Depth filter medium

Abstract

This invention provides an improved depth filtration medium with reduced extractant for DSP filtration and clarification applications. Furthermore, it provides an improved depth filtration medium that is compatible with gamma irradiation-based filter sterilization processes. [Solution] A depth filtration medium is provided for clarifying the recovered material from a feed stream of a cell culture containing therapeutic biomolecules. The depth filtration medium is composed of a sintered mixture of a polymer adsorbent and a thermoplastic binder, has extremely low extractability, and does not require pre-washing before use. Furthermore, the constituent materials used in the sintered depth filter medium exhibit high stability to gamma rays and are compatible with gamma-based pre-use sterilization processes.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 237,680, filed on 27 August 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Current biopharmaceutical manufacturing processes utilize a series of filtration and chromatographic purification steps to isolate and purify therapeutic biomaterials from mammalian cell culture suspensions. These therapeutic biomaterials may include, among other things, monoclonal antibodies, enzymes, antibody fragments, proteins, and fusion proteins. The series of filtration and chromatographic purification steps used to isolate and purify therapeutic biomaterials are commonly referred to as downstream purification (DSP). The first step of a DSP is often a clarification step aimed at removing whole cells, cell debris, and colloidal material from the feed stream containing the therapeutic biomaterial. Clarification steps used in a DSP may include centrifugation, microfiltration (by either tangential flow filtration or normal flow filtration), or more recently, depth filtration.

[0003] Conventional depth filters used in DSP clarification processes are often based on a wet-laid mixture of cellulose, diatomaceous earth, and a wet-strength binder resin. Other conventional depth filters used in DSP clarification processes are based on a wet-laid mixture of synthetic fibers, silica gel filter aids, and a wet-strength binder resin. The above types of depth filters may also include a synthetic nonwoven fiber layer for the purpose of clarifying cell culture feed streams pretreated by agglomeration or other means, or for the removal of whole cells and cell debris. Selected examples of this type of conventional depth filter include MILLISTAK+(R), MILLISTAK+(R) HC, MILLISTAK+(R) HC Pro, or CLARISOLVE(R) depth filters, all of which are commercially available from EMD Millipore Corporation (Burlington, Massachusetts).

[0004] Conventional depth filters manufactured by wet-laid processes typically require extensive pre-use washing with high-purity water to reduce organic and inorganic extracts.

[0005] Organic and inorganic extracts have various sources, which are typically introduced into the depth filter medium during the wet-laid manufacturing process. Naturally derived constituent materials such as cellulose and diatomaceous earth are also likely to be sources of certain organic and inorganic extractable components. In the DSP process, depth filter extracts can contaminate the feed stream containing therapeutic bio-derived materials, and these extracts must be removed from the depth filter before use in the DSP filtration process.

[0006] Yavorsky (U.S. Patent No. 7,673,757, '757 Patent) discloses a filter medium for removing biological contaminants from process liquids. Yavorsky's filters are made from an adsorbent material fused with a water-insoluble thermoplastic binder. Yavorsky's filters are described as having reduced levels of foreign contaminants (total organic carbon: TOC) (requiring pre-washing removal before use) compared to cellulose-containing filters. However, the inventors have found that the filters of the '757 Patent still have unacceptably high levels of organic and inorganic contaminants requiring pre-washing, and furthermore, may lack the stability essential for gamma irradiation used for filter sterilization, which is required in the bioprocessing field. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 7,673,757 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] There is a current need for improved depth filtration media with reduced extracts for DSP filtration and clarification applications. Furthermore, there is a current need for improved depth filtration media that are compatible with gamma irradiation-based filter sterilization processes. [Means for solving the problem]

[0009] In this specification, the inventors report the development of a novel and non-trivial totally synthetic depth filtration medium comprising a thermal fusion mixture of a polyethylene binder and a high-surface-area synthetic filter aid. The totally synthetic depth filtration medium of the present invention exhibits significantly reduced organic and inorganic extracts compared to prior art depth filters. The totally synthetic depth filtration medium of the present invention is prepared by a thermal process that fuses the polyethylene binder with a high-surface-area synthetic filter aid. This process forms a robust filter sheet containing the binder and the high-surface-area filter aid, eliminating the need for a wet-laid filter media manufacturing process. The wet-laid filter manufacturing process is a potential source of increased organic and inorganic extracts in conventional depth filtration media. In some embodiments of the present invention, the wet-laid filter manufacturing process is specifically excluded as a means of manufacturing the filter of the present invention. Furthermore, the thermal process utilized in the present invention also eliminates the need for wet-strength binder resins currently used in conventional depth filtration media. Wet-strength binder resins are another possible source of increased organic extracts in conventional depth filtration media. In one aspect of the present invention, the use of a wet-strength binder resin in the depth filter medium of the present invention is specifically excluded as an embodiment or aspect of the depth filter of the present invention.

[0010] Importantly, the novel deep-layer medium of the present invention also provides a reduction in organic and inorganic contamination after exposure to gamma irradiation used for sterilization. This is in stark contrast to prior art filter materials that "remove" the increased amount of contaminants after gamma irradiation, thereby requiring further increases in pre-washing time and water volume.

[0011] The high-surface-area filter aids used in constructing the depth filtration medium of the present invention remove whole cells, submicron particles, colloidal materials, soluble impurities (HCP and DNA), and cellular debris through adsorption mechanisms including electrostatic and hydrophobic interactions. Such species cannot be captured by particle sieving without the high-surface-area filter aids. The use of depth filters containing filter aids for these purposes has been previously reported (Nguyen et al., Biotechnol.J.2018, 1700771).

[0012] In a preferred embodiment, the synthetic adsorbent of the present invention is polystyrene. Examples of suitable polystyrenes known to those skilled in the art include PrAOH (polystyrene crosslinked with divinylbenzene, anion, hydroxide form, Purolite Corp., King of Prussia, Pennsylvania), PrCH (polystyrene crosslinked with divinylbenzene: cation, hydrogen form, Purolite Corp., King of Prussia, Pennsylvania), MB1 / 1h (polystyrene crosslinked with divinylbenzene, cation / anion hydrogen form, Purolite Corp., King of Prussia, Pennsylvania), polystyrene adsorbent (Medapore P787 (>63 μm), Merck KGaA, Darmstadt, Germany), poly(4-vinylbenzene) crosslinked with 2% divinylbenzene (MilliporeSigma, Burlington, Massachusetts), poly(4-vinylpyridine-co-ethylvinylbenzene) crosslinked with 25% divinylbenzene (MilliporeSigma, Burlington, Massachusetts), and one or more of these, but not limited to these. Further adsorbents can include ion exchange resins such as Amberlite™, Amberlite™ XAD™, and AmberChrom™ ion exchange resins (DuPont, Wilmington, Delaware). Suitable high surface area filter aids for the present invention can also include one or more of various chromatography resins, especially ESHMUNO® ion exchange chromatography resins and FRACTOGEL® ion exchange chromatography resins (both available from EMD Milipore, Burlington, Massachusetts). Those skilled in the art will recognize that suitable synthetic high surface area filter aids can be selected as long as they exhibit sufficient thermal stability to withstand the sintering process utilized in forming depth filter media sheets. In another aspect of the present invention, diatomaceous earth, silica, porous glass, zeolite, activated carbon, non-synthetic chromatography media, and cellulose-based materials are specifically excluded as embodiments or aspects of the depth filters of the present invention, either alone or in combination.

[0013] The all-synthetic depth filtration media of the present invention is also composed of constituent materials that exhibit high stability against gamma rays. As a result, the all-synthetic depth filtration media of the present invention can be sterilized by gamma rays or other radiation sterilization procedures such as X-rays and electron beams without adversely affecting the depth filtration performance in downstream purification process (DSP) clarification applications. Furthermore, the high gamma-ray stability of the constituent materials of the all-synthetic depth filtration media of the present invention provides very low organic and inorganic extracts even after sterilization by gamma-ray irradiation, unlike prior art filter media.

[0014] Thus, the all-synthetic depth filter of the present invention provides a depth filter that contains substantially no organic and inorganic extracts even after gamma-ray irradiation and requires little or no pre-washing before use. It is well known that polymer materials used in conventional depth filtration media such as cellulose and polyacrylic fibers can undergo bond breakage in the presence of ionizing radiation sources such as gamma rays, X-rays, and electron beams. These molecular changes often result in chain scission, recombination, and / or crosslinking, which can change the mechanical and / or chemical properties of the original material and "leach" it into the process fluid as external extracts. The compatibility of various polymers with gamma-ray sterilization doses has been previously evaluated. See "Effects of Gamma Irradiation on Polyethylene, Polypropylene, and Polystyrene" by Kawamura, Y. in Chapter 16 of Irradiation of Food and Packaging, ACS Symposium Series, American Chemical Society, Washington D.C., 2004, 262-276. In another aspect of the present invention, there is no need for pre-washing before use, except for filling the filtration device containing the filtration media with water or the solution intended for filtration. In yet another aspect of the present invention, pre-washing the depth filter of the present invention, except for a liquid sufficient to wet the filter, is explicitly excluded as an embodiment of the present invention.

[0015] In one aspect, the present invention envisions a depth filter comprising: a) a sintered depth filter medium containing a thermally fused mixture of polyethylene binders; and b) one or more adsorbents selected from the group consisting of PrAOH, PrCH, MB1 / 1h, polystyrene adsorbent, poly(4-vinylbenzene) crosslinked with 2% divinylbenzene, and poly(4-vinylpyridine-co-ethylvinylbenzene) crosslinked with 25% divinylbenzene. In another aspect of the present invention, the present invention envisions the depth filter medium to be significantly homogeneous. In yet another aspect of the present invention, the present invention envisions the depth filter to be about 1 mm to about 4 mm thick or about 2 mm to about 3 mm thick.

[0016] In another embodiment, the present invention intends to specifically exclude wet strength binder resins.

[0017] In another embodiment, the present invention intends for the polyethylene to adsorbent ratio to be about 1:10 to about 10:1, about 1:5 to about 5:1, about 1:2 to about 2:1, or the aforementioned polyethylene to adsorbent ratio to be about 1:1.

[0018] In another aspect, the present invention intends that the size of the polystyrene adsorbent is approximately 10 μm to 120 μm in diameter, approximately 30 μm to 100 μm in diameter, approximately 50 μm to 80 μm in diameter, approximately 60 μm to 70 μm in diameter, or approximately 63 μm in diameter.

[0019] In another embodiment, the present invention envisions a method for producing a sintered depth filter medium, comprising mixing a polyethylene binder with an adsorbent selected from the group consisting of PrAOH, PrCH, MB1 / 1h, polystyrene adsorbent, poly(4-vinylbenzene) crosslinked with 2% divinylbenzene, and poly(4-vinylpyridine-co-ethylvinylbenzene) crosslinked with 25% divinylbenzene to produce a mixture, spreading the mixture to a substantially uniform thickness, and heating the mixture at about 165°C for about 60 minutes.

[0020] In another embodiment, the present invention relates to a method for clarifying a feed stream containing a target protein, comprising a) a depth filter comprising a sintered depth filter medium comprising a polyethylene binder along with an adsorbent selected from the group consisting of PrAOH, PrCH, MB1 / 1h, a polystyrene adsorbent, poly(4-vinylbenzene) crosslinked with 2% divinylbenzene, and poly(4-vinylpyridine-co-ethylvinylbenzene) crosslinked with divinylbenzene, and b) a method for increasing the concentration of target protein to contaminants in the permeate by at least 20% compared to the ratio in the feed stream, by bringing the depth filter into contact with the feed stream such that the target protein passes through the depth filter and is retained in the permeate and contaminants are adsorbed by the depth filter.

[0021] In another embodiment, the present invention aims to increase the concentration of target protein to contaminant in the permeate by at least 50% compared to the ratio in the feedstream.

[0022] In another aspect of the present invention, the present invention aims to increase the concentration of target protein to contaminant in the permeate by at least 100% compared to the ratio in the feedstream. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 shows a photograph of the sintered depth filter medium of the present invention. The sintered depth filter medium was prepared as described in Example 3, Example ID 3-2. For comparison, a photograph of a cellulose-based benchmark sample, MILLISTAK+(R) CE25 filter media (MilliporeSigma, Burlington, Massachusetts), is shown. [Figure 2]Figure 2(A-D) shows cross-sectional SEM images of the sintered depth filter medium of the present invention. The sintered depth filter medium was prepared as described in Example 3, Example ID3-2. SEM images are provided at 100x (A and B) and 500x (C and D) magnifications. The SEM images show that large polystyrene beads exceeding 63 μm are captured by much smaller fused polyethylene particles. [Figure 3] Figure 3 shows the TOC flashout curve of a 23 cm² minicap filter apparatus containing a single layer of the sintered depth filter medium of the present invention as described in Example 3. [Figure 4] Figure 4 shows the TOC flashout curves of a 23 cm² mini-cap filter apparatus containing a single layer of the sintered depth filter medium of the present invention as described in Example 3, before and after gamma irradiation. [Figure 5] Figure 5 shows the metal extract data from a 23 cm² minicap filter apparatus containing a single layer of the sintered depth filter medium of the present invention as described in Example 3. [Figure 6-1] Figure 6 shows the metal extract data for the selected extracts, regardless of whether or not gamma irradiation was performed, using a 23 cm² minicap filter apparatus containing a single layer of the sintered depth filter medium of the present invention as described in Example 3. [Figure 6-2] Figure 6 shows the metal extract data for the selected extracts, regardless of whether or not gamma irradiation was performed, using a 23 cm² minicap filter apparatus containing a single layer of the sintered depth filter medium of the present invention as described in Example 3. [Figure 7] Figure 7 shows the filter resistance profile for an application test using a sintered depth filter medium for clarification of CHO cell culture samples. Details of this application / experiment are provided in Example 6. [Figure 8]Figure 8 shows the filter resistance profile for an application test using a sintered depth filter medium for clarification of a Peptone HY-SOY(R) T (Milliporesigma, Burlington, Massachusetts) model feed stream. Details of this application / experiment are provided in Example 7. [Figure 9] Figure 9 shows a comparison of the conductivity of aqueous filtrates from the '757 patent for various prior art filter media during washing. [Figure 10] Figure 10 shows the conductivity of the aqueous filtrate of the filter material of the present invention during washing, compared with two prior art filter materials before and after gamma ray irradiation. [Figure 11] Figure 11 shows the increase in conductivity of the aqueous filtrate during washing of the prior art CE25 medium after gamma irradiation. [Figure 12] Figure 12 shows the increase in conductivity of the aqueous filtrate during washing of the prior art DE40 medium after gamma irradiation. [Figure 13] Figure 13 shows that there is no increase in the conductivity of the aqueous filtrate during washing of the medium of the present invention after gamma ray irradiation. [Figure 14] Figure 14 predictively shows the decrease in conductivity of the aqueous filtrate during washing of the filter material of the present invention against various known prior art media, regardless of whether or not it is exposed to gamma radiation. [Modes for carrying out the invention]

[0024] <Definition> Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains. The following reference, namely *Process Scale Bioseparations for the Biopharmaceutical Industry*, edited by Abhinav A. Shukla, Mark R. Etzel, and Shishir Gadam, provides general definitions of many of the terms used herein. Where used herein, the following terms have the meanings thereof unless otherwise specified.

[0025] When introducing elements of this disclosure or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there is one or more elements. The terms “comprising,” “including,” and “having” are intended to mean comprehensive and that there may be additional elements other than those listed.

[0026] The transitional phrases “contain,” “essentially consist of,” and “consist of” have the meanings given in MPEP 2111.03 (Manual of Patent Examining Procedure, United States Patent and Trademark Office). All claims using the transitional phrase “essentially consist of” are understood to enumerate only the essential elements of the invention, and any other elements enumerated in a dependent claim are understood to be not essential to the invention as enumerated in the dependent claim.

[0027] For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers used herein to represent quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values ​​should be understood in all cases, whether expressly indicated or not, as being modified by the term "approximately." The term "approximately" generally refers to a range of numbers that are considered equivalent to (i.e., having the same function or result as) the listed values. Often, the term "approximately" may include numbers rounded to the nearest significant figure.

[0028] Therefore, unless otherwise indicated, the numerical parameters described in the following specification and the appended claims are approximations that may vary depending on the desired characteristics to be obtained by the present invention. 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 interpreted by applying ordinary rounding techniques, at least in light of the number of significant figures reported. Furthermore, all ranges disclosed herein should be understood to encompass all subranges contained therein. In other words, all numerical ranges are considered to include all digits within the range, as if they were explicitly enumerated.

[0029] Before describing the present invention in further detail, some terms are defined. The use of these terms is not intended to limit the scope of the present invention, but rather to facilitate its explanation.

[0030] All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole, to the same extent as each individual publication, patent, or patent application is specifically and individually referenced, notwithstanding the foregoing or otherwise. All publications, patents, and patent applications referenced herein are also typical of what a person skilled in the art would understand in the art of the present invention at the time of the present invention.

[0031] The term "bubbling point pore diameter" or "BP" refers to the pore diameter of the largest pore in the filter material.

[0032] As used herein, the term “cell culture” includes cells, cell debris and colloidal particles, biomolecules of interest, HCPs, and DNA.

[0033] The terms “Chinese hamster ovary cell protein” and “CHOP,” when used interchangeably herein, refer to a mixture of host cell proteins (“HCP”) derived from Chinese hamster ovary (“CHO”) cell cultures. HCP or CHOP generally exists as an impurity in cell culture media or lysates (e.g., a collected cell culture medium containing the protein or polypeptide of interest (e.g., an antibody or immunoadhesion expressed in CHO cells)). Generally, the amount of CHOP present in a mixture containing the protein of interest provides a measure of the purity of the protein of interest. Typically, the amount of CHOP in a protein mixture is expressed in ppm relative to the amount of the protein of interest in the mixture.

[0034] The terms “clarification process” or simply “clarification,” as used herein, generally refer to one or more steps used first in the purification of biomolecules. A clarification process generally includes the removal of cells and / or cellular debris using one or more steps, including, for example, centrifugation and depth filtration, tangential flow filtration, microfiltration, precipitation, aggregation and sedimentation, either alone or in various combinations thereof. In some embodiments, the present invention provides improvements over conventional clarification processes commonly used in various purification schemes. A clarification process generally includes the removal of one or more undesirable entities and is typically performed before steps involving the capture of desired target molecules. Another aspect of clarification is the removal of soluble and insoluble components in a sample (which may later lead to fouling of sterile filters in the purification process), thereby making the overall purification process more economical. A clarification process often includes one or more upstream primary clarification steps and one or more downstream secondary clarification steps. Clarification of cell culture harvests and high-solids feedstocks from modern manufacturing batch bioreactors (less than 25,000 L) and high cell density yields often requires primary and secondary clarification steps before any subsequent chromatographic operations.

[0035] When used herein, the terms “coarse filtration” or “coarse / medium filtration” generally refer to the removal of nearly all cells and some cellular debris in the purification of biomolecules.

[0036] As used herein, the term "microfiltration" generally refers to the removal of most cellular debris, colloidal particles, and soluble impurities such as HCPs, DNA, endotoxins, viruses, and lipids in the purification of biomolecules.

[0037] Filter throughput values ​​are generally expressed in "liters / square meter" or "L / m²". 2 While expressed as "column volume," for comparable comparisons, "column volume" or "CV" is used to account for large differences in thickness between samples.

[0038] The terms “contaminants,” “impurities,” and “residues” are used interchangeably herein and refer to any foreign or undesirable substances, including biological macromolecules such as DNA, RNA, one or more host cell proteins (HCP or CHOP), endotoxins, viruses, and lipids, that may be present in a sample containing the protein or polypeptide of interest (e.g., an antibody). These additives are separated from the foreign or undesirable molecules using the depth filter according to the present invention.

[0039] When the host cell is another mammalian, non-mammalian, or bacterial cell type, such as E. coli, yeast, insect, or plant, HCP is understood to refer to proteins other than the target protein found in the lysate of the host cell.

[0040] As used herein, the terms “monoclonal antibody” or “mAb” refer to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies constituting the population are identical except for any naturally occurring mutations that may be present in small amounts.

[0041] As used herein, the term “average flow pore size” or “MFP” refers to the pore size at a pressure drop where the flow through the wet filter media is 50% of the flow through the dry filter media.

[0042] As used herein, the term “organic extracts” refers to contaminants that may potentially migrate or be extracted from materials used to construct a filter medium or membrane, such as a porous depth filter medium, in the presence of water or other aqueous solutions used during washing. These contaminants may also include the constructing material itself, which may potentially detach from the filter during use, thereby requiring pre-washing of the filter before use to remove the organic extracts.

[0043] The terms "Total Organic Extract" and "TOC" refer to measurements of organic molecules present in aqueous solutions, such as water, measured as carbon content. Analytical techniques used to measure TOC typically involve the oxidation of all organic molecules in a solution to carbon dioxide, the measurement of the resulting CO2 concentration, and the correlation of this response with known carbon concentrations.

[0044] The term "inorganic extract" refers to trace metal species, including heavy metals, that can be extracted from a filter into the process fluid. These metal species can be measured by analytical techniques such as inductively coupled plasma atomic emission spectrometry (ICP-OES), ICP mass spectrometry (ICP-MS), and graphite furnace atomic absorption spectrometry (GFAAS).

[0045] "Pre-washing" is defined herein as washing the filter before use, usually with sterile water, to remove organic and inorganic extracts from the filter.

[0046] The terms "parts per million" or "ppm" are used interchangeably herein.

[0047] Pore ​​diameter is usually given as a nominal value. In some cases, manufacturers provide the mean flow pore (MFP) diameter or the foaming point (BP) pore diameter. Both MFP and BP can be measured using a capillary flow porometer.

[0048] The terms “target molecule,” “target biomolecule,” “desired target molecule,” and “desired target biomolecule” are used interchangeably herein and generally refer to one or more undesirable entities that may be present in a sample containing the polypeptide or product of interest, for example, the polypeptide or product of interest that is desired to be purified or separated from one or more impurities (e.g., monoclonal antibodies).

[0049] As used herein, the term "throughput" means the volume filtered through the filter divided by the surface area of ​​the filter. Throughput is calculated as: Volume of filtered fluid (L) / m² of filter area 2 It is represented as follows.

[0050] As used herein, the term “dirt retention capacity” is equivalent to the filter throughput of a given cell culture fluid, either directly collected or previously clarified. Higher throughput indicates higher dirt retention capacity.

[0051] As used herein and as will be understood by those skilled in the art, “wet-strength binder resin” is a resin that enables the formation of a filter sheet by binding fibers and / or particles together, increases the wet strength of the filter, and, depending on the binder used, can impart unique charge properties. Examples of wet-strength resins include resins containing synthetic polymers of urea or melamine-formaldehyde polymers, polyaminopolyamide-epichlorohydrin (PAE) polymers, and glyoxalized polyacrylamide (GPAM) resins.

[0052] <Sintered filter of the present invention>

[0053] The filters of the present invention are referred to herein as “sintered filters.” The term “sintered” is known to those skilled in the art and refers to articles manufactured by or subjected to sintering, which is a process of bonding powdered materials into a solid or porous mass by heating without liquefaction. The filters of the present invention include (or “essentially consist of” or “consist of” a high surface area synthetic filter aid bonded by a polyethylene binder by thermal fusion. The high surface area synthetic filter aid is 10 m 2 It is defined as having a BET specific surface area greater than / g. In some cases, the BET specific surface area of ​​a synthetic filter aid is 100m². 2 It may be greater than / g. BET measures surface area based on gas adsorption (Adsorption of Gases in Multimolecular Layers, Brunauer, Emmett, Teller, J.Am.Chem.Soc., February 1938, Vol. 60, 309). Alternative binders may also include thermoplastic powders, including nylon 6 powder (PA6, MilliporeSigma, Burlington, Massachusetts) and nylon 12 powder (PA12, MilliporeSigma, Burlington, Massachusetts). Other possible alternative binders include acrylic staple fibers, Fybrel(R) synthetic wood pulp, and Short Stuff(R) fibrillated HDPE (Minifibers, Inc.).

[0054] In preferred embodiments, the filters of the present invention are "homogeneous" or "fairly homogeneous," meaning that the ratio of synthetic filter additive to binder is the same or substantially the same throughout the filter, and the composition with respect to the ratio of synthetic filter additive to binder varies only within manufacturing tolerances.

[0055] The filter retention properties of the depth filter of the present invention may vary depending on the selected polymer, mixture, and ratio. Generally, the depth filter of the present invention exhibits increased permeability and decreased retention of fine particles as the packing of high-surface-area synthetic filter aids increases. Conversely, when the packing of high-surface-area synthetic filter aids in the sintered depth filter of the present invention decreases, it exhibits decreased permeability and increased retention of fine particles. In addition, those skilled in the art will consider the trade-off between filter permeability and filter retention of the sintered filter sheet in light of the decrease in mechanical integrity of the sintered filter sheet at high packing values ​​of high-surface-area synthetic filter aids exceeding 70% by weight.

[0056] The permeability of a depth filter sheet can be evaluated by determining the pressure drop in the PSI of a filtration system containing a depth filter sheet at various water flux velocities in LMH. A higher pressure drop indicates lower permeability of the filter sheet.

[0057] Process flux is the volumetric flow rate of the fluid being filtered (m³). 2 The process flux is expressed as L / m² divided by the frontal area of ​​the filtration device. 2 Expressed in L / hour (LMH). Typical process fluxes for biopharmaceutical depth filtration processes range from 75 to 300 LMH. The pressure drop of the sintered filter media of the present invention is less than 1.1 psi at a flux of 574 LMH.

[0058] As described above, the filters of the present invention are manufactured by thermal fusion. An example of the thermal fusion process is given in the following section on examples. Thermal fusion, sometimes called heat fusion, is a process known in the art, for example, used to join thermoplastics together. For example, instead of using adhesives, solder, or mechanical connections and gaskets, thermal fusion actually makes two plastic pieces into one solid piece by sufficiently softening and / or melting one or more thermoplastic materials so that when cooled, they bond together with other thermoplastic materials. The molten sintered filters of the present invention maintain distinction between components, i.e., the synthetic filter aids and binders are still visible as components when magnified. See Figure 2.

[0059] The synthetic sintered depth filter of the present invention is not limited by thickness, but in preferred embodiments, the thickness may be in the range of 0.1 mm to 5.0 mm, 0.5 mm to 4.0 mm, 1.0 mm to 4.0 mm, 1.0 mm to 3.0 mm, and 2.0 mm to 3.0 mm.

[0060] The synthetic sintered depth filter of the present invention is not limited by the ratio of the binder to the synthetic adsorption filter aid. In one embodiment, the ratio may be 1:10 to 10:1 binder:synthetic adsorption filter aid, 1:5 to 5:1 binder:synthetic adsorption filter aid, 1:2 to 2:1 binder:synthetic adsorption filter aid, or about 1:1 binder:synthetic adsorption filter aid. Those skilled in the art can determine a ratio suitable for any particular use without excessive experimentation using the guidance provided herein.

[0061] The synthetic adsorption filter aids of the present invention are typically spherical or slightly elliptical, having diameters of approximately 10 μm to 120 μm, 30 μm to 100 μm, 50 μm to 80 μm, 60 μm to 70 μm, or 63 μm. Those skilled in the art can determine a size suitable for any particular use without excessive experimentation using the guidance provided herein.

[0062] <Method for manufacturing a sintered depth filter according to the present invention>

[0063] The present invention also provides a method for producing the sintered depth filter of the present invention. Preferred methods are provided in the following Exemplary Methods section. In one embodiment, a polyethylene binder and a polystyrene adsorbent are mixed in a desired ratio. The mixture is evenly distributed onto a large metal baking sheet and flattened to 2 mm with a drawbar. The baking sheet is then baked at approximately 165°C for approximately 60 minutes. After cooling, the sheet of filter material is cut to the desired size. Those skilled in the art can determine the time and temperature for producing the synthetic depth filter of the present invention using different binders or different adsorbents without excessive experimentation using the guidance provided by the present invention. Similarly, those skilled in the art can determine the time and temperature for producing the synthetic depth filter of the present invention thicker or thinner than 2 mm without excessive experimentation using the guidelines provided by the present invention.

[0064] Another method for manufacturing depth filters is to use automated machinery, which allows for the efficient production of large quantities of filter media.

[0065] <Method of using the sintered depth filter of the present invention>

[0066] This invention relates to a method of using the synthetic sintered depth filter of the present invention. For example, the sintered depth filter of the present invention may be used to filter a cell culture medium (i.e., a feed stream) on which a bioproduct has been produced. Depending on the processing stage of the feed stream, filters of different pore sizes, porosity, and flow grades may be used. Those skilled in the art can use the teachings herein to determine the correct pore size, porosity, and flow grade for a particular use. In one embodiment, the synthetic depth filter of the present invention may be used to clarify a feed stream prior to further downstream processing. The clarification step removes cell culture residue such as whole cells, burst cells, large host cell proteins (HCPs), and other contaminants, while allowing the target protein to pass through the filter. In one embodiment, the target protein is a monoclonal antibody, a humanized monoclonal antibody, a CAR-T cell-producing antibody, etc. The target protein may also be another genetically engineered protein or a naturally occurring protein, for example, produced by cells transfected with an expression vector engineered for the expression of the desired target protein, or naturally expressed by a given cell type.

[0067] The result of passing a cell culture feed stream through the depth filter of the present invention is to increase the relative proportion of the target protein to contaminants (one or more) in the permeate (i.e., the feed stream passing through the filter). In this context, contaminants may include whole cells, cell debris, and colloidal particles. In one embodiment, the concentration of the target protein relative to the contaminants increases by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, and at least 1000% compared to the feed stream. In one embodiment, the concentration of the target protein relative to the contaminants increases by up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 100%, up to 200%, up to 500%, and up to 1000% compared to the feed stream. In one embodiment, the concentration of the target protein relative to the contaminant increases by 10% to 1000% and 50% to 500%.

[0068] The present invention intends that the depth filter of the present invention is not pre-washed before use for filtering a feed stream. In one embodiment, pre-washing of the depth filter of the present invention before filtering a feed stream is specifically excluded. [Examples]

[0069] [Example 1] Preparation of the sintered depth filter medium of the present invention. A polyethylene binder PE (MIPELON® XM-221u polyethylene binder, Mitsui Chemicals America, Inc.) and a polystyrene adsorbent PS (Medapore P787, over 63 μm, Merck KGaA (Darmstadt, Germany)) were mixed in the ratios shown in Table 1 below. The powder mixture was distributed onto a large metal baking sheet with a PTFE release film. Using a drawbar, the powder mixture was evenly distributed to a thickness of 2 mm. The baking sheet was placed in a preheated oven at 165°C for 60 minutes. The sample was removed from the oven and allowed to cool to room temperature. The sintered depth filter sheet was then placed in a 23 cm² oven.2 It was cut into disks of 2 and incorporated into a mini-cap filter test apparatus for an application test. The use of a mini-cap filter test apparatus in the evaluation of depth filter performance has been reported previously (Lutz, H et al., Biotechnol. Prog., 2015, 31, 6, 1542 - 1550, which is an example of the knowledge of those skilled in the art on this subject).

[0070] [Table 1]

[0071] [Example 2]

[0072] Preparation of the sintered depth filter medium of the present invention. A polyethylene binder PE (MIPELON™ XM - 221u polyethylene binder, Mitsui Chemicals America, Inc.) and a MICROLITE® ion exchange adsorbent (PrAOH, PrCH, and MBI / 1H (PUROLITE® Corporation, Bala Cynwyd, Pennsylvania)) materials were mixed at the ratios described in Table 2 below. For Examples 2 - 4, 2 - 5, and 2 - 6 (shown in Table 2 below), the MICROLITE® ion exchange adsorbent was dried at 125 °C for 3 to 18 hours. The powder mixture was dispensed onto a large metal baking sheet with a PTFE release film. Using a draw bar, the powder mixture was evenly dispensed to a thickness of 3 mm. The baking sheet was placed in a preheated oven at 165 °C for 60 minutes. The sample was taken out of the oven and cooled to room temperature. Then, the sintered depth filter sheet was cut into disks of 2 and incorporated into a filter test apparatus of 2 for an application test. 2 of 2 2 for an application test.

[0073] [Table 2]

[0074] [Example 3]

[0075] Preparation of the sintered depth filter medium of the present invention. A polyethylene binder PE (MIPELON® XM-221u polyethylene binder, Mitsui Chemicals America, Inc.) and a polystyrene adsorbent PS (Medapore P787, over 63 μm, Merck KGaA (Darmstadt, Germany)) were mixed in the ratios shown in Table 3 below. The powder mixture was distributed onto a large metal baking sheet with a PTFE release film. Using a drawbar, the powder mixture was evenly distributed to a thickness of 2 mm. The baking sheet was placed in a preheated oven at 165°C for 60 minutes. The sample was removed from the oven and allowed to cool to room temperature. The sintered depth filter sheet was then placed in a 23 cm² oven. 2 The material was cut into discs and incorporated into a mini-cap filter test apparatus for application testing. The use of a mini-cap filter test apparatus in evaluating depth filter performance has been previously reported (Lutz, H et al., Biotechnol. Prog., 2015, 31, 6, 1542-1550, which is an example of the knowledge of those skilled in the art in this subject).

[0076] [Table 3]

[0077] The filter media prepared according to this example are shown in Figures 1 and 2. Figure 1 shows a sintered depth filter medium prepared as in Example ID3-2(A). For comparison (B), a photograph of a cellulose-based benchmark sample (MILLISTAK(R) CE25 filter media) is shown.

[0078] Figure 2 is a cross-sectional SEM micrograph of the sintered depth filter medium of the present invention. The sintered depth filter medium shown in Figure 2 was prepared according to Example ID3-2. SEM micrographs are provided at 100x (A and B) and 500x (C and D) magnifications.

[0079] The sintered depth filter medium was prepared as described in Example ID3-7. As shown in Figure 3, a 100 L / m³ filter medium was introduced into the mini-cap filter apparatus at 600 LMH. 2 Milli-Q (MilliporeSigma, Burlington, Massachusetts) water was run through each of the following: 25 L / m³ 2 TOC extract samples were collected after washing. TOC extract values ​​recorded for the test apparatus containing the sintered depth filter medium of Examples ID3-7 were compared to the typical TOC flashout target (100 L / m³) of MILLISTAK+(R) HC (MilliporeSigma, Burlington, Massachusetts). 2 The TOC is far below 3 ppm in the flashout.

[0080] The sintered depth filter medium was prepared as described for Example ID 3-7. As shown in Figure 4, 23 cm 2 One of the mini-cap filter devices (square) was exposed to a gamma ray irradiation dose of 40-60 kGy, while the other (round) was not exposed. Each mini-cap filter device was treated with 100 L / m³ of 600 LMH. 2 Milli-Q water was poured through each at a rate of 25 L / m². 2 TOC extract samples were collected after washing. TOC extract values ​​recorded for the test apparatus containing the sintered depth filter medium of Examples ID3-7 were typical TOC flashout targets (100 L / m³) for MILLISTAK+(R) HC. 2 The TOC level was well below 3 ppm in flashout. No significant increase in TOC extract was observed in equipment exposed to 40-60 kGy of gamma rays. These results indicate that the sintered depth filter medium of the present invention would not require pre-use equipment cleaning for typical clarification operations used in biopharmaceutical manufacturing. Furthermore, the constituent materials of the sintered depth filter medium exhibit good stability against gamma rays.

[0081] The sintered depth filter medium was prepared as described in Example ID3-7. As shown in Figure 5, a 100 L / m³ filter medium was introduced into the mini-cap filter apparatus at 600 LMH.2 Milli-Q water was poured through each at a rate of 25 L / m². 2 Metal extract samples were collected after washing. The metal extracts were measured by inductively coupled plasma atomic emission spectroscopy (ICP-OES). The metal extract values ​​recorded for the test apparatus containing the sintered depth filter medium of Example ID3-7 were below the instrument's detection limit (0.02 ppm) for all metals except silicon and sodium.

[0082] The sintered depth filter medium was prepared as described for Example ID3-7. As shown in Figure 6, 23 cm 2 One of the mini-cap filter devices was subjected to a gamma ray irradiation dose of 40-60 kGy. Each mini-cap filter device was supplied with 100 L / m³ of 600 LMH. 2 Milli-Q water was poured through each at a rate of 25 L / m². 2 Metal extract samples were collected after cleaning. The metal extracts were measured by inductively coupled plasma atomic emission spectroscopy (ICP-OES). No significant increase in metal extracts was observed in the apparatus exposed to 40-60 kGy of gamma rays. These results indicate that the sintered depth filter medium of the present invention would not require pre-use apparatus cleaning for typical clarification operations used in biopharmaceutical manufacturing. Furthermore, the constituent materials of the sintered depth filter medium exhibit excellent stability against gamma rays.

[0083] [Example 4]

[0084] Preparation of the sintered depth filter medium of the present invention. Polyethylene binder PE (MIPELON(R) XM-221u polyethylene binder, Mitsui Chemicals America, Inc.) and crosslinked poly(4-vinylpyridine) adsorbent PVP. The PVP adsorbent contains poly(4-vinylpyridine) crosslinked with 2% divinylbenzene and poly(4-vinylpyridine-co-ethylvinylbenzene) crosslinked with 25% divinylbenzene, both purchased from MilliporeSigma in St. Louis, Missouri, USA. These materials were mixed in the ratios shown in Table 4 below. The powder mixture was distributed onto a large metal baking sheet with a PTFE release film. Using a drawbar, the powder mixture was evenly distributed to a thickness of 2 mm. The baking sheet was placed in a preheated oven at 165°C for 120 minutes. The sample was removed from the oven and allowed to cool to room temperature.

[0085] [Table 4]

[0086] [Example 5]

[0087] Water flow rate test of sintered depth filter medium after gamma ray irradiation. 23 cm³ containing the sintered depth filter medium described in Example 1. 2 A mini-cap test apparatus was prepared. It contained the sintered depth filter medium of the present invention, with a capacity of 23 cm². 2 One of the mini-cap test devices was subjected to gamma ray irradiation at a dose of 40-60 kGy. The described mini-cap device was attached to a peristaltic pump, and deionized water was pumped into the device. The flow rate was measured using a scale and timer. The pressure drop was measured using an electronic pressure transducer. The results of the water flow rate test are shown in Table 5 below. The filtration device containing the sintered depth filtration medium of the present invention exhibits a sufficiently low pressure drop at the relevant flux for application in the operation of a recovered material clarification unit.

[0088] [Table 5]

[0089] [Example 6]

[0090] Clarification of the recovered CHO cell culture. 23 cm³ containing the sintered depth filter medium described in Example 3. 2 A mini-cap test apparatus was prepared. It contained the sintered depth filter medium of the present invention, with a capacity of 23 cm². 2 One of the mini-cap test devices was subjected to gamma ray irradiation at a dose of 40-60 kGy. The filter test device was subjected to 25.7 × 10 6 The challenge was performed with CHO cell culture harvested with a total cell density (89% viability) of cells / ml. The described mini-cap device was attached to a peristaltic pump, and the cell culture harvested was pumped into the device. The volume of the filtrate was continuously recorded using a scale system and data recorder. Pre-use cleaning of the device containing the sintered depth filter medium described in Example 3 was not required. The filter pressure drop was measured using an electronic pressure transducer. The filtration resistance profile as a function of the filter throughput was plotted (see Table 6 and Figure 7), and these were compared with a conventional cellulose-based depth filter (MILLISTAK+(R) CE25 filter medium) tested as a control sample. Several observations can be made from the results of this experiment. (1) No significant increase in system resistance was observed for the filter medium of Example ID6-6. Increased PE binder packing resulted in increased filter retention, as indicated by the higher filter resistance profiles of ID6-2 to 6-5, for example. (2) The filter resistance profiles of Examples ID6-4 and 6-5 were similar. These results indicate that the sintered depth filter media of Example ID3-7 were not significantly decomposed by gamma radiation doses of 40-60 kGy. (3) The observed filter resistance profile and retention characteristics are comparable to those of conventional cellulosic depth filtration media (MILLISTAK+(R) CE, MilliporeSigma, Burlington, Massachusetts) in the recovery clarification unit operation.

[0091] [Table 6]

[0092] [Example 7]

[0093] Clarification of the model supply flow (Peptone HY-SOY(R) T). 23 cm³ containing the sintered depth filter medium described in Example ID3-2. 2 A mini-cap test apparatus was prepared. The filter test apparatus was set up with a flow rate of 600 LMH and 100 L / m³. 2 Milli-Q water was passed through the filter. The filter test apparatus was challenged with a model feed stream containing a 15 g / L suspension of Peptone HY-SOY(R) T (P6463, MilliporeSigma) in DI (distilled, deionized) water. The turbidity of the model feed solution was measured to be 179 NTU at a 10-fold dilution. This model feed stream was selected to approximate the depth filter clogging characteristics encountered in typical secondary clarification applications. Secondary clarification processes are generally used for particulate removal or turbidity reduction for certain downstream intermediates in selected applications, including post-centrifugation, post-perfusion bioreactors, post-cell retention apparatuses, post-primary depth filtration, and post-protein A. The described mini-cap apparatus was mounted on a peristaltic pump, and the Peptone HY-SOY(R) T feed stream was pumped into the apparatus, with the filtrate volume continuously recorded by scale and data recorder. The filter pressure drop was measured using an electronic pressure transducer. The filtration resistance profile was plotted as a function of filter throughput. Please refer to Table 7 and Figure 8. Several observations can be made from the results of this experiment. The observed filter resistance profile and retention characteristics (filtrate turbidity reduction) are comparable to the performance of conventional single-layer diatomaceous earth-based depth filtration media grades (MILLISTAK+(R) DE type filter media, MilliporeSigma, Burlington, Massachusetts) in secondary clarification unit operations.

[0094] [Table 7]

[0095] [Example 8]

[0096] Commercial prior art media (e.g., DE and CE media) exhibit high inorganic extractability (US Patent No. 7,673,757, the '757 patent is incorporated herein by reference in its entirety). As a result, 50 L / m³ 2 After washing, a high conductivity value of 8-12 μS / cm was measured for the filtrate. The medium described in the '757 patent showed a decrease in inorganic extract content, and the conductivity value of the filtrate was 58 L / m³. 2 After washing, it decreases to 2 μS / cm. See the prior art reference diagram, Figure 9, where the medium in the '757 patent is indicated by the name "Celpure". Based on the data provided in the '757 patent, a person skilled in the art can use CE or DE filter media for 50 L / m 2 You will recognize that a pre-use cleaning volume exceeding this amount is required. However, at least 58 L / m³ is needed. 2 Although the reduced pre-use cleaning volume is sufficient for the medium of the '757 patent, this required cleaning volume is still considered unacceptable.

[0097] In contrast, the PE:PS 1:1 filter media described in our specification exhibits produces very little inorganic extract from this filter material, thus significantly reducing the pre-use washing volume or eliminating the need for washing altogether. The conductivity of the filtrate is 25 L / m³. 2 The concentration is less than 0.5 μS / cm after washing (see Figure 13). Those skilled in the art will recognize, in view of the teachings herein, that filter media having such low inorganic extracts do not require pre-use washing.

[0098] In subsequent studies, samples of CE media, DE media, and PE:PS 1:1 filter media were exposed to a sterile dose of gamma rays (25-40 kGy). Both CE and DE media showed an increase in the conductivity of the filtrate after gamma irradiation (see Figures 11 and 12, respectively), but no increase was observed in the PE:PS 1:1 filter material described herein (Figure 13). Furthermore, the conductivity of the filtrate of the PE:PS 1:1 filter media was observed to be 1 / 50 to 1 / 100 of that of the tested CE and DE filter media samples.

[0099] [Example 9]

[0100] In this prophetic embodiment, a composite material of the type described in Example 3 of the '757 patent is washed with clean deionized water (MILLIQ(R), MilliporeSigma, Burlington, Massachusetts) at a specified washing volume of 58 L / m². 2 The conductivity of the subsequent effluent is measured. The conductivity value is interpreted as representing the level of soluble metals present in the filter medium. Figure 14 shows the conductivity values ​​obtained for various Celapure / polyethylene composite material samples of the '757 patent compared to the values ​​measured for the PE:PS 1:1 material of the present invention (see Figure 13). Conductivity data recorded for MILLISTAK+(R) CE25 (Figure 11) and DE40 depth filter media (Figure 12) are also provided for comparison. The conductivity data for the effluent of the Celapure / polyethylene composite material samples of the '757 patent is 58 L / m 2After washing, it shows a level of approximately 2 μS / cm. Since the conductivity value of the effluent represents the level of soluble metals present in the filter medium, these values ​​are not expected to increase or decrease in the prior art Celpure / polyethylene composite after exposure to gamma rays. Page 7, column 2, line 14 of the '757 patent states, "The extracts (inorganic substances) that cause conductivity do not originate solely from diatomaceous earth, or primarily from it, and in fact, the composite material of the present invention, which does not contain cellulose and thermosetting binders, results in a 75-90% reduction in effluent conductivity compared to conventional cellulose media." However, while the effluent conductivity is substantially reduced with the filter material of the '757 patent, the contribution of metal extracts cannot be excluded from the diatomaceous earth filter aid used in the composite material described in the reference. In contrast, when using the PE:PS 1:1 filter medium of the present invention, the effluent conductivity is further reduced to 95% compared to conventional cellulose media such as DE40, and a fraction of that compared to the material of the '757 patent. Depth filter materials such as the PE:PS 1:1 filter media described in the present invention will not require "pre-use washing to reduce the level of organic or inorganic contaminants to an acceptable level before use," as required by prior art filter materials including the filter media of the '757 patent ('757 patent, column 1, line 53).

Claims

1. It is a depth filter, a) A sintered depth filter medium containing a thermally fused mixture of polyethylene binders, b) One or more adsorbents selected from the group consisting of PrAOH, PrCH, MB1 / 1h, polystyrene adsorbent, poly(4-vinylbenzene) crosslinked with 2% divinylbenzene, and poly(4-vinylpyridine-co-ethylvinylbenzene) crosslinked with 25% divinylbenzene. A depth filter that includes this filter.

2. The depth filter according to claim 1, which is significantly homogeneous.

3. The depth filter according to claim 1, having a thickness of approximately 1 mm to approximately 4 mm.

4. The depth filter according to claim 3, having a thickness of approximately 2 mm to approximately 3 mm.

5. The depth filter according to claim 1, wherein the wet strength binder resin is specifically excluded.

6. The depth filter according to claim 1, wherein the ratio of polyethylene to the adsorbent is about 1:10 to about 10:

1.

7. The depth filter according to claim 1, wherein the ratio of polyethylene to the adsorbent is about 1:5 to about 5:

1.

8. The depth filter according to claim 1, wherein the ratio of polyethylene to the adsorbent is about 1:2 to about 2:

1.

9. The depth filter according to claim 1, wherein the ratio of polyethylene to the adsorbent is approximately 1:

1.

10. The depth filter according to claim 1, wherein the size of the polystyrene adsorbent is approximately 10 μm to approximately 120 μm in diameter.

11. The depth filter according to claim 1, wherein the size of the polystyrene adsorbent is approximately 30 μm to 100 μm in diameter.

12. The depth filter according to claim 1, wherein the size of the polystyrene absorbent is approximately 50 μm to 80 μm in diameter.

13. The depth filter according to claim 1, wherein the size of the polystyrene absorbent is approximately 60 μm to 70 μm in diameter.

14. The depth filter according to claim 1, wherein the size of the polystyrene absorbent is approximately 63 μm in diameter.

15. A method for producing a sintered depth filter medium, comprising: mixing a polyethylene binder with an adsorbent selected from the group consisting of PrAOH, PrCH, MB1 / 1h, polystyrene adsorbent, poly(4-vinylbenzene) crosslinked with 2% divinylbenzene, and poly(4-vinylpyridine-co-ethylvinylbenzene) crosslinked with 25% divinylbenzene to produce a mixture; spreading the mixture to a substantially uniform thickness; and heating the mixture at about 165°C for about 60 minutes.

16. A sintered depth filter medium produced by the method of claim 15, which is significantly homogeneous.

17. A sintered depth filter medium having a thickness of approximately 1 mm to approximately 4 mm, manufactured by the method described in claim 15.

18. A sintered depth filter medium having a thickness of approximately 2 mm to approximately 3 mm, manufactured by the method described in claim 17.

19. A sintered depth filter medium prepared by the method of claim 15, wherein the wet strength binder resin is particularly excluded.

20. A sintered depth filter medium prepared by the method of claim 15, wherein the ratio of polyethylene to the adsorbent is about 1:10 to about 10:

1.

21. A sintered depth filter medium prepared by the method of claim 15, wherein the ratio of polyethylene to the adsorbent is about 1:5 to about 5:

1.

22. A sintered depth filter medium prepared by the method of claim 15, wherein the ratio of polyethylene to the adsorbent is about 1:2 to about 2:

1.

23. A sintered depth filter medium prepared by the method of claim 15, wherein the ratio of polyethylene to the adsorbent is approximately 1:

1.

24. A sintered depth filter medium prepared by the method of claim 15, wherein the size of the adsorbent is approximately 10 μm to approximately 120 μm in diameter.

25. A sintered depth filter medium prepared by the method of claim 24, wherein the size of the adsorbent is approximately 30 μm to 100 μm in diameter.

26. A sintered depth filter medium prepared by the method of claim 25, wherein the size of the adsorbent is approximately 50 μm to 80 μm in diameter.

27. A sintered depth filter medium prepared by the method of claim 26, wherein the size of the adsorbent is approximately 60 μm to 70 μm in diameter.

28. A sintered depth filter medium prepared by the method of claim 27, wherein the size of the adsorbent is approximately 63 μm in diameter.

29. A method for clarifying a supply stream containing a target protein, a) Prepare a depth filter containing a sintered depth filter medium that includes a polyethylene binder together with an adsorbent selected from the group consisting of PrAOH, PrCH, MB1 / 1h, polystyrene adsorbent, poly(4-vinylbenzene) crosslinked with 2% divinylbenzene, and poly(4-vinylpyridine-co-ethylvinylbenzene) crosslinked with 25% divinylbenzene. b) A method in which the depth filter is brought into contact with the feed stream such that the target protein passes through the depth filter and is retained in the permeate, and contaminants are adsorbed by the depth filter, thereby increasing the concentration of the target protein to contaminants in the permeate by at least 20% compared to the ratio in the feed stream.

30. The method according to claim 29, wherein the concentration of the target protein relative to the contaminant in the permeate is increased by at least 50% compared to the ratio in the feed stream.

31. The method according to claim 29, wherein the concentration of the target protein relative to the contaminant in the permeate is increased by at least 100% compared to the ratio in the feed stream.

32. The method according to claim 29, wherein the depth filter medium is significantly homogeneous.

33. The method according to claim 29, wherein the depth filter has a thickness of about 1 mm to about 4 mm.

34. The method according to claim 33, wherein the depth filter has a thickness of approximately 2 mm to approximately 3 mm.

35. The method according to claim 29, wherein the wet strength binder resin is particularly excluded.

36. The method according to claim 29, wherein the ratio of polyethylene to adsorbent is about 1:10 to about 10:

1.

37. The method according to claim 29, wherein the ratio of polyethylene to adsorbent is about 1:5 to about 5:

1.

38. The method according to claim 29, wherein the ratio of polyethylene to adsorbent is about 1:2 to about 2:

1.

39. The method according to claim 29, wherein the ratio of polyethylene to adsorbent is approximately 1:

1.

40. The method according to claim 29, wherein the size of the adsorbent is approximately 10 μm to approximately 120 μm in diameter.

41. The method according to claim 40, wherein the size of the adsorbent is approximately 30 μm to 100 μm in diameter.

42. The method according to claim 41, wherein the size of the adsorbent is approximately 50 μm to 80 μm in diameter.

43. The method according to claim 42, wherein the size of the adsorbent is approximately 60 μm to 70 μm in diameter.

44. The method according to claim 43, wherein the size of the adsorbent is approximately 63 μm in diameter.

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