Headspace for Depth Filter and Method of Using the Same

Abstract

A depth filter with optimized headspace, a method for optimizing the headspace of a depth filter, and a method of filtration using a depth filter with optimized headspace are provided. The method includes clarification of a feedstream, the pretreatment of which can include either lowering the pH of the cell culture, adding a polymer (uncharged or charged), or adding salt to precipitate solubilized impurities that result in high insoluble biomass.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 825,505, filed Mar. 28, 2019, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The embodiments disclosed herein relate to depth filters, particularly depth filters having an optimized headspace, particularly encapsulated or sealed depth filters utilized in applications using high solids feeds, most commonly associated with clarified and virus inactivated products containing precipitate. Embodiments also relate to methods of optimizing depth filters and methods of filtering a sample having an optimized depth filter.

Background Art

[0003] Depth filters are ideal for a wide variety of primary and secondary clarification applications, including cell cultures, yeast and bacteria, such as E. coli lysates after centrifugation, E. coli refolding, media, vaccines, plasma proteins, aggregation by low pH, salt aggregation, polymer aggregation, and serum. Depth filters perform filtration by utilizing their depth or thickness. The filter media is typically a material constructed with a gradient density, generally having larger pores near the top and smaller pores at the bottom. Depth filters, unlike absolute filters, retain particles throughout the porous media, allowing both larger and smaller particles than the pore size to be retained. In particle retention in depth filtration, various particle retention methods are recognized, including size exclusion, hydrophobic adsorption, ion adsorption, and other interactions. These various retention methods allow the retention of both larger and smaller particles than a defined pore size.

[0004] Often, depth filters can be operated continuously such that most of the coarser particles are removed during the first filtration stage and finer particles are filtered out at the second stage. Thus, in cell cultures with a wide range of particle sizes from cells and cell debris etc., depth filters are intended to retain most of the suspended particles.

[0005] The three-dimensional matrix of the depth filter creates a tortuous path through which the sample passes. In various embodiments, the filter membrane or sheet can be made of wound cotton, polypropylene, rayon cellulose, glass fiber, sintered metal, ceramic, diatomaceous earth, or other known components such as silica, polyacrylic fiber (HC Pro media, manufactured by EMD Millipore Corporation, MA, USA). The composition containing the depth filter media can be chemically treated so that the filter media can capture charged particles such as DNA, host cell proteins, or aggregates by ionic or electrostatic interactions.

[0006] In conventional depth filtration, the limiting factor that suppresses the packing capacity of the depth filter is fouling of the depth filter media. Thus, it was not known that the headspace volume or physical fouling capture capacity (also known as cake filtration) within the depth filtration device plays an important role. The fouling mechanism of the depth filter may include pore blockage, cake formation and / or pore constriction. Recent improvements in cell culture have led to high biomass with increased cell viability. These feed streams can result in a decrease in the level of cell debris or colloidal substances, which generally leads to fouling of the depth filtration media.

[0007] Conventionally, encapsulated depth filters have been designed to minimize headspace in order to reduce the system's hold-up volume and overall device dimensions, improve recovery, and limit the flush volume (buffer and water). Therefore, the focus when improving the performance of depth filters has been on reducing the hold-up volume and the device footprint, which minimizes the device dimensions and consequently indirectly reduces the available headspace. However, the inventors have found that reducing the headspace can negatively impact the performance (loading capacity) of depth filters, especially when the filter is used for clarifying feeds with high solids content. SUMMARY OF THE INVENTION PROBLEM TO BE SOLVED BY THE INVENTION

[0008] Accordingly, an object of the embodiments disclosed herein is to optimize the headspace of depth filters to improve performance. MEANS FOR SOLVING THE PROBLEM

[0009] The problems of the prior art are addressed by the embodiments disclosed herein, which relate to depth filters having an optimized headspace, a method for optimizing the headspace of depth filters, and a method of filtration using a depth filter having an optimized headspace.

[0010] The inventors have unexpectedly discovered that, for example, for the design of a closed or encapsulated depth filter used for the removal of cells, cell debris, aggregated cell cultures or precipitates, the headspace volume available within the depth filter is an important variable. Thus, the headspace volume of the filter or the contaminant capture capacity (cake filtration capacity) is an important design variable for the depth filter encapsulation device. The influence of this variable is particularly pronounced for feed streams having a high solids (cell / biomass) concentration and for low fouling feed streams where a significantly large amount of feed can be processed without fouling of the filter media. For suitable applications where the depth filter capacity can be controlled by the headspace, the pretreatment includes either a decrease in the cell culture pH, the addition of a polymer (uncharged or charged), or the addition of a salt to precipitate the solubilized impurities that result in a highly insoluble biomass, including clarification of the aggregated feed stream. The variable is also important for scalability assessment.

[0011] In some embodiments, the optimized headspace is for a primary clarification device for cell culture clarification using a product containing 25 million to 100 million cells per mL, corresponding to approximately 3.5 - 11.5% biomass.

[0012] Accordingly, in some embodiments, there is provided a depth filter having an enclosed housing with an inlet, an outlet spaced from the inlet, and an internal volume containing a filter medium, and a headspace within the internal volume, the volume of the headspace being 4 - 14 liters per square meter of filter medium area, preferably 4 - 10 liters per square meter of filter medium area. In some embodiments, the filter medium comprises a composite of a graded layer of non-woven fibers, cellulose, and diatomaceous earth, or polyacrylic fibers and silica (synthetic), or activated carbon, polypropylene, nylon, glass fibers, and combinations thereof. In some embodiments, the minimum depth filter medium area of the depth filtration device is 0.1 m 2is larger. In some embodiments, the depth filtration media area is 0.1 m 2 ~1.1 m 2 .

[0013] In some embodiments, a process is provided for clarifying a feed containing a target biomolecule of interest, as well as a plurality of cell debris and / or colloidal particles, by depth filtration. In some embodiments according to the present disclosure, the process comprises providing a depth filtration device having a headspace with a volume of 4 to 14 liters per square meter of depth filter media area within the device, and a porous depth filter media; providing a feed containing a target biomolecule of interest, as well as a plurality of cell debris and / or colloidal particles; introducing the feed into the headspace and contacting the depth filter media with the feed; and separating the target biomolecule of interest from the cell debris and colloidal particles in the feed. In some embodiments, the process is performed without using a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step. In some embodiments, the target biomolecule of interest includes one or more of monoclonal antibodies (mAbs), enzymes, viruses, conjugated polysaccharides, biological agents, polyclonal antibodies, and other bimolecular cell substances expressed in mammalian cell cultures, plant cell cultures, bacterial cell cultures, insect cell cultures, and similar cultures of interest.

[0014] In some embodiments, a method is provided for determining the optimal area of a depth filter required to filter a quantity of cell culture, the cell culture including a plurality of cells having a cell diameter (d) and having a total cell density, the method comprising the following. 1. Calculating the volume (V c ) occupied by a single cell. 2. Multiplying V c by the total cell density to determine the cell volume per volume of cell culture (cells L / cell culture L). 3. Determining the headspace per unit area of the filter (headspace L / depth filter area m 2 ). 4. Divide the filter headspace per depth filter area by the cell volume per volume of the cell culture to determine the filter capacity (cell culture L / depth filter area m 2 ).

[0015] In some embodiments, the void volume occupied by the cells is taken into account when determining the volume occupied by the cells.

[0016] Optimization of the headspace of depth filters according to embodiments disclosed herein promotes the utilization of the total capacity of the depth filter media and can result in a 70% improvement with respect to non-fouling media compared to conventional designs. Since the area required is reduced, there are process economic improvements such as a reduction in the required amount of buffer and an increase in the product recovery rate.

[0017] These and other non-limiting aspects and / or features of the present disclosure are described in more detail below. To better understand the embodiments disclosed herein, reference is made to the accompanying drawings and description which form a part of the present disclosure.

[0018] The embodiments disclosed herein can take the form of various components and arrangements of components, as well as various process operations and arrangements of process operations. The drawings illustrate embodiments and should not be construed as limiting. The present disclosure includes the following drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

Figure 1A

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Figure 7D

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Figure 8B

Figure 8C

Mode for Carrying Out the Invention

[0020] The components, processes, and devices disclosed in this specification can be more fully understood by referring to the accompanying drawings. The drawings are merely schematic diagrams based on the convenience and ease of explanation of the present disclosure, and thus are not intended to show the relative sizes and dimensions of the device or its components and / or to define or limit the scope of the exemplary embodiments.

[0021] In the following description, specific terms are used for the purpose of clarity, but these terms are intended to refer only to the specific structures of the embodiments selected for the description in the drawings and are not intended to define or limit the scope of the present disclosure. It should be understood that in the drawings and the following description, like numbers refer to components with like functions.

[0022] For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers representing amounts of ingredients, ratios or proportions of materials, reaction conditions, and other numerical values used in this specification and the claims shall be understood to be modified in all instances by the term "about" or "approximately", whether or not explicitly indicated. The terms "about" or "approximately" generally refer to a range of numbers that are considered to be equivalent to the recited value (i.e., having the same effect or result). In many cases, these terms can include numbers that are rounded to the nearest significant digit.

[0023] Accordingly, unless the contrary is shown, the numerical parameters set forth in this specification and the claims are approximations that may vary depending on the desired properties required to be obtained by the embodiments disclosed herein. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be construed in light of the reported number of significant digits and by applying ordinary rounding techniques.

[0024] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0025] As used herein, various devices and components may be described as "comprising" other components. The terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and their variants are intended, as used herein, to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional components.

[0026] All ranges disclosed herein include the recited endpoints and can be combined independently (e.g., the range of "2 inches to 10 inches" includes the endpoints 2 inches and 10 inches, and all intermediate values).

[0027] As used herein, words of approximation can be applied to modify any quantitative expression that can vary without resulting in a change in the relevant basic function. Accordingly, values modified by terms such as "about" and "substantially" may or may not be limited to the specified exact value in some cases. The modifier "about" should also be considered as disclosing a range defined by the absolute values of two endpoints. For example, the expression "about 2 to about 4" also discloses the range of interpolated values of "2 to 4".

[0028] Note that many of the terms used herein are relative terms. For example, the terms "upper" and "lower" are relative to each other, i.e., the upper component is located at a higher altitude than the lower component, and should not be construed as requiring a particular orientation or position of the structure. As a further example, the terms "inner", "outer", "inward", and "outward" are relative to a center and should not be construed as requiring a particular orientation or position of the structure.

[0029] The terms "upper" and "bottom" are with respect to an absolute reference, i.e., the earth's surface. In other words, the upper position is always at a higher altitude than the bottom position with respect to the earth's surface.

[0030] The terms "horizontal" and "vertical" are used to indicate directions with respect to an absolute reference, i.e., the ground. However, these terms should not be interpreted as requiring that the structures be perfectly parallel or perfectly perpendicular to each other.

[0031] The term "gram" can be abbreviated as gm, the term "liter" can be abbreviated as L, the term "milliliter" can be abbreviated as mL, and the term "cubic centimeter" can be abbreviated as cm 2 and can be abbreviated as cm, the term "liter" can be abbreviated as L, and the term "liter per square meter" can be abbreviated as L / m 2 and can be abbreviated as L / m, and the term "liter per square meter per hour" can be abbreviated as L / m 2 / H or LMH.

[0032] As used herein, the term "depth filter" (e.g., a gradient density depth filter) achieves filtration within the depth of the filter material. A common class of such filters comprises a random matrix of bundled (otherwise fixed) fibers that forms a maze of complex and serpentine flow paths. Particle separation in these filters generally results from capture by or adsorption to the fiber matrix. The fiber-based filter material may be in the form of a mat or pad. The depth filter media most frequently used for the bioprocessing of cell culture broths and other feedstocks consists of cellulose fibers, filter aids such as diatomaceous earth (DE), and a resin binder. Depth filter media, unlike absolute filters, retain particles throughout the porous media and are capable of retaining both particles larger and smaller than the pore size. Particle retention is thought to include both size exclusion and adsorption by hydrophobic, ionic, and other interactions. The fouling mechanism may include pore blocking, cake formation, and / or pore constriction. Depth filters are advantageous in that they remove contaminants and eliminate validation issues by being disposable (i.e., single-use)

[0033] The primary clarification depth filter can remove whole cells and cell debris and thus achieve primary clarification of a feed containing the target biomolecule of interest as well as multiple cell debris and colloidal particles without using a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step.

[0034] Suitable cellulose depth filters, such as the MILLISTAK(R)+ filter commercially available from EMD Millipore Corporation, are composite filters that include layers of densely structured cellulose depth media and can be optimized for specific applications, such as retention of colloidal particles and cell debris, or retention of whole cells and larger debris. They combine two or more successive grades of media within a single filter cartridge or housing. These filters are most commonly used in polishing or secondary clarification processes to remove small amounts of suspended material from aqueous product (protein) streams. One function of these filters is to protect or extend the service life of more expensive downstream separation processes, such as sterile filtration and affinity chromatography. That is, the general use of these filters is as "prefilters" to protect the downstream process capacity (the amount of fluid that can pass through the filter before it clogs) from colloidal contaminants and other cell debris, thereby significantly extending the life of the downstream process. Additionally, such depth filters are also used to protect virus clearance filters by removing trace amounts of aggregated protein.

[0035] In conventional depth filtration applications, fouling of the depth filter media affects how much cell culture product can be processed / filtered by a "closed" or "encapsulated" depth filter of a given area. This amount is referred to as the depth filter loading capacity and is typically expressed in liters per square meter of depth filter bed area. 2) It is displayed as. Due to the recent increase in cell density, the solid content / cell population / biomass filling in the cell culture medium has increased. Furthermore, through the optimization of depth filter media designs including, but not limited to, novel depth media compositions and gradient layered depth filters, the amount of cell culture fluid that can be processed using these depth filter media has increased significantly. These two factors, either independently or in combination with sealed or encapsulated depth filter designs, can result in complete occupancy of the available upstream headspace by cells / biomass, limiting the overall capacity and not fully utilizing the depth filter media itself. Additionally, differences in headspace between different scale devices can pose scalability challenges. Lack of headspace can also result in media compression, which can be detrimental to the performance of the depth filter.

[0036] In the embodiments disclosed herein, any depth filtration system available to those skilled in the art may be used. In certain embodiments, the depth filter may be the MILLISTAK+(R) Pod Filter System available from EMD Millipore Corporation, which is a scalable, disposable or single-use format, for example, a modular design scalable to 5 to 12,000 liters or more. These depth filters incorporate multiple stages of density layers and adsorption filter media. For example, MILLISTAK+(R) DE is composed of selected grade cellulose fibers and diatomaceous earth. The MILLISTAK+(R) CE series is a single layer media containing cellulose fibers suitable for rough filtration applications (e.g., 1 to 30 microns). The MILLISTAK+(R) HC series specializes in improving productivity by combining two different technologies to improve filter capacity and retention rate. The media of some embodiments according to the present disclosure can be charged or uncharged. Also suitable is the CLARISOLVE(R) depth filter, commercially available from EMD Millipore and useful as a clarification tool having a gradient density structure specially designed to match the particle size distribution of the pretreated feed stream.

[0037] Commercially available depth filtration devices are available for clarification applications. As noted above, these include the Millistak+(R) HC, Millistak+(R) HC Pro, and Clarisolve(R) depth filters from EMD Millipore Corporation. These depth filtration devices are available in multiple device sizes depending on the intended application and the amount of process fluid to be filtered. For small amounts (<3L), a 23 cm 2 micro-pod device is used. For medium amounts (3 to 10L), laboratory-scale devices are available in device sizes of 135 cm 2 , 270 cm 2 , and 540 cm 2 . For pilot-scale and production-scale amounts (10 to 2000L), process-scale devices are 0.11 m2 、 0.33 m 2 、 0.55 m 2 、 0.77 m 2 、 and 1.1 m 2 are available in the device sizes of.

[0038] In some embodiments, the depth filter can be used in a method for clarifying a feed. In certain embodiments, depth filtration for clarification of a feed, feed stream, feedstock, cell culture broth, etc. is provided, which utilizes a depth filtration device with an optimized headspace without optionally using a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step. In some embodiments, primary clarification depth filtration of a chemically treated feed in which cell populations have aggregated into larger aggregates is provided. In certain embodiments, clarification of such feeds is performed upstream of one or more chromatographic operations.

[0039] In some embodiments, the depth filter media is housed within a housing (e.g., an enclosed housing where the housing is part of a filter module) having a fluid inlet and a fluid outlet spaced from the fluid inlet. As used herein, a filtration module is a self - contained assembly that includes depth media enclosed within a storage container, such as a plastic storage container, and has flow paths designed to perform filtration. The module can be used independently or with a holder. In some embodiments, the filter module is designed to be directly attached to piping, etc. and used for laboratory - scale filtration, such as in a pod. Larger modules can be assembled within a holder. The housing and any suitable seal ensure that the fluid being filtered passes through the depth filter media before exiting the housing through the outlet. In some embodiments, an integral filtration device is formed where all of the fluid being filtered must pass through the depth filter media from the inlet to the outlet. The inlet can be located on the outer surface of the housing or within a portion located at the center of the housing, and the outlet is located away from the inlet to provide a fluid path as described above. In some embodiments, the housing may be a molded plastic pressure vessel. In some embodiments, the housing may be a cartridge as known to those skilled in the art.

[0040] Figure 1A is a perspective view of a depth filter 10 according to some embodiments. Referring now to FIGS. 1A and 1B, a depth filter 10 according to some embodiments is shown. The depth filter 10 includes a suitable housing 12 having an inlet 13 and an outlet 14 spaced from the inlet 13. In some embodiments, the inlet 13 is formed by a cylindrical body 13A that extends axially from the upper surface of the disk-shaped body member 12A and terminates at an opening at its free end. The outlet 14 is formed by a cylindrical body 14A that extends axially from the bottom surface of the disk-shaped body member 12A and terminates at an opening at its free end. The housing 12 can be formed of any suitable material that is not harmful to the process, such as a polyolefin, e.g., polyethylene, polypropylene, and blends including polyethylene and / or polypropylene. The housing 12 may be a cartridge. Those skilled in the art will understand that the shape or configuration of the housing 12 shown in FIGS. 1A and 1B is merely exemplary, and other shapes or configurations, including pods, are within the scope of the embodiments disclosed herein. The depth filter may be a single-use device.

[0041] Figure 1B is a cross-sectional view of the depth filter 10 of FIG. 1A. FIG. 1B shows the internal volume of the housing 12 including a headspace 15, a depth filter matrix 16, and a downstream hold-up volume 17 in the direction of flow (indicated by arrows 19A, 19B) during filtration from the inlet 13 to the outlet 14, with the headspace 15 being upstream of the depth filter matrix 16 and the downstream hold-up volume 17.

[0042] Figure 2 is a front cross-sectional view of an alternative depth filter, e.g., a lens-shaped encapsulated depth filter according to some embodiments of the present disclosure. Figure 2 shows a depth filter with an encapsulated lens-shaped design, and the stacked disk filter is encapsulated within housing 12'. The internal volume of housing 12' includes a headspace 15', a depth filter matrix 16', and a hold-up volume 17'. The position of the inlet 13' in the case of an encapsulated device may be a concentric ring around the outlet 14', but other designs are possible and are within the scope of the embodiments disclosed herein. In some embodiments, the feed enters through inlet 13', is filtered by the depth filter media 16', and then the filtered fluid flows into the hold-up volume region 17'. As can be seen, regardless of the design / arrangement of the depth filter media 16, 16' and the hold-up volume 17, 17', the feed first crosses the headspace 15, 15'.

[0043] In some embodiments, the headspace can be measured based on the physical dimensions of the filter or by using an experimental approach. The physical dimensions of the filter can be measured using devices such as Vernier calipers, rulers, or, in the case of more complex filter shapes such as encapsulated lens-shaped depth filters and MILLISTAK(R) Pod filters, can be derived from the computer-aided design (CAD) drawings of the filter generated by software such as Solidworks (Dassault Systemes), Pro / Engineering, Creo (PTC).

[0044] For example, the calculation of the headspace of the depth filter shown in Figure 1A is as follows.

[0045] Headspace volume of the device =

Number

[0046] Headspace per unit area =

Number

[0047] In some embodiments, the experimental approach for quantifying the headspace can be carried out using a slurry of particles of known concentration. The particles used in this operation have a particle size such that they form a semi-porous cake on the upper surface of the depth filter without fouling the depth media. Suitable latex or polystyrene microspheres or bentonite of appropriate particle size are examples of particles that can be used. The appropriate particle size may be in the range of 15 to 100 microns. The lower end of the range depends on the grade / porosity of the filter.

[0048] A preferred example of experimentally quantifying the headspace is as follows. Two enclosed depth filter devices of known area (each "A" m 2 ) are pre-weighed, and their weights are W1 and W2 respectively. The depth media should be equivalent in each device and have a similar flow rate distribution. An aqueous slurry containing particles of appropriate size is filtered through both enclosed depth filter devices until a final pressure of 25 (pounds per square inch) psi is reached. To ensure a uniform distribution of solids, the particle concentration must be low (less than 5%), and a filtration flux of about 150 liters / m 2 ·hour must be maintained. The second device may be a scaled-down version of the first device that facilitates disassembly for recovering the slurry from the headspace.

[0049] In the first device, an air blowdown is performed to remove all the water that has entered the filter media and the downstream hold-up. After the air blowdown, the device is dried in an environment of controlled temperature and humidity. When a stable weight is achieved, the final weight of the device is measured (W3).

[0050] The second device is disassembled and slurry is recovered from the headspace. The weight of the wet slurry is measured (W4), dried in a controlled temperature and humidity environment, and its final weight is measured (W5). The headspace can be calculated using Equations 2 and 3.

[0051] Empty weight of Device 1 = W1 Empty weight of Device 2 = W2 Final weight of Device 1 after air blowdown and drying = W3 Wet weight of slurry = W4 Dry weight of slurry = W5 Weight of dried particulate matter from Device 1 = W3 - W1 Slight water content of slurry = ((W4 - W5) / W5) (weight of water / weight of dry slurry) Equation 2: Total headspace H of the device = (W3 - W1) * (1 + (W4 - W5) / W5) Equation 3: Headspace per unit area = H / A = ((W3 - W1) * (1 + ((W4 - W5) / W5)))) / A Table 1 shows a numerical example of the experimental method for determining the headspace volume.

[0052]

Table 1

[0053] Clarification uses generally involve a high content or supply flow of a cell population or biomass that limits or does not cause fouling of the depth filter media at all. In these cases, during filtration, the headspace may ultimately be exhausted. As a result, the headspace is a factor that limits the amount of processable feed rather than typical media fouling. Thus, by optimizing the headspace according to the embodiments disclosed herein, an apparatus can be designed that does not exhibit capacity limitations due to the headspace and, as a result, maximally utilizes the depth media. This is also useful when designing scaled-down devices because an inconsistent headspace-to-area ratio can result in scalability defects in the device.

[0054] In some embodiments, the optimal headspace for a primary clarification device can be defined using the following procedure.

[0055] Quantify the cell population content in the feed stream as a function of cell density.

[0056] The cells are assumed to be rigid spheres with an average diameter of 13 microns. The volume of each cell is as follows.

[0057] Volume of cell = V =

Number

[0058] This results in a correlation between the total cell density and the volume of the cell population per unit volume of the feed. Different cell lines can have different diameters. Thus, Cell volume V cells = V * TCD = 1.15 * 10 -9 ml / cell * 12 * 10 6 cells / ml of cell culture = 0.0138 ml of cells / ml of cell culture Therefore, the biomass % is V cells * 100 = 1.38% volume of cells / volume of cell culture as calculated.

[0059] Furthermore, if the cell population cannot be assumed to be spherical, or if there can be a significantly wide particle distribution with various sphericities, the biomass / cell population can be experimentally quantified by centrifuging a known volume of feed material and calculating the volume of the sedimented biomass (separated as a pellet at the bottom) relative to the total volume of the feed (e.g., sediment, aggregate, etc.). Figure 3 plots the correlation between the total cell density and the biomass / cell population volume percent in the feed stream.

[0060] Assume that the entire cell population is retained in the headspace of the filter device (if one or more top layers of the filter media have a very high porosity and an open structure for accommodating the biomass, the void volume of one or more top layers can also be quantified under the available headspace). When particles of a defined shape are randomly packed into a limited space, there is a void volume associated with that packing. For a randomly injected packing having a spherical shape, a void volume fraction of 0.375 was assumed. The appropriate void volume fraction ф for this assumption includes 0.5 to 0.38. Therefore, the volume occupied by the cells is V cells * (1 + φ) = 0.0138 * (1 + 0.375) = 0.0189 ml / ml of cell culture The headspace volume of the depth filter is X, and is expressed in units of headspace L / depth filter area m 2 of.

[0061] In this example, the headspace volume (X) is headspace 2 L / depth filter area m 2 is assumed to be.

[0062] The volume of the cell culture that can be processed through the filter, the filter capacity, is cell culture L / depth filter area m 2 is expressed in units of.

[0063] This value may be calculated as follows. That is, (filter capacity) = X / V cells * (1 + ф) = 2 / 0.0189 = 105.8 L of cell culture / depth filter area m 2 .

[0064] Similarly, when the volume of the headspace is 4 L headspace / depth filter area m 2 is, the volume of the cell culture that can be processed = X / V cells * (1 + φ) = 4 / 0.0189 = 211.8 L cell culture / depth filter area m 2 .

[0065] Similarly, when the volume of the headspace is 12 L headspace / depth filter area m 2 is, the volume of the cell culture that can be processed = X / V cells * (1 + φ) = 12 / 0.0189 = 634.9 L cell culture / depth filter area m 2 .

[0066] Next, identify the area required to filter a 2000 L batch.

[0067] A = V batch / (X / V cells ) = 2000 / 105.8 = 18.9 m 2 (headspace 2 L / m 2 ) = 2000 / 211.8 = 9.44 m 2 (headspace 4 L / m 2 ) = 2000 / 634 = 3.15 m 2(Headspace 12 L / m 2 ) Therefore, the reduction rate of the depth filter area with the increase of the headspace is as follows.

[0068] (A 2l / m2 -A 2l / m2 )*100 / A 2l / m2 =(18.9 - 18.9)*100 / 18.9 = 0% (A 2l / m2 -A 4l / m2 )*100 / A 2l / m2 =(18.9 - 9.44)*100 / 18.9 = 50% (A 2l / m2 -A 12l / m2 )*100 / A 2l / m2 =(18.9 - 3.15)*100 / 18.9 = 83%

[0069] The maximum filling capacity of the feed is plotted as a function of the headspace and the total cell density (or biomass) and is shown in Figure 4. The maximum filling capacity shown assumes that the filter media exhibits limited fouling or no fouling and that the capacity is restricted by the available headspace. These filling capacities were used in the calculation of the area required to process 2000 L. The batch volume is arbitrarily selected. The results are obtained by dividing the batch volume by each filling capacity and are plotted in Figure 5.

[0070] 2 L / m 2 The reduction in the depth filter media area to process a 2000 L batch that can be achieved by increasing the headspace for a headspace of 2 L / m was calculated. Since the percentage is independent of the total cell density, only one curve is shown. This area reduction rate is shown in Figure 6.

[0071] The results show that the optimal headspace volume is from approximately 4 liters of headspace per square meter of depth filter area (4 L / m 2 ) to approximately 14 liters of headspace per square meter of depth filter area (14 L / m2 ) is preferably about 4 L / m 2 to about 10 L / m 2 has been demonstrated. Other optimal headspace volumes are in the following ranges (all in L / m 2 ), 4 - 13, 4 - 12, 4 - 11, 4 - 10, 4 - 9, 4 - 8, 4 - 7, 4 - 6, 4 - 5; 5 - 14, 5 - 13, 5 - 12, 5 - 11, 5 - 10, 5 - 9, 5 - 8, 5 - 7, 5 - 6; 6 - 14, 6 - 13, 6 - 12, 6 - 11, 6 - 10, 6 - 9, 6 - 8, 6 - 7; 7 - 14, 7 - 13, 7 - 12, 7 - 11, 7 - 10, 7 - 9, 7 - 8; 8 - 14, 8 - 13, 8 - 12, 8 - 11, 8 - 10, 8 - 9; 9 - 14, 9 - 13, 9 - 12, 9 - 11, 9 - 10; 10 - 14, 10 - 13, 10 - 12, 10 - 11; 11 - 14, 11 - 13, 11 - 12; 12 - 14, 12 - 13; and 13 - 14 L / m 2 are included. If the ratio of the headspace volume to the depth filter area is too high, problems such as flow rate distribution and excessive weight of the device will occur.

[0072] (Example 1) The clarified CHO - S cell culture product of the aggregated cell culture was aggregated by the addition of a water - soluble polymer. At least one suitable water - soluble polymer is a poly(dimethyldiallylammonium chloride) (pDADMAC) polymer flocculant. The aggregated cell culture was filtered using six depth filtration devices described in Table 2. In these experiments, by assembling an upper part including an upper flow distributor and a suction hose barb, a filter media layer, and a bottom part including a lower flow distributor and a discharge hose barb, a depth filtration device of 23 cm 2 was constructed. The headspace of the device was changed by installing a plastic spacer ring between the upper part of the filter device and the bottom part of the filter device. A thermoplastic overmold jacket was utilized to put the components together and seal them watertight. The internal volume of the device was determined as follows. The assembled and dried device was weighed, completely filled with water, and weighed again. The depth filter media volume is the total thickness of the media layer (1.6 - 2.0 cm) and the filter front area (23 cm 2) was determined as the product with. The headspace of the device is calculated as half of the difference between the internal volume of the device and the depth filter media volume. The headspace of the device is defined as the internal volume of the device located above the layer of depth filter media in the depth filtration device. The tailspace of the device is defined as the internal volume of the device located below the layer of depth filter media in the depth filtration device.

[0073] The aggregated CHO-S cell culture product was processed through each depth filtration device at a flow rate of 150 LMH until a final pressure of 20 psi was obtained. Figures 7A through 7F are the pressure profile curves of various depth filtration devices according to Example 1.

[0074] In Example 1-1, a filter capacity of 462 L / m 2 was obtained. This device has no available headspace, and a slight compression of the filter media is calculated. The device of Example 1-2 utilized spacer rings to place the internal volume of the device (tailspace) below the layer of depth filter media. The filter capacity was 574 L / m 2 . In contrast, the device of Example 1-4 utilized the same number of spacer rings to place the internal volume of the device above the layer of depth filter media. In this device, an increase in depth filtration capacity of 675 L / m 2 was observed. Examples 1-3, 1-4, 1-5, and 1-6 represent the progression of increased device headspace and the corresponding effect on device filtration capacity. Figures 7A through 7F show a plot of device resistance versus filter throughput for the filter devices of Examples 1-1 through 1-6, as shown in Table 2. The process flux was 150 LMH. These plots show that the filter devices of Examples 1-2 and 1-3 provide a slightly higher filter capacity for the aggregated feed stream compared to the filter device of Example 1-1. The filter devices of Examples 1-4, 1-5, and 1-6 further increase the filter capacity for the aggregated feed stream compared to the filter device of Example 1-1.

[0075]

Table 2

[0076] (Example 2) Clarification of the aggregated cell culture. The CHO-S cell culture product was aggregated by the addition of a water-soluble polymer. At least one suitable water-soluble polymer is the poly(dimethyldiallylammonium chloride) (pDADMAC) polymer flocculant. The aggregated cell culture was filtered using three depth filtration devices described in Table 3. In these experiments, by assembling the top including the top flow distributor and the inlet hose barb, the filter media layer, and the bottom including the bottom flow distributor and the outlet hose barb, a 23 cm 2 depth filtration device was constructed. The headspace of the device was varied by installing a plastic spacer ring between the top of the filter device and the bottom of the filter device. A thermoplastic overmold jacket was utilized to enclose the components together and seal them watertight. The internal volume of the device was determined as follows. The assembled and dried device was weighed, completely filled with water, and weighed again. The depth filter media volume was determined as the product of the total thickness of the media layer (1.9 - 2.0 cm) and the filter front area (23 cm 2 ). The headspace of the device was calculated as one half of the difference between the internal volume of the device and the depth filter media volume.

[0077] The aggregated CHO-S cell culture product was processed through each depth filtration device at a flow rate of 150 LMH until a final pressure of 20 psi was obtained. Figures 8A through 8C are the pressure profile curves of various depth filtration devices according to Example 2.

[0078] In Example 2-1, 544 L / m 2The filter capacity was obtained. This device has no available headspace, and a slight compression of the filter media is calculated. In Examples 2-2 and 2-3, an increase in the depth filtration capacity was observed. These two devices show an increase in headspace, and the compression of the filter media is minimal. Figures 8A through 8C show plots of the device resistance versus the filter throughput of the filter devices of Examples 2-1, 2-2, and 2-3, as shown in Table 3. The process flux was 150 LMH. These plots show that the filter devices of Examples 2-2 and 2-3 provide a higher filter capacity for the agglomerated feed stream than the filter device of Example 2-1.

[0079]

Table 3

Claims

1. A depth filter comprising a housing having a suction port, a discharge port spaced apart from the suction port, an internal volume containing a filtration medium, and a headspace within the internal volume, wherein the volume of the headspace is 4 to 14 liters per square meter of the area of the filtration medium within the internal volume, The depth filter, wherein the liquid filtered by the depth filter is a supply stream of a cell culture using a product containing 25 million to 100 million cells per mL, corresponding to 3.5 to 11.5% biomass.

2. The depth filter according to claim 1, wherein the volume of the headspace is 4 to 10 liters per square meter of the area of the filtration medium within the internal volume.

3. The depth filter according to claim 1, wherein the filtration medium comprises a composite of a stepped layer of non-woven fiber, cellulose, and diatomaceous earth.

4. The area of the smallest filter medium is 0.1 m ２ The depth filter according to claim 1, wherein the area is 0.1 m

5. A filter assembly for filtering particles from a liquid, A housing having a suction port for introducing the liquid to be filtered and a discharge port for the filtered liquid, the discharge port being spaced apart from the suction port, A depth filter medium within the housing, A headspace within the housing and upstream of the depth filter medium in the direction of the flow of the fluid during filtration, the volume of the headspace being 4 to 14 liters per square meter of the area of the depth filter medium within the housing, The filter assembly, wherein the liquid to be filtered is a supply stream of a cell culture using a product containing 25 million to 100 million cells per mL, corresponding to 3.5 to 11.5% biomass.

6. The filter assembly according to claim 5, wherein the volume of the headspace is 4 to 10 liters per square meter of the area of the depth filter medium within the housing.

7. The filter assembly according to claim 5, wherein the depth filter medium comprises a composite of a stepped layer of non-woven fiber, cellulose, and diatomaceous earth.

8. A method for removing impurities from a biological sample, comprising filtering the sample through a depth filter having a housing with a volume, a medium within the volume, and a headspace within the volume, wherein the volume of the headspace is 4 to 14 liters per square meter of the area of the depth filter medium, The method wherein the liquid filtered by the depth filter is a feed stream of a cell culture using a product containing 25 million to 100 million cells per mL, corresponding to 3.5 to 11.5% biomass. Claim 9 A method for clarifying a feed containing a target biomolecule of interest and a plurality of cell debris and / or colloidal particles by depth filtration, a) providing a depth filtration device having a headspace and a porous depth filter medium, wherein the volume of the headspace is 4 to 14 liters per square meter of the area of the depth filter medium within the device, b) providing a feed containing a target biomolecule of interest and a plurality of cell debris and / or colloidal particles, c) introducing the feed into the headspace and contacting the depth filter medium with the feed, and d) separating the target biomolecule of interest from cell debris and colloidal particles in the feed, comprising The method wherein the liquid filtered by the depth filter is a feed stream of a cell culture using a product containing 25 million to 100 million cells per mL, corresponding to 3.5 to 11.5% biomass. Claim 10 The method according to claim 9, wherein the depth filtration is carried out without using a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step. Claim 11 The method according to claim 9, wherein the volume of the headspace is 4 to 10 liters per square meter of the area of the depth filter medium within the device. Claim 12 A method for optimizing the performance of a depth filter, wherein the depth filter has a housing having a fluid inlet, a fluid outlet spaced from the fluid inlet, an internal volume, a porous depth filter media within the internal volume, and a headspace between the fluid inlet and the porous depth filter media, and configuring the volume of the headspace to be 4 to 14 liters per square meter of the porous depth filter media within the housing, The method, wherein the liquid filtered by the depth filter is a supply stream of a cell culture using a product containing 25 million to 100 million cells per mL, corresponding to 3.5 to 11.5% biomass.

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