Surface treatment of purification membranes

Abstract

2.4-3.4% hydroxypropyl acrylate and 1.1-2.1% polyethylene glycol diacrylate; 2.5-3.5% bisphenol A ethoxylate diacrylate; 3.0-4.0% bisphenol A ethoxylate diacrylate and 1.5-2.5% hydroxypropyl acrylate; 1.5-2.5% hydroxypropyl acrylate and 2.0-3.0% polyethylene glycol diacrylate; 0.25-2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0-5.0% polyethylene glycol diacrylate; or 3.0-4.0% bisphenol A ethoxylate diacrylate. A filtration device for use with biological fluids, comprising: a membrane; and a crosslinked coating on the membrane to form a surface modification or surface treatment, the crosslinked coating being formed from a coating solution comprising a crosslinkable diacrylate.

Description

[Technical field]

[0001] FIELD OF THE DISCLOSURE Embodiments of the present disclosure relate to bioprocessing. More specifically, embodiments disclosed herein include the use of excipients and / or surface treatments on filtration membranes to enhance flux. [Background technology]

[0002] Biological fluids are processed in systems that include vessels, i.e. stainless steel vessels and / or disposable vessels or bioreactors, e.g. polymeric bags. These vessels are sterile and are used for the processing of biological materials, whether in batch, semi-continuous or continuous mode. The biological material(s) include cell cultures, empty capsids or viral vectors, plasmids, adeno-associated viruses and monoclonal antibodies (mAbs), among other substances. These biological materials must be purified using several steps, e.g. various chromatography, dead-end filtration and tangential flow filtration using porous membranes, to remove metabolic by-products and waste products, viruses, etc. By-products, material aggregates and waste products vary greatly in size, making filter fouling a problem during bioprocessing.

[0003] Several manufacturers offer filters in which viruses, such as parvoviruses, are retained. The filters include membranes made from various base polymers, such as poly(vinylidene fluoride), poly(ether sulfone), and some cellulosic materials. The surface of the membrane is often modified to be hydrophilic and / or relatively low protein binding, etc. Surface modification can be a chemical coating process that can change the pore size of the membrane, as well as imparting hydrophilicity, etc. Typically, it is not desirable to modify the pore size of the membrane, which can be a result of the coating process.

[0004] Some coating processes involve the formation of a polymeric coating containing polymeric units derived from monomers and crosslinking these polymeric units via one or more crosslinking approaches, e.g., chemical crosslinkers in the presence of an energy source such as gamma radiation, UV light, and / or E-beam. In some processes, the monomer itself can function as the crosslinker. Polymeric coatings used for this type of surface modification are often based on acrylic and allylic monomers and crosslinkers. The chemical and physical properties of the monomer / crosslinker ultimately control the surface properties of the final filter.

[0005] There are known examples of process streams of biological fluids that cause relatively low initial flux and / or ongoing flux decline, a fouling mechanism for virus filters during filtration of protein solutions. Adsorption of proteins within the biological fluid onto the surface of the membrane as a causative mechanism for fouling is a potential challenge during bioprocessing.

[0006] Some past prior art attempts at virus filtration processes have involved the use of excipients. For example, at least one method involved stabilizing downstream monoclonal antibody process intermediates by focusing on preventing large permanent aggregates that would permanently block the filter pores. The theory that concentration polarization occurs in some cases and is the primary cause of the flux behavior has been postulated. However, adsorption of aggregates to the surface of the pores as an alternative explanation has been dismissed as unlikely due to the hydrophilic coating of the membrane.

[0007] The use of excipients, especially viscosity-reducing excipients, is commonly known for multiple purposes to promote drug delivery and long-term storage stability in final formulation processes with higher protein concentrations. Excipient molecules are known to suppress protein-protein interactions. However, the use of excipients is limited. Many excipients, despite being effective for other purposes, do not provide significant filtration flux improvement. One potential reason for their ineffectiveness may be the addition of conductivity to the biofluid, typically via charged molecular species, such as formulations containing sulfonic acid derivatives. This excess conductivity masks ionic interactions that could otherwise eliminate adsorption interactions. These previous excipients do not significantly interfere with protein-protein and protein-membrane interactions that limit filtration flux.

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

[0009] It is an advancement in the art to provide membranes with surface treatment chemistry that improves use and performance with significantly increased flux. Without intending to be bound by theory, it is believed that these advances are due to an unexpectedly high reduction in protein-membrane interactions. A further advancement of the embodiments described in this disclosure, without intending to be bound by theory, also provides electrically neutral excipients that interact through π electrons and contribute little or nothing to electrical conductivity. The reduction in protein-protein interactions reduces the effective size of the proteins being filtered, and in some embodiments, in conjunction with the reduction in protein-membrane interactions, reduces pore constriction to increase processing flux. These two advances in the art each provide important advantages. Furthermore, together, they provide a synergistic advantage of increasing filter flux in filtering viruses from various biological processes and biological materials. Summary of the Invention [Means for solving the problem]

[0010] Some embodiments of filtration membranes comprising novel surface treatments formed from diacrylate solutions are disclosed as substantially shown in and / or described in connection with at least one of the drawings and as more fully described in the claims. Some embodiments of the present disclosure include a filtration device for filtering biological fluids for use with an excipient, a polyethersulfone membrane; and a crosslinked coating on the membrane to form a surface modification, the crosslinked coating being formed from a coating solution comprising a crosslinkable diacrylate, the excipient providing π-electron interactions. Some embodiments of the use of filtration membranes and excipients comprising novel surface treatment chemistries are disclosed. Some embodiments include methods of using various excipients that provide π-electron interactions to increase filter flux. It should be understood that the filtration membranes have novel surface treatment chemistries and / or the use of π-electron interacting excipients, which can be used to filter solutions containing mAbs as well as other biological modalities. Furthermore, the inventors have unexpectedly discovered, without intending to be limited by theory, that the concentration and interaction between proteins and virus filtration membranes are considered to be important factors in membrane fouling. Some embodiments of the present disclosure include a surface treatment for the membrane, the surface treatment is used to create a crosslinked coating and includes a coating solution further comprising at least one of the following: 1) 2.4-3.4% hydroxypropyl acrylate and 1.1-2.1% polyethylene glycol diacrylate; 2) 2.5-3.5% bisphenol A ethoxylate diacrylate; 3) 3.0-4.0% bisphenol A ethoxylate diacrylate and 1.5-2.5% hydroxypropyl acrylate; 4) 1.5-2.5% hydroxypropyl acrylate and 2.0-3.0% polyethylene glycol diacrylate; 5) 3.0-4.0% bisphenol A ethoxylate diacrylate; and 6) 0.25-2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0-5.0% polyethylene glycol diacrylate.

[0011] Various provisions, aspects and novel and inventive features of the present disclosure, as well as details of illustrative embodiments thereof, will become more fully understood from the following description and drawings. [Brief description of the drawings]

[0012] [Figure 1] 1 is a graph plotting throughput (kilograms per square meter) versus time for surface treated membranes according to embodiments of the present disclosure. [Diagram 2] 1 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 filter membrane according to an embodiment of the present disclosure. [Diagram 3] 1 is a graph plotting throughput (liters / meter squared) versus time for three virus filter membrane devices filtering mAbs according to embodiments of the present disclosure. [Figure 4] 1 is a graph plotting filtrate volume (mL) vs. excipient concentration in millimoles / liter (mM) after 10 minutes at 2000 relative centrifugal force (RCF) according to embodiments of the present disclosure. [Diagram 5] 1 is a graph plotting filtration flux (LMH) versus throughput (kilograms / meter squared) of surface-treated membranes for mAb according to embodiments of the present disclosure. [Figure 6] 1 is a graph plotting throughput (kilograms / square meter) versus time for a surface-treated virus filter membrane for solutions containing mAb at mAb concentrations of 10 g / L and 20 g / L, with and without a caffeine excipient, according to embodiments of the present disclosure. [Figure 7] 1 is a graph plotting surface-treated virus filter membrane throughput (kilograms / meter squared) versus time for a solution containing mAb and 300 mM pyridoxine excipient for three different virus filtration devices according to embodiments of the present disclosure. [Figure 8]1 is a table showing model Immunoglobulin G (IgG) protein nonspecific adsorption levels (micrograms) of various surface-treated virus filter membranes versus membrane effective filtration area (EFA) (square centimeters), according to embodiments of the present disclosure. [Figure 9] 1 is a first graph plotting throughput performance versus test pressure (psi) using a model test stream in accordance with an embodiment of the present disclosure. [Figure 10] 1 is a second graph plotting filtration flux (LMH) versus throughput (liters per square meter) of a surface treated virus filter membrane for a model stream according to embodiments of the present disclosure. [Figure 11] 13 is a third graph plotting filtration flux (LMH) versus throughput (liters per square meter) of a surface treated virus filter membrane for a model stream according to embodiments of the present disclosure. [Figure 12] 4 is a fourth graph plotting filtration flux (LMH) versus throughput (liters per square meter) of a surface treated virus filter membrane device for a model stream according to embodiments of the present disclosure. [Figure 13] FIG. 5 is a fifth graph plotting filtration flux (LMH) versus throughput (liters per square meter) of a surface treated virus filter membrane device for a model stream according to embodiments of the present disclosure. [Figure 14] 6 is a sixth graph plotting filtration flux (LMH) versus throughput (liters per square meter) of a surface treated virus filter membrane device for a model stream according to embodiments of the present disclosure. [Figure 15] FIG. 7 is a seventh graph plotting filtration flux (LMH) versus throughput (liters per square meter) of a surface treated virus filter membrane device for a model stream according to embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Therefore, in a manner that allows the features disclosed herein to be understood in detail, a more particular description of the embodiments of the present disclosure briefly summarized above can be obtained by referring to the attached drawings. It should be noted, however, that the attached drawings illustrate only typical embodiments of the present disclosure, and therefore should not be considered as limiting its scope, since the described and illustrated embodiments may admit of other equally effective embodiments. It should also be understood that elements and features of one embodiment may be found in other embodiments without further recitation, and the same reference numerals may be used to indicate equivalent elements common to the figures.

[0014] The term wt% is defined as weight percent, ie weight of solute / weight of solvent multiplied by 100.

[0015] The terms "bioreactor," "bag," and "container" are generally used interchangeably within this disclosure. A flexible bioreactor, bag, or container includes a flexible vessel that can, for example, contain a biological fluid, and that can be folded, collapsed, expanded, etc. A single-use bioreactor, bag, or container is a vessel that is typically flexible and is disposed of after a single use.

[0016] The terms "coating," "surface treatment," and "surface modification" are used interchangeably throughout this disclosure.

[0017] In some embodiments, the filtration device may represent a membrane capable of filtering biological fluids. In some embodiments, the filtration device may represent one or more membranes housed in a housing having at least one inlet and at least one outlet for biological fluids and optionally a vent. In some embodiments, the filtration device is a virus filtration device.

[0018] The term "sterile" is defined as free from contaminants, particularly within the bioprocessing industry, and free from bacteria, pathogens and other microorganisms.

[0019] The term "adjuvant" within this disclosure is defined as a substance that enhances the body's immune response to, for example, an antigen.

[0020] The term "upstream" is defined as a state that is physically located before another component in terms of the direction of fluid flow. For example, upstream may refer to a process that takes place before a cell culture process in a bioreactor. In some embodiments, the term "upstream" refers to a process for culturing cells in a bioreactor. The term "downstream" is defined as a state that is physically located after another component or process in terms of the direction of fluid flow in a chain of processes or unit operations. From upstream to downstream, the series of unit operations in bioprocessing may include cell culture, capture chromatography, viral inactivation, an intermediate column chromatography step, ultrafiltration / diafiltration, a second chromatography step, a viral filtration step, and a purification / concentration step 2.

[0021] By way of example, embodiments of the present disclosure include a virus filter, such as a virus filter having a membrane pore structure similar to VIRESOLVE® PRO (VPro), a virus filter device manufactured by EMD Millipore Corporation (Burlington, MA, USA) with improved surface treatment chemistry (ISC), and the use of novel excipients in the feed for improved filtration flux. Apart from this, the improved surface treatment provides significantly improved filtration flux. Also, the use of excipients with π-electron or hydrophobic interactions significantly improves filtration flux. Virus filtration filters using novel surface treatments and excipients provide synergistic improvements in filtration flux when used together. Also, combination embodiments provide economical and practical processing at a commercial scale. Also, excipients useful for improving flux through some membranes included at least one aromatic group. Aromaticity is believed to promote π-electron or hydrophobic interactions, which may play a role in preventing protein-protein and protein-membrane interactions. Additionally, excipients that allow minimal electrical conductivity were useful excipients in terms of improving transmembrane flux.

[0022] In some process streams, the virus filtration flux through the protein solution is much lower than the buffer flux and / or is dependent on the protein concentration.

[0023] Operation at typical protein concentration values, e.g., about 10 g / L, can be impractically slow. Processing time is limited by overhead costs for operating manufacturing equipment as well as other time-dependent manufacturing considerations. In this limited time, the costs incurred are proportional to the filter sizing required to process a set amount of feed. In addition, there are practical limitations such as the amount of associated buffers prepared for run and product recovery, which are also adjusted to the filter installation membrane area. It is also understood that filters containing membranes and / or using excipients described herein are also useful for continuous processes with longer run times, but can further benefit from higher process flux and reduced filter footprint.

[0024] 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 to remove microorganisms, particularly viruses, from biological fluids and, in some embodiments, purified monoclonal antibody solutions. In some embodiments, the biological fluid is a chemically defined medium used to culture cells expressing a protein of interest. In some embodiments, the biological fluid is a fractionated plasma preparation.

[0025] The compositions described herein are based on surface modifications of membranes that result in membranes that, when biological fluids having a particular modality, such as mAbs, fractionated plasma products, or chemically defined cell culture media, are filtered through the membrane, exhibit reduced fouling by one or more components in such fluids or media.

[0026] In some embodiments, the compositions described herein are directed to the porous membrane using an energy source. Exemplary energy sources include, but are not limited to, heat, electron beam, ultraviolet light, and gamma radiation.

[0027] Also described herein are methods of using the described surface-treated membranes to remove viral contaminants from a monoclonal antibody solution, for example, by filtering the antibody solution through the modified or surface-treated membrane.

[0028] Surface treatment of the membrane is accomplished by the following steps: First, a chemical mixture or protocol was prepared by mixing monomers and crosslinkers in specific ratios in an appropriate solvent(s), e.g., water, to form a coating solution. The range of concentrations generally used for monomers and crosslinkers varied from 0 to 5 weight percent. The chemical mixture / coating solution was applied to the membrane surface by immersing the membrane in the corresponding chemical mixture or otherwise painting or coating the membrane.

[0029] This film is further exposed to ultraviolet (UV) light, E-beam, gamma radiation, heat, etc. for polymerization and crosslinking reactions resulting in a crosslinked coating of chemicals on the film.

[0030] Many possible formulations are possible using a range of different agents for coating. Some example embodiments are given below. Also, the ratios given below are not intended to limit the embodiments. EXAMPLES

[0031] The possible range of the first protocol formulated in ISC1 includes 2.4-3.4% hydroxypropyl acrylate (HPA) and 1.1-2.1% polyethylene glycol diacrylate (PEGDA), with the ratio of HPA to PEGDA ranging from 2.2 to 1.6.

[0032] The possible range of the second protocol formulated in ISC2 includes 2.5-3.5% bisphenol A ethoxylate diacrylate.

[0033] The possible range for the third protocol formulated in ISC3 includes 3.0-4.0% bisphenol A ethoxylate diacrylate and 1.5-2.5% hydroxypropyl acrylate (HPA). The ratio of bisphenol A ethoxylate diacrylate to HPA ranges from 2.0 to 1.6.

[0034] The possible range of fourth protocols formulated in ISC4 includes 1.5-2.5% hydroxypropyl acrylate (HPA) and 2.0-3.0% polyethylene glycol diacrylate (PEGDA), with the ratio of HPA to PEGDA ranging from 0.75 to 0.83.

[0035] A possible range for the fifth protocol formulated in ISC5 includes 3.0-4.0% bisphenol A ethoxylate diacrylate.

[0036] The possible range of protocols for the sixth formulation, as formulated in ISC6, includes 0.25-2.5% 2-hydroxy-3-phenoxypropyl acrylate (HPPA) and 2.0-5.0% polyethylene glycol diacrylate (PEGDA), with the ratio of HPPA to PEGDA ranging from 0.12 to 0.62.

[0037] Other ranges and combinations are listed without protocol numbering as a simplified reference. For example, embodiments within the scope of the present disclosure include possible ranges including: 0.25-2.5% (2-hydroxy-3-phenoxypropyl acrylate and 2.0-5.0% polyethylene glycol diacrylate; 0.2-3.0% tetrahydrofurfuryl acrylate and 1.0-2.5% polyethylene glycol diacrylate; 0.2-3.0% (5-ethyl-1,3-dioxan-5-yl) methyl acrylate and 1.0-2.5% polyethylene glycol diacrylate; or 0.25-2.5% propoxylated tetrahydrofurfuryl acrylate and 2.0-3.0% polyethylene glycol diacrylate.

[0038] As tested, ISC1-ISC6 were formulated as follows: ISC1: 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, number average molecular weight 600 grams / mole (PEGDA600) (ratio of HPA / PEGDA600: 1.75).

[0039] ISC2 was formulated as 3 wt% Bisphenol A ethoxylate diacrylate, Cas#64401-02-1.

[0040] ISC3 was formulated as 3.5 wt% bisphenol A ethoxylate diacrylate and 2.0 wt% HPA.

[0041] ISC4 was similar to ISC1 but had a different ratio of components: 2.0 wt% HPA and 2.5 wt% PEGDA600.

[0042] ISC5 was similar to ISC2 but contained slightly more monomer, e.g., 3.5 wt % bisphenol A ethoxylate diacrylate.

[0043] ISC6 was formulated as 0.25-2.5 wt% hydroxy-3-phenoxypropyl acrylate and 2.0-5.0 wt% polyethylene glycol diacrylate. In some embodiments, 1.0 wt% HPPA and 3.75 wt% PEGDA were used. In some embodiments, 1.0 wt% HPPA and 2 wt% PEGDA were used. In yet other embodiments, 0.25 wt% HPPA and 2 wt% PEGDA were used.

[0044] To demonstrate the performance improvements possible with the present invention, monoclonal antibody test solutions were selected. These mAbs have several characteristics that make them particularly challenging for filtration using conventional virus filters. They have high hydrophobicity that causes protein-protein interactions and / or protein-membrane interactions. Dilution can mitigate both of these interactions, but commercial-scale production requires increased costs and equipment size for bioprocessing. Furthermore, after virus filtration, the product needs to be reconcentrated for storage and ultimately administration to patients. Depending on how extreme the interactions are for any given protein, they may only become significant when operating the purification process at conventional concentrations or under enhanced conditions where the concentrations are increased. Known approaches to mitigate these interactions in the field of high concentration formulations include altering the pH and conductivity of the buffer system to enhance ionic repulsion. While some benefits have been demonstrated with this approach, further unexpected improvements have been made using embodiments of the instruments and methods described herein. Additionally, dynamic light scattering (DLS) to measure the interaction parameter (kD), defined by the slope of the linear regression plotted from the diffusion coefficient versus protein concentration, is a method to measure protein-protein interactions. The method was found to be a useful technique for predicting the success of protein-protein interaction reduction approaches in improving viral filtration throughput. This technique has the advantage of requiring less feed material than a throughput run, and therefore can be used to screen more options. Even if small-scale samples are prepared by performing buffer exchange in a centrifugal filter unit, the flow rate of the protein solution can be correlated with the final throughput performance in the viral filtration operation. This was found to provide an additional approach to improving screening performance by manipulating buffers and using additives.

[0045] A model stream was utilized for routine screening of a wide variety of improved surface chemistry iterations. The model stream for routine chemistry screening was prepared from commercially available human immunoglobulin G (IgG) purified from plasma and lyophilized. In addition to selecting and preparing the feed to minimize fouling of the virus filter with large aggregates, the process pressure was reduced to allow more time for adsorptive fouling that contributes to overall fouling.

[0046] Therefore, routine screening was performed on monolayers of hydrophobic base membranes modified with the experimental chemistries. Volumetric throughput was measured versus time at a constant 10 psi feed pressure, with the end point being 90% flux decay versus protein-free buffer or 4 hours, whichever occurs first for any given sample. In Figure 9, it is shown that the two chemistries can be distinguished from the VPro control at 10 psi, but not at 30 psi or 50 psi. For the model stream, this specification was useful, but for other feed streams, the effect of surface chemistry can be seen at any typical operating pressure. Because of the possibility of day-to-day variation in the tests, the results of each test are normalized to the throughput of the control device(s).

[0047] FIG. 1 is a graph plotting throughput (kilograms per square meter) versus time for surface-treated membranes according to an embodiment of the present disclosure. FIG. 1 demonstrates improved mass flux of mAb1 using a caffeine excipient and an ISC1 device with a chemical surface treatment on the membrane, allowing faster filtration and higher throughput without fouling. The feed from the preceding purification step has no measurable flow at 10 g / L in VPro. Dilution without changing pH or conductivity demonstrates measurable flow rate in VPro. Titration to pH 7.5 at 10 g / L allows for improved flux in ISC1. Complete buffer exchange using a tris(hydroxymethyl)aminomethane (Tris) and bicine buffer system to a much lower conductivity of 0.2 millisiemens per centimeter (mS / cm) at 20 mM total molarity and up to pH 8.2 further enhances ionic repulsion and allows for improved flux in VPro with 50 mM caffeine added. This improvement requires an extra tangential flow filtration unit operation to achieve, thereby making this improvement undesirable. Simple titration with Tris to increase pH to 7.5 at a concentration of 2 moles / liter also appears to provide some improvement in ionic interactions while being an acceptable modification since it can be achieved without additional unit operations. In contrast, when run on the ISC1 according to an embodiment of the present disclosure, measurable flux is achieved at 10 g / L. When titration is run on the ISC1 in combination with the addition of 50 mM caffeine excipient, the flux increases dramatically, providing the best flux while maintaining concentration and avoiding additional unit operations.

[0048] Figure 2 shows the data from Figure 1 replotted as filtration flux (LMH) versus throughput. By replotting in this manner, the volumetric flux advantage for ISC1 when combined with the use of appropriate excipients can be seen.

[0049] The following conditions were applied for the trials depicted in Figures 1-2: Prior to application of the feed solution to the virus filtration device, it was treated with a multimodal adsorptive membrane prefilter designed to remove large protein aggregates that would otherwise block the pores of the virus filter. After prefiltration, the fluid was forced through the virus filter at a constant pressure of 50 pounds per square inch (PSI).

[0050] The VPro filtration device with the novel surface treatment is sufficient to demonstrate significantly superior performance in terms of filtration flux, even without the use of the novel excipient. Although the use of either the excipient or the ISC1 membrane alone demonstrates enhanced filtration flux, the use of both the surface treatment and the excipient results in a flux of approximately 600 liters per square meter of membrane per hour (LMH), which is 75% of the maximum possible value (buffer flux of 865 LMH at the same 50 PSI). This is a 40-fold improvement over the steady-state flux of approximately 15 LMH with either alone. The positive impact of the modified surface chemistry is an unexpected finding, given the previously published understanding of polarization as being dependent only on pore size and structure. The effective pore size can be reduced by adding surface chemistry to the base structure. Notably, in this case, there was no significant change in the size or structure of the pores. Similar pore size, similar starting membrane structure after placing the novel surface treatment on top of it is confirmed by similar buffer flux. Although excipients are known to reduce protein-protein interactions, their effect on protein-membrane interactions has not been independently established, and the synergistic improvement when combining surface treatment with the use of excipients is even more unexpected.

[0051] It is further understood that many virus filters can incorporate the surface treatments described herein that are applied to the membranes used in their construction and / or can use novel excipients to modify the feed stream and realize their synergistic effects, for example, virus filter devices such as VIRESOLVE® PRO, Viresolve® NFP and other membranes manufactured by EMD Corporation (Burlington, MA, USA), and other virus filters such as VIROSART HF and PEGASUS PRIME.

[0052] Although the examples presented here use caffeine (which has aromatic character) as a novel excipient, it is believed that other aromatic excipient(s) that promote pi-electron interactions (and do not add significant conductivity) will also show similar synergistic effects, i.e., significantly enhanced filtration flux. At least two other suitable aromatic excipients are pyridoxine and / or phenylalanine and their derivatives.

[0053] Additional aromatic excipients that may be effective include, for example, pyridoxal, pyridoxamine, theobromine, theophylline, xanthine, tyrosine, tryptophan, histidine, histamine, ascorbic acid, folic acid, and naphthalene-1-sulfonic acid and derivatives thereof. The concentration of aromatic excipients used in the biofluid is between 5 mM and 300 mM. Also, in some embodiments, combinations of aromatic excipients are used. For example, some embodiments include using pyridoxine and histidine together.

[0054] FIG. 3 is a graph plotting throughput (liters per square meter) versus time for three virus filter devices filtering mAb2 according to an embodiment of the present disclosure. Prior to application of the feed solution to the virus filter device, a synthetic depth filter containing silica was used to remove large protein aggregates that would otherwise block the pores of the virus filter. The improvement of the surface chemistry on the VPro-based membrane alone may be sufficient to obtain very good performance. In other words, the improvement of the VPro device incorporating the novel surface-treated membrane shows a greater throughput. Furthermore, the VPro device with the novel surface-treated membrane and using aromatic excipients has an even greater throughput. In this example, two different versions of the improved chemistry approach are presented: ISC1; ISC2. Without changing the feed solution conditions from the preceding purification step, the ISC filter maintained a higher flux and achieved a higher throughput during a 60-minute run time when run at a constant pressure of 30 pounds per square inch (PSI). Two different embodiments of the improved chemistry approach are presented. The two separate surface modifications, i.e., surface treatment chemistries, are carried out by different chemical moieties: The first surface modification (ISC1) 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).

[0055] Hydroxypropyl acrylate (HPA), e.g. Cas#25584-83-2

[0056] [ka]

[0057] Polyethylene glycol diacrylate (PEGDA), e.g., Cas#26570-48-9

[0058] [ka]

[0059] The second surface modification was performed with a single component (eg, bisphenol A ethoxylate diacrylate, e.g., Cas#64401-02-1) that acts as both a monomer and a crosslinker.

[0060] Bisphenol A ethoxylate diacrylate, e.g., Cas#64401-02-1

[0061] [ka]

[0062] In both cases, the appropriate chemical formulation was identified from a series of tests conducted under different formulation conditions. In both surface modifications, e-beam was used as the energy source to initiate the polymerization and crosslinking steps. Other energy sources known to those skilled in the art are also contemplated herein.

[0063] FIG. 4 is a graph plotting filtrate volume vs. excipient concentration in millimoles per liter (mM) after 10 minutes at 2000 relative centrifugal force (RCF) according to an embodiment of the present disclosure. After buffer exchange into buffers containing different excipients at various concentrations was completed, the amount of buffer passing through the membrane was measured at fixed time points according to an embodiment of the present disclosure. In this laboratory method, the volume of buffer passing at a fixed time correlated with the flux performance of the virus filter, where the mAb passed through the virus filter, even though the mAb was retained by the membrane. Higher buffer flux through the membrane in the centrifuge device predicted improved flux on virus filters, such as those used in bioprocessing applications. This method was used as a convenient screening method before performing more complex flux runs with virus filters. FIG. 4 shows the results of testing additional neutral cyclic excipients, as can be seen. At higher concentrations, e.g., 300 mM, pyridoxine nearly reached the performance of caffeine at 50 mM. Phenylalanine had a relatively small benefit over increasing concentrations, i.e., the range 50-150 mM, but still exceeded the control. Sodium chloride (NaCl) was added to a base buffer of 20 mM Tris-bicine to reach a conductivity of 3 mS / cm, matching the conditions that could be achieved by titration on a scale such as those shown in Figures 1 and 2.

[0064] FIG. 5 is a graph plotting filtration flux (LMH) versus throughput (kilograms per square meter) of surface-treated membranes for mAb according to an embodiment of the present disclosure. As shown, L-phenylalanine did not perform as well as the other examples. 300 mM pyridoxine performed even better than caffeine; the ISC3 variant sample was equal to or better than ISC1. One further embodiment of an improved chemical approach (ISC3) is presented beyond those shown in the other figures. This surface modification was achieved by using a combination of an acrylic monomer (e.g., hydroxypropyl acrylate (HPA), e.g., Cas#25584-83-2) and a component that can act as both a monomer and a crosslinker (e.g., bisphenol A ethoxylate diacrylate, e.g., Cas#64401-02-1). The synergistic improvement between the excipients and the improved surface chemistry allowed the very difficult to filter mAb1 to be processed at an increased concentration of 20 g / L at 300 LMH.

[0065] 6 is a graph plotting throughput (kilograms per square meter) versus time for surface-treated membranes for mAb-containing solutions at 10 g / L and 20 g / L mAb1 using a caffeine excipient according to an embodiment of the present disclosure. The improved surface chemistry of ISC1 allows for measurable flux at 20 g / L mAb1, but at higher concentrations of this more adsorption-prone mAb, the flux is significantly improved when combined with an excipient such as caffeine. At the lower concentration of 10 g / L mAb1, the flux is further reduced with this combination, as seen by the high mass flux.

[0066] 7 is a graph plotting surface treated membrane throughput (kilograms per square meter) versus time for a solution containing mAb1 and using 300 mM pyridoxine excipient for three different virus filtration devices according to embodiments of the present disclosure. FIG 7 shows a difficult to filter mAb1 in combination with an excipient and an improved surface chemistry virus filter.

[0067] ISC1 alone is not enough to make mAb1 perform at 20 g / L. 300 mM pyridoxine alone and standard VPro are better than ISC1 alone. Using both pyridoxine excipient and ISC1 gives the best performance.

[0068] Caffeine:

[0069] [ka]

[0070] L-Phenylalanine:

[0071] [ka]

[0072] Pyridoxine:

[0073] [ka]

[0074] Figure 8 is a table showing the nonspecific adsorption levels of a model immunoglobulin G (IgG) protein. Specifically, a 1 g / L protein solution of goat gamma globulin was spiked with approximately 106 counts per minute (cpm) of 125I-goat anti-rabbit IgG labeled protein in a phosphate buffered saline (PBS) pH 7.4 solution. Triplicate samples of the membrane were measured and the results reported relative to the membrane effective filtration area (EFA). The associated reduction in protein adsorption is confirmed by this analytical method, which shows a reduction in ISC3, which has the lowest protein binding.

[0075] FIG. 9 is a graph plotting throughput performance versus test pressure (psi) using a model test stream according to an embodiment of the present disclosure. The plotted data shows why subsequent tests using the model stream are performed at 10 psi, which allows better discrimination between samples. FIG. 9 shows the throughput of tests of a filtration device containing a single layer of surface-modified membrane, with the test endpoint defined as 90% flux decay against protein-free buffer or 4 hours, whichever occurs first for any given sample device. Due to possible day-to-day variations in testing, the results of each test are normalized to the throughput of a control device or the average throughput of multiple control devices. The model stream was prepared from commercially available human IgG purified from plasma and lyophilized. It shows why the two improved surface chemistries according to an embodiment of the present disclosure can be differentiated from the VPro control at 10 psi, but not at 30 psi or 50 psi. As shown, the membranes prepared with hydroxypropyl acrylate (HPA) and polyethylene glycol diacrylate (PEGDA) achieved higher throughput than the VPro control when tested at 10 psi.

[0076] 10 is a graph plotting the filtration flux (LMH) of a single layer of a surface-treated membrane versus throughput (liters per square meter) for a model stream operated at 10 psi according to an embodiment of the present disclosure. As shown, membranes prepared with a component that can act as a monomer and crosslinker (e.g., bisphenol A ethoxylate diacrylate, e.g., Cas#64401-02-1) with or without a range of concentrations of HPA (e.g., hydroxypropyl acrylate (HPA), e.g., Cas#25584-83-2) achieved higher throughput than the VPro control.

[0077] 11 is a graph plotting monolayer filtration flux (LMH) versus throughput (liters per square meter) for a model stream operated at 10 psi according to an embodiment of the present disclosure. As shown, membranes prepared with 2-hydroxy-3-phenoxypropyl acrylate (HPPA) and polyethylene glycol diacrylate (PEGDA) achieved higher throughput than the VPro control.

[0078] 2-Hydroxy-3-phenoxypropyl acrylate (HPPA), e.g., Cas#16969-10-1

[0079] [ka]

[0080] 12 is a graph plotting the filtration flux (LMH) of a single layer versus throughput (liters per square meter) for a model stream operated at 10 psi according to an embodiment of the present disclosure. As shown, membranes prepared with hydroxypropyl acrylate (HPA) and polyethylene glycol diacrylate (PEGDA) achieved higher throughput than the VPro control.

[0081] 13 is a graph plotting the filtration flux (LMH) of a single layer of surface-treated membranes versus throughput (liters per square meter) for a model stream operated at 10 psi according to an embodiment of the present disclosure. As shown, membranes prepared with tetrahydrofurfuryl acrylate (Cas#2399-48-6) and polyethylene glycol diacrylate (PEGDA) achieved higher throughput than the VPro control.

[0082] Tetrahydrofurfuryl acrylate, e.g., Cas#2399-48-6

[0083] [ka]

[0084] 14 is a graph plotting the filtration flux (LMH) of a single layer of surface-treated membranes versus throughput (liters per square meter) for a model stream operated at 10 psi according to an embodiment of the present disclosure. As shown, membranes prepared with (5-ethyl-1,3-dioxane-5-yl)methyl acrylate (Cas#66492-51-1) and polyethylene glycol diacrylate (PEGDA) achieved higher throughput than the VPro control.

[0085] (5-Ethyl-1,3-dioxan-5-yl)methyl acrylate, for example, Cas#66492-51-1

[0086] [ka]

[0087] 15 is a graph plotting the filtration flux (LMH) of a single layer of surface-treated membranes versus throughput (liters per square meter) for a model stream operated at 10 psi according to an embodiment of the present disclosure. As shown, membranes prepared with bisphenol A ethoxylate diacrylate (Cas#64401-02-1) alone or with hydroxypropyl acrylate (HPA), polyethylene glycol diacrylate (PEGDA) and one of 2-hydroxy-3-phenoxypropyl acrylate (HPPA), hydroxypropyl acrylate (HPA), tetrahydrofurfuryl acrylate (Cas#2399-48-6), or (5-ethyl-1,3-dioxane-5-yl) methyl acrylate (Cas#66492-51-1) achieved higher throughput than the VPro control.

[0088] The embodiments of the present disclosure include a filtration device. In some embodiments, the filtration device is a polyethersulfone membrane, which is an asymmetric porous membrane, optionally having a surface modification. In some embodiments, the surface modification is a crosslinked coating on the membrane, the crosslinked coating being formed from a coating solution that includes a crosslinkable diacrylate.

[0089] In some embodiments, the solution used to create the crosslinked coating disposed on the membrane further comprises at least one of the following: 2.4-3.4% hydroxypropyl acrylate and 1.1-2.1% polyethylene glycol diacrylate; 2.5-3.5% bisphenol A ethoxylate diacrylate; 3.0-4.0% bisphenol A ethoxylate diacrylate and 1.5-2.5% hydroxypropyl acrylate; 1.5-2.5% hydroxypropyl acrylate and 2.0-3.0% polyethylene glycol diacrylate; 3.0-4.0% bisphenol A ethoxylate diacrylate and 1.5-2.5% hydroxypropyl acrylate; 1.5-2.5% hydroxypropyl acrylate and 2.0-3.0% polyethylene glycol diacrylate; 0.25-2.5% (2-hydroxy-3-phenoxypropyl acrylate and 2.0-5.0% polyethylene glycol diacrylate; 0.2-3.0% tetrahydrofurfuryl acrylate and 1.0-2.5% polyethylene glycol diacrylate; 0.2-3.0% (5-ethyl-1,3-dioxan-5-yl)methyl acrylate and 1.0-2.5% polyethylene glycol diacrylate; or 0.25-2.5% propoxylated tetrahydrofurfuryl acrylate and 2.0-3.0% polyethylene glycol diacrylate.

[0090] In some embodiments, the filtration device can be used to filter a biological fluid using an aromatic excipient, which in some embodiments 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.

[0091] It is understood that membranes having a crosslinked surface coating can be used in conjunction with the use of excipients as described herein. Furthermore, all of the filtration and / or purification methods for mAbs or fractionated plasma products described herein can use both membranes having a surface treatment and excipients, either separately or in conjunction therewith.

[0092] All ranges of the compositions listed herein include the ranges therebetween, and may include or exclude the endpoints. Optionally included ranges are from the integer values ​​therebetween (or one original endpoint included) to the recited place or the next lower place. For example, if the lower limit is 0.2, the optional included endpoints can be 0.3, 0.4, 1.1, 1.2, etc., as well as 1, 2, 3, etc., and if the upper limit is 8, the optional included endpoints can be 7, 6, etc., as well as 7.9, 7.8, etc. One-sided boundaries such as 3 or more also include the corresponding boundary (or range) starting from the recited place integer value or the next lower place. For example, 3 or more includes 4 or 3.1 or more.

[0093] Throughout this specification, references to "one embodiment," "a particular embodiment," "one or more embodiments," "some embodiments," or "an embodiment" indicate that a feature, structure, material, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of phrases such as "in one or more embodiments," "in a particular embodiment," "in one embodiment," "some embodiments," or "in an embodiment" throughout this specification are not necessarily referring to the same embodiments.

[0094] Although some embodiments have been described above, other implementations and applications are within the scope of the following claims. Although the specification has been described with reference to certain embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It should thus be further understood that numerous modifications can be made to the exemplary embodiments, and that other arrangements and patterns can be devised without departing from the spirit and scope of the embodiments according to the present disclosure. Furthermore, the particular features, structures, materials, or characteristics can be combined in any suitable manner in any one or more of the embodiments.

[0095] The patent applications and patent publications and other non-patent references cited herein are hereby incorporated by reference in their entirety, in the entirety of the portion cited, as if each individual publication or reference was specifically and individually indicated to be incorporated by reference herein as if fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references.

Claims

1. 1. A filtration device for use with a biological fluid, comprising: Membrane; and a cross-linked coating on said membrane to form a surface modification; A filtration device wherein the crosslinked coating is formed from a coating solution that includes a crosslinkable diacrylate.

2. 10. The filtration device of claim 1, wherein the coating solution used to create the crosslinked coating comprises at least one of the following: 2.4-3.4% hydroxypropyl acrylate and 1.1-2.1% polyethylene glycol diacrylate; 2.5-3.5% bisphenol A ethoxylate diacrylate; 3.0-4.0% bisphenol A ethoxylate diacrylate and 1.5-2.5% hydroxypropyl acrylate; 1.5-2.5% hydroxypropyl acrylate and 2.0-3.0% polyethylene glycol diacrylate; 0.25-2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0-5.0% polyethylene glycol diacrylate; or 3.0-4.0% bisphenol A ethoxylate diacrylate.

3. The filtration device of claim 1 , wherein the membrane is a porous membrane.

4. 2. The filtration device of 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 filtration device of claim 1 further comprising an air vent.

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

8. 1. A method for making a membrane for filtering a biological fluid, comprising: applying a crosslinkable diacrylate solution to a porous membrane; and initiating curing of the crosslinkable solution to form a cured coating on the porous membrane; wherein the cured coating enhances a high flux filtration device.

9. 9. The method of claim 8, wherein the crosslinkable diacrylate solution used to make the crosslinked coating comprises at least one of the following solutions: 2.4-3.4% hydroxypropyl acrylate and 1.1-2.1% polyethylene glycol diacrylate; 2.5-3.5% Bisphenol A ethoxylate diacrylate; 3.0-4.0% Bisphenol A ethoxylate diacrylate and 1.5-2.5% hydroxypropyl acrylate; 1.5-2.5% hydroxypropyl acrylate and 2.0-3.0% polyethylene glycol diacrylate; 0.25-2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0-5.0% polyethylene glycol diacrylate; or 3.0-4.0% 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. 9. The method of claim 8, wherein the method of initiating the crosslinking reaction is application of E-beam, UV light, or high energy radiation.

12. The method of claim 8 , wherein the membrane is contained within a housing further comprising a biofluid inlet and a biofluid outlet.

13. 1. A method for filtering a biological fluid, comprising: Containing a surface-treated porous membrane within a filtration device having an inlet and an outlet, the surface treatment comprising a crosslinked diacrylate coating; introducing a biological fluid into the inlet of the filtration device; and collecting filtrate from said outlet; The method includes:

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

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

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

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

18. 1. A filtration device for use with a biological fluid, comprising: Membrane; and a crosslinked coating disposed on said membrane to form a crosslinked copolymer surface modification or treatment, said crosslinked coating being formed from a coating solution comprising a crosslinkable diacrylate, said coating solution being 2.4-3.4% hydroxypropyl acrylate and 1.1-2.1% polyethylene glycol diacrylate or 3.0-4.0% bisphenol A ethoxylate diacrylate and 1.5-2.5% hydroxypropyl acrylate; or 1.5-2.5% hydroxypropyl acrylate and 2.0-3.0% polyethylene glycol diacrylate, or 0.25-2.5% 2-hydroxy-3-phenoxypropyl acrylate and 2.0-5.0% polyethylene glycol diacrylate; A filtration device comprising:

19. 20. The filtration device of claim 18, further comprising an air vent.

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

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