CN116744986A

CN116744986A - Fibrinogen sterilization filtration

Info

Publication number: CN116744986A
Application number: CN202080108137.2A
Authority: CN
Inventors: 余爱彬; S·陈; H·李; X·张; Y·李
Original assignee: Aixikang Co ltd; Guangzhou Bioseal Biotech Co Ltd
Current assignee: Aixikang Co ltd; Guangzhou Bioseal Biotech Co Ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2023-09-12
Also published as: WO2022133973A1

Abstract

A method of aseptic filtration of a fibrinogen solution is disclosed, the method comprising filtering the solution with one or more filters selected from the group consisting of glass fiber prefilters and aseptic filters made of polysulfone or derivatives thereof. Further disclosed herein are the sterilized fibrinogen solutions obtained by the method.

Description

Fibrinogen sterilization filtration

Technical Field

In particular, the invention relates to a method for the sterile filtration of fibrinogen solutions.

Background

Natural fibrinogen (factor I) is a glycoprotein complex that circulates in the blood of vertebrates.

Fibrinogen is an essential protein for blood coagulation. It polymerizes to insoluble fibrin formed at the end of the cascade, thereby controlling clotting, resulting in the formation of a clot that closes the vascular cleft responsible for bleeding. Thus, placement of the clot is necessary to ensure that bleeding stops.

Providing compositions comprising fibrinogen, particularly for therapeutic purposes, requires purification techniques that yield a product that is sufficiently purified from contaminants of various nature.

U.S. patent No. 5259971 discloses a method for purifying native, intact fibrinogen from a liquid sample containing contaminants having molecular weights higher and/or lower than that of fibrinogen. The method includes filtering the sample using one or more filters having a molecular weight cut-off that allows for separation of native, intact fibrinogen from contaminants.

U.S. patent No. 10493133 discloses a method for preparing a highly concentrated fibrinogen solution comprising adding an amino acid or amino acid derivative and/or salt to a low concentrated fibrinogen solution followed by ultrafiltration concentration.

U.S. patent No. 7816495 discloses a method of purifying fibrinogen and easily dissolvable fibrinogen preparations.

Us patent No. 7309428 discloses a method and apparatus for separating plasma, the apparatus having a mixing unit in the form of a first injection, the mixing unit having a first connecting tube and a first piston to provide a compartment for a mixture of plasma and protein precipitant to be separated; and the separation unit is composed of a filter pipe and is used for separating and storing the separated solid substances.

Us patent No. 9561478 discloses a separation membrane comprising a polymer, characterized in that a functional layer is formed on a surface of one side of the membrane, a peak area percentage of carbon derived from an ester group measured by Electron Spectroscopy for Chemical Analysis (ESCA) on a surface of the aforementioned functional layer is 0.1% (in atomic number) or more but not more than 10 (atomic number%), and a peak area percentage of carbon derived from an ester group measured by Electron Spectroscopy for Chemical Analysis (ESCA) on a surface opposite to the functional layer is not more than 10 (atomic number%). A separation membrane module with little adhesion of organic substances, proteins, platelets, etc. is provided with a separation membrane as a built-in membrane.

Japanese patent application No. 2007215569 discloses a plasma component separator which is a hollow fiber membrane type plasma component separator wherein a hollow fiber membrane is composed of polysulfone polymer and polyvinylpyrrolidone, the permeability of bovine plasma immunoglobulin G is 80% or more and 100% or less, the permeability of bovine plasma fibrinogen is 10% or more and 30% or less, the liquid surface elevation value of the hollow fiber membrane measured by a capillary elevation method is 80mm or more and 120mm or less when converted into a hollow fiber membrane having an inner diameter of 200 μm, and the round conversion diameter of the hole diameter of the inner surface of the hollow fiber membrane is 13nm to 64nm.

Disclosure of Invention

The fibrinogen concentration in typical formulations reaches 60mg/ml to 120mg/ml. Fibrinogen molecules have a typical molecular weight of about 340kDa, have a rod-like shape with dimensions 9nm by 47.5nm by 6nm, and readily form multimeric particles in aqueous solution. Such particles do not dissolve and can easily accumulate on the surface of the filter membrane used in the sterilizing filtration operation, thereby clogging the membrane and making filtration difficult. In the conventional method, a polyvinylidene fluoride (PVDF) filter membrane is used, and a one square meter membrane can only filter about 5L to 10L of fibrinogen solution due to clogging of the filter membrane. In addition, the filter needs to be replaced a plurality of times during the filtering operation, the operation is complicated, and the risk of microbial contamination is high.

It is an object of the present invention to develop a sterile filtration method for fibrinogen solutions which allows to obtain sterile fibrinogen compositions with an increased filtration capacity (over about 90%). The present inventors have developed a new filtration process which surprisingly provides an extraordinary filtration capacity of fibrinogen formulation liquid, e.g. 50L/m ² To 100L/m ² Films that are easy to implement on an industrial scale at an acceptable industrial cost price.

According to one aspect of the present disclosure, there is provided a method of sterile filtration of a solution comprising a protein of interest, the method comprising: the solution is filtered with at least one filter selected from the group consisting of a glass fiber prefilter and a sterile filter membrane comprising polysulfides or derivatives thereof.

In some embodiments of any aspect provided herein, the derivative of polysulfone is selected from the group consisting of Polyethersulfone (PES), polyphenylsulfone (PPSU), and mixtures thereof. In some embodiments, the polysulfone comprises PES.

In some embodiments of any aspect provided herein, the method comprises filtering the solution with a prefilter and polyvinylidene fluoride (PVDF).

In some embodiments of any aspect provided herein, the method comprises filtering the solution with polysulfone.

In some embodiments of any aspect provided herein, the filter comprising polysulfone or derivative thereof has a removal rating of 0.15 μm to 0.25 μm.

In some embodiments of any aspect provided herein, the glass fiber prefilter has a removal rating of 0.35 μm to 0.55 μm.

In some embodiments of any aspect provided herein, the method comprises filtering the solution with a glass fiber prefilter prior to filtering with polysulfone.

In some embodiments of any aspect provided herein, the filtration is performed at a pressure of 2psi to 30 psi.

In some embodiments of any aspect provided herein, the solution is obtained from a plasma fraction. In some embodiments, the plasma fraction is obtained from pigs.

In some embodiments of any aspect provided herein, the protein of interest comprises fibrinogen.

In some embodiments of any aspect provided herein, the method is characterized by a filtration capacity for a solution (e.g., fibrinogen solution) of at least 5kg/m ² . In some embodiments of any aspect provided herein, the method is characterized by a filtration capacity for a solution (e.g., fibrinogen solution) of at least 15kg/m ² . In some embodiments of any aspect provided herein, the method is characterized by a filtration capacity for a solution (e.g., fibrinogen solution) of at least 40kg/m ² 。

In some embodiments of any aspect provided herein, the method is characterized by a fibrinogen recovery of at least 85%.

In some embodiments of any aspect provided herein, the at least one filter has a non-uniform pore size distribution.

According to one aspect of the present invention, there is provided a sterile filtration of a fibrinogen containing solution comprising a first filtration over a filter having a removal rating of 0.35 μm to 0.55 μm and a second filtration over a filter having a removal rating of 0.15 μm to 0.25 μm.

In some embodiments, there is provided a sterile fibrinogen solution obtained by a method according to any aspect provided herein.

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the operation or testing of embodiments of the present invention, exemplary methods and materials are described below. In case of conflict, the patent specification and its definitions will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting as to the necessity.

Drawings

Some embodiments of the invention are described herein, by way of example, with reference to the accompanying drawings. Referring now specifically to the drawings, it is emphasized that the details shown are by way of example and are shown for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings make apparent to those skilled in the art how the embodiments of the present invention may be practiced.

In the drawings:

FIG. 1 shows comparative filtration capacities (kg/m) of several batches of fibrinogen solutions using the following in each triad ² ) Is a bar graph of (2): polyvinylidene fluoride ("PVDF"; left column), glass fiber prefilter ("PRE"; middle column), and polyethersulfone ("PES"; right column).

FIG. 2 shows comparative filtration capacities (kg/m) of several batches of fibrinogen solution as given in FIG. 1 ² ) And further shows the filtering capacity (right column, added in each quad) when using pre+pes filters.

Detailed Description

It is an object of the present invention to provide an efficient method for the sterilization and filtration of fibrinogen from solution.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the examples. The invention is capable of other embodiments or of being practiced or of being carried out in various ways.

In one aspect of the present disclosure, there is provided a method of sterile filtration of a mixture or solution comprising a protein of interest, the method comprising: the mixture or solution is filtered with one or more filters selected from the group consisting of glass fiber prefilters and aseptic filters comprising polysulfone.

Those skilled in the art will appreciate that filters or membranes are employed to produce the product. In the absence of a filter or membrane module used in the methods disclosed herein, the methods will not yield the desired product with the desired purity, concentration, etc., or increased capacity. A filter or membrane assembly is located within the fluid process stream and is capable of filtering the above-mentioned mixture or solution as it passes therethrough.

The term "filtration" includes all of those separation processes as well as any other process utilizing a filter to separate substances. In an exemplary embodiment, the step of passing the fibrinogen-containing solution through a filter allows micro-flocs that may be present in the solution to be removed therefrom. In some embodiments, the filtration of the present disclosure is not an ultrafiltration step or process. Generally, the term "ultrafiltration" refers to a process in which a liquid is separated into fractions by pressure driven flow through a semipermeable membrane having a molecular weight cut-off in the range of 200 to about 330,000 and a pore diameter of about 10 to 1000 angstroms.

The term "filter" (also referred to as a "filter membrane") may refer to a screen, sheet wire mesh or mesh structure used for filtration. The filter may be designed, for example, as a planar membrane or a planar screen. The filter may be osmotically selective and used to filter particles from the fluid, particularly for producing high purity proteins of interest.

In some embodiments, "filtering a solution" means the step of passing a solution (e.g., a source solution containing a protein of interest (e.g., fibrinogen)) through a filter to remove micro-flocs that may be present therefrom. That is, fibrinogen may pass through the filter along with contaminants (e.g., bacteria) to become trapped in or on the filter. In some embodiments, the source solution is fresh, or in some embodiments, the source solution has been frozen and thawed prior to filtration, which may generally have more multimers than fresh solution (i.e., not frozen), which may reduce filtration capacity.

As used herein, the term "mixture" refers to, but is not limited to, a combination of components in any physical form (e.g., blend, solution, suspension, dispersion, etc.).

The terms "sterile filtration", "aseptic filtration" or any grammatical variations thereof are a functional description of the filtration of a formulation through a sterile filter to remove contaminants such as bacteria and/or mycoplasma.

The term "sterile" as used in accordance with the present invention refers to a material that is free or substantially free of microbial and/or viral contamination. In this regard, the term "contaminant" means a substance other than the desired protein of interest.

In some embodiments, the step of sterilizing filtration may achieve at least a 1-log, at least a 2-log, at least a 3-log, at least a 4-log, or at least a 5-log reduction of microorganisms in the source solution.

Typically, but not exclusively, glass fiber filters are made of resin bonded glass fibers (such as but not limited to glass wool), however, rock wool, long glass fibers (filaments) or glass fiber cloth may be used. In general, acid-resistant or alkali-resistant glass fibers containing basic oxides can be used as the glass fibers of the filter layer. When considering pressure loss, the glass fiber filter may have a density of 150kg/m ³ -250kg/m ³ And the thickness of the glass fiber filter may be in the range of 40mm to 60 mm.

In an exemplary embodiment, the mixture is a liquid sample comprising fibrinogen. Most preferably, fibrinogen is present at a concentration of 15mg/ml or less. Alternatively, the purified protein (e.g., fibrinogen) may be precipitated using chemical reagents, followed by centrifugation or lyophilization. Without further dilution, the sample may be concentrated by filtration.

The term "prefilter" refers to a filter that may prevent relatively large sized solid particles (typically exceeding 100 microns). Typically, but not exclusively, the pre-filter is positioned upstream of one or more primary, primary filters.

In some embodiments, the mixture comprising the protein of interest comprises a source solution. "source solution" broadly refers to a combination, mixture and/or blend of ingredients having at least one liquid component and a protein of interest. The solution typically contains at least one solvent in an amount or volume greater than the solute. Typical solvents include water. In some embodiments, the source solution comprises a mixture of proteins. In some embodiments, the source solution comprises plasma, typically blood plasma or a fraction thereof. In some embodiments, the plasma comprises oxalate-treated plasma. In some embodiments, the source solution comprises plasma harvested from a mammal. In some embodiments, the mammal is selected from the group consisting of humans, horses, cows, and pigs. In an exemplary embodiment, the source solution comprises porcine plasma.

Thus, the plasma proteins may include one or more coagulation factors (F), such as fibrinogen, prothrombin, thrombin, FX, FXa, FIX, FIXa, FVII, FVIIa, FVIII, FVIIIa, FXI, FXIa, FXII, FXIIa, FXIII, FXIIIa, fengwei, the libland (von Willebrand) factor, and the like; transport proteins such as albumin, transferrin, ceruloplasmin, haptoglobin, heme binding proteins, and the like; protease inhibitors such as beta-antithrombin, alpha 2-macroglobulin, cl-inhibitors, tissue Factor Pathway Inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), protein C, protein S, etc.; anti-angiogenic proteins, such as potential antithrombin, etc.; highly glycosylated proteins including alpha-1-acid glycoprotein, antichymotrypsin, meta-alpha-trypsin inhibitor, alpha-2-HS glycoprotein, C-reactive protein, and the like; other proteins such as histidine-rich glycoproteins, mannan-binding lectin, C4-binding proteins, fibronectin, GC-globulin, plasminogen; blood factors such as erythropoietin, interferon, tumor factors, tPA, gCSF, and derivatives and muteins thereof.

In some embodiments, the protein of interest comprises fibrinogen. As used herein and in the art, the term "fibrinogen" refers to the precursor protein of the blood clot matrix. Fibrinogen has a molecular weight of about 340,000 daltons and has 3 pairs of different polypeptide chains aα, bβ and γ linked together by disulfide bonds. Typically, fibrinogen has a three-nodular structure: two identical D-terminal globular domains and a central E globular domain connected by a supercoiled α -helix.

In some embodiments, the fibrinogen is derived from fibrinogen concentrate. In some embodiments, the fibrinogen is a blood-derived fibrinogen concentrate.

Throughout this document, the terms "derived from" or "derived from" are used interchangeably and refer to the origin or source of a related component, which may include naturally occurring, recombinant, processed, unpurified or purified molecules (e.g., related proteins).

In some embodiments, the source solution comprises a plasma fraction obtained from pre-purified plasma.

By "plasma fraction obtained from pre-purified plasma" is meant any fraction or sub-fraction of human plasma that has been the subject of one or more purification steps. Such plasma fractions thus include the supernatant of cryogenically precipitated plasma, the cryogenically precipitated (resuspended) plasma, fraction I obtained by ethanol fractionation (according to the method of Cohn or ki Dan Le with nieman (Kistler & Nitschmann)), the eluate of the chromatography and the unadsorbed fraction.

In one embodiment of the invention, the fibrinogen composition subjected to the method of the invention is subjected to an additional chromatography step. Thus, according to one embodiment, the fibrinogen composition subjected to the method according to the invention is a chromatographic eluate or non-adsorbed fraction from a chromatographic column (including multi-column chromatography).

The term "polysulfone" is generally used to describe a polymer containing the general formula- (Ar-SO) ₂ -Ar) -one or more dipentylsulfone groups (e.g. monomers), wherein Ar is a substituted or unsubstituted aryl group such as phenyl, biphenyl, bisphenol or any other aryl group containing aromatic hydrocarbons or heteroaromatic rings. In some embodiments, the polysulfone is selected from the group consisting of Polysulfone (PSU), polyethersulfone (PES), polyphenylsulfone (PPSU), and mixtures or derivatives thereof.

The term "derivative" or "chemical derivative" refers to a subject molecule that has been chemically modified but remains largely unchanged, e.g., a subject molecule that has been substituted with additional or different substituents, a subject molecule in which a portion has been oxidized or hydrolyzed, and so forth.

The term "polymer" describes an organic substance consisting of a plurality of repeating structural units (monomer units) covalently linked to each other.

In some embodiments, the method includes filtering the source solution with a glass fiber prefilter and a polyvinylidene fluoride (PVDF) filter downstream or below it. In some embodiments, the method includes filtering the source solution with a glass fiber prefilter and a polysulfone filter downstream or below the glass fiber prefilter.

In some embodiments, the glass fiber prefilter may have a removal rating of 0.3 μm to 0.6 μm, such as 0.4 μm, 0.5 μm, or 0.6 μm, including any values and ranges therebetween, meaning that particles greater than a specified size are effectively trapped by the filter. The filter may have a pore size of about 1 μm or less or about 0.5 μm or less. It is understood that the removal rating and pore size are different parameters, the removal rating reflects the performance of the filter, and the pore size is a property of the filter membrane, although these parameters may affect each other and be closely related.

In some embodiments, the glass fiber prefilter is characterized by a maximum operating pressure of 20psi to 70psi at about room temperature. In some embodiments, the glass fiber prefilter is characterized by a maximum operating pressure of 20psi, 30psi, 40psi, 50psi, 60psi, or 70psi, including any values and ranges therebetween, at about room temperature.

The term "maximum operating pressure" refers to the highest pressure that does not damage the filter. Differential pressure is the pressure difference between the filter inlet and the filter outlet.

In some embodiments, the operating pressure is controlled by a pump, for example, to form differential pressure output and input sides of the filter, with the output side being at a greater pressure. The term "pump" refers to any device that moves a fluid by applying suction or pressure, such as by compressed air.

By "about room temperature" is meant at least one temperature value in the range of 10 ℃ to 40 ℃ or 15 ℃ to 37 ℃, such as 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 37 ℃ or 40 ℃, including any value and range therebetween.

The progress of the filtration process may be monitored using a variety of means, such as by detecting the concentration of contaminants on the filter.

As shown in the examples section below, it has been demonstrated that the use of glass fiber prefilters makes it possible to increase the filtration capacity compared to the old process using only PVDF filters, thus, for example, simplifying the industrial implementation of the process.

In some embodiments, the filtration capacity of the fibrinogen solution is increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% when using the glass fiber prefilter as compared to an old process using only PVDF filters.

In some embodiments, the filtration capacity is increased by 30% to 150% when using a glass fiber prefilter, as compared to conventional methods using PVDF filters alone.

In the present disclosure, "filtration capacity" means the maximum load of protein of interest (e.g., fibrinogen) per membrane area, in kg protein/m of the relevant filter (or combination of filters) ² Is represented by a film.

In some embodiments, the filtration capacity is at least 5kg/m when a PVDF filter is used after the glass fiber prefilter is used ² At least 5.5kg/m ² Or at least 6kg/m ² . In some embodiments, the filtration capacity is 4.5kg/m when a PVDF filter is used after the glass fiber prefilter is used ² To 15kg/m ² . In some embodiments, the filtration capacity is 4.5kg/m when a PVDF filter is used after the glass fiber prefilter is used ² 、5kg/m ² 、5.5kg/m ² 、6kg/m ² 、6.5kg/m ² 、7kg/m ² 、7.5kg/m ² 、8kg/m ² 、8.5kg/m ² 、9kg/m ² 、9.5kg/m ² 、10kg/m ² 、10.5kg/m ² 、11kg/m ² 、11.5kg/m ² 、12kg/m ² 、12.5kg/m ² 、13kg/m ² 、13.5kg/m ² 、14kg/m ² 、14.5kg/m ² Or 15kg/m ² Including any values and ranges therebetween.

Accordingly, there is provided a method for increasing the filtration capacity of a PVDF filter by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% in the aseptic filtration of a solution comprising a protein of interest (e.g. fibrinogen), the method comprising filtering the solution with at least one prefilter comprising glass fibers prior to PVDF filtration.

In some embodiments, the solution comprising the protein of interest comprises fibrinogen that has not been previously frozen and/or thawed prior to the filtration process. Typically, the frozen and thawed material may have more multimeric, insoluble, aggregated or partially denatured material, thereby reducing the filtration capacity (when prefilter is used) by about 50% as compared to fresh solution (i.e., previously unfrozen and/or thawed solution).

In some embodiments, the fibrinogen recovery is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% when using a PVDF filter after using a glass fiber prefilter. In some embodiments, fibrinogen recovery is from about 88% to about 100% using a PVDF filter after the glass fiber prefilter is used. In some embodiments, fibrinogen recovery is about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, including any values and ranges therebetween, when using a PVDF filter after using a glass-fiber prefilter.

In some embodiments, fibrinogen recovery (fibrinogen recovery from fibrinogen source solution) is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, or at least 12% when using a PVDF filter after using a glass fiber prefilter, as compared to a method using a PVDF filter without a prefilter. In some embodiments, fibrinogen recovery from the fibrinogen solution is increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%, including any value and range therebetween, when the PVDF filter is used after the glass fiber prefilter is used, as compared to a method using the PVDF filter without the prefilter.

The term "fibrinogen recovery" (also referred to as "fibrinogen activity recovery" and "filtration yield") describes the concentration (content) of fibrinogen found after the filtration process divided by the concentration of fibrinogen in the solution prior to the filtration process.

In some any of the embodiments of any of the aspects provided herein, the method is characterized by a protein recovery of at least about 95%. In some any of the embodiments of any of the aspects provided herein, the method is characterized by a protein recovery of about 95% to about 98%.

The term "protein recovery" (also referred to as "protein yield") describes the amount of protein found after a filtration process divided by the amount of protein before the filtration process.

"protein content" refers to the amount of protein contained in a substance. Indeed, the aseptic filtration in the disclosed method of removing microorganisms from a fluid stream (without adversely affecting product quality) allows nearly all proteins (including fibrinogen) to pass through the filter.

In some embodiments, the method includes filtering the source solution with a polysulfone filter.

Exemplary methods for measuring protein recovery and fibrinogen recovery are provided in the examples section and are known in the art.

As further shown in the examples section that follows, it has been demonstrated that the use of polysulfone (e.g. PES) filters makes it possible to increase the filtration capacity compared to the old (traditional) method using only PVDF filters, thus simplifying the industrial implementation of the method.

In some embodiments, the filtration capacity is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 6-fold when using a polysulfone filter as compared to an old process using a PVDF filter. In some embodiments, the filtration capacity is increased 2-fold to 6-fold when polysulfone (e.g., PES) is used, as compared to the old method of using PVDF filters.

In some embodiments, when a polysulfone (e.g., PES) filter is used, the filtration capacity is at least 10kg/m ² At least 12kg/m ² Or at least 15kg/m ² . In some embodiments, when a polysulfone (e.g., PES) filter is used, the filtration capacity is 15kg/m ² To 30kg/m ² . In some embodiments, when a polysulfone (e.g., PES) filter is used, the filtration capacity is 15kg/m ² 、16kg/m ² 、17kg/m ² 、18kg/m ² 、19kg/m ² 、20kg/m ² 、21kg/m ² 、22kg/m ² 、23kg/m ² 、24kg/m ² 、25kg/m ² 、26kg/m ² 、27kg/m ² 、29kg/m ² 、29kg/m ² Or 30kg/m ² Including any values and ranges therebetween.

In some embodiments, when using a polysulfone (e.g., PES) filter, the fibrinogen recovery from the fibrinogen solution is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

In some embodiments, the method includes prefiltering with glass fibers and thereafter filtering the source solution with a polysulfone (e.g., PES) filter (i.e., downstream thereof).

As further shown in the examples section that follows, it has been demonstrated that the use of a glass fiber prefilter makes it possible to further increase the filtration capacity compared to the sulfone filter alone. In some embodiments, the filtration capacity of the fibrinogen solution is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, or at least 7-fold when using the glass fiber prefilter as compared to the sulfone filter alone (i.e., without using the glass fiber prefilter).

In some embodiments, the filtration capacity is increased by 30% to 150% when using a glass fiber prefilter as compared to a sulfone filter alone.

Thus, there is provided a method of increasing the filtration capacity of a polysulfone (e.g. PES) filter by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold or at least 7-fold compared to the use of a sulfone filter alone in the sterile filtration of a solution comprising a protein of interest (e.g. fibrinogen), the method comprising filtering the solution with at least one prefilter comprising glass fibers.

In some embodiments, the fibrinogen recovery is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 89% when a polysulfone (e.g., PES) filter is used after the glass fiber prefilter is used. In some embodiments, fibrinogen recovery is from about 88% to about 100% using a PVDF filter after the glass fiber prefilter is used. In some embodiments, when a polysulfone (e.g., PES) filter is used after the glass fiber prefilter is used, the fibrinogen recovery is about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 89%, including any value and range therebetween.

The shape of the pores or holes of the filter may affect the filtering capacity of the filter. It is assumed that an asymmetric pore filter has a higher capacity for interception of small particles than a filter having a uniform (symmetric) pore shape. The pore shape and size of the old PVDF filter appears to be much more uniform than that of the glass fibers and PES. At the same time, a 0.45 micron glass fiber prefilter may intercept large particles in solution to help increase the filtration flux of the final filter.

Accordingly, in one aspect of the present disclosure, there is provided a method of sterile filtration of a mixture comprising a protein of interest (e.g., fibrinogen), the method comprising: the mixture is filtered with at least one filter having a gradient pore size from one planar surface to another planar surface or a gradient pore size within the membrane. Thus, in some embodiments, the filter membrane, or in some embodiments, the plurality of filter membranes, form a generally porous member that is asymmetrically porous, having pores that gradually decrease in diameter as one progresses from the upper (feed) surface to the lower surface, such that the generally membrane pores form a "V" shape, are non-uniform, and are not easily blocked by particles in solution. Additionally, or alternatively, the overall membrane pores are linear and non-uniform.

In this context, "asymmetric" or "non-uniform" means a non-uniform or broad pore size distribution. In one embodiment, the term refers to the median pore size or removal rating of the upper (feed) surface being from 50% to 400% higher than the median pore size or removal rating of the lower surface, or in some embodiments from 100% to 200% higher, such as 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, or 400%, including any value and range therebetween. Alternatively, these terms mean a pore size change in at least one surface of the membrane of 50% to 400%, or in some embodiments 100% to 200%, such as 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390% or 400%, including any value and range therebetween.

By "size" is meant at least one dimension, such as a diameter, of a pore, typically a micropore.

Generally, the sterile filtration method of this aspect comprises forming a gradient pore size by using at least one filter selected from the group consisting of a glass fiber prefilter and a sterile filter comprising polysulfone. Embodiments of "filters," "glass fiber prefilters," "sterile filters," "polysulfones," "proteins of interest," and "mixtures" are described and incorporated herein in their entirety.

In one aspect of the disclosure, a method of sterile filtration of a fibrinogen-containing solution is provided that includes a first filtration over a filter having a removal rating of about 0.35 μm to about 0.55 μm (e.g., 0.35 μm, 0.4 μm, 0.45 μm, 0.50 μm, or 0.55 μm, including any values and ranges therebetween), and a second (e.g., subsequent) filtration over a filter having a removal rating of about 0.15 μm to about 0.25 μm (e.g., 0.15 μm, 0.20 μm, or 025 μm, including any values and ranges therebetween). In some embodiments of this aspect, the filtration capacity for the solution is at least 15kg/m ² To at least 85kg/m ² For example 15kg/m ² 、20kg/m ² 、30kg/m ² 、35kg/m ² 、40kg/m ² 、45kg/m ² 、50kg/m ² 、55kg/m ² 、60kg/m ² 、65kg/m ² 、70kg/m ² 、75kg/m ² 、80kg/m ² Or 85kg/m ² Including any values and ranges therebetween.

In some embodiments, there is provided a sterile solution of fibrinogen obtained by the methods disclosed herein according to any of the aspects or embodiments described above.

As used herein, the term "about" refers to ± 10%.

The terms "comprising," including, "" containing, "" implying, "" containing, "" having, "" with, "and variations thereof mean" including but not limited to. The term "consisting of … …" means "including and limited to". The term "consisting essentially of …" means that the composition, method, or structure may comprise additional ingredients, steps, and/or components, provided that the additional ingredients, steps, and/or components do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

The word "exemplary" is used herein to mean "serving as an example, instance, or illustration. Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and/or as comprising no features of other embodiments.

The word "optionally" is used herein to mean "provided in some embodiments and not provided in other embodiments. Any particular embodiment of the invention may include a plurality of "optional" features unless such features conflict.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds (including mixtures thereof).

Throughout the present application, various embodiments of the present application may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a fixed limitation on the scope of the present application. Accordingly, the description of a range should be considered to have all possible subranges as well as individual values within the range explicitly disclosed. For example, descriptions of ranges such as 1 to 6 should be considered to have the explicitly disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual values within the range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

Whenever numerical ranges are indicated herein, it is intended to include any recited number (fractional or integer) that is within the indicated range. The phrase "range between" a first indicated number and a second indicated number and "range from" a first indicated number "to" a second indicated number "is used interchangeably herein and is meant to include the first indicated number and the second indicated number, as well as all fractions and integers therebetween.

As used herein, the term "method" or "process" interchangeably used herein refers to the manner, means, technique and procedure used to accomplish a given task, including but not limited to those known to or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, analytical, pharmacological, biological, biochemical and medical arts.

As used herein, and unless otherwise indicated, the terms "by weight," "w/w," "weight percent," or "wt%" are used interchangeably herein to describe the concentration of a particular substance in the total weight of the corresponding mixture, solution, formulation, or composition.

In those instances where a convention analogous to "at least one of A, B and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a composition having at least one of A, B and C" would include but not be limited to compositions having a alone, B alone, C, A and B together alone, a and C together, B and C together, and/or A, B and C together, etc.). It will be further understood by those within the art that, in fact, whether in the detailed description, claims, or drawings, disjunctive words and/or phrases presenting two or more alternative terms should be understood to encompass the possibility of including one of the terms, either of the terms, or both. For example, the phrase "a or B" will be understood to include the possibilities of "a", "B" or "a and B".

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered as essential features of those embodiments unless the embodiments do not function without those elements.

Various embodiments and aspects of the invention as described above and as claimed in the claims section below are experimentally supported in the following examples.

Examples

Reference is now made to the following examples, which illustrate, in a non-limiting manner, some embodiments of the invention, along with the above description.

The aim of this study was to explore a new method for safely and effectively increasing the filtration flux of fibrinogen formulation liquid to achieve the aim of improving the yield of the final product and saving filter consumables.

Material and equipment

Material

Fibrinogen solution: fibrinogen liquid formulations were manufactured by Guangzhou double embroidery biotechnology Co., ltd (Guangzhou Bioseal biotechnology Co, ltd), contained 30mg/ml to 50mg/ml fibrinogen, and were typically stored at-20℃to about-30℃and thawed prior to performing filtration experiments. Various batches were used as described below.

Equipment

The equipment details with relevant parameters are summarized in table 1 below:

TABLE 1

Example 1: experimental design I

Experimental design I was performed, in particular to investigate the feasibility of filtering fibrinogen liquid formulations using various filter designs compared to conventional filters (referred to as "F1").

Three parallel filtration process lines were designed to filter fibrinogen liquid formulation simultaneously and the filtered liquids were weighed to compare the filtration capacities of the different filtration lines. Filtration temperature: room temperature, 20-26 deg. Filtration pressure: 2psi-30psi, using compressed air to provide the desired pressure. The experimental parameters of the process line were as follows:

line 1: a millboro polyvinylidene fluoride (PVDF) filter was used. Filtration area: 500cm ² The method comprises the steps of carrying out a first treatment on the surface of the Catalog number: MPGL10CL3. This is the same kind of filter used in the old process.

Line 2: glass fiber prefilter (also referred to as "PRE") was added before the millbo PVDF final filter, filter area: 900cm ² The method comprises the steps of carrying out a first treatment on the surface of the Catalog number: DFA3001UBC.

Line 3: sterilizing and filtering by using a Polyethersulfone (PES) final filter instead of a Miibo PVDF filter; area of filter: 230cm ² The method comprises the steps of carrying out a first treatment on the surface of the Catalog number: KA02ECV2FT.

Results

The results of the various batches are summarized in table 2 below and further illustrated in fig. 1.

TABLE 2

As can be seen in Table 2, the PVDF has an average filtration capacity of about 4.7kg/m ² A membrane; the average filtration capacity of PRE+PVDF is 8.7kg/m ² Membranes, and the average filtration capacity of PES is much higher: 20.0kg/m ² And (3) a film.

The data clearly show that adding a pre-filter or replacing the PVDF filter with a PES filter can significantly improve the filtration capacity of the fibrinogen liquid formulation.

Based on these results, the filtration capacity of the combination method (glass fiber prefilter+pes final filter) was further explored as follows.

Example 2: experimental design II

Four parallel filtration process lines were designed to simultaneously filter fibrinogen formulation liquid and the filtered liquid was weighed to compare the filtration capacities of the different filtration lines. Filtration temperature: room temperature, 20-26 ℃; filtration pressure: 2psi-30psi, as described above. Experimental process line 1 to experimental process line 3 and parameters as described above, line 4 was added: sterile filtration was performed using a glass fiber prefilter ("PRE") + PES final filter, with the same pattern and filtration area as described above.

The results are summarized in table 3 below and further illustrated in fig. 2.

TABLE 3 Table 3

Comparing these two experimental results, it can be easily seen that the combined filtration method of PRE+PES (as the final filter in the filtration process) can further improve the filtration capacity for fibrinogen preparation liquid, and the average filtration capacity can be up to about 80kg/m ² Average value of the film.

The data clearly show that adding a prefilter or replacing PVDF filter with a PES filter can significantly improve the filtration capacity for the main preparation solution.

"904", "205" and "607" are three batches of fibrinogen preparation liquid used as starting materials in this experiment. Fibrinogen activity and protein concentration can be seen in table 4 below.

TABLE 4 Table 4

The reason why the results of '904 and' 205 are different may be due to the fact that: the material of '904 has been frozen and then reconstituted, and upon freezing and then thawing, more multimers may be formed, which may affect the filtration capacity, while' 205 comprises fresh material.

Without being bound by any particular theory or mechanism, it is hypothesized that the pore (pore) shape of the filter membrane affects the filtration capacity of the filter. Asymmetric pore filters have a higher capacity to intercept small particles than filters with uniform pore shapes. The pore shape of PVDF filters is much more uniform than that of glass fibers and PES. The uniform membrane may be completely blocked by particles in the solution. Meanwhile, the 0.45 micron glass fiber prefilter can intercept large particles in the solution. In glass fiber prefilters, the membrane pores are linear and non-uniform and are not easily clogged by particles in the solution. And this may help to increase the filtration flux of the final filter. PES membranes have "V" -shaped pores and are also non-uniform and therefore are not easily blocked by particles in solution.

Selection of filter specifications for production

Based on the above experimental data of the filtering capability, the average filtering capability of PRE+PES can reach about 80kg/m ² And larger. If the density of the fibrinogen formulation liquid is considered to be 1g/ml, the average filtration capacity of PRE+PES may reach about 80L/m ² . The' 904 fibrinogen formulation liquid has been frozen and thereafter reconstituted and under this worse condition the filtration capacity of the process also reaches 54.26L/m ² 。

The very 20 inch PES final filter had a 2.08m ² And the batch size of fibrinogen formulation liquid used was 70L, it was sufficient to filter the entire batch using a 20 inch pi PES filter. Thus, the sterilization filter to be selected is a Por 20 inch PES filter (catalog number: AB2UECV7PH 4).

Regarding the selection of prefilters, 900-cm was selected ² Used for experiments, rapidly and smoothly filters 5L of liquid fibrinogen solution. A0.45 micron 20 inch fiberglass filter was selected as the filter surface area of 1.30m ² Is used (catalog number: AB2UB7PH 4), however, the size of the prefilter can be adjusted according to actual production.

Example 3: fibrinogen activity

This set of experiments was performed in order to investigate the effect of the filter on the protein of the fibrinogen liquid formulation during the new filtration process.

The experimental procedure was the same as the filter filtration capacity study experiment mentioned above. After filtration, the fibrinogen liquid formulation is sampled before and after filtration to detect fibrinogen ("FIB") activity and determine protein content to assess adsorption of the target protein by the new filter.

Protocols for detecting FIB Activity

FIB activity was determined by claus method (Clauss method), widely used in clinical laboratories. Guidelines for this procedure are approved by the national clinical laboratory standards committee (National Committee for Clinical Laboratory Standard, NCCLS). Here, stago STA-compact (an automatic coagulation analyzer based on the Claus method) is used for fibrinogen detection. The assay uses a diluted fibrinogen sample in which the reagent thrombin is used in high concentration to initiate coagulation. Calibration curves were drawn using serial dilutions of reference fibrinogen standard. The test plasma or fibrinogen samples were diluted, incubated, and FIB detector containing phospholipids was used. Thrombin and calcium were added and timing started with the addition of FIB detector. The time taken for clot formation is compared to an internal reference calibration curve to derive the fibrinogen concentration (e.g., mg/mL) of the test sample.

Protocol for detecting protein content

"protein content" refers to the amount of protein contained in a substance. In fact, aseptic filtration is a process that removes microorganisms from a fluid stream without adversely affecting product quality, meaning that the process will allow nearly all proteins (including fibrinogen) to pass through the filter.

Protein content was determined by a bicinchoninic acid (BCA) protein assay, which is a BCA-based detergent compatible formulation for colorimetric detection and quantification of total protein. Albumin standards were prepared at concentrations of 0 μg/mL, 25 μg/mL, 125 μg/mL, 250 μg/mL, 750 μg/mL, 1000 μg/mL, 1500 μg/mL, 2000 μg/mL, after which 25 μl of the replicated or unknown sample was pipetted into microplate wells (working range = 20 μg/mL-2000 μg/mL). Next, an amount of 200 μl of BCA working solution was added to each well, and the plate was thoroughly mixed on a plate shaker for 30 seconds. The plates were then covered and incubated at 37℃for 30 minutes. The plate was then cooled to room temperature and absorbance at or near 562nm was read on a plate reader.

The results for each sample are given in tables 5-7 below.

TABLE 5

/>

TABLE 6

TABLE 7

Based on the above data, the average values were calculated and summarized in table 8 below.

TABLE 8

It can be seen that the protein recovery and fibrinogen activity recovery of the new filtration method is superior to the old filtration method.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations as fall within the spirit and broad scope of the appended claims.

Claims

1. A method of sterile filtration of a solution comprising fibrinogen, the method comprising: filtering the solution with at least one filter selected from the group consisting of a glass fiber prefilter and a sterile filter comprising polysulfone or a derivative thereof.

2. The method of claim 1, wherein the polysulfone derivative is selected from the group consisting of Polyethersulfone (PES), polyphenylsulfone (PPSU), and combinations or copolymers thereof.

3. The method of claim 1 or 2, wherein the polysulfone comprises PES.

4. A method according to any one of claims 1 to 3, comprising filtering the solution with the pre-filter and thereafter with polyvinylidene fluoride (PVDF).

5. A method according to any one of claims 1 to 3, comprising filtering the solution with the filter comprising polysulphone.

6. The method of claim 5, comprising filtering the solution with the glass fiber prefilter prior to the filtering with the filter comprising polysulfone.

7. The method of any one of claims 1-6, wherein the polysulfone or derivative thereof has a removal grade of 0.15 μιη to 0.25 μιη.

8. The method of any one of claims 1 to 7, wherein the glass fiber prefilter has a removal rating of 0.35 μιη to 0.55 μιη.

9. The method of any one of claims 1 to 8, wherein the filtering is performed at a pressure of 2psi to 30 psi.

10. The method according to any one of claims 1 to 9, wherein the solution is obtained from a plasma fraction.

11. The method of claim 10, wherein the plasma fraction is obtained from pigs.

12. The method of claim 4 having at least 5kg/m ² Is used for filtering the solution.

13. The method of claim 5 having at least 15kg/m ² Is used for filtering the solution.

14. The method of claim 6, having at least 40kg/m ² And up to about 85kg/m ² Is used for filtering the solution.

15. The method of any one of claims 1 to 14, having a fibrinogen recovery of at least 85%.

16. The method of any one of claims 1 to 15, wherein the at least one filter has a non-uniform pore size distribution.

17. A method of sterile filtration of a fibrinogen containing solution, the method comprising a first filtration over a filter having a removal rating of 0.35 μm to 0.55 μm and a second filtration over a filter having a removal rating of 0.15 μm to 0.25 μm.

18. The method of claim 17, having at least 15kg/m ² Is used for filtering the solution.

19. The method of claim 17 or 18, having a fibrinogen recovery of at least 85%.

20. A sterilized fibrinogen solution obtained by the method of any one of claims 1 to 19.