KR20190143499A - Agarose ultrafiltration membrane composites for size based separations - Google Patents

Abstract

Embodiments described herein relate to agarose ultrafiltration membrane composites and methods for making and using the same.

Description

Agarose Ultrafiltration Membrane Composites for Size-Based Separation {AGAROSE ULTRAFILTRATION MEMBRANE COMPOSITES FOR SIZE BASED SEPARATIONS}

This application claims priority to US Provisional Patent Application No. 62 / 205,859, filed August 17, 2015, and US Provisional Patent Application No. 62 / 268,220, filed December 16, 2015, which are incorporated herein by reference in their entirety. have.

Embodiments described herein relate to a novel ultrafiltration composite structure comprising an agarose layer on a porous support membrane and a method for making such a superfiltration composite structure. Also described herein are methods for using such ultrafiltration complex structures, for example to remove viruses from biopharmaceutical solutions.

Ultrafiltration membranes are typically used in pressure-driven filtration processes. Virus removal membrane filters are widely used in the biotech industry to provide the necessary safety of manufactured healing products. These filters are believed to retain a high percentage of viruses that may be present in the feed containing the healing product, and the product flows through the membrane.

Ultrafiltration (UF) membranes are primarily used to concentrate or diafilter soluble polymers such as proteins, DNA, viruses, carbohydrates, and natural or synthetic polymers. For many applications, ultrafiltration is carried out in tangential flow filtration (TFF) mode where the feed solution is passed across the membrane surface and molecules smaller than the pore size of the membrane are passed (filtered) and the residue (retentate) Remains on the upstream side of the membrane. As the fluid passes through the membrane, it needs to be recycled or added to the sample flow to maintain efficient TFF operation. One advantage of using the TFF method is that the fluid constantly sweeps across the face of the membrane, which causes fouling and polarization of the solute at or near the membrane surface leading to the membrane's long life. ) Tends to decrease.

Superfiltration membranes are generally exposed asymmetric membranes formed mostly on the support, which often become a permanent part of the membrane structure. The support may be a non-woven fabric, or a preformed membrane. UF membranes are manufactured by an immersion casting method and are exposed and asymmetric. Early commercial applications involved protein concentrations and membranes were rated by the molecular weight of the protein, for example, allowing them to maintain the molecular weight cutoff rating of the membrane (MWCO).

Superfiltration membrane ratings based on tests with proteins continue to be performed, while conventional methods use non-protein macromolecules with narrow molecular weight distributions, such as polysaccharides (Dextrans) or polyethylene glycols (e.g., , "A rejection profile test for ultrafiltration membranes and devices", Biotechnology 9 (1991) 941-943)).

Methods of making ultrafiltration membranes by immersion casting are well known. A concise statement is given to “Microfiltration and ultrafiltration: Principles and Applications”; Marcel Decker (1996); L. J. Zeman and A. J. Gidney, editors. One exemplary manufacturing method consists of the following steps: Are described.

a) preparation of specific and well controlled polymer solutions;

b) casting the polymer solution in the form of a thin film on the substrate;

c) solidifying the resulting film of the polymer solution in a nonsolvent;

d) optionally drying the ultrafiltration membrane.

Controlling the pore size in the ultrafiltration membrane is generally not straightforward. Not only does the solids content of the casting solution affect the membrane porosity, but the pore size also affects the relative proportion of non-solvent and solvent remaining in the casting solution. If the non-solvent enters the film before the solvent remains, the polymer is precipitated around a large volume of solvent (functioning as a pore former) which results in a high porosity and large pore size UF membrane. The opposite is true if the solvent remains in the film faster than the inflow of the non-solvent, and the resulting UF membrane has low porosity and small pores. Temperature adjustments for both, as well as additives to non-manufacturing or casting solutions, are often used to control the removal of solvent from the cast film and the relative proportion of cosmetic entry.

Agarose is a natural polysaccharide that is widely used to make porous beads. These beads find numerous applications in chromatographic separations. Recent techniques describing the formation of agarose beads (for chromatographic applications) have used warm, water-insoluble solvents in which agarose is emulsified before gel formation by cooling. See, eg, Gerten, S. Biochim, Biophys, Acta 1964, 79: 393-398; And Benzsson et al., S. Biochim, Biophys, Acta 1964, 79: 399. As described in US Pat. No. 4,647,536, another method for forming agarose beads is to drop the agarose emulsion into the cooled oil. This method is also described in “Methods in“ Enzymology ”(Vol. 135 Part B, page 401, Academic Press, 1987). The polymer must be heated above its melting temperature of about 92 ° C. and dissolve in the presence of water. Above this temperature, the polymer melts, and then the molten polymer is solvated with water to form a solution. The polymer is molten in water as long as the temperature is above the gelling point of the polymer at about 45 ° C. Below the gel point, the polymer phase separates and hydrogels so that the solution takes some shape just before gelling. In addition, when agarose approaches its gelling point, the viscosity of the solution becomes high and becomes higher as the gel begins to form into the lid.

Traditionally, for polysaccharide beads, they are used in chromatography media, and the heated solution is kept above its gel point and stirred into unmixed heated fluid, such as mineral or vegetable oil. Next, the two-phased material (beads of agarose in the unmixed fluid) is cooled and the beads are recovered. The beads themselves are diffusely porous and can be used as preparation for size exclusion chromatography. In addition, they can be further progressed by crosslinking, addition of various capture chemistries such as affinity chemistry or ligands, positive or negative charges, hydrophobicity, or the like and crosslinking and chemical bonding to enhance their capture ability.

Agarose is widely used to form porous beads, and targets and / or impurities travel into and out of the pores in the diffusion / drive process.

The embodiments described herein relate to a novel ultrafiltration composite structure comprising agarose. Agarose beads may be found in the prior art, but previously, agarose may be employed as a filtration membrane such that the target and / or impurities travel into and through the pores in a pressure-driven process as described above. It could not be used to create a continuous, flat porous structure.

The terms “aragose ultrafiltration composite structure” and “aragose ultrafiltration membrane composite”, as used interchangeably herein, refer to at least one supporting porous membrane (also referred to as a substrate). And multilayer porous structures comprising a layer of porous agarose deposited on the membrane.

The ultrafiltration membrane composites described herein have several advantages over the currently available ultrafiltration membranes. In particular, the ultrafiltration membrane composites described herein avoid the use of harmful organic solvents in the manufacturing process. This helps to carry out simpler, safer, cheaper and more environmentally friendly manufacturing processes. In addition, the forming process can be simplified due to its ability to easily control the pore size of the ultrafiltration membrane by treating fewer process steps and agarose concentration. In addition, the ultrafiltration membrane complexes described herein exhibit low protein binding, stability under high pH conditions, and also do not depend on the presence of extractable and / or releasable compositions for forming porous structures.

In some embodiments, the ultrafiltration membrane composites described herein are substantially cellulose compounds. In some embodiments, a method of preparing an agarose ultrafiltration membrane composite is provided, the method comprising: a) having an average pore size in the range of 0.01 μm to 1 μm and an average thickness in the range of 10 μm to 500 μm (also referred to as a substrate). As a step of providing a porous support membrane, the porous support membrane is polyester, polyolefin, polyethylene (PE), polypropylene, polyamide, polyethylene terephthalate (PET), polyether-ether ketone (PEEK), polysulfone, A polymer selected from the group consisting of polyethersulfone (PES), aromatic polymers and fluorinated polymers such as polytetrafluoroethylene and polyvinylidene fluoride (PVDF); b) providing an agarose solution; c) casting a layer of agarose solution on the porous support membrane at a temperature in the range of 20 to 90 ° C. to form an agarose coated porous support membrane; And d) immersing the agarose coated porous support membrane in a water bath at a temperature below the gelling point of the agarose solution, thereby forming an agarose superfiltration membrane.

In some embodiments, the agarose ultrafiltration membrane composite has a pore size of less than 0.1 μm. In some embodiments, the bath comprises ice water.

The method described above is useful for the preparation of agarose ultrafiltration membrane composites having porous supports, wherein the membrane composites have a molecular weight cut-off (MWCO) value of 10-1000 kDa (R90).

In some embodiments, the agarose ultrafiltration membrane composite comprises a porous support formed from a polymer selected from the group consisting of polyvinylidene fluoride (PVDF), polyethylene (PE), and polyethersulfone (PES). In certain embodiments, the porous support comprises polyvinylidene fluoride (PVDF) or ultra high molecular weight polyethylene (UHMW-PE). In some embodiments by the method described herein, an agarose solution having a concentration of agarose in the range of 1-12% by weight is used. In another embodiment, an agarose solution having a concentration of agarose in the range of 5-11 wt% is used. In certain embodiments, the method is carried out by providing an agarose solution comprising ZnCl ₂ as a stabilizer at concentrations up to 15% by weight.

In some embodiments described herein, a crosslinking agent is added to the agarose solution, thereby stabilizing the agarose layer under positive transmembrane pressure (TMP), referred to as the differential pressure between the up and down pressures of the membrane. Thus, in some embodiments, the agarose solution is used in the process comprising a cross-linker or cross-linking agent having a concentration in the range of 0.01% to 1% by weight. Exemplary crosslinkers or crosslinkers are divinylsulphone (DVS). In certain embodiments described herein, the agarose solution is heated to a temperature in the range of 20-90 ° C. or 45-75 ° C. In certain embodiments, the agarose solution is heated to a temperature of 70 ° C.

In some embodiments, the heated agarose solution is applied to the porous support membrane, as described herein, and the porous support membrane is also heated to a temperature, such as in the range of 20 to 90 ° C or 45 to 75 ° C. In certain embodiments, the porous support membrane is heated to a temperature of 70 ° C. The temperature at which the agarose solution and the porous support membrane are heated may be the same temperature or may be different temperatures.

Following application of the agarose solution onto the porous support membrane, the agarose coated porous support membrane is then immersed in the bath at a temperature below the gelling point of the agarose solution. The gelling point of agarose can be predetermined using those described in the present invention and those known in the art. It will be clear to the person skilled in the art how to determine the gelation point of the agarose solution. In some embodiments, the bath is cooled to a temperature below room temperature. In some embodiments, the temperature is in the range of 5-60 ° C. In another embodiment, the temperature is in the range of 10-40 ° C.

In some embodiments, the porous support membrane has an average thickness in the range of 100 μm to 200 μm and an average pore size of less than 0.2 μm. In some embodiments, the porous support membrane has a thickness in the range of 10 μm to 500 μm.

The methods described herein are useful for preparing ultrafiltration membrane composites comprising a layer of agarose on a porous support membrane. In some embodiments, the porous support membrane layer penetrates or penetrates into the porous support membrane, resulting in a membrane composite having high resistance to delamination. A penetration thickness of 1 to 15 μm was found to be a membrane suitable for filtration and to have high resistance to delamination.

The agarose superfiltration membrane composites described herein generally comprise agarose layers having a thickness in the range of 1 μm to 100 μm or in the range of 1 μm to 20 μm. In certain embodiments, the thickness of the agarose layer is in the range of 10 μm to 20 μm. In various embodiments, the agarose layer is deposited on the porous support membrane. In some embodiments, deposition of the agarose layer on the porous support membrane comprises infiltration or infiltration of the agarose solution into the lower porous support membrane, gelation and then solidification of the agarose solution by cooling. Agarose penetrates into the supporting membrane in the range of 1-15 micrometers.

In some embodiments, the porous support membrane is polyester, polyolefin, polyethylene (PE), polypropylene, polyethylene terephthalate (PET), polyether-ether ketone (PEEK), polyethersulfone (PES), and polytetra A woven or nonwoven fabric formed of a polymer selected from the group consisting of fluorinated polymers such as fluoroethylene or polyvinylidene fluoride (PVDF). In some embodiments, the porous support membrane comprises polyvinylidene fluoride (PVDF), polyethylene (PE), polyethersulfone (PES), polyethylene terephthalate (PET), and polycaprolactam and poly (hexamethylene adipamide Woven or nonwoven polymeric fibers formed from a polymer selected from the group consisting of polyamides).

As evidenced by the experimental data provided herein, the ultrafiltration membrane composites described herein comprise a porous support membrane having an average thickness of greater than 10 μm. In some embodiments, the ultrafiltration membrane composites described herein comprise a porous support membrane having a thickness in the range of 20 μm to 500 μm. In certain embodiments, the porous support membrane has an average thickness of 100 μm.

The present invention also describes a process for using an agarose ultrafiltration membrane composite. In some embodiments, the membrane complexes described herein are useful for removing viral particles from a sample containing a protein of interest. In some embodiments, the protein of interest is a recombinant protein. In certain embodiments, the protein of interest is a monoclonal antibody.

The membrane composites described herein can be used under normal flow tangential flow conditions or tangential flow filtration conditions and can also be employed in processes such as purification and / or concentration of protein solutions. In some embodiments, the membrane composites described herein are embedded in a suitable filtration device.

In various embodiments, the agarose ultrafiltration membrane composites described herein are used for size based separation. In some embodiments, the membrane complexes described herein can be used for the removal of viral or viral particles from a solution containing a protein of interest, eg, by size based separation or size exclusion.

1 (FIGS. 1A and 1B) includes a description of the devices used for the preparation of the agarose membrane composites described herein, and FIG. 1A shows a poly positioned on a hot or heated plate 30 at 70 ° C A device 10 having a microporous substrate 20 (ie, porous support membrane) formed from vinylidene fluoride (PVDF) is shown, wherein the agarose casting solution is deposited on the glass plate 50 by tape 40. Pour over the supported substrate 20.
FIG. 1B includes a microporous substrate (PVDF) 20 (ie, a porous support membrane) positioned on a hot or heated plate 30 at 70 ° C. and has a shim 80 and a plastic sheet 70. A device 60 sandwiched between the shims 80 is used to gap the micrometer adjustable knife 90 used to disperse agarose casting solution, but with a substrate 20 and a plastic sheet. Before pouring the agarose casting solution between the 70s, a micrometer adjustable knife 90 is then used to disperse the agarose so that the agarose membrane composite is quickly separated from the hot plate and Contact with ice for at least 2 minutes, followed by immersion in a water bath maintained at 20 ° C., plastic sheet 70 is carefully peeled off.
FIG. 2 shows a high magnification SEM micrograph (field emission scanning electron microscope) of agarose ultrafiltration membrane composites prepared as described in Example 5, where SEM was performed and analyzed under cryogenic conditions to maintain pore structure. Samples are cooled to a temperature below 150 ° C. and FIGS. 2A and 2B are cross-sectional views showing the top and bottom of the membrane composite (ie, the interface between the microporous substrate and the agarose layer), and FIG. 2C shows the surface image. The membrane is shown to be slightly asymmetric with a top open more than the bottom.
FIG. 3 shows a rejection curve illustrating the fractional molecular weight of the representative agarose membrane composites described herein, the X-axis shows the fractional molecular weight and the Y-axis the rejection, analysis as described in Example 4. It is done together.
4 is a graph showing the dependence of the flux and fractional molecular weight of the agarose membrane composite on the concentration of the agarose casting solution employed, and R90 and water flux data are described in Examples 3 and 4. As determined, the X axis represents the agarose concentration, the right Y axis represents the permeation amount in LMH / PSI, and the left Y axis represents R90.
FIG. 5 is a graph showing the dependence of the fractional molecular weight of the agarose membrane composite described herein on the ionic strength of the agarose casting solution employed, wherein the R90 numbers are determined as described in Example 7. Where the X axis represents the concentration of ZnCl ₂ used and the right Y axis and the left Y axis represent R90 values of 7% and 10% by weight agarose solutions, respectively, and higher, regardless of the concentration of agarose in the casting solution. The ionic strength of the casting solution results in a more open superfiltration membrane, and as expected, at any given ionic strength, a more open superfiltration membrane is obtained with a lower agarose casting solution concentration.
FIG. 6 is a graph illustrating crosslinking of DVS and agarose layers to prevent from compression under pressure, illustrating that 20% loss in 15 psi transmission can be avoided by crosslinking agarose layers with 0.1% DVS solution. , The X-axis represents the pressure at PSI and the Y-axis represents the standardized transmission in LMH / PSI.
FIG. 7 is a graph showing that drying of the wet agarose membrane composite in a 20% glycerin solution (in water) does not negatively affect the fractional molecular weight of the membrane, ie, the drying process shows the pore structure of the agarose layer. Indicates not to collapse.
FIG. 8 is a graph showing the penetration depth of the agarose layer into the top layer of the microporous PVDF substrate, with basic analytical data obtained using a SEM-EDS Instrument (INCA300, Oxford Instrument, UK) (on a SEM insert). Microregion composition analysis (indicated by an asterisk) is performed at 5 μm intervals in the Z direction starting from the surface of the microporous substrate and extending 40 μm into the substrate, while the fluorine signal is due to the PVDF microporous substrate, while the oxygen signal is agarose In addition, most agarose is localized in the first layer to a depth of 10 μm of the microporous substrate, with the X axis representing depth (micrometers) and the Y axis representing the ratio (O / F).
FIG. 9 is a graph showing the increase in viscosity of a representative agarose solution (7% by weight) when the gel point is reached, where a rotational viscometer is used to perform the analysis as described herein, with the X-axis being temperature (Celsius). ) And the Y axis represents the viscosity.
FIG. 10 is a graph depicting the characteristic permeation attenuation of the agarose agarose ultrafiltration membrane complex and the Viresolve® Pro membrane, with virus-spiked cell culture media flowing through each device at a constant pressure of 10 psi. Membrane permeation is measured and throughput is monitored by measuring the permeation volume at various time points, with the X axis representing throughput and the Y axis representing J / Jo values.

Embodiments described herein relate to agarose ultrafiltration membrane composites and methods of making and using the same. In particular, the ultrafiltration membrane composites described herein are prepared by casting a layer of agarose solution onto a porous support membrane (also referred to as a substrate). The agarose solution penetrates into the porous support membrane, resulting in an agarose ultrafiltration membrane composite that is highly resistant to interlaminar peeling.

As used herein, the term “superfiltration membrane” or “UM membrane” refers to Pure Appl. Based on the definition of the International Union of Pure and Applied Chemistry (IUPAC) terminology, published in Chem. (1996), 68, 1479; According to him, microfiltration is defined as a pressure-driven membrane-based separation process in which particles larger than 0.1 μm and dissolved macromolecules are rejected, and ultrafiltration is particles smaller than 0.1 μm and larger than about 2 nm and It is defined as a pressure-driven membrane-based separation process in which dissolved macromolecules are rejected.

Thus, ultrafiltration membranes are capable of concentrating or diafiltering soluble macromolecules having a size in a solution of less than about 0.1 μm and also in tangential flow mode for extended periods, typically up to 4 hours and up to 24 hours. It is defined as continuously operable. In contrast, microporous membranes can remove particles larger than 0.1 μm and are used in dead-end filtration applications. Microporous membranes generally allow soluble macromolecules to pass through the membrane.

The methods described herein combine a solution with a thermal phase reversal technique and eliminate the use of organic solvents in casting solutions or in cost preparation. In addition, the methods described herein allow for simpler measures to control the pore size. This is at least in part due to the fact that it is generally easy to control the pore size of agarose gels for gels of other compounds such as crosslinked (crosslinked) dextran or polyacrylamide gels. For example, according to US Pat. No. 3,527,712A, the pore size of the agarose gel depends on the agarose concentration of the gel. It is known in the art that when the concentration of agarose decreases, the effective pore size of the gel increases. Thus, according to the specific concentration of agarose, it becomes possible to classify molecules having a molecular weight between 10 Da and 700 kDa. To treat agarose, the agarose polymer must be heated above its melting temperature of about 92 ° C. in the presence of water. The polymer melts at that temperature and the molten polymer is then solvated with water to form a solution. The polymer is dissolved in water when the temperature is higher than the gelling point of the polymer and the gel point is in the range of 20 to 43 ° C. depending on the type and concentration of agarose used. At and below the gel point, the polymer phase separates and becomes a hydrogel. The form of agarose used to prepare the membrane composites described herein exhibits a gelation point in the range of about 35-40 ° C. or in the range of 37-39 ° C.

In some embodiments, the agarose superfiltration membranes described herein comprise a) a heated stainless steel roll with or without a film thereon; And b) a hot support solution of agarose and a porous support membrane between the two nip rolls formed by the rotating cylinder :.

The degree of agarose penetration into the porous support membrane and the thickness of the agarose layer are controlled by the solution pressure at the inlet into the space between the nipple rolls, the durometer (hardness) and diameter of the roll, the solution viscosity and the process speed.

In another embodiment, the agarose solution can be applied to the porous support membrane by conventional knife-over-roll or slot die coating methods.

The porous support membrane is then contacted with a nonsolvent for agarose, such as water, at or below its gelling temperature. In some embodiments, the agarose layer can be done in additional steps such as crosslinking, chemical derivatization using functional chemistry, and the like.

In another embodiment, the agarose ultrafiltration membrane composite is formed by spreading the agarose solution between the porous support membrane and the non-stick nonpolymer sheet. Dispersion of the agarose solution may be achieved by any suitable means. Exemplary embodiments include rubber rollers or bird knives, doctor blades, and the like. The spreading step can be done on a hot plate at a temperature above the gelling point of the agarose solution, thereby using thermal phase inversion to form an ultrafiltration layer over the entire surface along the length and width of the porous support membrane. Next, leaving the porous support membrane coated with a thin layer of agarose in the ultrafiltration range, the agarose coated porous support membrane is coated with water to prevent damage to the agarose layer while the non-tacky polymer sheet is removed. Contact. Optionally, the formed agarose layer can then be done in additional steps such as crosslinking, derivatization using functional chemistry and the like.

Porous support membranes, also referred to as substrates in the present invention, can be used to prepare the agarose ultrafiltration membrane composites described herein with polymers such as polyethylene, polypropylene, polyether-ether ketone (PEEK) And may be used where there are various solvents such as DMSO, DMF and NMP.

The porous support membrane or substrate is preferably made porous to resist flow, mechanical strength, flexibility and resistance to swelling or dissolution by organic solvents. The porous support membrane may comprise a woven or nonwoven fabric, which may be polyester, polyamide, polycaprolactam, poly (hexamethylene adipamide), polyolefin, polyethylene (PE), polypropylene, aromatic polymer, polyethylene terephthalate (PET), polyether-ether ketones (PEEK), or fluorinated polymers such as polytetrafluoroethylene or polyvinylidene fluoride (PVDF), polysulfone or polyethersulfone (PES), halogenated polymers or polytetrafluoro And polymers such as fluorinated polymers such as ethylene and polyvinylidene fluoride (PVDF).

In some embodiments, a porous support membrane that is microporous is used. Such microporous membranes can be made of ultra high molecular weight polyethylene (UPE) as described in US Pat. No. 4,778,601 A. These microporous membranes are generally made of ultra high molecular weight polyethylene. This method employs the extrusion of a solution of porogen and UHMW-PE through the forming dye followed by thermal phase separation of the polymer and the pore polymer. The microporous structures are then created by removal of the porosity polymer.

The preparation of such microporous membranes generally includes the steps of forming a mixture of ultra high molecular weight polyethylene and a porepolymer. This mixture is heated to elevated temperature to form a solution. The solution is extruded through the forming die under appropriate shear, thus forming a membrane. The extrudate is cooled to phase separate into polymer-rich, porogen-poor and polymer-poor and porogen-rich phases in the membrane. The removal of the pore-producing polymer creates a microporous structure in the membrane. The microporous membrane formed according to the above is then dried. A detailed description of this process can be found, for example, in US Pat. No. 4,778,601, the entire contents of which are incorporated herein by reference. In some embodiments, these microporous membranes are suitable as substrates or supporting membranes for the production of layered ultrafiltration membranes, as described herein.

In addition to the polymers described above, it is also possible to use porous membranes formed of other woven or nonwoven polymers for the production of the ultrafiltration membrane composites described herein.

In some embodiments, suitable porous support membranes useful for the preparation of the porous agarose membrane composites described herein have an average layer thickness of greater than 10 μm. In other embodiments, these support membranes have a thickness in the range of 20 μm to 500 μm. In another embodiment, porous support membranes having a thickness of up to 120 μm are employed. In certain embodiments, the porous support membrane has an average thickness of 100 μm and an average pore size of about 0.2 μm.

In some embodiments, the agarose ultrafiltration membrane composites described herein can be prepared, for example, as shown in FIGS. 1A and 1B. In order to achieve good adhesion between the porous support membrane and the agarose, it has been found that it is preferred if the polymer support membrane is heated to an elevated temperature before contact with the agarose solution. In some embodiments, the porous support membrane is heated to a temperature in the range of about 20-90 ° C., or a temperature in the range of 45-75 ° C. In certain embodiments, the porous support membrane is heated to 70 ° C. In addition, the agarose solution is heated to a temperature elevated to, for example, a temperature in the range of 20 to 90 ° C., or a temperature in the range of 45 to 75 ° C., before contacting the agarose solution. When the agarose solution is cast and dispersed on the porous support membrane, it is desirable to control the depth of penetration of the agarose solution into the lower support membrane. Thus, the porous support membrane treated with agarose is cooled and converted to the solid state as soon as possible. For the cooling step, the agarose coated porous support membrane is contacted with ice water and then contacted the water bath, which is maintained at a temperature below 25 ° C. to effect gelation. The agarose coated membrane may be in direct contact with water without ice. In some embodiments, the temperature of the bath is in the range of 5 to 60 ° C. In some embodiments, the temperature of the water is maintained at a temperature in the range of 10-40 ° C.

For the preparation of the agarose layer, the agarose solution is prepared comprising agarose having a concentration in the range of 1-12% by weight or in the range of 5-11% by weight. In some embodiments, commercially available agarose forms such as HD2, HR, ES 3: 1 or LE are used. Examples of commercial sources of agarose are Hispanagar, S. sp. a. to be. Agarose powder is mixed with water at room temperature and then heated to elevated temperature until agarose solution is formed. When the agarose solution reaches a temperature of about 92 ° C., the melting point of the agarose, the solution is stirred by centrifugal force and the gas is removed.

Generally, degassing is performed after cooling to a centrifugal force of 3500 rpm and a temperature in the range of 20 to 90 ° C. or a temperature in the range of 45 to 75 ° C. In certain embodiments, the temperature is about 70 ° C. The agarose solution is maintained at this temperature until used to cast the agarose film.

The applied agarose solution may also further comprise an additive such as, for example, zinc chloride as a stabilizer. The stabilizer can be mixed with agarose powder and added to the aqueous solution at an appropriate ratio. In this way, for example, the casting solution may be prepared comprising a stabilizer such as zinc chloride at a concentration up to 15% by weight. In some embodiments, the agarose solution comprises ZnCl ₂ at a concentration of up to 10% by weight. However, solutions comprising less than 5% ZnCl ₂ may also be used.

To ensure penetration of the agarose solution into the porous support membrane, the porous support membrane is heated to a temperature of about 20-90 ° C. before the agarose solution is cast on the membrane. In some embodiments, the porous support membrane is heated to a temperature in the range of 45-75 ° C. or a temperature of about 70 ° C.

When the agarose solution is applied to the porous support membrane, it is important that an extremely accurate amount of agarose solution penetrates into the porous support membrane. It is also undesirable to block pores of the porous support membrane by the agarose solution. When the agarose solution is cast on the porous support membrane, it is passed by appropriate means uniformly over the surface of the membrane. Suitable means include, but are not limited to, for example, a bird knife, or a doctor blade with a gap. The coated membrane then immediately drops from the heating source and the agarose solution is cooled and gelled. To achieve this, the coated membrane is immersed in a water bath and maintained at a temperature in the range of 15-20 ° C. or 18-20 ° C. In certain embodiments, the temperature is about 20 ° C. Cooling may also be achieved by subsequent dipping in ice baths and / or water baths.

In addition, the agarose layer can be crosslinked to increase pressure resistance. Furthermore, surface modification of the agarose coated membrane can also be done, depending on the desired application of such a membrane, for example for use as an ion exchange membrane.

To crosslink the agarose layer, the prepared agarose coated membrane may be contacted in a suitable manner with a solution comprising a crosslinking agent or crosslinking material. In another embodiment, the crosslinking agent may be added to the agarose solution before the agarose layer is cast on the porous support membrane. In some embodiments, the crosslinking agent is included in the agarose solution at a concentration ranging from 0.01% to 1% by weight. Direct addition of the crosslinking agent ensures uniform crosslinking of the agarose. Various crosslinking methods known in the art and described herein can be used.

As noted above, the crosslinking reaction may involve a reaction between the dissolved crosslinker and the agarose of the membrane composite. Suitable reaction solutions may be based on water soluble or organic solvents, or aqueous-organic mixtures. Non-limiting examples of organic solvents include N-methyl pyrrolidone, dimethyl acetamide, dimethyl sulfoxide, dimethyl formamide and similar solvents.

Exemplary crosslinkers include, for example, two or multifunctional epoxides such as epichlorohydrin, butanediol diglycidyl ether (BUDGE), ethylenediol diglycidyl ether (EDGE), polyethyleneglycol diglycidyl ether and Such as butane diepoxide. Multifunctional N-methyl methoxy compounds may also be used as crosslinking reagents. For example, Cymel 385 and Powerlink 1174, all of which are available from Cytec Industries of West Paterson, New Jersey. It has been found that the crosslinking reaction can be carried out with a solution in which the crosslinking agent is present at a concentration ranging from about 5% to about 60% or from about 10% to about 40% by weight.

In certain embodiments, divinylsulfone or DVS is used as the crosslinking agent. DVS has proved to be particularly suitable for crosslinking the agarose layer described herein. DVS may be added to the agarose solution prior to casting of the agarose layer and may also be applied at a very low concentration relative to the concentration of crosslinkers commonly used as described above.

One skilled in the art can readily determine appropriate reaction conditions, for example, to employ appropriate concentrations, temperatures, pressures and settings of the equipment used. In general, the reaction is expected to occur at a higher rate at higher temperatures: however, the reaction temperature should be chosen such that the composite material described in the present invention is modified as flexibly as possible. In addition, one of ordinary skill in the art can easily determine the influence of the reaction scale. For example, larger reaction vessels will require longer time to reach the reaction temperature and also to cool down. In addition, higher pressures can be used to increase the reaction rate. Depending on the reaction vessel, the expert may use a continuous flow reactor or other suitable means to increase the contact of the reactants with the membrane, thereby controlling the reaction. Higher concentrations generally increase the reaction rate. The type of crosslinker applied and the solvent selected can play a role in determining the required reaction time. Hydroxyl ion activity is another important reaction condition.

Generally, the reaction time used is in the range of about 2 to about 100 hours, but a reaction time of about 4 to about 24 hours is most common. The reaction can be done at room temperature and up to about 60 ° C. In some embodiments, the reaction is conducted at a temperature in the range of about 25 ° C to about 50 ° C. One skilled in the art can alter or reduce the time, for example, by using a continuous roll for a roll process to increase the mass transfer rate, or by adjusting the temperature, concentration or any other parameters to further increase the reaction rate. You can.

When the multifunctional epoxy compound is used as the crosslinking agent, the reaction is carried out under basic conditions. For example, sodium or potassium hydroxide. Typically, about 0.1 M to about 1 M hydroxide solution is used. One skilled in the art can readily determine a method for balancing the reaction of agarose to alkaline degradation. Higher hydroxide concentrations and higher reaction temperatures generally accelerate alkaline degradation, while lower hydroxide concentrations and lower reaction temperatures generally slow the rate of crosslinking reaction as well as the degradation rate.

Powderlink 1174, Cymel 385 and similar crosslinking agents (multifunctional N-methyl methoxy compounds) crosslink with agarose via hydroxyl to agarose with an acid catalyst such as toluene sulfonic acid. Other similar acid catalysts are organic sulfonic acids and non-oxidizing inorganic acids. About pH or a suitable acidic condition of about 2 to 4 is generally suitable. Preferred catalysts are Cycyt 4040, sulfonic acid catalysts available from Cytec industries. It will be very apparent to those skilled in the art that strong acid conditions cannot be recommended due to membrane degradation.

The reaction between the agarose membrane and the crosslinking reactant can be carried out, for example, in an aqueous solution such as water or 100% water mixed with a solvent such as methylethyl ketone, methylpentanediol, acetone or other ketones. However, this list of aqueous solutions is exemplary. In certain embodiments, the crosslinking step is performed in an alkaline environment.

The ultrafiltration agarose membrane composites described herein can be modified using any suitable technique known in the art or described herein. In some embodiments, a membrane suitable as the ion exchange material may be formed.

As described herein, the term “ion exchange material” refers to a high molecular weight matrix having covalently charged charge substituents immobilized thereon. For overall charge neutrality, non-covalent counterions are bound to charged substituents by interactions of ions. "Ion exchange material" has the ability to exchange ions of non-covalent counterions or surrounding solutions for similarly charged binding partners. Depending on the charge of its exchangeable counterion, the "ion exchange material" is referred to as "cationic exchange material" or "anion exchange material". Depending on the nature of the charged group (substituent), the “ion exchange material” is referred to as sulfonic acid or sulfopropyl or carboxyl resin, for example in the case of cation exchange materials. Additionally, depending on the chemistry of the charged group / substituent, the “ion exchange material” may be classified as a strong or weak ion exchange material, for example based on the strength of the covalent charged substituents. For example, in some embodiments, the strong cation exchange material may include sulfonic acid groups (eg, sulfopropyl groups) as charged substituents. Exemplary weak cation exchange materials include carboxylic acid groups (eg, carboxymethyl groups) as charged substituents. Exemplary strong anion exchange materials include quaternary ammonium groups, while exemplary weak anion exchange materials include diethylaminoethyl groups as charged substituents.

Moreover, the surface of the membrane composites prepared according to the methods described herein comprises a negative charge introduced through a one or two step process. In the case of a one-step process, charge alteration reactants are added to the crosslinking solution. In the case of the two-step method, the charge addition reaction is carried out before or after the crosslinking reaction.

Suitable reactants for forming negatively charged membrane complexes include, for example, compounds of structure X (CH ₂ ) mA or alkali metal salts thereof, wherein X is halogen, preferably chloride or bromide; A is carboxyl or sulfonate. One or more of the following, eg, reaction time, reactant concentration, pH, and temperature, can be controlled to adjust the amount of negative charge added to the surface of the membrane.

In certain embodiments described herein, divinylsulfone is used as a crosslinking agent under reaction conditions as described in US Pat. No. 4,591,640, which is incorporated herein by reference. In some embodiments, the crosslinking reaction is carried out at room temperature to pH> 11 in the presence of a reducing agent, and the unreacted secret groups are inactivated by neutral hydrophilic inerts, including some hydroxyl groups.

In some embodiments, the positive charge is imparted to the membrane using a glycidyl quaternary ammonium compound or quaternary ammonium alkyl halide. In some embodiments, the halide molecule has a structure of Y (CH 2) m B wherein Y is halogen and B is a positive charge component.

In any of the described embodiments, the crosslinking reaction is done before adding charged groups to the membrane. By carrying out the crosslinking reaction prior to adding charged groups, charge repulsion between like-charged groups can be avoided, thus preventing swelling of the polymer and membrane. In another embodiment, crosslinking is done simultaneously with the addition of charge. In this example, it is desirable to control the crosslinking reaction at a rate that avoids swelling when charge is added.

Based on the knowledge of the art combined with the knowledge of the present application, one skilled in the art will understand that the solvent agarose ultrafiltration membrane composites described herein can be prepared having the desired pore size, charge and other material properties.

Using the method described above, superfiltration membrane composites are obtained, which comprise a layer of agarose and a porous support membrane, wherein the agarose penetrates at least a portion of the thickness of the porous support membrane, thereby Results in a highly resistant agarose ultrafiltration membrane composite. For example, in some embodiments described, agarose penetrates into the porous support membrane, thereby reaching a penetration depth of 1-15 μm.

In some embodiments, the agarose layer has a thickness in the range of 1-100 μm or in the range of 1-40 μm. In certain embodiments, the agarose layer has a thickness in the range of 10-20 μm or in the range of 15-20 μm. The thickness of the agarose layer includes agarose on top of the porous support membrane as well as penetrating into the porous support membrane. In the various embodiments described, the agarose layer penetrates at least a portion of the thickness of the porous support membrane and then gels and solidifies by cooling the agarose.

The agarose ultrafiltration membrane composite has several uses. Membrane complexes can be used in biological manufacturing on an industrial scale as well as in analytical applications. In some embodiments, the membrane composite is employed in the device.

As noted above, the described agarose ultrafiltration membrane complexes are well suited for filtration of molecules of any size, as well as useful for virus purification in a normal flow or tangential flow format.

The membrane complexes described herein are high resolution useful for removing viruses from recombinant protein containing solutions. The use of such membrane complexes to remove viruses is, for example, large capacity (eg, as measured by the amount of protein of interest processed through the unit area of the membrane complex) and high efficiency (eg, log reduction value, As measured by LRV).

For example, when filtering a water soluble protein solution through a membrane to remove the virus, the membrane generally has a pore size small enough to retain the viruses while allowing the protein of interest to pass through the membrane. In general, such membranes preferably have high throughput and high viral retention.

Virus retention is defined as the Log Reduction Value (LRV), which is defined as the algorithm of the ratio of titer (titer concentration) in the supply to the filtered liquid. Throughput is defined as the volume of protein solution that can pass through a region of the membrane before complete fouling occurs. As used herein, the term “complete fouling” refers to the concentration of the membrane where less than 10% of the original flux of the membrane is observed when filtration through the membrane to obtain viral retention of at least 3.5 LRV. It is generally observed that both high plus through the membrane and low protein binding of the membrane surface lead to high throughput. Throughput values of a given membrane vary greatly depending on the type and concentration of the protein solution, pressure, ionic strength and other test conditions. Under typical process conditions, a satisfactory ultrafiltration membrane has a throughput of at least about 1000 L / m ² .

A more representative performance gauge of viral maintenance membranes is the membrane area, calculated according to the Vmax method. All details regarding this method can be found in De 1 775 016 B1, the description of which is incorporated herein by reference.

In some embodiments, removal of the virus is a filtration containing the agarose ultrafiltration membrane complex described herein under conditions that permit passage of the recombinant protein through the membrane while preventing passage of the virus through the membrane complex. It involves the flow of the recombinant protein containing solution through the device.

In some embodiments described herein, the agarose ultrafiltration membrane complex used for removal of the virus is substantially hydrophilic, ie, easily wettable in water. In various embodiments described herein, the agarose layer in the membrane forms membrane hydrophilicity. As noted above, the agarose ultrafiltration membrane complex, when used for virus removal, preferably allows passage of the virus through the membrane complex while allowing passage of the protein of interest. This is accomplished, at least in part, by having agarose layer on the porous support membrane when the pores of the applied agarose layer are small enough to retain the virus while allowing passage of the protein of interest through the membrane.

In some embodiments, the membrane composites described herein are embedded in the device. In some embodiments, the device comprises a corrugated tube formed of one, two or three agarose ultrafiltration membrane composites described herein, in some embodiments, such a device is used for the removal of viruses. However, without wishing to be bound by theory, it is contemplated that any suitable device format that implements the membrane composites described herein may be used.

In some embodiments, the membrane complexes described herein (either alone or employed in a suitable device) may be used in LRV greater than 6 or relatively small viruses (eg parvovirus) for relatively large viruses (eg murine leukemia virus). Can be used to effectively remove the virus at LRV greater than 4.

In some embodiments, the device used to remove the virus from the protein containing solution includes a housing suitable for containing the membrane complex filtration material described herein, and also includes an inlet and a filtered liquid for containing the liquid to be filtered. The filter material further comprises one, two or three composite void-free membranes, the upstream layer being oriented such that its tightest face faces downstream.

In general, by employing a number of asymmetric superfiltration membranes in which the membrane is “tight side downstream” oriented and arranged in a pleated configuration, the resulting filter capsule has good virus retention capability while maintaining good flux.

In certain embodiments, the filtration capsule used to remove the virus comprises a tubular housing and a pleated filtration tube substantially coaxially enclosed within the housing. The tubular housing of the filtration capsule may be configured to contain and send a fluid process stream therethrough, thereby providing a fluid inlet and a filtrate outlet. Upstream of the pleated filtration tube, the fluid process stream is introduced into the filtration capsule through the fluid inlet. Downstream of the pleated filtration tube, the fluid process stream exits the filtrate outlet from the filtration capsule.

For further details on the construction and operation of such devices, reference may be made to EU 2 163 296 A1 and US Pat. No. 5,736,044 A1, the disclosures of which are incorporated herein by reference. In particular, aspects of the filter cartridge can be employed in the construction of filter capsules employing the membrane composites described in the present invention without departing from the spirit and scope of the present invention.

To remove the virus from the protein solution, a solution containing one or more protein (s) of interest and one or more forms of viruses undergoes a filtration step using one or more agarose ultrafiltration membranes, the filtration being in TFF mode or NFF. Can be done in mode. In either mode, filtration is performed under conditions to maintain the virus, generally having a diameter of 20 to 100 nanometers (nm), while allowing passage of one or more protein (s) through the membrane complex. Moreover, when filtration of the solution is complete, the membrane is washed with water or aqueous buffer solution to remove any retained proteins. The use of a wash step allows to obtain a high yield of protein solution substantially free of virus.

The present invention, combined with the knowledge of the art, may enable one or more persons skilled in the art to understand the methods described herein.

Moreover, needless to say to those skilled in the art, in the examples and the remainder of the description, the amount of ingredients present in the composition at all times is added up to only 100% or 100% by mole, based on the composition as a whole, and higher than the indicated percentage range. Even if viruses are caused, they cannot be exceeded. Unless otherwise indicated, the% data are% and mole% by weight, with the exception of the proportions, they are presented in volumetric data such as eluents for the preparation where solvents in any volume are used in the mixture.

The temperature in the examples, the detailed description and the claims is always ° C.

Embodiments are further illustrated by the following embodiments, which are to be considered illustrative. All references, patents and contents of published patent applications and figures cited in this application are incorporated herein by reference.

Example

Example 1 Preparation of Agarose Solution

It is added 10% by weight of agarose solution, 10 g of agarose powder (type HD2 obtained from Hispanagar and type HR, ES, 3: 1 and LE obtained from Aquapor) to 90 g of water and mixed at room temperature for 30 minutes. The hydrated agarose mixture is then heated in the microwave until agarose solution is formed. The solution is degassed by centrifugal force at about 3500 rpm and 70 ° C. for 5 minutes. The free flowing properties of the solution are maintained at this temperature. The solution is maintained in a hybrid oven at 70 ° C. until used to cast a thin agarose film or layer. Other agarose solution concentrations are prepared in a similar manner using appropriate relative ratios of agarose and water.

Example 2: Preparation of Agarose Solution with Other Additives

The agarose solution was initially prepared as described in Example 1 and hybrid oven was heated to 70 ° C. until 10 g of zinc chloride (98% obtained from Acro) was added to the 10% by weight agarose solution and the salt dissolved. Are mixed in. The free flowing properties of the solution are maintained under these conditions. The solution is then maintained in a hybrid oven at 70 ° C. until used to cast a thin agarose film or layer. Other agarose solution concentrations, including other zinc chloride concentrations, are prepared in a similar manner using appropriate relative ratios of agarose, zinc softened and water.

Example 3: Determination of Water Permeation of Agarose Superfiltration Membrane Composites

Water flux measurements are performed using an Amicon (EMD Millipore Corporation, Billerica) stirred cell. A wet membrane (50:50 IPA (in water mixture)) is placed in the cell. The cell is filled with deionized water, pressurized to 25 psig, connected to a subscribed air supply. The effluent is collected over standard test times and the permeate is calculated using known membrane areas. Alternatively, an automatic permeation test instrument can be used to test the membrane composite.

Example 4: Determination of Fractional Molecular Weight of Agarose Filtration Membrane Composites

Rejection of model solutes is the most common method for evaluating the performance of ultrafiltration membranes. Accordingly, nominal molecular weight limitations can be determined with various solutes; Proteins are frequently used. The NMWL of an UF membrane is generally the molecular mass of the smallest protein that the membrane typically rejects at a selected level or rejection of 90-95%. Other solutes that can be used to characterize UF membranes include dextran, which are available in various molecular weights. The overall rejection spectrum from molecules of about 1000 Da molecular weight to about 2,000,000 Da can be measured in a single test.

Fractional molecular weight measurements, among others, were described in Separation Science and Technology, 16 (3), p. L. 275-290 (1981). Zeman and M. Based on methods published by Wales. Membrane composites to be characterized are problematic with solutions containing polydisperse dextran at molecular weights ranging from 1000 to 2,000,000 Da in suitable devices. The transmission during the test is controlled with a low transmission to minimize the concentration difference polarization. Feed and permeate streams are sampled and analyzed by size exclusion chromatography (SEC) and the chromatographic data is used to calculate rejection as a function of dextran molecular mass.

The rejection (R) with dextran molecular mass is R = 1-Cp / Cf, where Cp and Cf are the dextran concentrations of the given molecular mass for feeding and permeation, respectively. The molecular weight at which the membrane maintains 90% of the dextran feed is 90% of the dextran rejection value (R90).

General NMWL data from this analysis for a representative agarose membrane composite is shown in FIG. 3.

Example 5: Method for preparing 65 kDa, 120 kDa and 250 kDa NMWL agarose ultrafiltration membrane composites

This example illustrates a process for preparing a 65 kDa nominal fractional molecular weight agarose ultrafiltration membrane composite.

Polyvinylidene fluoride (PVDF) microporous membranes having an average pore size of 0.2 μm and an average thickness of 100 μm were employed as the microporous membrane substrate. The microporous substrate was attached to a hot plate and kept at a temperature of 70 ° C.

A polymer solution containing 10% by weight agarose (type ES) prepared in Example 1 was cast on the heated microporous PVDF membrane using a micrometer adjustable knife with a 25 μm gap. The agarose coated membrane was then quickly removed from the heated plate and immersed in a water bath maintained at a temperature of 20 ° C. (shown in FIG. 1A).

* Mebrain imaging was performed on a cryo stage manufactured by Gatan, model Alto 2500. Alto 2500 has a sophisticated manufacturing chamber attached directly to a high resolution scanning electron microscope (SEM). The chamber includes a cooled fracture knife that ensures controlled depth fracture. A separate 2-port quick freeze station was used to freeze samples. Cross sections of the prepared membrane composites are shown in FIGS. 2A-2C. The thickness of the agarose coating on the surface of the agarose substrate is about 15 μm.

The membrane composite is free of defects and has the permeation and retention characteristics (determined as described in Examples 3 and 4) shown in Table 1.

A 120 kDa NMWL agarose ultrafiltration membrane composite was prepared as follows. Polyvinylidene fluoride (PVDF) microporous membranes having an average pore size of 0.2 μm and an average thickness of 100 μm were employed as the microporous membrane substrate. The microporous substrate was attached to a hot plate and kept at a temperature of 70 ° C.

A polymer solution containing 10% by weight agarose (type 3: 1) prepared as described in Example 1 was placed on the heated microporous PVDF membrane using a bud or micrometer adjustable knife with a 25 μm gap. Cast. The agarose coated membrane was then quickly removed from the hot plate and immersed in a water bath maintained at a temperature of 20 ° C.

As measured by SEM, the thickness of the agarose coating on the surface of the microporous substrate is about 18 μm. The membrane composite is free of defects and has the permeation and retention properties (determined as described in Examples 3 and 4) shown in Table 1. Finally, a 250 kDa NMWL agarose ultrafiltration membrane composite was prepared as follows.

Polyvinylidene fluoride (PVDF) microporous membranes having an average pore size of 0.2 μm and an average thickness of 100 μm were employed as the microporous membrane substrate. The microporous substrate was attached to a hot plate and kept at a temperature of 70 ° C.

A polymer solution containing 10% by weight agarose (type HR) prepared as described in Example 1 was cast on the heated microporous PVDF membrane using a bird knife with a 25 μm gap. The coated membrane was then quickly removed from the hot plate and immersed in a water bath maintained at a temperature of 20 ° C.

As measured by SEM, the thickness of the agarose coating on the surface of the microporous substrate is about 15 μm. The membrane composite is free of defects and has the permeation and retention characteristics (determined as described in Examples 3 and 4) shown in Table 1.

Example 6: Dependence of Fractional Molecular Weight and Water Permeability of Agarose Superfiltration Membrane Composite on Concentration of Agarose in Casting Solution

The apparatus used to prepare the agarose membrane composite is shown in FIG. 1. First, the nonporous plastic sheet 70 is adhered under a hot or heated plate 30 maintained at 70 ° C. The PVDF microporous membrane substrate (pore size rating 0.2 μm) was cured in Zara by placing it on top of the plastic sheet 70 and adhering it to the plastic sheet 70. A polymer solution containing 5, 7 and 10% by weight agarose (type ES) prepared as described in Example 1 was used with a PVDF membrane substrate 20 using a micrometer adjustable knife having a gap of about 35 μm. Scattered as a thin layer between the plastic sheets 70. The agarose coated membrane sheet pair was then removed from the hot or heated plate 30 and placed in an ice bath to undergo agarose kelization and form a superfiltration layer by thermal phase inversion. The membrane sheet pair is then contacted with water (in a bath maintained at 20 ° C.) to damage the agarose ultrafiltration layer while the plastic sheet 70 is removed leaving a microporous substrate coated with a thin layer of agarose. I did not let you.

As shown in FIG. 4, the membrane fraction molecular weight and permeate are a function of agarose concentration in the casting solution. R90 of 25 kDa (1.2 LMH / PSI), 47 kDa (3.7 LMH / psi), and 1200 kDa (5.4 LMH / PSI) is obtained in 10, 7 and 5 wt% agarose solutions, respectively.

Example 7 Dependence of Agarose Ultrafiltration Membrane Composite Fraction Molecular Weight on Zinc Chloride Concentration in Casting Solution

Representative membrane composites were prepared as described in Example 6 with the difference of using the agarose casting solution (7 and 10% by weight HR type agarose) prepared as described in Example 2. As shown in FIG. 5, the high salt concentration in the casting solution results in a more open UF membrane. At 7 wt% agarose casting solution concentrations comprising 0, 5, 10, 12 and 14 wt% ZnCl 2, each of R90 was obtained: 35 kDa, 31 kDa, 44 kDa and 300 kDa, respectively. At 10 wt% agarose casting solution concentrations comprising 0, 5, 10, 15 wt% ZnCl ₂ , each of R90 was obtained: 15 kDa, 16 kDa, 26 kDa and 1350 kDa, respectively.

Example 8 Crosslinking of DVS and Agarose Membrane Composites to Prevent Agarose Layer Compression Under Pressure

Representative agarose membrane composites were prepared as described in Example 5. Three membrane composite membrane disks (each 25 mm in diameter) were placed in 20 ml sodium carbonate solution (0.5 M at pH 11) containing 0.1, 0.5 and 1% solution of mercaptoethanol (obtained from Sigma-Aldrich) in DI water. Wet (all together) and mix for 6 hours. Next, the membrane was tested as described in Example 3.

As shown in FIG. 6, when the composite membrane becomes compressible by pressure, it causes a 20% loss of water permeation at 15 psi. Crosslinking with DVS improves the mechanical properties of the agarose gel layer and prevents pressure damage. Only 0.1 wt% DVS is needed.

Example 9: Drying of the Agarose Membrane Composite

This example illustrates the drying of the agarose membrane composite in a 20% glycerin solution (in water).

Representative agarose membrane composites were prepared as described in the examples above. Fractional molecular weight and permeation amount of the membrane complex are determined as described in Examples 3 and 4. The membrane complex was removed from the test stirred cell, washed with water, and treated with 20 vol% glycerin in an aqueous solution, functioning as a humectant. The membrane composite was then dried under atmospheric conditions for 3 days.

As shown in the ratio shown in FIG. 7, the fractional molecular weight of the membrane composite does not change after drying. The permeation rate of the membrane composite before and after drying does not change to 0.7 LMH / psi.

Example 10 Evaluation of Penetration of Agarose into Substrates

This example illustrates the evaluation of the depth of agarose penetration into the microporous PVDF substrate.

Some penetration of agarose into the microporous substrate is necessary to obtain a sufficiently strong bond between the substrate and the ultrafiltration agarose layer and to become an agarose membrane composite with high resistance to interlaminar peeling. Penetration depth analysis is performed using Scanning Electron Microscope-Energy Dispersive Spectroscopy (SEM-EDS) Instrument (INCA300, Oxford Instruments, UK). Microarea complex analysis is performed at 5 μm intervals in the Z direction starting from the surface of the microporous substrate and extending 40 μm into the substrate.

Representative agarose membrane composites were prepared as described in Example 5. Basic analytical data obtained from SEM-EDS is used to determine the concentration of oxygen and fluorine atoms present in the sample. The fluorine signal is due to the PVDF microporous substrate, while the oxygen signal is due to the agarose. As shown in FIG. 8, the depth of agarose penetration into the PVDF substrate is approximately 10 μm.

Example 11 Determination of Gelling Points of Different Grades of Agarose

This example illustrates the determination of gelling points of different grades of agarose using a rotational viscometer.

A representative 7 wt% agarose solution was prepared as described in Example 1 and stored at 70 ° C. in a hybrid oven. The Brookfield viscosity standard (Lot # 112305) was used for inspection for instrument calibration: recording and observation, 489/502 ± 6cp. (Leg guard set boundary conditions, 50 RPM speed, LV2 spindle) To be used).

16 ml of agarose solution is poured into a sample chamber attached to a water jacket mounted on a Brookfield Viscometer Model DV-II + Pro (Brookfield Labs Inc.). The temperature of the sample chamber is set to 55 ° C. using a circulating temperature bath (containing water, VWR Scientific Products Model # 11301-1), and the chamber temperature is directly Reading is provided. Does each sample of agarose solution initiate the collection of data points? Allow to equilibrate at 55 ° C. for 10 minutes before in the temperature control chamber. The measurement is obtained using Wingather V3.0 software (Brookfield Engineering Labs Inc.) with an SC4-25 spindle with a rotational speed of 2 rpm and a starting torque of 2%.

The temperature of the chamber containing the agarose solution is lowered by continuously adding ice to the circulation temperature bath until the gel point is reached, as evidenced by the first increase in viscosity as shown in FIG. 9. If the torque is extended by 95%, data collection is stopped and the viscometer is off.

The apparatus temperature of gelation is reported in Table 2 for a representative set of different agarose grades at 7% by weight.

Example 12: Throughput and Viral Retention Characteristics of Agarose Superfiltration Membrane Complexes Using Solutions Containing Cell Culture Media

For throughput and virus maintenance with cell culture media, the agarose superfiltration membrane complex was prepared in Example 1 using 7% by weight agarose (type 3: 1) and GEHP (0.2μ hydrophobic PES) as the microporous substrate. Prepared as described. The membrane composite has a permeability of R90 and 4 LMH / psi of 485 kDa. The membrane composite disc (25 mm) was cut and placed in a Swinnex ^® filter holder with a filter area of 4.5 cm ² . One layer of polyester nonwoven is used on the bottom / outlet side of the device. A 25mm Viresolve ^® Pro device is used as the control. The membrane composite has a water permeability of R90 and 14 LMH / psi of 100 kDa.

Permeability, distribution culture medium throughput and virus maintenance were tested in a constant pressure setup with load cells. For the growth of Chinese Hamster Ovary (CHO) cells, a chemically defined cell culture medium of EM Millipore Corporation was used for the study.

Model virus, Bacterial phage PhiX-174 is added to acetate buffer at a concentration of 1.4 * 107 PFU / ml, pH 5, conductivity 13.5 mS / cm. The device was washed with buffer for 10 minutes and the feed was switched to the viral spike vessel and the viral spike cell culture medium was flowed through each device at a constant pressure of 10 psi. Throughput was monitored by measuring the penetration volume at various time points.

Samples for virus analysis were collected at each throughput (15, 250 and 500 L / m ² ) and LRV results are shown in Table 3. FIG. 10 shows permeation reduction of the agarose superfiltration membrane composite and the Viresolve ^® Pro membrane. The agarose ultrafiltration membrane composites described herein exhibit plugging behavior different from Viresolve ^® Pro, and the permeation rate decreases rapidly before the membrane dose is reached.

The specification is to be understood in terms of references cited in the specification, which is heretofore incorporated by reference herein. The embodiments in the specification are provided for illustration of embodiments and should not be construed as limiting the scope. Those skilled in the art will readily recognize that many other embodiments are encompassed by the present invention. All publications and references were incorporated by reference in their entirety. For the range in which the materials employed for reference contradict or contradict the invention, this specification will replace any such materials. Citation of any reference herein does not mean that such reference is prior art.

Unless otherwise indicated, all numbers indicating the amounts of components, cell culture, treatment conditions, and the like used in the specification are to be understood as being modified in all instances by the term “about”. Thus, unless otherwise indicated to the contrary, the numerical parameters are approximate and may vary depending upon the desired characteristic aspects obtained by the embodiments described herein. Unless otherwise indicated, the term "at least" preceding a series of conveying elements should be understood to refer to all components of the series. Those skilled in the art will recognize equivalents to the specific embodiments described herein, or may confirm using only routine experimentation. Such equivalents are to be understood as included in the following claims.

Many modifications and variations of the embodiments described herein may be made without departing from the spirit and scope of the invention, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only and are not meant to be limiting in any way. The detailed description and examples are to be considered as illustrative only, with a true scope and spirit of the invention being indicated by the following claims.

Claims (17)

Agarose ultrafiltration membrane composite having a pore size of less than 0.1 μm,
The composite includes a porous support membrane having an agarose layer on the porous support membrane, the agarose layer penetrates into the porous support membrane at 1-15 μm, and the agarose layer has a thickness in the range of 1-100 μm. An agarose casting solution comprising a target or impurity, or a target and impurities running into and through the pores, wherein the agarose layer further comprises 7-10 wt%, 5-15 wt% ZnCl ₂ Including, agarose ultrafiltration membrane composite. The agarose ultrafiltration membrane composite of claim 1, wherein the agarose layer penetrates into at least a portion of the thickness of the porous support membrane. The agarose ultrafiltration membrane composite of claim 1, wherein the composite is resistant to delamination. The agarose ultrafiltration membrane composite of claim 1, wherein the agarose layer comprises a thickness in the range of 1-20 μm. The agarose ultrafiltration membrane composite of claim 1, wherein the porous support membrane comprises a woven or nonwoven polymeric material. 6. The woven or nonwoven polymeric material of claim 5, wherein the woven or nonwoven polymeric material comprises polyester, polyamide, polyolefin, polyethylene (PE), polycaprolactam, poly (hexamethylene adipamide), polypropylene, aromatic polymer, polyethylene terephthalate ( PET), polyether-ether ketone (PEEK), polysulfone, polyethersulfone (PES), halogenated polymers and fluorinated polymers. The agarose ultrafiltration membrane composite of claim 6, wherein the fluorinated polymer is selected from polytetrafluoroethylene and polyvinylidene fluoride (PVDF). The agarose ultrafiltration membrane composite of claim 1, wherein the porous support membrane comprises an average thickness in the range of 20 μm to 500 μm. The agarose superfiltration membrane composite of claim 2, wherein a portion of the thickness of the porous support membrane through which the agarose penetrates is 1-15 μm. As a method for producing an agarose ultrafiltration membrane composite, the method
i. Providing a porous support membrane having an average pore size in the range of 0.01 μm to 1 μm and an average thickness in the range of 10 μm to 500 μm,
ii. Providing an agarose solution,
iii. Casting a layer of agarose solution on the porous support membrane, and
iv. Immersing the agarose coated porous support membrane in a water bath at a temperature below the gelling point of the agarose solution, whereby agarose ultrafiltration membrane composite having an average pore size of less than 0.1 μm ,
The agarose solution in step (ii) comprises agarose in a concentration ranging from 1-12 wt% or 5-11 wt%,
The agarose solution in step (ii) further comprises ZnCl ₂ at a concentration of up to 15% by weight,
The agarose solution in step (ii) comprises a crosslinking agent in a concentration ranging from 0.01% by weight to 1% by weight. The method of claim 10, wherein the porous support membrane is polyethylene, polypropylene, polycaprolactam, poly (hexamethylene adipamide), polyethylene terephthalate, polyether-ether ketone, polysulfone, polyethersulfone, polytetrafluoro A process for producing an agarose ultrafiltration membrane composite comprising a polymer selected from the group consisting of roethylene, and polyvinylidene fluoride (PVDF). The method of claim 10, wherein the porous support membrane is polyvinylidene fluoride (PVDF), ultra high molecular weight polyethylene (UHMW-PE), polycaprolactam, poly (hexamethylene adipamide), polysulfone, and polyethersulfone. A method for producing an agarose ultrafiltration membrane composite, comprising a polymer selected from the group consisting of: The method of claim 10, wherein the agarose solution in step (ii) comprises ZnCl _{2 at} a concentration of up to 15% by weight, and divinylsulphone (DVS) as a crosslinking agent. Method for producing ultrafiltration membrane composites. The method of claim 10, wherein the agarose solution in step (ii) is heated to a temperature in the range of 20 to 90 ° C. or 45 to 75 ° C. 12. The method of claim 10, wherein the porous support membrane in step (iii) is heated to a temperature in the range of 20-90 ° C. or in the range of 45-75 ° C. 12. The method of claim 10, wherein in step (iv), the agarose coated porous support membrane is immersed in a water bath at a temperature in the range of 5-60 ° C or in the range of 10-40 ° C. The method of claim 10, wherein in step (i) the porous support membrane is provided to have an average thickness between 100 μm-200 μm and an average pore size of 0.2 μm.

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