Surface treatment of membranes for purification

Abstract

The present invention relates to the surface treatment of a film having a crosslinkable diacrylate solution for the preparation of a crosslinked coating comprising a solution of at least one of 2.4 to 3.4% of hydroxypropyl acrylate and 1.1 to 2.1% of polyethylene glycol diacrylate; from 2.5% to 3.5% of a bisphenol A ethoxylate diacrylate; from 3.0 to 4.0% of a bisphenol A ethoxylate diacrylate, and from 1.5 to 2.5% of hydroxypropyl acrylate; 1.5% to 2.5% of hydroxypropyl acrylate and 2.0% to 3.0% of polyethylene glycol diacrylate; from 0.25% to 2.5% of 2-hydroxy-3-phenoxypropyl acrylate and from 2.0% to 5.0% of polyethylene glycol diacrylate; or from 3.0 to 4.0% of a bisphenol A ethoxylate diacrylate. The present invention relates to a filtration device for use with a biological fluid, having a membrane; and a cross-linked coating on the membrane to form a surface modification or treatment, wherein the cross-linked coating is formed from a coating solution comprising a cross-linkable diacrylate.

Description

Technical Field

[0001] Embodiments of the present disclosure relate to bioprocessing. More particularly, embodiments disclosed herein include the use of excipients and / or surface treatments on filtration membranes to increase flux. Background Art

[0002] Biofluids are processed in systems including containers (i.e., stainless steel containers and / or disposable containers) or bioreactors (e.g., polymer bags). These containers are sterile and used to process biological products, whether in batches, semi-continuously, or in continuous mode. One or more of the biological products include cell cultures, empty capsids or viral vectors, plasmids, adeno-associated viruses and monoclonal antibodies (mAbs) and other products. These biological products must be purified using several methods (e.g., various chromatography, dead-end filtration using porous membranes and tangential flow filtration) to remove metabolic byproducts and waste, viruses, etc. Because the byproducts, product aggregates, and waste vary greatly in size, the fouling of the filter is a problem during bioprocessing.

[0003] Several manufacturers offer a variety of filters that retain viruses, such as parvovirus. The filters include membranes made from a variety of base polymers, such as poly(vinylidene fluoride), poly(ethersulfone), and some cellulosics. The surface of the membrane is typically modified to be hydrophilic and / or relatively low in protein binding, etc. The surface modification can be a chemical coating process that, in addition to imparting hydrophilic properties, etc., can also change the pore size of the membrane. Typically, changing the pore size of the membrane is not required and can be a result of the coating process.

[0004] Some coating processes involve: forming polymer coatings from monomers, the polymer coatings comprising polymer units; and cross-linking these polymer units via one or more cross-linking means, such as chemical cross-linking agents in the presence of an energy source such as gamma radiation, ultraviolet light, and / or electron beams. In some processes, the monomers themselves can serve as the cross-linking agent. The polymer coatings used for this type of surface modification are typically based on acrylic and allylic monomers and cross-linking agents. The chemical and physical properties of the monomers / cross-linking agents ultimately control the surface properties of the final filter.

[0005] There are known cases of process streams of biological fluids that result in lower initial flux and / or sustained flux decline, namely fouling mechanisms of virus filters during filtration of protein solutions. As a causative mechanism of fouling, adsorption of proteins within biological fluids on the surface of the membrane is a potential challenge during bioprocessing.

[0006] Some prior art attempts at virus filtration processes in the past include the use of excipients. For example, at least one method includes steps for stabilizing downstream monoclonal antibody process intermediates by focusing on preventing large permanent aggregates that permanently block filter pores. A theory has postulated that concentration polarization occurs and explains flux behavior in certain cases. However, adsorption of aggregates to the pore surface is misunderstood as an alternative explanation that is unlikely due to the hydrophilic coating of the membrane.

[0007] Excipients, particularly the use of viscosity-reducing excipients, are generally known in the higher final formulation processing of protein concentration, and are used to promote multiple purposes of drug delivery and long-term shelf stability. Excipient molecules are known to inhibit protein-protein interactions. However, the use of excipients has been restricted. Although effective for other purposes, many excipients cannot provide significant filtration flux improvements. Their invalid potential reason may be that conductivity (conductivity) is usually added to biofluids via charged molecular species such as preparations (such as preparations containing sulfonic acid derivatives). This excessive conductivity shields (shield) ion interactions, otherwise the ion interactions can repel adsorption interactions. These previous excipients do not significantly prevent protein-protein interactions and protein-membrane interactions, and the interaction can limit filtration flux.

[0008] Although many monoclonal antibodies (mAbs) have minimal protein-protein interactions and protein-membrane interactions that limit filtration flux at current bioprocessing concentrations, filtration issues at low concentrations still exist. In addition, advances in other unit operations are pushing concentrations to higher levels where these interactions may even more frequently become limiting factors.

[0009] Providing a membrane with a significantly increased flux with a surface treatment chemistry is an advancement in the art, and the surface treatment chemistry enhances uses and performance. Without intending to be bound by theory, it is believed that these advancements are due to the unexpected height reduction of protein-membrane interactions. Without intending to be bound by theory, the additional advancement of the embodiments described in the present disclosure also provides an electrically neutral excipient, which interacts via π-electrons and hardly contributes to conductivity or does not contribute to conductivity. Reducing protein-protein interactions reduces the effective size of the protein to be filtered, and in some embodiments, this reduces pore contraction in combination with the reduction of protein-membrane interactions to achieve an increase in processing flux. These two advancements in the art each provide significant advantages. In addition, combining to provide synergistic advantages, thereby providing increased filter flux when filtering viruses from various biological processes and products. Summary of the invention

[0010] Some embodiments of filter membranes are disclosed, the filter membranes comprising a novel surface treatment formed from a diacrylate solution, substantially as shown in at least one of the accompanying drawings and / or described in conjunction with at least one of the accompanying drawings, as more fully set forth in the claims. Some embodiments of the present disclosure include a filter device for filtering biological fluids, the filter device is used with: an excipient, a polyethersulfone membrane; and a cross-linked coating on the membrane to form a surface modification, wherein the cross-linked coating is formed from a coating solution comprising a cross-linkable diacrylate, and wherein the excipient provides π-electron interactions. Some embodiments of filter membranes are disclosed, the filter membranes comprising novel surface treatment chemistry and the use of excipients. Some embodiments include methods of using various excipients, the excipients providing π-electron interactions to increase filter flux. It should be understood that the filter membranes have novel surface treatment chemistry and / or the use of π-electron interaction excipients; the filter membranes can be used to filter solutions containing mAbs and other biological models. In addition, without intending to be limited by theory, the inventors have surprisingly found that concentration and interaction between proteins and virus filter membranes are considered important factors in membrane fouling. Some embodiments of the present disclosure include surface treatment of the membrane, wherein the surface treatment comprises a coating solution for preparing a cross-linked coating, the cross-linked coating further comprising at least one of the following: 1) 2.4 to 3.4% hydroxypropyl acrylate and 1.1 to 2.1% polyethylene glycol diacrylate; 2) 2.5 to 3.5% bisphenol A ethoxylate diacrylate; 3) 3.0 to 4.0% bisphenol A ethoxylate diacrylate and 1.5 to 2.5% hydroxypropyl acrylate; 4) 1.5 to 2.5% hydroxypropyl acrylate and 2.0 to 3.0% polyethylene glycol diacrylate; 5) 3.0 to 4.0% bisphenol A ethoxylate diacrylate; and 6) 0.25 to 2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0 to 5.0% polyethylene glycol diacrylate.

[0011] Various provisions, aspects, and novel and inventive features of the present disclosure, as well as details of exemplary embodiments thereof, will be more fully understood from the following description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows a graph plotting treatment throughput (in kilograms per square meter) versus time for a surface treated film according to an embodiment of the present disclosure;

[0013] Figure 2Shown is a graph plotting filtration flux (in liters per square meter per hour (LMH)) versus throughput (in kilograms per square meter) for a surface treated virus filtration membrane according to an embodiment of the present disclosure;

[0014] Figure 3 shows a graph plotting the throughput (in liters per square meter) versus time for three virus filtration membrane devices filtering mAbs according to embodiments of the present disclosure;

[0015] Figure 4 shows a graph plotting filtrate volume (mL) after ten minutes at 2000 relative centrifugal force (RCF) versus excipient concentration (in millimoles per liter (mM)) according to embodiments of the present disclosure;

[0016] Figure 5 Shown is a graph plotting the filtration flux (in LMH) of a surface treated membrane for mAb according to an embodiment of the present disclosure versus the throughput (in kg / m2);

[0017] Figure 6 shows a graph plotting throughput (in kilograms per square meter) versus time for a surface treated virus filtration membrane for a solution containing mAb at mAb concentrations of 10 g / L and 20 g / L, with or without a caffeine excipient, according to an embodiment of the present disclosure;

[0018] Figure 7 shows a graph plotting throughput (in kilograms per square meter) of a surface treated virus filtration membrane versus time for a solution comprising mAb and 300 mM of a pyridoxine excipient for three different virus filtration devices according to embodiments of the present disclosure;

[0019] Figure 8 A table showing the level of non-specific adsorption of a model immunoglobulin G (IgG) protein (reported in micrograms) relative to the membrane effective filtration area (EFA) (in square centimeters) for various surface treated virus filtration membranes according to embodiments of the present disclosure is shown;

[0020] Fig. 9 shows a first graph plotting throughput performance versus test pressure (in psi) for a model test flow according to an embodiment of the present disclosure;

[0021] Fig.10 showing a second graph plotting filtration flux (in LMH) versus throughput (in liters per square meter) for a surface treated virus filtration membrane for a model flow according to an embodiment of the present disclosure;

[0022] Fig.11 showing a third graph plotting filtration flux (in LMH) of a surface treated virus filtration membrane versus throughput (in liters per square meter) for a model flow according to an embodiment of the present disclosure;

[0023] Fig.12 showing a fourth graph plotting filtration flux (in LMH) of a surface treated virus filtration membrane device versus throughput (in liters per square meter) for a model flow according to an embodiment of the present disclosure;

[0024] Fig.13 showing a fifth graph plotting filtration flux (in LMH) of a surface treated virus filtration membrane device versus throughput (in liters per square meter) for a model flow according to an embodiment of the present disclosure;

[0025] Fig.14 showing a sixth graph plotting filtration flux (in LMH) versus throughput (in liters per square meter) for a surface treated virus filtration membrane device for a model flow according to an embodiment of the present disclosure; and

[0026] Fig.15 A seventh graph is shown plotting filtration flux (in LMH) versus throughput (in liters per square meter) for a surface treated virus filtration membrane device for a model flow according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0027] Therefore, by referring to the accompanying drawings, a more specific description of the embodiments of the present disclosure briefly summarized above, in order to understand in more detail the manner in which the features disclosed herein are understood, can be obtained. However, it should be noted that the accompanying drawings illustrate only typical embodiments of the present disclosure and should not be considered as limiting its scope, as the embodiments described and shown may admit of other equally effective embodiments. It should also be understood that the elements and features of one embodiment may be present in other embodiments without further description, and the same reference numerals are sometimes used to indicate comparable elements common to the drawings.

[0028] The term "wt %" is defined as weight percent, ie, weight of solute / weight of solvent multiplied by 100.

[0029] The terms "bioreactor," "bag," and "container" are generally used interchangeably within this disclosure. A flexible bioreactor, bag, or container implies a flexible container that can be folded, collapsed, and unfolded, etc., and is capable of containing, for example, a biological fluid. A single-use bioreactor, bag, or container, which is also generally flexible, is a vessel that is used once and then disposed of.

[0030] The terms "coating," "surface treatment," and "surface modification" are used interchangeably throughout this disclosure.

[0031] In some embodiments, a filtration device may refer to a membrane capable of filtering a biological fluid. In some embodiments, a filtration device may refer to one or more membranes contained within a housing having at least one input and at least one output for a biological fluid, and optionally a vent. In some embodiments, the filtration device is a virus filtration device.

[0032] The term "sterile" is defined as the condition of being free from contaminants, particularly within the bioprocessing industry, bacteria, germs and other microorganisms.

[0033] The term "adjuvant" within the present disclosure is defined as a substance that enhances the body's immune response, such as the immune response to an antigen.

[0034] The term "upstream" is defined as a condition in a physical position before another component relative to the flow direction of the fluid. For example, upstream can indicate a process that occurs before a cell culture process in a bioreactor. In some embodiments, the term "upstream" indicates a process for culturing cells in a bioreactor. The term "downstream" is defined as a condition in a physical position after another component or process relative to the flow direction of the fluid within a series of processes or unit operations. From upstream to downstream, a series of unit operations in bioprocessing can include cell culture, capture chromatography, virus inactivation, intermediate column chromatography steps, ultrafiltration / diafiltration, a second chromatography step, a virus filtration step, and a purification / concentration step 2.

[0035] By way of example, embodiments of the present disclosure include virus filters, e.g., having PRO (VPro) (virus filter device manufactured by EMD Millipore Corporation, Burlington, MA, USA) similar membrane pore structure virus filter, the virus filter has improved surface treatment chemistry (ISC) and the use of novel excipients in the feed solution for improving filtration flux. Individually, the improved surface treatment provides significantly improved filtration flux. Moreover, the use of excipients with π-electron or hydrophobic interaction significantly improves filtration flux. When used in combination, the virus filtration filter using the novel surface treatment and excipient provides a synergistic improvement in filtration flux. In addition, the combined embodiment provides economical and practical processing on a commercial scale. And, the excipient that can be used to improve the flux through certain membranes contains at least one aromatic group. It is believed that aromaticity promotes π-electron or hydrophobic interaction, which can play a role in preventing protein-protein and protein-membrane interactions. In addition, in terms of improving transmembrane flux, excipients that allow minimum conductivity are useful excipients.

[0036] For some process streams, the virus filtration flux for protein solution is much lower than buffer flux, and / or depends on protein concentration. With typical protein concentration value (e.g., about 10g / L) operation, it may be too slow to be implemented. Processing time is subject to the limitation of indirect cost of operating manufacturing facilities and other time-dependent manufacturing considerations. In this limited time, the cost generated is proportional to the filter size required for the feed material of the processing set amount. In addition, there are practical limitations, such as the volume of the associated buffer for preparing to run and preparing to recover the product, and the volume is also proportional to the filter installation membrane area (scale). It should also be understood that the filter comprising film and / or when using the excipient described herein can also be used for continuous processing with longer running time, but can still benefit from higher processing flux and reduced filter space.

[0037] In some embodiments, the membrane comprises a polyethersulfone (PES) membrane. In some embodiments, the PES membrane is a porous membrane. In some embodiments, the porous membrane comprises a symmetric membrane or an asymmetric membrane. Embodiments disclosed herein provide novel compositions for surface treatment, which are used to remove microorganisms, particularly viruses, from a biological fluid and a purified monoclonal antibody solution in some embodiments. In some embodiments, the biological fluid is a chemically defined culture medium for culturing cells expressing a protein of interest. In some embodiments, the biological fluid is a fractionated plasma product.

[0038] The compositions described herein are based on surface modification of membranes, resulting in membranes that exhibit reduced fouling by one or more components in biological fluids or culture media having a specific pattern, such as mAbs, fractionated plasma products, or chemically defined cell culture media, when such fluids or culture media are filtered through the membrane.

[0039] In some embodiments, the compositions described herein involve porous membranes using an energy source. Exemplary energy sources include, but are not limited to, heat, electron beam, ultraviolet light, and gamma radiation.

[0040] Also described herein are methods of using the described surface-treated membranes for removing viral contaminants from a monoclonal antibody solution, for example, by filtering the antibody solution through the modified or surface-treated membrane.

[0041] The surface treatment of film is achieved by the following steps. Initially, by mixing monomers and cross-linking agents in a specific ratio to form a coating solution in one or more suitable solvents such as water, a chemical mixture or scheme is prepared. The range of concentrations commonly used for monomers and cross-linking agents varies from 0 to 5 weight %. By dipping the film in the corresponding chemical mixture or otherwise applying or coating the film, the chemical mixture / coating solution is applied on the film surface. The film is further exposed to ultraviolet rays (UV), electron beams, gamma radiation, heat, etc., for polymerization and cross-linking reactions, thereby generating a cross-linked coating of the chemical substance (achemstry) on the film.

[0042] For coatings, a variety of potential formulations using various ranges of agents are possible. Examples of some embodiments are as follows. Moreover, the ratios expressed below should not be considered as limitations on the embodiments.

[0043] A possible range for the first approach to formulating ISC 1 includes: 2.4 to 3.4% hydroxypropyl acrylate (HPA) and 1.1 to 2.1% polyethylene glycol diacrylate (PEGDA). The ratio of HPA to PEGDA is in the range of 2.2 to 1.6.

[0044] A possible range for the second approach to formulating ISC 2 includes: 2.5 to 3.5% bisphenol A ethoxylate diacrylate.

[0045] A possible range for formulating a third version of ISC 3 includes: 3.0 to 4.0% bisphenol A ethoxylate diacrylate and 1.5 to 2.5% hydroxypropyl acrylate (HPA). The ratio of bisphenol A ethoxylate diacrylate to HPA is in the range of 2.0 to 1.6.

[0046] A possible range for the fourth approach to formulating ISC 4 includes: 1.5 to 2.5% hydroxypropyl acrylate (HPA) and 2.0 to 3.0% polyethylene glycol diacrylate (PEGDA). The ratio of HPA to PEGDA is in the range of 0.75 to 0.83.

[0047] A possible range for formulating a fifth version of ISC 5 includes: 3.0 to 4.0% bisphenol A ethoxylate diacrylate.

[0048] Possible ranges for formulating the sixth version of ISC 6 include: 0.25 to 2.5% 2-hydroxy-3-phenoxypropyl acrylate (HPPA) and 2.0 to 5.0% polyethylene glycol diacrylate (PEGDA). The ratio of HPPA to PEGDA ranges from 0.12 to 0.62.

[0049] Other ranges and combinations are described without numbering these schemes for ease of reference. For example, embodiments within the scope of the present disclosure include possible ranges comprising: 0.25 to 2.5% (2-hydroxy-3-phenoxypropyl acrylate and 2.0 to 5.0% polyethylene glycol diacrylate; 0.2 to 3.0% tetrahydrofurfuryl acrylate and 1.0 to 2.5% polyethylene glycol diacrylate; 0.2 to 3.0% (5-ethyl-1,3-dioxan-5-yl) methyl acrylate and 1.0 to 2.5% polyethylene glycol diacrylate; or 0.25 to 2.5% propoxylated tetrahydrofurfuryl acrylate and 2.0 to 3.0% polyethylene glycol diacrylate.

[0050] As tested, ISC 1 to ISC 6 were formulated as follows:

[0051] ISC 1: 4.5 wt% total chemicals. 2.86 wt% hydroxypropyl acrylate Cas#25584-83-2 (HPA) and 1.64 wt% polyethylene glycol diacrylate Cas#26570-48-9 (PEGDA600) having a number average molecular weight of 600 g / mol (ratio HPA / PEGDA600: 1.75).

[0052] ISC 2 was formulated as 3 wt% bisphenol A ethoxylate diacrylate, Cas #64401-02-1.

[0053] ISC 3 was formulated as 3.5 wt% bisphenol A ethoxylate diacrylate and 2.0 wt% HPA.

[0054] ISC 4 is similar to ISC 1 but has a different ratio of components, namely 2.0 wt % HPA and 2.5 wt % PEGDA600.

[0055] ISC 5 is similar to ISC 2, but has slightly more monomer, for example 3.5 wt % bisphenol A ethoxylate diacrylate.

[0056] ISC 6 is formulated with 0.25 to 2.5 wt% hydroxy-3-phenoxypropyl acrylate and 2.0 to 5.0 wt% polyethylene glycol diacrylate. In some embodiments, 1.0 wt% HPPA and 3.75 wt% PEGDA are used. In some embodiments, 1.0 wt% HPPA and 2 wt% PEGDA are used. In still other embodiments, 0.25 wt% HPPA and 2 wt% PEGDA are used.

[0057] Monoclonal antibody test solutions are selected to demonstrate possible performance improvements of the present invention. These mAbs have several features that make them particularly challenging to filter with conventional virus filters. They have high hydrophobicity that causes protein-protein interaction and / or protein-membrane interaction. Dilution can alleviate these interactions of all two types, but in commercial-scale manufacturing, the increase in cost and equipment size is required for bioprocessing. In addition, after virus filtration, the product needs to be re-concentrated for storage and ultimately to the patient. Depending on the extreme degree of interaction for any given protein, they may become significant at conventional concentrations or only when operating the purification process under conditions of enhanced concentration increase. Known means to alleviate these interactions in the field of high-concentration preparations include changes in the pH and conductivity of the buffer system to enhance ion rejection. Although some benefits have been demonstrated by this means, additional and unexpected improvements have been made using the embodiments of the apparatus and methods described herein. In addition, measuring the interaction parameter (k D Dynamic light scattering (DLS) is a method for measuring protein-protein interactions, and the interaction parameters are defined by the linear regression slope drawn by the diffusion coefficient and the protein concentration. It has been found that this method is a useful technology to predict the success of protein-protein interaction reduction means in improving the virus filtration throughput. Compared to the throughput operation, this technology has the advantage of requiring less feed material, so it can be used to screen more options. Even when preparing small-scale samples by performing buffer exchange in a centrifugal filter unit, the flow rate of the protein solution can also be associated with the final throughput performance in the virus filtration operation. It has been found that this provides additional means to screen performance improvements by manipulating buffer and using additives.

[0058] For routine screening of various improved surface chemistry iterations, a model flow was utilized. The model flow for routine chemistry screening was prepared from commercially obtained human immunoglobulin G (IgG) purified from plasma and lyophilized. In addition to selecting the feed and preparing the feed in such a way as to minimize viral filter fouling due to large aggregates, the processing pressure was also reduced to allow more time for adsorptive fouling to contribute to overall fouling.

[0059] Therefore, conventional screening was performed on a monolayer phobic base membrane modified with the experimental chemistry. Volumetric throughput was measured versus time at a constant feed pressure of 10 psi, with the endpoint being 90% flux decay relative to protein-free buffer or 4 hours, whichever occurred first for any given sample. Fig. 9 In the example, it is shown how two chemistries can be distinguished from the VPro control at 10 psi, but not as well as at 30 psi, or 50 psi. For the model stream, this specification is valid, although for other feed streams, the effect of surface chemistry can be observed at any typical operating pressure. Due to the potential day-to-day variability in the testing, the results of each test were normalized to the throughput of one or more control units.

[0060] Figure 1 Shown is a graph plotting treatment throughput (in kilograms per square meter) versus time for surface treated films according to embodiments of the present disclosure. Figure 1The improvement of mass flux of mAb 1 using caffeine excipient and ISC 1 device with chemical surface treatment on membrane is demonstrated, thereby allowing faster filtration and more throughput without scaling. As from the feed VPro of the previous purification step, there is no measurable flow at 10g / L. By diluting without changing the pH value or conductivity, measurable flow is demonstrated with VPro. At 10g / L, titration to pH 7.5 provides improved flux of ISC 1. Using tris (hydroxymethyl) aminomethane (Tris) and Bicine buffer system at a total molar concentration of 20mM, up to pH 8.2, complete buffer exchange to a much lower conductivity of 0.2 millisiemens / centimeter (mS / cm) further enhances ion rejection, thereby allowing improvement of flux with VPro with the addition of 50mM caffeine. This improvement will require additional tangential flow filtration unit operation to achieve, making it undesirable. A simple titration with 2 mol / L concentration of Tris to increase to pH 7.5 would also otherwise make some improvements in ionic interactions, while being an acceptable change since it can be achieved without additional unit operations. In stark contrast, when run with ISC 1 according to embodiments of the present disclosure, measurable flux was achieved at 10 g / L. When the titration was combined with the addition of 50 mM caffeine excipient and run on ISC 1, the flux increased dramatically, providing optimal flux while maintaining concentration and avoiding additional unit operations.

[0061] Figure 2 The results are replotted as filtration flux (in LMH) versus throughput from Figure 1 By replotting the data in this manner, an advantage in volumetric flux can be observed for ISC 1 when combined with the use of appropriate excipients.

[0062] Apply the following conditions to Figure 1 to Figure 2 The test shown in . Before the feed solution was applied to the virus filtration device, it was processed through a multi-mode adsorption membrane pre-filter, which was designed to remove large protein aggregates that would otherwise clog the pores of the virus filter. After pre-filtration, the fluid was flowed through the virus filter at a constant pressure of 50 pounds per square inch (PSI).

[0063] The VPro filter device with novel surface treatment is also enough to show remarkable excellent performance in filtration flux even without using novel excipients. Although excipients or ISC 1 membranes are used alone to show enhanced filtration flux, when surface treatment and excipients are used, the flux of about 600 liters / square meter of membrane / hour (LMH) obtained is 75% of the maximum possible value (buffer flux of 865LMH under the same 50PSI). Compared to the stable flux of about 15LMH in the case of either one alone, this is an improvement of 40X. In view of the past disclosed understanding that polarization depends only on pore size and structure, the positive impact of modified surface chemistry is an unexpected discovery. By increasing surface chemistry to the basic structure, the effective pore size can be reduced. It is worth noting that in this case, there is no significant change in pore size or structure. The similar pore size after the novel surface treatment is set on a similar starting membrane structure has been confirmed by similar buffer flux. While excipients are known to reduce protein-protein interactions, the effect on protein-membrane interactions has not been determined independently, with the synergistic improvements resulting when surface treatment and excipient use are combined being even more surprising.

[0064] It should be further understood that many virus filters may contain the surface treatments described herein applied to the membranes used in their construction and / or use novel excipients to modify feed flows and achieve their synergistic effects. For example, a virus filter device such as that manufactured by EMD Corporation, Burlington, MA, USA PRO, NFP and other membranes, as well as other virus filters such as VIROSARTHF and PEGASUS PRIME.

[0065] Although the examples shown here use caffeine (it has aromaticity) as a novel excipient, it is believed that other one or more aromatic excipients that promote π-electron interaction (without increasing significant conductivity) show similar synergy, that is, significantly enhanced filtration flux. At least two other suitable aromatic excipients are pyridoxine and / or phenylalanine and their derivatives. Additional aromatic excipients that can be effective include, for example, pyridoxal, pyridoxamine, theobromine, theophylline, xanthine, tyrosine, tryptophan, histidine, histamine, ascorbic acid, folic acid and naphthalene-1-sulfonic acid and their derivatives. The concentration of the aromatic excipient used in the biological fluid is: 5mM to 300mM. And, in some embodiments, a combination of aromatic excipients is used. For example, some embodiments include using pyridoxine and histidine together.

[0066] Figure 3A graph plotting the throughput (in liters per square meter) versus time of three virus filter devices filtering mAb 2 according to embodiments of the present disclosure is shown. For the purpose of removing large protein aggregates that would otherwise clog the pores of the virus filter, the feed solution was processed through a synthetic depth filter containing silica before being applied to the virus filtration device. There are cases where improvements to the surface chemistry on the VPro base membrane alone are sufficient to obtain very good performance. In other words, improvements to the VPro device comprising a novel surface treated membrane exhibited greater throughput. In addition, the VPro device with the novel surface treated membrane and using an aromatic excipient had an even greater throughput. In this example, two different forms of improved chemical means are proposed - ISC 1; ISC 2. With no changes to the feed solution conditions from the previous purification step, the ISC filter maintained higher flux and achieved higher throughput during a 60-minute run time when operated at a constant pressure of 30 pounds per square inch (PSI). Two different embodiments of the improved chemical method are proposed. Two independent surface modifications, i.e., surface treatment chemistries, were performed by different chemical components. The first surface modification (ISC 1) was achieved by using a combination of an acrylic monomer (e.g., hydroxypropyl acrylate (HPA), e.g., Cas#25584-83-2) and an acrylic crosslinker (e.g., polyethylene glycol diacrylate (PEGDA), e.g., Cas#26570-48-9).

[0067] Hydroxypropyl acrylate (HPA), e.g. Cas#25584-83-2

[0068]

[0069] Polyethylene glycol diacrylate (PEGDA), such as Cas#26570-48-9

[0070]

[0071] The second surface modification is performed with a single component (eg, bisphenol A ethoxylate diacrylate, such as Cas#64401-02-1) that acts as both a monomer and a crosslinker.

[0072] Bisphenol A ethoxylate diacrylate, e.g. Cas#64401-02-1

[0073]

[0074] In both cases, suitable chemicals were identified from a series of studies performed under different formulation conditions. In both surface modifications, electron beams were used as energy sources to initiate polymerization and crosslinking steps. Other energy sources known to those skilled in the art are also contemplated herein.

[0075] Figure 4 A graph plotting the volume of filtrate after ten minutes at 2000 relative centrifugal force (RCF) versus excipient concentration in millimoles per liter (mM) according to an embodiment of the present disclosure is shown. According to an embodiment of the present disclosure, the amount of buffer passing through the membrane is measured at a fixed time point after completing the buffer exchange into buffers containing different excipients at different concentrations. Even in this experimental method, the volume of buffer passed through in a fixed time is also associated with the viral filter flux performance when the mAb passes through the viral filter. Higher buffer flux through the membrane in the centrifugal device indicates improved flux on the viral filter, such as will be used in bioprocessing applications. This method is used as a convenient screening method before running more complex flux runs with the viral filter. It can be seen that Figure 4 Results of testing additional neutral cyclic excipients are shown. Although at higher concentrations, such as 300 mM, pyridoxine almost reaches the performance of 50 mM caffeine. Phenylalanine has a relatively smaller benefit at increasing concentrations (i.e., in the range of 50 to 150 mM), but is still superior to the control. The 20 mM Tris-Bicine base buffer is supplemented with sodium chloride (NaCl) to achieve a conductivity of 3 mS / cm to match such conditions, which are obtained by 1 and Figure 2 Titration on the scale shown in can be achieved.

[0076] Figure 5 A graph is shown plotting the filtration flux (in LMH) of a surface treated membrane for mAb according to an embodiment of the present disclosure versus the throughput (in kg / m2). As shown, L-phenylalanine is not as effective as the other examples; 300 mM pyridoxine is even better than caffeine; ISC 3 sample variants are equivalent to or better than ISC 1. In addition to those shown in the other figures, an additional embodiment (ISC 3) of the improved chemical approach is presented. The surface modification is achieved by using an acrylic monomer (e.g., hydroxypropyl acrylate (HPA), such as Cas#25584-83-2) in combination with a component that can act as a monomer as well as a crosslinker (e.g., bisphenol A ethoxylate diacrylate, such as Cas#64401-02-1). With the synergistic improvement between the excipient and the improved surface chemistry, mAb 1, which is very difficult to filter, can be processed at 300 LMH at an increased concentration of 20 g / L.

[0077] Figure 6 A graph plotting the throughput (in kg / m2) of a surface treated membrane versus time at 10 g / L and 20 g / L of mAb 1 using a caffeine excipient for mAb-containing solutions according to an embodiment of the present disclosure is shown. The improved surface chemistry of ISC 1 allowed measurable flux at 20 g / L of mAb 1, but for a mAb with such a strong adsorption tendency and at higher concentrations, the flux was greatly improved when combined with an excipient such as caffeine. Even this combination had reduced flux, as can be observed by the higher mass flux of mAb 1 at the lower concentration of 10 g / L.

[0078] Figure 7 Shown are graphs plotting throughput (in kg / m2) versus time for a solution containing mAb 1 for three different virus filtration devices using 300 mM pyridoxine excipient for surface treated membranes according to embodiments of the present disclosure. Figure 7 The difficult to filter mAb 1 is shown - combining excipients with an improved surface chemistry virus filter.

[0079] ISC 1 by itself was not sufficient to run 20 g / L of mAb 1. Pyridoxine at 300 mM by itself was better with standard VPro than ISC 1 by itself. Using both pyridoxine excipient and ISC 1 gave the best performance.

[0080] caffeine:

[0081]

[0082] L-Phenylalanine:

[0083]

[0084] Pyridoxine:

[0085]

[0086] Figure 8A table showing the level of non-specific adsorption of a model immunoglobulin G (IgG) protein is shown. Specifically, a protein solution of 1 g / L of Goat Gamma Globulin in phosphate buffered saline (PBS) pH 7.4 solution was spiked with approximately 106 counts / minute (cpm) of 125I-goat anti-rabbit IgG labeled protein. Triplicate membrane samples were measured, with results reported relative to the membrane effective filtration area (EFA). Reduction in adsorption of the relevant protein was confirmed by this analytical method, which showed a reduction with ISC 3 having the lowest protein binding.

[0087] Fig. 9 A graph plotting throughput performance versus test pressure (in psi) for a model test flow according to an embodiment of the present disclosure is shown. The plotted data illustrates why subsequent tests with the model flow were run at 10 psi, where the samples could be better differentiated. Fig. 9 The throughput of a test of a filtration device containing a single layer of surface modified membrane is shown, where the test endpoint is defined as 90% flux decay relative to a protein-free buffer or 4 hours, whichever occurs first for any given sample device. Due to potential day-to-day variability in the test, the results of each test were normalized to the throughput of the control device or the average of multiple control devices. The model flow was prepared from commercially obtained human IgG purified from plasma and lyophilized. It shows how two improved surface chemistries can be distinguished from the VPro control at 10psi, but not as well as at 30psi, or 50psi, according to embodiments of the present disclosure. As shown, when tested at 10psi, membranes prepared with hydroxypropyl acrylate (HPA) and polyethylene glycol diacrylate (PEGDA) achieved higher throughput compared to the VPro control.

[0088] Fig.10 A graph is shown plotting the filtration flux (in LMH) of a single layer of surface treated membranes according to embodiments of the present disclosure for a model flow operating at 10 psi versus throughput (in liters per square meter). As shown, membranes prepared with a component capable of acting as both a monomer and a crosslinker (e.g., bisphenol A ethoxylate diacrylate, e.g., Cas#64401-02-1) achieved higher throughput compared to the VPro control with and without a range of concentrations of HPA (e.g., hydroxypropyl acrylate (HPA), e.g., Cas#25584-83-2).

[0089] Fig.11A graph plotting the filtration flux (in LMH) of a single layer of surface treated membranes according to embodiments of the present disclosure for a model flow operating at 10 psi is shown versus the throughput (in liters per square meter). As shown, membranes prepared with 2-hydroxy-3-phenoxypropyl acrylate (HPPA) and polyethylene glycol diacrylate (PEGDA) achieved higher throughput compared to the VPro control.

[0090] 2-Hydroxy-3-phenoxypropyl acrylate (HPPA), e.g. Cas#16969-10-1

[0091]

[0092] Fig.12 A graph is shown plotting the filtration flux (in LMH) of a single layer of surface treated membranes according to embodiments of the present disclosure for a model flow operating at 10 psi versus throughput (in liters per square meter). As shown, membranes prepared with hydroxypropyl acrylate (HPA) and polyethylene glycol diacrylate (PEGDA) achieve higher throughput than the VPro control.

[0093] Fig.13 A graph is shown plotting the filtration flux (in LMH) of a single layer of surface treated membranes according to embodiments of the present disclosure for a model flow operating at 10 psi versus throughput (in liters per square meter). As shown, membranes prepared with tetrahydrofurfuryl acrylate (Cas#2399-48-6) and polyethylene glycol diacrylate (PEGDA) achieve higher throughput than the VPro control.

[0094] Tetrahydrofurfuryl acrylate, e.g. Cas#2399-48-6

[0095]

[0096] Fig.14 A graph is shown that plots the filtration flux (in LMH) of a single layer of surface treated membranes according to embodiments of the present disclosure against the throughput (in liters per square meter) for a model flow operated at 10 psi. As shown, membranes prepared with (5-ethyl-1,3-dioxan-5-yl)methyl acrylate (Cas#66492-51-1) and polyethylene glycol diacrylate (PEGDA) achieve higher throughput than the VPro control.

[0097] Acrylic acid (5-ethyl-1,3-dioxane-5-yl)methyl ester, such as Cas#66492-51-1

[0098]

[0099] Fig.15 A graph plotting the filtration flux (in LMH) of a single layer of surface treated membranes according to embodiments of the present disclosure for a model flow operating at 10 psi is shown versus throughput (in liters per square meter). As shown, higher throughput was achieved compared to the VPro control with membranes prepared with Bisphenol A ethoxylate diacrylate (Cas#64401-02-1) alone or with hydroxypropyl acrylate (HPA), polyethylene glycol diacrylate (PEGDA) with one of 2-hydroxy-3-phenoxypropyl acrylate (HPPA), hydroxypropyl acrylate (HPA), tetrahydrofurfuryl acrylate (Cas#2399-48-6), or (5-ethyl-1,3-dioxan-5-yl)methyl acrylate (Cas#66492-51-1).

[0100] Embodiments of the present disclosure include a filtration device. In some embodiments, the filtration device is a polyethersulfone membrane, and the polyethersulfone membrane is optionally an asymmetric porous membrane with a surface modification. In some embodiments, the surface modification is a cross-linked coating on the membrane, wherein the cross-linked coating is formed by a coating solution comprising a cross-linkable diacrylate.

[0101] In some embodiments, the coating solution used to prepare the cross-linked coating disposed on the membrane includes at least one of: 2.4 to 3.4% hydroxypropyl acrylate and 1.1 to 2.1% polyethylene glycol diacrylate; 2.5 to 3.5% bisphenol A ethoxylate diacrylate; 3.0 to 4.0% bisphenol A ethoxylate diacrylate and 1.5 to 2.5% hydroxypropyl acrylate; 1.5 to 2.5% hydroxypropyl acrylate and 2.0 to 3.0% polyethylene glycol diacrylate; 3.0 to 4.0% bisphenol A ethoxylate diacrylate and 1.5 to 2.5% hydroxypropyl acrylate; 0.25 to 2.5% of propoxylated tetrahydrofurfuryl acrylate and 2.0 to 5.0% of polyethylene glycol diacrylate; 0.2 to 3.0% of tetrahydrofurfuryl acrylate and 1.0 to 2.5% of polyethylene glycol diacrylate; 0.2 to 3.0% of (5-ethyl-1,3-dioxan-5-yl)methyl acrylate and 1.0 to 2.5% of polyethylene glycol diacrylate; or 0.25 to 2.5% of propoxylated tetrahydrofurfuryl acrylate and 2.0 to 3.0% of polyethylene glycol diacrylate.

[0102] In some embodiments, the filtration device can be used to filter biological fluids, wherein an aromatic excipient is used. In some embodiments, the aromatic excipient includes at least one of caffeine, pyridoxine, phenylalanine, pyridoxal, pyridoxamine, theobromine, theophylline, xanthine, tyrosine, tryptophan, histidine, histamine, ascorbic acid, folic acid, or naphthalene-1-sulfonic acid.

[0103] It should be understood that membranes with cross-linked surface coatings can be used in combination with the use of excipients described herein. In addition, all filtration and / or purification methods of mAbs or fractionated plasma products described herein can use membranes with surface treatments and excipients, either alone or in combination.

[0104] All ranges of the preparation disclosed herein include the range between them, and may include or not include endpoints. The optional included range is from the integer value (or including an original endpoint) in the order of magnitude described or the next smaller order of magnitude between them. For example, if the lower limit range value is 0.2, the optional included endpoint can be 0.3, 0.4, ..., 1.1, 1.2, etc., and 1, 2, 3, etc.; if the upper limit range is 8, the optional included endpoint can be 7, 6, etc., and 7.9, 7.8, etc. One-sided boundaries (such as 3 or more) are similarly included in the consistent boundaries (or scopes) starting from the integer value of the described order of magnitude or a lower order of magnitude. For example, 3 or more include 4 or 3.1 or more.

[0105] References throughout this specification to "one embodiment," "specific embodiments," "one or more embodiments," "some embodiments," or "an embodiment" indicate that features, structures, materials, or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present disclosure. Thus, throughout this specification, appearances of phrases such as "in one or more embodiments," "in specific embodiments," "in one embodiment," "some embodiments," or "in an embodiment" do not necessarily refer to the same embodiment.

[0106] Although some embodiments have been discussed above, other specific implementations and applications are also within the scope of the following claims. Although the specification is described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. Therefore, it should be further understood that various modifications may be made to the illustrative embodiments, and other arrangements and modes may be designed without departing from the spirit and scope of the embodiments according to the present disclosure. In addition, in any one or more embodiments, specific features, structures, materials, or characteristics may be combined in any suitable manner.

[0107] The entire contents of the publications of patent applications and patents and other non-patent references cited in this specification are hereby incorporated by reference in their entirety, as if each individual publication or reference was specifically and individually indicated to be incorporated by reference in its entirety as fully set forth herein. Any patent application to which this application claims priority is also incorporated by reference in the manner described above for publications and references.

Claims

1. A filtration device for use with a biological fluid, comprising: membrane; and Crosslinking the coating on the membrane to form a surface modification, The cross-linked coating is formed by a coating solution, and the coating solution contains a cross-linkable diacrylate.

2. The filtration device according to claim 1, wherein the coating solution used to prepare the cross-linked coating comprises at least one of the following: 2.4 to 3.4% hydroxypropyl acrylate and 1.1 to 2.1% polyethylene glycol diacrylate; 2.5 to 3.5% bisphenol A ethoxylate diacrylate; 3.0 to 4.0% bisphenol A ethoxylate diacrylate and 1.5 to 2.5% hydroxypropyl acrylate; 1.5 to 2.5% hydroxypropyl acrylate and 2.0 to 3.0% polyethylene glycol diacrylate; 0.25 to 2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0 to 5.0% polyethylene glycol diacrylate; or 3.0 to 4.0% bisphenol A ethoxylate diacrylate. The filtration device according to claim 1 , wherein the membrane is a porous membrane. The filtration device according to claim 1 , wherein the membrane is a symmetric porous membrane or an asymmetric porous membrane.

5. The filtration device of claim 1, wherein the membrane comprises a polyethersulfone material.

6. The filter device of claim 1, further comprising a vent.

7. The filtration device of claim 1, wherein the filtration device is a virus filtration device.

8. A method for preparing a membrane for filtering a biological fluid, comprising: applying a crosslinkable diacrylate solution to the porous membrane; and Curing of the cross-linkable solution is initiated to form a cured coating on the porous membrane, wherein the cured coating facilitates a high flux filtration device.

9. The method of claim 8, wherein the crosslinkable diacrylate solution used to prepare the crosslinked coating comprises a solution of at least one of: 2.4 to 3.4% hydroxypropyl acrylate and 1.1 to 2.1% polyethylene glycol diacrylate; 2.5 to 3.5% bisphenol A ethoxylate diacrylate; 3.0 to 4.0% bisphenol A ethoxylate diacrylate and 1.5 to 2.5% hydroxypropyl acrylate; 1.5 to 2.5% hydroxypropyl acrylate and 2.0 to 3.0% polyethylene glycol diacrylate; 0.25 to 2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0 to 5.0% polyethylene glycol diacrylate; or 3.0 to 4.0% bisphenol A ethoxylate diacrylate.

10. The method of claim 8, wherein the coating solution is applied to the membrane by a dipping or spraying step.

11. The method according to claim 8, wherein the method for initiating the crosslinking reaction is the application of electron beams, ultraviolet light, or high-energy radiation.

12. The method of claim 8, wherein the membrane is housed in a housing, the housing further comprising a biological fluid input and a biological fluid output.

13. A method for filtering a biological fluid, comprising: containing a surface-treated porous membrane in a filtration device having an input end and an output end, wherein the surface treatment comprises a cross-linked diacrylate coating; introducing a biological fluid into the input end of the filtration device; and The filtrate is collected from the output end.

14. The method of claim 13, wherein the membrane comprises a polyethersulfone material.

15. The method of claim 13, wherein the filtration device is a dead-end filter or a tangential flow filter.

16. The method of claim 13, wherein the biological fluid comprises a monoclonal antibody solution, a protein-containing solution, a fragment antigen-binding fragment-containing solution, a recombinant protein-containing solution, or a fractionated plasma product.

17. Surface treatment of the membrane, comprising: A crosslinkable diacrylate solution for preparing a crosslinked coating, wherein the crosslinked coating comprises a solution of at least one of: 2.4 to 3.4% hydroxypropyl acrylate; and 1.1 to 2.1% polyethylene glycol diacrylate; 2.5 to 3.5% bisphenol A ethoxylate diacrylate; 3.0 to 4.0% bisphenol A ethoxylate diacrylate and 1.5 to 2.5% hydroxypropyl acrylate; 1.5 to 2.5% hydroxypropyl acrylate and 2.0 to 3.0% polyethylene glycol diacrylate; 0.25 to 2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0 to 5.0% polyethylene glycol diacrylate; or 3.0 to 4.0% bisphenol A ethoxylate diacrylate.

18. A filtration device for use with a biological fluid, comprising: membrane; and A cross-linked coating is provided on the film to form a cross-linked copolymeric surface modification or treatment, wherein the cross-linked coating is formed by a coating solution containing a cross-linkable diacrylate, wherein the coating solution is 2.4 to 3.4% hydroxypropyl acrylate and 1.1 to 2.1% polyethylene glycol diacrylate, or 3.0 to 4.0% bisphenol A ethoxylate diacrylate and 1.5 to 2.5% hydroxypropyl acrylate; or 1.5 to 2.5% hydroxypropyl acrylate and 2.0 to 3.0% polyethylene glycol diacrylate, or 0.25 to 2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0 to 5.0% polyethylene glycol diacrylate.

19. The filter device of claim 18, further comprising a vent.

20. The filtration device of claim 18, wherein the filtration device is a virus filtration device.

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