Determination of pore size in microfilters

Abstract

The pore profile of the microfilter is determined by: (210) providing a saturated porous microfilter membrane including a first surface, a second surface, and a matrix disposed therebetween, the matrix being saturated with a storage solution; (220) contacting the saturated porous microfilter membrane with an intermediate solvent such that the storage solution dissolves in the intermediate solvent, thereby desaturating the membrane; (230) resaturating the membrane by applying a test fluid to the desaturated membrane; (240) applying a pressure to the first surface of the resaturated membrane, the pressure being applied by contacting the first surface with a gas or liquid; (250) detecting a flow of gas, liquid, and / or test fluid from the second surface in response to the pressure; and (260) determining a pore size profile of the porous microfilter membrane based on the level of pressure that results in the flow of gas, liquid, and / or test fluid from the second surface. Subsequently, the filter used to filter 281 the cell culture product can be selected by selecting 171 a filter with an appropriate pore size profile.

Description

[Technical field]

[0001] SUMMARY OF THE DISCLOSURE Embodiments herein relate to determining the pore size of a microfilter and methods for filtering cell culture products.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 254,468, filed October 11, 2021, which is incorporated by reference in its entirety. [Background technology]

[0003] Filters are used in many pharmaceutical and other industrial manufacturing applications. In the production of therapeutic proteins such as monoclonal antibodies, the therapeutic protein is harvested from the bioreactor in which it is produced. Harvesting often utilizes microfilters such as hollow fiber membrane (HFM) or flat sheet membrane (FSM) filters that separate the produced protein from the cells that produce it. Summary of the Invention [Means for solving the problem]

[0004] In some embodiments, a method of determining a pore size profile of a microfilter is described. The method may include providing a saturated porous microfilter membrane including a first surface, a second surface, and a matrix disposed therebetween, the matrix being saturated with a storage solution. The method further includes contacting the saturated porous microfilter membrane with an intermediate solvent to desaturate the membrane by dissolving the storage solution in the intermediate solvent. The method further includes applying a test fluid to the desaturated membrane to resaturate the membrane. The method further includes applying a pressure to the first surface of the resaturated membrane, the pressure being applied by contacting the first surface with a gas or liquid. The method further includes detecting a flow of the gas, liquid, and / or test fluid from the second surface in response to the pressure. The method further includes determining a pore size profile of the fine-pored microfilter membrane based on a level of pressure that results in a flow of the gas, liquid, and / or test fluid from the second surface.

[0005] In some embodiments, a method of filtering a cell culture product containing a therapeutic protein is described. The method may include providing a saturated porous microfilter membrane including a first surface, a second surface, and a matrix disposed therebetween, the matrix being saturated with a storage solution. The method further includes contacting the saturated porous microfilter membrane with an intermediate solvent to desaturate the membrane by dissolving the storage solution in the intermediate solvent. The method further includes applying a test fluid to the desaturated membrane to resaturate the membrane. The method further includes applying a pressure to the first surface of the resaturated membrane, the pressure being applied by contacting the first surface with a gas or liquid. The method further includes detecting a flow of the gas, liquid, and / or test fluid from the second surface in response to the pressure, and a pore size profile of the membrane is determined based on a level of pressure that results in the flow of the gas, liquid, and / or test fluid from the second surface. The method further includes selecting a membrane for filtration only if the determined pore size profile of the membrane is within a specified range, the specified range corresponding to the passage of the therapeutic protein through the porous microfilter membrane. The method further includes filtering the cell culture product, including the therapeutic protein, through the selected microfilter membrane or a microfilter membrane from the same batch as the selected microfilter membrane. In some embodiments, the specified range corresponds to the passage of molecules having a molecular weight of up to 50 kDa, 75 kDa, 100 kDa, 150 kDa, 200 kDa, 500 kDa, 750 kDa, or 1000 kDa through the porous microfilter membrane. For example, the specified range can be 5-120 nanometers, 60-100 nanometers, 5-100 nanometers, or 60-120 nanometers. In some methods of filtering a cell culture product, the cell culture product includes cellular debris and host cell proteins in addition to the therapeutic protein.For any of the methods of filtering a cell culture product described herein, the cell culture product can be of a cell culture selected from the group consisting of: mammalian cells, such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), or human epithelial kidney 293 cells; insect cells, such as Sf21 / Sf9 or Trichoplusia ni Bti-Tn5bl-4; yeast cells, such as Saccharomyces or Pichia; plant cells; chicken cells; and prokaryotic cells, such as Escherichia coli cells. In any of the methods of filtering a cell culture product described herein, the therapeutic protein is selected from the group consisting of: an antibody, an antigen-binding antibody fragment, an antibody protein product, a Bispecific T Cell Engager (BiTE®) molecule, a multispecific antibody, an Fc fusion protein, a recombinant protein, and an active fragment of a recombinant protein.

[0006] Any of the methods described herein can further include cutting the porous microfilter membrane to specified dimensions, e.g., a length of 5 to 15 cm, prior to contacting the saturated microfilter membrane with the intermediate solvent. For example, the microfilter membrane can comprise a flat sheet fiber, and the specified dimensions can include a specified length and a specified width.

[0007] For any of the methods described herein, the microfilter membrane can be an ultrafiltration membrane or a portion thereof.

[0008] For any of the methods described herein, the microfilter membrane may comprise, consist essentially of, or consist of polysulfone, polyethersulfone, polyvinylidene fluoride, or cellulose.

[0009] For any of the methods described herein, the storage solution may include or consist of a water-soluble non-volatile solution, such as water, benzyl alcohol, or a polyol. As an example, the polyol may include or consist of glycerol, such as glycerin. For any of the methods described herein, the storage solution may be soluble in an intermediate solvent, which does not dissolve the microfilter membrane, and which evaporates at 20° C. at 1 atmosphere.

[0010] For any of the processes described herein, the intermediate solvent may comprise or consist of an alcohol, such as isopropyl alcohol.

[0011] For any of the methods described herein, the method may further include drying the desaturated microfilter membrane prior to applying the test fluid.

[0012] For any of the methods described herein, desaturating the microfilter membrane includes, for example, having the reservoir solution below the limit of detection by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy when the ATR-FTIR spectrum of the first surface and / or the second surface matches a reference spectrum of the pure material (such as polysulfone or polyethersulfone) from which the microfilter membrane is made.

[0013] For any of the methods described herein, drying may be carried out until the intermediate solvent is below the detection limit by ATR-FTIR spectroscopy.

[0014] For any of the methods described herein, drying is carried out until the amount of storage solution in the matrix is ​​less than or equal to 1%, 0.5%, 0.1%, or 0.01% of saturation.

[0015] For any of the methods described herein, the test fluid has a surface tension of 70 mN m -1 is less than.

[0016] For any of the methods described herein, the test fluid may comprise an organic solvent or a mixture of organic solvents and / or the test fluid comprises or consists of a Porofil® product, a Fluorinert® product, a Porefil® product, a Porewick® product, or a Galwick® product.

[0017] For any of the methods described herein, the contact angle of the test fluid with the first surface may be sufficient to saturate the filter with the test fluid, for example an angle of 15° or less, for example an angle of 0°.

[0018] For any of the methods described herein, the microfilter membrane may include hollow fibers, and applying pressure may include limiting the pressure to a level that does not cause rupture of the fibers of the microfilter membrane.

[0019] For any of the methods described herein, the microfilter membrane may include a flat sheet membrane, and applying pressure may include limiting the pressure to a level that does not cause rupture or destruction of the flat sheet membrane of the microfilter membrane.

[0020] For any of the methods described herein, the pore size profile may be inversely proportional to the level of pressure that results in the release of a test fluid from the pores of a porous microfilter membrane, where the applied pressure (P), the surface tension of the test fluid (γ), the contact angle of the test fluid with the membrane surface (θ), and the diameter of the pore at its narrowest point (D) are: P = 4 * γ * (cosθ) / D [Formula I] It is related as.

[0021] For any of the methods described herein, determining the pore size profile comprises measuring the pore size profile according to [Equation I]: P=4*γ*(cosθ) / D This may include using: [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram of a porous microfilter of some embodiments. [Figure 2A] 2A-B are flow diagrams: Figure 2A is a flow diagram illustrating a method of determining a pore size profile of a microfilter according to some embodiments; and Figure 2B is a flow diagram illustrating a method of filtering a cell culture product according to some embodiments. [Figure 2B] 2A-B are flow diagrams: Figure 2A is a flow diagram illustrating a method of determining a pore size profile of a microfilter according to some embodiments; and Figure 2B is a flow diagram illustrating a method of filtering a cell culture product according to some embodiments. [Figure 3A] 3A-B are graphs showing ATR-FTIR spectroscopy spectra obtained for a fiber and for a microfilter according to methods of some embodiments. [Figure 3B] 3A-B are graphs showing ATR-FTIR spectroscopy spectra obtained for a fiber and for a microfilter according to methods of some embodiments. [Figure 4A] Figures 4A-D are field emission scanning electron microscope (FE-SEM) micrographs of the pore morphology before (Figures 4A and 4C) and after (Figures 4B and 4D) the storage solution (glycerol in this example) removal and drying procedure. Images were acquired at 20,000x magnification (Figures 4A-B) and 50,000x magnification (Figures 4C-D). [Figure 4B] Figures 4A-D are field emission scanning electron microscope (FE-SEM) micrographs of the pore morphology before (Figures 4A and 4C) and after (Figures 4B and 4D) the storage solution (glycerol in this example) removal and drying procedure. Images were acquired at 20,000x magnification (Figures 4A-B) and 50,000x magnification (Figures 4C-D). [Figure 4C]Figures 4A-D are field emission scanning electron microscope (FE-SEM) micrographs of the pore morphology before (Figures 4A and 4C) and after (Figures 4B and 4D) the storage solution (glycerol in this example) removal and drying procedure. Images were acquired at 20,000x magnification (Figures 4A-B) and 50,000x magnification (Figures 4C-D). [Figure 4D] Figures 4A-D are field emission scanning electron microscope (FE-SEM) micrographs of the pore morphology before (Figures 4A and 4C) and after (Figures 4B and 4D) the storage solution (glycerol in this example) removal and drying procedure. Images were acquired at 20,000x magnification (Figures 4A-B) and 50,000x magnification (Figures 4C-D). [Diagram 5] FIG. 5 is a graph showing that measurements of pore size profiles according to embodiments herein were reproducible, with the pore size profiles measured being consistent with expected trends. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The pore size distribution of a microfilter is an attribute that affects the performance of the filter and, consequently, the yield of therapeutic proteins from a bioreactor. Thus, the variability of the pore size profile between filters has the potential to significantly alter process performance. Many microfilter products are often provided saturated in a storage solution, sometimes referred to as a wetting agent, such as glycerin. It is observed herein that the storage solution can interfere with the accuracy and reliability of the measurement of the pore size profile. However, conventional approaches to removing the storage solution can disrupt the pore structure of the microfilter, affecting the microfilter performance. Thus, conventional approaches to assess the pore size of a microfilter can cause inaccuracies that can lead to the use of filters with pore size distributions that are not suitable for filtering cell culture products of therapeutic proteins.

[0024] Described herein are methods for determining the pore size profile of a microfilter and for filtering cell culture products. The methods described herein can accurately ascertain the pore size profile of a microfilter. In the methods, the microfilter can be provided saturated in a storage solution. The methods can include contacting (e.g., immersing) the microfilter with an intermediate solvent to remove the storage solution from the microfilter by dissolving the storage solution in the intermediate solvent, and desaturating the microfilter so that no storage solution remains (although it is believed that trace amounts of storage solution may still be present). The microfilter can optionally be dried after contact with the intermediate solvent to further remove residual storage solution and / or intermediate solvent. The pore size profile of the microfilter can then be calculated based on the level of pressure that results in the flow of gas or liquid through the microfilter. A test fluid can be applied to the desaturated microfilter. Pressure can then be applied to a first surface of the desaturated microfilter, for example, by applying a gas or liquid to the first surface. The pressure may result in a flow of gas or liquid through the microfilter such that the gas or liquid may be detected emerging from a second surface of the microfilter opposite the first surface. The method may include increasing the pressure until it results in a flow of gas or liquid through the microfilter. Based on the determined pore size of the microfilter, the microfilter may be selected or rejected for filtering a cell culture product pool containing a therapeutic protein.

[0025] As used herein, "pore size profile" refers to the pore size distribution of a microfilter. The pore size profile may be expressed as a mathematical distribution, as a range, or as a single numerical value, such as an average or median, possibly accompanied by a characterization of the distribution, such as a standard deviation or quartiles. Of course, a microfilter will generally contain many pores that are not each exactly the same size, so specifying a pore size profile with one or more numerical values ​​does not mean that every single pore of the microfilter is exactly the same size. For brevity, "pore size profile" may be referred to as "pore size" herein. Unless expressly stated otherwise or clear from the context, it should be understood that "pore size" refers to the pore size profile without the implication that every single pore of the microfilter is the same size. A rigorous characterization of the pore size profile of a microfilter may be achieved by the methods described herein and is beneficial to ensure consistent process performance in the application in which the filter is used.

[0026] Capillary Flow Porometry (CFP) Capillary flow porometry (CFP) is an established analytical technique for measuring the pore size of hollow fiber (and flat sheet) microfiltration (MF) membranes. The method was described by A. Einstein in 1923 (A. Einstein and H. Muehsam. Deutsche medizinische Wochenschrift. v49, no. 31 (1923): 1012-1013, which is incorporated herein by reference in its entirety).

[0027] For any of the methods described herein, the pore size profile may be determined by capillary flow porometry (CFP). In CFP, a membrane (such as a microfilter) is first filled with a test fluid. The membrane is then exposed to an increasing gas pressure, causing the test fluid, which is initially held in place in the membrane pores by capillary forces, to be forced out of the pores. Gas flow across the membrane is measured as the gas forces the liquid out of the pores. The test fluid is a function of the applied pressure (P), the surface tension of the test fluid (γ), the contact angle (θ) between the membrane surface and the test fluid, and the diameter of the pore at its narrowest point (D), according to the Young-Laplace equation: P = 4γcosθ / D [Formula I] As a result, it is expelled from the pores.

[0028] Thus, for the methods described herein, the pore size profile may be inversely proportional to the level of pressure that results in the ejection of a test fluid (and / or the expulsion of gas) from the pores of the porous microfilter, as described in [Equation I]. In some embodiment methods, [Equation 1] is used to determine the pore size profile of the microfilter.

[0029] The determination of the pore size profile may be based on the level of pressure that results in the flow of gas or liquid from the second surface as described herein.

[0030] The performance of a CFP can include uniform wetting of the membrane to be tested by an appropriately selected test fluid, such as a test fluid described herein.

[0031] To remove a storage solution such as glycerol trapped inside the pores of a membrane (such as an HFM) in a microfilter, a significant amount of mechanical pressure is typically required to create motion in the liquid due to the flow resistance caused by the high surface tension of the liquid and the small pore size of the membrane, which can be on the order of 5-200 nm. A practice by some membrane suppliers is to utilize a very low surface tension liquid, such as a Porolfil® product (or other liquids, such as Fluorinert® products, Porefil® products, Porewick® products, or Galwick® products), to "wet" the membrane (fill the membrane pores) in the hope that the glycerol will automatically be displaced or pumped out of the membrane by the low surface tension liquid. CFP measurements are then performed directly on the membrane, assuming it is uniformly wetted with the Porofil® product. The low surface tension liquid can wet or fill the pores of a membrane such as an HFM when there is no liquid trapped within the pores. Because the low surface tension liquid simply displaces the air trapped in the pores. The viscosity of air is less than 1000 times that of the liquid, so the airflow resistance is negligible compared to the liquid. However, the highly wettable low surface tension liquid cannot displace another liquid inside the membrane pores without additional mechanical pressure. This conventional practice typically causes errors and inconsistencies in CFP measurements. A more delicate procedure, for example in the method described herein, can remove glycerol in the membrane, such as HFM or flat sheet, before CFP measurement to preserve the pore structure.

[0032] Microfilter As used herein, a "microfilter" refers to a filter membrane that includes or consists of a flat sheet membrane (FSM) or a hollow fiber membrane (HFM). Because a microfilter contains pores, it may also be referred to as a "porous microfilter." A membrane may be specified with a MWCO, although the precise determination of the exact molecular weight cut-off (MWCO) may be a complex analytical challenge. In the absence of a standardized method for determining MWCO, membrane manufacturers often simply follow manufacturing procedures that follow recipes known to yield membranes within the appropriate MWCO range. A microfilter may include or consist of a polymer, such as polysulfone, polyethersulfone, polyvinylidene fluoride, or cellulose, or a combination of two or more of the listed items. In some embodiments, a microfilter is an ultrafiltration (UF) filter. Because a microfilter refers to a type of membrane, a "microfilter" may also be referred to as a "microfilter membrane" herein. Thus, a "porous microfilter" may also be referred to as a "porous microfilter membrane." The membrane may comprise or consist of HFM or FSM.

[0033] FIG. 1 shows a schematic diagram of a microfilter membrane 100 with a first surface 101 and a second surface 102 filled with a test fluid 110 and containing pore structures 120a, 120b, 120c. The narrowest point of each pore structure 120a, 120b, 120c is indicated by a white arrow. When pressure is applied to the first surface 101 (black arrow, 130), the test fluid is emptied from the largest pores first toward the second surface 102, and as the pressure increases, successively smaller diameter pores are subsequently emptied (manifested as an increase in flow measured from the second surface). The material between the first surface 101 and the second surface 102 can be referred to as a "matrix" 103.

[0034] A microfilter from which all or substantially all of the storage solution has been removed may be referred to as a "de-saturated" or "desaturated" microfilter. If any storage solution in the microfilter is below the detection limit as measured by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, all or substantially all of the storage solution has been removed. That is, the ATR-FTIR spectrum may be matched to the spectrum of the pure microfilter material alone. For example, if the microfilter is made of polysulfone or polyethersulfone, the ATR-FTIR spectrum of the microfilter may be compared to the spectrum of pure polysulfone or polyethersulfone (as appropriate) to determine if any storage solution or other wetting liquid can be detected. As used herein, a "dry" microfilter refers to a de-saturated microfilter.

[0035] In some embodiments, a microfilter is selected to have a specified MWCO. For example, a microfilter may be selected for use in filtering only if the determined pore size profile of the microfilter is within a specified range, e.g., for molecules with a molecular weight of up to 50 kDa, 75 kDa, 100 kDa, 150 kDa, 200 kDa, 500 kDa, 750 kDa, or 1000 kDa passing through the microfilter. In some embodiments, a microfilter is selected for use in filtering only if the determined pore size profile of the microfilter is compatible with molecules with a molecular weight of less than 50 kDa, 75 kDa, 100 kDa, 150 kDa, 200 kDa, 500 kDa, 750 kDa, or 1000 kDa passing through the microfilter.

[0036] In some embodiments, the microfilter is selected to have a pore size profile in which the average diameter of the pores falls within a specified range, for example, 5-120 nanometers, 5-100 nanometers, 60-120 nanometers, or 60-100 nanometers.

[0037] Stock solutions Microfilters are often provided saturated in a storage solution, which may also be referred to as a storage liquid or wetting agent. UF HFM or FSM microfilters are typically impregnated to saturation with a storage solution, glycerin or other non-volatile liquid, to protect the pore structure against collapse or other physical deformation during drying of the water present in the membrane during fiber production. Such storage solutions typically have a surface tension higher than that essential for the performance of CFP on UF membranes. Thus, preparation of microfilter samples for CFP measurements is advantageously by the methods described herein, which (A) remove the storage solution and replace it with the selected CFP test fluid, and (B) leave the pore structure of the membrane substantially undisturbed. The pore structure is of course "substantially" undisturbed if no perturbation is detected in the sample, e.g., a sample observed by SEM, and / or if at least 85%, 90%, or 95% of the pores in the sample retain their structure from before removal of the storage solution.

[0038] In some embodiment methods, the storage solution comprises or consists of a water-soluble non-volatile solution, such as water, an aqueous liquid, benzyl alcohol, or a polyol. For example, the polyol can comprise or consist of glycerin or glycerol.

[0039] Intermediate Solvent The intermediate solvent for some embodiment methods herein comprises or consists of a solvent that 1) dissolves the storage solution, 2) does not dissolve the filter membrane, and 3) can be removed by drying (evaporation) or by replacement with the test fluid.

[0040] Advantageously, the intermediate solvent has a surface tension similar to that of water (approximately 70 mN m -1The lower the temperature, the lower the evaporation temperature, which may cause weaker capillary forces to be exerted on the pore structure and allow evaporation to proceed without distorting the pores. Examples of intermediate solvents suitable for the methods herein include alcohols such as isopropyl alcohol. In some embodiment methods, the intermediate solvent comprises or consists of a solvent that 1) dissolves the storage solution, 2) does not dissolve the filter membrane, and 3) can be removed by drying (evaporation) or by replacement with the test fluid.

[0041] Test Fluid Suitable test fluids for the methods herein include fluids that can completely wet the surface of the microfilter. Typically, fluids that have low or zero contact angles with the filter matrix material can usually completely wet the matrix of the microfilter. Thus, for the methods described herein, the contact angle of the test fluid with the surface (e.g., the first surface described herein) is low enough to saturate the microfilter with the test fluid. For example, the contact angle of the test fluid may be 15° or less, 10° or less, or 0° or less.

[0042] Low surface tension can be advantageous for test fluids of the methods described herein. For the small pore sizes typical of UF membranes, the pressure to complete a CFP measurement can be higher than the burst pressure of the membrane (HFM or FSM). Thus, a test fluid with low surface tension can make it possible to perform a CFP measurement at a lower applied pressure, thus avoiding bursting of the membrane. As an example, the test fluid has a surface tension lower than that of water. The surface tension of water is 70 mN m -1 Therefore, the test fluid has a surface tension of 70 mN m -1 It may be less than.

[0043] Other beneficial features of test fluids suitable for the methods described herein include test fluids that do not dissolve, swell, and / or otherwise interact with the membrane material of construction for the microfilter.

[0044] Test fluids for the methods described herein may comprise or consist of an organic solvent or mixtures thereof. In some embodiment methods, the test fluid comprises or consists of Porofil® products, Fluorinert® products, Porefil® products, Porewick® products, or Galwick® products.

[0045] Therapeutic Proteins As used herein, "therapeutic protein" and variations of this root term have their ordinary and accustomed meaning as understood by those of skill in the art in light of this disclosure. It refers to a polypeptide for medical use in a subject, such as a human subject, or for veterinary use in a non-human mammal. A therapeutic protein may be a protein for medical use, such as a candidate for medical use or a protein approved for medical use by a governmental authority, such as the FDA or EMA.

[0046] In the methods described herein, the therapeutic protein may be selected from the group consisting of an antibody, an antigen binding protein, an antibody protein product, a bispecific T cell engager (BiTE®) molecule, a multispecific antibody (such as a bispecific or trispecific antibody), an Fc fusion protein, a recombinant protein, a synthetic peptide, and an active fragment of a recombinant protein.

[0047] "Antibody" has its customary and ordinary meaning as understood by those of skill in the art in light of this disclosure. It refers to an immunoglobulin that specifically binds to a target antigen, including, for example, chimeric antibodies, humanized antibodies, and fully human antibodies. By way of example, an antibody may be a monoclonal antibody. By way of example, a human antibody may be of a particular isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE. A human IgG antibody generally comprises two full-length heavy chains and two full-length light chains. An antibody may be derived from only a single source, or may be "chimeric," i.e., different portions of the antibody may be derived from two or more different antibodies from the same or different species. Of course, once an antibody is obtained from a source, the antibody may be subjected to further manipulation, for example to enhance stability and folding. Thus, it will be understood that a "human" antibody can be derived from a source and may have been further engineered, for example in the Fc region. An engineered antibody may still be referred to as a type of human antibody. Similarly, unless otherwise specified, variants of human antibodies, such as those that have been affinity matured, will also be understood to be "human antibodies." In some embodiments, an antibody comprises, consists essentially of, or consists of a human antibody, a humanized antibody, or a chimeric monoclonal antibody.

[0048] The "heavy chain" of an antibody, antigen binding protein, antibody protein product, bispecific T cell engager molecule, or multispecific antibody comprises a variable region ("VH") and three constant regions, namely CH1, CH2, and CH3. The "light chain" of an antibody, antigen binding protein, antibody protein product, bispecific T cell engager molecule, or multispecific antibody comprises a variable region ("VL") and a constant region ("CL"). Human light chains include kappa and lambda chains. Exemplary light chain constant regions suitable for antigen binding proteins include the human lambda constant region and the human kappa constant region.

[0049] In various embodiments, the therapeutic protein is an antibody protein product. The term "antibody protein product" as used herein refers, in various instances, to any one of several antibody surrogates that are based on the structure of an antibody but are not found in nature. In some embodiments, the antibody protein product has a molecular weight in the range of at least about 12 kDa to about 250 kDa. In certain embodiments, the antibody protein product has a valency (n) ranging from monomer (n=1) to dimer (n=2), trimer (n=3), to tetramer (n=4), unless of higher valency. In some embodiments, the antibody protein product is based on the complete antibody structure and / or mimics an antibody fragment that retains complete antigen-binding ability, such as scFv, Fab, and VHH / VH (described below). The smallest antigen-binding antibody fragment that retains a complete antigen-binding site is the Fv fragment, consisting entirely of the variable (V) region. A soluble, flexible amino acid peptide linker is used to link the V region to a scFv (single chain fragment variable) fragment to stabilize the molecule, or a constant (C) domain is added to the V region to generate a Fab fragment (fragment antigen-binding). Both scFv and Fab fragments can be easily produced in host cells, for example prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody formats such as diabodies, triabodies, and tetrabodies, or minibodies (miniAbs), including disulfide bond stabilized scFv (ds-scFv), single chain Fab (scFab), and different formats consisting of scFv linked to oligomerization domains. The smallest fragments are the VHH / VH of camelid heavy chain Abs and single domain Abs (sdAbs). The building block most frequently used to generate new antibody formats is the single variable (V) domain antibody fragment (scFv), which contains the V domains (VH and VL domains) from the heavy and light chains linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of peptibodies consists of a biologically active peptide grafted onto the Fc domain. Peptibodies have been well described in the art.See, e.g., Shimamoto et al., mAbs 4(5):586-591 (2012). Bispecific T cell engager molecules, such as those that contain half-life extending moieties, are also examples of antibody protein products.

[0050] Therapeutic proteins suitable for the methods described herein may include polypeptides including those that bind to one or more of the following: CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34; such as those that interfere with receptor binding. HER receptor family proteins, such as HER2, HER3, HER4, and EGF receptor. Cell adhesion molecules, such as LFA-I, MoI, pl50, 95, VLA-4, ICAM-I, VCAM, and alpha v / beta 3 integrin. Growth factors such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian inhibitory substance, human macrophage inflammatory protein (MIP-I alpha), erythropoietin (EPO), nerve growth factors such as NGF-beta, platelet derived growth factor (PDGF), fibroblast growth factors such as aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGFs) such as TGF-α and TGF-β, e.g., TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, among others, insulin-like growth factors-I and II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors. Insulin and insulin-related proteins, such as insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins. Coagulation and coagulation-related proteins, such as factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin, and thrombopoietin, among others; (vii) other blood and serum proteins, including, but not limited to, albumin, IgE, and blood group antigens. Colony-stimulating factors and their receptors, such as M-CSF, GM-CSF, and G-CSF, among others, and their receptors, such as the CSF-1 receptor (c-fms).Receptors and receptor-associated proteins, such as flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor ("TPO-R", "c-mpl"), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptor, such as c-Kit, and other receptors. Receptor ligands, such as OX40L, which is a ligand for the OX40 receptor. Neurotrophic factors, such as bone-derived neurotrophic factor (BDNF), and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Relaxin A chain, relaxin B chain, and prorelaxin; interferons and interferon receptors, such as interferon-α, -β, and -γ, and their receptors. Interleukins and interleukin receptors, such as IL-1 to IL-33 and IL-1 to IL-33 receptors, among others, such as the IL-8 receptor. Viral antigens, e.g., AIDS envelope viral antigens. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor alpha and beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptide, DNAse, inhibin, and activin. Integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies. Myostatin, TALL proteins, e.g., TALL-I, amyloid proteins, e.g., but not limited to, amyloid beta protein, thymic stromal lymphopoietin ("TSLP"), RANK ligand ("RANKL" or "OPGL"), c-kit, TNF receptors, e.g., TNF receptor type 1, TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing.

[0051] Examples of therapeutic proteins suitable for the methods described herein include antibodies or variants thereof comprising an IgG1 constant region comprising one or more of the following mutations selected from the group consisting of L242C, A287C, R292C, N297G, V302C, L306C, and K334C, numbered according to the EU system. For example, infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, altinumab, atolizumab ... imiumab), tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab , cc49, cedelizumab, certolizumab pegol, cetuximab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, durigotumab, dupilumab, ecromeximab, eculizumab, edovacomab, edreco Mab, efalizumab, efungumab, elotuzumab, ersilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab situxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exibirmab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab,Figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomilikimab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inlacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab , inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimuumab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab passudotox, muromonab -cd3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narutumumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, Panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placumab, ponezumab, priliximab, pritumumab, PRO140, kirisumab, racotumomab, radletumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab,satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, TGN1412, tremelimumab, ticilimumab, thilimumab Rudrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, beparimomab, besenkumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, dillalimumab, or zolimomab alitox.

[0052] In some embodiments, the therapeutic protein is a BiTE® molecule. A BiTE® molecule is an engineered bispecific antigen-binding construct that directs the cytotoxic activity of T cells against cancer cells. It is a fusion of two single chain variable fragments (scFv) of different antibodies or amino acid sequences from four different genes onto a single peptide chain of about 55 kDa. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to tumor cells via a tumor-specific molecule. Blinatumomab (BLINCYTO® product) is an example of a BiTE® molecule specific for CD19. Modified BiTE® molecules (such as those modified to extend half-life) can also be used in the disclosed methods. In various aspects, the polypeptide is an antigen-binding protein, e.g., a BiTE® molecule. In some embodiments, the antibody protein product comprises a BiTE® molecule.

[0053] Cell culture and cell culture products The therapeutic proteins described herein can be produced by cell culture, and therefore can of course be constituted by cell culture products.Cell culture products that can be filtered according to the methods herein can include those of cell cultures selected from the group consisting of mammalian cells, such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. Hep G2), or human epithelial kidney 293 cells; insect cells, such as Sf21 / Sf9 or Trichoplusia ni Bti-Tn5bl-4 cells; yeast cells, such as Saccharomyces or Pichia cells; plant cells; avian cells, such as chicken cells; and prokaryotic cells, such as Escherichia coli cells.

[0054] Method for determining pore size profile In some embodiments, a method of determining a pore size profile is described. The method may include providing a saturated porous microfilter. The porous microfilter may include a first surface, a second surface, and a matrix disposed between the first surface and the second surface. The matrix may be saturated with a storage solution. The method may further include contacting the saturated porous microfilter with an intermediate solvent to desaturate the microfilter by dissolving the storage solution in the intermediate solvent. The method may further include applying a test fluid to the desaturated microfilter to resaturate the microfilter. The method may further include applying a pressure to a first surface of the resaturated microfilter. The pressure may be applied by contacting the first surface with a gas or a liquid. The method may further include detecting a flow of the gas, liquid, and / or test fluid from the second surface in response to the pressure. The method may further include determining a pore size profile of the microfilter based on a level of pressure that results in a flow of the gas, liquid, and / or test fluid from the second surface.

[0055] A method of determining a pore size profile according to some embodiments is illustrated in FIG. 2A. A saturated porous microfilter is provided that includes a first surface, a second surface, and a matrix disposed therebetween, the matrix being saturated with a storage solution 210. The saturated porous microfilter is contacted with an intermediate solvent, where the storage solution dissolves in the intermediate solvent to desaturate the microfilter 220. For example, contacting the microfilter with the intermediate solvent may include immersing the microfilter in the intermediate solvent. A test fluid is then applied to the desaturated microfilter to resaturate the microfilter 230. A pressure is applied to the first surface of the resaturated microfilter 240. The pressure may be applied by contacting the first surface with a gas or a liquid. After the pressure is applied, a flow of gas, liquid, and / or test fluid from the second surface in response to the pressure is detected 250. For example, the release of the test fluid itself used to apply the pressure, or of the gas and / or liquid, from the second surface may be detected. The pressure may be gradually increased (e.g., continuously or in steps) until a flow of gas, liquid, and / or test fluid from the second surface is detected. Once a flow of gas, liquid, and / or test fluid from the second surface is detected, a pore size profile of the microfilter may be determined based on the level of pressure that results in the flow of gas, liquid, and / or test fluid from the second surface 260. The pore size profile of the microfilter may be determined. For example, the pore size profile may be determined by CFP as described herein. For example, Equation 1 may be used to determine the pore size profile based on the level of pressure. In some embodiments, a microfilter may be selected for filtration only if the determined pore size profile of the microfilter is within a specified range. The specified range may be a range that corresponds to the passage of a therapeutic protein through the porous microfilter. For example, the therapeutic protein may be a specified therapeutic protein as described herein.The method may further include filtering the cell culture product containing the therapeutic protein through the selected microfilter (or a microfilter from the same batch as the selected microfilter). It will be understood that one or more of the noted portions of the method may be repeated, omitted, or performed in a different order as required in the context. In some embodiments, the method includes drying the desaturated microfilter 230 prior to applying the test fluid. Drying may be performed until the intermediate solvent is below the detection limit by ATR-FTIR spectroscopy.

[0056] In some embodiment methods, the method is performed on a microfilter sample that is pulled from the HFM production line before the storage solution is introduced. Of course, for such methods, 210 and 220 may be omitted.

[0057] Method for filtering cell culture products In some embodiments, a method of filtering a cell culture product containing a therapeutic protein is described. The method may include providing a saturated porous microfilter including a first surface, a second surface, and a matrix disposed therebetween, the matrix being saturated with a storage solution. The method may include contacting the saturated porous microfilter with an intermediate solvent to desaturate the microfilter by dissolving the storage solution in the intermediate solvent. The method may include resaturating the microfilter by applying a test fluid to the desaturated microfilter. The method may include applying a pressure to a first surface of the resaturated microfilter, the pressure being applied by contacting the first surface with a gas or liquid. The method may include detecting a flow of the gas, liquid, and / or test fluid from the second surface in response to the pressure, and a pore size profile of the microfilter is determined based on a level of pressure that results in the flow of the gas, liquid, and / or test fluid from the second surface. A microfilter may be selected for filtration only if the determined pore size profile of the microfilter is within a specified range corresponding to passage of the therapeutic protein through the porous microfilter. The method may further include filtering the cell culture product, including the therapeutic protein, through a selected microfilter. The cell culture product may be a product of a cell culture as described herein. Of course, filtering the cell culture product through a selected microfilter does not require filtering the cell culture through an exact fraction of the material processed according to the method, but may also include filtering the cell culture product through a microfilter from the same batch as the microfilter being tested.

[0058] A method of filtering a cell culture product according to some embodiments is illustrated in FIG. 2B. A saturated porous microfilter is provided 211, including a first surface, a second surface, and a matrix disposed therebetween, the matrix being saturated with a storage solution. The saturated porous microfilter is contacted with an intermediate solvent, where the storage solution dissolves in the intermediate solvent to desaturate the microfilter 221. For example, contacting the microfilter with the intermediate solvent may include immersing the microfilter in the intermediate solvent. A test fluid is then applied to the desaturated microfilter to resaturate the microfilter 231. Pressure is applied to a first surface of the resaturated microfilter 241. Pressure may be applied by contacting the first surface with a gas or a liquid. After pressure is applied, a flow of gas, liquid, and / or test fluid from the second surface in response to the pressure is detected 251. For example, the release of the test fluid itself used to apply pressure, or of the gas and / or liquid, from the second surface may be detected. The pressure may be gradually increased (e.g., continuously or in steps) until a flow of gas, liquid, and / or test fluid from the second surface is detected. Once a flow of gas, liquid, and / or test fluid from the second surface is detected, a pore size profile of the microfilter may be determined based on the level of pressure that results in the flow of gas, liquid, and / or test fluid from the second surface 261. The pore size profile of the microfilter may be determined. For example, the pore size profile may be determined by the CFP described herein. For example, Equation 1 may be used to determine the pore size profile based on the level of pressure. A microfilter may be selected for filtration only if the determined pore size profile of the microfilter is within a specified range 271. The specified range may correspond to the passage of the therapeutic protein through the porous microfilter. The method may further include filtering a cell culture product containing the therapeutic protein through the selected microfilter or a microfilter of the same batch as the selected microfilter 281.It will be understood that the microfilter for which the pore size profile is determined may be a sample of a larger microfilter, and that microfilters not actually used to determine the pore size profile, or portions of other microfilters from the same batch, may be used to filter cell culture products and be understood to have a pore size profile consistent with the microfilter actually tested. It will be understood that one or more of the noted portions of the method may be repeated, omitted, or performed in a different order, as required by the context. The cell culture product may be a product of a cell culture as described herein. In some embodiments, the method includes drying the desaturated microfilter 231 before applying the test fluid. Drying may be performed until the intermediate solvent is below the detection limit by ATR-FTIR spectroscopy.

[0059] In some embodiments of the methods of filtering a cell culture product, the specified ranges correspond to the passage of molecules through the porous microfilter having a molecular weight of up to 50 kDa, 75 kDa, 100 kDa, 150 kDa, 200 kDa, 500 kDa, 750 kDa, or 1000 kDa. In some embodiments of the methods of filtering a cell culture product, the specified ranges are average pore diameters of 5-120 nanometers, 60-100 nanometers, 5-100 nanometers, or 60-120 nanometers.

[0060] According to some embodiment methods of filtering a cell culture product, the cell culture product may contain cells and / or cell components in addition to the therapeutic protein. For example, the cell culture product may contain cell debris and / or host cell proteins in addition to the therapeutic protein. Filtration may separate the therapeutic protein from some or all of the other materials in the cell culture product by allowing the therapeutic protein to pass through a microfilter while not allowing other portions of the cell culture product to pass through.

[0061] Examples of suitable cell culture products may include: mammalian cells, such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. Hep G2) or human epithelial kidney 293 cells; insect cells, such as Sf21 / Sf9 or Trichoplusia ni Bti-Tn5bl-4 cells; yeast cells, such as Saccharomyces or Pichia cells; plant cells; avian cells, such as chicken cells; and prokaryotic cells, such as Escherichia coli cells, of cell cultures selected from the group consisting of:

[0062] The methods of filtering a cell culture product described herein may be used to filter a cell culture product that includes a therapeutic protein that is a candidate for medical use or has been approved for medical use by a governmental agency such as the FDA or EMA. In some embodiments of the methods of filtering a cell culture product, the therapeutic protein is selected from the group consisting of: an antibody, an antigen-binding antibody fragment, an antibody protein product, a bispecific T-cell engager (BiTE®) molecule, a multispecific antibody, an Fc fusion protein, a recombinant protein, and an active fragment of a recombinant protein.

[0063] Additional options for the methods described herein Of course, for the methods described herein, samples from a batch of microfilters can be tested to determine whether the batch of microfilters can be used to filter cell culture products containing therapeutic proteins. Also, samples of membranes can be tested directly prior to assembly of the membranes into a filter device. Of course, for any of the methods described herein, the microfilters tested in the method do not have to be full-sized microfilters, but can be samples of larger microfilters (so that the larger microfilters, and / or other microfilters from the same batch, can then be used to filter cell culture products as described herein). In some methods described herein, the method includes cutting the fibers of the porous microfilter to a specified size prior to contacting the saturated microfilter with the intermediate solvent. The specified dimensions may be, for example, a length of 5-10 cm, 5-15 cm, 5-20 cm, 10-15 cm, or 10-20 cm, or may be a specified length and width (e.g., a flat sheet filter may be cut to a specified length and width, e.g., 10 cm x 10 cm, 10 cm x 20 cm, or 20 cm x 20 cm).

[0064] In some methods described herein, the microfilter may be provided in a storage solution, for example, glycerin. The method may include extracting the storage solution from the microfilter with an intermediate solvent that is miscible with glycerin but does not dissolve or excessively deform the membrane. This may be accomplished by simple immersion of the membrane in the solvent. The method may include removing the microfilter from the intermediate solvent and drying the microfilter. Upon drying, the intermediate solvent may evaporate. The intermediate solvent has a lower surface tension than water. The microfilter (now free of any liquid) can now be easily contacted with the test fluid by simply immersing the microfilter in the test fluid, allowing the test fluid to wet the microfilter. In some cases, the microfilter may be contacted with the test fluid before or after mounting the microfilter in a specialized sample holder for CFP.

[0065] For any of the methods described herein, the microfilter may be an ultrafiltration membrane or a portion thereof. In these methods, the microfilter may comprise, consist essentially of, or consist of polysulfone, polyethersulfone, polyvinylidene fluoride, or cellulose.

[0066] For any of the methods described herein, the storage solution can be soluble in an intermediate solvent, which does not dissolve the microfilter, and which evaporates at 20° C. at 1 atmosphere. For any of the methods described herein, the storage solution can include or consist of a water-soluble non-volatile solvent, such as water, benzyl alcohol, or a polyol. For example, the polyol can include or consist of glycerin or glycerol.

[0067] For any of the processes described herein, the intermediate solvent may comprise or consist of an alcohol, such as isopropyl alcohol.

[0068] For any of the methods described herein, the microfilter is desaturated when the reservoir solution in the microfilter (or a sample thereof) is below the detection limit by ATR-FTIR spectroscopy. It can be concluded that the microfilter is desaturated when the ATR-FTIR spectrum of the first surface and / or the second surface matches a reference spectrum of the pure material (such as polysulfone or polyethersulfone) from which the microfilter is made.

[0069] While it is believed that sufficient storage solution is dissolved by the intermediate solvent to allow for the determination of the pore size profile without further processing, it is further believed that additional amounts of storage solution and / or intermediate solvent may be removed by drying the desaturated microfilter before applying the test fluid. Thus, in some embodiments, the methods described herein further comprise drying the desaturated microfilter before applying the test fluid. In some embodiment methods, the drying is performed until the intermediate solvent is below the detection limit by ATR-FTIR spectroscopy. In some embodiment methods, the drying is performed until the amount of storage solution in the matrix of the microfilter is 1%, 0.5%, 0.1%, or 0.01% or less of saturation.

[0070] For any of the methods described herein, the microfilter comprises a hollow fiber membrane or a flat sheet membrane, and applying pressure comprises limiting the pressure to a level that does not cause rupture of the fibers or sheets of the microfilter. As described herein, a membrane having low surface tension (70 mN m -1 The test fluids (e.g., less than 1000 psi) allow the CFP to be completed at relatively low pressures, thereby avoiding rupture of the fibers or sheet. EXAMPLES

[0071] Example 1: Sample preparation UF HFM fiber samples were obtained from Cytiva (fiber type 750E, MWCO 750 kDa). Fibers were cut to the desired length (12 cm) using microspheres. The cut segments were placed in a suitable container filled with solvent (isopropyl alcohol) to remove the storage solution (glycerin). The container with the segments and extraction solvent was subjected to agitation (orbital shaker, 200 rpm, 1 hour). The segments were removed from the solvent and allowed to dry (1 hour). At that point, the segments (referred to as "dried" in figures and descriptions herein) were ready for the introduction of the CFP test fluid (Porofil®) into the membrane. This was achieved by simply immersing in Porofil.

[0072] Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and field emission scanning electron microscopy (FE-SEM) measurements were carried out at different stages of sample preparation.

[0073] ATR-FTIR spectroscopy was performed on the inner surface of the HFM samples to demonstrate the removal of the storage solution and replacement with the selected test fluid. Figures 3A-B show the spectra obtained for an untreated fiber (glycerin can be easily detected), a "born dry" fiber (glycerin cannot be detected and the spectrum matches a reference spectrum of pure polysulfone, the polymer used to make the HFM), and a fiber backfilled with Porofil® product, a mixture of fluorocarbons (Porofil® product can actually be detected). Reference spectra for glycerin and polysulfone are included.

[0074] Attempts to replace the glycerin directly with Porofil® product (i.e., by immersing untreated fibers in Porofil® product) did not remove the glycerol. Spectra of fibers prepared in this manner still showed the presence of large amounts of glycerol (data not shown). To demonstrate that the sample preparation procedure avoids significantly disturbing the membrane pore structure, high-resolution SEM images were obtained from the HFM inner surface before glycerin removal (Figures 4A and 4C) and after achieving a "bone-dry" condition (Figures 4B and 4D). Representative images are shown in Figures 4A-4D, which show no evidence of damage to the HFM pore structure. Figures 4A-4B show images obtained at 20,000x magnification, and Figures 4C-4D show images obtained at 50,000x magnification.

[0075] It is therefore concluded that the methods herein remove all or substantially all of the storage solution from the microfilter without substantially perturbing the pore size profile, rendering the microfilter suitable for CFP.

[0076] Example 2: CFP measurements of prepared UF HFM samples CFP measurements were performed on the HFM samples after preparation of these samples according to the procedure in Example 1. The results obtained showed reproducible measurements, with pore sizes measured according to expected trends, as shown in Figure 5. This result demonstrates the validity of the sample preparation procedure described herein.

Claims

1. 1. A method for filtering a cell culture product containing a therapeutic protein, comprising: providing a saturated porous microfilter membrane comprising a first surface, a second surface, and a matrix disposed therebetween, the matrix being saturated with a storage solution; contacting the saturated porous microfilter membrane with an intermediate solvent to desaturate the membrane by dissolving the storage solution in the intermediate solvent; resaturating the membrane by applying a test fluid to the desaturated membrane; applying pressure to the first surface of the resaturated membrane, the pressure being applied by contacting the first surface with a gas or a liquid; detecting a flow of the gas, liquid, and / or test fluid from the second surface in response to the pressure, wherein the pore size profile of the membrane is determined based on the level of the pressure that results in the flow of the gas, liquid, and / or test fluid from the second surface; selecting the membrane for use in filtration only if the determined pore size profile of the membrane falls within a designated range, the designated range corresponding to passage of the therapeutic protein through the porous microfilter membrane; filtering the cell culture product containing the therapeutic protein through the selected microfilter membrane or a microfilter membrane from the same batch as the selected microfilter membrane; A method comprising:

2. 10. The method of claim 1, wherein the specified range corresponds to the passage of molecules through the porous microfilter membrane having a molecular weight of up to 50 kDa, 75 kDa, 100 kDa, 150 kDa, 200 kDa, 500 kDa, 750 kDa, or 1000 kDa.

3. The method of claim 1 , wherein the specified range is 5 to 120 nanometers, 60 to 100 nanometers, 5 to 100 nanometers, or 60 to 120 nanometers.

4. 10. The method of claim 1, wherein the cell culture products include, in addition to the therapeutic protein, cell debris and host cell proteins.

5. 2. The method of claim 1, wherein the therapeutic protein is selected from the group consisting of: an antibody, an antigen-binding antibody fragment, an antibody protein product, a bispecific T cell engager molecule, a multispecific antibody, an Fc-fusion protein, a recombinant protein, and an active fragment of a recombinant protein.

6. A method for determining a pore size profile of a microfilter, comprising: providing a saturated porous microfilter membrane comprising a first surface, a second surface, and a matrix disposed therebetween, the matrix being saturated with a storage solution; contacting the saturated porous microfilter membrane with an intermediate solvent to desaturate the membrane by dissolving the storage solution in the intermediate solvent; resaturating the membrane by applying a test fluid to the desaturated membrane; applying pressure to the first surface of the resaturated membrane, the pressure being applied by contacting the first surface with a gas or a liquid; detecting a flow of the gas, liquid, and / or test fluid from the second surface in response to the pressure; determining the pore size profile of the micro-porous filter membrane based on the level of the pressure that results in the flow of the gas, liquid, and / or test fluid from the second surface; A method comprising:

7. 10. The method of claim 1 or 6, further comprising cutting the porous microfilter membrane to specified dimensions comprising a length of 5 to 15 cm prior to contacting the saturated microfilter membrane with the intermediate solvent.

8. 8. The method of claim 7, wherein the microfilter membrane comprises a flat sheet of fabric and the specified dimensions include a specified length and a specified width.

9. 10. The method of claim 1 or 6, wherein the storage solution comprises a water-soluble, non-volatile solution.

10. The method of claim 9, wherein the storage solution contains glycerol.

11. 10. The method of claim 1 or 6, wherein the storage solution is soluble in the intermediate solvent, the intermediate solvent does not dissolve the microfilter membrane, and the intermediate solvent evaporates at 20°C under 1 atmosphere.

12. 10. The method of claim 1 or 6, wherein the intermediate solvent comprises or consists of an alcohol.

13. 10. The method of claim 1 or 6, further comprising the step of drying the desaturated microfilter membrane before applying the test fluid.

14. 10. The method of claim 1 or 6, wherein desaturating the microfilter membrane comprises, for example, ensuring that the stock solution is below the detection limit by ATR-FTIR spectroscopy when the ATR-FTIR spectrum of the first surface and / or the second surface matches a reference spectrum of a pure material (such as polysulfone or polyethersulfone) from which the microfilter membrane is made.

15. 10. The method of claim 1 or 6, wherein the drying step is carried out until the intermediate solvent is below the detection limit by ATR-FTIR spectroscopy.

16. 10. The method of claim 1 or 6, wherein the drying step is carried out until the amount of storage solution in the matrix is ​​1%, 0.5%, 0.1%, or 0.01% or less of saturation.

17. The method of claim 1 or 6, wherein the test fluid comprises an organic solvent or a mixture of organic solvents.

18. 10. The method of claim 1 or 6, wherein the contact angle of the test fluid with the first surface is sufficient to saturate the filter with test fluid, for example an angle of 15° or less, for example an angle of 0°.

19. 10. The method of claim 1 or 6, wherein the microfilter membrane comprises hollow fibers, and the step of applying pressure comprises limiting the pressure to a level that will not cause rupture of the fibers of the microfilter membrane.

20. The pore size profile is inversely proportional to the level of pressure that results in the release of the test fluid from the pores of the porous microfilter membrane, where the applied pressure (P), the surface tension of the test fluid (γ), the contact angle of the test fluid with the membrane surface (θ), and the diameter of the pore at its narrowest point (D) are: P=4*γ*(cosθ) / D [Formula I] 10. The method of claim 1 or 6, wherein

Images