System and method for combining biological collection and capture

Abstract

Tangential flow filtration systems and methods for capturing and collecting biological products and related compositions are provided.

Description

Technical Field

[0001] This invention relates to bioprocess systems and methods for manufacturing biological products produced from cultured cells. Background Technology

[0002] In the biotechnology and pharmaceutical industries, many different process operations are commonly used to purify cell-derived bioproducts from bioreactor systems. These operations may include centrifugation, multiple filtration operations, and one or more chromatographic operations. Typically, each purification operation reduces product yield. Methods are needed to streamline purification processes to improve yield and reduce time and associated costs while maintaining high product yield and purity. Summary of the Invention

[0003] This invention provides a method for performing a combined capture and collection operation, which is initiated by directly adding a capture resin to cell culture medium or lysis buffer without prior clarification steps, such as prior centrifugation and / or filtration. Subsequent steps utilize a combination of percolation and concentration operations through a tangential flow filtration device to produce the bioproduct in high yield and high purity. In some aspects, the direct addition of resin to cell culture medium or lysis buffer containing the bioproduct includes contacting the cell culture medium or lysis buffer with the resin in a secondary container or in the recirculation retentate stream loop of a tangential flow filtration module, which for simplicity may also be referred to herein as a "recirculation loop".

[0004] In one aspect, a method is provided for separating biological products from a process fluid characterized by high cell density and / or high turbidity, comprising: performing a capture operation by contacting the process fluid with a capture resin in a retentate stream loop of a filtration module for a period of time sufficient to bind the biological products to the resin; performing a washing operation by separating the process fluid and resin mixture into a permeate stream and a retentate stream of the filtration module, and recirculating the retentate stream through the retentate stream loop to a first number of permeate volumes (DV), while directing the permeate stream to a waste container outside the fluid loop, thereby producing a clarified retentate stream, and allowing... The clarified retentate is concentrated in the retentate loop; an elution operation is performed by contacting the clarified retentate with a certain volume of elution buffer in the retentate loop for a period of time sufficient to separate the bioproducts from the resin; a collection operation is performed by circulating the clarified retentate and resin mixture through a filter module, thereby separating the mixture into a permeate stream containing the bioproducts and a retentate stream containing the resin; and the retentate stream is recirculated through the retentate loop for a second number of DVs, while the permeate stream is directed to a recovery container outside the fluid loop, thereby separating the bioproducts in the recovery container.

[0005] In one aspect, a method is provided for separating biological products from a process fluid characterized by high cell density and / or high turbidity, wherein the method includes: performing a capture operation by contacting the process fluid with a capture resin in a process vessel for a period of time sufficient to bind the biological products to the resin; performing a washing operation by circulating the process fluid and resin mixture through a filter module to separate the mixture into a permeate stream and a retentate stream, wherein the filter module and the process vessel are interconnected in a retentate stream loop, and the retentate stream is recirculated through the retentate stream loop for a first number of permeate volumes (DV), while simultaneously directing the permeate stream outside the fluid loop. The waste container of the part generates a clarified retentate stream, which is optionally concentrated in a process container and eluted. The clarified retentate stream is then contacted with a certain volume of elution buffer for a period of time sufficient to separate the bioproducts from the resin. The mixture of clarified retentate stream and resin is circulated through a filter module to separate the mixture into a permeate stream containing bioproducts and a retentate stream containing resin. The retentate stream is then recirculated through the retentate stream loop for a second number of DVs, while the permeate stream is directed to a recovery container outside the fluid loop, where the bioproducts are separated.

[0006] In one aspect, a method is provided for separating biological products from a process fluid characterized by high cell density and / or high turbidity, wherein the method includes: performing a capture operation by contacting the process fluid with a capture resin in a process vessel for a period of time sufficient to bind the biological products to the resin, while simultaneously circulating a mixture of the process fluid and resin through a filtration module to separate the mixture into a permeate stream and a retentate stream, wherein the filtration module and the process vessel are interconnected in a closed retentate stream loop and an open permeate stream loop, such that the retentate stream is prevented from leaving the filtration module to retain the resin, while the permeate stream is guided back to the process vessel through the permeate stream loop for a washing operation, and by opening the retentate stream loop to circulate the mixture of the process fluid and resin through the filtration module. The retentate stream is recirculated through the retentate stream loop for a first number of percolation volumes (DV), while the percolation stream loop is closed and the percolation stream is directed to a waste container outside the fluid loop, thereby producing a clarified retentate stream. Optionally, the clarified retentate stream is concentrated in a process vessel and eluted. A collection operation is performed by contacting the clarified retentate stream with a certain volume of elution buffer sufficient to separate the bioproducts from the resin for a period of time. The mixture of clarified retentate stream and resin is circulated through the module, thereby separating the mixture into a percolation stream containing bioproducts and a retentate stream containing resin. The retentate stream is then recirculated through the module for a second number of DV, while the percolation stream is directed to a recovery container outside the fluid loop, thereby separating the bioproducts.

[0007] In one aspect, a method is provided for separating biological products from a process fluid characterized by high cell density and / or high turbidity, wherein the method comprises: performing a capture operation by (i) contacting the process fluid with a capture resin in a primary process vessel for a period of time sufficient to bind the biological products to the resin, and (ii) circulating a mixture of the process fluid and resin through a secondary process vessel in fluid communication with a filtration module for a second period of time, thereby separating the mixture into a permeate stream and a retentate stream, wherein the filtration module and the secondary process vessel are interconnected in a retentate stream loop, and the primary process vessel, the secondary process vessel, and the filtration module are interconnected in a permeate stream loop, such that the retentate stream is recirculated in the retentate stream loop between the secondary process vessel and the filtration module, while the permeate stream is recirculated in the permeate stream loop between the primary process vessel, the secondary process vessel, and the filtration module, and washing is performed. The process involves circulating a mixture of process fluid and resin through a filtration module by closing the permeate flow loop, recirculating the retentate flow through the retentate flow loop for a first number of permeate volumes (DV), and simultaneously directing the permeate flow to a waste container outside the fluid loop, thereby producing a clarified retentate flow. Optionally, the clarified retentate flow is concentrated in a secondary process vessel for elution. The clarified retentate flow is then contacted with a certain volume of elution buffer for a period sufficient to separate the bioproducts from the resin, and a collection operation is performed. The clarified retentate flow and resin mixture are then circulated through the filtration module, separating the mixture into a permeate flow containing bioproducts and a retentate flow containing resin. The retentate flow is then recirculated through the retentate flow loop for a second number of DV, while the permeate flow is directed to a recovery container outside the fluid loop, thereby separating the bioproducts.

[0008] In some aspects of any of the methods described herein, the filtration module comprises a macroporosity tangential flow filtration (TFF) filter medium composed of nonwoven fibers with pore sizes in the range of 50-200 micrometers. In some aspects, the filter medium may be in the form of a tubular / spiral plate, referred to herein as a tangential flow chromatography filter (filter) or “TFCF”. In some aspects, the filter medium is composed of a nonwoven polypropylene / polyethylene polymer with a pore size of 50-200 micrometers and is formed into a tubular membrane, for example, by spirally wound plate membranes, such as… Figure 27 As shown. Suitable membranes include nonwoven wet-laid membranes. In some respects, TFCF media are not formed by extrusion.

[0009] The method may further include a capture operation in a secondary container fluidly connected to the process container. This may include transferring a first volume of process fluid from the process container to the secondary container, wherein the secondary container contains resin, or adding resin to the secondary container, and performing capture and washing operations to obtain a clarified retentate stream in the secondary container.

[0010] The method may further include: performing a second or further capture operation prior to the elution operation by transferring a second or further volume of process fluid to a secondary process vessel containing a clarified retentate stream, and performing a second or further capture and washing operation to obtain a second or further clarified retentate stream in the secondary vessel; optionally, repeating the capture and washing operation with a third or other volume of process fluid prior to the elution and collection operation.

[0011] In all cases, it should be understood that washing operations may include percolation and concentration steps.

[0012] The method may also include replenishing fluid lost in the permeate stream during one or both of the washing and collection operations to maintain a constant volume of fluid in the process vessel and / or secondary vessel in a batch or continuous process.

[0013] The method may also include, during one or both of the washing and collection operations, not replenishing the fluid lost in the permeate stream, in order to concentrate the fluid in the process vessel.

[0014] The method may include, after the collection operation, performing an additional filtration step on the collected cell products, which includes filtration through a tangential flow depth filter medium.

[0015] The method may further include, for insect or mammalian cells, the process fluid being characterized by a live cell density (VCD) or total cell density (TCD) of 10E5 to 10E9 cells / ml, or for bacterial cells, the process fluid being characterized by an optical density (OD) of 1-350 at 600 or 620 nanometers (nm).

[0016] The method may further include wherein, prior to contact with the resin, the process fluid is characterized by a turbidity of 100-30,000 nephelometric turbidity units (NTU) or 200-1,000 NTU.

[0017] The method may further include the process fluid being characterized by a viscosity of about 1.5-30 centipoise (cP).

[0018] The method may further include a washing operation sufficient to remove 95-99% of cells and / or cellular proteins and nucleic acids from the entrapped material stream.

[0019] The method may further include a washing operation sufficient to achieve an average reduction of 2-5 logarithmic levels in cellular proteins and nucleic acids in the retained material stream.

[0020] The method may further include the following: after the collection operation, at least 90% of the resin is retained in the retrieval fluid.

[0021] The method may further include a filter module comprising a filter medium including one or more hollow fiber elements forming a hollow fiber depth filter medium, each hollow fiber element consisting of a porous wall with a thickness of 2-10 millimeters (mm) defining an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of about 50-90%, and a pore size class of 10-50 micrometers.

[0022] The method may further include each of one or more hollow fiber elements forming the hollow fiber depth filter medium comprising a porous wall with a thickness of 2-10 mm, the porous wall defining an inner cavity with an ID of 1-12 mm, a porosity of about 60-90%, and a pore size class of 10, 20, 30, 40, or 50 micrometers.

[0023] The method may further include a filtration module comprising a macroporous tangential flow chromatography or "TFC" filter medium comprising one or more hollow fiber elements composed of nonwoven fibers with a pore size in the range of 50-200 micrometers. In some aspects, the TFC filter medium may be in the form of a flat plate helically wound into a tubular shape, which may also be referred to as "tubular / helical winding". Exemplary methods for preparing TFC filter media are as follows: Figure 27 In some aspects, the TFC filter media is composed of a nonwoven polypropylene / polyethylene polymer with a pore size of 50-200 micrometers. In some aspects, the TFC filter media includes a porous wall with a thickness of 0.1 to 0.5 mm, which defines an inner cavity with an ID of 1 to 12 mm, a porosity of about 60 to 90%, and a pore size class of 10, 20, 30, 40, 50, 100, 150, or 200 micrometers.

[0024] The method may further include wherein the biological product is an antibody, recombinant protein, or viral particle.

[0025] The method may further include functionalizing the resin with an Fc-binding ligand or a ligand that binds to virus particles. Other technical features will be apparent to those skilled in the art from the following figures, description, and claims.

[0026] In one aspect, a system is provided for separating biological products from a process fluid characterized as defined herein by high cell density and / or high turbidity, wherein the system comprises: a process vessel containing the process fluid; at least one filtration module in fluid communication with the process vessel, the filtration module comprising a tangential flow filtration (TFF) or tangential flow depth filtration (TFDF) module, wherein the filtration module comprises one or more hollow fiber elements forming a filter medium and a housing, each hollow fiber element comprising a porous wall with a thickness of 0.5-10 mm, the porous wall defining an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of about 50-90%, and a pore size class of 10-50 micrometers, the housing being adapted to separate the process fluid into a permeate stream and a retentate stream as the process fluid flows through the filter medium, the housing having an inlet, a permeate outlet, and a retentate outlet; and at least one pump.

[0027] In one aspect, a system is provided for separating biological products from a process fluid characterized as defined herein by high cell density and / or high turbidity, wherein the system comprises: a process container containing the process fluid; at least one filter module in fluid communication with the process container, wherein the filter module comprises one or more hollow fiber elements forming a TFC filter medium and a housing, each hollow fiber element comprising a porous wall with a thickness of 0.1-0.5 mm defining an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of about 50-90%, and a pore size class of 10-200 micrometers, the housing being adapted to separate the process fluid into a permeate stream and a retentate stream as it flows through the filter medium, the housing having an inlet, a permeate outlet, and a retentate outlet; and at least one pump. In some aspects, the TFC filter media is composed of a nonwoven polypropylene / polyethylene polymer with a pore size of 50-200 micrometers and a porous wall thickness of 0.1-0.5 mm, which defines an inner cavity with an ID of 1-12 mm, a porosity of about 60-90%, and a pore size class of 10, 20, 30, 40, 50, 100, 150, or 200 micrometers.

[0028] The system may also include at least two modules connected in series or in parallel with the process vessel.

[0029] The system may also include a retardant pump and a permeate pump.

[0030] The system may also include a permeate container and a product recovery container.

[0031] The system may also include a fluid or buffer supply source connected in a feed relationship to the process vessel. Other technical features will be apparent to those skilled in the art from the following figures, description, and claims.

[0032] In one aspect, a tangential flow filtration (TFF) or tangential flow depth filtration (TFDF) module is provided, comprising one or more hollow fiber elements and a housing forming a hollow fiber filter medium or a hollow fiber depth filtration medium. Each hollow fiber element consists of a porous wall with a thickness of 0.5-10 mm defining an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of about 50-90%, and a pore size class of 10-50 micrometers. The housing is adapted to separate a process fluid into a permeate stream and a retentate stream as the process fluid flows through the filter medium. The housing is provided with an inlet, a permeate outlet, and a retentate outlet.

[0033] In one aspect, a tangential flow filtration (TFF) module is provided, comprising one or more hollow fiber elements forming a TFC filter medium and a housing. Each hollow fiber element consists of a porous wall with a thickness of 0.1-0.5 mm, defining an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of about 50-90%, and a pore size class of 10-200 micrometers. The housing is adapted to separate a process fluid into a permeate stream and a retentate stream as the filter medium flows through it. The housing has an inlet, a permeate outlet, and a retentate outlet. In some aspects, the TFC filter medium is composed of a nonwoven polypropylene / polyethylene polymer with a pore size of 50-200 micrometers and a porous wall thickness of 0.1-0.5 mm, defining an inner cavity with an ID of 1-12 mm, a porosity of about 60-90%, and a pore size class of 10, 20, 30, 40, 50, 100, 150, or 200 micrometers. Attached Figure Description

[0034] 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 drawings are provided for illustrative purposes only, and the scale, position, order, and relative dimensions reflected in the drawings may vary. In the drawings, identical or substantially identical or equivalent elements are generally indicated by the same reference numerals, and similar elements are generally indicated by similar reference numerals, with redundant descriptions omitted. For the purposes of clarity and simplicity, not every element is labeled in every figure, nor is every element of every embodiment shown, and some of these illustrations are not essential for enabling those skilled in the art to understand the present disclosure.

[0035] Figure 1This is a flowchart depicting a capture / collection operation according to one aspect of the invention, which illustrates four main processes: capture, washing, elution, and collection.

[0036] Figure 2 A system according to one aspect of the invention is shown that can be used in conjunction with capture / acquisition operations.

[0037] Figure 3 A system according to one aspect of the invention is shown that can be used in conjunction with "constant feed concentration" operation.

[0038] Figure 4 A system according to one aspect of the invention is shown that can be used in conjunction with "dead end capture".

[0039] Figure 5 It is a line graph showing the relationship between pressure (psi) and time (minutes).

[0040] Figure 6 The results of the model are shown, which indicate that the membrane with a porosity of 67% will not clog at an average permeation flux of 4,000 LMH.

[0041] Figure 7A This is a line graph showing the RNA (ng / ul) removed from the process fluid (step 1) through 9 percolation volumes (DV, steps 2-10) in the permeate (triangle) fluid stream and the retentate (square) fluid stream through a TFDF membrane with a porosity of 67%.

[0042] Figure 7B This is a line graph showing the host cell DNA (HCD, ng / ul) removed from the process fluid (step 1) through 7 percolation volumes (DV, steps 2-8) in the permeate (triangle) fluid stream and the retained (square) fluid stream through a TFDF membrane with a porosity of 67%.

[0043] Figure 8A This is a line graph showing the reduction in turbidity (NTU) through nine percolation volumes (DV, steps 2-10) in the permeate (triangular) and retentate (square) fluid streams passing through a TFDF membrane with a porosity of 67%, starting from the process fluid (step 1). In the permeate stream, the turbidity is reduced to almost zero. In the retentate stream, the remaining turbidity is caused by the resin (approximately 1.5% resin slurry).

[0044] Figure 8B This is a line graph of the absorbance (AU) of the permeate at 260 nm (triangle) and 280 nm (square) during a percolation process through a TFDF membrane with a porosity of 67%, starting from the process fluid (step 1) and passing through 9 percolation volumes (DV, steps 2-10).

[0045] Figure 9 This is a line graph showing the change of absorbance (AU) of permeate (triangle) and retentate (square) fluid streams at 977 nm over time (min) through a TFDF membrane with a porosity of 67%.

[0046] Figure 10 A system according to one aspect of the invention is shown that can be used in conjunction with "constant feed concentration" operation.

[0047] Figure 11 This is a bar graph showing the results of an acquisition and capture operation according to one aspect of the present invention.

[0048] Figure 12 A system according to one aspect of the present invention is shown.

[0049] Figure 13 A system according to one aspect of the present invention is shown.

[0050] Figure 14 It is a line graph showing the relationship between the percentage (%) of immunoglobulin G (IgG) binding and the percentage (%, w / v) of protein A resin for three concentrations of IgG (1 mg / ml IgG (top line), 3 mg / ml IgG (middle line), and 10 mg / ml IgG (bottom line)).

[0051] Figure 15 It is a line graph showing the relationship between the percentage (%) of AAV coating (AAV) bound to resin 1 and resin 2 and the percentage (%, w / v) of resin solids.

[0052] Figure 16 This is a line graph showing the host cell DNA removed during a process using HEK293 cells.

[0053] Figure 17 This is a graph showing the amount of lysate removed as the turbidity in the permeate decreases in a process using HEK293 cells.

[0054] Figure 18 This is a bar graph showing the product recovery of a 1-liter scaled-down model of monoclonal antibody (mAb) harvested from Chinese hamster ovary (CHO) cells.

[0055] Figure 19 It shows Figure 18 Pressure curves of a scaled-down model were collected and captured using a 1-liter mAb sample.

[0056] Figure 20This is a bar graph showing the product recovery of a 1-liter scaled-down model of AAV9 harvested from HEK293 cells.

[0057] Figure 21 It shows Figure 20 Pressure curves of a scaled-down model were acquired using a medium-1 liter AAV acquisition system.

[0058] Figure 22 This is a line graph showing the relationship between the capture of monoclonal antibodies (mAbs) (in mg IgG per ml of resin) and time (minutes). The top curve represents the measurement obtained from the online capture retentate at a specified time; the bottom curve represents the measurement obtained from the batch capture retentate.

[0059] Figure 23 This is a bar chart showing AAV captured using batch capture or online capture processes, with data acquired at a single 3-hour time point.

[0060] Figure 24 This is a bar chart showing the percentage (%) of AAV in the solution used for online single-pass processes for capture / collection.

[0061] Figure 25A The pressure profile of the untreated pyrolysis solution is shown.

[0062] Figure 25B The pressure profile of the pyrolysis solution treated under acidic conditions is shown.

[0063] Figure 25C The pressure profile of the lysis buffer treated with endonuclease is shown.

[0064] Figure 26A This is a bar graph showing the cleared DNA obtained by lysis buffer treated with acidic conditions.

[0065] Figure 26B This is a bar graph showing the cleared host cell proteins obtained by lysate treated with acidic conditions.

[0066] Figure 27 This is a schematic diagram of a method for forming a high-porosity tangential flow filtration (TFF) filter medium composed of nonwoven fibers in the form of tubular / spiral flat sheets with pore sizes ranging from 50 to 200 micrometers. As shown, the nonwoven flat strip (50-200 micrometer pore size) is wound into a spool (e.g., by spiral winding) and then thermally or ultrasonically welded to itself in a spiral manner to form a tubular filter element. In some aspects, the nonwoven fibers are polypropylene / polyethylene polymers. Detailed Implementation

[0067] Recovering cell-derived biological products (which may include antibodies and other recombinant proteins, viral particles, viral vectors (including adeno-associated virus (AAV) particles or lentivirus (LV) particles or AAV vectors or LV vectors), and other nucleic acid vectors (including eukaryotic or bacterial plasmid vectors, and other nucleic acid-based products (including antisense oligonucleotides, mRNA, siRNA, shRNA, cDNA, etc.)) involves physically separating the biological products from cell debris in the producing cells or host cells and / or cell culture medium or lysis buffer (hereinafter referred to as "CF"). Product recovery operations typically begin when the producing cells reach a predetermined cell density (characterized by viable cell density (VCD) or total cell density (TCD)). Typically, the CF at this stage contains a high density of cells and cell debris. In the process of lysing cells to release biological products, CF will contain high concentrations of host cell proteins (HCP) and host cell DNA (hcDNA), as well as other cellular debris and / or viral particles, and will typically also have a viscosity greater than 1 centipoise (cP) (e.g., in the range of 2–30 cP, or 2–20 cP, or 2–10 cP). In either case, at the initial stage of product recovery from CF, 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, CF typically undergoes multiple clarification and concentration operations (including centrifugation, filtration, and percolation) to obtain a fluid with sufficient purity and concentration for further purification by column chromatography (including affinity chromatography).

[0068] Compared to existing methods, the present invention advantageously utilizes a single tangential flow filtration operation to capture and collect bioproducts from CF without requiring a prior clarification step (including, for example, a prior centrifugation step and / or a prior filtration step). Instead, as described in detail below, a suitable capturing resin (e.g., a suitably functionalized affinity resin, ion exchange resin, hydrophobic interaction chromatography (HIC) resin, multimode chromatography (MMC) resin, or immobilized metal affinity chromatography (IMAC) resin) is directly contacted with the unclarified CF under conditions that allow the bioproducts to bind to the resin during the capture operation. The capture operation may also include an optional concentration step. The capture operation can be performed in a variety of ways, as discussed in detail below, followed by a washing operation (which may be preceded by an optional concentration step) performed by percolation through a filtration module. The filtration module includes a filter medium that may include tangential flow filtration (TFF) media, tangential flow depth filtration (TFDF) media, or tubular / spiral plate filter media, referred to herein as a tangential flow chromatography filter or "TFCF". In practice, the washing operation may include a series of percolation and concentration steps, as described below. The bioproduct is then eluted from the capture resin and collected in a collection operation, as described below, which may also include a series of percolation and concentration steps. The collection operation also separates the resin, which can then be regenerated. The methods and systems described herein advantageously reduce processing time, lower costs, increase efficiency, and provide high product yields, at least in part due to the reduction in the number of required operation steps. Furthermore, the methods described herein provide bioproducts of high purity. For example, in the case where the bioproduct is recombinant viral particles, as described in detail below, the recovered viral particles have low levels of contaminating host cell proteins and nucleic acids. Alternatively, in the case where the bioproduct is a recombinant protein (e.g., an antibody or monoclonal antibody), the recovered antibody has low levels of contaminants (including viruses).

[0069] Figure 1 This is a flowchart illustrating the steps of a biological product capture / collection operation according to one aspect of the invention. The term "biological product" refers to a product produced by cells. Exemplary biological products include recombinant proteins, antibodies, nucleic acid vectors (including viral vectors such as AAV or lentiviral (LV) vectors), and viral particles (including AAV and LV particles and virus-like particles (VLPs)). The producing cells can be bacterial cells, yeast cells, insect cells, or mammalian cells. The biological product may be secreted from or released from the cell into a cell culture medium (CF), for example, by cell lysis. In cases where cells are lysed to release the biological product, the CF may also be referred to as a "lysate."

[0070] An important aspect of the method described herein is that the CF undergoes no clarification process prior to contact with the capturing resin. As mentioned above, the CF at this stage is 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 CF may have a cP of 1.5–30 cP, or 1.5–20 cP, or 1.5–10 cP, or a cP of about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20. In the discussion below, the CF is referred to as the “process fluid.” Thus, at the start of a capture / collection operation according to the method described herein, the process fluid is characterized by high cell density and / or high turbidity, and optionally a viscosity greater than 1 cP, as defined in more detail in the following paragraphs.

[0071] In aspects where the process fluid is characterized by high cell density, cell density can be measured as viable cell density or “VCD” (including VCD pre-lysis, where cells are lysed to release cell products to be recovered) or total cell density “TCD” (which includes both viable and non-viable cells). For example, the VCD or TCD of a process fluid can be from 1 x 10^5 (10E5) to 10E9 cells / ml. In some aspects, for mammalian and insect cells, the VCD or TCD of the process fluid can be about 10E5 to 10E6 cells / ml, or about 10E6 to 10E7 cells / ml, or about 10E8 to 10E9 cells / ml. In other aspects, such as when the cells are bacterial cells, cell density can be measured in units of optical density (OD). For example, when the cells are E. coli cells, the OD of the process fluid at 600 or 620 nm can be 1–350, or 30–300, or 30–250.

[0072] In aspects where the process fluid is characterized by high turbidity, the turbidity can be measured in scattering turbidity units (NTU). In some aspects, the turbidity of the process fluid can be about 100-30,000 NTU. In some aspects, the turbidity of the process fluid can be about 100-10,000 NTU, or about 100-5,000 NTU, or about 100-2,500 NTU, or about 100-1,000 NTU, or about 100-500 NTU. In some aspects, the turbidity of the process fluid can be about 200-1,000 NTU, or about 300-1,000 NTU, or about 400-1,000 NTU. In some aspects, the turbidity of the process fluid can be about 300, about 400, about 500, about 600, about 700, about 800, or about 900 NTU. In this document, the turbidity of the process fluid refers to its turbidity before the addition of the capturing resin. Generally, resins can increase the turbidity of a fluid by approximately 3,000–6,000 NTU or more, depending on the amount of resin added. The methods and systems described herein allow for the capture of biological products from highly turbid process fluids without the need for prior clarification steps, such as pre-centrifugation or filtration operations.

[0073] In cases where the process fluid is characterized by a viscosity greater than 1 cP, the viscosity of the process fluid can be about 5-100 cP, or 5-50 cP, or 5-25 cP. In some aspects, the viscosity of the process fluid can be about 2, about 5, about 10, or about 15 cP. The process fluid is characterized by a viscosity greater than 1, for example, in cases where cells that produce biological products are lysed to release the biological products into the process fluid before capture.

[0074] In some respects, the process fluid is characterized by one or more of the following: high cell density (which may be high live cell density (VCD), high total cell density (TCD) or high optical density (OD)), high turbidity and / or viscosity greater than 1, wherein high cell density, high turbidity and viscosity are defined by the ranges above.

[0075] Reference Figure 1 The method described herein begins with a capture operation 102. During the capture process, as described above, a process fluid containing the biological product and cells and / or cell debris, or including cell lysates (where cells are lysed to release the biological product), and having a high cell concentration and / or high turbidity, is contacted with the capture resin for a period of time under conditions suitable for the binding of the biological product to the resin. This time period is selected according to the specific resin, as recommended by the manufacturer. Suitable binding conditions, as recommended by the resin manufacturer, may include, for example, a specific temperature.

[0076] In the aspect of lysing cells to release biological products, cells can be lysed using any suitable method (preferably mechanical, such as using back pressure from a centrifugal pump) before the capture operation begins.

[0077] According to the methods described herein, contact for the capture operation can be achieved by adding the capture resin directly to the primary process vessel (which may be a bioreactor), or by any other suitable method, such as by adding the process fluid to a secondary process vessel containing the resin, or by recycling the resin in the flow path of the filtration module (also known as the retentate flow path). Thus, in some aspects, the capture operation can be achieved by adding the capture resin directly to the bioreactor or directly to a secondary process vessel in fluid communication with the bioreactor. Alternatively, the resin may be present in a retentate flow loop of the filtration module, which is fluidly connected to the bioreactor or the secondary process vessel. According to any of the foregoing configurations, the retentate flow loop may also be fluidly connected to one or more of a wash buffer tank, an elution buffer tank, and a regeneration buffer tank. As mentioned above, the term "process fluid" refers to CF, characterized by high cell density or high turbidity, or both, and may also have a viscosity greater than 1 cP, since the fluid is not subjected to any prior clarification process (including prior centrifugation or filtration).

[0078] According to the methods described herein, suitable resins for capturing biological products are in the form of beads or other particles with an average particle diameter approximately 1.5 to 10 times larger than the average pore size of the filter medium. Suitable capturing resins include affinity resins, ion exchange resins, hydrophobic interaction chromatography (HIC) resins, multimode chromatography (MMC) resins, and immobilized metal affinity chromatography (IMAC) resins. In some aspects of the methods described herein, the resin may have an average particle size of about 20 micrometers, about 30 micrometers, or about 50 micrometers. In some aspects, the average particle size of the resin may be up to 200 micrometers, for example, 20-50 micrometers or 50-200 micrometers, or about 100 micrometers, about 150 micrometers, or about 200 micrometers. Suitable affinity resins include chemical substances or ligand chemicals capable of binding biological products to the resin with high affinity. For example, in some aspects, the resin is functionalized with a ligand (e.g., Staphylococcus aureus Protein A or a derivative thereof) capable of binding to the Fc region of an antibody or other Fc-containing protein. In some respects, the resin is functionalized with ligands capable of binding viral particles (e.g., AAV or LV particles). In some respects, the ligands are capable of binding one or more AAV capsid proteins. Suitable resins that can be used include resins formed from discrete polymer particles functionalized with affinity ligands, wherein these polymer particles can be made from polysaccharides such as agar, agarose, dextran, starch, cellulose, pullulan, etc., and their stabilized variants and derivatives; or wherein these particles are made from synthetic polymers (e.g., polystyrene, polyvinyl ether, polyvinyl alcohol, polyacrylate, polymethacrylate, polyacrylamide, etc.). Suitable affinity resins include regenerated resins and single-use or disposable resins. Suitable affinity resins are commercially available. For example, Repligen Corp.'s (Waltham MA) AVIPure® AAV affinity resin and CaptivA® protein A affinity resin provide capture of AAV viral particles (sometimes referred to in the art as AAV vectors) and Fc-containing proteins, respectively. Other suitable resins include those made with protein A or protein A derivatives (e.g., Eshmuno) ® A (MilliporeSigma) and MabCaptureC ® (Thermo Fisher Scientific) functionalized resins; and metal chelates (including those for separating recombinant proteins, especially histidine-tagged proteins, such as Fractogel) with other ligands. ® Metal chelates (MilliporeSigma) or Capto ®Chelating (Cytiva) functionalized resins; and other ligand-functionalized resins for capturing virus particles, such as POROS for isolating AAV. ® CaptureSelect AAV (Thermo Fisher Scientific) and Capto ® AVB (Cytiva), or CaptureSelect Lenti VSVG (Thermo Fisher Scientific) for capturing VSV-G pseudo-LV particles.

[0079] Under conditions suitable for binding the bioproduct to the resin, after incubating the captured resin with the process fluid for a period of time, a washing 104 operation is initiated to separate the resin particles from the process fluid and wash away cells, cell debris, and other contaminants, including, for example, host cell nucleic acids (including DNA and RNA), host cell proteins (HCP), and viral particles, wherein the bioproduct is not a viral particle, and is percolated through a TFF module interconnected in the fluid loop with the process vessel as described herein. In some aspects, the flow-through or percolated material stream is returned to the bioreactor or secondary process vessel. In other aspects, the flow-through or percolated material stream is directly delivered to the waste container. Suitable buffers for the washing operation include, for example, Tris, Tween, HEPES, and citrate buffer. In some aspects, the washing buffer does not include phosphate buffer. In some aspects, the washing buffer may include 0.15 M sodium chloride (NaCl), neutral pH. In some aspects, the washing step may be used to verify a predetermined log reduction in contaminating viruses to be removed from the final bioproduct. The ability to remove small viruses (i.e., viral particles with a size in the 20-nanometer range) is determined by... Figure 24 The bar graph illustrates this. It shows that AAV viruses easily pass through a large-pore size filter. Bars labeled "strip" indicate that no detectable virus remains on the resin after elution. Therefore, the large-pore size filter described herein can also allow other viruses of similar size to AAV to pass through, reducing the viral load in the final product. Furthermore, the wash buffer may include virus inactivation conditions (e.g., low pH) to inactivate enveloped viruses.

[0080] The TFF module used in the systems and methods described herein comprises one or more hollow fiber elements forming a filter medium encapsulated within a filter housing. As used herein, the term "hollow fiber" can refer to both "fiber" (characterized in industry as typically having an inner lumen with a diameter less than 2 mm) and "tube" (the term can be used for cases where the inner lumen diameter is greater than 2 mm, for example, in the range of 2-12 mm). Therefore, the term "hollow fiber" is used herein to refer to either a fiber or tubular filter element that collectively comprises an inner lumen with a diameter (also referred to as the internal diameter of the filter element or "ID") in the range of 1-12 mm. In some aspects, the pore size of the hollow fiber element, which is composed of nonwoven fibers, is in the range of 50-200 micrometers. In some aspects, the filter medium can be in the form of a flat plate spirally wound into a tubular shape, which may also be referred to as "tubular / spiral winding" or, in the context of this invention, a tangential flow chromatography filter or "TFCF". In some aspects, the filter medium is composed of a nonwoven polypropylene / polyethylene polymer with a pore size of 50-200 micrometers. Suitable membranes include nonwoven wet-laid membranes. In some respects, TFCF media are not formed by extrusion.

[0081] The filter housing of the TFF module includes a process fluid inlet for introducing process fluid into the upstream or near-end housing of the module and a retentate outlet for leading retentate out of the housing from the downstream or far-end housing of the module. The filter housing will also include at least one permeate outlet for leading permeate out of the housing. The housing may include other ports, such as vent ports and exhaust ports. In some aspects, the filter media is encapsulated within the filter housing to provide an integral device that can be a single-use or disposable unit. In some aspects, the single-use or disposable unit can be sterile. In some aspects, the single-use or disposable unit can be sterilized by ethylene oxide gas sterilization or by radiation (e.g., X-ray radiation, gamma radiation, or electron beam radiation).

[0082] Each hollow fiber element of the filter media consists of multiple nonwoven polymer fibers characterized by pore sizes of 10-50 micrometers, or 20-50 micrometers, or about 30 micrometers, about 40 micrometers, or about 50 micrometers. In some cases, the polymer fibers are sintered. In other cases, the polymer fibers are melt-blown.

[0083] In some aspects, the hollow fiber filter media is a "depth filtration" media, and each of the plurality of hollow fiber elements is defined by a thick porous wall that defines an inner cavity having an inner diameter (ID). In some aspects, the inner diameter (ID) is about 1-12 mm, or about 3-6 mm, or about 4-5 mm. In some aspects, the thickness of the porous wall is about 2-10 mm, or about 4-10 mm, or about 4-6 mm, or about 2-6 mm. In some aspects, the porosity of the wall is in the range of about 50-70% (0.50-0.70). In some aspects, the pore size of one or more hollow fiber elements is 10-50 micrometers. In some aspects, the length of one or more hollow fiber elements is 5-150 cm. In some aspects, the length of one or more hollow fiber elements is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 cm. In some aspects, the length of one or more hollow fiber elements is about 20 cm or about 110 cm.

[0084] Porosity (P) is calculated as a weight percentage based on the density (d) of one or more hollow fiber elements, measured in grams per cubic centimeter (g / cc). When one or more hollow fiber elements consist of more than one type of polymer, the term "dA" takes into account the aggregated or mixed density (dA) of the material, such that the porosity of the aggregated material is calculated as follows:

[0085] P = 1-(d / dA).

[0086] For example, when one or more hollow fiber elements are made of a two-component material, dA is calculated as the sum of the densities of each polymer multiplied by its weight percentage in the material. Therefore, for a material composed of two polymers, P1 and P2, with contents of 70% and 30% by weight respectively, and polymer densities d1 and d2 respectively, the aggregation density is calculated as follows:

[0087] dA = (0.70 x d1) + (0.3 x d2)

[0088] In some aspects, the hollow fiber element is defined by a porous wall about 0.075-10 mm thick (or about 0.075-0.5 mm thick for tangential flow filtration (TFF) media and about 2-10 mm thick for tangential flow depth filtration (TFDF) media), wherein the porous wall defines an inner cavity with a diameter of about 0.5-12 mm, and wherein the porosity of the wall is in the range of about 50-80% (0.50-0.80) or about 50-70%. In some aspects, the pore size of one or more hollow fiber elements is 10-50 micrometers, or 20-50 micrometers, or 30-50 micrometers. In some aspects, the pore size of one or more hollow fiber elements is about 30 micrometers, about 40 micrometers, or about 50 micrometers.

[0089] In some aspects, hollow fiber tangential flow filtration (TFF) elements are defined by thin porous walls approximately 0.075-0.3 mm thick. In embodiments, the porous walls define an inner cavity with an ID of approximately 0.5 to 6 mm in diameter or approximately 0.5-2 mm in diameter, wherein the porosity of the walls is in the range of approximately 50-80% (0.50-0.80) or approximately 50-70%. In some aspects, the pore size of one or more hollow fiber elements is in the range of 10-50 micrometers, 20-50 micrometers, or 30-50 micrometers. In some aspects, the pore size of one or more hollow fiber elements is in the range of approximately 30 micrometers, approximately 40 micrometers, or approximately 50 micrometers.

[0090] In some aspects, the hollow fiber depth filtration (TFDF) element is defined by a porous wall about 2-10 mm thick, or about 2-6 or 4-10 mm thick, defining an inner cavity with an ID of about 1-12 mm in diameter, or about 3-6 mm or about 4-5 mm in diameter, wherein the porosity of the wall is in the range of about 50-80% (0.50-0.80) or about 50-70%. In some aspects, the pore size of one or more hollow fiber elements is 10-50 micrometers, or 20-50 micrometers, or 30-50 micrometers. In some aspects, the pore size of one or more hollow fiber elements is about 30 micrometers, about 40 micrometers, or about 50 micrometers.

[0091] Hollow fiber depth filter media can be defined by their cross-sectional area and the number of hollow fiber elements constituting the media, or by parameters of one or more hollow fiber elements forming the media (e.g., ID, wall thickness, porosity, and length of one or more hollow fiber elements). In some aspects, hollow fiber depth filter media can be defined by their permeability, specifically by their normalized water permeability (NWP) measured in LMH / psi meters. In some aspects, the NWP of hollow fiber depth filter media is from about 7,000 to about 12,000 LMH / psi.

[0092] In aspects where the filter media is a tangential flow chromatography (TFC) filter media, the hollow fiber element is composed of nonwoven fibers with a pore size ranging from 50 to 200 micrometers. In some aspects, the TFC filter media may be in the form of a flat plate spirally wound into a tubular shape, referred to herein as a tangential flow chromatography filter or "TFCF". In some aspects, the TFCF media is composed of a nonwoven polypropylene / polyethylene polymer with a pore size of 50 to 200 micrometers. In some aspects, the TFCF media includes a porous wall with a thickness of 0.1 to 0.5 mm, defining an inner cavity with an ID of 1 to 12 mm, a porosity of about 60 to 90%, and a pore size class of 10, 20, 30, 40, 50, 100, 150, or 200 micrometers. Suitable membranes include nonwoven wet-laid membranes. In some aspects, the TFCF media is not formed by extrusion.

[0093] In some aspects, the hollow fiber elements of TFF or TFDF filter media are made of one or more materials including polysulfone, polyethersulfone (PES), or modified polyethersulfone (mPES). In embodiments, the polysulfone, PES, or mPES has an anisotropic structure.

[0094] Hollow fibers used in filter units can be formed from a variety of materials using various processes. For example, hollow fibers can be formed by assembling numerous particles, filaments, or combinations 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 used to assemble the hollow fibers. The pore size and distribution of hollow fibers formed from filaments will also depend on the density of the filaments used to assemble the hollow fibers. For example, an average pore size ranging from 0.5 micrometers to 50 micrometers can be achieved by varying the filament density.

[0095] Suitable particles and / or filaments include inorganic and organic particles and / or filaments. In some embodiments, the particles and / or filaments may be monocomponent particles and / or monocomponent filaments. In some embodiments, the particles and / or filaments may be multicomponent (e.g., bicomponent, tricomponent, etc.) particles and / or filaments. For example, bicomponent particles and / or filaments having a core formed of a first component and a coating or sheath formed of a second component can be used, and many other possibilities exist.

[0096] In various embodiments, the granules and / or filaments can be made of polymers. For example, the granules and / or filaments can be polymer-monocomponent granules and / or filaments formed from a single polymer, or they can be polymer-multicomponent (i.e., bicomponent, tricomponent, etc.) granules and / or filaments formed from two, three, or more polymers. A variety of polymers can be used to form monocomponent and multicomponent granules and / or filaments, including polyolefins (e.g., polyethylene and polypropylene), polyesters (e.g., polyethylene terephthalate and polybutylene terephthalate), polyamides (e.g., nylon 6 or nylon 66), fluoropolymers (e.g., polyvinylidene fluoride (PVDF) and polytetrafluoroethylene (PTFE)), etc. Suitable polyethylene polymers include, but are not limited to, high-density polyethylene (HDPE) and high molecular weight or ultra-high molecular weight polyethylene (UHMWPE).

[0097] The granules can be formed into a tubular shape using, for example, a tubular mold. Once formed into a tubular shape, the granules can be bonded together using any suitable process. For example, the granules can be bonded together by heating them to a temperature at which they partially melt and bond together at different contact points (a process known as sintering), optionally while also compressing the granules. As another example, the granules can be bonded together by using a suitable adhesive to bond them together at different contact points, optionally while also compressing the granules.

[0098] Filament-based manufacturing techniques that can be used to form tubular shapes include, for example, simultaneous extrusion from multiple extrusion dies (e.g., melt extrusion, solvent-based extrusion, etc.), or electrospinning or electrospraying onto a rod-shaped substrate (which is then removed), and so on.

[0099] Filaments can be bonded together using any suitable process. For example, filaments can be bonded together by heating them to a temperature at which they partially melt and bond together at different contact points, optionally while also compressing them. As another example, filaments can be bonded together by using a suitable adhesive to bond them together at different contact points, optionally while also compressing them.

[0100] In certain implementations, many fine extruded filaments can be joined together at different points to form hollow fibers, for example, by forming tubular shapes from extruded filaments and heating the filaments to join them together, as well as other possible solutions.

[0101] In some aspects, the hollow fiber elements of depth filtration media are formed from sintered or melt-blown polymer fibers. In the context of “polymer fibers” or “polymer filaments,” the terms “fiber” and “filament” are used interchangeably herein. Polymers that can be used include polyolefins (e.g., polyethylene and polypropylene), polyesters (e.g., polyethylene terephthalate and polybutylene terephthalate), polyamides (e.g., nylon 6 or nylon 66), fluoropolymers (e.g., polyvinylidene fluoride (PVDF) and polytetrafluoroethylene (PTFE)), and so on. Suitable polyethylene polymers include high-density polyethylene (HDPE) and high-molecular-weight or ultra-high-molecular-weight polyethylene (UHMWPE). In some aspects, the polymer is selected from polypropylene, polyesters, and mixtures thereof.

[0102] The term "sintering" as used herein refers to the use of heat and, optionally, pressure in a bonding process. In this method, polymer fibers are heated to a temperature at which the filaments partially melt and bond together at different points of contact, optionally while the filaments are also compressed. Thus, sintering bonds the fibers at the contact points, creating void spaces between the fibers. Many fine extruded filaments can be bonded together at different points to form hollow fibers, for example, by forming a tubular shape from the extruded filaments and heating the filaments to bond them together.

[0103] The term "meltblown" refers to the use of a gaseous stream at the exit of a filament extrusion die to draw or thin molten filaments. Meltblown filaments are described, for example, in Berger's US 5607766. When monocomponent or bicomponent filaments exit the extrusion die, they can be drawn using known meltblowing techniques to produce filament assemblies. These filament assemblies can then be bonded together in the form of hollow fibers.

[0104] In some aspects, hollow fibers in the filter media used in the filter modules described herein can be formed by combining bicomponent filaments having a sheath of a first material that can be bonded at temperatures below the melting point of the core material. For example, hollow fibers can be formed by combining bicomponent extrusion technology with meltblown drawing technology to produce an entangled web of biocomponent filaments, and then shaping and heating the web, for example in an oven or using a heated fluid (e.g., steam or heated air), to bond the filaments at their contact points. An example of a sheath-core meltblown die is schematically shown in US 5,607,766, in which molten polymer forming the sheath and molten polymer forming the core are fed into the die and extruded together. Extruding the molten bicomponent sheath-core filaments into a high-speed airflow draws the filaments thin, thereby enabling the production of fine bicomponent filaments. Berger's US 3,095,343 discloses an apparatus for aggregating and heat-treating multi-filament webs to form a continuous tubular body (e.g., hollow fiber) composed of filaments predominantly randomly oriented longitudinally, wherein the filament tubular body is longitudinally aligned as a whole and generally in a parallel orientation, but has short portions extending randomly along non-parallel divergent and convergent directions. In this way, for example by using a conical nozzle with a central channel forming member, a web of sheath-core bicomponent filaments can be drawn into a confined area, where it is aggregated into a tubular rod shape and heated or cured to bond the filaments.

[0105] Hollow fiber depth filter media do not have a defined pore size. However, the pore size can be determined using methods known in the art, such as the "bubble point test." The bubble point test is based on the fact that, for a given fluid and pore size, under constant wetting conditions, the pressure required to force bubbles through the pores is inversely proportional to the pore diameter. In practice, this means that the maximum pore size of a filter can be determined by wetting the filter with the fluid and measuring the pressure at which a continuous flow of bubbles is first observed downstream of the wetted filter. The point at which the first flow of bubbles emerges from the filter reflects one or more of the largest pores in the filter, and the relationship between pressure and pore size is based on Poiseuille's law, which simplifies to P = K / d, where P is the gas pressure at which the bubble flow appears, K is an empirical constant depending on the filter, and d is the pore diameter. In this respect, the pore size determined experimentally can be measured using a device such as the POROLUX™ 1000 porosimeter (Porometer NV, Belgium) or a similar device.

[0106] In practice, given the macropore size of the filter media used in the methods described herein, a pass / retention test can be used to determine the pore size instead of a bubble point test. According to the methods described herein, the average pore size of the hollow fiber elements forming the filter media is selected to retain resin particles used to capture biological products. In some aspects, the average pore size of the material forming the porous walls of the hollow fiber elements is 1.5 to 10 times smaller than the average diameter of the resin, or about 2 to 5 times smaller. In some aspects, the walls of one or more hollow fiber elements are characterized by a porosity of about 50-70% (0.50-0.70), or about 55-70%. In some aspects, the pore size grades of one or more hollow fiber elements are 10-50 micrometers, or about 20 micrometers, about 30 micrometers, about 40 micrometers, or about 50 micrometers, depending on the size of the resin particles.

[0107] In some respects, the hollow fiber elements thus formed can be additionally coated with a suitable coating material (such as PVDF) on the inside or outside of the fiber, and this coating process can also reduce the pore size of the hollow fiber.

[0108] In some aspects, the average diameter of the captured resin beads is 20 micrometers, 50 micrometers, or 75 micrometers, and the filter media includes hollow fiber elements in which the porosity of the walls is in the range of about 50-90% (0.50-0.90) or about 50%, about 60%, about 70%, about 80%, or about 90%. In some aspects, the pore size of one or more hollow fiber elements is in the range of 10-50 micrometers or about 10 micrometers, about 20 micrometers, about 30 micrometers, about 40 micrometers, or about 50 micrometers.

[0109] Back Figure 1 The washing 104 operation includes multiple perfiltration steps sufficient to remove cells, cell debris, and associated impurities from the resin using a perfiltration buffer, such as phosphate-buffered saline (PBS) or other suitable buffer. The extent of resin washing during the washing operation can be measured by the perfiltration volume (DV). A single DV is the initial volume of fluid in the process vessel after capture 102. Therefore, one DV has been processed when the permeate volume equals the initial volume. Perfiltration can be performed in batch or continuous processes. Perfiltration is performed using 5–20 DV, according to some aspects of the method described herein. The perfiltration operation is preferably performed at an average process throughput of about 500–4000 LMH or about 1000–4000 LMH.

[0110] Washing 104 may also include concentrating the resin. In some aspects, the resin may be concentrated to, for example, about 1 to 80% by volume (% v / v), or about 10-70% v / v, or about 20-60% v / v. In some aspects, the resin may be concentrated 10 to 100 times (10-100X).

[0111] Back Figure 1 Following wash 104, elution 106 is initiated by adding elution buffer to the process vessel or secondary vessel containing the resin-bound bioproduct, or to the retentate stream loop containing the resin-bound bioproduct. The elution buffer is prepared to disrupt the high-affinity binding between the resin and the bioproduct and will be selected according to the manufacturer's instructions for the specific resin used. The resin is incubated with the elution buffer for a period of time under conditions suitable for maximally dissociating the bioproduct from the resin. The time and conditions may include specific pH and salt concentrations, or may be determined according to the manufacturer's instructions for the resin. After elution 106, collection 108 is initiated by passing the resin and dissociated bioproduct through a filtration module and collecting the product from the permeate stream. Collection continues through multiple DVs via the filtration module as needed to achieve the desired product yield. Collection operations are preferably performed at an average permeate flux of approximately 500–4000 LMH or approximately 1000–4000 LMH.

[0112] In some respects, an additional filtration step using a 0.8 / 0.2 micron or other similar gradient membrane filter (e.g., a 0.45 / 0.2 micron glass fiber or polymer capsule filter) can be added during the process of allowing the eluent to leave the filter and enter the collection container to remove any large particles that may have left the TFF filter during the elution step.

[0113] It should also be understood that multiple acquisition / capture paths and modules, as described herein, can be connected in parallel. In one aspect, at least two paths and modules are connected in parallel such that the resin in the first module can be regenerated, while the resin in the second parallel path / module continues to undergo the capture and elution steps, thereby enabling continuous operation.

[0114] Figure 2 A system for capturing / collecting biological products according to one aspect of the invention is illustrated schematically. For example... Figure 2 As shown, a unique and advantageous feature of the system and method described herein is that the capture / collection operation is performed using a single fluid loop that interconnects the process vessel 202 and the filter module 206, rather than using multiple fluid loops for these operations. Figure 1 The capture step 102 mentioned above is performed in process container 202, which can be a primary process container, for example... Figure 2 The bioreactor shown, or as Figure 3 The secondary process vessel 324 is shown. Return to... Figure 2The figure illustrates a fluid conduit 228 for process vessel 202, which can be used to add resin or process fluid to the process vessel according to various methods described herein. For example, in the case where the capture 102 step is performed by adding resin directly to a primary process vessel (e.g., a bioreactor), the conduit can be used to transfer the resin to the primary process vessel. Alternatively, in cases where a secondary process vessel contains resin, such as... Figure 3 As shown, the conduit can be used to transfer process fluids into a secondary container.

[0115] As described above, during the capture process, the process fluid and resin are incubated together for a period of time under conditions suitable for binding the bioproducts with the resin. During this time, the process fluid and resin can be mixed using any suitable method. In one aspect of the method described herein, mixing is performed by operating the impeller of the process vessel 202. According to this aspect, valves 226, 214, and 216 remain closed. In another aspect, the process fluid and resin can be mixed by operating pump 210 to recirculate them through the filter module 206. According to this aspect, valves 214 and 216 are opened, while valve 218 remains closed.

[0116] After capture 102, a washing process is initiated. If necessary, the washing operation may include a series of concentration and percolation steps to remove contaminants and impurities from the resin containing the bound products. During washing, valves 214, 216, 218, and 220 are opened, while valves 222 and 226 remain closed. Fluid is circulated through the filtration module 206 by operating the retentate pump 210. The retentate pump may be, for example, a low-shear centrifugal pump. The filtration module 206 separates the fluid into a retentate stream containing the resin containing the bound products and a percolate stream containing cells and / or cell lysates and other debris. The percolate stream may be directed to the waste container 204 by operating the percolate pump 212, or, in an alternative not shown in the figure, to a secondary container, for example, to capture production cells. The percolate pump 212 may be, for example, a peristaltic pump. The retentate stream returns to the process container 202 via a single retentate fluid loop. Buffer, cell culture medium, or optional additional process fluids as described below can be added to process vessel 202, for example, via conduit 228, to replenish the fluid volume lost in the permeate flow.

[0117] After washing, the bioproduct is eluted from the resin in the process vessel. During elution, the fluid in the process vessel is isolated by closing valves 218 and 220 and shutting off the permeate pump 212. Elution buffer is added to the process vessel 202, for example, through conduit 228, and the resin bound to the product is incubated for a period of time to provide maximum dissociation of the bioproduct from the resin. During elution, the mixture of the resin-bound product and the buffer can be mixed using any suitable method, such as by operating an impeller in the process vessel or by recirculating through the filter module 206, as described above regarding binding capture.

[0118] After elution, valves 218 and 222 are opened to activate permeate pump 212, collecting the biological products in the permeate stream into product recovery container 208. Similar to washing, the recovery operation may include a series of concentration and permeation steps through filter module 206 until the desired amount of product is collected. Permeation buffer may be added to the system, for example, through conduit 228.

[0119] In an optional step, the captured resin can be regenerated within process container 202, or collected offline via conduit 224 through valve 226 into a separate collection container. If regeneration is performed within process container 202, regeneration buffer can be added via conduit 228. Resin regeneration can be performed according to the manufacturer's instructions.

[0120] The system may also include one or more of a flow meter, a pressure sensor, and a controller.

[0121] The method for capturing / collecting biological products according to the present invention can be carried out in a variety of ways. For example, in one aspect, a "constant feed concentration" or "CFC" operation can be utilized. According to this aspect, capture is performed by directly adding resin to a primary process vessel (e.g., a bioreactor) containing process fluid. As described above, according to all the methods described herein, the process fluid is characterized by a high cell concentration and / or high turbidity. As described above, optionally, in the case of mixing, the resin and process fluid are incubated together for a period of time under conditions suitable for maximizing the binding of cell products with the resin. After capture, a portion of the fluid mixture is moved to a secondary vessel, where a washing process is initiated in "constant feed concentration" or "CFC" mode. In this operating mode, the secondary vessel is kept at a constant volume by replenishing the fluid lost in the permeate stream with additional process fluid and resin mixture from the primary process vessel. CFC operation continues until all process fluid mixture from the primary vessel is added to the secondary vessel and concentrated to the desired volume. At this point, the contents of the secondary vessel are washed in a process that may include a combination of concentration and percolation operations until the resin is sufficiently free of contaminants and at a concentration suitable for elution.

[0122] Elution is initiated by adding elution buffer to a secondary container containing the bioproduct bound to the resin. As described above, after incubation for a period of time, the collection operation is initiated under conditions suitable for maximally eluting the bioproduct from the resin, and the cell product is collected by percolation through a filtration module. As described above, collection can be performed in concentrated, percolated, or a combination of both modes until the desired amount of product is collected. Optionally, the capture resin can be regenerated within the secondary container or collected in a separate collection container for offline regeneration.

[0123] Figure 3 A system for bioproduct capture / collection using CFC operation is schematically illustrated according to one aspect of the invention. According to this aspect, capture is initiated by adding capture resin directly to a primary process vessel 302 (e.g., a bioreactor) containing process fluid via conduit 330. As described above, the resin and process fluid mixture is optionally incubated for a period of time, under conditions suitable for maximizing the binding of cell products with the resin, and optionally in the case of mixing.

[0124] Following capture, for example, a portion of the mixture from primary process vessel 302 is transferred to secondary process vessel 324 by opening valve 328, which can be facilitated by operating pump 326. During this initial transfer process, valves 340, 338, 334, 314, and 316 remain closed. After the initial transfer is complete, a washing process is initiated via percolation through filter module 306. Valve 328 between the primary and secondary process vessels is closed, valves 314 and 316 between secondary process vessel 324 and filter module 306 are opened, as are valves 318 and 320 between the percolation outflow and percolation waste containers of the filter module. During washing, valve 322 leading to product recovery container 308 remains closed. Fluid recirculation between secondary process vessel 324 and filter module 306 is achieved by operating pump 310, while percolation outflow is directed to percolation waste container 304 by operating percolation pump 312. During this process, the rate at which fluid flows from the primary process vessel 302 into the secondary process vessel 324 is regulated by operating pump 326 to maintain a constant volume of fluid in the secondary process vessel 324. For example, the inflow rate is regulated by operating a fluid control mechanism to match the outflow rate of the permeate from the system.

[0125] After the complete mixture of process fluid and resin is added from the primary process vessel to the secondary process vessel, valve 328 can be closed again and pump 326 can be shut off, while the fluid continues to be recirculated through filter module 306, thereby concentrating the fluid in the secondary process vessel 324 due to fluid loss in the permeate stream.

[0126] After the mixture of process fluid and resin is sufficiently concentrated in a secondary process vessel, an additional washing process is initiated by adding percolation buffer to the secondary process vessel, for example, by opening valve 338 and allowing the buffer to flow into the secondary process vessel via conduit 336. Washing is performed through a process that may include combining a concentration operation with a percolation operation through filtration module 306 until the resin is sufficiently free of contaminants and at a concentration suitable for elution.

[0127] For elution, the fluid loop is closed, for example, by shutting off all valves except valves 314 and 316, and elution is initiated by adding elution buffer to the secondary process vessel 324, for example by opening valve 338 and allowing the buffer to enter the system via conduit 336. After adding a sufficient volume of buffer, valve 338 is closed, and the mixture is incubated for a period of time, optionally as described above, in the case of mixing, under conditions suitable for maximizing the elution of the bioproducts from the resin.

[0128] After elution, the collection operation is initiated by opening valves 318 and 322 (valve 320 remains closed) and by operating pump 310 to recirculate the fluid through filter module 306. As described above, the bioproducts are recovered from the permeate stream by percolation through filter module 206 and collected in product recovery container 308. Also as described above, collection can be performed using a series of percolation and concentration processes until the desired amount of product is collected. In an optional step, the captured resin can be regenerated in a secondary container or collected in a separate collection container, for example, by flushing the resin from the secondary container through valve 334 and conduit 332, where the resin can be collected and regenerated offline. In the case of resin regeneration in a secondary container, a regeneration buffer can be added to the container, for example, via conduit 336 or other suitable conduit or port.

[0129] It should be understood that Figure 3The system shown can also be used in batch processes, where discrete volumes or "batches" of process fluid are captured and washed separately. For example, in an exemplary batch process, valve 328 is opened to allow a first volume of process fluid from primary process vessel 302 to enter secondary process vessel 324 containing captured resin, for example, by operating pump 326. Capture is performed as described above. After capture, a washing process is initiated as described above, except that there is no continuous feed from primary process vessel 302 to secondary process vessel 324, as in CFC operation. Instead, valve 328 remains closed until the first volume of process fluid and resin has been adequately washed and concentrated by percolation through filter module 306. After this operation is completed, valve 328 is opened to add a second volume of process fluid to the resin in secondary process vessel 302. Capture and washing are repeated in the same manner as described for the first volume of process fluid. Capture and washing are repeated in batches until all fluid from primary vessel 302 has been added to secondary vessel 324 and processed. Elution, collection, and optional regeneration of the captured resin are performed as described above.

[0130] On the other hand, "dead end" capture can be used for capture / acquisition operations. Figure 4 A system for "dead-end" capture operation is shown. According to this aspect, capture is initiated in the same manner as described above, i.e., by direct contact between the process fluid and the capture resin, for example, by adding the resin directly to a primary process vessel containing the process fluid, or alternatively by adding a volume of process fluid to a secondary process vessel containing the resin. Unlike the method discussed above, valve 416 is closed here to prevent the entrapment flow, and a fluid loop is formed between process vessel 402 and filter module 406 operating in "dead-end" mode by opening valves 414, 418, and 428 via the permeate flow. Since valves 420 and 422 also remain closed, the permeate is collected and transported back to the process vessel. After capture, valves 416 and 420 are opened while valve 428 is closed, and valve 422 remains closed to form a fluid loop between process vessel 402 and filter module 406 via the entrapment flow. As described above, washing is performed by percolating the resin containing the product through the filter module 406, wherein the resin-containing process fluid is retained in the retrieval material stream and recycled through the module, while the percolated material is removed from the system and collected in a waste container. Resin elution, collection, and optional regeneration are performed as described above.

[0131] Figure 5This is a line graph showing the relationship between pressure (psi) and time (min), illustrating the critical process flux at point C of 4700 LMH (2 liters / min (LPM)). Critical process flux is defined using a representative feed stream (e.g., a process fluid containing cell culture medium and / or cell lysate with resin) or other non-aqueous feed stream that provides the maximum operating flux for a given feed stream. In practice, the critical process flux will be significantly lower than the membrane's NWP and must be determined experimentally. In this paper, we used different feed flow rates for analysis to determine a suitable range of operating fluxes, which are determined to be in the range of approximately 500–7000 LMH.

[0132] Figure 6 The model results are shown, demonstrating that a TFDF membrane with a porosity of 67% will not clog at an average permeation flux of 4,000 LMH. The model predictions were tested as described in the following experiments.

[0133] Figure 7A The performance of a 67% porosity TFDF membrane in removing RNA from cell lysate is demonstrated. The initial cell density before lysis was 6.7E6 cells / ml. This experiment showed 100% RNA removal. Both the retained and permeate streams showed equal and stable RNA reduction in this process (2x initial concentration, step 1; 9x permeate volume wash, steps 2–10). These data indicate that RNA can be removed from source fluids with minimal or no enzymatic degradation using endonucleases such as Benzonase® or Decontaminase®.

[0134] Figure 7B The performance of a 67% porosity TFDF membrane in removing host cell DNA (HCD) from a mixture of cell lysis buffer and affinity resin (1% AviPure) is demonstrated. The initial cell density before lysis was 9E6 cells / ml. This experiment showed approximately 100% removal of DNA. Both the retained and permeate streams showed equal and stable reductions in HCD during this process (2x initial concentration, step 1; 7x permeate volume washes, steps 2–8). These data indicate that HCD can be removed from source liquids with minimal or no enzymatic degradation using endonucleases such as Benzonase® or Decontaminase®.

[0135] Figure 8AThe performance of a TFDF membrane with a porosity of 67% in reducing the turbidity of a mixture of cell lysate and affinity resin is shown. The initial cell density before lysis was 6.7E6 cells / ml. The initial turbidity of the cell lysate was 509 NTU. Adding affinity resin to the lysate increased the turbidity to 4670 NTU, indicating that the resin alone generated approximately 4000 NTU. After completing the initial 2x concentration cycle (step 1 in the figure) by passing the process fluid and resin mixture through the TFDF module, the turbidity of the retained material stream increased from approximately 4000 to approximately 8000 NTU, indicating that the TFDF module highly retains the resin. The fact that the turbidity of the retained material remained at approximately 8000 NTU during the subsequent percolation step further demonstrates the high resin rejection rate. Furthermore, in the first concentration step, the percolate turbidity steadily decreased from 509 NTU to approximately 260 NTU, indicating that turbidity from the cell lysate readily passed through the filter. During the 9 DV period, the turbidity value steadily decreased, indicating that cell lysates were not retained and could easily pass through the TFDF tube with a porosity of 67%.

[0136] Figure 8B The performance of a 67% porosity TFDF membrane in removing cell lysates is shown. The initial cell density before lysis was 6.7E6 cells / ml. The removal performance of the cell lysates was measured by UV-Vis at 260 nm and 280 nm before and after the addition of affinity resin. The affinity resin absorbed at 975 nm and had minimal effect on the A260 and A280 values ​​(data not shown). Since AAV was absent in this experiment, the decrease in A260 and A280 values ​​only indicates the removal of lysate components such as host cell proteins, DNA, and RNA. After the first concentration step (2x CF), there was no change or a slight decrease in absorbance, indicating that the cell lysates passed freely through the 67% porosity TFDF tube with minimal retention. If any retention of cell lysate components occurred during the concentration step, an increase in signal would have been observed. This was further confirmed by the continuous decrease in absorbance values ​​throughout the percolation step, indicating a high permeability of the cell lysates through the 67% porosity TFDF tube.

[0137] Figure 9 The high resin rejection rate of the TFDF membrane with a porosity of 67% is shown. The rejection rate of Poros 50HQ resin (50 μm average diameter) was tested in 1X PBS on a TFDF tube with a porosity of 67% in full recirculation mode. The feed flow rate was set to 800 mL / min (~1400 s). -1The permeate pump was set to 100 mL / min (2000 LMH). Data collected showed that the 67% porosity TFDF tube retained all resin during the 30-minute experiment, as no significant absorbance was detected in the permeate. After complete recirculation for 30 minutes, the permeate pump was turned off, and the two retentate readings were measured, yielding a total yield of 91%.

[0138] Figure 10 A "constant feed concentration" system according to one aspect of the invention is illustrated. This system is used to validate a concept experiment in which the bioproduct is AAV viral particles (vp). Briefly, the capture operation is initiated by adding approximately 12 mL of affinity resin to 1-L lysed HEK293 cells containing 3.4E10 vp / mL AAV 9 in process vessel 1008. The resin is incubated in process vessel 1008 for 2 hours, then transferred to a 200 mL secondary process vessel 1024 via a peristaltic pump (pump not shown), incubated for another 1 hour, and mixed via recirculation through filter module 1012. During this additional 1-hour incubation (which may be referred to as the "full recirculation mode"), the permeate is returned to process vessel 1008, and the returned permeate material is continuously fed into secondary process vessel 1024 to ensure maximum binding with the resin. Simultaneously, the affinity resin is retained in the retentate stream of filter module 1012 in the recirculation fluid loop and is not returned to process vessel 1008. After capture, the concentration and washing processes are initiated. First, the resin / lysis buffer mixture is concentrated to ~200 mL by operating the filtration module 1012 in CFC mode. Then, the mixture is washed through the filtration module 1012 via percolation with 8 DV of washing buffer (phosphate-buffered saline or PBS). Following percolation, in the final concentration step, the 200 mL working volume is concentrated to ~150 mL to accommodate the elution buffer volume. For elution, 50 mL of elution buffer (100 mM glycine, 150 mM NaCl, 0.01% P-188, pH 2) is added directly to the secondary process vessel 1024 (not shown in the figure) using a peristaltic pump (addition time <1 min). Percolation with the elution buffer is immediately initiated using vacuum. The eluted virus particles were collected in a Product Recovery 1014 container pre-filled with ~170 mL (10% of the final volume) of neutralization buffer (1 M Tris base, pH 9) to equilibrate the freshly eluted virus to pH ~7. The eluted virus was washed with 8 DV elution buffer to obtain a final volume of approximately 1.7-L.

[0139] The results are as follows Figure 11As shown. The initial count of viral particles in the 1-L primary process container was determined to be 3.40E13 by ELISA, shown in the first bar on the left labeled “Cell Lysis Buffer (Feed, Start)”. After the initial two hours of capture in the primary process container (where affinity resin was added directly to the cell lysis buffer), only 3% of unbound viral particles were found in the solution, indicating a high degree of binding to the resin. As described above, after 1 hour of rebinding through the filtration module in full recirculation mode, the amount of viral particles in the solution increased to 8%. During washing, minimal viral particles were found in the wash buffer after 3 and 8 DV, indicating strong binding to the resin. After elution and washing with 8 DV of elution buffer as described above, the viral particles in the product recovery container were quantified by ELISA. As shown in the last bar on the left labeled “Final Collected Product (Eluted Permeate)”, more than 3.00E13 viral particles were recovered, indicating that a total recovery rate of 92% was achieved by this method of directly collecting and capturing viral particles from crude cell lysate without any prior washing steps.

[0140] Figure 12 Another configuration of the system according to the invention is shown. In this configuration, the resin is recirculated within the flow path 1210 of the filtration module (which may also be referred to as the "retained material loop"). Capture is performed by introducing cell culture medium or lysis buffer into the fluid loop via conduit 1216 and recirculating it with the resin for a period of time to allow the target biological product to bind to the resin. In this capture step, both the retained material stream and the permeate stream are recirculated within the fluid loop. After capture, a concentration and washing process is initiated. During this process, the washing buffer, along with the cells, debris, and cell culture medium or lysis buffer in the permeate, is directly transferred to waste, while the resin with bound biological products remains within the retained material loop 1210. The concentration step may be performed before and / or after the washing process. After the concentration and washing process, an elution process is performed to release the biological product from the resin by introducing elution buffer, for example, via conduit 1218, into the fluid loop 1210. The biological product now in the permeate 1214 is collected in a product recovery container.

[0141] In one aspect, the filtration module comprises a macroporous filter media consisting of nonwoven fibers with pore sizes ranging from 50 to 200 micrometers. In other aspects, the filter media may be in the form of tubular / spiral plates, also referred to herein as tangential flow chromatography filters or "TFCF". In some aspects, the TFCF is composed of a nonwoven polypropylene / polyethylene polymer with a pore size of 50-200 micrometers and is formed into a tubular membrane, for example, by spirally winding a plate-shaped TFCF membrane. Suitable membranes include nonwoven wet-laid membranes. In some aspects, the TFCF media is not formed by extrusion.

[0142] In some respects, multiple filtration modules may be included in series or in parallel. In cases where the lysate contains bioproducts, acid pretreatment of the lysate can be performed before it is introduced into the retentate stream loop. In some respects, additional optional filtration of the permeate containing bioproducts can be performed after elution from the resin.

[0143] As shown in the figure, there is no recirculation tank. However, recirculation tanks may be included for larger-scale resin slurries.

[0144] Figure 13 Depicting Figure 12 An alternative configuration of the system, wherein a secondary process vessel is included in the fluid loop 1314 to receive cell culture medium or lysis buffer from the process vessel at appropriate points in the acquisition-capture process, as well as wash buffer, elution buffer and regeneration fluid from an external vessel.

[0145] In one aspect, the filtration module includes a high-porosity filter. In some aspects, multiple filtration modules may be included in series or in parallel. In aspects where the lysate contains bioproducts, acid pretreatment of the lysate may be performed before it is introduced into the retentate stream loop. In some aspects, additional optional filtration may be performed on the permeate containing bioproducts after elution from the resin.

[0146] The test was performed using protein A resin that binds IgG and two types of AAV resins. Figure 12 The system. In the initial experiments, the binding affinity of each resin to its target ligand was evaluated using batch adsorption studies, which are described in detail in the following two paragraphs.

[0147] like Figure 14As shown, the affinity of protein A resin for binding IgG was measured using a batch adsorption study. Briefly, aliquots of protein A resin (% protein A resin varying between 1-5% w / v) were incubated with PBS solutions of IgG at concentrations ranging from 1 to 10 mg / mL for 1 hour, followed by centrifugation and collection of the supernatant. The amount of IgG adsorbed on each resin aliquot was calculated based on the equilibrium concentration of IgG in the supernatant. For 3% resin and 1 mg / mL IgG, more than 85% of IgG was bound at 30 minutes and more than 90% at 60 minutes. The resin loading was approximately 70 mg IgG / mL resin.

[0148] The affinity of resins 1 and 2 for AAV binding to AAV8 and AAV9 was measured using batch adsorption studies. Briefly, aliquots of the resins (% resin varying between 1-5% w / v) were incubated with AAV8 or AAV9 at a titer of approximately 1E12 viral particles (vp) / ml for 3 hours, followed by centrifugation and collection of the supernatant. The amount of AAV adsorbed on each resin aliquot was calculated based on the equilibrium concentration of AAV in the supernatant. Figure 15 As shown, the data indicate different binding preferences between different serotypes and resins. Resin loading ranged from 1E14 vp / ml to 1E15 vp / ml.

[0149] Figure 16 It shows the use of according to Figure 12 The system reduced host cell DNA (HCD) in the feed / retentate lines and permeate. Initially at nearly 120 ng / µl, HCD decreased to almost zero after concentration and 2–8 permeate volumes (DV). Data showed a very high HCD clearance rate of approximately 98%.

[0150] Figure 17 The diagram shows the difference between the feed / retention material (the darker line at the top) and the product obtained through 2x concentration, followed by utilization based on... Figure 12 The system was subjected to 1-10 percolation volumes (DV), and the turbidity of the percolate (light-colored line) decreased. Data showed that the turbidity of the percolate decreased by approximately 99%. Due to the presence of resin retained in the retentate recirculation loop, the turbidity of the retentate remained at approximately 8000 NTU.

[0151] Figure 18 It shows the use of according to Figure 12 The system was used to collect monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells at a 1-liter scale-up collection-capture scale-down assay, achieving product recovery. CHO cells were at a concentration of 50E6 cells / ml, and the mAb concentration was 4.4 mg / ml. Protein A resin 1 was used at 5% w / v, with a filter surface area of ​​4.3 cm². 2The permeate flux was 2000 LMH. The antibody capture rate was greater than 90% at 60 minutes (total incubation time was 120 minutes). The final yield was approximately 92%.

[0152] Figure 19 It shows the use of according to Figure 12 The system in Figure 18 Pressure curves from a 1-liter scale mAb acquisition-capture scaled-down experiment were obtained. The data show that the process pressure remained unchanged throughout the run, indicating consistent flux and no filter clogging.

[0153] Figure 20 It shows the use of according to Figure 13 The system was used to collect AAV9 from HEK293 cells at a 1-liter scale-down product recovery rate in a capture-capture experiment. The HEK293 cell concentration was 10E6 cells / ml, and the viral capsid titer was approximately 3E11 vp / ml. AAV9 resin 1 was used at 0.5% w / v, and the filter surface area was 3.0 cm². 2 The permeate flux was 2000 LMH. The AAV capture rate at 120 minutes was greater than 95% (total incubation time was 120 minutes). The final yield was approximately 90%.

[0154] Figure 21 It shows Figure 20 Pressure profiles for a 1-liter scale AAV acquisition-capture model. The data show no change in process pressure throughout the operation, indicating consistent throughput and no filter clogging.

[0155] According to some aspects of the method described herein, the capture step can be performed in one of two ways. In the first way, capture is performed by adding resin to a primary or secondary process vessel, which may also be referred to as a "feed vessel". This method may also be referred to herein as "batch capture". In the second way, capture is performed by preloading resin into the retentate flow loop 1210 of the filtration module, which may also be referred to as a "retentate recirculation loop" or a "fluidized bed". This method may also be referred to herein as "in-line capture". In the in-line capture process, the resin slurry within the flow path is maintained at a constant volume. This is achieved by maintaining the same flow rate between the feed stream 1216 (which has a high concentration of product) and the permeate stream leaving the flow path (which has a low concentration of product). Experiments were conducted to compare the capture efficiency of the batch method and the in-line method using monoclonal antibodies and AAV as target biological products. In the in-line capture process, the permeate is recycled back to the feed vessel. In terms of waste treatment, this is an ideal capture method, but it may increase processing time because the feed is diluted by the low-concentration waste stream returned.

[0156] Figure 22 The capture of monoclonal antibodies (mAbs) over time (minutes) is shown (in mg IgG / ml resin). The top curve represents online capture, and the bottom curve represents batch capture. In this experiment, the online capture process was more efficient than batch capture, achieving higher saturation. Experiments were performed using 200 mL of Chinese hamster ovary (CHO) cell supernatant with an initial protein titer of 2.39 mg / mL. For both batch and online capture, 5 g of protein A affinity resin was used. This means that in the batch process, the resin was present at a lower concentration of 2.5% wt / vol (5 g resin / 200 ml liquid), compared to a resin slurry concentration maintained at 40% wt / vol (5 g resin / 12.5 ml liquid) in the online process. The loop feed / discharge rate for the online process was 10 mL / min.

[0157] A second experiment was conducted using AAV9 as the target bioproduct. In this experiment, only a single final data point was collected after three (3) hours of contact with the resin. The results are as follows: Figure 23 As shown. In this experiment, batch capture was superior to online capture, likely due to the higher initial feed volume while the feed / discharge rate remained constant at 10 mL / min. Therefore, in this experiment, compared to the above regarding... Figure 22 Compared to the experiments discussed, unbound products had a lower chance of binding to the resin. The data also showed that the same amount of virus was captured online at half the resin loading ratio (0.5% vs. 0.25%). This suggests that less resin is needed to capture the same amount of virus using this method. However, this result may be due to the failure to reach equilibrium after three hours.

[0158] Experiments were performed using 1000 mL of AAV 9 with an initial titer of 2.32E11 vp / mL. The affinity resin was 5 g or 2.5 g of AVIPure AAV 9 resin. The loop feed / discharge rate was the same as in the monoclonal antibody capture experiment, at 10 mL / min. Batch capture: 0.5% solids; online capture: 0.5% solids; 0.25% solids. Solids are v / v% of the resin added relative to the initial liquid volume. Concentration ratios: 40% and 20%, where the concentration ratio is the ratio of liquid to solid slurry in the flow path during capture. This parameter indicates the resin concentration in the flow path during online capture. The main difference between the batch and online methods is that during online capture, the resin is concentrated in the flow path loop, while during batch capture, the resin is uniformly dispersed in the feed container.

[0159] In an alternative process known as the "single-pass process," instead of returning the permeate stream to the process vessel, it is directly fed into the waste via a 1220. For the single-pass process, the resin requires sufficient residence time to capture the target biological product, similar to traditional column chromatography.

[0160] Results of a non-optimized, fully single-pass online process for capturing / acquiring AAV, such as Figure 24 As shown. (Refer to...) Figure 12 In this single-pass process, the permeate stream is not returned to the process vessel; instead, it is directly fed into the waste via 1220. The initial viral titer is set to 100% (leftmost bar). The next six bars are labeled "Permeate Online Hours #", representing hourly measurements of viral products in the permeate stream after 1, 2, 3, 4, 5, or 6 hours of capture. The data show that the amount of product lost to the waste is the same at each time point, indicating an excess of resin binding sites relative to the maximum binding allowed by the residence time. The column labeled "Waste" represents a pooled sample of all product waste from the capture and washing steps. The final column, "Pooled Products," represents the AAV recovered from the collection operation. Overall, the data demonstrate the feasibility of the process. For example, the yield could be improved by optimizing the elution buffer composition or resin saturation.

[0161] This experiment utilized 1 liter of AAV9 lysis buffer, a 5-minute residence time, a gentle feed flow, and a resin loading ratio of 3.7E13 vp / ml. Residence time refers to the average time of interaction between any product particles and the resin in the flow path, calculated by dividing the liquid volume in the fluid loop by the permeate flow rate.

[0162] An additional series of experiments were conducted to investigate the conditions under which filter clogging occurred and to implement additional processes to reduce clogging. HEK cells were mechanically lysed on an orbital shaker in a buffer containing 50 mM Tris base, 2 mM MgCl2, and 1% polysorbate 20 (Tween 20). After lysis, the lysate was (1) left untreated (control), (2) treated with citric acid (in buffer for 20 min, where the pH was lowered to 4.1 by adding citric acid), or (3) treated with 50 units / ml of endonuclease. Incubation at room temperature for 2 h in the absence of resin was used to simulate batch capture. The lysate was then single-pass filtered through a 50 μm wet-laid nonwoven polypropylene / polyethylene polymer filter at a recirculation rate of 500 ml / min and a flux of 2000 LMH. Membrane clogging was assessed by pressure (psi) versus volumetric output (L / m³). 2 The relationship diagram is used to represent it. Figure 25AThe pressure curves for the control (lysate without acid or endonuclease treatment) are shown. In the control, both the feed flow without filtration and the retained material flow exhibited pressures exceeding 2000 L / m³. 2 Constant pressure for flux. In contrast, permeate pressure and transmembrane pressure (TMP) range from 1000 to 1500 L / m. 2 All of these data began to deviate from the baseline, indicating inconsistent flux and membrane clogging. These data suggest that the standard HEK lysis procedure produces large cell debris that cannot pass through the 50 μm filter and causes clogging during concentration.

[0163] Figure 25B The pressure profiles of the lysis buffer subjected to low pH conditions by treatment with citric acid are shown. Here, compared to the control run, pressures are above 4000 L / m³. 2 The process pressure remained unchanged before the flux was increased. These data suggest that citric acid treatment prevented clogging during the concentration process, but clogging was observed during the subsequent percolation process.

[0164] Figure 25C The pressure profile of the lysis buffer treated with endonuclease is shown. Here, the process pressure remained constant throughout the experiment, ranging from over 4500 L / m³. 2 The throughput. These data indicate that endonuclease treatment prevents clogging during the concentration and percolation processes.

[0165] like Figure 26A and Figure 26B As shown, citric acid treatment also leads to DNA loss during the percolation washing step. Figure 26A ) and protein ( Figure 26B Good removal of ).

[0166] Although the invention disclosed herein has been described by way of specific embodiments and applications, those skilled in the art can make various modifications and variations thereto without departing from the scope of the invention as set forth in the claims.

[0167] It should be understood that the invention has been described in this application at different levels of detail. In some cases, details that are unnecessary for those skilled in the art to understand the invention, or that make other details difficult to understand, may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the appended claims. Unless otherwise defined, the technical terms used herein should be understood as those commonly understood by those skilled in the art to which this disclosure pertains.

[0168] Various features of the process system can be used independently or in combination. It should be understood that the system disclosed herein can be implemented in different forms and should not be construed as being limited to the implementation shown in the accompanying drawings.

[0169] It should be understood that, as described herein, "implementation" (e.g., shown in the accompanying drawings) can refer to an illustrative representation of an environment, article of manufacture, or component in which the disclosed concepts or features may be provided or embodied, or to a representation of a manner in which only the concepts or features may be provided or embodied. However, such illustrated embodiments should be understood as examples (unless otherwise stated), and other ways of embodying the described concepts or features (e.g., those that a person of ordinary skill in the art would understand upon learning the concepts or features from this disclosure) are within the scope of this disclosure. Furthermore, it should be understood that while the accompanying drawings may illustrate one or more embodiments of a concept or feature together in a single embodiment incorporating such a concept or feature, such concepts or features should be understood (unless otherwise stated) to be independent and separate from each other, and shown together for convenience, not intended to be limited to their coexistence or use together. For example, a feature shown or described as part of an embodiment may be used alone or together with one or more other features to produce yet another embodiment. Therefore, this subject matter is intended to cover such modifications and variations that fall within the scope of the appended claims and their equivalents.

[0170] In view of the foregoing, it should be understood that the various embodiments illustrated in the figures have multiple separate and independent features, each of which individually has unique benefits that are required, but not essential, for the containers, systems, and related methods of this disclosure. Therefore, it is not necessary for all of the multiple individual features described herein to be present in order to achieve at least some of the desired characteristics and / or benefits described herein.

[0171] The foregoing discussion has broad application and is presented for illustrative and descriptive purposes, and is not intended to limit this disclosure to the one or more forms disclosed herein. It should be understood that various additions, modifications, and substitutions can be made to the embodiments disclosed herein without departing from the concept, spirit, and scope of this disclosure. In particular, those skilled in the art will appreciate that the principles of this disclosure can be implemented in other forms, structures, arrangements, proportions, and with other elements, materials, and components without departing from the concept, spirit, or scope, or characteristics of the invention. For example, for the purpose of simplifying this disclosure, various features of this disclosure may be combined in one or more aspects, embodiments, or configurations. However, it should be understood that various features of certain aspects, embodiments, or configurations of this disclosure may be combined in alternative aspects, embodiments, or configurations. Although this disclosure is presented according to embodiments, it should be understood that not all of the various individual features of this subject matter need to be present in order to achieve at least some of the desired features and / or benefits, or such individual features. Those skilled in the art will understand that this disclosure can be used with numerous modifications or alterations to the structure, arrangement, proportions, materials, components, etc., used in the practice of this disclosure, which are particularly suited to specific environments and operational requirements without departing from the principles, spirit, or scope of this disclosure. For example, an element shown as integrally formed may be composed of multiple parts, or an element shown as multiple parts may be integrally formed; the operation of an element may be reversed or otherwise altered; and the size or scale of an element may be changed. Similarly, although operations or processes are described in a specific order, this should not be construed as requiring such a specific order, or that all operations or processes must be performed to achieve the desired result. Furthermore, other embodiments are also within the scope of the preceding claims. In some cases, the operations described in the claims may be performed in a different order and still achieve the desired result. Therefore, embodiments of this disclosure are to be considered illustrative rather than restrictive in all respects, and the scope of the claimed subject matter is indicated by the appended claims and is not limited to the foregoing description or the specific embodiments or arrangements described or shown herein. In view of the foregoing, any feature of any embodiment may be used and may be claimed individually or in combination with the features of that embodiment or any other embodiment. The scope of this subject matter is indicated by the appended claims and is not limited to the foregoing description.

[0172] In the foregoing description and the preceding claims, the following will be understood: 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 conjunctive and disjunctive in practice. The terms “a,” “an,” “the,” “first,” “second,” etc., do not exclude multiple entities. For example, an entity referred to herein as “a” or “an” means one or more of that entity. Therefore, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein. All directional references (e.g., proximal, distal, up, down, upward, downward, left, right, lateral, longitudinal, front, back, top, bottom, above, below, vertical, horizontal, radial, axial, clockwise, counterclockwise, and / or similar) are used solely for identification purposes to aid the reader's understanding of this disclosure, and / or to distinguish areas of related elements from one another, and do not limit the related elements, particularly regarding their location, orientation, or use in this disclosure. Connecting references (e.g., attachment, coupling, connection, and joining) should be interpreted broadly and may include intermediate members between sets of elements and relative movement between elements, unless otherwise stated. Thus, connecting references do not necessarily infer that two elements are directly connected and fixed to each other. Identifying references (e.g., primary, secondary, first, second, third, fourth, etc.) are not intended to imply importance or priority, but are used to distinguish one feature from another.

[0173] In the claims, the term "comprises" does not exclude the presence of other elements, components, features, regions, integrals, steps, operations, etc. Furthermore, although individual features may be included in different claims, these features can be advantageously combined, and inclusion in different claims does not imply that the combination of features is infeasible and / or disadvantageous. Moreover, singular references do not exclude plural references. Reference marks in the claims are provided merely as illustrative examples and should not be construed as limiting the scope of the claims in any way.

Claims

1. A method for separating biological products from a process fluid characterized by high cell density and / or high turbidity, comprising: The capture operation is performed by contacting the process fluid with the capture resin in the retrieval flow loop of the filtration module for a period of time sufficient to bind the bioproducts to the resin. A washing operation is performed by separating the process fluid and resin mixture into a permeate stream and a retentate stream for the filtration module, and recirculating the retentate stream through the retentate stream loop for a first number of permeate volumes (DV), while simultaneously guiding the permeate stream to a waste container outside the fluid loop, thereby producing a clarified retentate stream, and optionally concentrating the clarified retentate stream in the retentate stream loop; The elution operation is performed by contacting the clarified retentate stream with a certain volume of elution buffer in the retentate stream loop for a period of time sufficient to separate the bioproduct from the resin. The collection operation involves circulating the clarified retentate stream and resin mixture through the filtration module, thereby separating the mixture into a permeate stream containing the bioproducts and a retentate stream containing the resin; and recirculating the retentate stream through the retentate stream loop a second number of DVs, while guiding the permeate stream to a recovery container outside the fluid loop, where the bioproducts are separated.

2. A method for separating biological products from a process fluid characterized by high cell density and / or high turbidity, comprising: The capture operation is performed by contacting the process fluid with the capture resin in the process vessel for a period of time sufficient to allow the bioproduct to bind to the resin. A washing operation is performed by circulating the process fluid and resin mixture through a filtration module, thereby separating the mixture into a permeate stream and a retentate stream, wherein the filtration module and the process vessel are interconnected in a retentate stream loop, and the retentate stream is recirculated through the retentate stream loop for a first number of permeate volumes (DV), while the permeate stream is directed to a waste container outside the fluid loop, thereby producing a clarified retentate stream, and optionally the clarified retentate stream is concentrated in the process vessel; The elution operation is performed by contacting the clarified retentate stream with a certain volume of elution buffer for a period of time sufficient to separate the biological product from the resin. The collection operation involves circulating the clarified retentate stream and resin mixture through the filtration module, thereby separating the mixture into a permeate stream containing the bioproducts and a retentate stream containing the resin; and recirculating the retentate stream through the retentate stream loop a second number of DVs, while guiding the permeate stream to a recovery container outside the fluid loop, where the bioproducts are separated.

3. A method for separating biological products from a process fluid characterized by high cell density and / or high turbidity, comprising: The capture operation is performed by contacting the process fluid with the capture resin in the process container for a period of time sufficient to bind the bioproduct to the resin, while simultaneously circulating the mixture of process fluid and resin through a filtration module to separate the mixture into a permeate stream and a retentate stream. The filtration module and the process container are interconnected in a closed retentate stream loop and an open permeate stream loop, such that the retentate stream is prevented from leaving the filtration module, thereby retaining the resin, while the permeate stream is guided back to the process container through the permeate stream loop. A washing operation is performed by opening the retentate flow circuit, circulating the process fluid and resin mixture through the filter module, and recirculating the retentate flow through the retentate flow circuit for a first number of percolation volumes (DV), while simultaneously closing the percolation flow circuit and directing the percolation flow to a waste container outside the fluid circuit, thereby producing a clarified retentate flow, which is optionally concentrated in the process container. The elution operation is performed by contacting the clarified retentate stream with a certain volume of elution buffer for a period of time sufficient to separate the biological product from the resin. The collection operation is performed by circulating the clarified retentate stream and resin mixture through the module, thereby separating the mixture into a permeate stream containing the bioproduct and a retentate stream containing the resin; and the retentate stream is recirculated through the module for a second number of DVs, while the permeate stream is directed to a recovery container outside the fluid loop, thereby separating the bioproduct.

4. A method for separating biological products from a process fluid characterized by high cell density and / or high turbidity, comprising: Perform a capture operation, through (i) The process fluid is brought into contact with the capture resin in the primary process vessel for a period of time sufficient to allow the bioproduct to bind to the resin; (ii) The mixture of process fluid and resin is circulated through a secondary process vessel in fluid communication with the filtration module during a second time period, thereby separating the mixture into a permeate stream and a retrieval stream. The filtration module and the secondary process container are interconnected in the entrapment flow loop, and the primary process container, the secondary process container, and the filtration module are interconnected in the permeate flow loop, such that the entrapment flow is recirculated between the secondary process container and the filtration module in the entrapment flow loop, and the permeate flow is also recirculated between the primary process container, the secondary process container, and the filtration module in the permeate flow loop. A washing operation is performed by closing the permeate flow circuit, circulating the mixture of process fluid and resin through the filtration module, and recirculating the retentate flow through the retentate flow circuit for a first number of permeate volumes (DV), while simultaneously guiding the permeate flow to a waste container outside the fluid circuit, thereby producing clarified retentate flow, and optionally concentrating the clarified retentate flow in the secondary process vessel; The elution operation is performed by contacting the clarified retentate stream with a certain volume of elution buffer for a period of time sufficient to separate the biological product from the resin. as well as The collection operation is performed by circulating the clarified retentate stream and resin mixture through the filtration module, thereby separating the mixture into a permeate stream containing the biological products and a retentate stream containing the resin; and the retentate stream is recirculated through the retentate stream loop for a second number of DVs, while the permeate stream is guided to a recovery container outside the fluid loop, thereby separating the biological products.

5. The method of claim 2, wherein the method includes performing the capture operation in a secondary vessel fluidly connected to the process vessel, the method comprising... A first volume of process fluid is transferred from the process vessel to the secondary vessel, wherein the secondary vessel contains the resin, or the resin is added to the secondary vessel. The capture and washing operations are performed to obtain a clarified entrapment stream in the secondary container.

6. The method of claim 5, wherein the method comprises performing a second capture operation or other capture operation prior to the elution operation by transferring a second volume or further volume of process fluid into the secondary process vessel containing the clarified retentate stream, and Perform a second capture and wash operation or other capture and wash operations to obtain a second clarified retentate stream or other clarified retentate stream in the secondary container, optionally repeating the capture and wash operations with a third volume or other volume of process fluid prior to performing the elution and capture operations.

7. The method according to any one of claims 1 to 6, wherein during one or both of the washing and collection operations, fluid lost in the permeate stream is replenished to maintain a constant volume of fluid in the process vessel and / or the secondary vessel in a batch or continuous process.

8. The method according to any one of claims 1 to 7, wherein during one or both of the washing and collection operations, the fluid lost in the permeate stream is not replenished to concentrate the fluid in the process vessel.

9. The method according to any one of claims 1 to 8, wherein for insect or mammalian cells, the process fluid is characterized by a live cell density (VCD) or total cell density (TCD) of 10E5 to 10E9 cells / ml, or for bacterial cells, the process fluid is characterized by an optical density (OD) of 1-350 at 600 or 620 nanometers (nm).

10. The method according to any one of claims 1 to 9, wherein the process fluid is characterized by a turbidity of 100-30,000 scattering turbidity units (NTU) or 200-1,000 NTU prior to contact with the resin.

11. The method according to any one of claims 1 to 10, wherein the process fluid is characterized by a viscosity of about 1.5-30 centipoise (cP).

12. The method according to any one of claims 1 to 11, wherein the washing operation is sufficient to remove 95-99% of cells and / or cellular proteins and nucleic acids from the entrapment material stream.

13. The method according to any one of claims 1 to 11, wherein the washing operation is sufficient to reduce the average amount of cellular proteins and nucleic acids in the entrapped material stream by 2-5 logarithmic orders.

14. The method according to any one of claims 1 to 13, wherein after the collection operation, at least 90% of the resin is retained in the retentate stream.

15. The method according to any one of claims 1 to 14, wherein after the collection operation, the collected biological products are filtered through a tangential flow depth filtration (TFDF) filter medium.

16. The method according to any one of claims 1 to 15, wherein the filtration module comprises a tangential flow depth filtration (TFDF) filter medium, comprising one or more hollow fiber elements forming the hollow fiber depth filtration medium, each hollow fiber element comprising a porous wall with a thickness of 2-10 millimeters (mm), the porous wall defining an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of about 50-90%, and a pore size class of 10-50 micrometers.

17. The method of claim 16, wherein each of the one or more hollow fiber elements forming the hollow fiber depth filter medium comprises a porous wall with a thickness of 2-10 mm, the porous wall defining an inner cavity with an ID of 1-12 mm, a porosity of about 60-90%, and a pore size class of 10, 20, 30, 40, or 50 micrometers.

18. The method according to any one of claims 1 to 15, wherein the filtration module comprises a macroporous tangential flow filtration (TFF) filter medium with a pore size in the range of 50-200 micrometers, made of nonwoven fibers.

19. The method of claim 18, wherein the filter medium is composed of a nonwoven polypropylene / polyethylene polymer, a polyester polymer, a polyamide polymer, or a fluoropolymer.

20. The method of claim 19, wherein the filter medium comprises a porous wall with a thickness of 0.1 to 0.5 mm, the porous wall defining an inner cavity with an ID of 1-12 mm and a porosity of about 60-90%.

21. The method of claim 20, wherein the filter medium is in the form of a flat plate spirally wound into a tubular shape, optionally thermally welded or ultrasonically welded to itself in a spiral manner to form a tubular filter element.

22. The method of claim 21, wherein the filter medium is manufactured using a wet web forming technique.

23. The method of claim 22, wherein the filter medium is not formed by extrusion.

24. The method according to any one of claims 1 to 19, wherein the biological product is an antibody, recombinant protein, or viral particle.

25. The method of claim 24, wherein the resin is functionalized with an Fc-binding ligand or a ligand that binds to virus particles.

26. A system for separating biological products from a process fluid characterized by high cell density and / or high turbidity, comprising: A process container containing the process fluid; At least one filtration module in fluid communication with the process vessel, the filtration module comprising: One or more hollow fiber elements forming a filter medium, each hollow fiber element comprising a porous wall with a thickness of 0.5-10 mm, the porous wall defining an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of approximately 50-90%, and a pore size class of 10-50 micrometers, and... A housing adapted to separate the process fluid into a permeate stream and a retentate stream when the process fluid flows through the filter medium, the housing having an inlet, a permeate outlet and a retentate outlet; as well as At least one pump.

27. The system of claim 26, wherein at least two filter modules are connected in series or in parallel with the process vessel, optionally including two or more units of the filter modules and the process vessel connected in series or in parallel.

28. The system of claim 26, wherein the system comprises a retentate pump and a permeate pump.

29. The system of claim 26, wherein the system comprises a permeate container and a product recovery container.

30. The system of claim 26, wherein the system includes a fluid or buffer supply source connected in a feed relationship to the process vessel.

31. A tangential flow filtering (TFF) module or a tangential flow depth filtering (TFDF) module, comprising: One or more hollow fiber elements forming a hollow fiber filter medium or a hollow fiber depth filter medium, each hollow fiber element comprising a porous wall defining an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of approximately 50-90%, and a pore size class of 10-50 micrometers, and... A housing suitable for separating a process fluid into a permeate stream and a retentate stream when the process fluid flows through the filter medium, the housing having an inlet, a permeate outlet, and a retentate outlet.

32. A tangential flow filtering (TFF) module, comprising: A nonwoven polypropylene / polyethylene polymer filter media, in the form of a flat plate spirally wound into a tubular shape, has a pore size of 50-200 micrometers and a porous wall with a thickness of 0.1 to 0.5 mm, wherein the porous wall defines an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of approximately 60-90%, and a pore size class of 40, 50, 100, 150, or 200 micrometers. A housing suitable for separating a process fluid into a permeate stream and a retentate stream when the process fluid flows through the filter medium, the housing having an inlet, a permeate outlet, and a retentate outlet.

33. A system for separating biological products from a process fluid characterized by high cell density and / or high turbidity, comprising: Process container containing the process fluid, At least one tangential flow filtration (TFF) module in fluid communication with the process vessel, the filtration module comprising: A nonwoven polypropylene / polyethylene polymer filter medium, in the form of a flat plate spirally wound into a tubular shape, has a pore size of 50-200 micrometers and a porous wall with a thickness of 0.1 to 0.5 mm. The porous wall defines an inner cavity with an internal diameter (ID) of 1-12 mm, a porosity of approximately 60-90%, and pore size grades of 40, 50, 100, 150, or 200 micrometers. A shell is provided to separate the process fluid into a permeate stream and a retentate stream as the process fluid flows through the filter medium. The shell has an inlet, a permeate outlet, and a retentate outlet. The recycling loop includes flexible conduits interconnected between the process vessel and the TFF module to form a entrapment flow loop, and At least one pump.

34. The system of claim 33, wherein at least two TFF modules are connected in series or in parallel with the process vessel, optionally including two or more units of the filter module and the process vessel connected in series or in parallel.

35. The system according to claim 33 or 34, wherein the system comprises a feed pump, a retentate pump, and a permeate pump.

36. The system according to any one of claims 33 to 35, wherein the system comprises one or more of a washing buffer, an elution buffer, and a regeneration buffer connected in a feed relationship to the recirculation loop.

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