Systems and methods for single-pass countercurrent diafiltration

Abstract

Systems and methods are provided for single-pass countercurrent diafiltration of a fluid feed material that operate to reduce the impact on biological products, reduce diafiltration buffer usage, and reduce equipment size compared to conventional systems.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 157,877, filed March 8, 2021, the entire contents of which are incorporated by reference herein as if set forth in their entirety.

[0002] Disclosed herein are systems and methods for diafiltration of biological products. More specifically, the diafiltration systems and methods operate to reduce the impact on the biological product, reduce diafiltration buffer usage, and reduce equipment size as compared to conventional systems. [Background technology]

[0003] Diafiltration is a dilution process that involves removing or separating components of a solution based on their molecular size by using small molecule permeable filters to obtain a highly pure solution. Diafiltration is performed for buffer exchange to achieve the appropriate buffer composition during the formulation of biological products.

[0004] Buffer exchange is typically performed in a system that uses a recirculation loop to and from a tangential flow filtration (TFF) membrane and a feed tank large enough to hold the entire batch volume. The TFF membrane allows components smaller than the molecular weight cut-off to pass through the filter and into the permeate stream that is sent to waste, while larger components are retained in the retentate stream and returned to the feed tank by circulation. The required amount of diafiltration buffer specified to achieve the desired clearance factor is added to the feed tank or retentate stream at the same rate that buffer is removed through the permeate to maintain a constant volume in the feed vessel.

[0005] Fixed volume batch diafiltration is the most common process configuration. Commercially available systems for batch diafiltration can contain hundreds of square meters of membrane area housed on a skid. Increasing concerns about cost of goods and manufacturing flexibility have led to increased interest in the potential of continuous bioprocessing.

[0006] U.S. Patent No. 10,183,108 to Pall Corporation describes a diafiltration system designed for continuous buffer exchange. Although these stages provide continuous buffer exchange, the system requires a larger buffer volume to achieve the same level of buffer exchange as a corresponding batch system, and the multi-head pumping system is more complex.

[0007] Rucker-Pezzini et al. describe a three-stage single-pass diafiltration system (see Rucker-Pezzini et al., “Single pass diafiltration integrated into a fully continuous mAb purification process.” Biotechnology and Bioengineering. 2018; 115:1949-1957).

[0008] Nambiar et al. described the use of single-pass tangential flow filtration (SPTFF) stages for continuous diafiltration (see Nambiar et al., "Countercurrent staged diafiltration for formulation of high value proteins." Biotechnology and Bioengineering. 2018; 115:139-144). To reduce buffer requirements, the individual stages were arranged in a countercurrent configuration. Summary of the Invention [Problem to be solved by the invention]

[0009] There remains a need for improved diafiltration systems and methods that reduce the impact on biological products, reduce consumption of diafiltration buffer, and reduce the size of the equipment required. [Means for solving the problem]

[0010] Disclosed herein are systems and methods for efficient diafiltration of biological products. The systems and methods can support continuous or batch processes and reusable or disposable processing.

[0011] The systems disclosed herein provide distinct advantages over conventional tangential flow filtration (TFF) systems used for diafiltration of biological products. In particular, diafiltration performed by systems according to embodiments operates such that the biological product experiences only one pump pass, compared to about 40 to about 80 pump passes in conventional TFF systems. In this manner, potential effects on the biological product are reduced.

[0012] Another advantage of the systems disclosed herein is a substantial reduction in the amount of diafiltration buffer used, for example, diafiltration buffer usage can be reduced by at least about 60% compared to conventional TFF systems, and when using a three-stage system, eight diavolumes of diafiltration can be achieved with three diavolumes of diafiltration buffer.

[0013] A further advantage of the systems disclosed herein is that the equipment size can be substantially reduced compared to conventional TFF systems. In some embodiments, the feed flow rate of the system is about 40 to about 80 times lower than the feed flow rate of conventional TFF systems. In certain embodiments, the system is more easily manufactured using disposable product contacting flow paths compared to conventional TFF systems. The size of conventional TFF systems may not permit the use of disposable construction materials.

[0014] The systems and methods described herein achieve buffer exchange, i.e., diafiltration, at a constant retentate concentration. The TFF cassettes used in the systems and methods each include a diafiltration channel, a flat first filtration membrane, a retentate channel, a flat second filtration membrane, and a permeate / diafiltration buffer collection channel, where (i) the flat first filtration membrane separates the diafiltration channel and the retentate channel from each other, (ii) the flat second filtration membrane separates the retentate channel and the permeate / diafiltration buffer collection channel from each other, (iii) the diafiltration channel is fluidically connected to at least one inlet for diafiltration medium, (iv) the retentate channel is fluidically connected to at least one inlet for feed fluid and at least one outlet for retentate, and (v) the permeate / diafiltration buffer collection channel is fluidically connected to at least one outlet for permeate / diafiltration buffer. The systems and methods described herein provide stepwise diafiltration of a feed medium containing a molecule of interest (e.g., a protein or other biological molecule). The feed medium is diafiltered against a diafiltration buffer across a suitable membrane, where diafiltration occurs several times (e.g., two, three, four or more times) in adjacent diafiltration cassettes. This stepwise diafiltration is characterized by a countercurrent flow of diafiltration buffer against the feed medium. The permeate (and diafiltration buffer, if any) leaving the system from the first diafiltration cassette (e.g., the leftmost cassette shown in FIG. 1) contains washed-out impurities, mixtures of ions, etc., whereas the retentate leaving the system after the last diafiltration cassette (e.g., the rightmost cassette shown in FIG. 1) contains the molecule of interest that has been exchanged into the diafiltration buffer.

[0015] In a first aspect, a system for single-pass countercurrent diafiltration of a fluid feed comprising: two, three, four or more filtration stages; conduits for facilitating fluid communication between manifolds in the filtration stages; conduits and at least one feed pump for providing the fluid feed to a first filtration unit; conduits and at least one buffer pump for providing diafiltration buffer to a final filtration stage; conduits and at least one buffer pump per filtration stage for providing permeate and diafiltration buffer from a subsequent filtration stage to the previous filtration stage; and conduits and filtrate pumps for removing permeate (and diafiltration buffer, if any) from the system, each filtration stage comprises (i) a manifold segment, and (ii) one or more TFF cassettes as described herein, the manifold segment comprising a first manifold (i.e., inlet) for receiving and conveying a fluid feed or retentate into the filtration stage, a second manifold (i.e., outlet) for receiving and conveying a retentate out of the filtration stage, a third manifold (i.e., inlet) for receiving and conveying a permeate and diafiltration buffer through the filtration stage, and a fourth manifold (i.e., outlet) for receiving and conveying a permeate and diafiltration buffer out of the filtration stage; the manifold segment is fluidly connected to one or more TFF cassettes as described herein; by connecting a second manifold in the manifold segment to a first manifold in the manifold segment in a subsequent filtration stage so that the retentate of one stage serves as the feed for the next filtration stage, and by connecting a fourth manifold in the manifold segment to a third manifold in the manifold segment in the previous filtration stage, the filtration stages being fluidly connected through the manifold segments to provide flow paths between the filtration stages; A system is disclosed in which a first filtration stage in the system receives a fluid feed from a conduit connected to a feed pump; a second manifold in the final (i.e., last) filtration stage is a retentate outlet that allows retentate to exit the system and is not connected to the previous filtration stage.

[0016] In certain embodiments, the system includes two filtration stages. In certain embodiments, the system includes three filtration stages.

[0017] In a second aspect, a method of filtering a fluid feed using the system described herein is provided.

[0018] In a third embodiment, there is provided a method for producing a biological product of interest, comprising the steps of: (I) culturing a eukaryotic cell expressing a biological product of interest in cell culture; (II) harvesting a biological product of interest from the cell culture in the form of a fluid feed comprising (a) the biological product of interest, and (b) one or more impurities and / or buffer components; (III) purifying a fluid feed containing the biological product of interest and one or more impurities and / or buffer components to separate the biological product of interest from the fluid feed; and (IV) optionally, formulating the biological product of interest into a pharma- ceutically acceptable formulation suitable for administration; The method further includes the steps of: (a) passing the fluid feed through a single-pass countercurrent diafiltration system; and (b) collecting the permeate (and diafiltration buffer, if any) and retentate from the system in separate containers. Includes; A method is provided, wherein the system is a system according to embodiments described herein.

[0019] Further advantages of the present technology will become readily apparent to those skilled in the art from the following drawings and detailed description, which are to be regarded as illustrative in nature and not as restrictive. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 illustrates an exemplary single-pass countercurrent diafiltration system. [Diagram 2] 1 is a graph of diavolume (buffer flow rate / feed flow rate) versus tracer clearance for a system with one TFF cassette according to an embodiment. [Diagram 3] 1 is a graph of the diafiltration efficiency of a two-stage single-pass countercurrent diafiltration system. [Figure 4] FIG. 1 is a graph of the natural log clearance of glucose versus diavolume of diafiltration buffer for a three-stage, single-pass countercurrent diafiltration system. The horizontal line (9.2 natural log clearance) marks the limit of detection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention In the following detailed description, numerous specific details are described to provide a thorough understanding of the present technology. However, it will be apparent to those skilled in the art that the present technology may be practiced without some of these specific details. In other instances, well-known structures and techniques are not shown in detail so as not to obscure the present technology. Preferences and options for any given aspect, feature, embodiment or parameter of the present invention should be considered as disclosed in combination with all preferences and options for all other aspects, features, embodiments and parameters of the present invention, unless the context indicates otherwise.

[0022] To facilitate the understanding of the present technology, several terms and phrases are defined below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] definition As used herein, the singular articles "one," "a," "an," and "the" are intended to include "at least one" or "one or more" unless otherwise indicated. These articles are therefore used herein to refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, "a component" means one or more components, and thus, more than one component is conceivable and can be used in the implementation of the described embodiments.

[0024] The term "about" generally refers to a small degree of error in a measurement, often stated as a range of values ​​that includes the true value within a certain confidence level (usually ±1σ with a 68% confidence interval). The term "about" may also be expressed as an integer and a value of ±20% of the integer.

[0025] The term "biological product" generally refers to a product of interest that is created through a biological process or through chemical or catalytic modification of an existing biological product. Biological processes include cell culture, fermentation, metabolism, respiration, etc. Biological products of interest include, for example, antibodies, antibody fragments, proteins, hormones, vaccines, fragments of natural proteins (e.g., fragments of bacterial toxins used as vaccines, such as tetanus toxoid), fusion proteins or peptide complexes (e.g., sub-stage vaccines), virus-like particles (VLPs), etc.

[0026] The term "system" refers to a single-pass countercurrent diafiltration system. Such a system is configured to operate in a single-pass mode, where the fluid passes through the system only once.

[0027] The term "countercurrent" refers to the backfeed of a filtrate stream to a retentate stream at a location (stage) upstream from where a particular filtrate was produced. Thus, countercurrent can refer to a multi-stage backfeed of a filtrate stream.

[0028] The term "TFF cassette" generally refers to a tangential flow filtration device that includes a membrane suitable for separating a feed solution containing a biological product into a retentate stream and a permeate stream. A "TFF cassette" refers to a plate-and-frame structure suitable for a TFF or diafiltration process that includes a filtration membrane and separate feed / retentate and permeate flow channels. In particular, the TFF cassettes used in the systems and methods each include a diafiltration channel, a flat first filtration membrane, a retentate channel, a flat second filtration membrane, and a permeate / diafiltration buffer collection channel, configured such that the flat first filtration membrane separates the diafiltration channel and the retentate channel from one another, and the flat second filtration membrane separates the retentate channel and the permeate / diafiltration buffer collection channel from one another. The diafiltration channel is fluidly connected to at least one inlet for diafiltration medium; the retentate channel is fluidly connected to at least one inlet for feed fluid and at least one outlet for retentate; and the permeate / diafiltration buffer collection channel is fluidly connected to at least one outlet for permeate / diafiltration buffer.

[0029] The term "filtration membrane" refers to a selectively permeable membrane for separating a feed into a permeate stream and a retentate stream using a TFF or diafiltration process. Exemplary filtration membranes include, but are not limited to, Hydrosart® membranes and polyethersulfone (PESU) membranes, such as those commercially available from Sartorius. Other filtration membranes may be suitable for the systems and methods described herein, and such membranes may be characterized by a stable polymer composition, a wide pH range, and / or a wide temperature range. Typically, the filtration membranes described herein have a pore size ranging from about 5 kilodaltons (kD) to about 300 kD or from about 0.0012 microns to about 0.075 microns. In specific embodiments, the filtration membranes have a pore size ranging from about 20 kD to about 40 kD or from about 25 kD to about 35 kD. In one embodiment, the filtration membrane has a pore size or average pore size of about 30 kD. In one embodiment, the filtration membrane has a pore size suitable for processing antibodies.

[0030] The term "feed" or "feed stream" refers to a solution sent to a filtration stage to be filtered. The feed sent to a filtration stage for filtration can be, for example, a feed from a feed vessel (e.g., vessel, tank) external to the system, or it can be retentate from a previous filtration stage in the same system.

[0031] The term "filtration" generally refers to the act of separating a feed into two streams, a permeate and a retentate.

[0032] The term "permeate" refers to the portion of the feed that permeates the membrane.

[0033] The term "filtrate" refers to any material that has permeated the membrane (permeate and / or diafiltration buffer).

[0034] The term "retentate" refers to the portion of the solution retained by the membrane. In certain embodiments, no enrichment (i.e., concentration) of the retentate occurs during the exemplary diafiltration process. According to embodiments, buffer exchange (diafiltration) is achieved at a constant retentate concentration.

[0035] "Feed conduit" refers to a conduit for carrying feed from a feed source (e.g., a feed vessel) to a filtration stage.

[0036] "Retentate conduit" refers to a conduit in a filtration assembly for carrying retentate.

[0037] "Permeate conduit" refers to a conduit in a filtration assembly for carrying permeate.

[0038] The term "flow path" refers to a channel that supports the flow of a fluid (or liquid) through all or a portion of the system. The fluid can be a feed, permeate, diafiltration buffer, or retentate. For example, a flow path through the entire system from a feed inlet to a retentate outlet; a flow path within a filtration stage (e.g., a flow path through a TFF cassette and / or manifold segment in a filtration stage); and a flow path between two or more adjacent filtration stages. A flow path can have any topology (e.g., straight, coiled, zigzag arranged) that supports the desired flow. A flow path can be parallel or serial. A flow path can also refer to a path that results in a single pass through a system.

[0039] "Filtration stage" refers to a unit in a system that contains a manifold segment and one or more TFF cassettes as described herein.

[0040] "Manifold segment" refers to a block having multiple manifolds (e.g., inlets and outlets), including a manifold for carrying feed, a manifold for carrying diafiltration buffer into the filtration stage, a manifold for carrying retentate, and a manifold for carrying permeate / diafiltration buffer out of the filtration stage.

[0041] The term "plurality," when used herein to describe system units, refers to two or more.

[0042] The term "fluidly connected" refers to two or more components of a system (e.g., two or more manifold segments, two or more TFF cassettes, a manifold segment and one or more TFF cassettes) being connected by one or more conduits (e.g., a feed conduit, a retentate conduit, a diafiltration buffer conduit, a permeate and diafiltration buffer conduit) so that fluid can flow from one component to another.

[0043] As used herein, the term "processing" refers to the act of filtering a feed material containing a biological product and then recovering the diafiltered product, which can be recovered by directing the retentate to a suitable collection vessel.

[0044] The terms "continuous process," "series process," "continuous operation," and "series operation" refer to the distribution of fluids or liquids in a system to one filtration stage at a time, with the retentate stream of the preceding unit serving as the feed stream for the subsequent adjacent unit.

[0045] The expressions "conversion" and "conversion per pass" are used herein to denote the fraction of the feed volume (expressed as a percentage of the feed stream volume) that permeates the membrane during a pass through the flow channel.

[0046] The term "residence time" refers to the hold-up volume on the feed side of the membrane divided by the flow rate.

[0047] The term "enrichment factor" as used herein refers to the amount by which a product is concentrated in a feed stream, which depends on both the volumetric enrichment factor and the retention rate.

[0048] The term "diafiltration" or "DF" is used to refer to a special class of filtration in which the retentate is simultaneously diluted with a solvent to reduce the concentration of soluble permeate components and refiltered.

[0049] The term "diavolume" is a measure of the degree of cleaning performed during the diafiltration process. It is based on the volume of diafiltration buffer introduced into a stage operation compared to the retentate volume.

[0050] The terms "downstream" or "downstream processing" generally refer to some or all of the steps required to capture a biological product from the original solution in which it was created, for purification of the biological product from undesirable components and impurities, for filtration or inactivation of pathogens (e.g., viruses, endotoxins), and for formulation and packaging.

[0051] The term "ultrafiltration" refers to filtration used to retain biological products and pass buffer components smaller than the molecular weight cutoff into the filtrate. Ultrafiltration membranes used to retain biological products generally have a molecular weight cutoff of about 5 kD to about 300 kD.

[0052] The terms "polypeptide", "polypeptide product", "protein" and "protein product" are used interchangeably herein and refer to a molecule comprised of two or more amino acids, e.g., at least one chain of amino acids linked via consecutive peptide bonds, as known in the art. In one embodiment, a "protein of interest" or "polypeptide of interest" is a protein encoded by an exogenous nucleic acid molecule transformed into a host cell, the exogenous DNA determining the amino acid sequence. In another embodiment, a "protein of interest" is a protein encoded by a nucleic acid molecule that is endogenous to the host cell.

[0053] The term "retention" as used herein refers to the percentage of a particular biological product (e.g., protein) that is retained by a membrane and can also be calculated as apparent retention or specific retention.

[0054] As used herein, the term "disposable" refers to an article that is suitable for being discarded after a single use, and a reusable article that is used only once in a process according to the present invention and is no longer used in the process. Such articles are also called "disposable".

[0055] The term "skid" refers to a system of components housed in a frame that allows the system to be easily transported. An individual skid can contain a complete process system or can contain systems that perform a particular aspect of a process. Multiple skids can be combined to create larger systems or entire portable plants.

[0056] The term "tangential flow filtration" or "TFF", also known as cross-flow filtration, refers to a process in which the feed stream flows parallel to the membrane surface. The application of pressure causes a portion of the stream to permeate the membrane (the filtrate) while the remainder is retained (the retentate).

[0057] The term "transmembrane pressure" or "TMP" refers to the average pressure exerted from the feed side to the filtrate side of a membrane.

[0058] The term "antibody" as used herein broadly refers to a protein capable of recognizing and binding to a specific antigen. The term specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments (e.g., Fab fragments, F(ab')2, Fv fragments or Fc fragments from truncated antibodies, scFv-Fc fragments, minibodies, diabodies or scFv), as well as double-chain and single-chain antibodies. The term also includes human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. Furthermore, the term includes genetically engineered derivatives of antibodies. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0059] As used herein, the terms "culture" and "cell culture" and "mammalian cell culture" refer to a population of cells, either surface-attached or in suspension, maintained or grown in a medium under conditions suitable for the survival and / or growth of the cell population. These terms can also refer to the cell population and the medium in which the population is suspended.

[0060] The term "impurities" as used herein refers to undesirable chemical or biological compounds produced during the culture process. Impurities can include, for example, ethyl alcohol, butyl alcohol, lactic acid, acetone, ethanol, gaseous compounds, peptides, lipids, ammonia, aromatic compounds, DNA and RNA fragments, and degradation products of medium components and biological products.

[0061] As used herein, the term "culture medium" or "cell culture medium" refers to a solution containing nutrients to nourish the cells being cultured. Typically, these solutions provide the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cells for minimal growth and / or survival. The solutions may also contain components that enhance growth and / or survival beyond minimum ratios, including hormones and growth factors. The solutions are formulated to have optimal pH and salt concentrations for cell survival and growth.

[0062] Diafiltration Systems Disclosed herein is a system for single-pass countercurrent diafiltration and a method of using the system to filter a fluid feed (eg, a liquid feed or solution feed).

[0063] In one embodiment, the systems and methods relate to diafiltration of a fluid feed, in which the fluid feed and diafiltration buffer are flowed through a single-pass diafiltration system as described herein and diafiltered, and a retentate is collected.

[0064] The diafiltration system includes multiple filtration stages fluidly connected such that retentate moves sequentially from a first filtration stage to subsequent filtration stages before reaching the final filtration stage, and permeate and diafiltration buffer flow countercurrently from the subsequent stages to the previous stages. Each "filtration stage" includes one or more TFF cassettes and a manifold segment including a block having multiple manifolds (e.g., inlets and outlets). In certain embodiments, one or more TFF cassettes are within or housed in the manifold segment. In certain embodiments, the system includes two or more filtration stages. In certain embodiments, the system includes three or more filtration stages. In certain embodiments, the system includes two, three, or four stages. In general, there is no particular limit to the number of stages that can be used in the system. However, the systems and methods are designed to achieve improved diafiltration with two, three, or four stages. In particular, the systems and methods are designed to achieve at least 8 diavolumes of diafiltration, which is typically required for biological products, with two, three, or four stages. In certain embodiments, the systems and methods achieve 8 diavolumes of diafiltration with two stages. In certain embodiments, the systems and methods achieve 8 diavolumes of diafiltration in three stages.In certain embodiments, the systems and methods achieve 8 diavolumes of diafiltration in four stages.

[0065] An exemplary system (10) is shown in FIG. 1. With reference to FIG. 1, a fluid feed from a feed source (100) flows into a feed inlet of a first filtration stage ("Stage 1") via feed conduits (101a, 101b). The fluid feed is forced through the feed conduits by a feed pump (i.e., "feed pump"). The feed passes through a TFF cassette (200), which separates the retentate from the permeate and diafiltration buffer present in the first filtration stage. The retentate flows into the subsequent (i.e., second) filtration stage via feed conduit (201). The permeate and diafiltration buffer flow into the first filtration stage via permeate-diafiltration buffer conduits (302a, 302b) which lead to permeate-diafiltration buffer inlets. The permeate and diafiltration buffer are forced from the second filtration stage to the first filtration stage by a buffer pump (also known as a filtrate pump) (i.e., "filtrate pump 2") through the permeate-diafiltration buffer conduits. The permeate (and diafiltration buffer, if any remainder) exits the first filtration stage via the outlet of the first filtration stage. The permeate (and diafiltration buffer, if any remainder) is forced through the permeate-filtrate conduits (202a, 202b) by a filtrate pump (i.e., "filtrate pump 3"). The permeate (and diafiltration buffer, if any remainder) can be discarded accordingly.

[0066] In the second filtration stage ("Stage 2") shown in FIG. 1, the retentate from Stage 1 passes through a TFF cassette (300), which separates the retentate from the permeate and diafiltration buffer present in the second filtration stage. The retentate from Stage 2 flows into the subsequent (i.e., third) filtration stage via feed conduit (301). Permeate and diafiltration buffer flow from the third filtration stage into the second filtration stage via permeate and diafiltration buffer conduits (402a, 402b) which lead to the permeate inlet. The permeate and diafiltration buffer are forced from the third filtration stage to the second filtration stage by a buffer pump (i.e., "Filtrate Pump 1") through the permeate and diafiltration buffer conduits.

[0067] As shown in FIG. 1, diafiltration buffer ("DF buffer") flows from a diafiltration buffer source (500) into the final (or third) filtration stage ("stage 3") through diafiltration buffer conduits (502a, 502b) that lead to the diafiltration buffer inlets of the final filtration stage. A buffer pump (or "DF buffer pump") pushes the diafiltration buffer through the diafiltration buffer conduits to the final filtration stage. In the final filtration stage, the retentate from stage 2 passes through a TFF cassette (400), which separates the retentate from the permeate and diafiltration buffer present in the third filtration stage. The retentate from stage 3 exits the system via a retentate outlet and retentate conduits (401a, 401b). The retentate may then be stored or sent to other downstream components for other processes. An optional valve (403) may be used to regulate backpressure.

[0068] As described above, each filtration stage includes a manifold segment that includes a first manifold (i.e., inlet) for receiving and conveying the feed (or retentate) into the filtration stage, a second manifold (i.e., outlet) for receiving and conveying the retentate through the filtration stage, a third manifold (i.e., inlet) for receiving and conveying the permeate and / or diafiltration buffer through the filtration stage, and a fourth manifold (i.e., outlet) for receiving and conveying the permeate and diafiltration buffer out of the filtration stage. The filtration stages are fluidly connected through the manifold segments by coupling the second manifold in the manifold segment to the first manifold in the manifold segment in the subsequent filtration stage so that the retentate of one stage serves as the feed for the next filtration stage, and by coupling the fourth manifold in the manifold segment to the third manifold in the manifold segment in the preceding filtration stage to provide a flow path between the filtration stages. The manifold segments in each stage are also fluidly connected to one or more TFF cassettes that can be stacked on one or both sides of the manifold segments. In addition, the system includes a feed inlet at a first stage in the system and a retentate outlet at a last stage in the system, such that the feed initially enters the system through the first filtration stage and the retentate (i.e., product) exits the system at the last filtration stage.

[0069] The systems described herein utilize countercurrent flow of permeate and diafiltration buffer; that is, the flow paths of permeate and diafiltration buffer move in the conduits and filtration stages in the direction from the last filtration stage to the first filtration stage. FIG. 1 shows, by way of example, a simple three-stage system in which each stage has a single TFF cassette, and each TFF cassette has a single filter cell with two membranes that separate a single diafiltration channel, a single feed / retentate channel, and a single permeate / buffer collection channel. As will be apparent to those skilled in the art, more complex arrangements (e.g., multiple TFF cassettes per stage, multiple filter cells per TFF cassette, etc.) can also be used, so long as the appropriate delineation between the diafiltration, feed / retentate, and permeate / buffer collection channels is maintained.

[0070] The filtration area of ​​the cassette can be any suitable filtration area. In certain embodiments, the system has a filtration area of ​​about 0.3 to about 80 m per cassette. 2 , about 0.3~3m 2 Or about 10 to about 80 m 2 In one embodiment, the system has a filtration area ranging from about 0.3 to about 3 m per cassette. 2 The filtration area is in the range of

[0071] The fluid feed can be any liquid (e.g., biological liquid) or solution containing a biological product or particle (e.g., viral particles, host cell proteins) to be filtered. For example, the fluid feed can contain a biological product (e.g., a target protein such as a recombinant protein) and one or more impurities (e.g., non-target proteins) and / or buffer components. Typically, the systems and methods are used for buffer exchange performed at or near the end of a biological product purification process. In certain embodiments, the buffer components removed during diafiltration are not proteins.

[0072] In certain embodiments, the fluid feed is obtained from a source of a biological product (e.g., a hybridoma or other host cell expressing a monoclonal antibody (MAb). In certain embodiments, the biological product in the fluid feed is an MAb present in an elution buffer from a chromatography step. In certain embodiments, the diafiltration buffer is a formulation buffer for a drug.

[0073] Operation of the system in a single-pass mode allows for direct flow-through diafiltration of the biological product in the absence of recirculation, which reduces the overall system size through elimination of mechanical parts and allows for continuous operation at high conversion levels.

[0074] In general, the systems of the present invention can be assembled and operated using standard, existing components (e.g., TFF systems) that are well known and commercially available, such as a cassette holder, conduits (e.g., tubing, pipes) for feed, retentate, permeate, and buffer, housings or enclosures, valves, gaskets, pump stages (e.g., pump stages including pump housings, diaphragms, and check valves), one or more reservoirs (e.g., process vessels for feed, retentate, permeate, and buffer), and pressure gauges.

[0075] TFF Cassette A TFF cassette suitable for use in the exemplary systems and methods includes a plate-and-frame structure suitable for a TFF or diafiltration process, including a filtration membrane and separate channels for the feed / retentate, permeate / diafiltration buffer entering the stage, and permeate / diafiltration buffer exiting the stage. In general, a TFF cassette includes multiple adjacent cross-flow filtration units (i.e., filter cells), which typically consist of a repeating array of a retentate channel for the feed fluid or retentate being filtered, two flat filtration membrane layers, a permeate / diafiltration buffer intake channel, and a permeate / diafiltration buffer collection channel. The permeate / diafiltration buffer intake and collection channels of a filter cell are separated from the retentate channel of the next filter cell by an additional flat membrane layer. Each retentate channel is fluidically connected to an inlet for the feed fluid to be filtered and an outlet for the retentate; each permeate / diafiltration buffer intake channel is fluidically connected to an inlet for the permeate / diafiltration buffer; and each permeate / diafiltration buffer collection channel is fluidically connected to an outlet for the permeate / diafiltration buffer.

[0076] In one embodiment, the TFF cassette includes a diafiltration channel, a flat first filtration membrane, a retentate channel, a flat second filtration membrane, and a permeate / diafiltration buffer collection channel, wherein (i) the flat first filtration membrane separates the diafiltration channel and the retentate channel from one another, (ii) the flat second filtration membrane separates the retentate channel and the permeate / diafiltration buffer collection channel from one another, (iii) the diafiltration channel is fluidly connected to at least one inlet for diafiltration medium, (iv) the retentate channel is fluidly connected to at least one inlet for feed fluid and at least one outlet for retentate, and (v) the permeate / diafiltration buffer collection channel is fluidly connected to at least one outlet for the permeate / diafiltration buffer.

[0077] In certain embodiments, the first filtration membrane and the second filtration membrane have respective pore sizes or respective molecular weight cut-offs; the pore size or molecular weight cut-off of the first filtration membrane is at least as large as the pore size or molecular weight cut-off of the second filtration membrane.

[0078] In certain embodiments, the first filtration membrane is a microfiltration or ultrafiltration membrane and / or the second filtration membrane is an ultrafiltration membrane.

[0079] In one embodiment, the first filtration membrane has a molecular weight cut-off (MWCO) in the range of 30 kD to 1,500 kD, and the second filtration membrane has a molecular weight cut-off in the range of 5 kD to 1,500 kD, about 5 kD to about 300 kD, about 20 kD to about 40 kD, or about 25 kD to about 35 kD.

[0080] In certain embodiments, the one or more TFF cassettes comprise a plurality of stacked arrays, each stacked array comprising a respective diafiltration channel, a respective first filtration membrane, a respective retentate channel, a respective second filtration membrane, and a respective permeate / diafiltration buffer collection channel, such that the stacked arrays combine to form a filter cassette.

[0081] In certain embodiments, the free volume of the diafiltration channel and / or the retentate channel decreases in the flow direction from an inlet for the feed fluid to an outlet for the retentate.

[0082] In certain embodiments, multiple layers of fibrous material are disposed above one another in the retentate channel such that the free volume of the retentate channel decreases in the flow direction.

[0083] In certain embodiments, the filtration membrane of one or more TFF cassettes is a stabilized cellulosic polymer filtration membrane. In certain embodiments, each TFF cassette comprises one or more stabilized cellulosic polymer filtration membranes, for example stabilized cellulosic polymer membranes optimized for the biotechnology and pharmaceutical industries. In certain embodiments, the stabilized cellulosic polymer membranes have a wide pH and temperature range and are hydrophilic. In certain embodiments, the stabilized cellulosic polymer membranes resist protein binding and fouling. In certain embodiments, the stabilized cellulosic polymer membranes can be sterilized by steam or autoclaving. In certain embodiments, the stabilized cellulosic polymer membranes can be regenerated or depyrogenated using NaOH, with or without heating. In certain embodiments, one or more TFF cassettes can be used to remove the following from a fluid feed: mammalian cells, such as CHO and BHK; bacteria, such as E. coli and Pasteurella C. diphtheria; yeast; and cell lysates. In certain embodiments, the filtration membrane or polymer membrane in the TFF cassette has a pore size ranging from about 5 kD to about 300 kD or from about 0.0012 microns to about 0.075 microns. In certain embodiments, the filtration membrane has a pore size ranging from about 20 kD to about 40 kD or from about 25 kD to about 35 kD. In one embodiment, the filtration membrane has a pore size of about 30 kD. In one embodiment, the filtration membrane has a pore size suitable for processing antibodies.

[0084] In certain embodiments, the filtration membrane or polymer membrane in the TFF cassette is about 0.3 to about 3.0 mm. 2 In certain embodiments, the filtration membrane or polymer membrane in the TFF cassette has a membrane area of ​​about 10 to about 80 m. 2 The filter has a filtering area of ​​100 mm.

[0085] In certain embodiments, the filtration membrane of one or more TFF cassettes is a polyethersulfone (PESU) filtration membrane. In certain embodiments, each TFF cassette comprises one or more PESU polymeric filtration membranes, for example, PESU polymeric membranes optimized for the biotechnology and pharmaceutical industries. In certain embodiments, the PESU membrane has a wide pH range and a wide temperature range and is hydrophilic. In certain embodiments, the PESU membrane resists protein binding and fouling. In certain embodiments, the PESU membrane can be sterilized by steam or autoclaving. In certain embodiments, the PESU membrane can be regenerated or depyrogenated using NaOH, with or without heating. In certain embodiments, the PESU filtration membrane in the TFF cassette has a pore size ranging from about 0.05 μm to about 0.2 μm or from about 0.1 μm to about 0.2 μm.

[0086] In certain embodiments, each diafiltration cassette has the same basic design, while in other embodiments, a combination of different diafiltration cassettes (e.g., different membrane materials, different pore sizes, etc.) can be used.

[0087] In one embodiment, a system for single-pass countercurrent diafiltration of a fluid feed includes two or more filtration stages; conduits for facilitating fluid communication between manifolds in the filtration stages; conduits and at least one feed pump for providing the fluid feed to a first filtration unit; conduits and at least one buffer pump for providing diafiltration buffer to a final filtration stage; conduits and at least one buffer pump per filtration stage for providing permeate and diafiltration buffer from a subsequent filtration stage to the previous filtration stage; and conduits and filtrate pumps for removing permeate (and diafiltration buffer, if any) from the system. Each filtration stage is (i) a manifold segment, and (ii) one or more TFF cassettes; the manifold segment includes a first manifold (i.e., inlet) for receiving and conveying a fluid feed or retentate to the filtration stage, a second manifold (i.e., outlet) for receiving and conveying a retentate from the filtration stage, a third manifold (i.e., inlet) for receiving and conveying a permeate and diafiltration buffer through the filtration stage, and a fourth manifold (i.e., outlet) for receiving and conveying a permeate and diafiltration buffer from the filtration stage; the manifold segment is fluidly connected to the one or more TFF cassettes; by connecting a second manifold in the manifold segment to a first manifold in the manifold segment in a subsequent filtration stage so that the retentate of one stage serves as the feed for the next filtration stage, and by connecting a fourth manifold in the manifold segment to a third manifold in the manifold segment in the previous filtration stage, the filtration stages being fluidly connected through the manifold segments to provide flow paths between the filtration stages; A first filtration stage in the system receives a fluid feed from a conduit connected to a feed pump; a second manifold in the final (i.e., last) filtration stage is a retentate outlet that allows retentate to exit the system and is not connected to the previous filtration stage.

[0088] In certain embodiments, the manifold segment in each stage is also fluidly connected to one or more TFF cassettes, hi certain embodiments, the one or more TFF cassettes are within the manifold segment and are stacked on one or both sides of the manifold segment.

[0089] Each manifold segment has a manifold structure or device that allows the segment to be fluidly connected to the manifold segments in the adjacent filtration stage. The manifold segments are connected in a manner that promotes serial flow of fluid from manifold segment to manifold segment. For example, adjacent manifold segments are arranged such that a first manifold in each manifold segment is connected to a second manifold in the adjacent manifold segment. As a result of this arrangement, the retentate of one stage (which exits the stage through a second manifold in the manifold segment) serves as the feed for the next stage (which is received in the first manifold of the manifold segment). The manifolds in the manifold segments provide separate paths (i.e., countercurrent flow of permeate and diafiltration buffer) for discharging permeate and diafiltration buffer from the filtration stage and receiving permeate and diafiltration buffer in the previous filtration stage in the series of filtration stages.

[0090] In certain embodiments, parallel flow between adjacent manifold segments is prevented using seals or valves (e.g., sanitary valves) to facilitate serial flow of fluids between stages. For example, seals or valves can be placed in the manifolds carrying the feed and retentate to prevent parallel flow of fluids into adjacent manifold segments. The use of seals or valves to prevent parallel flow is particularly desirable when the manifold segments are fully perforated such that the first, second, and third manifolds each pass completely through the manifold segment.

[0091] Seals (e.g., mechanical seals) suitable for placement in the manifold include, but are not limited to, rings (e.g., O-rings, metal rings), moldings, packings, sealants, and gaskets. Preferably, the seal is a gasket, such as a gasket that closes the opening or has a length sufficient to close the dead volume between the opening and the first passageway in the manifold. Preferably, the gasket is flexible and sanitary (e.g., low-strip, washable, sanitizable, low extractable gaskets). The gasket may comprise an elastomeric material or a metal (e.g., metal foil).

[0092] The use of valves instead of seals provides greater operational flexibility by allowing parallel flow between manifold segments when the valves are open and serial flow when the valves are closed. Valves suitable for use in the manifold include, for example, pinch valves (e.g., diaphragm valves). Preferably, the valves are low shear and sanitary (e.g., conformable, non-toxic, disinfectable, low-strip). As used herein, a "sanitary valve" is a valve that is capable of maintaining a sterile connection whether open or closed. Typically, sanitary valves are conformable, non-toxic, disinfectable, and low-strip.

[0093] The manifold segment in each filtration stage is also fluidly connected to one or more TFF cassettes, as described herein. For example, the manifold segment can be fluidly connected to the TFF cassettes via a flow channel extending from a first, i.e., feed manifold, in the manifold segment through the plurality of TFF cassettes, and a retentate flow channel that passes through the plurality of TFF cassettes and returns to a second, i.e., retentate channel, in the manifold segment.

[0094] The TFF cassettes can be located (e.g., stacked) on one or both sides of the manifold segment. Typically, each filtration stage is approximately 10 m on each side of the manifold segment.2 It can accommodate a membrane area of ​​up to 20 m per stage, for a total area of ​​approximately 20 m 2 In general, the number of cassettes that can be stacked on each side of a manifold segment depends on the membrane area of ​​the cassettes, among other things. In certain embodiments, each filtration stage in the system contains the same number and arrangement of TFF cassettes.

[0095] In one embodiment, the TFF cassettes (e.g., about one, two, three, four, five, six, seven, eight, nine, ten or more TFF cassettes) are located on both sides of the manifold segment. In another embodiment, the TFF cassettes (e.g., about one, two, three, four, five, six, seven, eight, nine, ten or more TFF cassettes) are located on only one side of the manifold segment. When TFF cassettes are located on both sides of the manifold segment, the number of TFF cassettes on each side of the manifold segment can be different or the same. In certain embodiments, the total number of TFF cassettes on each side of the manifold segment is the same.

[0096] In certain embodiments, the system may include one or more filtration stages having cassettes configured for parallel processing and one or more filtration stages having cassettes configured for serial processing (e.g., using valves, gaskets, or diverter plates). Preferably, the filtration stage having a cassette configured for parallel processing precedes the filtration stage having a cassette configured for serial processing in the system. In certain embodiments, all filtration stages in the system have cassettes configured for parallel processing, except for the last or final filtration stage having a cassette arranged for serial processing.

[0097] End plates or cassette holders are typically used to hold, i.e., seal, the TFF cassettes during the filtration stages. End plates and cassette holders can be attached for use with a particular cassette.

[0098] The system of the present invention typically includes a feed inlet and a retentate outlet. The feed inlet is generally located at the first filtration stage in the system, with one end connected to a conduit (e.g., pipe, tube) connected to a feed tank and the other end connected to a first manifold in a manifold segment in the first stage for receiving the feed into the system. The retentate outlet is generally located at the last or final filtration stage in the system, with one end connected to a second manifold in a manifold segment in the last stage and the other end connected to a conduit (e.g., pipe, channel) connected to a retentate vessel.

[0099] The systems described herein may further include one or more additional components useful for performing a diafiltration process, examples of which are known in the art, including: one or more sampling ports, T-lines (e.g., for in-line buffer addition), pressure sensors, diaphragms for pressure sensors, valve sensors and flow meters that indicate whether valves in the system are open or closed. In certain embodiments, the system includes a sampling port (e.g., a sanitary sampling port) at one or more locations in the system. For example, the sampling port can be included in the retentate line, the permeate / diafiltration buffer line, or at the end of both. Typically, the sampling port is located in a manifold segment in the filtration stage. In one embodiment, the system does not have a diverter plate.

[0100] In some embodiments, one or more components of the system may be disposable.

[0101] In certain embodiments, the system includes a flow meter on the diafiltration buffer line. In certain embodiments, the system includes a flow meter on the filtrate line. In certain embodiments, the system includes a flow meter on the product feed line. In certain embodiments, the system includes a flow meter on the retentate line. In certain embodiments, the system includes automation for controlling the relative flow rates between the product and the diafiltration buffer and / or filtrate.

[0102] Typically, all buffer (or filtrate) pumps are controlled (mechanically or by automation) to operate at the same flow rate. This can be achieved at laboratory scale by using multi-head peristaltic pumps to achieve the same flow rate through each pump while still maintaining the ability to vary the overall flow rate.

[0103] method Disclosed herein is a method of filtering a fluid feed using the single-pass countercurrent diafiltration system described herein.

[0104] In one embodiment, a method relates to diafiltration of a fluid feed, in which the fluid feed and a diafiltration buffer are passed through a single-pass diafiltration system described herein to separate and recover retentate and permeate portions of the fluid feed.

[0105] In one embodiment, the method includes passing the fluid feed through a single-pass countercurrent diafiltration system and collecting the permeate (and diafiltration buffer, if any) and retentate from the system in separate vessels. In certain embodiments, the permeate / buffer and retentate are not recirculated through the system (i.e., the method occurs in a single pass through the system).

[0106] The methods described herein include a step of performing diafiltration (e.g., to remove or reduce the concentration of salts or solvents in the fluid feed or to achieve buffer exchange). In certain embodiments, diafiltration is performed by a process known in the art as discontinuous or batch diafiltration, in which the fluid feed is concentrated to reduce the diafiltration volume and then the feed is returned to its starting volume by adding diafiltration solution. In another embodiment, diafiltration is performed by adding diafiltration solution to the retentate to increase the diafiltration volume and then concentrating the sample back to its original volume. In yet another embodiment, diafiltration is performed by a process known in the art as continuous or constant volume diafiltration, in which diafiltration solution is added to the unfiltered feed at the same rate as permeate is removed from the system. Suitable diafiltration solutions are well known and include, for example, water and various aqueous diafiltration buffer solutions.

[0107] To perform diafiltration, the system can include a reservoir or container for diafiltration solution and one or more conduits for carrying the diafiltration solution from the diafiltration solution container to a final filtration stage.

[0108] To avoid extreme concentration and in-line dilution as part of the diafiltration process, it is preferable to pump the diafiltrate through the filtration assembly to maintain the flow rate of the retentate section at the same flow rate as the initial feed. This requires that the rate of diafiltrate buffer addition be matched to the rate of permeate removal. Because each pump has closely matched pumping speeds, the process is balanced and maintains efficient buffer exchange.

[0109] In certain embodiments, a two-stage system can be used to achieve diafiltration equivalent to at least 8 diavolumes used for batch TFF.

[0110] Methods for manufacturing or producing a biological product of interest known in the art can be used in combination with the systems and methods for filtering a fluid feed material described herein. For example, those skilled in the art know how to manufacture or produce a biological product, such as a recombinant protein, using fermentation. In certain embodiments, the production of the biological product of interest comprises 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 can comprise maintaining the eukaryotic cells in a suitable medium and under conditions that allow growth and / or protein production / expression. The biological product of interest can be produced by fed-batch or continuous cell culture. For example, the eukaryotic cells can be cultured in fed-batch or continuous cell culture, preferably in continuous cell culture.

[0111] In certain embodiments, the eukaryotic host cell is a yeast cell. In one embodiment, the eukaryotic host cell is a mammalian cell. A mammalian cell as used herein is a mammalian cell line suitable for the production of recombinant secreted therapeutic proteins and therefore may also be referred to as a "host cell". In certain embodiments, the mammalian cell is a rodent cell, such as a hamster cell. The mammalian cell is an isolated cell or a 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 a BHK21, BHK TK-, Jurkat cell, 293 cell, HeLa cell, CV-1 cell, 3T3 cell, CHO, CHO-K1, CHO-DXB11 (also called CHO-DUKX or DuxB11), CHO-S cell or CHO-DG44 cell or a derivative / progeny of any of such cell lines. In a particular embodiment, the mammalian cell is a CHO cell, such as CHO-DG44, CHO-K1 and BHK21, more preferably CHO-DG44 and CHO-K1 cells. In a particular embodiment, the mammalian cell is a CHO-DG44 cell. Also included are glutamine synthetase (GS) deficient derivatives of mammalian cells, particularly CHO-DG44 and CHO-K1 cells. In one embodiment, the mammalian cell is a Chinese Hamster Ovary (CHO) cell, such as CHO-DG44 cell, CHO-K1 cell, CHO DXB11 cell, CHO-S cell, CHO GS deficient cell or derivatives thereof.

[0112] In certain embodiments, the host cell may further comprise one or more expression cassettes encoding a heterologous protein, such as a therapeutic protein, e.g., a recombinant secreted therapeutic protein. In certain embodiments, the host cell may also be a murine cell, e.g., a murine myeloma cell, such as NS0 and Sp2 / 0 cells, or any derivative / progeny of such cell lines.

[0113] Expression of the biological product or recombinant protein of interest occurs in cells that contain a DNA sequence encoding the biological product or recombinant protein of interest, which is transcribed and translated into a protein sequence including post-translational modifications to produce the biological product or recombinant protein of interest in cell culture.

[0114] Provided herein is a method for producing a biological product of interest, comprising the steps of: (I) culturing a eukaryotic cell expressing a biological product of interest in cell culture; (II) harvesting a biological product of interest from the cell culture in the form of a fluid feed comprising (a) the biological product of interest, and (b) one or more impurities and / or buffer components; (III) purifying a fluid feed containing the biological product of interest and one or more impurities and / or buffer components to separate the biological product of interest from the fluid feed; and (IV) Optionally, formulating the biological product of interest into a pharma- ceutically acceptable formulation suitable for administration. Including, moreover, (a) passing a fluid feed through a single-pass countercurrent diafiltration system; and (b) collecting the permeate (and diafiltration buffer, if any) and retentate from the system in separate containers. Including, A method is disclosed, wherein the system is a system according to embodiments described herein.

[0115] In certain embodiments, the biological product of interest is a recombinant protein. In certain embodiments, the step of culturing the eukaryotic cell expressing the biological product of interest in the cell culture is carried out in a fed-batch cell culture. In certain embodiments, the step of culturing the eukaryotic cell expressing the biological product of interest in the cell culture is carried out in a continuous cell culture.

[0116] The following examples are offered for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0117] Example 1 The flow rates of multiple buffer (or filtrate) pumps were controlled using a multi-head peristaltic pump to achieve the same flow rate through each pump while maintaining the ability to vary the overall flow rate. 2 A Sartopure® 30 kd Hydrosart® TFF cassette and antibody solutions from Boehringer Ingelheim Fremont Incorporated (BIFI) were used. A single cassette was run over a range of flow rates. Table 1 shows the corresponding flow rates of the feed, retentate, permeate and diafiltration buffer for each experiment.

[0118] [Table 1]

[0119] The feed materials were approximately 6 g / L glucose and 153 g / L monoclonal antibody solution. The glucose concentration of the retentate samples was measured using the GLC3D and GLC3B methods for the Cedex cell counter Bio HT glucose test. Table 2 shows the relationship between the dilution factor of glucose in the antibody solution and the glucose concentration measured by the Bio HT glucose test method.

[0120] [Table 2]

[0121] The data confirm that the test method can accurately measure glucose concentrations equivalent to diafiltration using 9 diavolumes of diafiltration buffer when starting with a glucose concentration of 153 g / L. Figure 2 shows the relationship between the ratio of diafiltration buffer flow rate to product feed flow rate (feed flow rate and retentate flow rate are the same) and the clearance of the tracer from the retentate stream. Figure 2 shows the expected clearance of glucose expressed as the natural logarithmic decrease in glucose concentration versus the number of diavolumes of diafiltration buffer used (df buffer flow rate / feed flow rate). The one-to-one linear relationship shown would be expected if there was uniform introduction of df buffer along the length of the membrane and complete mixing of the df buffer with the retentate. The experimental data shows that when run at the experimental flow rates, the clearance is higher than these expected values ​​when the df buffer flow rate is about 3 times lower than the feed flow rate, and lower than expected values ​​when the ratio of df buffer flow rate:feed flow rate is higher than about 3.5:1 (see Table 1). Based on the data obtained in Figure 2, a system with three stages could be operated with a df buffer flow rate:feed flow rate ratio of 3:1 and utilize countercurrent flow to achieve a natural log clearance of greater than 8. At this flow rate ratio and normalized flow rate ratios similar to those in Table 1, the concentration in the retentate should remain constant throughout the run.

[0122] The experimental data in FIG. 3 show that by using a df buffer flow rate:feed (or retentate) flow rate ratio of less than 4:1 in a two-stage system, a natural log diafiltration (glucose reduction) of greater than 8 can be achieved. The experiment with a df buffer flow rate:retentate flow rate ratio of 3.2:1 was performed by starting the df buffer flow first and then starting the retentate side flow. The experiment with a df buffer flow rate:retentate flow rate ratio of 3.7:1 was performed by starting the retentate side flow first and then starting the df buffer flow. The clearance is lower when the system is started in this manner first than when the system is fully primed with df buffer and the df buffer flow is started first. In both cases, the system eventually reaches the same steady state over time. The final clearance achieved with a df buffer flow rate to retentate flow rate ratio of 3.7 in FIG. 3 represents the minimum clearance compared to the actual antibody production process conditions that would begin operation with a df buffer prime and flow. The data show that diafiltration of greater than 8 diavolumes can be achieved at countercurrent flow rates of less than 4:1 (df buffer / feed) in a two-stage system. For an eight-diavolume diafiltration operation typical in the manufacture of biological products, this would reduce the diafiltration buffer used by 50%. Table 3 compares the study data from the two-stage system with calculations for a conventional TFF system diafiltering the same amount of monoclonal antibody product in the same amount of time. The data demonstrate the improved efficiency of the multi-stage countercurrent system. In particular, the data support an approximately 40- to approximately 80-fold reduction in pump passes for an exemplary single-pass countercurrent diafiltration system.

[0123] [Table 3]

[0124] The experimental data in Figure 4 show that by using a df buffer flow rate:feed (or retentate) flow rate ratio of less than 3:1 in a three-stage system, a natural log of diafiltration (glucose reduction) of greater than 8 can be achieved. For an eight-diavolume diafiltration operation, typical in the manufacture of biological products, this would reduce the diafiltration buffer used by 63%. Table 4 compares the study data from the three-stage system with calculations for a conventional TFF system diafiltering the same amount of monoclonal antibody product in the same amount of time. The data demonstrate the improved efficiency of the multi-stage countercurrent system. In particular, the data support an approximately 40- to approximately 80-fold reduction in pump passes for an exemplary single-pass countercurrent diafiltration system.

[0125] [Table 4]

[0126] In the foregoing specification, various embodiments have been described with reference to examples. However, it will be apparent that various modifications and changes may be made thereto to realize additional embodiments without departing from the broader scope of the exemplary embodiments as set forth in the following claims. Accordingly, the specification and drawings are to be regarded in an illustrative sense, rather than a restrictive sense.

Claims

1. 1. A system for single-pass countercurrent diafiltration of a fluid feed, comprising: two or more filtration stages; conduits for facilitating fluid communication between manifolds of the filtration stages; conduits and at least one feed pump for providing the fluid feed to a first filtration stage; conduits and at least one buffer pump for providing diafiltration buffer to a final filtration stage; conduits and at least one buffer pump per filtration stage for providing permeate and diafiltration buffer from a subsequent filtration stage to a previous filtration stage; and conduits and filtrate pumps for removing permeate (and diafiltration buffer, if any) from the system, Each filtration stage is (i) a manifold segment, and (ii) one or more tangential flow filtration (TFF) cassettes; the manifold segment including a first manifold (i.e., inlet) for receiving and conveying the fluid feed or retentate to the filtration stage, a second manifold (i.e., outlet) for receiving and conveying retentate through the filtration stage, a third manifold (i.e., inlet) for receiving and conveying permeate and diafiltration buffer through the filtration stage, and a fourth manifold (i.e., outlet) for receiving and conveying permeate and diafiltration buffer through the filtration stage; the manifold segment is fluidly connected to the one or more TFF cassettes; by connecting the second manifold in the manifold segment to the first manifold in the manifold segment in the subsequent filtration stage so that the retentate of one stage serves as a feed for the next filtration stage, and by connecting the fourth manifold in the manifold segment to the third manifold in the manifold segment in the previous filtration stage, the filtration stages are fluidly connected through the manifold segments to provide flow paths between the filtration stages; the first filtration stage in the system receives the fluid feed from a conduit connected to a feed pump; the second manifold of the final filtration stage is a retentate outlet not connected to a previous filtration stage that allows the retentate to exit the system; 11. The TFF cassette of claim 10, wherein the TFF cassette comprises a diafiltration channel, a flat first filtration membrane, a retentate channel, a flat second filtration membrane, and a permeate / diafiltration buffer collection channel, wherein (i) the flat first filtration membrane separates the diafiltration channel and the retentate channel from one another, (ii) the flat second filtration membrane separates the retentate channel and the permeate / diafiltration buffer collection channel from one another, (iii) the diafiltration channel is fluidly connected to at least one inlet for diafiltration medium, (iv) the retentate channel is fluidly connected to at least one inlet for a feed fluid and at least one outlet for the retentate, and (v) the permeate / diafiltration buffer collection channel is fluidly connected to at least one outlet for the permeate / diafiltration buffer.

2. 2. The system of claim 1, wherein the system has exactly two filtration stages.

3. 10. The system of claim 1 comprising three filtration stages.

4. The system according to any one of claims 1 to 3, wherein the first filtration membrane has a molecular weight cut-off in the range of 30 kD to 1,500 kD.

5. The system according to any one of claims 1 to 4, wherein the second filtration membrane has a molecular weight cut-off in the range of 5 kD to 1,500 kD.

6. 10. A method for filtering a fluid feed comprising the steps of: (i) passing the fluid feed through a single-pass countercurrent diafiltration system according to any one of claims 1 to 5; and (ii) collecting permeate and retentate from the system in separate vessels.

7. 7. The method of claim 6, which can be used to achieve diafiltration equivalent to at least 8 diavolumes.

8. 8. A method according to claim 6 or 7, in which there are precisely two filtration stages in the system.

9. 8. The method of claim 6 or 7, wherein the system comprises three filtration stages.

10. The method of any one of claims 6 to 9, wherein the fluid supply comprises a biological product and one or more impurities or buffer components.

11. The method according to any one of claims 6 to 10, which is carried out at or near the end of a purification process of a biological product.

12. 12. The method of claim 10 or 11, wherein the fluid supply is obtained from a source of the biological product.

13. The method of any one of claims 10 to 12, wherein the biological product in the fluid supply is a monoclonal antibody present in an elution buffer from a chromatography step.

14. 1. A method for producing a biological product of interest, comprising: (I) culturing a eukaryotic cell expressing a biological product of interest in a cell culture; (II) harvesting the biological product of interest from the cell culture in the form of a fluid feed comprising: (a) the biological product of interest; and (b) one or more impurities and / or buffer components; (III) purifying the fluid feed containing the biological product of interest and the one or more impurities and / or buffer components to separate the biological product of interest from the fluid feed; and (IV) formulating the biological product of interest into a pharma- ceutically acceptable formulation suitable for administration. Including, moreover, (a) passing the fluid feed through a single-pass countercurrent diafiltration system; and (b) collecting the permeate (and diafiltration buffer, if any) and retentate from the system in separate containers. Including, the single-pass countercurrent diafiltration system includes two or more filtration stages; conduits for facilitating fluid communication between manifolds of the filtration stages; conduits and at least one feed pump for providing the fluid feed to a first filtration stage; conduits and at least one buffer pump for providing diafiltration buffer to a final filtration stage; conduits and at least one buffer pump per filtration stage for providing permeate and diafiltration buffer from a subsequent filtration stage to the previous filtration stage; and conduits and filtrate pumps for removing permeate (and diafiltration buffer, if any) from the system, Each filtration stage is (i) a manifold segment, and (ii) one or more tangential flow filtration (TFF) cassettes; the manifold segment including a first manifold (i.e., inlet) for receiving and conveying the fluid feed or retentate to the filtration stage, a second manifold (i.e., outlet) for receiving and conveying retentate from the filtration stage, a third manifold (i.e., inlet) for receiving and conveying permeate and diafiltration buffer through the filtration stage, and a fourth manifold (i.e., outlet) for receiving and conveying permeate and diafiltration buffer from the filtration stage; the manifold segment is fluidly connected to the one or more TFF cassettes; by connecting the second manifold in the manifold segment to the first manifold in the manifold segment in the subsequent filtration stage so that the retentate of one stage serves as a feed for the next filtration stage, and by connecting the fourth manifold in the manifold segment to the third manifold in the manifold segment in the previous filtration stage, the filtration stages are fluidly connected through the manifold segments to provide flow paths between the filtration stages; the first filtration stage in the system receives the fluid feed from a conduit connected to a feed pump; the second manifold of the final filtration stage is a retentate outlet not connected to a previous filtration stage that allows the retentate to exit the system; the TFF cassette comprises a diafiltration channel, a flat first filtration membrane, a retentate channel, a flat second filtration membrane, and a permeate / diafiltration buffer collection channel, wherein (i) the flat first filtration membrane separates the diafiltration channel and the retentate channel from one another, (ii) the flat second filtration membrane separates the retentate channel and the permeate / diafiltration buffer collection channel from one another, (iii) the diafiltration channel is fluidly connected to at least one inlet for diafiltration medium, (iv) the retentate channel is fluidly connected to at least one inlet for a feed fluid and at least one outlet for the retentate, and (v) the permeate / diafiltration buffer collection channel is fluidly connected to at least one outlet for the permeate / diafiltration buffer.

15. A method for producing a biological product of interest, comprising: (I) culturing a eukaryotic cell expressing a biological product of interest in a cell culture; (II) harvesting the biological product of interest from the cell culture in the form of a fluid feed comprising: (a) the biological product of interest; and (b) one or more impurities and / or buffer components; (III) purifying the fluid feed containing the biological product of interest and the one or more impurities and / or buffer components to separate the biological product of interest from the fluid feed; Including, moreover, (a) passing the fluid feed through a single-pass countercurrent diafiltration system; and (b) collecting the permeate (and diafiltration buffer, if any) and retentate from the system in separate containers. Including, the single-pass countercurrent diafiltration system includes two or more filtration stages; conduits for facilitating fluid communication between manifolds of the filtration stages; conduits and at least one feed pump for providing the fluid feed to a first filtration stage; conduits and at least one buffer pump for providing diafiltration buffer to a final filtration stage; conduits and at least one buffer pump per filtration stage for providing permeate and diafiltration buffer from a subsequent filtration stage to the previous filtration stage; and conduits and filtrate pumps for removing permeate (and diafiltration buffer, if any) from the system, Each filtration stage is (i) a manifold segment, and (ii) one or more tangential flow filtration (TFF) cassettes; the manifold segment including a first manifold (i.e., inlet) for receiving and conveying the fluid feed or retentate to the filtration stage, a second manifold (i.e., outlet) for receiving and conveying retentate from the filtration stage, a third manifold (i.e., inlet) for receiving and conveying permeate and diafiltration buffer through the filtration stage, and a fourth manifold (i.e., outlet) for receiving and conveying permeate and diafiltration buffer from the filtration stage; the manifold segment is fluidly connected to the one or more TFF cassettes; by connecting the second manifold in the manifold segment to the first manifold in the manifold segment in the subsequent filtration stage so that the retentate of one stage serves as a feed for the next filtration stage, and by connecting the fourth manifold in the manifold segment to the third manifold in the manifold segment in the previous filtration stage, the filtration stages are fluidly connected through the manifold segments to provide flow paths between the filtration stages; the first filtration stage in the system receives the fluid feed from a conduit connected to a feed pump; the second manifold of the final filtration stage is a retentate outlet not connected to a previous filtration stage that allows the retentate to exit the system; the TFF cassette comprises a diafiltration channel, a flat first filtration membrane, a retentate channel, a flat second filtration membrane, and a permeate / diafiltration buffer collection channel, wherein (i) the flat first filtration membrane separates the diafiltration channel and the retentate channel from one another, (ii) the flat second filtration membrane separates the retentate channel and the permeate / diafiltration buffer collection channel from one another, (iii) the diafiltration channel is fluidly connected to at least one inlet for diafiltration medium, (iv) the retentate channel is fluidly connected to at least one inlet for a feed fluid and at least one outlet for the retentate, and (v) the permeate / diafiltration buffer collection channel is fluidly connected to at least one outlet for the permeate / diafiltration buffer.

16. 16. The method of claim 14 or 15, wherein the biological product of interest is a recombinant protein.

17. The method according to any one of claims 14 to 16, wherein the step of culturing eukaryotic cells expressing the biological product of interest in a cell culture is carried out in a fed-batch cell culture.

18. The method according to any one of claims 14 to 16, wherein the step of culturing eukaryotic cells expressing the biological product of interest in cell culture is carried out in continuous cell culture.

19. A method according to any one of claims 14 to 18, wherein steps (a) and (b) are used to achieve diafiltration equivalent to at least 8 diavolumes.

20. A method according to any one of claims 14 to 19, wherein the system has precisely two filtration stages.

21. The method of any one of claims 14 to 19, wherein the system comprises three filtration stages.

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