recirculation vessel 202 such that the inlet conduit is not submerged.

[0101] Following the wash phase 106, the resin in the recirculation loop is isolated in a settle phase 108. When using the filter 204 to concentrate and wash cells and cell debris out of the system, the filter 204 should operate in TFF mode to prevent filter plugging or fouling. However, after large debris and other foulants have been removed from the system, TFF is no longer required to prevent filter fouling. The resin may be settled directly in the flowpath to efficiently elute and regenerate the resin.

[0102] In an example, the filter 204 is positioned below the recirculation vessel 202 to encourage fluid to drain out the filter 204 with gravity. In this example, the flow through the filter 204 is downwards vertically. This design may use a positive displacement pump as the recirculation pump 212 below the filter 204 to effectively move either liquid, solid, or gas around the recirculation loop. The recirculation pump 212 is also oriented in such a way to pull fluid down through the filter 204 to avoid high pressure situations that can form when pumping against a solid interface.

[0103] The resin can be settled in any suitable manner and location depending on the design of the flowpath. The settling should be configured to displace the slurry in the primary recirculation line and trap the resin in the recirculation vessel 202 above the filter 204. In other aspects, the resin may be settled in any other suitable location, vessel, or column. For example, the resin may be settled in an external fritted column, as described in FIG. 4 infra.Docket No. 1580.00231WO

[0104] In systems in which the return line in the primary recirculation loop is above the liquid level as described herein, a peristaltic pump can be reversed to pull air into the recirculation loop and push fluid back up into the recirculation vessel 202 (or any other vessel used for settling). In another example, an auxiliary line can be added to the primary recirculation loop to pump air or liquid into the loop displacing the resin back into the recirculation vessel 202. For example, if a Levitronix pump is used at large scale trapping the resin on one side of the pump will require additional valves, clamps, and auxiliary lines. For example, valve 234 may be closed to prevent flow of fluids through the outline line of the Levitronix pump.

[0105] Once the resin is trapped in the recirculation vessel 202, the recirculation pump 212 can be stopped to encourage the resin to settle naturally.

[0106] In another example, the resin may be settled in the recirculation vessel 202 and also in the filter 204. That is, the recirculation pump 212 may be stopped and / or a valve such as valve 216 may be closed to keep the resin from flowing through the recirculation loop. The resin is thus settled in the recirculation vessel 202, the filter 204, and any lines connecting the process elements from the pump to the recirculation vessel 202. In an example, the recirculation pump 212 may be operated after the closing of valve 216 to cause the resin in the line after the recirculation pump 212 to be deposited in the recirculation vessel 202. In this example, a separate liquid feed line, such as from a separate feed vessel, provides fluid below closed valve 216 to pump the resin remaining in the recirculation loop into the recirculation vessel 202.

[0107] The fluid that is used to settle the resin in the recirculation vessel 202 may be any of the process fluids described herein. For example, the wash buffer may be suspending the resin at the time that the resin is gathered in the recirculation vessel 202. The resin suspended in the wash buffer is pumped in any of the described methods into the recirculation vessel 202 and a valve closed to trap the resin. In another example, the resin is not gathered in the recirculation vessel 202 until the elution buffer is added to the system. For example, the elution buffer is added as described in the elution phase 110 before the resin is gathered in the recirculation vessel 202. The resin is thus suspended in the elution buffer, pumped into the recirculation vessel 202, trapped in the recirculation vessel 202, and then settled.

[0108] After the recirculation loop has been stopped by stopping the recirculation pump 212 and the resin is trapped in the recirculation vessel 202, the excess liquids may be removed from the settled resin by opening valve 218 and turning on the permeate pump 214. The permeate pump 214 may remove any excess liquids that pass through the filter membrane. For example,Docket No. 1580.00231WO the wash buffer from the wash phase 106 may be removed along with any cells, debris, or other contaminants. If an agitator is in the recirculation vessel 202, the agitator may be stopped. The remaining resin settles into a settled arrangement and is no longer suspended in a slurry. Tn this manner, the resin in the recirculation loop or the recirculation vessel 202 may act in a manner similar to a chromatography column in an elution phase 110.

[0109] Following the settle phase 108, the biologic product is eluted from the resin in an elution phase 110. During elution, if an elution buffer has not already been added to settle the resin, an elution buffer is added to the recirculation vessel 202, for example via a feed pump 210. The elution buffer is allowed to contact any portion of the recirculation loop that has resin, such as the recirculation vessel 202, the filter 204, and any conduits connecting process elements.

[0110] Product-bound resin is incubated for a period of time to provide maximum dissociation of the biologic product from the resin. During the elution phase 1 10, the resin is not pumped around the recirculation loop via the recirculation pump 212. Because the resin is not being pumped, the recirculation loop does not require a particular amount of liquids sufficient to keep the resin in suspension. Only an amount of elution fluid is required sufficient to extract the desired percentage of the product from the resin.[OHl] After the elution phase 110, the product is recovered in a harvest operation 112. In an example, valve 218 is opened.

[0112] The elution buffer and the product pass through the filter membrane as a permeate. The permeate pump 214 may be started and valve 226 opened to extract the permeate from the filter and transfer the permeate to a product vessel 232. Additional elution buffer may be added to the recirculation vessel 202 as the permeate is being extracted in a dynamic process. However, the amount of fluid with the product in the product vessel 232 is significantly lower than an amount of fluids in a permeate recovered in a conventional diafiltration harvest capture process.

[0113] If the resin was settled only in the recirculation vessel 202 before elution and not in the filter 204, then two phases would exist in the recirculation vessel 202 after a complete elution. The settled resin would be in one phase and the elution buffer with the product would be in a second phase. If valve 222 was closed to retain the resin in the recirculation vessel 202, then valve 222 is opened. The resin, the elution buffer, and the product are allowed to fill the filter 204 and the lines above the valve 216 or the recirculation pump 212. The settled resin will break apart and drain into the filter 204. In certain examples, performing an elution on resin that is only settled in the recirculation vessel 202 does not allow the elution buffer to distribute asDocket No. 1580.00231WO completely as when the resin is settled evenly throughout the filter 204, the recirculation tank 202, and any interconnected lines.

[0114] In an optional step, capture resin can be regenerated within the process vessel 202 or collected into a separate collection vessel and regenerated off-line. If regenerating within the recirculation vessel 202, regeneration buffer may be added through feed pump 210. Regeneration of the resin may be accomplished based on the manufacturer’s instructions.

[0115] The resin in the recirculation vessel 202 may be resuspended in a subsequent product stream or otherwise forced into a slurry or solution by any suitable process such as by starting an agitator or pumping air into the recirculation vessel 202. The resuspended resin may be used for a subsequent harvest capture process. The resin may be resuspended by receiving liquid pumped into the recirculation vessel 202. The liquid may be pumped into the recirculation vessel from an external source or by the components described herein. An example configuration to pump liquid into recirculation vessel 202 to resuspend the resin is described below with respect to FIG. 3.

[0116] The system may also comprise one or more of a flowmeter, a pressure sensor, and a controller. In an optional step, an analysis device, such as a Repligen FlowVPX system, may be mounted either in a process line of the system or in an external location. For example, the FlowVPX may be mounted in the permeate line such as after the permeate pump 214, before the permeate pump 214, in the recirculation loop, or in any suitable location. Any type of device may be used to measure characteristics of the permeate flow, such as the concentration of product in the fluid.

[0117] FIG. 3 illustrates a system that may be utilized in connection with a capture / harvest operation in accordance with an aspect of the invention.

[0118] In the example of FIG. 3, a valve 220 is provided on a vent 308 that allows air to enter or leave the conduit from the recirculation pump 212 to the recirculation vessel 202. Conduit sealed by the valve 220 may be open to the atmosphere on one end and connected to the recirculation loop on the opposing end. When the valve is open, air may be pulled into the recirculation loop. By operating with the valve 220 open to the vent 308, the recirculation pump 212 can pump the resin in the recirculation loop back up into the recirculation vessel 202 through the filter using the pumped air through vent 308. Providing a vent 308 to allow air to enter the line allows the conduit that returns fluids to the recirculation vessel 202 to be submerged. Pumping the fluids into the recirculation vessel 202 from a submerged inlet line preventsDocket No. 1580.00231WO excessive foaming from the fluids. Alternatively, instead of a vent 308 with a valve 220, the vent 308 may be a J-tube above the liquid level.

[0119] FIG. 3 further illustrates a feed addition vessel 206 and a feed pump 210 that are capable of feeding directly into the recirculation loop. By closing valve 310 and opening valve 312, the feed pump 210 may deliver fluids such as elution buffer, product feed, wash buffer, or other fluids into the recirculation loop. By closing valve 312 and opening valve 310, the feed pump 210 may deliver fluids directly to the top of the recirculation vessel 202.

[0120] Feed pump 210 and feed addition vessel 206 may also be used to deliver liquids to the bottom of recirculation vessel 202. The liquids may be pumped into the bottom inlet / outlet to resuspend the resin. In an example, valves 310 and 234 are closed. Valves 312, 222, and 216 are opened. Feed pump 210 is turned on to deliver liquids from feed addition vessel 206. In some examples, the recirculation pump 212 is engaged in a direction that is reversed from the typical flow path of the recirculation loop. With certain pump types, the liquid will pass through recirculation pump 212 without the pump being engaged. The liquid passed upwards through the filter 204 and into the bottom of the recirculation vessel 202.

[0121] Other functions of FIG. 3 may be substantially the same as the functions described with respect to FIG. 2.

[0122] FIG. 4 illustrates a system that may be utilized in connection with a capture / harvest operation in accordance with an aspect of the invention.

[0123] In FIG. 4, a fritted column is used for the elution phase 110. In this example, the capture phase 104 and the wash phase 106 are performed as described with respect to FIG. 2. However, the resin is settled into a separate vessel, such as a fritted column 416, during the settle phase 108. An example fritted column 416 is illustrated and described in FIG. 5 infra. Instead of forcing the resin back into the recirculation vessel 202, the resin is pumped to the fritted column 416 by operation of the recirculation pump 212 and by opening valve 216 and valve 222. Valve 422 may be a three-way valve, an assembly of multiple valves, a manifold, or any other process to direct flow in multiple paths. In this example, valve 422 is configured to allow resin to be pumped to the fritted column 416 and not back to the recirculation vessel 202.

[0124] When the resin is pumped into the fritted column 416, the wash buffer or any other remaining fluids may be extracted from the fritted column 416 by starting pump 408 and opening valve 410. The extracted fluids may be transferred to a waste vessel 230.Docket No. 1580.00231WO

[0125] After the resin is settled in the fritted column 416, elution fluid may be pumped to the fritted column 416 by starting pump 406. Pump 406 pumps elution fluid from the second feed addition vessel 414 to the fritted column 416. The elution phase 110 operates in a similar manner as the elution phase 110 described in FIG. 2. The fritted column may be any type of column or vessel that allows the elution buffer to extract the product from the resin. The fritted column 416 allows the product to be pumped from the fritted column 416 by the pump 408 while retaining the resin. For example, the fritted column 416 may be a filter similar to the filter 204, a vessel with a screen or frit before an outlet line, or any other type of process equipment that separates the product from the resin. The product is pumped to the product vessel 232 when the valve 412 is opened and the pump 408 is started.

[0126] In another example, the fritted column 416 can also be a reservoir with a second filter similar to the filter 204. The system may pump the resin into the secondary reservoir and use the second filter as the fritted column during elution in a similar manner as described with filter 204 and recirculation vessel 202. This process alternative allows fewer contaminants into the system during the elution phase, which allows the filter to remain cleaner and less prone to clogs. As illustrated in FIG. 4, the elution phase may be performed in any suitable vessel or location. For example, the elution may be performed in the recirculation vessel 202, the recirculation loop including the filter 204, or an external column such as the fritted column 416. In some examples, the recirculation vessel 202 may be a fritted column or a process vessel. In each case the resin can be retained with a fritt, a filter, a screen, or other device.

[0127] In another aspect, a positive column pressure is maintained in the fritted column 416. To achieve this pressure, the process uses a positive displacement pump as pump 406 to apply positive pressure (0<x<30psi) to the column. In an example, the positive pressure is between 0 and 30 PSI. To fill the body of the fritted column 416, a vent is positioned on the fritted column 416. The vent is opened during filling while the valves on the outlet, valves 412 and 410, are closed. To wash or settle the resin at constant volume the vent on the fritted column 416 is closed and valve 410 is opened to allow waste to proceed to the waste vessel 230. To perform elution at constant volume, the vent on the fritted column 416 is closed and valve 412 is opened to allow product to proceed to the product vessel 232. Because of the positive pressure of pump 406, the outlet pump 408 may not be required to extract fluids from the fritted column 416.

[0128] In another example, multiple cycles may be performed to recycle the resin. Recycling resin may be useful when the volume of resin needed to capture all the target biologic product inDocket No. 1580.00231WO the process fluid is greater than the resin in the system. An economical election may be made to use only a fraction of the total resin that would be required for sufficient capture in a single pass. In a multiple cycle process, the resin is used, cleaned, and reused to process all the biologic product.

[0129] To perform multiple cycles on an application using a fritted column 416, the process may have an outlet recirculation line returning the resin from the fritted column 416 back to the recirculation vessel 202. The recirculation line may have a valve on the outlet of the fritted column 416 and a pump to transfer the resin. After the biologic product has been eluted and transferred to the product vessel 232, an additional third addition vessel and pump may be used to provide additional buffers and / or air to fritted column 416 to resuspend the resin. The suspended resin is then pulled from the fritted column by the pump and transferred to the recirculation tank to be used in a subsequent capture phase as another cycle is performed as described infra. The recycled resin may be used on a subsequent feed of product or to remove additional biologic product from a product feed after an incomplete harvest capture.

[0130] In an additional aspect, for applications in which the resin is recycled to complete a capture of the product, at least a second fritted column may be used in parallel with the first fritted column 416. Two or more fritted columns placed in parallel can reduce idle time of the filter 204 and process the feed material more efficiently. Clean resin and fresh product feed can be introduced to the recirculation vessel 202 once bound resin is transferred out of the flow path to a first fritted column 416. Because the resin is transferred out of the flow path of the recirculation vessel 202 and the filter 204, an additional quantity of resin and product can begin a new cycle while resin is in the fritted column 416. The second cycle of resin and product may be transferred to the second fritted column for processing while the resin from the first fritted column 416 is recycled to the recirculation vessel 202 or otherwise disposed.

[0131] FIG. 5 is an image of an example fritted column 416. The fritted column 416 has an inlet 502 that allows the resin, elusion buffer, or any other fluids or slurries to be pumped or fed into the fritted column 416. The fritted column 416 is illustrated with a screw cap 510 with a sealing gasket. The screw cap 510 may represent any type of closing and sealing mechanism, such as a cap that is sealed with bolts, clasps, clamps, or other devices. The fritted column 416 is illustrated with a chamber 506 for liquid and resin. The chamber 506 holds the resin and liquids in the body of the fritted column 416 during process functions such as the elution phase.Docket No. 1580.00231WO

[0132] The fritted column 416 is illustrated with a bottom fritt 508. The bottom frit 508 in a fritted column 416 may be a porous, stationary layer that provides structural support and maintains the column's geometry. The bottom frit 508 also serves to distribute fluid flow evenly across the column's cross-sectional area, preventing plugging and ensuring efficient fluid passage. The bottom frit 508 may include a porous material such as glass beads, ceramic, fritt glass, or other material. The bottom frit 508 may retain solids such as the resin in the fritted column 416 while allowing liquids to pass to the outlet 504. The fritted column 416 may include an outlet 504 that allows liquids or other materials to flow out of the fritted column 416.

[0133] FIG. 6 is a graph of product concentration in the permeate outlet for the capture / harvest operation compared to conventional harvest capture in accordance with one embodiment.

[0134] The concentration of a product, such as antibodies, in the permeate stream was measured for two embodiments of the present technology and an example of a conventional harvest system. For each of the three examples, a concentration of the product in the permeate line was measured by an analysis device, such as a Repligen FlowVPX system. The VPX system may be mounted in the permeate line, such as after the permeate pump 214. This device or any other suitable type of device may be used to measure characteristics of the permeate flow, such as the concentration of product in the fluid.

[0135] In the graph, the concentration of product in g / L is displayed on the Y axis. The Permeate / Resin Volume (P / R) on the X axis is a ratio that represents the amount of liquid collected relative to the amount of resin. As described herein, the present technology allows a higher concentration of product to be recovered in the permeate stream with a lower volume of other fluids or water. The P / R ratio is important as users desire to keep the ratio as low as possible. The greater the P / R ratio, the more liquid is being sent onto the next step, which may require additional time, unit operations, and greater storage capacity.

[0136] In the example represented by the set with neutral buffer pH 7 line, the resin was settled in the reservoir using wash buffer. Once the resin was pumped or recirculated or otherwise gathered in the recirculation vessel 202, wash buffer was used as a neutral buffer to allow the resin to settle in the reservoir. As described, settled resin is no longer in suspension in the liquid, but is settled into a lowest point of the vessel. Since the product was settled with the wash buffer, the product will not release from the resin. Approximately 1 P / R volume of wash buffer was used to settle the resin. In an example, during this step the recirculation pump is off, the permeate pump and addition lines are on at 3 mL / min. After 1 P / R of wash buffer the wash buffer providedDocket No. 1580.00231WO by the addition line was stopped and elution buffer was provided by the addition line. The total P / R volume for this process was 2.4 and 98% of the captured antibody was recovered in the permeate.

[0137] In the example represented by the set with elution buffer pH 2 line, the resin was settled in the reservoir using elution buffer. For this embodiment, the wash buffer was drained from the flow path prior to settling the resin with the elution fluid. The resin was resuspended in elution buffer by adding the elution buffer and stopping the permeate pump 214. The resin was gathered in the reservoir. Elution buffer was used to settle the resin in the reservoir. The recirculation pump 212 is stopped and the permeate and addition lines were started at 3 mL / min. In the first P / R volume of elution buffer the concentration in the permeate is steady at approximately 18g / L before rising sharply. The total P / R volume for this run was 2.2 and 89% of the captured antibody was recovered.

[0138] In the example represented by the Diafiltration line, a conventional diafiltration harvest capture method was used. The resin was maintained suspended in a slurry and the resin was never trapped in the reservoir. To initiate the elution step, a bolus of IM citric acid was added to the batch at 10% of the liquid volume in the flowpath. Following the bolus lOOmM citric acid was used to continue the elution step. The Diafiltration line in Figure 5 illustrates that the conventional method is less efficient at recovering the product. The Diafiltration line has a long “tail,” where the product is still being collected at a low concentration. To compare, the set with neutral buffer pH 71ine and the set with elution buffer pH 2 line contain relatively sharp peaks where the product is released in a small amount of volume. The P / R volume for this run was around 6-7 and 73% of the captured antibody was recovered.

[0139] Both settling methods using the present technology (neutral buffer pH 7and elution buffer pH 2) recovered a higher percentage of the product in a lower volume of liquids than the diafiltration method.EXAMPLE

[0140] An example experiment utilizing the Harvest Capture processes described herein was performed. The results of the experiment are provided herein.

[0141] The clarification and purification of Adeno-Associated Virus (AAV) from cell culture typically involves multiple sequential unit operations, extending over several days and yielding around 80% under optimal conditions. These processes impose high costs, energy demands, andDocket No. 1580.00231WO operational burdens, particularly at manufacturing scale. This study evaluates clarification and purification of AAV from cell culture media in a streamlined workflow in accordance with an embodiment as described herein. This process is referred to as Harvest Capture in this example. Using AAV9 produced in HEK cells, Harvest Capture was compared to a conventional industrial process involving depth filtration, tangential flow filtration (TFF), and affinity chromatography. Metrics included yield, processing time, volumetric throughput, and impurity reduction. Harvest Capture achieved an 83% yield with a total process time of 200 minutes, maintained low operating pressure, and provided significant host cell protein (HCP) and DNA clearance. In contrast, the conventional method required multiple steps, took substantially longer, and experienced cumulative product loss. These results suggest Harvest Capture offers a viable, efficient alternative to traditional workflows for AAV purification.

[0142] Current industrial strategies for AAV purification rely on sequential operations - chemical lysis, enzymatic digestion, depth filtration, TFF concentration, and affinity chromatography - often spanning several days. While effective, these workflows impose significant demands in terms of time, labor, equipment footprint, and consumables. Furthermore, high-capacity depth filters often suffer from fouling, leading to reduced throughput and potential filter changes mid-process. The Harvest Capture approach in this example is designed to simplify virus recovery by combining clarification and purification into a single unit operation. In this method, affinity resin is added directly to unclarified cell culture, enabling direct capture of the virus. Large-porosity filtration coupled with a TFF module removes cell debris while retaining the resin-virus complex. Bound AAV is subsequently recovered through low-pH elution. The streamlined design aims to reduce processing time, increase volumetric throughput, and maintain or improve product yield relative to conventional methods. This study compares the performance of Harvest Capture with a conventional process using depth filtration and column chromatography for the recovery of AAV9 from HEK lysate.HEK Cell Lysis

[0143] HEK cells (2.7 L culture volume) transfected with AAV9 were lysed in a shake flask. Lysis was performed by adding 300 mL of 10x Tween 20 lysis buffer (500 mM Tris base, 20 mM MgCb, 10% Tween 20) and 25 U / mL of endonuclease. The mixture was incubated at 37 °C for 2 hours. The lysate was split into two batches: 1 L for Harvest Capture and 2 L for conventional depth filtration.Docket No. 1580.00231WOHarvest Capture Workflow

[0144] 1. Capture

[0145] Six mL of AVIPure AAV9 resin were added to the 1 L of HEK lysate. The slurry was incubated at room temperature on a shaker (150 RPM) for two hours.

[0146] 2. Resin Concentration and Wash

[0147] The slurry was transferred to a recirculation reservoir and processed at a recirculation rate of 500 mL / min (0.42 m / s linear velocity) and permeate flux of 2000 LMH (10 mL / min) using a 6 cm2Harvest Capture filter. The resin was concentrated to the minimum hold-up volume, washed with 12 diavolumes of 150 mM NaCl, and then transferred to a fritted column.

[0148] 3. Elution and Regeneration

[0149] Elution was performed using a glycine buffer (100 mM glycine, 150 mM NaCl, 0.01 % pol oxamer p-188, pH 2), applied for 4 resin volumes. The product was neutralized with 1 M Tris base (pH 9). The resin was stripped with 0.1 M NaOH (3 resin volumes) and reequilibrated in 150 mM NaCl (3 resin volumes).Alternative Harvest and Purification Procedure

[0150] 1. Depth Filtration

[0151] The 2 L lysate was processed using a primary depth filter (20 cm2D0SP) followed by a secondary depth filter (23 cm2X0SP). Filters were primed and flushed with 150 mM NaCl prior to use, operated at 200 LMH. Due to high pressure, two 23 cm2 X0SP filters were required to process the entire batch.

[0152] 2. Tangential Flow Filtration (TFF) (Optional)

[0153] One liter of clarified material was concentrated using a D02-E100-05 hollow fiber filter (115 cm2surface area) to a final volume of 40 mL, achieving a 20* concentration factor. The process was run at 4000 s ' shear rate and 8 psi TMP.

[0154] 3. Affinity Column Chromatography

[0155] Affinity capture was performed using 5 mL of AVIPure AAV9 resin in a 1.13 cm diameter * 5 cm bed height column. Unconcentrated feed (I L) was processed in two cycles (3 min residence time per load step), while concentrated feed (62 mL) was processed in a single cycle (5 min residence time). Each cycle included equilibration, load, wash, elution, acid strip, base CIP, and re-equilibration (See Appendix Table 1 for steps, buffers, column volumes (CV), and residence time.Docket No. 1580.00231WOResultsHarvest Capture

[0156] During Harvest Capture using TFF module concentration and diafiltration, pressure remained constant at ~0.5 psi, indicating stable filter performance without fouling. Processing 1 L required 70 minutes of total filtration time. This result is illustrated in FIG. 7.

[0157] FIG. 7 represents a pressure profile during a Harvest Capture TFF step.

[0158] Transmembrane pressure (psi) was recorded throughout the concentration and diafiltration of 1 L HEK cell lysate bound to AVIPure AAV9 resin using a 6 cm2Harvest Capture filter. The process was operated at a recirculation rate of 500 mL / min (linear velocity 0.42 m / s) and a permeate flux of 2000 LMH (10 mL / min) with 150 mM NaCl wash buffer. The X axis of FIG. 7 represents process time (minutes), and the Y axis represents transmembrane pressure (psi). Pressure remained constant at approximately 0.5 psi throughout the run, indicating stable filter performance without significant fouling.

[0159] The resin slurry was transferred from the filtration flowpath to a 20 mL fritted column. The flowpath was chased with 150 mM NaCl to collect any remaining resin that may have stayed in the flowpath during transfer. An additional three column volumes of 150 mM NaCl was used to settle the resin. A glycine-based elution buffer (pH 2) was continuously added to elute the resin. The resin transfer and elution were completed within 20 minutes.

[0160] Samples were taken from the feed, waste, and product streams to determine product yield and reduction of host cell protein and DNA impurities. A Progen AAV9 ELISA kit was used to measure viral capsid concentration. Host cell proteins were quantified using a Cygnus HCP assay, and DNA concentration was determined with Thermo Quant-iT PicoGreen assay.

[0161] Table 1 represents the Harvest Capture Capsid, HCP and DNA results.TABLE 1

[0162] The total workflow time post lysis was 200 minutes. Capsid yield was 83%, with HCP and DNA log reductions of 4.3 and 2.4, respectively.Depth FiltrationDocket No. 1580.00231WO

[0163] The depth filtration train was processed in two separate batches, with primary clarification performed first followed by secondary clarification. This approach, rather than the continuous sequence commonly used in industry, was selected to enable independent evaluation of each filtration stage. The primary depth filter approached 1000 L / m2without exceeding 30 psi, while the secondary filters reached 30 psi at 503 L / m2and 336 L / m2, respectively (Figure 2). Virus recovery was 91% after primary clarification and 81% after secondary clarification, with an additional 5% loss observed following sterile filtration.

[0164] FIG. 8 is a graph representing pressure profiles for primary and secondary depth filtration during conventional AAV clarification. Inlet pressure (psi) was monitored during filtration of 2 L HEK cell lysate using a 20 cm2D0SP primary depth filter followed by 23 cm2X0SP secondary depth filters, operated at 200 LMH with 150 mM NaCl buffer. The X axis represents volumetric throughput (L / m2) and the Y axis represents inlet pressure (psi). The primary filter (DOSP) approaches 1000 L / m2without reaching 30 psi, while the first (XOSP 1) and second (XOSP 2) secondary filters reached the 30-psi limit at 503 L / m2and 336 L / m2, respectively, indicating faster fouling in secondary clarification.

[0165] The total process time for depth filtration using this method was around 2 hours for 2 liters of material. To accurately compare the duration for depth filtration to Harvest Capture we can estimate that 1 liter of material processed in a connected train would be completed in a quarter of the time or 2.5-3 hours.Tangential Flow Filtration (TFF)

[0166] One liter of material was concentrated using a D02-E100-05. Filter parameters included mPES hollow fibers, 63 fibers, 5 mm inner diameter, 20 cm effective length, 115 cm2 surface area. The final concentration factor was 16.7x. The crossflow shear rate was 4000s- 1 and a backpressure valve maintained 8 psi TMP. Buffer exchange was not performed during this step.

[0167] The process flux reached a maximum of 60 LMH in the beginning of the run and decreased to less than 5 LMH towards the end of the run.

[0168] FIG. 9 is a graph illustrating a flux profile during TFF of clarified AAV material. Process flux (LMH) was measured during concentration of 1 L clarified HEK lysate to 40 mL using a D02-E100-05 Repligen hollow fiber filter (115 cm2 surface area) at 4000s-l shear rate and 8 psi TMP. The x-axis represents volumetric throughput (L / m2) and the y-axis represents flux (LMH). Flux declined steadily as throughput increased, dropping below 5 LMH near the end of the run.Docket No. 1580.00231WO

[0169] After the TFF step was complete, the concentrate was harvested from the flowpath and 20 mL of 150 mM NaCl was chased through the flowpath to maximize the recovery of virus. The chase and concentrated material were combined and sterile filtered to prepare for affinity chromatography column. An ELISA assay measured an 8% decrease in viral particles. This duration for this step was 6.5 hours.Affinity Chromatography

[0170] Column affinity chromatography was performed on both unconcentrated and concentrated AAV9 material using a small-scale column packed with 5.014 mL of AVIPure- AAV9 affinity resin (1.13 cm diameter, 5 cm bed height). Both feed streams had been clarified and sterile filtered as described in the steps above. The unconcentrated feed was processed in two sequential cycles, with a residence time of 3 minutes during each load step. Each cycle lasted approximately 365 minutes and included equilibration, loading, chase wash, elution, acid strip, base CIP, and re-equilibration. In contrast, the concentrated feed prepared via TFF ultrafiltration, was processed in a single cycle with a 5-minute residence time. This significantly reduced the overall processing time to just 2.3 hours.

[0171] Product recovery based on capsid quantification was unexpectedly high in both cases: 120% for the unconcentrated material and 140% for the concentrated. These values likely reflect analytical variability or systematic error in titer measurements rather than the true yield, as recovery exceeding 100% is not physically plausible. Nonetheless, both processes demonstrated that the AVIPure-AAV9 resin platform provides high recovery of product, likely exceeding 95% under ideal conditions.

[0172] The affinity step also provided effective impurity reduction. Host cell protein (HCP) clearance measured by the Cygnus HCP assay, resulted in a 4.8 log reduction for the unconcentrated batch and 3.7 log reduction for the concentrated batch. DNA clearance assessed using the Thermo Quant-iT PicoGreen assay showed log reductions of 2.9 and 3.0, respectively.Volumetric Throughput

[0173] Harvest Capture used a large porosity filter in conjunction with tangential flow filtration module to prevent the formation of a cake layer and allows cell culture species that are typically retained by depth filters to pass directly through the membrane. FIG. 7 shows Harvest Capture filtration approaching 2500 L / m2 without pressure increasing. The volumetric throughput boundaries for Harvest Capture are not well defined and are limited by the volume of feed material rather than filter health. Comparing FIG. 7 and FIG. 8, the volumetric throughput forDocket No. 1580.00231WO depth filters are significantly less. The observed pressure rise with increasing volumetric throughput suggests progressive formation of a cake layer of retained particles, which increases resistance and reduces filtrate permeability.

[0174] From FIG. 8 it was observed that the primary depth filter had a high volumetric throughput, able to exceed 1000 L / m2 without reaching 30 psi. The secondary depth filters had a much lower volumetric throughput, reaching 30 psi for the first and second X0SP at 503 L / m2 and 336 L / m2, respectively. It is common in industry to oversize depth filters to avoid high pressure situations that may lead to failure, filter swapping, or breakthrough.Capsid Loss

[0175] In conventional AAV purification workflows, product loss accumulates across multiple unit operations, with each step such as depth filtration, tangential flow filtration, sterile filtration, and column chromatography contributing incremental yield reductions. Even small losses per operation can compound significantly when the process involves several sequential steps. In contrast, the Harvest Capture strategy consolidates clarification and purification into a single unit operation, which eliminates intermediate transfers and minimizes opportunities for product loss. As a result, the overall recovery is higher, reflecting the reduced number of loss-induced interfaces and handling events inherent to the streamlined process.

[0176] FIG 10 is a graph illustrating AAV9 capsid yield for Harvest Capture. FIG 10 illustrates the product for the single unit Harvest Capture process, with capsid yield around 81%.

[0177] FIG. 11 is a graph illustrating the standard multi-step clarification and purification process 9. FIG. 11 illustrates the yield after each step of the conventional process, including primary depth filtration, secondary depth filtration, sterile filtration (0.2um), and tangential flow filtration (UF / DF). This data illustrates the cumulative reduction in capsid recovery across multiple unit operations.Process Time

[0178] The total duration for the Harvest Capture process was approximately 200 minutes, consisting of three distinct stages: two hours for capture, 70 minutes for filtration, and less than 20 minutes for transfer and elution. It is important to note that Harvest Capture processed only 1 L of material with a 6 cm2 filter, whereas the standard method processed 2 L using depth filters with surface areas of -20-23 cm2. This difference in filter surface area and volume makes direct time comparison challenging, however, scaling the depth filtration process to a 1 L batch in series would still be estimated at about 3 hours, excluding subsequent operations. Once depth filtrationDocket No. 1580.00231WO was complete, the batch was split to evaluate affinity capture with and without prior concentration. Tangential flow filtration (TFF) was used to concentrate one liter of material and required another 6.5 hours to complete 87 L / m2. Affinity chromatography varied between 2.33 hours for the concentrated material and over 12 hours for unconcentrated material when using 5mL of AVIPure AAV9 affinity resin. While there are other strategies to reduce the process time including oversizing filters, affinity columns, or high flow through resins, Harvest Capture offers an efficient and scalable approach to clarification and purification.

[0179] Accordingly, this study compared the performance of Harvest Capture and a conventional multi-step purification workflow for AAV9 recovery from HEK lysate. Harvest Capture completed clarification and affinity purification in a single integrated process, requiring approximately 200 minutes from virus loading to final elution, and achieved an overall yield of 83% with 4.3-log HCP reduction and 2.4-log DNA reduction. The conventional workflow, consisting of depth filtration, TFF concentration, and affinity chromatography, required multiple unit operations with extended processing times and cumulative virus losses at each step, resulting in 71-78% recovery.

[0180] Table 2 below provides data of the affinity column process specifications.Docket No. 1580.00231WOTABLE 2Example Processes for Harvest Capture of Recombinant Protein Using Ion Exchange or HIC Chromatographic Resin

[0181] In some aspects, a harvest capture process as described herein may include an ion exchange resin, for example, a cation exchange resin or an anion exchange resin. The harvest capture process performed with a cation exchange resin may be particularly advantageous, for example, where the target protein has an isoelectric point greater than or equal to 4 and where the protein is soluble and stable at acidic pH. A harvest capture process performed with an anion exchange resin may be particularly advantageous, for example, where the target protein has an isoelectric point less than or equal to 9 and where the protein is soluble and stable at basic pH.

[0182] In an exemplary process of performing harvest capture with an ion exchange resin, the resin may be added to a cell lysate, for example a pH adjusted cell lysate. Typically, where the resin is a cation exchange resin, the pH of the cell lysate is adjusted to be at least about 0.5-2 pH units below the isoelectric point of the target protein. Where the resin is an anion exchange resin, the pH of the cell lysate is adjusted to be at least about 0.5-2 pH units above the isoelectric point of the target protein. In some aspects, the lysate may be subjected to a heat treatment prior to pH adjustment. In some aspects, the conductivity of the lysate may be reduced, for example via dilution or diafiltration, in order to facilitate binding of the target protein to the ion exchange resin.

[0183] One or more optional steps may be performed prior to addition of the ion exchange resin to the pH adjusted cell lysate. For example, a step of cell lysis may be performed. The cells may be any type of cell, for example mammalian, bacterial or yeast cells. Lysis may be performed, for example, utilizing homogenization, including high-pressure homogenization, and / or chemical lysis.

[0184] Following addition of the ion exchange resin to the pH adjusted cell lysate, the mixture is incubated for a period of time to allow for binding of target protein to the resin. Following binding, the resin mixture is passed through a harvest capture filter as described herein, e.g. as described in one or more of FIG. 2 through FIG. 4 and accompanying text, to removeDocket No. 1580.00231WO contaminating protein, nucleic acids, cell debris, etc., followed by recovery and transfer of the ion exchange resin to a column. The resin is then washed to remove bound impurities while retaining the target protein on the resin. In a typical wash process, the resin may be washed with several column volumes of a wash buffer, for example 5-10 column volumes of wash buffer. The wash buffer is typically a salt buffer, for example a buffer containing from about 50-150 mM sodium chloride (NaCl). The wash step is followed by elution of the target protein from the resin with a suitable elution buffer. For example, the elution buffer may comprise an increasing salt gradient and / or a shift in pH. For cation exchange resins, for example, the elution buffer may comprise an increase in pH, moving toward or above the isoelectric point of the target protein. For anion exchange resins, the elution buffer may comprise a decrease in pH, moving toward or below the isoelectric point of the target protein.

[0185] In another example, a harvest capture process as described herein may include a Hydrophobic Interaction Chromatography (HIC) resin. The process includes adding a HIC resin to a cell lysate comprising the target protein. Prior to addition of the HIC resin, the lysate may be adjusted to provide optimal conditions for target protein binding to the resin. Adjustments may include one or more of heat treatment, a pH adjustment, for example to a more acidic or basic pH, and / or addition of salt, such as sodium chloride (NaCl), sodium sulfate, ammonium sulfate, etc. Generally, high conductivity promotes the HIC interaction and is necessary for binding of the target protein to the resin.

[0186] One or more optional steps may be performed prior to adjustments made to the lysate and addition of the HIC resin to the cell lysate. For example, a step of cell lysis may be performed. The cells may be any type of cell, for example mammalian, bacterial or yeast cells. Lysis may be performed using any suitable method, for example utilizing homogenization, including high- pressure homogenization, and / or chemical lysis.

[0187] Following addition of the HIC resin to the cell lysate, the mixture is incubated for a period of time to allow for binding of target protein to the resin. Following binding, contaminating protein, nucleic acids, cell debris, etc. are removed by passing the mixture through a harvest capture filter as described herein, e.g. as described in one or more of FIG. 2 through FIG. 4 and accompanying text. Following filtration, the HIC resin is recovered and transferred to a column. The resin is then washed to remove bound impurities while retaining the target protein on the resin. In a typical process, the resin is washed with several column volumes of wash buffer, for example 5-10 column volumes of wash buffer. The wash buffer is typically aDocket No. 1580.00231WO high salt buffer, for example a buffer containing from about 0.5-2 M ammonium sulfate or similar. The wash step is followed by an elution step where the target protein is removed from the resin with an elution buffer. For example, the elution buffer may include lower amounts of salt, e.g., ammonium sulfate, for example less than 1 M, or less than 0.5 M, or less than 0.1 M ammonium sulfate.

[0188] In another example, a harvest capture process as described herein may include an ion exchange resin or HIC resin as discussed above where instead of adding the ion exchange or HIC resin to a cell lysate, it is instead added to a solution comprising a secreted target protein, for example host cell culture fluid, including clarified host cell culture fluid or unclarified host cell culture fluid or conditioned medium. In accordance with this aspect, the target protein may include, e.g., immunoglobulins or other proteins secreted into the host cell culture fluid, for example in mammalian, bacterial, fungal or insect cells used for recombinant protein production. The remaining steps in the method, including optional steps, are as described above, including the post-resin binding step of filtration through a harvest capture filter as described herein, e.g. as described in one or more of FIG. 2 through FIG. 4 and accompanying text.

[0189] In another example, a harvest capture process as described herein may include an ion exchange resin or HIC resin as discussed above where the resin is added to cell extract or homogenate, or similar solution comprising a target cellular protein, instead of adding the resin to a cell lysate. This may be the case, for example where the target protein is a non-recombinant natural product isolated from biological cells or tissues. The remaining steps in the method, including optional steps, are as described above, including the post-resin binding step of filtration through a harvest capture filter as described herein, e.g. as described in one or more of FIG. 2 through FIG. 4 and accompanying text.

[0190] While the invention herein disclosed has been described by means of specific embodiments and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims.

[0191] It will be appreciated that the present invention is set forth in various levels of detail in this application. In certain instances, details that are not necessary for one of ordinary skill in the art to understand the invention, or that render other details difficult to perceive may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. Unless definedDocket No. 1580.00231WO otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0192] Various features of a process system may be used independently of, or in combination, with each other. It will be appreciated that a system as disclosed herein may be embodied in different forms and should not be construed as limited to the illustrated embodiments of the figures.

[0193] It should be understood that, as described herein, an “embodiment” (such as illustrated in the accompanying Figures) may refer to an illustrative representation of an environment or article or component in which a disclosed concept or feature may be provided or embodied, or to the representation of a manner in which just the concept or feature may be provided or embodied. However, such illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. In addition, it will be appreciated that while the Figures may show one or more embodiments of concepts or features together in a single embodiment of an environment, article, or component incorporating such concepts or features, such concepts or features are to be understood (unless otherwise specified) as independent of and separate from one another and are shown together for the sake of convenience and without intent to limit to being present or used together. For instance, features illustrated or described as part of one embodiment can be used separately, or with one or more other features to yield a still further embodiment. Thus, it is intended that the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0194] In view of the above, it should be understood that the various embodiments illustrated in the figures have several separate and independent features, which each, at least alone, has unique benefits which are desirable for, yet not critical to, the presently disclosed vessel, system, and associated method. Therefore, the various separate features described herein need not all be present in order to achieve at least some of the desired characteristics and / or benefits described herein.

[0195] The foregoing discussion has broad application and has been presented for purposes of illustration and description and is not intended to limit the disclosure to the form or forms disclosed herein. It will be understood that various additions, modifications, and substitutions may be made to embodiments disclosed herein without departing from the concept, spirit, andDocket No. 1580.00231WO scope of the present disclosure. In particular, it will be clear to those skilled in the art that principles of the present disclosure may be embodied in other forms, structures, arrangements, proportions, and with other elements, materials, and components, without departing from the concept, spirit, or scope, or characteristics thereof. For example, various features of the disclosure are grouped together in one or more aspects, embodiments, or configurations for the purpose of streamlining the disclosure. However, it should be understood that various features of the certain aspects, embodiments, or configurations of the disclosure may be combined in alternate aspects, embodiments, or configurations. While the disclosure is presented in terms of embodiments, it should be appreciated that the various separate features of the present subject matter need not all be present in order to achieve at least some of the desired characteristics and / or benefits of the present subject matter or such individual features. One skilled in the art will appreciate that the disclosure may be used with many modifications or modifications of structure, arrangement, proportions, materials, components, and otherwise, used in the practice of the disclosure, which are particularly adapted to specific environments and operative requirements without departing from the principles or spirit or scope of the present disclosure. For example, elements shown as integrally formed may be constructed of multiple parts or elements shown as multiple parts may be integrally formed, the operation of elements may be reversed or otherwise varied, the size or dimensions of the elements may be varied. Similarly, while operations or actions or procedures are described in a particular order, this should not be understood as requiring such particular order, or that all operations or actions or procedures are to be performed, to achieve desirable results. Additionally, other implementations are within the scope of the following claims. In some cases, the actions recited in the claims can be performed in a different order and still achieve desirable results. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the claimed subject matter being indicated by the appended claims, and not limited to the foregoing description or particular embodiments or arrangements described or illustrated herein. In view of the foregoing, individual features of any embodiment may be used and can be claimed separately or in combination with features of that embodiment or any other embodiment, the scope of the subject matter being indicated by the appended claims, and not limited to the foregoing description.

[0196] In the foregoing description and the following claims, the following will be appreciated. The term “about” refers to a range of 1-10% around the stated value. The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open-ended expressions that are both conjunctiveDocket No. 1580.00231WO and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. All directional references (e.g., proximal, distal, upper, lower, upward, downward, left, right, lateral, longitudinal, front, back, top, bottom, above, below, vertical, horizontal, radial, axial, clockwise, counterclockwise, and / or the like) are only used for identification purposes to aid the reader’s understanding of the present disclosure, and / or serve to distinguish regions of the associated elements from one another, and do not limit the associated element, particularly as to the position, orientation, or use of this disclosure. Connection references (e.g., attached, coupled, connected, and joined) are to be construed broadly and may include intermediate members between a collection of elements and relative movement between elements unless otherwise indicated. As such, connection references do not necessarily infer that two elements are directly connected and in fixed relation to each other. Identification references (e.g., primary, secondary, first, second, third, fourth, etc.) are not intended to connote importance or priority but are used to distinguish one feature from another.

[0197] In the claims, the term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. In addition, singular references do not exclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way.

Claims

Docket No. 1580.00231WOCLAIMSWhat is claimed is:

1. A method for isolating a biologic product from a process fluid, comprising contacting a process fluid comprising a target biologic product in a recirculation vessel with a capture resin to form a resin mixture in the recirculation vessel; circulating the resin mixture in a first direction through a tangential flow filtration (TFF) module with a wash buffer, thereby separating the mixture into permeate and retentate fluid streams, wherein the TFF filtration module and the recirculation vessel are interconnected in a retentate fluid circuit, and recirculating the retentate fluid stream through the retentate fluid circuit for a first number of diafiltration volumes (DVs) while directing the permeate stream to a waste container outside the fluid circuit, thereby producing a clarified retentate fluid containing the resin-bound target biologic product; stopping the recirculation and extracting the process fluid and wash buffer through the TFF module by operation of a permeate pump leaving the resin to settle in the clarified retentate fluid; contacting the clarified retentate fluid with a volume of elution buffer; and extracting a second permeate fluid stream comprising the eluted biologic product through the TFF module by operation of the permeate extraction pump while the capture resin is retained in the retentate stream.

2. The method of claim 1, wherein the capture resin is an affinity resin, a hydrophobic interaction chromatography resin, or an ion exchange resin.

3. The method of claim 1, wherein the retentate fluid enters the recirculation vessel at a top surface of the recirculation vessel such that an entrance of the retentate fluid is not submerged in the retentate fluid in the recirculation vessel.

4. The method of claim 3, further comprising: reversing a flow of a recirculation pump to pump the clarified retentate fluid in a second direction opposite the first direction to force the retentate fluid into the recirculation vessel.

5. The method of claim 4, wherein when a flow of the recirculation pump is reversed, air is pumped in the second direction to force the clarified retentate fluid into the recirculation vessel.Docket No. 1580.00231WO6. The method of claim 4, wherein the retentate fluid is retained in the recirculation vessel with a frit.

7. The method of claim 4, wherein the retentate fluid is retained in the recirculation vessel with a valve.

8. The method of claim 1, wherein the waste permeate stream from the wash operation comprises wash fluid, debris, and contaminants from the process fluid.

9. The method of claim 1, wherein a process line in which the retentate fluid enters the recirculation vessel at a top surface of the recirculation vessel is configured such that an entrance of the retentate fluid is submerged in the retentate fluid in the recirculation vessel, and wherein the process line comprises a vent.

10. The method of claim 9, wherein the TFF module utilizes a tubular / spiral flat sheet filter medium.

11. The method of claim 1, wherein the circulation is stopped by stopping a recirculation pump and / or closing a valve below the TFF module.

12. The method of claim 1, wherein during the wash operation, fluid lost to the permeate stream is not replaced in order to concentrate the fluid in the process vessel.

13. The method of claim 1, wherein the second permeate fluid stream comprising the eluted biologic product is pumped by the permeate pump into a product vessel.

14. The method of claim 15, further comprising adding an additional amount of the process fluid comprising the target biologic product to the capture resin in the recirculation vessel and repeating the remaining steps of claim 1.

15. The method of claim 1, further comprising pumping air into the recirculation vessel to resuspend the capture resin in solution.

16. The method of claim 1, wherein the biologic product is an antibody, a recombinant protein or a virus particle.Docket No. 1580.00231WO17. The method of claim 1, wherein the resin is functionalized with Fc-binding ligands or ligands that bind to particles, optionally Adeno-Associated Virus particles or Lentivirus particles.

18. A method for isolating a biologic product from a process fluid, comprising contacting the process fluid with a capture resin in a process vessel for a period of time sufficient to allow binding of the biologic product to the capture resin; adding a wash buffer to the process vessel from a first feed addition vessel; circulating the process fluid and resin mixture in a first direction through a tangential flow filtration (TFF) module, thereby separating the mixture into permeate and retentate fluid streams, wherein the filtration module and the recirculation vessel are interconnected in a retentate fluid circuit, and recirculating the retentate fluid stream through the retentate fluid circuit for a first number of diafiltration volumes (DVs) while directing the permeate stream to a waste container outside the fluid circuit, thereby producing a clarified retentate fluid, and optionally concentrating the clarified retentate fluid in the process vessel; stopping the circulation of the process fluid and resin mixture through the TFF module; extracting the process fluid and wash buffer through the TFF module by a permeate pump to allow the capture resin to settle in the clarified retentate fluid; pumping the clarified retentate fluid to an elution vessel;; performing an elution operation by contacting the clarified retentate fluid with a volume of elution buffer; and performing a harvest operation by extracting a permeate fluid stream comprising the biologic product through a filtration process from the second elution vessel while the capture resin is retained in the elution vessel.

19. The method of claim 18, wherein one or more of the elution buffer, the wash buffer, cleaning fluid, and storage buffer is pumped into the elution vessel from a second feed addition vessel.

20. The method of claim 18, wherein the clarified retentate fluid is pumped to a fritted column by directing a three-way valve in the recirculation circuit to open in a direction of the elution vessel and close a direction in the recirculation circuit.

21. The method of claim 18, wherein the elution vessel is a fritted column.

22. The method of claim 18, wherein capture resin is an affinity resin, a hydrophobic interaction chromatography resin, or an ion exchange resin.

Images