WO2025216784A1 - Vgt depth filtration media - Google Patents

Abstract

The present invention is to depth filter media that are suitable for the clarification of cell culture media streams used for viral and gene therapy (VGT) applications and that also have compatibility with radiation based sterilization methods.

Description

VGT DEPTH FILTRATION MEDIA

Cross-Reference to Related Applications

[0001] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63/631 ,795, filed April 9, 2024, the entire contents of which is incorporated herein by reference.

Background

[0002] Due to the growing demand for viral and gene therapies, improved filtration and purification technologies for the downstream bioprocessing of these products are needed in order to improve product recovery and to reduce manufacturing costs. Commercially available depth filtration products are well-suited for use in many biopharmaceutical applications, especially for the production of monoclonal antibodies (mAbs). Monoclonal antibodies and viral vectors, such as adeno-associated virus (AAV) and lentivirus (LV), may present different surface charges at a given application pH, due to their different isoelectric points. As a result, an increased binding of the negatively charged viral vectors such as AAV or LV to the positively charged depth filter media can occur. The increased binding can result in a decreased yield of the viral vector product. In addition, it is desireable for the depth filtration media used in viral gene therapy (VGT) harvest applications to be compatible with a pre-use sterilization or bioburden-reduction step to minimize the risk of contamination. This is particularly important in processes where a sterilizing- grade 0.22 pm filtration step is not feasible due to the large size of the viral vector. The materials of construction typically used in the depth filtration media known in the art (cellulose, polyacrylic fibers, wet-strength binder resins) may be unstable when exposed to high doses of radiation, such as with gamma or X-ray-based sterilization methods. In these situations, an increased extractibles profile, degradation of material physical properties, polymer embrittlement, or changes in the filtration performance of the depth filtration media may be observed. As a result of these needs, improved depth filtration media are desired to enable improved VGT product recovery and compatibility with radiation based sterilization methods. Summary of the Invention

[0003] This specification describes depth filter media compositions and their use in the clarification of cell culture feedstreams. The depth filter media compositions are intended for viral and gene therapy (VGT) applications. Depth filter media known in the art are not necessarily designed for these applications and demonstrate a decreased product recovery due to the binding of negatively charged viral particles to a positively charged depth filter media.

[0004] Moreover, the materials of construction used to prepare the prior art depth filter products may be incompatible with radiation-based sterilization methods, such as gamma ray or X-ray irradiation. The depth filter media described herein demonstrate increased compatibility with radiation-based sterilization methods and increase recovery of VGT products such as AAV2 and lentiviruses (LV) as well as monoclonal antibodies (mAb)as comared to prior art depth filter media.

[0005] It is contemplated that the present invention comprises, consists essential of or consists of a depth filter medium suitable for high virus recovery and resistance to sterilizing radiation, said depth filter media comprising: a) i) a polymer fiber comprising cellulose, ii) a silica filter aid, iii) a wet-strength binder resin and iv) carboxymethyl cellulose (CMC); b) wherein, the polymer fiber is at from about 40% to about 60%, the silica filter aid is from about 40% to about 60%, the wet-strength binder resin is at from about 2% to about 3% and having a zeta-potential of about 0 mV to -100 mV, -20mV to - 80mV or -60 to -70 mV, at a pH of about 7.0 to about 7.5, and the CMC is at about 0.5% or less; c) the filter medium has been sterilized by gamma irradiation, and; d) wherein, when used to recover viruses, virus recovery is at least 60 percent.

[0006] It is further contemplated that the virus to be recovered is lentivirus. [0007] It is still further contemplated that the virus recovery it at least 75%. [0008] It is still further contemplated that virus recovery is at least 90%.

[0009] It is still further contemplated that the polymer fiber is from about 45% to 55%.

[0010] It is still further contemplated that the silica filter aid is from about 45% to 55%. [0011] It is contemplated that the present invention comprises, consists essentially of or consists of a depth filter medium suitable for high virus recovery and resistance to sterilizing radiation, said depth filter media comprising: a) i) a polymer fiber comprising polyethylene, ii) a silica filter aid and iii) a wet-strength binder resin; b) wherein, the polymer fiber is at from about 70% to about 95%, the silica filter aid is from 0% to about 25 % and the wet-strength binder resin is at from about 2% to 3% and has a zeta-potential of about 300 mV to 400 mV, at a pH from about 5.0 to about 6.5; c) the filter medium has been sterilized by gamma irradiation, and; d) wherein, when used to recover viruses, virus recovery is at least 60 percent.

[0012] It is further contemplated that the depth filter media explicitly excludes CMC.

[0013] It is still further contemplated that the silica filter aid is from more than 0% to about 25%.

[0014] It is still further contemplates that the virus to be recovered is adeno- associated virus, for example, AAV2.

[0015] It is still contemplated that the virus recovery it at least 75%.

[0016] It is still further contemplated that the virus recovery is at least 90%. [0017] It is contemplated that the present invention comprises, consists essentially of or consists of a depth filter medium suitable for high monoclonal antibody recovery and resistance to sterilizing radiation, said depth filter media comprising: a) i) a polymer fiber comprising polyethylene, ii) a silica filter aid and iii) a wet-strength binder resin; b) wherein, the polymer fiber is at from about 40% to about 60%, the silica filter aid is from about 40% to about 60% and the wet-strength binder resin is at from about 2% to about 3% and has a high ionic charge as defined by having a zeta-potential of about 300 mV to about 400 mV at a pH from about 5.0 to about 5.5, and; c) the filter medium has been sterilized by gamma irradiation, and; d) wherein, when used to recover monoclonal antibodies (mAb), antibody recovery is at least 60 percent.

[0018] It is further contemplated that the depth filter media explicitly excludes CMC.

[0019] It is still further contemplated that the mAb recovery it at least 75%. [0020] It is still further contemplated that the mAb recovery is at least 90%. [0021] It is still further contemplated that the polymer fiber of the depth filter media is from about 45% to 55%.

[0022] It is still further contemplated that the silica filter aid of the depth filter media is from about 45% to 55%.

[0023] It is contemplated that the present invention comprises, consists essentially of or consists of a method of increasing the purity of a viral suspension, the method comprising: a) providing, i) a virial suspension with impurities in need of purification, ii) a depth filter having a media comprising cellulose polymer fiber at from about 40% to about 60%, silica filter aid from about 40% to about 60%, wet-strength binder resin is at from about 2% to about 3% and having a zeta-potential of about mV to about 0 mV to -100 mV, -20mV to -80mV or -60 to -70 mV at pH about 7.0 to about 7.5, and the CMC is at about 0.5% or less; b) filtering said viral suspension through said filter media; c) wherein, said recovery of viruses in said viral suspension is at least 60% and said impurities are decreased by at least 50%.

[0024] It is further contemplated that the virus to be recovered is lentivirus. [0025] It is still further contemplated that the virus recovery it at least 75%. [0026] It is still further contemplated that the virus recovery is at least 90%. [0027] It is still further contemplated that the polymer fiber of the depth filter media is from about 45% to 55%.

[0028] It is still further contemplated that the silica filter aid of the depth filter media is from about 45% to 55%.

[0029] It is contemplated that the present invention comprises, consists essentially of or consists of a method of increasing the purity of a viral suspension, the method comprising: a) providing, i) a virial suspension with impurities in need of purification, ii) a filter comprising polyethylene polymer fiber at from about 70% to about 95%, silica filter aid from about 40% to about 60% and wet-strength binder resin at from about 2% to about 3% and having a high ionic charge as defined by having a zeta-potential of about 100 mV to about 300 mV at a pH from about 5.0 to about 5.5; b) filtering said viral suspension through said filter media; c) wherein, said recovery of viruses in said viral suspension is at least 60% and said impurities are decreased by at least 50%. [0030] It is further contemplated that the depth filter media explicitly excludes CMC.

[0031] It is still further contemplated that the silica filter aid is from more than 0% to about 25%.

[0032] It is still further contemplated that the virus is adeno-associated virus, for example AAV2.

[0033] It is still further contemplated that the virus recovery it at least 75%. [0034] It is still further contemplated that the virus recovery is at least 90%. [0035] It is still further contemplated that the polymer fiber of the depth filter media is from about 45% to 55%.

[0036] It is still further contemplated that the silica filter aid of the depth filter media is from about 45% to 55%.

[0037] It is contemplated that the present invention comprises, consists essentially of or consists of a method of increasing the purity of a monoclonal antibody (mAb) suspension, the method comprising: a) providing, i) a mAb suspension with impurities in need of purification, ii) a filter comprising a polyethylene polymer fiber at from about 40% to about 60%, the silica filter aid at from about 40% to about 60% and the wet-strength binder resin at from about 2% to about 3% and has a high ionic charge as defined by having a zeta-potential of about 300 mV to about 400 mV at a pH from about 5.0 to about 5.5; b) filtering said mAb suspension through said filter media; c) wherein, said recovery of mAb in said mAb suspension is at least 60% and said impurities are decreased by at least 50%.

[0038] It is further contemplated that CMC is explicitly excluded from the depth filter media.

[0039] It is still further contemplated that the virus recovery it at least 75%. [0040] It is still further contemplated that the virus recovery is at least 90%. [0041 ] It is still further contemplated that the polymer fiber of the depth filter media is from about 45% to 55%.

[0042] It is still further contemplated that the silica filter aid of the depth filter media is from about 45% to 55%. Description of the Figures

[0043] Fig. 1 shows examples of nanopod filters (Fig. 1 A) with a filtration area of 5 cm² and Micro20 (Fig. 1 B) with a filtration area of 20 cm².

[0044] Figs. 2A - F show the ^-potential of new-developed filter sheets grouped by pre-/post-gamma irradiation. (A) Un-irratiated cellulose-based filter media. (B) Gamma-irradiated cellulose filer media. (C) Un-irradiated PE-based filter media. (D) Gamma-irradiated PE-based filter media. (E) Unirradiated PA-based filter meida. (F) Gamma-irradiated PA-based filter media.

[0045] Figs. 3A - F show the ^-potential of new-developed filter sheets grouped by different positive charged resin. (A) High positive charged cellulose-based filters. (B) Low positive charged cellulose-based filters. (C) High positive charged PE-based filters. (D) Low positive charged PE-based filters. (E) High positive charged PA filters. (F) Low positive charged PA filters.

[0046] Figs. 4A - H show trends for total organic carbon (TOC) extractables for depth filter media of Examples 1-1 to 1-23 as a function of pre-use flush volume. (A) Un-irratiated cellulose-based filter media. (B) Gamma-irradiated cellulose filer media. (C) Un-irradiated PE-based filter media. (D) Gammairradiated PE-based filter media. (E) Un-irradiated PA filter meida. (F) Gamma-irradiated PA filter media (G) Un-irradiated benchmark filter media. (H) Gamma irradiated benchmark filter media.

[0047] Figs. 5A - F show filter resistance profiles for cellulose and polyethylene-based depth filters from Example 1-1 to 1-12. (A) Un-irradiated cellulose-based filters in 5 cm² nanopod devices; (B) gamma-irradiated cellulose-based filters in 5 cm² nanopod devices; (C) un-irradiated PE/Si filters using 20 cm² Micro20 devices; (D) gamma-irradiated PE/Si filter using 20 cm² Micro20 devices; (E) un-irradiated filter media in 5 cm² nanopods; (F) gamma-irradiated benchmark filter media in 5 cm² nanopods.

[0048] Figs. 6A - F show phiX (bacteriophage (pX174 (PhiX174)) yield for depth filtration media un- and post-gamma irradiation. (A) Un-irradiated cellulose-based filter in 5 cm² nanopod devices; (B) gamma-irradiated cellulose-based filters in 5 cm² nanopod devices; (C) un-irradiated PE/Si filters using 20 cm² Micro20 devices; (D) gamma-irradiated PE/Si filter using 20 cm² Micro20 devices; (E) un-irradiated benchmark filter media in 5 cm² nanopod devices; (F) gamma-irradiated benchmark filter media in 5 cm² nanopod devices.

[0049] Figs. 7A - F show turbidity reduction using a bacteriophage model feedstream. (A) un-irradiated cellulose-based filter in 5 cm² nanopod devices; (B) gamma-irradiated cellulose-based filters in 5 cm² nanopod devices; (C) un-irradiated PE/Si filters using 20 cm² Micro20 devices; (D) gammairradiated PE/Si filter using 20 cm² Micro20 devices; (E) un-irradiated benchmark filter media in 5 cm² nanopods; (F) gamma-irradiated benchmark filter media in 5 cm² nanopods.

[0050] Figs. 8A - B show filtration performance of high-charged PE/Si filter 20 cm² Micro20 devices. (A) Filter capacities; (B) turbidity reduction.

[0051] Figs. 9A - C shows AAV2 filtration performance on single layer benchmark filter media. (A) resistance profile; (B) turbidity breakthrough curve; (C) combined results of AAV2 recovery with filter capacity.

[0052] Figs. 10A - D show filtration performance on high-charge filters using an AAV2 feedstream. (A) Resistance profile; (B) turbidity reduction; (C) AAV2 recovery; (D) combined results of filter capacity (bars) and filtrate AAV2 recovery (dots).

[0053] Figs. 11 A - C show filtration performance on medium-charge PE filters challenged with an AAV2 feedstream. (A) Resistance vs. throughput; (B) AAV2 recovery vs. throughput; (C) turbidity breakthrough curve.

[0054] Figs. 12A - C show filtration performance on PA filters challenged with an AAV2 feedstream, (a) Resistance profile; (b) AAV2 recovery of filtrate; (c) turbidity of filtrate.

[0055] Figs. 13A - C show filtration performance on X-ray irradiated filters challenged with an AAV2 feedstream. (A) Resistance profile; (B) AAV2 recovery of filtrate; (C) turbidity breakthrough curve. [0056] Figs. 14A - C show lentivirus filtration performance on benchmark filter media. (A) Resistance profile; (B) turbidity breakthrough curve; (C) combined results of lentivirus recovery and filter capacity.

[0057] Figs. 15A - C show filtration performance on cellulose-based filter sheets challenged with a lentivirus feedstream. (A) Resistance profile; (B) turbidity breakthrough curve; (C) combined results of lentivirus recovery and filter capacity(lnput feed turbidity: 464 NTU (nephelometric turbidity units).

[0058] Figs. 16A - E show filtration performance on PE-based filters challenged with a lentivirus feedstream. (A) Resistance profile on high and low charged PE filters; (B) turbidity breakthrough curve on high and low charged PE filters; (C) resistance profile on medium charged PE filters; (D) turbidity breakthrough curve on medium charged PE filters; (E) lentivirus recovery on PE filters.

[0059] Figs. 17A - C shows filtration performance on PA-based filters challenged with a lentivirus feedstream. (A) Resistance profile; (B) lentivirus infectiveness recovery; (C) turbidity and capacity.

[0060] Figs. 18A - C show lentivirus filtration performance on X-ray irradiated filters. (A) Resistance profile on X-ray irradiated filters; (B) turbidity breakthrough curves; (C) combined results of lentivirus recovery and filter capacity.

[0061] Figs. 19A - C show secondary clarification of a CHO centrate feedstream (mAbO2) including the filtration performance of unirradiated and gamma-irradiated experimental depth filter media. (A) Resistance profile; (B) turbidity breakthrough curve; (C) mAb titer recovery.

[0062] Figs. 20A - C show AAX/2 filtration performance on (A) medium-high charged polyethylene filters, (B) medium-low charged polyethylene filters and (C) low charged polyethylene filters.

[0063] Fig. 21 shows a chart of data points used in Figs. 2 and 3 and Example 2. Detained Description of the Invention

[0064] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following reference will provide one of skill with a general definition of many of the terms used in this invention: Process Scale Bioseparations for the Biopharmaceutical Industry, edited by Abhinav A. Shukla, Mark R. Etzel, and Shishir Gadam. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0065] When introducing elements of the present disclosure or the preferred embodiments(s) thereof, the articles "a," "an," "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0066] The transitional phrases “comprising,” “consisting essentially of” and “consisting of” have the meanings as given in MPEP 2111.03 (Manual of Patent Examining Procedure; United States Patent and Trademark Office, 9^th Ed., Revision Feb 2023 [R-07.2022]). Any claims using the transitional phrase “consisting essentially of” will be understood as reciting only essential elements (/.e., the basic and novel characteristics) of the invention and any other elements recited in dependent claims are understood to be non- essential to the invention recited in the claim from which they depend.

Likewise, any additional elements over those claimed that are described in a prior art reference(s) are excluded from the claims by use of the transitional phrase “consisting essentially of.”

[0067] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about" whether or not explicitly indicated. The term "about" generally refers to a range of numbers that one would consider equivalent to the recited value (/.e., having the same function or result). In many instances, the term "about" may include numbers that are rounded to the nearest significant figure. [0068] Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Moreover, all ranges disclosed herein are to be understood to encompass all sub ranges subsumed therein. In other words, any ranges of numbers are considered to include all numbers within the range as if they were explicitly recited.

[0069] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. All publications, patents and patent applications cited herein are also representative of what one of ordinary skill in the art would understand with regard to the field of the invention at the time of the invention.

[0070] Technical Definitions

[0071] Before describing the present invention in further detail, a number of terms will be defined. Use of these terms does not limit the scope of the invention but only serve to facilitate the description of the invention.

[0072] The term “depth filter” is a filter that achieves filtration within the depth of the filter material. Particle separation in depth filters results from entrapment by and/or adsorption to, the fiber and filter aid matrix comprising the filter material. In other words, depth filters are filters that use a porous filtration medium to retain particles throughout the medium by entrapment and/or adsorption, rather than just on the surface of the medium.

[0073] As used herein the phrase "cell culture" includes cells, cell debris and colloidal particles, biomolecule of interest, host cell proteins (HCP) and DNA. Cell cultures may be of any cells or cell lines typically or non-typically used by one of ordinary skill in the art for the production of, for example, a desired protein or other bio-molecule. For example, the product may be an antibody, a virus, viral vector or viral particle (collectively, “viral agents”). “Cell culture fluid” includes cells, cell debris and colloidal particles, biomolecule of interest, host cell proteins (HCP) and DNA deposited into the fluid by culturing cells in cell culture media.

[0074] One example of a typical cell used in the art is the Chinese hamster ovary cell (CHO). Thus, the terms "Chinese hamster ovary cell protein" and "CHOP" as used interchangeably herein, refer to a mixture of host cell proteins ("HCP") derived from a Chinese hamster ovary ("CHO") cell culture. The HCP or CHOP is generally present as an impurity in a cell culture medium or lysate (e.g., a harvested cell culture fluid containing a protein or polypeptide of interest (e.g., an antibody or viral agent expressed in a CHO cell). Generally, the amount of CHOP present in a mixture comprising a protein of interest provides a measure of the degree of purity for the protein of interest. Typically, the amount of CHOP in a protein mixture is expressed in parts per million relative to the amount of the protein of interest in the mixture. [0075] The term "clarification step" or simply "clarification," as used herein, generally refers to one or more steps used initially in the purification of biomolecules. The clarification step generally comprises removal of cells and/or cellular debris using one or more steps including any of the following alone or various combinations thereof, e.g., centrifugation and depth filtration, tangential flow filtration, microfiltration, precipitation, flocculation and settling. In some embodiments, the present invention provides an improvement over the conventional clarification step commonly used in various purification schemes. The clarification step generally involves the removal of one or more undesirable entities and is typically performed prior to a step involving capture of the desired target molecule. Another aspect of clarification is the removal of soluble and insoluble components in a sample which may later on result in the fouling of a sterile filter in a purification process, thereby making the overall purification process more economical. The clarification step often includes a primary clarification step(s) upstream and a secondary clarification downstream. The clarification of cell culture harvests and high-solids feedstocks from large harvest volumes from modern production batch bioreactors (<25,000 L) and high cell densities often require primary, as well as secondary clarification steps prior to any subsequent chromatography operations and the like. [0076] The terms "coarse filtration" or "coarse/medium filtration," as used herein, generally refer to the removal of mostly whole cells and some cellular debris in the purification of biomolecules.

[0077] The term "fine filtration," as used herein, generally refers to the removal of mostly cellular debris, colloidal particles and soluble impurities such as HCP, DNA, endotoxins, viruses and lipids in the purification of biomolecules. [0078] Filter throughput values are generally expressed in terms of "liters/square meter" or "L/m²" though for equivalent comparisons, "column volume" or "CV" is used to account for large differences of thickness between samples.

[0079] The terms "contaminant," "impurity" and "debris" are used interchangeably herein and refer to any foreign or objectionable material, including a biological macromolecule such as a DNA, an RNA, one or more host cell proteins (HCPs or CHOPs), endotoxins, lipids and one or more additives which may be present in a sample containing a protein, antibody, viral agent, or polypeptide of interest being separated from one or more of the foreign or objectionable molecules using a depth filter according to the present invention.

[0080] It is understood that where the host cell is another mammalian, nonmammalian, or bacterial cell type, for example, E. coli, yeast cell, insect or plant, HCP refers to the proteins, other than target proteins, found in a lysate of the host cell.

[0081] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, /.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. [0082] As used herein the term "organic extractable(s)" refers to contaminants that in the presence of water or other aqueous solutions used during flushing, can potentially migrate or be extracted from materials used to make filter media or membranes, such as porous depth filter media. These contaminants may also include the materials of construction themselves which could potentially shed from the filter during use thereby requiring pre-flushing of the filter prior to use of the filter to remove said organic extractable(s). [0083] The term "total organic carbon" and "TOC" refers to the measurement of organic molecules present in an aqueous solution such as water and measured as carbon content. Analytical techniques used to measure TOC typically involve oxidation of all organic molecules in solution to carbon dioxide, measuring the resultant CO2 concentration, and correlating this response to a known carbon concentration.

[0084] The term “inorganic extractable” refers to trace metallic species, including heavy metals that may be extracted from the filter into the process fluid. These metallic species may be measured by analytical techniques such as inductively coupled plasma optical emission spectrometry (ICP-OES), ICP mass spectrometry (ICP-MS), and graphite furnace atomic absorption spectrometer (GFAAS) techniques.

[0085] “Pre-flush” is defined herein as flushing a filter prior to use, usually with sterile water, to remove organic and inorganic extractables from the filter prior to use.

[0086] The term "parts per million" or "ppm" are used interchangeably herein. [0087] The terms "target molecule," "target biomolecule," "desired target molecule" and "desired target biomolecule” are used interchangeably herein and generally refer to a polypeptide or product of interest (e.g., a monoclonal antibody) which is desired to be purified or separated from one or more undesirable entities, e.g., one or more impurities, which may be present in a sample containing the polypeptide or product of interest.

[0088] As used herein the term "throughput" means the volume filtered through a filter divided by the frontal area of the filter. The throughput is expressed in terms of L of fluid filtered I m² of filter area.

[0089] As used herein the term "dirt holding capacity" is equivalent to filter throughput of a given cell culture fluid, either from direct harvest or previously clarified. Higher throughput represents higher dirt holding capacity.

[0090] As used herein and as understood to one of ordinary skill in the art, “wet-strength binder resin” is a resin that allows for the formation of filter sheets 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 are resins comprising synthetic polymers of urea or melamine-formaldehyde based polymers, polyaminopolyamide-epichlorohydrin (PAE) polymers and glyoxalated polyacrylamide (GPAM) resins.

[0091] The term “^-potential” (zeta-potential) is defined in the art as the electrical potential at the slipping plane. This plane is the interface which separates mobile fluid from fluid that remains attached to the surface. Zeta potential is a scientific term for electrokinetic potential. More specifically, The -potential is the potential difference across phase boundaries between solids and liquids. It's a measure of the electrical charge of particles are that are suspended in liquid. Zeta potential is measured in volts or millivolts (mV).

[0092] Depth Filters of the Present Invention

[0093] The depth filters and depth filter media of the present invention comprise, consist essentially of or consist of unique and hitherto unknown combinations of polymer fiber, filter aid, wet strength resin and, in some instances, carboxymethyl cellulose. The wet strength resin used in the depth filter media of the present invention has a defined ^-potential (/.e., the electrical potential of the interfacial double layer at the location of the slipping plane relative to a point in the bulk fluid away from the interface - The zetapotential is caused by the net electrical charge contained within the region bounded by the slipping plane.). The electric charge influences the attraction of repulsion of components of the fluid stream and, while the present invention is not limited by theory, is believed to influence attraction and binding of impurities in the filter stream and repulsion of desired component(s) of the filter stream. In this regard, the depth filter media, the filters made therewith, and processes and methods utilizing said depth filter media and said depth filters, allow for the increased purification of the target components of the present invention (e.g., viruses and monoclonal antibodies) while also maintaining integrity and effectiveness after gamma irradiation.

[0094] Gamma irradiation is commonly used to sterilize devices used in the medical and biotechnology industry and is a form or ionizing radiation sterilization. While commonly used, gamma irradiation is not without limitations or problems. One such limitation is that it can cause detrimental effects to items exposed to it including rendering filter media less suitable and less effective for particular desired uses. The inventors of the present invention have found that the unique formulations for the depth filter media of the present invention combine both effective and industry desired filtration properties while also being resistant to the detrimental effects of gamma irradiation.

[0095] The depth filter media of the present invention combines polymer fiber with a filter aid and a wet strength resin. In some instances carboxymethyl cellulose is also included. In other instances, carboxymethyl cellulose is specifically excluded. The polymer resin can be any resin known to one of skill in the art but in a preferred embodiment, the resin used in making the polymer fiber is polyethylene. Size or size range (length and diameter) of the fiber is selected based on the desired use, desired porosity and desired liquid flow characteristics through the filter media. One of skill in the art can select the suitable fiber size or size range based on desired uses of the depth filter media. In some instances, more than one fiber size range may be selected for use in the depth filter media of the present invention.

[0096] The filter aid likewise can be any filter aid known to one of skill in the art. Filer aids known in the art include, but are not limited to, perlite, porous glass, zeolite, silica gel, activated carbons and magnesium silicate. Preferred filer aids for use in the present invention are, for example, silica gel or magnesium silicate.

[0097] As used herein and as understood to one of ordinary skill in the art, “wet-strength binder resin” is a resin that allows for the formation of filter sheets 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. Non-limiting examples of wet-strength resins are resins comprising synthetic polymers of urea or melamine-formaldehyde based polymers, polyaminopolyamide-epichlorohydrin (PAE) polymers and glyoxalated polyacrylamide (GPAM) resins.

[0098] Representative examples of the wet strength resin of the present invention are given, supra. Additionally, several commercially available wet strength resins known in the art. These are given in Table 5, below, with the chemistry type indicated in the first column and the surface charge type and relative (-potential indicated in the second column. [0099] Table 5

[0100] An exemplary list of commercially available wet strength resins known in the art includes, but is not limited to: Seiko PMC WS4020, WS4030, WS4027, TS4070, Buckman Bubond® 650, Bubond® 818, Bubond® 5928, Solenis Kymene™ 557, Kymene™ 725, Kymene™ 830, Kymene™ X-Cel, Kymene™ XRV20-AO, Kymene™ 777LX, Kymene™ 888ULX, Chang Chun Group W5500, W5600, W5500HLC, CHT Group Quimex 8090, Quimex T 9090, Kemira Fennostrength™ PA21 , Fennostrength™ 4063, Korfez Kimya PPR-150, Ecolab Metrix Armor+, Georgia Pacific AMRES® 8855, AMRES® 1110E, and AMRES® HP-100E. Details of the wet strength resins can be found, for example, at https://www.seikopmc.co.jp/en/, https://www.ecolab.com/offerings/wet-strength-additives, https://www.thermaxglobal.com/chemicals/, https://www.ccp.com.tw/ccpweb.nsf/homepage7openagent , https://www.cht.com/en/, https://www.kemira.com/products/wet-strength- additives/, https://www.solenis.com/en/products-and-services/pulp- paper/strength-additives/ and in their product brochures: Kymene™ Wet Strength Resin Innovations. Further details can also be found in US 7,683,121 B2 (Ecolab) and US 7,781 ,501 B2 (Georgia Pacific Chemicals, LLC). One of skill in the art can determine the -potential of the wet-strength resin of any filter media made therefrom, if needed.

[0101] Further details of wet strength resins suitable for the present invention are also given in US 7,252,735 and EP 1 632 604 B1 , both which are representative of what is known by one of skill in the art at the time of this invention and are incorporated herein by reference

[0102] The designations of High, Medium High, Medium Low and Low with regard to “Type of Charge” in the chart, above, translate into (-potential ranges of approximately: over 200 mV, 100 - 200 mV, 0 - 100 mV and zero mV and below, respectively, at a pH of about 7.0 for the filter media of the present invention which is made with the referenced wet-strength resin.

[0103] Methods of Making the Depth Filters of the Present Invention [0104] The depth filters of the present invention may be made by a wet-laid process. In an non-limiting example, polymer fibers are dispersed into water or other solution(s); afterwards, filter aids and resins are blended into the mixture. Then, the liquid is drawn through a vacuum flask, and the solid disk is dried in an oven. The process results in a non-woven material that can have varied physical characteristics based on the materials used.

[0105] In addition to the wet-laid method, discussed above, depth filters may also be made by a dry process. In one aspect, a binder and sorbent are mixed at the desired ratio. The mixture is, for example, distributed evenly onto large metal baking sheet and leveled with a drawbar a desired height. The baking sheet is then baked for about 60 minutes at about 165°C. See, also for example, WO 2023/025906 A1 to EMD Millipore, which is representative of what one of skill in the art is knowledgeable of at the time of the present invention with regard to certain methods of making depth filters. It is incorporated herein by reference in its entirety. After cooling, the sheets of filter material were cut to desired size. One of skill in the art will be able to determine times and temperatures for making the depth filters of the present invention that utilize a different binder or binders or different sorbent or sorbents, without undue experimentation with the guidance provided by the present invention. Likewise, one of skill in the art will be able to determine times and temperatures for making the depth filters of the present invention that are thicker or thinner than 2 mm, without undue experimentation with the guidance provided by the present invention.

[0106] Preferred methods of making the depth filters of the present invention are provided in the exemplification section, infra.

[0107] Methods of use of the Depth Filters of the Present Invention

[0108] The present invention contemplates methods of use for the depth filters of the present invention. For example, the depth filters of the present invention may be used for filtering cell culture media (/.e., a feed stream) in which a bioproduct has been produced. The bioproduct may be, for example, a viral vector(s) or viruses. In another aspect, the bioproduct may be an antibody(ies) or other desired protein(s) including glycoproteins. Depending on the stage of processing of the feed stream, filters of differing pore size, porosity, and flow grade may be used. One of skill in the art will be able to determine the correct pore size, porosity and flow grade for a particular use with the aid of the teachings of this specification. In one aspect, the synthetic depth filter of the present invention may be used to clarify a feed stream prior to further downstream processing. A clarification step would remove, for example, cell culture debris such as whole cells, ruptured cells, large host cell proteins (HOP), and other contaminants, etc., while allowing the bioproduct, e g., the target proteins to pass through the filter. In one embodiment, the bioproduct is a target protein and may be a monoclonal antibody, humanized monoclonal antibody, CAR-T cell produced antibody, etc. In another embodiment, the bioproduct is a virus or viral vector. The bioproduct may also be other genetically engineered or naturally occurring proteins, for example, produced by a cell transfected with an expression vector engineered for expression of the desired target protein or naturally expressed by a given cell type. Likewise, the bioproduct may be an engineered virus or viral vector. [0109] The result of passing a cell culture feed stream through a depth filter of the present invention is to increase the relative proportion of target bioproduct (for example, viruses or mAbs) to the contaminant(s) in the permeate (/.e., the feed stream that passes through the filter). In this context, the contaminants may include whole cells, cellular debris, host cell proteins (HCP) and colloidal particles. In one aspect, the concentration of target bioproduct to contaminant(s) is increased 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%, as compared to the feed stream. In one aspect, the concentration of target bioproduct to contaminant(s) is increased 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% as compared to the feed stream. In one aspect, the concentration of target bioproduct to contaminant(s) is increased by from 10% - 1000%, from 50% - 500% and from 10% - 100%. In another aspect, the percent of contaminant is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or higher.

[0110] The present invention contemplates that the depth filter media of the present invention is suitable for improved VGT product recovery and antibodies while also having compatibility with radiation based sterilization methods such as, but not limited to, gamma irradiation.

[0111] As various changes could be made in the above-described and below- exemplified compositions, devices and processes without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

Exemplification

[0112] Example 1 - Depth Filtration Media

[0113] In a general process, the components of depth filtration media include wet-laid cellulose or polyethylene fiber, filter aids such as diatomaceous earth (DE), silica(s) or perlite, and a resin binder with a positive charge. (Pieracci, John P., et al., Chapter 9 - Industry Review of Cell Separation and Product Harvesting Methods. Biopharmaceutical Processing, s.l. : Elsevier, 2018, pp. 165-206). A wet-laid process is a common process to be used to form the filter media. Polymer fibers are dispersed into water or other solutions; afterwards, filter aids and resins are blended into the mixture. Then, the liquid is drawn through a vacuum flask, and the solid disk is dried in an oven. Depth filtration media were prepared using the formulation components as described in Table 1a below. As a first component, synthetic fibers such as polyethylene and polyaramid were selected. These materials of construction demonstrate a high compatibility to gamma or X-ray radiation. As a second component, filter aids such as DE, perlite, porous glass, zeolite, silica gel, activated carbons or magnesium silicate were selected. (Yavorksy, David P. Adsorbent Filter Media for Removal of Biological Comtaminants in Process Liquids. US 2007/0193938 A1 United States of America, 2007). Loading levels were around 50 wt% of the depth filter media formulation (but in practice may vary depending on the application - one of skill in the art, with the teaching of this specification, would be able to determine a suitable wt% for an identified use). As a third component, wet-strength binder resins comprising a low, medium or high positive charged resin were evaluated (see, Table 5, above). These resins with different surface charges are believed to affect the adsorption ability of the products or impurities from different viral vector feedstreams, which further influence the product yield and impurity clearance. As an optional fourth component, carboxymethyl cellulose (CMC), was included for better formation of filter sheets as described.

[0114] After formation, the filters sheets were built into small filter test devices (nanopod) with a filtration area of 5 cm² or 20 cm² Micro20 filter devices as seen in Fig. 1.

[0115] To evaluate the effect of gamma ray or X-ray radiation, test devices containing the filter media of the present invention were irradiated at 25-40 kGy dose levels and were compared with control devices not exposed to radiation. From the literature, (Gamma Compatible Materials. [Online] 2014. [Cited: August 16, 2023.] https://www.nordian.com/wp- comtent/uploads/2014/10/GT_Gamma_Compatible_Materials.pdf) cellulose, polyethylene, and polyaramid are compatible with 25-40 kGy gamma/X-ray irradiation. Thus, the experimental filter media comprising cellulose fiber, polyethylene (PE) fiber or polyaramid (PA) fiber with other components were built into filter handsheets as listed in Table 1a and Table 2. As a comparative examples, single filter layers from commercial Millistak+®, Millistak® HC Pro, Clarisolve®, and Polygard® (all from MilliporeSigma, Burlington, MA) devices were also prepared (by the manufacturer) as described below. Table 1a Formulations of new-developed filter sheets.

[0116] Other than the compositions of filter materials, devices formats for the different examples, flow rate, and gamma irradiation conditions are also listed in Table 1b. Un-irradiated samples were referred as Example 1-X-a, gammairradiated samples were number as Example 1-X-b, and X-ray-irradiated samples were number as Example 1-X-c.

Table 1b. Other properties of experimental filter media.

N = no radiation

Table 2 Formulations of 2^nd group of new-developed polyethylene filter media.

[0117] Example 2 - ^-Potential of Depth Filter Media

[0118] The choice of wet-strength binder resin can influence the surface charge of the depth filter media. In addition, depending on the materials of construction, acidic and basic functional groups on the surface of the component fibers, the filter aid, and the binder resin can impact the surface charge depending on the solution pH. These changes in surface charge can influence the adsorption properties of the viral vector or mAb product, host cell proteins, DNA, colloidal matter, solids, as well as other charged or uncharged impurities. Zeta-potential (^-potential) measurements were made to monitor the charge behavior of the depth filter media grades at various solution pH values. A general procedure for the zeta potential measurement is as follows: 20 g filter sheets were blended in 1 L tap water. Half of the mixture was titrated with 1 M NaOH to adjust the solution pH from neutral to basic; while the other half was titrated with 1 M HCI to adjust the solution pH from neutral to acidic. The ^-potential at each pH value was tested by Mutek SZP-10 system zeta potential instrument from BTG Americas Inc., Pointe-Claire QC. All the cellulose, PE, and PA handsheets pre-/post-gamma irradiation were tested, and the trends were shown in Figs. 2 and 3. The results in Fig. 2 are grouped by different polymer fibers and pre-/post-gamma irradiation, while the results in Fig. 3 are grouped by different surface charge on the filters. In general, the filter sheets comprising a binder resin with a stronger positive charge resin showed higher (-potential. After gamma irradiation, there were some changes in the trends of -potential to pH observed.

[0119] For some of the cellulose-based experimental filter sheets (/.e., Example 1-1 and 1-4), as seen in Figs. 2A, 2B, 3A and 3B, a decrease in (- potential was observed after gamma irradiation, while other samples showed little change in (-potential (/.e., Examples 1-2 and 1-3). Examples 1-1 and 1-4 contain CMC, which is a cellulose derivative with carboxymethyl groups (- CH2-COOH) for better filter sheets formation. Upon gamma irradiation, the CMC in Examples 1-1 and 1-4 may undergo crosslinking reactions, affecting the measured (-potential. (Radiation crosslinking of carboxymethylcellulose of various degree of substiturtion at high concentration in aqueous solutions of natural pH, Wach, Radoslaw A., et a!., 5, s.l. : Radiation Physics and Chemistry, 2003, Vol. 68. 771-779). Examples 1-7 and 1-8 comprising of magnesium silicate presented higher (-potential after gamma irradiation. [0120] For the PE-containing filter sheets, as seen in Fig. 2C, the trend from high-charged to low-charged is clear. But Example 1-12, comprising medium- charged resin and CMC, showed lower (-potential than Example 1-10 without CMC, which was comparable to low-charged filter media (/.e., Examples 1-8 and 1-11). The low (-potential on Example 1-12 might be due to the existence of negatively charged CMC. The trend with charged resin in Fig. 2D changed little after gamma irradiation. When it is compared with the samples pre-/post-gamma irradiation, in Figs. 3C and 3D, it appears that high-charged PE-based experimental filter media demonstrated no significant change in (-potential indicating good stability post-gamma irradiation. For low-/medium-charged PE-based experimental filter media, only Example 1-9 comprising medium-charged resin gave a decreased (-potential, others (Examples 1-8, 1-11 , and 1-12) showed an increase in (-potential after gamma irradiation. While not wishing to be limited by theory, the decrease in (-potential on Example 1-9 might be ascribed to the degradation of charged binder resins after gamma irradiation. [0121] For the PA-containing filter sheets, the ^-potential of the highly charged filter Example 1-13 and 1-14 decreased at pH=7 after gamma irradiation, while the ^-potential increased on low charged filter Example 1 -15 and 1-16 after gamma irradiation. While not wishing to be limited by theory, the reason might be the degradation of charged binder resin after gamma irradiation.

[0122] Example 3 - Depth Filter Media Extractables

[0123] Depth filter media prepared using synthetic fibers can demonstrate lower extractables than depth filters made from naturally derived materials of construction such as cellulose and diatomaceous earth (DE). (Woo, Maybelle, et al., High Capacity Composite Depth Filter Media with Low Extractables. US 2016/0114272 A1 United States of America, 2016). In the extractable TOC study, the tested filters were built into Micro20 devices with a filter area of 20 cm². Milli-Q water was flushed through the filters at 300 LMH (10 mL/min), which is the recommended pre-use flush flow rate in filtration experiments. Total organic carbon (TOC) Samples were collected at 20, 40, 60, 80, and 100 L/m² throughput into a 40 mL sample vial with septa cap. For Examples 1-1 to 1-16, the TOC level is lower than some of the benchmark materials, e.g., Millistak+® filter media, Examples 1-17 and 1-18 comprising cellulose or cellulose/DE. A reduced extractables profile for synthetic depth filters can enable a significant reduction in pre-use flush volumes. The TOC extractables profile as a function of pre-use flush volume are shown in Fig. 4. In general, the TOC extractables level decreases with increasing the water flush volumes. Table 3 lists the measured TOC level at throughputs of 60 and 100 L/m² for the various depth filter media grades from Example 1-1 to 1 - 22. All the depth filter media grades demonstrate an elevated TOC level after gamma irradiation. Filter sheets comprising synthetic materials PE and PA (Example 1 -7-b to 1-16-b) showed lower TOC levels compared to cellulose- based filter sheets (Examples 1-1 -b to 1-4-b) indicating a lower volume of preflush liquid needed before filtration. The polyaramid-containing filter media from Examples 1 -13-b to 1-16-b showed the lowest TOC levels after gamma radiation, indicating improved radiation stability for this material at 25-40 kGy gamma irradiation dose levels. Table 3 TOC level at 60 and 100 L/m² for depth filter media of Examples 1-1 to 1-22.

[0124] Example 4 - General Procedure for Depth Filtration

[0125] Filter test devices were attached to PendoTech pressure transducers from PendoTECH, (West Windsor Township, NJ) and a peristaltic pump using 1/16-inch tubing. The devices were flushed with deionized (DI) water at a flowrate of 300 LMH until 50 L/m² throughput, which was 2.5 mL/min for 5 cm² nanopod and 10 mL/min for 20 cm² Micro20 for 10 min. The cell culture harvest feedstream was then pumped through the devices at a flowrate of 150 LMH and the filtrate was collected individually from each device. The filter pressure was continuously monitored and recorded by means of a data recorder (PendoTech, Model PDKT-PCS-NFFSS). The filtration testing was stopped when the system pressure reached 20 psi. A sample of the filtrate was collected every 50 L/m² throughput for turbidity testing and phage recovery assay. Filtrate turbidity measurements were performed using a turbidity meter (HACH 2100Q Portable Turbidity Meter).

[0126] Example 5 - Filtration Testing Using a Bacteriophage <pX174 Model Feedstream

[0127] As a preliminary screening evaluation, the filter media candidates from Example 1 were evaluated with a bacteriophage cpX174 (PhiX174) feedstream, because <pX174 bacteriophage shows similar properties as AAV2 including virus size (Electron microscopic studies of bacteriophage phiX 174 intact and “eclipsing” particles and the genome by the staining and shadowing method. Yazaki, K. 3, s.l.: Journal of Virology Methods, 1981 , Vol. 2. 159-167; Adeno-associated virus strctural biology as a tool in vector development. Drouin, L. M. and Agbandje-McKenna, M. 12, s.l.: Future Virology, 2013, Vol. 8. 1183-1 199) and isoelectric point (Isoelectric points of viruses. Michen, B. and Graule, T. 2, s.l.: Isoelectric points of viruses, 2010, Vol. 109. 388-397). A bacteriophage feedstream derived from human embryonic kidney 293T cells (HEK293T) was used for these experiments. After cell growth, a buffer solution including Triton X-100, magnesium chloride (MgCh), BENZONASE®endonuclease (MilliporeSigma, Burlington, MA), and sodium chloride (NaCI) was added into the feedstream to lyse the cells. The lysed feedstream was then spiked by <pX174 bacteriophage before filtration.

[0128] The filter capacities for the cellulose and polyethylene-based (PE- based) filters are shown in Fig. 5. From this data, we find that cellulose- based experimental filter media achieved high filtration capacities with minimal pressure build up for capacities as high as 300 L/m². For PE-based filter media (Examples 1-7 to 12), 20 cm² Micro20 devices were tested until 150 L/m² throughput due to the limited volume of the feedstream. Filtration data for several commercial depth filter media grades is also provided in Fig. 5E and 5F. The filter throughputs at 20 psi for the cellulose-based and PE- based experimental filters (Example 1-1 to 1-12) compare favorably to the filter media layers used in commercial Millistak+® and Mi llistak+® HC Pro depth filters. Cellulose- and PE-based experimental filter sheets showed higher capacities than single layer components in commercial Millistak+®, Millistak+® HC Pro, Clarisolve®, or Polygard® (Examples 1-17 to 1-23). After gamma irradiation, increased pressure was observed at capacities < 300 L/m² on cellulose-based experimental filter media (Examples 1-1 to 1-4) indicating a small reduction in filtration capacity after gamma irradiation. PE-based filter sheets (Examples 1-7 to 1-12) showed no difference in filter capacity post gamma irradiation until 150 L/m² indicating better gamma tolerance than cellulose-based filter media.

[0129] In addition to the assessment of the depth filtration capacity, the percent recovery of the bacteriophage model is another important performance consideration. (pX174 yields above 80% are acceptable for the purposes of this study. In Fig. 6A, all the cellulose-based filters provided yields greater than 80% yield. After gamma irradiation, Example 1-1 , 1-2, and 1-4 post-gamma irradiation met the requirement as in Fig. 6B indicating the stability with 25-40 kGy gamma irradiation.

[0130] In this study, the filtrate clarity was also assessed with turbidity samples collected every 50 L/m². To permit comparison of filter retention performance between individual bioreactor harvests, the turbidity data was normalized into turbidity reduction in percentage, and these results are shown in Fig. 7 For the cellulose-based experimental filters, Examples 1-4-a, 1-5-a, and 1-6-a were able to remove at least 80% cell debris without gamma irradiation, while Examples 1-1 -b, 1-2-b, 1-3-b, and 1-4-b showed better turbidity reduction performance with gamma irradiation. From this study, high- charged cellulose-based experimental filter media, Example 1-4, gave comparable performance pre- and post-gamma irradiation.

[0131] As seen in Figs. 5C and 5D, the depth filter devices built no pressure until at least up to 150 L/m². No change was observed post-gamma irradiation. When comparing with the filtrate clarity, Examples 1-7 and 1-10 with high charged resin removed more cells or cell debris. After exposing to gamma irradiation, high-charged filters, Examples 1-7 and 1-10 also showed the highest turbidity reduction.

[0132] Based on the preliminary results from the bacteriophage model feedstream, depth filter media Examples 1-7 and 1-10 were tested to a throughput 300 L/m² using Micro20 devices to measure their filtration capacities. In Fig. 8A, we find that all the tested filters gave capacities greater than 300 L/m² and the resistances on the filters post-gamma irradiation at 300 L/m² were higher than controlled filters (/.e., 210% for Example 1-7, and 167% for Example 1-10). Filtrate samples were analyzed for the final phage yield and achieved 100% recovery for the high-charge PE/Si filters. The turbidity breakthrough results in Fig. 8B indicate a good performance in removing cell or cell debris on the high-charge filters. The results also indicate that Example 1-10 showed no significant difference pre- /post-gamma irradiation. Comparing to cellulose-based experimental filters (Examples 1-1-a to 1-4-a) in Figs. 5B and 7B, Example 1-10-b post-gamma irradiation showed lower resistance (/.e., about 0.01 LMH/psi) and comparable turbidity drop level. Moreover, Example 1-10 showed little difference pre- /post-gamma irradiation implying the stability of this material toward gamma irradiation.

[0133] Example 6 - AAV2 Feedstream Study

[0134] The depth filter media described in Examples 1 -4, 1 -7, and 1 -10 were tested with an AAV2 feedstream. HEK293 transfected by AAV2 was used as the feedstream. Similar to the bacteriophage feeds, the AAV2 feed was lysed by a buffer solution before pumping through the test filters. 10.5% TWEEN® 20 (polysorbate 20), 1 M MgCL, and 100U/mL BENZONASE® endonuclease were added into to the AAV2 transfected HEK293T. The feedstream was mixed in a shaker at 37 °C and 155 rpm for 2 hours. Afterwards, the feedstream was agitated at the same condition for another 1 hour after adding 5 M NaCI.

[0135] AAV2 filtration performance on benchmark single layer media was carried out as a comparison. The results are shown in Fig. 9.

[0136] The filtration performance of the experimental depth filter media using the AAV2 feed is shown in Fig. 10. Example 1-4 displayed the highest filtration capacity reaching to about 180 L/m² as shown in Fig. 10A, filtration capacity was unchanged post gamma-irradiation. A significant turbidity reduction from the feedstream is shown in Fig. 10B. From Figs. 10C and 10D, it is shown that the cellulose-containing depth filter media Example 1-4 gave the highest filtration capacity (about 180 L/m²) and the highest AAV2 recovery (> 70%). Moreover, no significant difference in filter performance was observed for this material pre-/post-gamma irradiation. The PE-based filters (Examples 1-7 and 1-10) showed reduced filter capacities compared with Example 1-4 material, and this may be attributed to a higher (-potential at pH=7. The medium-charged PE filter, Examples 1-9 and 1-12 are suggested to be tested for AAV2 clarification. Although Example 1-4 showed a higher filter capacity than Examples 1-7 and 1-10, the PE-based filter media is preferable due to all-synthetic materials of construction.

[0137] Depth filter media containing medium-charged binder resin and PE fibers (Examples 1-9 and 1-12) were challenged using an AAV2 feedstream. The results are shown in Fig. 11. Example 1-9 depth filter media comprising polyethylene, silica gel, and a medium-charge binder resin achieved a high filter capacity (/.e., 300 L/m²) as shown in Fig. 11 A. The AAV2 recovery was approximately 68% and this value is higher than the benchmark filter media (Example 1-19, Millistak+® DE40) and slightly lower than the cellulose-based experimental depth filter, Example 1-4 (/.e., 74%) as shown in Fig. 11B. The turbidity of the filtrate was slightly higher than either Example 1-4 or 1-12, as shown in Fig. 11C

[0138] A third group of filter media comprising fibrillated polyaramid, silica gel, high or low-charge binder resin, and/or CMC was evaluated. The filtration performance is shown in Fig. 12. When compared to the benchmark commercial filter media or other filter media grades described above, the PA filter media gave the lowest filter capacities ranging between 28-50 L/m² as shown in Fig. 12A. A decreased AAV2 product recovery for the PA filter media was also observed, ranging between 57-70% as shown in Fig. 12B. The filter retention for the PA filter media is good, with filtrate turbidities lower than 20 NTU as shown in Fig. 12C. In the benchmark or the cellulose or PE containing experimental filter media described above, the AAV2 recovery and solid retention were observed to be related to the surface -potential, in which high-charged filters presented better filtrate turbidity but lower AAV2 recovery. In contrast, the AAV2 recovery and filtrate turbidity of PA filter media did not correlate to the ^-potential indicating that the pore structure of PA filter media was tight. In addition, no significant drop in filter performance was shown pre- /post-gamma irradiation.

[0139] X-ray irradiation was also evaluated for biopharmaceutical sterilization applications. Thus, candidates comprising different materials of construction including Examples 1-4, 1-9, and 1-13 were irradiated by 25-40 kGy X-ray. Both control and X-ray irradiated filters were then challenged with an AAV2 feedstream. The evaluation results are shown in Fig. 13. From the results, no significant difference was observed on the tested filter media pre-/post-X-ray irradiation. Examples 1-4 and 1-9 achieved both high filtration capacities (Fig. 13A) and AAV2 recovery (up to 80%) (Fig. 13B), while Example 1-13 exhibited a reduced filter capacity (Fig. 13B). This result is similar to the results shown for the gamma-irradiated filters. Based on the turbidity measurements, all the tested filters showed good performance for filter retention. Therefore, 25-40 kGy X-ray irradiation showed little effect on the tested filters.

[0140] After the investigation of the first group of new developed filter media, we found that medium charged polyethylene filters (SPE5, Examples 1-9- a/b/c) displayed a low filter capacity but a high AAV2 recovery and a high filtrate clarity. The low filter capacity might be ascribed to the existence of filter aids which might adsorb more impurities leading to the low capacity. Thus, a second group of new-developed polyethylene filter media (Table 2) with different filter aids loading were tested as shown in Fig. 20. Different charged resins, medium-high, medium-low, and low charged resins, were investigated in this test. From the figure, it is clear that the filter capacity of the new-developed filter media with no filter aids was the highest. As the filter aids loading increased, the filter capacity decreased as shown in Fig. 20A. The AAV2 recovery also decreased as the increase of the filter aids loading due to the binding of AAV2 with silica gel. When the filter aids loading was 0%, the AAV2 recovery was the highest as shown in Fig. 20B. On the contrary, the filtrate clarity went higher as the increase of filter aids loading, which is because the filter aids could bind the impurities. Comparing with the different charged resins, the medium-high charged filter achieved the highest filter capacity while medium-low charged filter showed the highest AAV2 recovery as shown in Fig. 20C.

[0141] Example 7 - Lentivirus Feedstream Study

[0142] The clarification of feedstreams containing lentivirus is challenging using existing commercially available products due to the significant yield loss attributed to charge-based product adsorption. The negatively charged lentivirus is believed to strongly adsorb to the positively charged depth filter sheets resulting in a significant loss in recovery. In this study, depth filter sheets with high, medium, and low charged binder resins were evaluated in lentivirus clarification applications.

[0143] For a comparison, benchmark single layer filter media (Examples 1-17 to 1-23) were tested with lentivirus transfected HEK293T feedstream, and the results are shown in Figs. 14A - 14C.

[0144] A HEK293T feed transfected by lentivirus was used for testing lentivirus filtration performance on tested filter media. From the group of cellulose-based filter media described in Examples 1-1 to 1-4, filter sheets comprising of a low charge resin binder (Example 1-1 and 1-2) and a high charge resin binder (Example 1-4) were evaluated using a lentivirus feedstream. Gamma irradiated samples were also evaluated. As seen in Figs. 15A and 15C, the highly positive-charged filter Example 1-4 gave the highest filtration capacity (e.g., 160 L/m² for controlled sample and 140 L/m² for gamma irradiated sample) and the lowest lentivirus infectiveness recovery. The increased lentivirus loss likely is due to the binding between the negatively charged LV and the positively charged depth filter. In contrast, Example 1-1 employs a low charge resin binder and this filter media achieved about 100% lentivirus recovery with a capacity of 130 L/m². The filtrate capacity, lentivirus recovery and filtrate clarity data are shown in Fig. 15C. The filtrate clarities of the cellulose-based filters were in a range of 2-11 NTU with a 100 L/m² throughput. Turbidity breakthrough was observed at a throughput of 150 L/m². Comparing with the commercial benchmark filter media (Example 1-17, Millistak+® CE50) in Fig. 14, Example 1-1 demonstrated a comparable filter capacity and lentivirus recovery, which were about 130 L/m² and 98% yield.

[0145] For the PE-based depth filter media of Examples 1-7 to 1-12, the filters were challenged using two separate lentivirus feedstreams. The input turbidity for the data presented in Fig. 16A and 16B was 1000+ NTU, while the input turbidity was 464 NTU for the data presented in Fig. 16C and 16D. Since a difference in cell density will impact the measured filtration capacities, the throughput curves and turbidity breakthrough curves are separated accordingly. In Figs. 16A and 16C, the high charged depth filters Examples 1- 7 and 1-10 gave increased filter capacities than the low charge filters Examples 1-8 and 1-11. After gamma irradiation, no significant drop in filtration capacity was observed between high- and low-charged filters. In addition, the filter capacities for the CMC-containing filters (Examples 1-10, 1- 11 , and 1-12) were slightly reduced versus Examples 1-7, 1-8, and 1-9. In Figs. 16B and 16D, all the filter grades afforded good solids retention based on the turbidity breakthrough data. The lentivirus recovery was shown in Fig. 16E, in which the recovery was related to surface charges on the filter media. Low-charged filter media, Examples 1-8 and 1-11 presented the highest lentivirus recovery, while high-charged filter media, Examples 1-7 and 1-10 showed the lowest lentivirus recovery. The reason is that lentivirus feedstream has negative charge, which could bind to the filter media with high positive resins. For the low-charged PE-based experimental filter media (Examples 1-8 and 1-11), the lentivirus recoveries were lower than Millitak+® CE50 as in Fig. 14, and high-/medium-charged PE-based experimental filter media (Examples 1-7 and 1-10) has lower filter capacities compared to Millistak+® CE50.

[0146] The group of depth filters from Examples 1-13 to 1-16 comprising fibril lated polyaramid (PA) was also evaluated using a lentivirus feedstream. The evaluation data is shown in Fig. 17. The PA filters demonstrated a low filtration capacity Fig. 17A. Like the results of the AAV2 study described in Example 6, above, low filter capacities between the range of 28 to 67 L/m² were observed for these samples. The high charged filter media achieved the higher filter capacities and no difference in solid retention performance was observed between the low- and high-charged filter media. Moreover, all the PA filter media showed good solid retention performance, and the filtrate turbidities were around 20 NTU, while the turbidity of the inlet feedstream was 1000+ NTU. Additionally, a slightly higher capacity on gamma-irradiated PA filter media was observed. Lentivirus yield is shown in Fig. 17B, which varied from 49% to 65% depending on different compositions. Low-charged filters, Examples 1-15 and 1-16 achieved higher lentivirus recoveries due to less product binding. In addition, lentivirus recovery on gamma irradiated filters were higher than controlled samples, which is consistent with the results of filter capacities. Table 4 Filter capacity and lentivirus infectiveness recovery values for different cellulose-, PE-, and PA- based filter sheets.

[0147] Similar experiments ran on X-ray irradiated filters with lentivirus feedstream. Candidate filter media with good lentivirus filtration performance in the previous experiment were selected for X-ray irradiation study including Example 1-17 from benchmark filters, Example 1-1 in cellulose-based experimental filters, Examples 1-8 and 1-11 in PE-based experimental filter, and Example 1-15 in PA-based experimental filter. As seen in resistance profile in Fig. 18A, the filters comprising CMC (/.e., Examples 1-1 , 1-11 and 1- 15) showed a stable performance post-X-ray irradiation, while the filter capacities on Examples 1-17 and 1-8 decreased after X-ray irradiation.

Moreover, cellulose-based filters including benchmark and experimental ones displayed higher filter capacities than PE or PA-based filters, which might be due to the open pores of the filters. Regarding the filtrate clarity, although the turbidity on cellulose-based filters, Examples 1-17 and 1-1 increased after X- ray irradiation, they were still able to remove the turbidity from about 800 NTU to less than 100 NTU in Fig. 18B. For the lentivirus recovery, it is clear in Fig. 18C that cellulose-based filters (Examples 1-17 and 1-1) achieved higher lentivirus recovery than PE- or PA-based filters (Examples 1-8, 1-11 , and 1- 15). Compared to the filter without CMC (Examples 1-17 and 1 -8), the filters comprising with CMC (Examples 1-1 , 1-11 , and 1-15) showed a stable filtration performance after X-ray irradiation.

[0148] Example 8 - Monoclonal antibody study

[0149] Current trends in the biopharmaceutical industry are directed towards the use of closed processing for downstream manufacturing processes. To meet these needs, pre-sterilized depth filtration media may be required. Many of the commercial depth filters currently used in these applications are not compatible with gamma- or x-ray-based irradiation methods. In this example, the performance of some of the gamma-compatible depth filtration materials described in this disclosure were evaluated using a mAb-containing CHO cell culture harvest feedstream. Examples 1-7, 1-13, and 1-19 were challenged using a CHO centrate feedstream (mAbO2) having an input turbidity of 250 NTU, and the resistance profile and turbidity breakthrough curve are shown in Fig. 19. As seen in resistance profile (Fig. 19A), Examples 1-13 (high- charged polyaramid filter) and 1-19 (Millistak+® DE40) showed no difference in the resistance profiles pre-/post-gamma irradiation. For Example 1-7, the high-charged polyethylene filter presented a lower resistance after gamma irradiation. For Example 1-13, the high-charged polyaramid filters gave a faster rise in filter resistance than the other samples indicating a tight pore structure, and these results are consistent with the results of AAV2 and lentivirus study. In Fig. 19B, the tested examples show a similar solid retention performance except Example 1-13-a, which showed turbidity breakthrough at throughput of approximately 150 L/m². In addition, no significant change in filtrate clarity was observed for Examples 1-7 and 1-13. The mAb recovery data is shown in Fig. 19C. The experimental filter media samples, Examples 1-7 and 1-13 also gave comparable mAb recovery to Millistak+® DE40 filter media.

Claims

CLAIMS \Ne Claim:

1 . A depth filter medium suitable for high virus recovery and resistance to sterilizing radiation, said depth filter media comprising: a. i) a polymer fiber comprising cellulose, ii) a silica filter aid, iii) a wetstrength binder resin and iv) carboxymethyl cellulose (CMC); b. wherein, the polymer fiber is at from about 40% to about 60%, the silica filter aid is from about 40% to about 60%, the wet-strength binder resin is at from about 2% to about 3% and having a zeta-potential of about zero mV to about -100 mV at a pH of about 7.0 to about 7.5, and the CMC is at about 0.5% or less; c. the filter medium has been sterilized by gamma irradiation, and; d. wherein, when used to recover viruses, virus recovery is at least 60 percent.

2. The depth filter media of Claim 1 , wherein said virus to be recovered is lentivirus.

3. The depth filter media of Claim 1 , wherein said virus recovery it at least 75%.

4. The depth filter media of Claim 1 , wherein said virus recovery is at least 90%.

5. The depth filter media of Claim 1 , wherein the polymer fiber is from about 45% to 55%.

6. The depth filter media of Claim 1 , wherein the silica filter aid is from about 45% to 55%.

7. A depth filter medium suitable for high virus recovery and resistance to sterilizing radiation, said depth filter media comprising: a. i) a polymer fiber comprising polyethylene, ii) a silica filter aid and iii) a wet-strength binder resin; b. wherein, the polymer fiber is at from about 70% to about 95%, the silica filter aid is from 0% to about 25 % and the wet-strength binder resin is at from about 2% to 3% and has a zeta-potential of about 100 mV to 300 mV at a pH from about 5.0 to about 6.5,; c. the filter medium has been sterilized by gamma irradiation, and; d. wherein, when used to recover viruses, virus recovery is at least 60 percent.

8. The depth filter media of Claim 7, wherein said depth filter media explicitly excludes CMC.

9. The depth filter media of Claim 7, wherein said silica filter aid is from more than 0% to about 25%.

10. The depth filter media of Claim 7, wherein said virus is adeno-associated virus (AAV2).

11 .The depth filter of Claim 7, wherein said virus recovery it at least 75%.

12. The depth filter of Claim 7, wherein said virus recovery is at least 90%.

13. A depth filter medium suitable for high monoclonal antibody recovery and resistance to sterilizing radiation, said depth filter media comprising: a. i) a polymer fiber comprising polyethylene, ii) a silica filter aid and iii) a wet-strength binder resin; b. wherein, the polymer fiber is at from about 40% to about 60%, the silica filter aid is from about 40% to about 60% and the wet-strength binder resin is at from about 2% to about 3% and has a high ionic charge as defined by having a zeta-potential of about 300 mV to about 400 mV at a pH from about 5.0 to about 5.5, and; c. the filter medium has been sterilized by gamma irradiation, and; d. wherein, when used to recover monoclonal antibodies (mAb), antibody recovery is at least 60 percent.

14. The depth filter media of Claim 13, wherein CMC is explicitly excluded.

15. The depth filter media of Claim 13, wherein said mAb recovery it at least 75%.

16. The depth filter media of Claim 13, wherein said mAb recovery is at least 90%.

17. The depth filter media of Claim 13, wherein the polymer fiber is from about 45% to 55%.

18. The depth filter media of Claim 13, wherein the silica filter aid is from about 45% to 55%.

19. A method of increasing the purity of a viral suspension, the method comprising: a. providing, i) a virial suspension with impurities in need of purification, ii) a depth filter having a media comprising cellulose polymer fiber at from about 40% to about 60%, silica filter aid from about 40% to about 60%, wet-strength binder resin is at from about 2% to about 3% and having a zeta-potential of about 0 mV to about -100 mV at pH about 7.0 to about 7.5, and the CMC is at about 0.5% or less; b. filtering said viral suspension through said filter media; c. wherein, said recovery of viruses in said viral suspension is at least 60% and said impurities are decreased by at least 50%.

20. The method of Claim 19, wherein said virus to be recovered is lentivirus.

21 .The method of Claim 19, wherein said virus recovery it at least 75%.

22. The method of Claim 19, wherein said virus recovery is at least 90%.

23. The method of Claim 19, wherein the polymer fiber is from about 45% to 55%.

24. The method of Claim 19, wherein the silica filter aid is from about 45% to 55%.

25. A method of increasing the purity of a viral suspension, the method comprising: a. providing, i) a virial suspension with impurities in need of purification, ii) a filter comprising polyethylene polymer fiber at from about 40% to about 60%, silica filter aid from about 40% to about 60% and wetstrength binder resin at from about 2% to about 3% and having a high ionic charge as defined by having a zeta-potential of about 100 mV to about 300 mV at a pH from about 5.0 to about 5.5; b. filtering said viral suspension through said filter media; c. wherein, said recovery of viruses in said viral suspension is at least 60% and said impurities are decreased by at least 50%.

26. The method of Claim 25, wherein said depth filter media explicitly excludes CMC.

27. The method of Claim 25, wherein said silica filter aid is from more than 0% to about 25%.

28. The method of Claim 25, wherein said virus is adeno-associated virus (AAV2).

29. The method of Claim 25, wherein said virus recovery it at least 75%.

30. The depth filter of Claim 25, wherein said virus recovery is at least 90%.

31 .A method of increasing the purity of a monoclonal antibody (mAb) suspension, the method comprising: a. providing, i) a mAb suspension with impurities in need of purification, ii) a filter comprising a polyethylene polymer fiber at from about 40% to about 60%, the silica filter aid at from about 40% to about 60% and the wet-strength binder resin at from about 2% to about 3% and has a high ionic charge as defined by having a zeta-potential of about 300 mV to about 400 mV at a pH from about 5.0 to about 5.5; b. filtering said mAb suspension through said filter media; c. wherein, said recovery of mAb in said mAb suspension is at least 60% and said impurities are decreased by at least 50%.

32. The method of Claim 31 , wherein CMC is explicitly excluded from the depth filter media.

33. The method of Claim 31 , wherein said mAb recovery it at least 75%.

34. The method of Claim 31 , wherein said mAb recovery is at least 90%.

35. The method of Claim 31 , wherein the polymer fiber is from about 45% to 55%.

36. The method of Claim 31 , wherein the silica filter aid is from about 45% to 55%.