Integrated and continuous method and system for virus filtration, concentration, and buffer replacement.

Abstract

A method and system for integrated and continuous viral filtration and concentration of a biological product includes an initial purification system coupled to a final purification system, the initial purification system including a viral reduction filtration (VRF) skid, and the final purification system including a single-pass tangential filtration-diafiltration (SPTFF-DF) skid.

Description

[Technical Field]

[0001] This specification discloses systems and methods for use in the production of biological products (e.g., proteins), and more specifically, single-use systems and methods for integrated and continuous viral filtration, concentration, and buffer exchange. In certain embodiments, the system is an integrated single-use system for use in processing a feed stream containing a biological product or material of interest, including a viral filtration unit operation coupled to a single-pass tangential flow filtration (SPTFF) and dialysis filtration (DF) unit operation. [Background technology]

[0002] A key challenge in the manufacture of therapeutic drugs is the ability to consistently deliver products with the required purity and efficacy, free from environmental and process-related contamination. The manufacture of biologics is particularly challenging, given the involvement of living cells.

[0003] The conventional manufacturing of biologics involves a series of similar unit operations and can be divided into two main parts: upstream and downstream. The upstream unit operations typically include cell culture and harvesting steps, while the downstream consists of multiple purification steps. Specifically, the final step in the downstream process typically includes a virus reduction filtration (VRF or VF) step, followed by ultrafiltration / dialysis filtration (UF / DF) for product concentration and buffer replacement.

[0004] Traditionally, VRF and UF / DF systems have been separated and often run consecutively on different days, utilizing large equipment that occupies considerable space. New technologies aim to shorten the process time of VRF systems across the production line using periodic / continuous filtration, but concerns about virus breakthroughs remain.

[0005] In this technological field, there is a need for improved processes, particularly VRF and UF / DF processes, that enable large-scale production in a small footprint, using single-use equipment, within a rapid timeframe, while minimizing the risk of viral breakthroughs. [Overview of the project]

[0006] This specification discloses systems and methods for integrated, continuous viral filtration, ultrafiltration, and dialysis filtration for use in the production of biological products such as monoclonal antibodies, particularly in the processing of feed streams generated by batch or continuous production of the biological product of interest.

[0007] In one embodiment, a single-use system is provided for the integrated, continuous processing of an initial biologic product, the system including a viral filtration unit operation coupled to a single-pass tangential flow filtration (SPTFF) and dialysis filtration (DF) unit operation.

[0008] In another embodiment, an integrated, continuous method is provided for providing a processed biological product, the method comprising: a) providing a feed stream (e.g., a fluid feed) containing an initial biological product; b) filtering the feed stream to remove viral contaminants; c) concentrating the initial biological product; and d) performing buffer exchange to produce a processed biological product.

[0009] In a third embodiment, a method for producing the target biological product is provided, the method being (I) In cell culture, eukaryotic cells that express the target biological product; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) Purifying the fluid feed containing the target biological product and one or more impurities or buffering components, and separating the target biological product from the fluid feed; and (IV) Optionally, formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; The method further includes passing the fluid feed through a single-use system for integrated continuous processing of initial biological products, The single-use system for integrated continuous processing of initial biological products includes a viral filtration unit operation coupled with single-pass tangential flow filtration (SPTFF) and dialysis filtration (DF) unit operations.

[0010] In a fourth embodiment, a method for producing the target biological product is provided, the method being (I) In cell culture, eukaryotic cells that express the target biological product; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) Purifying the fluid feed containing the target biological product and one or more impurities or buffering components, and separating the target biological product from the fluid feed; and (IV) Optionally, formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; The above method further, a) To provide a feed stream (e.g., fluid feed) containing initial biological products; b) Filtering the supply stream to remove viral contaminants; c) Concentrating the initial biological products; and d) including buffer exchange to produce the processed biological product.

[0011] In a fifth aspect, a virus filtration - ultrafiltration and diafiltration (VF - UFDF) system is disclosed, the system includes an initial purification unit operation and a final purification unit operation, and the unit operations are connected.

[0012] In one embodiment, the initial purification unit operation includes at least one virus filtration membrane for removing virus particles, and the final purification unit operation includes a single - pass tangential flow filtration (SPTFF) and diafiltration (DF) system for concentration and buffer exchange.

[0013] In one embodiment, the initial purification unit operation includes a pump, at least one pre - filter, and one or more virus reduction filtration membranes.

[0014] In one embodiment, the final purification unit operation includes one or more SPTFF membranes, a DF mixing tank, a DF membrane, sensors, pumps, or combinations thereof.

[0015] In one embodiment, the material is a protein.

[0016] In a specific embodiment, the material is a monoclonal antibody.

[0017] In one embodiment, the process is performed within a time frame that is approximately 50% shorter compared to conventional processing systems.

[0018] In one embodiment, the process is performed within a time of approximately 24 hours or less.

[0019] In one embodiment, the process is performed within a time of approximately 12 hours or less.

[0020] In one embodiment, the process results in a ten - fold increase in the concentration of the material.

[0021] In one embodiment, the system further includes a feed storage tank connected to the initial purification assembly.

[0022] In a specific embodiment, the feed storage tank holds purified and polished monoclonal antibodies at a concentration of approximately 5 to 20 g / L, more specifically, approximately 8 to 12 g / L.

[0023] The features and other aspects of the present invention can be best understood by referring to the following description of specific exemplary embodiments of the present invention in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows a schematic diagram of an integrated, single-use system for final purification, based on an exemplary embodiment, which includes viral filtration (VF) as the final step in the initial purification suite, coupled with single-pass tangential flow filtration (SPTFF) and dialysis filtration (DF) systems for concentration and buffer exchange in the final purification suite. [Figure 2] Figure 2 shows a schematic diagram of a virus filtration system used in the integrated single-use system of Figure 1, based on an exemplary embodiment. [Figure 3] Figure 3 shows a schematic diagram of the final purification system used in the integrated single-use system of Figure 1, based on an exemplary embodiment. [Figure 4] Figure 4 shows a graphical representation of the operating time of an integrated single-use system when operated in Mode 1 or Mode 2, based on an exemplary embodiment. [Figure 5A] Figure 5A shows a graph of the volume conversion coefficient flux variation profile (molecule 1) of the Pall 4-in-series based on an exemplary embodiment, showing the volume conversion coefficient (VCF) versus supply flux of molecule 1 at various starting concentrations using Pall's 4-in-series membrane for SPTFF membranes. [Figure 5B]Figure 5B shows a graph of the volume conversion coefficient flux variation profile (molecule 2) of the Pall 9-in-series based on an exemplary embodiment, showing the volume conversion coefficient (VCF) versus supply flux of molecule 2 at various starting concentrations using Pall's 9-in-series membrane for SPTFF membranes. [Figure 5C] Figure 5C shows a graph of the volume conversion coefficient flux variation profile (molecule 3) of the Pall 9-in-series based on an exemplary embodiment, showing the volume conversion coefficient (VCF) versus supply flux of molecule 3 at various starting concentrations using Pall's 9-in-series membrane for SPTFF membranes.

[0025] The drawings merely illustrate exemplary embodiments of the present invention and should not be considered to limit its scope; other equally effective embodiments may also be permitted. [Modes for carrying out the invention]

[0026] To produce biological products from fluids (e.g., cell culture media or clarified cell culture media), purification and / or particle separation may be necessary. Conventional approaches involve many steps, each performed in a separate instrument system. Process costs typically include the cost of individual instruments and space for control and processing hardware associated with each step.

[0027] This specification discloses methods, systems, and apparatus used for filtering molecules, such as proteins, and more specifically, methods and systems for integrated and continuous viral filtration, ultrafiltration, and dialysis filtration of molecules. Advantageously, the systems and methods disclosed herein enable viral filtration, ultrafiltration, and dialysis filtration in a compact form, i.e., with a smaller footprint than conventional approaches.

[0028] While exemplary embodiments of the present invention are described below, along with the use of a particular apparatus, other embodiments of the present invention are applicable to other types of apparatus used in a process that perform the same or similar functions and are overall more space-saving compared to conventional systems.

[0029] I. Definition The terms "biologic product" or "biologic material" generally refer to the product of interest produced through a biological process or through the chemical or catalytic modification of an existing biological product. Examples of biological processes include cell culture, fermentation, metabolism, and respiration. Examples of the biological product of interest include antibodies, antibody fragments, proteins, hormones, vaccines, fragments of natural proteins (fragments of bacterial toxins used as vaccines, e.g., tetanus toxoid), fusion proteins or peptide complexes (e.g., subunit vaccines), and virus-like particles (VLPs).

[0030] As used herein, the term “continuous” refers to two or more integrated (physically connected) continuous unit operations with a minimum retention volume between them. Such processes are also referred to as fully continuous or end-to-end continuous. When a process consists of both batch and continuous unit operations, for example, a continuous upstream process (cell culture and target protein synthesis) and a batch downstream process (purification and formulation of proteins into active pharmaceutical ingredients or drug products), the process is hybrid. In the specific context of the associated unit operations described herein, the term “continuous” refers to constant or aperiodic liquid transport. In one embodiment, the methods and systems described herein enable continuous viral filtration, concentration, and buffer exchange for a batch of proteins.

[0031] The terms "diafiltration" or "DF" are used to mean buffer exchange, that is, the exchange of one set of buffer salts for another set.

[0032] The term "diavolume" or "DV" is a measure of the degree of washing performed during the diafiltration process. It is based on the amount of diafiltration buffer introduced into the unit operation relative to the volume of the retained material.

[0033] The terms “downstream” or “downstream processing” generally refer to some or all of the processes necessary for the capture of the biological product from the original solution in which it was produced, the purification of the biological product by removing undesirable components and impurities, the filtration or inactivation of pathogens (e.g., viruses, endotoxins), and formulation and packaging.

[0034] The term "highly concentrated" refers to a concentration higher than the starting concentration, preferably significantly higher than before. The increase in concentration depends, for example, on the selected biomolecules and media, as well as the conditions and parameters of the ultrafiltration and diafiltration apparatus used. In certain embodiments described herein, the final protein concentrations are about 1 to about 80 g / L, about 10 to about 80 g / L, about 20 to about 80 g / L, about 20 to about 70 g / L, about 30 to about 70 g / L, or more specifically, about 1 to about 10 g / L, about 10 to about 20 g / L, about 20 to about 30 g / L, about 30 to about 40 g / L, about 40 g / L to about 50 g / L, about 50 to about 60 g / L, about 60 to about 70 g / L, and about 70 to about 80 g / L. In certain embodiments, the final protein concentration is higher than about 80 g / L. In certain embodiments, the final protein concentration increases by 2, 3, 4, 5, or 10 times or more compared to the protein concentration in the feed. In a specific embodiment, a material with 10 g / L in 100 L increases to 100 g / L in 10 L, i.e., the concentration increases tenfold.

[0035] The terms “supply,” “supply sample,” and “supply stream” refer to the solution delivered (e.g., sequentially, as a batch) to a unit operation to be filtered (e.g., viral filtration, SPTFF).

[0036] As used herein, the term “filtration” refers to a pressure-driven separation process that uses a membrane to separate components in a solution or suspension according to their size differences. As a result of filtration, at least a portion (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%) of undesirable biological contaminants (e.g., mammalian cells, bacteria, yeast cells, viruses, or mycobacteria) and / or particulate matter (e.g., precipitated proteins) are removed from the liquid (e.g., liquid culture medium or fluid present in any of the systems or processes described herein).

[0037] As used herein, the term “filtrate” means a fluid containing a detectable amount of recombinant antibody released from a filter (e.g., a pre-filter or a viral filter).

[0038] As used herein, the term “flow channel” refers to a channel that supports the flow of a liquid (e.g., feed, retain, permeate) through all or part of a system or subsystem.

[0039] In this specification, the term “integrated” in relation to a system or process means a system or process in which structural elements work together to achieve a specific result (e.g., the production of monoclonal antibodies from liquid culture media).

[0040] The term "microfiltration" refers to a type of filtration used to separate intact cells from relatively large debris or protein aggregates from a mixture, using pore sizes ranging from approximately 0.05 μm to 1 μm in diameter.

[0041] As used herein, the term “perfusion cell culture” refers to perfusion culture carried out by continuously supplying fresh culture medium to a bioreactor and constantly removing used medium that does not contain cells while retaining cells in the reactor. Therefore, since cells are retained in the reactor via a cell retention device, a higher cell density can be obtained in perfusion culture compared to continuous culture. The perfusion rate depends on the requirements of the cell line, the concentration of nutrients in the feed, and the level of toxination.

[0042] The terms “polypeptide,” “polypeptide product,” “protein,” and “protein product” are used interchangeably herein and refer to molecules consisting of two or more amino acids, as known in the art, for example, at least one amino acid chain linked by a continuous peptide bond. In one embodiment, the “protein of interest” or “polypeptide of interest” is a protein encoded by an exogenous nucleic acid molecule transformed into a host cell, where the exogenous DNA determines the amino acid sequence. In another embodiment, the “protein of interest” is a protein encoded by a nucleic acid molecule that is endogenous to the host cell.

[0043] As used herein, the term “prefilter” refers to a filter upstream of the viral filtration membrane. The purpose of the prefilter is to selectively retain clogging components before the filtration step that reduces the virus, while allowing the passage of the biological products of interest.

[0044] As used herein, the term “retained material” refers to a fraction of a specific biological product (e.g., a protein) that is retained by a membrane. This can be calculated as either apparent or intrinsic retention.

[0045] As used herein, the term “single-use” refers to articles suitable for single use and subsequent disposal, and reusable articles used only once in the processes according to the present invention and not subsequently in those processes. Such articles are also referred to as “disposable.”

[0046] The term "skid" refers to a system of components contained within a framework designed to facilitate the transport of a system. Individual skids can include a complete process system or a system performing a specific aspect of a process. Multiple skids can also be combined to form a larger system or an entire transportable installation.

[0047] The term "single-pass tangential flow filtration" or "SPTFF" refers to a type of tangential flow filtration in which the feed flow is directed to pass through the filter in a single pass without recirculation.

[0048] Tangential flow filtration, or TFF, also known as cross-flow filtration, refers to a process in which the feed flow flows parallel to the membrane surface. When pressure is applied, a portion of the flow passes through the membrane (filtrate / permeate), while the remainder is retained (retained material). In conventional TFF, the retained material is recycled to a feed storage tank.

[0049] The term "membrane pressure" or "TMP" refers to the average pressure applied from the feed to the filtrate side of the membrane.

[0050] As used herein, the terms “ultrafiltration” or “UF” refer to any technique of subjecting a solution or suspension to a semipermeable membrane that allows a solvent and small solute molecules to pass through while retaining polymers. Ultrafiltration may be used to increase the concentration of polymers in a solution or suspension. In one embodiment, ultrafiltration is used to increase the concentration of proteins in water. Membrane evaluation may be expressed, for example, by nominal molecular weight (NMW) and in the range of about 1 kD to about 1,000 kD.

[0051] The term "unit operation" refers to a functional process that may be carried out in the process of producing biological substances from liquid culture media.

[0052] The terms “virus reduction filtration,” “VRF,” or “VF” refer to a common unit operation in biomanufacturing aimed at reducing viral contamination. This process retains viral particles on the surface and within the pores of a filter and is carried out based on the size of the virus. Virus filters can be positioned at various locations in a typical protein purification process. In one embodiment, the virus filter is positioned immediately upstream of the UF / DF. The virus reduction level is calculated by comparing the amount of virus in a pre-treated load with the amount of virus in a post-treated sample. The level is typically expressed as the logarithm of the reduction (log10). Virus removal filters are broadly classified into two categories: filters that remove large viruses, typically endogenous retroviruses of 80–100 nm, with a reduction of >4 or >6 log10; and filters that remove small and large viruses (larger than parvoviruses of 18–24 nm) with a reduction of >4 log10. The reduction in the number of virus particles can be about 1% to about 99%, preferably about 20% to about 99%, more preferably about 30% to about 99%, more preferably about 40% to about 99%, even more preferably about 50% to about 99%, even more preferably about 60% to about 99%, even more preferably about 70% to about 99%, even more preferably about 80% to about 99%, and even more preferably about 90% to about 99%. In certain non-limiting embodiments, the amount of virus in the purified antibody product, if present, is less than the ID50 of the virus (the amount of virus that infects 50 percent of the target population), preferably less than at least one-tenth of the ID50 of the virus, more preferably less than at least one-hundredth of the ID50 of the virus, and even more preferably less than one-thousandth of the ID50 of the virus.

[0053] The disclosed systems and methods can be better understood by reading the following description of non-limiting illustrative embodiments with reference to the accompanying drawings, which are briefly described below, and which show corresponding parts of each figure identified by the same reference numerals. II. Systems

[0054] The systems disclosed herein are suitable for processing quantities of material (biological products such as monoclonal antibodies) produced by any suitable biological manufacturing process, including continuous or batch production.

[0055] In one embodiment, the system enables the processing of a certain amount of material (biological product) produced by a system comprising one or more integrated sequential upstream operations, such as continuous (perfusion) cell culture, capture, virus inactivation, polishing, or a combination thereof.

[0056] In certain embodiments, the systems and methods disclosed herein are suitable for use in small suites using single-use channels, in conjunction with iSKID (see, for example, International Publication No. 2020 / 205559). Where referred to herein, “iSKID” is a protein synthesis platform that performs initial purification, virus inactivation, and polishing steps sequentially over a perfusion operation (e.g., a two-week high-intensity perfusion operation). The systems and methods disclosed herein are not limited to applications using iSKID, but are any systems used to produce biological products such as monoclonal antibodies.

[0057] In one embodiment, a VF-UFDF system (also known as a VF-TFF system) is provided, which includes both an initial purification assembly and a final purification assembly, these assemblies being connected, linked, or otherwise integrated. In certain embodiments, the system is for single use.

[0058] The VF-UFDF system may further include a feed storage tank, for example, a protein pool tank that holds purified and polished proteins and, in certain embodiments, is connected to the initial purification unit operation. The feed storage tank is thoroughly mixed. The capacity of the protein pool tank may vary. In one embodiment, the protein pool tank has a capacity between about 200 liters and about 5,000 liters. In certain embodiments, the protein pool tank stores up to 40 kg of purified and polished monoclonal antibodies, or purified and polished monoclonal antibodies at concentrations between about 5 to about 20 g / L, or between about 5 to about 15 g / L, or between about 8 to about 12 g / L, or between about 9 to about 11 g / L or about 10 g / L.

[0059] Optionally, the system may include a pre-filtration step (e.g., microfiltration) to remove larger impurities or contaminants such as protein aggregates.

[0060] In one embodiment, a system is provided comprising two skid bodies, including a first virus reduction filtration (VRF) skid in the initial purification suite and a second single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) skid in the final purification suite.

[0061] In certain embodiments, the VRF skid includes a VRF pump, at least one VRF prefilter, and one or more VRF filters. In certain embodiments, one or more VRF filters are located within a VRF manifold. By applying pressure, a portion of the fluid is fed through the filter membrane into the filtrate flow. In certain embodiments, the pump is replaced by a pressure supply vessel.

[0062] VRF pumps may vary. In one embodiment, the flow rate of the VRF pump is designed for 40 kilograms of product, more specifically, the flow rate is about 80 liters / hour to 680 liters / hour, about 400 liters / hour to 560 liters / hour, or about 440 liters / hour to 520 liters / hour. Generally, the operating range of the flow rate of such a pump is in the range of about 5 liters / hour to 1,200 liters / hour.

[0063] One or more VRF membranes may be different. During the operation, the product enters the permeate freely through the pores of the VRF membrane, while virus particles, if present, are retained by the membrane.

[0064] The virus filter can remove at least a portion (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99%, or 100%) of the virus from a fluid containing recombinant antibodies (e.g., a liquid culture medium, or any fluid present in any process described herein) as the fluid flows through the filter.

[0065] In VRF, various filters can be used depending on the filtration mode, membrane area, membrane pore side, membrane material, module configuration, and test method.

[0066] Examples of typical, non-limiting membrane materials include polymer materials such as polyethylene, polypropylene, ethylene vinyl acetate copolymer, polytetrafluoroethylene, polycarbonate, polyvinyl chloride, polyester, cellulose acetate, regenerated cellulose, cellulose composite materials, polysulfone, polyethersulfone, polyarylsulfone, polyphenylsulfone, polyacrylonitrile, polyvinylidene fluoride, nonwoven and woven fiber materials, or inorganic materials.

[0067] Generally, the required membrane area depends on how much (i.e., volume or mass) of filtration is intended. In specific embodiments, the filter may be approximately 1 to 10 m². 2 , or approximately 1 to 8 meters 2, or approximately 8m 2 , about 6m 2 , about 4m 2 , or approximately 2m 2 The following applies: In one embodiment, the filter is approximately 4 m³ per 15 kg. 2 The following applies, or approximately 8m for 40kg. 2 The following applies:

[0068] The pore sizes of the film may vary, and in one embodiment, they are approximately 10 to 100 nm, more specifically approximately 15 to 50 nm, even more specifically approximately 20 to 30 nm, and even more specifically approximately 20 nm.

[0069] In certain embodiments, the VRF membrane is pre-sterilized. In other embodiments, the VRF membrane is formed from a material suitable for sterilization. Examples of commercially available VRF membranes include Viresolve® Pro (Millipore), Planova 20N (Asahi Kasei), and Virosart (Sartorius).

[0070] In one embodiment, one or more VRF membranes are terminal filters. In the terminal filters, the flow of the solution or suspension to be separated (or supplied) is perpendicular to the membrane.

[0071] The capabilities of VRF systems may vary. Generally, viral reduction is measured by the ratio of the viral titer in the feedstock to the fraction of the associated product, and is called the log10 reduction factor (LRF). In one embodiment, the VRF can be greater than about 6 LRF, greater than about 5 LRF, greater than about 4 LRF, greater than about 3 LRF, or greater than about 2 LRF. The overall LRF of a single manufacturing process is based on the individual LRF of each process step. In certain embodiments, no viral breakthrough is observed.

[0072] In certain embodiments, the VRF assembly consists of a pre-filter attached to a VRF filter manifold and connected to a final formulation break tank via a single-use sterile connector.

[0073] Generally, VRF operations are optimized to identify conditions that maximize throughput, minimize processing time, and ensure reliable virus removal.

[0074] In one embodiment, the throughput is from about 200 to about 1,000 L / m 2 and more specifically from about 400 to about 600 L / m 2 In certain embodiments, the throughput is about 400 to about 450, about 450 to about 500, about 500 to about 550, or about 550 to about 600 L / m 2 In certain embodiments, the throughput is about 400 to about 450, about 450 to about 500, about 500 to about 550, or about 550 to about 600 L / m.

[0075] In one embodiment, the mass throughput is from about 1 to about 10 kg / m 2 and more specifically about 3 to about 5 kg.

[0076] In certain embodiments, the processing time is about 8 hours or less for up to 40 kg. For example, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour or less.

[0077] In certain embodiments, the processing time is about 8 hours or less for 15 kg. For example, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour or less.

[0078] The SPTFF-DF assembly functions as a component for the final purification of the system and is composed of one or more SPTFF membranes, a DF mixing tank, a DF membrane, sensors, and pumps. In this second skid, a concentrated buffer diluted in-line with water is supplied to save space.

[0079] In one embodiment, the assembly includes a break tank, a buffer concentrate for diafiltration (DF), water for injection (WFI), an SPTFF-DF skid, UF / DF pool supply tubing, and a UFDF pool tank.

[0080] According to this embodiment, the break tank is designed to provide pressure break and safety when the flow rates between the virus filtration skid and the SPTFF-DF skid do not perfectly match. In one embodiment, the break tank has a capacity of approximately 20 liters to approximately 100 liters. The break tank is connected to both the VRF skid and the SPTFF-DF skid.

[0081] The buffer concentrate for diafiltration (DF) and WFI are designed to be mixed together so that the DF buffer concentrate is diluted to an appropriate concentration for buffer exchange.

[0082] The WFI is designed to flush downstream equipment and provide water to adjust the concentration of the DF buffer concentrate to the appropriate concentration / strength used in the process.

[0083] The SPTFF-DF skid is a skid unit that is pre-mounted on a skid and is easily transportable, and has several sterile connections for fluidly connecting the SPTFF-DF skid to other devices and downstream of a virus filtration skid. In one embodiment, the SPTFF-DF skid includes an SPTFF pump, an SPTFF1 membrane, a DF pool 1 tank, a DF pool 2 tank, a DF buffer pump, a WFI pump, an in-line mixer, a DF pool 1 pump, a DF pool 2 pump, a DF membrane, and an optional SPTFF2 membrane. Additional devices may be used without departing from the scope and spirit of the exemplary embodiment. Furthermore, certain devices may be combined, but the combined devices may retain the same or similar functions without departing from the scope and spirit of the exemplary embodiment.

[0084] In one embodiment, after the first tank is filled with the concentrated product, the material begins diafiltration through a conventional TFF membrane while the SPTFF process continues filling the second tank. In one embodiment, emptying the first pool and / or concentrating into the final UFDF pool does not need to be completed before the second pool begins diafiltration (i.e., Mode 1). In another embodiment, once the first tank is finished (the material has been diafiltration and is empty / concentrated toward the final UFDF pool) and the second tank is filled, the second tank begins diafiltration (i.e., Mode 2). Advantageously, this reduces the required time by almost half, completing the operation within a 12-hour timeframe. In certain embodiments, the UF / DF time is reduced by approximately 50% compared to the conventional operation, allowing for operations that process more material within the same timeframe. Also advantageous, the demands on the pump are reduced at any time, allowing for the use of a smaller pump system for the same total amount of material. Furthermore, using single-use fluid channels reduces the time and resources required for system cleaning after operation is complete.

[0085] This system can be operated in one of two modes: (i) manual / partially automatic mode, or (ii) fully automatic mode. In both operating modes, the VRF operates with the same automation, the only differences being the flow rate and membrane area.

[0086] Depending on the load and membrane performance, multiple VRF membranes may be configured for operation within the filter manifold and switched as needed.

[0087] Referring here to Figures 1-3, the integrated single-use system 100 for performing an integrated virus filtration, concentration, and dialysis filtration process 105 includes an initial purification system 200 and a final purification system 300.

[0088] The initial purification system 200 includes a protein pool tank 210, a virus reduction filter (VRF) wash 220, a water for injection (WFI) tank 230, a virus filtration skid 240, and a break tank supply pipe 290. While certain devices are included as part of the initial purification system 200 in this specification, additional devices may be used or combined without departing from the scope and spirit of the exemplary embodiments.

[0089] The protein pool tank 210 is a tank designed to hold purified and polished proteins and, according to some exemplary embodiments, has a capacity of 500 liters to about 5,000 liters. However, the capacity of the protein pool tank 210 may differ in other embodiments. The protein pool tank 210 stores about 5 kilograms to about 40 kilograms of purified and polished mAB and is a single-use mixer. The protein in the protein pool tank 210 is in the range of 5 grams / liter to about 15 grams / liter, preferably about 10 grams / liter. The protein pool tank 210 is fluidly connected to the virus filtration skid 240 via a protein pool tank discharge line 212, which connects to the virus filtration skid 240 at the protein pool tank sterile connection section 241.

[0090] The VRF cleaner 220 is designed to flush the integrated single-use system 100, in particular the virus filter 270 and the virus filtration skid 240. The VRF cleaner 220 is fluidly connected to the virus filtration skid 240 via a VRF cleaner discharge line 222, which connects to the virus filtration skid 240 at the VRF cleaner sterile connection section 244.

[0091] The WFI230 is designed to supply water for flushing the filter, and for flushing the virus filter or pre-filter as needed. The WFI230 is fluidly connected to the virus filtration skid 240 via the WFI discharge line 232, which connects to the virus filtration skid 240 at the WFI sterile connection 247.

[0092] The virus filtration skid 240 is a skid unit pre-installed on a skid, ready for easy transport, and has several sterile connections for fluid connection to other devices. The virus filtration skid 240 includes a VRF pump 250, a VRF prefilter 260, and one or more VRF membranes 270 optionally placed within a VRF manifold 271. While certain devices are included herein as part of the virus filtration skid 240, additional devices may be used or combined without departing from the scope and spirit of the exemplary embodiments.

[0093] The VRF pump 250 is fluidly connected to the protein pool tank sterile connection 241 via a VRF pump suction line 242, which includes a VRF pump suction line control valve 243 located between the VRF pump 250 and the protein pool tank sterile connection 241. The VRF pump 250 is also fluidly connected to the VRF washing sterile connection 244 via a VRF washing supply line 245, which extends from the VRF washing sterile connection 244 to a VRF pump suction tube joint 251 located between the VRF pump suction line control valve 243 and the VRF pump 250, and includes a VRF washing supply line control valve 246 located between the VRF pump suction tube joint 251 and the VRF washing sterile connection 244. Furthermore, the VRF pump 250 is fluidly connected to the WFI sterile connection 247 via the WFI supply line 248, which extends from the WFI sterile connection 247 to the VRF pump suction tube joint 251 and includes a WFI supply line control valve 249 positioned between the VRF pump suction tube joint 251 and the WFI sterile connection 247. The VRF pump is a single-use pump head incorporated into the flow path. According to some exemplary embodiments, the VRF pump 250 is either a QF1200SU Quattroflow low-shear pump with a supply flow rate of 80 liters / hour to 680 liters / hour (operating range of 10 liters / hour to 1,200 liters / hour) or a Watson Marlow 600 pump with a supply flow rate of approximately 480 liters / hour (operating range of 5 liters / hour to 950 liters / hour), although other types of pumps may be used in other embodiments.

[0094] The VRF prefilter 260 is fluidly connected to the VRF pump 250 via the VRF pump discharge line 254.

[0095] The VRF membrane 270 is fluidly connected to the VRF prefilter 260 via the VRF prefilter discharge line 262. According to some exemplary embodiments, multiple VRF membranes 270A, 270B, 270C (or more) are arranged in parallel with each other and optionally on the VRF manifold 271 to fluidly connect to the VRF prefilter 260. The size of the VRF membrane 270 ranges from 1 square meter to 4 × 4 square meters (16 square meters). In certain embodiments, the VRF membrane 270 is Planova 20N, Planova BioEX, or Viresolve Pro, having flux ranges of 20 LMH to 70 LMH, 35 LMH to 170 LMH, or 100 to 350 LMH, respectively. The VRF membrane 270 is fluidly connected to the break tank sterile connection 278 via the VRF membrane discharge line 276, which includes a VRF membrane discharge line control valve 277 located between the VRF membrane 270 and the break tank sterile connection 278. The VRF waste discharge line 273 is connected to the VRF membrane discharge line 276 at a VRF membrane discharge pipe joint 272 located between the VRF membrane discharge line control valve 277 and the VRF membrane 270, and includes a VRF waste discharge line control valve 274 located between the VRF membrane discharge pipe joint 272 and the waste 280.

[0096] The virus filtration skid 240 completes the initial purification system 200. The break tank sterile connection 278 is fluidly connected to the final purification system 300 via the break tank supply pipe 290. According to some exemplary embodiments, this break tank supply pipe 290 passes through a mouth hole in a wall (not shown) that separates the initial purification suite 200 from the final purification suite 300.

[0097] The final purification system 300 includes a break tank 310, a dialysis filtration (DF) buffer concentrate 320, water for injection (WFI) 330, a single-pass tangential flow filtration and dialysis filtration (SPTFF-DF) skid 340, a UFDF pool supply pipe 390, and a UFDF pool tank 395. While certain devices are included as part of the final purification system 300 in this specification, additional devices may be used or combined without departing from the scope and spirit of the exemplary embodiments.

[0098] The break tank 310 is a tank designed to provide pressure break and safety in case the flow rates between the virus filtration skid 240 (Figure 2) and the SPTFF-DF skid 340 do not perfectly match. According to some exemplary embodiments, the break tank has a capacity of 20 liters to about 100 liters, although the capacity of the break tank 310 may differ in other embodiments. The break tank 310 is fluidly connected to the virus filtration skid 240 (Figure 2) via the break tank supply pipe 290. The break tank 310 is also fluidly connected to the SPTFF-DF skid 340 via the break tank discharge line 312, which connects to the SPTFF-DF skid 340 at the break tank sterile connection section 341.

[0099] The diafiltration (DF) buffer concentrate 320 and WFI 330 are designed to be mixed together so that the DF buffer concentrate 320 is diluted to an appropriate concentration. The DF buffer concentrate 320 is fluidly connected to the SPTFF-DF skid 340 via the DF buffer concentrate discharge line 322, which connects to the SPTFF-DF skid 340 at the DF buffer concentrate sterile connection section 344.

[0100] The WFI330 is designed to flush downstream equipment and provide water to adjust the concentration of the DF buffer concentrate 320 to an appropriate level. The WFI330 is fluidly connected to the SPTFF-DF skid 340 via the WFI discharge line 332, which connects to the SPTFF-DF skid 340 at the WFI sterile connection 347.

[0101] The SPTFF-DF skid 340 is a skid unit that is pre-mounted on the skid and readily transportable, and has several sterile connections for fluidly connecting the SPTFF-DF skid 340 to other devices downstream of the virus filtration skid 240. The SPTFF-DF skid 340 includes an SPTFF pump 3000, an SPTFF1 membrane 3010, a DF pool 1 tank 3020, a DF pool 2 tank 3030, a DF buffer pump 3040, a WFI pump 3050, an in-line mixer 3060, a DF pool 1 pump 3070, a DF pool 2 pump 3080, a DF membrane 3090, and an optional SPTFF2 membrane 3100. In this specification, certain devices are included as part of the SPTFF-DF skid 340, but additional devices may be used or combined without departing from the scope and spirit of the exemplary embodiments.

[0102] The SPTFF pump 3000 is fluidly connected to the break tank sterile connection 341 via the SPTFF pump suction line 342, which includes an SPTFF pump suction line control valve 343 located between the SPTFF pump 3000 and the break tank sterile connection 341. The SPTFF pump 3000 is a single-use pump head incorporated into the flow path. According to some exemplary embodiments, the SPTFF pump 3000 is a QF1200SU quattroflow low-shear pump having a supply flow rate range of 20 liters / hour to 1,200 liters / hour.

[0103] The DF buffer pump 3040 is fluidly connected to the DF buffer concentrate sterile connection 344 via the DF buffer pump suction line 345, and the DF buffer pump suction line 345 includes a DF buffer pump suction line control valve 346 located between the DF buffer pump 3040 and the DF buffer concentrate sterile connection 344. The DF buffer pump 3040 is a single-use pump head incorporated into the flow path. According to some exemplary embodiments, the DF buffer pump 3040 is a QF1200SU Quattroflow low-shear pump having an operating range of 20 liters / hour to 1,200 liters / hour.

[0104] The WFI pump 3050 is fluidly connected to the WFI sterile connection 347 via the WFI pump suction line 348, which includes a WFI pump suction line control valve 349 located between the WFI pump 3050 and the WFI sterile connection 347. The WFI pump 3050 is a single-use pump head incorporated into the flow path. According to some exemplary embodiments, the WFI pump 3050 is a QF1200SU Quattroflow low-shear pump having an operating range of 20 liters / hour to 1,200 liters / hour.

[0105] The inline mixer 3060 is fluidly connected to the DF buffer pump 3040 via the DF buffer pump discharge line 3042. The inline mixer 3060 is also fluidly connected to the WFI pump 3050 via the WFI pump discharge line 3052, which extends from the WFI pump 3050 to the WFI pump discharge pipe joint 3041, located along the DF buffer pump discharge line 3042 between the inline mixer 3060 and the DF buffer pump 3040. The inline mixer 3060 is an inline dilution system using WFI 330 for the DF buffer concentrate 320 and includes a helical inline mixer.

[0106] The SPTFF1 membrane 3010 is fluidly connected to the SPTFF pump 3000 via the SPTFF pump discharge line 3002. The SPTFF1 membrane 3010 is also fluidly connected to the inline mixer 3060 via the SPTFF1 membrane flush line 3062, which extends from the inline mixer 3060 to the SPTFF pump discharge pipe joint 3001 and includes an SPTFF1 membrane flush control valve 3063, which is located along the SPTFF pump discharge line 3002 between the SPTFF pump 3000 and the SPTFF1 membrane 3010. According to some exemplary embodiments, the SPTFF1 unit 3010 consists of a series of membranes (e.g., Centrasette cassettes stacked on a Centrastak 100) having a size capacity ranging from about 0.9 square meters to about 20 square meters. According to some exemplary embodiments, the SPTFF1 units 3010 are installed in a 9-unit configuration. The size of the SPTFF1 membrane 3010 is up to 20 square meters when designed for 40 kilograms of product via an integrated single-use system 100. The SPTFF1 membrane 3010 is fluidly connected to waste 396 via an SPTFF permeate waste line 3011, which includes an SPTFF permeate waste control valve 3012 for controlling the flow of permeate from the SPTFF1 membrane 3010 to the waste 396.

[0107] The DF pool 1 tank 3020 is fluidly connected to the SPTFF 1 membrane 3010 via the retaining pool 1 line 3014, which includes a retaining pool 1 line control valve 3015 located between the SPTFF 1 membrane 3010 and the DF pool 1 tank 3020. The DF pool 1 tank 3020 can be operated in first and second modes, which will be described in further detail in connection with the operation of the integrated single-use system 100. According to some exemplary embodiments, the DF pool 1 tank 3020 has a tank capacity of 20 to 100 liters. In certain embodiments, the skid connection is aseptic. The DF Pool 1 tank 3020 is also fluidly connected to the inline mixer 3060 via the DF buffer pool 1 line 3064, which extends from the DF Pool 1 tank 3020 to the inline mixer discharge pipe joint 3061 located between the inline mixer 3060 and the SPTFF1 membrane flush control valve 3063, and includes the DF Pool Tank Control Valve 3065 adjacent to the inline mixer discharge pipe joint 3061 and the DF Pool 1 Tank Control Valve 3066 adjacent to the DF Pool 1 tank 3020.

[0108] The DF Pool 2 tank 3030 is also fluidly connected to the SPTFF 1 membrane 3010 via the Retaining Pool 2 line 3016, which extends from the DF Pool 2 tank 3030 to the Retaining Pool 1 piping joint 3013 located between the SPTFF 1 membrane 3010 and the Retaining Pool 1 line control valve 3015 along the Retaining Pool 1 line 3014, and includes the Retaining Pool 2 line control valve 3017 located between the Retaining Pool 1 piping joint 3013 and the DF Pool 2 tank 3030. The DF Pool 2 tank 3030 can be operated in first and second modes, which will be described in more detail in connection with the operation of the integrated single-use system 100. According to an exemplary embodiment, the DF Pool 2 tank 3030 is similar to the DF Pool 1 tank 3020. Furthermore, the SPTFF1 membrane retainer waste line 3018 extends from the retainer pool 1 piping joint 3013 to the waste 396 and includes an SPTFF1 membrane retainer waste line control valve 3019 for controlling the flow of retainer to the waste 396. The DF pool 2 tank 3030 is also fluidly connected to the inline mixer 3060 via the DF buffer pool 2 line 3067, which extends from the DF buffer pool 1 piping joint 3068, located between the DF pool tank control valve 3065 and the DF pool 1 tank control valve 3066, to the DF buffer pool 2 piping joint 3069, located between the DF pool 2 tank 3030 and the residue pool 2 line control valve 3017, and includes a DF pool 2 tank control valve 3160.

[0109] The DF Pool 1 pump 3070 is fluidly connected to the DF Pool 1 tank 3020 via the DF Pool 1 pump suction line 3022. The DF Pool 1 pump 3070 is a single-use pump head incorporated into the flow path. According to some exemplary embodiments, the DF Pool 1 pump 3070 is a QF4400SU Quattroflow low-shear pump with an operating range of 150 liters / hour to 5,000 liters / hour, a QF5050SU with an operating range of 50 liters / hour to 5,000 liters / hour, or another pump with an appropriate capacity depending on the embodiment. The DF Pool 2 pump 3080 is fluidly connected to the DF Pool 2 tank 3030 via the DF Pool 2 pump suction line 3032. The DF Pool 2 pump 3080 is a single-use pump head incorporated into the flow path. According to some exemplary embodiments, the DF Pool 2 pump 3080 is identical or similar to the DF Pool 1 pump 3070.

[0110] The DF membrane 3090 is fluidly connected to the DF pool 1 pump 3070 via the DF pool 1 pump discharge line 3072 and includes a DF pool 1 pump control valve 3073. The DF membrane 3090 is also fluidly connected to the DF pool 2 pump 3080 via the DF pool 2 pump discharge line 3082, which extends from the DF pool 2 pump 3080 to a DF membrane pool pump piping joint 3074 located between the DF membrane 3090 and the DF pool 1 pump control valve 3073 and includes a DF pool 2 pump control valve 3083. The DF membrane 3090 is also fluidly connected to the inline mixer 3060 via the DF membrane flush line 3161, which extends from the inline mixer discharge pipe joint 3061 to the DF membrane flush piping joint 3162 located between the DF membrane pump pool pump piping joint 3074 and the DF membrane 3090 along the DF pool 1 pump discharge line 3072, and includes a DF membrane flush control valve 3163. The DF membrane 3090 can be operated in a first mode and a second mode, which will be described in more detail in connection with the operation of the integrated single-use system 100. According to some exemplary embodiments, the DF membrane 3090 is a Centrastak 100 membrane having a size capacity from 0.9 square meters to 20 square meters. The DF membrane 3090 is fluidly connected to the waste 397 via the DF permeate waste line 3091, which includes a DF permeate waste control valve 3092 for controlling the flow of permeate from the DF membrane 3090 to the waste 397.

[0111] The DF membrane 3090 is fluidly connected to the DF pool 1 tank 3020 via the DF pool 1 retained material recycling line 3093, which extends from the DF membrane 3090 to the DF pool 1 tank 3020 and includes a DF pool 1 retained material recycling control valve 3094. The DF membrane 3090 is also fluidly connected to the DF pool 2 tank 3030 via the DF pool 2 retained material recycling line 3095, which extends from the DF pool tank retained material piping joint 3096, located between the DF membrane 3090 and the DF pool 1 retained material recycling control valve 3094 along the DF pool 1 retained material recycling line 3093, to the DF pool 2 tank 3030 and includes a DF pool 2 retained material recycling control valve 3097. The retained portion of the DF membrane 3090 is fluidly connected to waste 397 via a DF membrane retained waste line 3098, which extends from the DF pool tank retained piping joint 3096 to the waste 397 and includes a DF membrane retained waste control valve 3099 for controlling the flow of retained material from the DF membrane 3090 to the waste 397. According to some embodiments, the DF membrane retained waste line 3098 may be led directly to the waste 397 or may be combined with another waste line such as a DF permeate waste line 3091.

[0112] The SPTFF2 membrane 3100 is fluidly connected to the DF pool 1 pump 3070 via the SPTFF2 membrane pool 1 supply line 3076, which extends from the SPTFF2 membrane 3100 to the SPTFF2 membrane pool 1 supply line joint 3075 located between the DF pool 1 pump 3070 and the DF pool 1 pump control valve 3073 along the DF pool 1 pump discharge line 3072, and includes the SPTFF2 membrane pool 1 supply line control valve 3077. The SPTFF2 membrane 3100 is also fluidly connected to the DF pool 2 pump 3080 via the SPTFF2 membrane pool 2 supply line 3086, which extends from the SPTFF2 membrane pool 2 supply pipe joint 3085 located between the DF pool 2 pump 3080 and the DF pool 2 pump control valve 3083 along the DF pool 2 pump discharge line 3082 to the second SPTFF2 membrane pool 2 supply pipe joint 3087 located between the SPTFF2 membrane 3100 and the SPTFF2 membrane pool 1 supply line control valve 3077 along the SPTFF2 membrane pool 1 supply line 3076, and includes the SPTFF2 membrane pool 2 supply line control valve 3088. The SPTFF2 membrane 3100 is also fluidly connected to an inline mixer 3060 via an SPTFF2 membrane flush line 3164, which extends from an inline mixer discharge pipe joint 3061 to an SPTFF2 membrane flush piping joint 3087 and includes an SPTFF2 membrane flush control valve 3165. The SPTFF2 membrane 3100 is optional. According to some embodiments, the SPTFF2 membrane 3100 is similar to the SPTFF1 membrane 3010. The SPTFF2 membrane 3100 is also fluidly connected to waste 397 via an SPTFF2 permeate waste line 3101, which includes an SPTFF2 permeate waste control valve 3102 for controlling the flow of permeate from the SPTFF2 membrane 3100 to the waste 397.

[0113] The SPTFF2 membrane 3100 is fluidly connected to the UFDF pool tank sterile connection 380 via the SPTFF2 retaining line 3105, which extends from the SPTFF2 membrane 3100 to the UFDF pool tank sterile connection 380 and includes an SPTFF2 retaining control valve 3106. The retaining portion of the SPTFF2 membrane 3100 is fluidly connected to the waste 397 via the SPTFF2 retaining waste line 3107, which extends from the SPTFF2 membrane retaining piping connection 3108, located between the SPTFF2 membrane 3100 and the SPTFF2 retaining control valve 3106 along the SPTFF2 retaining line 3105, to the waste 397 and includes an SPTFF2 membrane retaining waste control valve 3109. According to some embodiments, the SPTFF2 retained waste line 3107 may be directly connected to the waste 397, or it may be combined with another waste line such as the DF permeate waste line 3091 or the SPTFF2 permeate waste line 3101.

[0114] The UFDF pool tank 395 is fluidly connected to the UFDF pool tank sterile connection 380 via the UFDF pool supply pipe 390. The UFDF pool tank 395 is designed to have a capacity of 100 liters to 500 liters. The UFDF pool tank 395 receives concentrated material, if present, from the SPTFF2 membrane 3100, or from the DF pool 1 tank 3020 and DF pool 2 tank 3030, respectively, after the DF membrane 3090 has recirculated the retained material back to the DF pool 1 tank 3020 and DF pool 2 tank 3030. After the UFDF pool tank 395 receives the concentrated material, the concentrated material undergoes final filtration and final formulation according to known processes and procedures not described herein.

[0115] Now that the schematic diagrams in Figures 1 to 3 have been explained, the operation of the integrated single-use system 100 will be described. According to a brief overview of the integrated single-use system 100, it is designed to collect a pool of 5 to 40 kg of single-pass polished monoclonal antibodies (mAbs) in a single batch in less than 12 hours (including non-operational setup and decomposition time) into a pre-final filtration (UFDF) pool by (i) virus reduction filtration (VRF) performed by the virus filtration skid 200, and subsequently by (ii) single-pass tangential flow filtration (SPTFF), buffer exchange by dialysis filtration (DF), and enrichment by an optional second enrichment by SPTFF, all of which are performed by the SPTFF-DF skid 300. This integrated single-use system 100 can be operated in one of two modes: a manual / partially automated mode, called Mode 1 or First Mode, which can process up to 15 kilograms in a single 12-hour batch; or a fully automated mode, called Mode 2 or Second Mode, which can process up to 40 kilograms in a single 12-hour batch. All channels, membranes, pump heads, and connectors are made from sterile, single-use materials.

[0116] The procedure described below has an estimated operating range for processing 5 to 40 kilograms of antibody in 12 hours, but those skilled in the art can modify these operating ranges to process more or less than 5 to 40 kilograms of antibody in 12 hours, or adjust the time. Before commencing the procedure, the VRF membrane 270, SPTFF1 membrane 3010, SPTFF2 membrane 3100 (if used), and DF membrane 3090, along with the associated lines and tanks, are flushed and prepared with VRF wash 220 and / or WFI 230 and / or DF buffer and / or WFI 330.

[0117] In both Mode 1 and Mode 2 operation modes, the virus filtration skid 240 is operated by the same automation, differing only in flow parameters and membrane area. Depending on the load and membrane performance, multiple VRF membranes 270 may be configured in parallel to operate within the VRF manifold 271 and switched as needed.

[0118] According to an exemplary embodiment, in the initial purification 200, 5 to 40 kilograms of purified and polished mAb are stored at approximately 10 g / liter (7 to 13 g / L) in a protein pool tank 210, which is a 200 to 5,000 L single-use mixer (SUM) or storage tank. A VRF pump 250 pumps the material from the protein pool tank to a VRF prefilter 260 and then to a VRF membrane 270 at a feed rate of 80 to 680 liters / hour. The VRF pump 250 is a QF1200SU Quattroflow low-shear pump with a single-use pump head integrated into the flow path and having an operating range of 20 liters / hour to 1,200 liters / hour. VRF membranes 270 ranging from 0.9 square meters to 8 square meters are used and loaded to a capacity of 385 liters / square meter, which can be up to 600 liters / square meter, with a target flux of 64 LMH, which can be up to 300 LMH. The flow continues from the VRF membranes 270 to the break tanks 310, which are 20-100 liter tanks that are part of the final purification 300. The initial purification 200 runs continuously for 6 hours, or until all the starting material stored in the protein pool tanks 210 has been processed and filling of the break tanks 310 has begun. During the operation of the initial purification 200, the VRF pump suction line control valve 243 and the VRF membrane discharge line control valve 277 are in the open position to allow flow through them, while the VRF wash supply line control valve 246, the WFI supply line control valve 249, and the VRF waste discharge line control valve 274 are in the closed position to prevent flow through them.

[0119] In a particular embodiment, after all the supply material has been loaded, the VRF membrane 270 is flushed with 10 liters / m² of VRF wash 220. When the initial purification 200 wash is performed, before and after the operation of the initial purification 200, the VRF pump suction line control valve 243 and the WFI supply line control valve 249 are set to the closed position to prevent flow through them, and the VRF wash supply line control valve 246 and the VRF membrane discharge line control valve 277 are set to the open position to allow flow through them. The wash fluid leaves the initial purification 200 and goes to the break tank 310.

[0120] When the break tank 310 begins filling, the SPTFF pump 3000 on the SPTFF-DF skid 340 starts dispensing material from the break tank 310 through the SPTFF1 membrane 3010 at a supply flow rate equivalent to that of the VRF pump 250, which is 80 liters / hour to 680 liters / hour.

[0121] The operation of the SPTFF-DF skid 340 changes from this point onward depending on whether it is operated based on Mode 1 or Mode 2, which differ significantly in operation. When the SPTFF-DF skid 340 is operated in the less automated Mode 1, it can process up to 15 kilograms of protein, dividing the material that was in the protein pool tank 210 into two consecutive DF pool tanks 3020 and 3030, which are DF pool 1 tank 3020 and DF pool 2 tank 3030. When processing more than 15 kilograms of protein, the SPTFF-DF skid 340 operates in Mode 2, performing many shorter diafiltration (DF) steps by alternately switching between the processing material in DF pool 1 tank 3020 and DF pool 2 tank 3030.

[0122] Here, the operation of the SPTFF-DF skid 340 in Mode 1 is described. The SPTFF1 membrane 3010 is configured in a 9-sequence configuration. In other embodiments, the sequence can be 4, 5, 6, 7, 8, or 9. A QF1200SU Quattroflow low-shear pump with a single-use pump head integrated into the flow path is used to operate the SPTFF-DF skid 340, with an operating range of 20 liters / hour to 1,200 liters / hour. When processing 15 kilograms of protein, the SPTFF1 membrane 3010 has a membrane area of ​​approximately 9 square meters, which varies between 3 square meters and 20 square meters in operation. To achieve a consistent volume concentration factor (variable), a constant flux, which can be 10 LMH to 50 LMH, is targeted, and if the permeate flux decreases during operation, the supply flux can be changed to maintain a constant VCF. For example, in molecule 1 using a four-membrane configuration with an initial concentration of 10 g / L, a feed flux of 25.5 LMH was targeted to achieve an 8x VCF and a target concentration of 80 g / L. The requirements for target concentration, flux, and membrane area must be determined before operation in development by flux variation experiments, as shown in molecules 1, 2, and 3 in Figures 5A, 5B, and 5C, respectively. The membrane area of ​​the SPTFF1 membrane 3010 can be increased or decreased depending on the molecular-specific properties to maintain a relatively constant flux that matches the VRF flow rate. The membrane holder is a Centralstak 100, which can accommodate membrane areas from 0.9 square meters to 20 square meters. The concentrated material, or the material held in the SPTFF1 membrane 3010, flows out of the SPTFF1 membrane 3010 into a DF pool 1 tank 3020, which is a single-use mixing tank with a maximum capacity of 100 liters used for diafiltration. During this time, the retaining pool 1 line control valve 3015 is in the open position, and the retaining pool 2 line control valve 3017 and the SPTFF1 membrane retaining waste line control valve 3019 are in the closed position. While processing the material from the initial purification 200 in the SPTFF1 membrane 3010, the retaining pool 1 line control valve 3015 switches to the closed position, and the retaining pool 2 line control valve 3017 switches to the open position, stopping the load on DF pool 1 tank 3020 and starting the load on DF pool 2 tank 3030.According to some exemplary embodiments, the DF pool 2 tank 3030 is a single-use mixing tank that is similar in size to or the same as the DF pool 1 tank 3020. When the break tank 310 is empty, the SPTFF1 membrane 3010 is flushed with twice the membrane holding capacity of the cleaning buffer, which is a mixture of the DF buffer concentrate 320 and the WFI wash 330.

[0123] During the operation of the SPTFF1 membrane 3010, when the retaining pool 1 line control valve 3015 switches to the closed position and the retaining pool 2 line control valve 3017 switches to the open position, the DF pool 1 tank 3020 begins dialysis filtration. The DF pool 1 pump 3070 begins pumping material from the DF pool 1 tank 3020 and passes the material through the DF membrane 3090, which has an area of ​​2 to 20 square meters according to some exemplary embodiments, at a target supply flow rate of 800 liters / hour to 7,000 liters / hour or 360 LMH. For the operation of the DF membrane 3090, the DF pool 1 pump 3070 is required to be a larger pump than the SPTFF supply pump. According to some exemplary embodiments, the DF pool 1 pump is a single-use pump head integrated into the flow path and is a QF4400SU quattroflow low-shear pump with an operating range of 150 liters / hour to 5,000 liters / hour. Similar to the area of ​​the SPTFF1 membrane 3010, the area of ​​the DF membrane 3090 can be adjusted on a per-molecule basis. The membrane holder is a Centrastak 100, which can accommodate areas from 0.90 square meters to 20 square meters. At a flow rate of 80 grams / liter, diafiltration is expected to have an average conversion rate of 10% or a flux of 36 LMH. The diafiltration buffer concentrate 320 is mixed in-line with WFI 330, and the resulting mixture is added to the DF pool 1 tank 3020 via the DF permeate waste line 3091 in an amount that automatically matches the flow rate of permeate exiting the DF membrane 3090. After 7-10 diavolutes (DV) of the mixture have been added to the DF pool 1 tank 3020, the material should be properly buffered, which can be detected via one or more in-tank / in-line sensors (not shown). To add 8 DV of the mixture, this process using the DF pool 1 tank 3020 and DF membrane 3090 is expected to take approximately 3 hours. The material held in the DF membrane 3090 is recycled and returned to the DF pool 1 tank 3020 via the DF pool 1 material recycling line 3093.Once the addition of the mixture of DF buffer concentrate 320 and WFI 330 to DF pool 1 tank 3020 is complete and the buffer exchange is properly completed, a similar process is repeated in DF pool 2 tank 3030 after the SPTFF1 membrane 3010 has finished its second phase of operation.

[0124] Any SPTFF2 membrane 3100 can be used to achieve the final target concentration (the final desired concentration determines the path length / membrane area required for this concentration process). The material present in DF pool 1 tank 3020 after buffering is pumped through DF pool 1 pump 3070 to SPTFF2 membrane 3100 via SPTFF2 membrane pool 1 supply line 3076. As the material flows through SPTFF2 membrane 3100, the concentrated material of the retained material flows into UFDF pool tank 395, which is a 100-500 liter tank, before final filtration and final formulation. Once the material from DF pool 1 tank 3020 has been processed through SPTFF2 membrane 3090, a similar process through SPTFF2 membrane 3100 is repeated with the material present in DF pool 2 tank 3030. In another embodiment where the SPTFF2 membrane 3100 is absent, the buffering material in DF pool 1 tank 3020 and DF pool 2 tank 3030 is sequentially transferred from tanks 3020 and 3030, respectively, to the UFDF pool tank 395.

[0125] Alternatively, the SPTFF-DF skid 340 can operate in Mode 2, as described below. The general operating principle of the SPTFF-DF skid 340 operating in Mode 2 is the same as when the SPTFF-DF skid 340 operates in Mode 1, except that instead of completely filling DF pool 1 tank 3020 and switching the material sent from protein pool tank 210 to fill DF pool 2 tank 3030, the filling of DF pool 1 tank 3020 and DF pool 2 tank 3030 is switched multiple times during the operation of the SPTFF-DF skid 340 in Mode 2, with many less filled DF pool tanks 3020 and 3030 being processed back and forth. While one of the tanks, DF pool 1 tank 3020 and DF pool 2 tank 3030, undergoes dialysis filtration by the DF membrane 3090DF and then sends the material to the SPTFF2 membrane 3100 (or directly to the UFDF pool tank 395), the other tank is filled. The more frequently the switching can be performed, the more material can be processed in the same amount of time. In this mode 2, additional automation may be required for valves (valve switching) that switch the fluid flow between DF pool 1 tank 3020 and DF pool 2 tank 3030 while monitoring and reacting the total volume, pH, conductivity, concentration, and flow rate.

[0126] Once the operation of Mode 1 or Mode 2 is complete, depending on the mode selected to operate the SPTFF-DF skid 340, the integrated single-use system 100 is then flushed and the single-use flow path is discarded.

[0127] Figure 4 shows a graphical time representation 400 of modes 1 and 2 comparing the operating times of the integrated single-use system when operating in mode 1 410 or mode 2 450 according to an exemplary embodiment. Referring to Figure 4, mode 1 410 operates with minimal automation and processes up to 15 kilograms of material, while mode 2 operates with additional automation and processes up to 40 kilograms of material. Mode 1 410 includes the operation of virus reduction filtration (VRF) 415 in the virus filtration skid 240, single-pass tangential flow filtration (SPTFF) 425 in part of the SPTFF-DF skid 340, and dialysis filtration (DF) 435 in part of the SPTFF-DF skid 340. VRF 415, SPTFF 425, and DF 435 collectively form the entire process of the integrated single-use system 100 (Figure 1). Therefore, VRF415 takes 5.8 hours to start the process and complete it in mode 1 410 for up to 15 kilograms of material. SPTFF425 in mode 1 410 starts immediately after VRF415 and takes 6 hours to complete. DF435 starts midway through SPTFF415 and takes 6 hours to complete, 3 hours in DF pool 1 tank 3020 (Figure 3) and 3 hours in DF pool 2 tank 3030 (Figure 3). Thus, the entire process of the integrated single-use system 100 (Figure 1) operating in mode 1 410 takes less than 10 hours to complete processing up to 15 kilograms of material, which is a sufficient amount to meet clinical requirements.

[0128] Mode 2 450 includes the operation of virus reduction filtration (VRF) 455 in the virus filtration skid 240, single-pass tangential flow filtration (SPTFF) 465 in part of the SPTFF-DF skid 340, and dialysis filtration (DF) 475 in part of the SPTFF-DF skid 340. VRF 455, SPTFF 465, and DF 475 collectively form the entire process of the integrated single-use system 100 (Figure 1). Thus, VRF 455 takes 8.5 hours for up to 40 kilograms of material to start the process and complete in Mode 2 450. VRF 455 in Mode 2 450 takes longer than VRF 415 in Mode 1 410 because the amount of material being processed is similarly increased. SPTFF 465 in Mode 2 450 starts immediately after VRF 455 and takes 9 hours to complete. The DF475, starting immediately after the SPTFF465, takes approximately 9 hours to complete 10 cycles each in DF Pool 1 tank 3020 (Figure 3) and DF Pool 2 tank 3030 (Figure 3) (each cycle slightly shorter than 55 minutes). Therefore, the entire process of the integrated single-use system 100 (Figure 1) operating in Mode 2 450 takes less than 10 hours to complete the processing of up to 40 kilograms of material, which is sufficient to meet both clinical and commercial requirements. Because the DF475 can be started immediately after the SPTFF465 and operates in short, repetitive cycles between DF Pool 1 tank 3020 (Figure 3) and DF Pool 2 tank 3030 (Figure 3), Mode 2 450 can process even more material than Mode 1 410.

[0129] Figure 5A shows graph 500 of the volume conversion coefficient (VCF, molecule 1) for flux fluctuations in the Pall 4-in-series, and based on an exemplary embodiment, it shows the volume conversion coefficient (VCF) 510 vs. feed flux 520 of molecule 1 using Pall 4-in-series membranes for single-pass tangential flow filtration (SPTFF) 1 membrane 3010 (Figure 3) at various starting concentrations. Essentially, what can be seen from this graph 500 is that the maximum potential conversion coefficient decreases at high starting concentrations, but lower fluxes also have a greater effect. When starting with high concentrations of material, lower fluxes result in higher concentrations. When starting with low concentrations of material, lower fluxes have less effect on the conversion coefficient. These experiments can be performed before large-scale enrichment of the product to determine the optimal flux of a given molecule for the desired conversion coefficient and final concentration. Thus, at the starting concentration (~10 g / liter) of the protein pool tank 210, the flux must be quite low (~10 LMH) to enrich to the desired 80 g / liter. To operate the integrated single-use system 100 (Figure 1) or the VFTFF system at the desired flux, a configuration of nine (or, in certain embodiments, fewer than nine, e.g., four or more but less than nine) or more than nine SPTFF1 films 3010 (Figure 3) is required.

[0130] Figure 5B shows a graph of the volume conversion coefficient (VCF, numerator 2) for flux fluctuations of the Pall 9-in-series, illustrating the volume conversion coefficient (VCF) 550 versus supply flux 560 of numerator 2 using Pall's 9-in-series film for SPTFF1 film (Figure 3) at various starting concentrations, based on an exemplary embodiment. Referring to Figure 5B, it can be seen that the maximum latent conversion coefficient decreases at high starting concentrations, but lower fluxes also have a greater impact. The 9-in-series SPTFF can achieve higher concentrations at similar fluxes as the 4-in-series, can be more easily combined with slower VF systems, and is an ideal candidate for SPTFF configuration in VF-TFF systems. One limitation is that at higher supply concentrations, the maximum supply flux of the system is lower due to the pressure threshold. If this cannot be addressed by using additional film area, especially if the concentration required in the DF process is low, a smaller system (e.g., 7-in-series or 4-in-series) can be used instead. This set of flux fluctuations demonstrates that the supply flux within the operating range of the integrated single-use system 100 (Figure 1) or the VFTFF system can achieve the desired enrichment coefficient with a reasonable supply flux.

[0131] Figure 5C shows graph 570 of the volume conversion coefficient (VCF, molecule 3) for the flux variation of the Pall 9-in-series, and based on an exemplary embodiment, it shows the volume conversion coefficient (VCF) 580 vs. supply flux 590 of molecule 3 using Pall's 9-in-series membrane for the SPTFF1 membrane (Figure 3) at various starting concentrations. Referring to Figure 5C, it can be seen that the maximum latent conversion coefficient decreases at high starting concentrations, but lower fluxes also have a greater impact. The 9-in-series SPTFF can achieve higher concentrations at similar fluxes as the 4-in-series, can be more easily combined with slower VF systems, and is an ideal candidate for SPTFF configuration in VF-TFF systems. If higher VF flow rates are used or higher supply concentrations are used, especially when the concentration required in the DF process is low, a smaller system (e.g., 7-in-series or 4-in-series) can be used instead. This set of flux variations demonstrates that the supply flux within the operating range of the integrated single-use system 100 (Figure 1) or VFTFF system can achieve the desired enrichment coefficient at a reasonable supply flux. Considering the above, the integrated single-use system 100 provides at least one of risk avoidance, space saving, and time saving when processing biological products (e.g., proteins) through a filtration process.

[0132] Regarding risk avoidance, one of the principles of this system is that all materials can be processed in a single batch. Performing virus filtration all at once minimizes the risk of viral leak-through, as well as operational problems caused by changes in flow rate and pressure, as opposed to continuous or periodic filtration. Furthermore, the absence of "sub-batches" avoids regulatory concerns regarding batch definitions.

[0133] Regarding space saving, by implementing SPTFF and VRF in parallel with matching the flow rates, the space required to hold the viral filtration material is reduced, thereby requiring only small break tanks with capacities of 20 to 100 liters. The tank size required for diafiltration (DF) is also minimized by targeting a high initial concentration factor, i.e., 8 times in some exemplary embodiments, to reduce the VRF pool from 10 g / liter to 80 g / liter. In specific embodiments, the tank is approximately 350 L. Space saving is also achieved by using an in-line dilution system for the DF buffer via a helical in-line mixer, as in some exemplary embodiments.

[0134] In a specific embodiment, the system operates in Mode 1, and the size of the DF tank is at least about one-sixth the size of the starting tank. For example, there are approximately 350L x 2 tanks for a 2,000L starting tank.

[0135] In a specific embodiment, the system operates in mode 2, and the size of the tank required for DF is at least about 8, at least about 10, at least about 12, at least about 14, at least about 16, or at least about 1 / 20th the size of the starting tank.

[0136] In terms of time savings, running VRF in parallel with SPTFF automatically saves at least one day, as these processes are traditionally performed on separate days (VRF on the first day and UFDF on the second). Time savings can also be achieved by using a two-tank DF system. After the first DF tank is filled with concentrated product, the material begins diafiltration through the conventional TFF membrane, while the SPTFF process continues filling the second DF tank. Once the first DF tank is empty (the material has been diafiltrationed) and the second tank is filled, the second DF tank begins diafiltration. This process can reduce the UF / DF time by almost half, allowing the operation to be completed within 12 hours when operating in mode 2 (Figure 4).

[0137] Another advantage is that this system reduces the demands on the pump at any given time, allowing for a smaller pump system and thereby reducing costs. Furthermore, the use of single-use fluid paths reduces the time and resources required for cleaning the system after operation is complete. III. Method

[0138] This specification also discloses a method for removing viruses by filtering and concentrating / exchanging a biological product (e.g., a protein) in an integrated, continuous manner.

[0139] In one embodiment, the method comprises (i) providing a biological product in a solution; and (ii) subjecting the solution to (a) virus reduction filtration (VRF), (b) concentration by single-pass tangential flow filtration (SPTFF), (c) buffer exchange by dialysis filtration (DF), and (d) optionally, a second concentration by SPTFF.

[0140] As described above, filtering out viruses that may be present in compositions containing biological products intended for use in biopharmaceuticals is an important aspect of quality control. Biological products can be, among many other substances, proteins, nucleic acids, carbohydrates, lipids, or biomaterials. Proteins can be, for example, therapeutic proteins such as antibodies, antibody fragments, antibody derivatives, cytokines, growth factors, hormones, enzymes, or blood coagulation factors, or vaccine proteins such as antigen proteins. Biological products can be produced by biological systems such as cells, tissues, or organisms, particularly mammalian cells, plant cells, or bacterial cells. Biological products can be produced by homogeneous processes such as suspension culture based on the use of agitated tank bioreactors, airlift bioreactors, or wave bioreactors, or heterogeneous processes such as adhesion culture based on microcarrier-based systems, packed-bed bioreactors, or hollow fiber bioreactors, carried out in discontinuous modes such as batch culture or fed-batch culture, or continuous modes such as continuous culture with perfusion, and carried out on any appropriate scale, such as laboratory, pilot, or production scale. The virus may be a bacterium (i.e., a bacteriophage, also called a "phage"), or in particular a virus capable of infecting humans and / or animals, such as individual humans or animals to which the biological product is intended to be administered. The virus may have been introduced into the composition containing the biological product from an exogenous source, such as due to inadequate failure to maintain sterility, or from an endogenous source, such as the biological system used to produce the biological product.

[0141] The method of the present invention can be used to reliably remove or eliminate viruses that may have been present during the production of a biological product, for example, due to viral contamination. As long as there may be multiple active particles of multiple different types of viruses and / or a predetermined type of virus, the method of the present invention can be used to remove multiple active particles of multiple different types and / or a predetermined type. Therefore, for example, the method of the present invention can be used to ensure that the biopharmaceutical, ultimately containing the biological product, does not contain active particles of any type of virus in any amount exceeding acceptable limits, for example, that the biopharmaceutical is free of viral active particles.

[0142] In certain embodiments, the method of the present invention includes viral filtration, concentration, and diafiltration in a single batch of less than 12 hours (including non-operational setup and decomposition time). In one embodiment, the method of the present invention requires less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, or 5 hours or less.

[0143] In one embodiment, (i) providing a biological product in solution includes providing about 5 to about 40 kg of purified and polished monoclonal antibodies (mAbs) stored at about 10 g / L (5 to 15 g / L) in a 500 to 2,000 L single-use mixer (SUM) or storage tank.

[0144] In one embodiment, (ii) providing the solution to (a) virus reduction filtration (VRF) includes the material passing through a prefilter to the VRF membrane (Planova BioEX) using a QF1200SU Quattroflow low-shear pump (operating range 20-1,200 L / hr) equipped with a single-use pump head incorporated into the flow path, at a supply flow rate of approximately 80-680 L / hr. 0.9-8 m 2 Using a VRF membrane, a target flux of 64 LMH (maximum 150 LMH) yielded 385 L / m³. 2 (Maximum 600L / m 2Load up to the capacity of ). This process is carried out continuously for 6 hours, or until all initial material has been processed. After all supply material has been loaded, the membrane will be 10 L / m 2 It is flushed with a cleaning buffer.

[0145] The flow continues to the 20-100L break tank in the final purification stage. Once the break tank begins filling, the SPTFF pump on the second skid starts feeding the material through the SPTFF membrane at a flow rate (80-680L / hr) equivalent to that of the VRF supply pump.

[0146] (b) The method of concentration by single-pass tangential flow filtration (SPTFF) may vary. That is, SPTFF-DF operates differently depending on the operating mode. When operating in mode 1, which is not highly automated, a maximum of 15 kg can be processed, and the VRF pool is divided into two consecutive DF pools. When processing quantities exceeding 15 kg in mode 2, many shorter DF steps are performed by switching alternately between DF pool 1 and DF pool 2.

[0147] Mode 1: The SPTFF membrane is configured in a 4-9 membrane configuration. A QF1200SU Quattroflow low-shear pump (operating range 20-1,200 L / hr) with a single-use pump head integrated into the flow path is used for SPTFF operation. Target concentration and flux must be determined before development operation by flux variation experiments similar to those shown in Figure 1. The membrane area can be increased or decreased depending on the specific properties of the molecules in order to maintain a relatively constant flux. The membrane holder is Centrastak 100, with a range of 0.9-20 m². 2 It can accommodate the following. The concentrated material flows out of the SPTFF and into the first of two 100L single-use mixing tanks for dialysis filtration. While processing the material from the VRF, a valve at the outlet of the SPTFF switches, initiating a load to the second 100L single-use mixing tank. After the break tank is empty, the membrane is flushed with cleaning buffer at twice the membrane's holding capacity.

[0148] During the SPTFF operation, when the SPTFF outlet valve switches to the second 100L tank, the first tank begins dialysis filtration. The pump pumps the material 0.9-20m 2 Start by passing the TFF membrane at a target flow rate of 800-7,000 L / hr or 360 LMH. DF operation requires a larger QF4400SU Quattroflow low-shear pump (operating range 150-5,000 L / hr) with a single-use pump head integrated into the flow path. Similar to SPTFF operation, the membrane area for DF operation can be adjusted on a per-molecule basis. The membrane holder is Centrastak100, 0.9-20 m². 2 It can accommodate the following. With 80 g / L diafiltration, an average conversion rate of 10% (flux of 36 LMH) is expected. The buffer concentrate for diafiltration is mixed in-line with water and added to the mixing tank in an amount that automatically matches the permeate flow rate. After 5-10 diavolutes (DV), the material needs to be properly buffered (detected by in-tank / in-line sensors). For 8 DV, this process is expected to take approximately 3 hours. After the SPTFF process is completed, this process is repeated in a second diafiltration tank.

[0149] An optional second SPTFF membrane can be used in UF2 to achieve the final target concentration (the final desired concentration determines the path length required for this concentration process). This concentrated material is then sent to a final 200-500 L pool before final filtration and final formulation.

[0150] Mode 2: The general operating principle is the same, but instead of completely filling the DF pool and switching it midway through the VRF pool, the DF pool is switched multiple times while many smaller DF pools are operating. One tank performs DF, and while the material is sent to the second SPTFF (or directly to the final pool), the other tank is filled. The more frequently the switching, the more material can be processed in the same amount of time.

[0151] The system is then flushed, and the single-use channels are discarded. The systems and methods disclosed herein may be used to provide aqueous formulations containing any biological product of interest (e.g., proteins).

[0152] The method of the present invention provides the aforementioned advantages with respect to the system, namely, risk avoidance, space saving, and time saving when processing biological products (e.g., proteins) through a filtration process.

[0153] Methods known in the art for producing or generating the biological product of interest can be used in combination with the systems and methods for filtering fluid feeds described herein. For example, those skilled in the art know of methods for producing or generating biological products, such as recombinant proteins, using fermentation. In certain embodiments, the generation of the biological product of interest includes culturing eukaryotic cells expressing the biological product of interest in a cell culture. Culturing eukaryotic cells expressing the biological product of interest in a cell culture may include maintaining the eukaryotic cells in appropriate media and conditions that enable growth and / or protein production / expression. The biological product of interest may be produced by fed-batch or continuous cell culture. Therefore, eukaryotic cells may be cultured in fed-batch or continuous cell culture, preferably in continuous cell culture.

[0154] In certain embodiments, the eukaryotic host cell is a yeast cell. In one embodiment, the eukaryotic host cell is a mammalian cell. The mammalian cells used herein are mammalian cell lines suitable for the production of secreted recombinant therapeutic proteins and are therefore also called “host cells”. In certain embodiments, the mammalian cell is a rodent cell such as a hamster cell. The mammalian cell is an isolated cell or cell line. In certain embodiments, the mammalian cell is a transformed and / or immortalized cell line. In certain embodiments, the mammalian cell is adapted for continuous passage in cell culture and does not include primary non-transformed cells or cells that are part of an organ structure. In certain embodiments, the mammalian cell is BHK21, BHK TK-, Jarcutt cells, 293 cells, HeLa cells, CV-1 cells, 3T3 cells, CHO, CHO-K1, CHO-DXB11 (also known as CHO-DUKX or DuxB11), CHO-S cells and CHO-DG44 cells, or a derivative / offspring of any of such cell lines. In certain embodiments, the mammalian cells are CHO cells, e.g., CHO-DG44, CHO-K1, and BHK21, and more preferably CHO-DG44 cells and CHO-K1 cells. In certain embodiments, the mammalian cells are CHO-DG44 cells. Mammalian cells, particularly glutamine synthetase (GS) deficient derivatives of CHO-DG44 cells and CHO-K1 cells, are also included. In one embodiment, the mammalian cells are Chinese hamster ovary (CHO) cells, e.g., CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, CHO-S cells, CHO-GS deficient cells, or derivatives thereof.

[0155] In certain embodiments, the host cell may further comprise one or more expression cassettes encoding heterologous proteins, such as therapeutic proteins, for example recombinant secretory therapeutic proteins. In certain embodiments, the host cell may also be mouse cells, such as mouse myeloma cells, such as NS0 and Sp2 / 0 cells, or derivatives / offspring of any such cell lines.

[0156] The expression of the target biological product or recombinant protein occurs within a cell containing the DNA sequence encoding the target biological product or recombinant protein, which is then transcribed and translated into a protein sequence including post-translational modifications, thereby generating the target biological product or recombinant protein in cell culture.

[0157] This specification discloses a method for producing a target biological product, and the method is described below. (I) In cell culture, eukaryotic cells that express the target biological product; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) Purifying the fluid feed containing the target biological product and one or more impurities or buffering components, and separating the target biological product from the fluid feed; and (IV) Optionally, formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; The method further includes passing the fluid feed through a single-use system for integrated continuous processing of initial biological products, The single-use system for integrated continuous processing of initial biological products includes a viral filtration unit operation coupled with single-pass tangential flow filtration (SPTFF) and dialysis filtration (DF) unit operations.

[0158] This specification discloses a method for producing a target biological product, and the method is described below. (I) In cell culture, eukaryotic cells that express the target biological product; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffering components; (III) Purifying the fluid feed containing the target biological product and one or more impurities or buffering components, and separating the target biological product from the fluid feed; and (IV) Optionally, formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; The above method further, a) To provide a feed stream (or fluid feed) containing initial biological products; b) Filtering the supply stream to remove viral contaminants; c) Concentrating the initial biological products; and d) including buffer exchange to produce the processed biological product.

[0159] In certain embodiments, the biological product of interest is a recombinant protein. In certain embodiments, the step of culturing eukaryotic cells expressing the biological product of interest in cell culture is performed using fed-batch cell culture. In certain embodiments, the step of culturing eukaryotic cells expressing the biological product of interest in cell culture is performed using continuous cell culture.

[0160] While the present invention has been described with reference to specific embodiments, these descriptions are not intended to be constrained. Various modifications of the disclosed embodiments and alternative embodiments of the invention will become apparent to those skilled in the art with reference to the description of the invention. It should be understood by those skilled in the art that the disclosed concepts and specific embodiments are readily available as a basis for modifying or designing other structures or methods to accomplish the same objectives as the present invention. It should also be understood by those skilled in the art that such equivalent structures do not depart from the spirit and scope of the invention as described in the accompanying claims. Therefore, the claims are considered to encompass all such modifications or embodiments that fall within the scope of the invention.

Claims

1. A single-use system for integrated continuous processing of early biological products, The process includes filtration, concentration, and buffer exchange. It includes two skid bodies, one of which is a single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operating skid and the other is a viral filtration (VRF) unit operating skid. The single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operating skid is connected downstream of the viral filtration (VRF) unit operating skid and includes a first DF tank and a second DF tank. This single-use system is configured such that while one of the first or second DF tanks performs DF and then sends the material to the final pool, the other tank is filled.

2. A single-use system for integrated continuous processing of initial biological products, The process includes filtration, concentration, and buffer exchange. It includes two skid bodies, one of which is a single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operating skid and the other is a viral filtration (VRF) unit operating skid. The single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operating skid is connected downstream of the viral filtration (VRF) unit operating skid and includes a first DF tank and a second DF tank. This single-use system is configured such that one of the tanks, the first DF tank or the second DF tank, performs DF and then sends the material to the final pool via the second SPTFF while the other tank is filled.

3. The single-use system according to claim 1 or 2, wherein the biological product is a protein.

4. The single-use system according to claim 1 or 2, wherein the biological product is a monoclonal antibody.

5. The single-use system according to claim 1 or 2, wherein the operation of the virus filtration unit includes a pump, at least one pre-filter, and one or more virus-reducing filtration membranes.

6. The single-use system according to claim 1 or 2, wherein the SPTFF-DF unit operation includes one or more SPTFF membranes, a DF mixing tank, a DF membrane, a sensor, a pump, or a combination thereof.

7. The single-use system according to claim 1 or 2, further comprising a supply storage tank connected to the operation of the virus filtration unit.

8. The single-use system according to claim 7, wherein the supply storage tank holds the purified and polished monoclonal antibody at a concentration of 5 to 20 g / L.

9. The single-use system according to claim 7, wherein the supply storage tank holds the purified and polished monoclonal antibody at a concentration of 8 to 12 g / L.

10. The single-use system according to claim 1 or 2, wherein the system performs integrated, continuous virus filtration, concentration, and buffer exchange within an 8-hour timeframe.

11. The single-use system according to claim 1 or 2, wherein the system performs integrated, continuous virus filtration, concentration, and buffer exchange within a time period of 24 hours or less.

12. The single-use system according to claim 1 or 2, wherein the system performs integrated, continuous virus filtration, concentration, and buffer exchange within a time of 12 hours or less.

13. The single-use system according to claim 1 or 2, wherein the system performs integrated, continuous virus filtration, concentration, and buffer exchange within a time of 8 hours or less.

14. The single-use system according to claim 1 or 2, wherein the system is capable of processing a process that increases the concentration of the biological product tenfold.

15. An integrated, continuous method for providing processed biological products, a) To provide a feed stream containing initial biological products; b) Filtering the supply stream to remove viral contaminants; c) Concentrating the initial biological products; and d) Performing a buffer exchange to produce the processed biological product, Steps b) to d) above are performed by operating a viral filtration (VRF) unit connected to the operation of a single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit. The single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operation is connected downstream of the viral filtration (VRF) unit operation and includes a first DF tank and a second DF tank. The method wherein, while one of the first or second DF tanks performs DF and then sends the material to the final pool, the other tank is filled.

16. An integrated, continuous method for providing a processed biological product, a) To provide a feed stream containing initial biological products; b) Filtering the supply stream to remove viral contaminants; c) Concentrating the initial biological products; and d) Performing a buffer exchange to produce the processed biological product, Steps b) to d) above are performed by operating a viral filtration (VRF) unit connected to the operation of a single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit. The single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operation is connected downstream of the viral filtration (VRF) unit operation and includes a first DF tank and a second DF tank. The method wherein, while one of the first DF tank or the second DF tank performs DF and then sends the material to the final pool via the second SPTFF, the other tank is filled.

17. The method according to claim 15, wherein the operation of the virus filtration unit includes a pump, at least one prefilter, and one or more virus reduction filtration membranes.

18. The method according to any one of claims 15 to 17, wherein the SPTFF-DF unit operation includes one or more SPTFF membranes, a DF mixing tank, a DF membrane, a sensor, a pump, or a combination thereof.

19. The method according to any one of claims 15 to 17, wherein the initial biological product is a protein.

20. The method according to any one of claims 15 to 17, wherein the initial biological product is a monoclonal antibody.

21. The method according to any one of claims 15 to 17, wherein the method involves performing integrated, continuous virus filtration, concentration, and buffer exchange within a time period of 24 hours or less.

22. The method according to any one of claims 15 to 17, wherein the method involves performing integrated, continuous virus filtration, concentration, and buffer exchange within a time period of 12 hours or less.

23. The method according to any one of claims 15 to 17, wherein the method involves a process that increases the concentration of the initial biological product tenfold.

24. A method for producing the target biological product, (I) Culturing eukaryotic cells that express the target biological product in cell culture; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffer components; (III) Purifying the fluid feed containing the target biological product and one or more impurities or buffer components, and separating the target biological product from the fluid feed; and (IV) Formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; Step IV of the above method further includes passing the fluid feed through a single-use system for integrated continuous processing of initial biological products, The process includes filtration, concentration, and buffer exchange. The single-use system for integrated continuous processing of early biological products comprises two skids, including a viral filtration (VRF) unit operating skid connected to a single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operating skid. The single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operating skid is connected downstream of the viral filtration (VRF) unit operating skid and includes a first DF tank and a second DF tank. The manufacturing method wherein, while one of the first or second DF tanks performs DF and then sends the material to the final pool, the other tank is filled.

25. A method for producing a target biological product, (I) Culturing eukaryotic cells that express the target biological product in cell culture; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffer components; (III) Purifying the fluid feed containing the target biological product and one or more impurities or buffer components, and separating the target biological product from the fluid feed; and (IV) Formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; Step IV of the above method further includes passing the fluid feed through a single-use system for integrated continuous processing of initial biological products, The process includes filtration, concentration, and buffer exchange. The single-use system for integrated continuous processing of early biological products comprises two skids, including a viral filtration (VRF) unit operating skid connected to a single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operating skid. The single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operating skid is connected downstream of the viral filtration (VRF) unit operating skid and includes a first DF tank and a second DF tank. The manufacturing method wherein, as a result, one of the tanks, the first DF tank or the second DF tank, performs DF and then sends the material to the final pool via the second SPTFF while the other tank is filled.

26. The method according to claim 24, wherein the target biological product is a recombinant protein.

27. The method according to any one of claims 24 to 26, wherein the step of culturing eukaryotic cells that express the target biological product in cell culture is performed using fed-batch cell culture.

28. The method according to any one of claims 24 to 26, wherein the step of culturing eukaryotic cells that express the target biological product in cell culture is performed in continuous cell culture.

29. A method for producing the target biological product, (I) Culturing eukaryotic cells that express the target biological product in cell culture; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffer components; (III) Purifying the fluid feed containing the target biological product and one or more impurities or buffer components, and separating the target biological product from the fluid feed; and (IV) Formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; Step IV of the above method further, a) To provide a feed stream containing initial biological products; b) Filtering the supply stream to remove viral contaminants; c) Concentrating the initial biological products; and d) Performing a buffer exchange to produce the processed biological product, Steps b) to d) above are performed by operating a viral filtration (VRF) unit connected to the operation of a single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit. The single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operation is connected downstream of the viral filtration (VRF) unit operation and includes a first DF tank and a second DF tank. The manufacturing method wherein, while one of the first or second DF tanks performs DF and then sends the material to the final pool, the other tank is filled.

30. A method for producing a target biological product, (I) Culturing eukaryotic cells that express the target biological product in cell culture; (II) Collecting the target biological product from the cell culture in the form of a fluid feed containing the target biological product and one or more impurities or buffer components; (III) Purifying the fluid feed containing the target biological product and one or more impurities or buffer components, and separating the target biological product from the fluid feed; and (IV) Formulating the biological product of the objective into a pharmaceutically acceptable formulation suitable for administration; Step IV of the above method further, a) To provide a feed stream containing initial biological products; b) Filtering the supply stream to remove viral contaminants; c) Concentrating the initial biological products; and d) Performing a buffer exchange to produce the processed biological product, Steps b) to d) above are performed by operating a viral filtration (VRF) unit connected to the operation of a single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit. The single-pass tangential flow filtration-dialysis filtration (SPTFF-DF) unit operation is connected downstream of the viral filtration (VRF) unit operation and includes a first DF tank and a second DF tank. The manufacturing method wherein, as a result, one of the tanks, the first DF tank or the second DF tank, performs DF and then sends the material to the final pool via the second SPTFF while the other tank is filled.

31. The method according to claim 29, wherein the target biological product is a recombinant protein.

32. The method according to any one of claims 29 to 31, wherein the step of culturing eukaryotic cells that express the target biological product in cell culture is performed in fed-batch cell culture.

33. The method according to any one of claims 29 to 31, wherein the step of culturing eukaryotic cells that express the target biological product in cell culture is performed in continuous cell culture.

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