CN114470969A

CN114470969A - Deep layer filtering medium and its use

Info

Publication number: CN114470969A
Application number: CN202011259137.0A
Authority: CN
Inventors: 冉曙光; 王强; 蒋德席
Original assignee: Sichuan Yuanda Shuyang Pharmaceutical Co Ltd
Current assignee: Sichuan Yuanda Shuyang Pharmaceutical Co Ltd
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2022-05-13

Abstract

The invention provides a deep filtering medium/deep filtering system and application thereof in filtering liquid to be filtered in a water phase, and belongs to the technical field of filtering. The filter aid of the depth filtration media comprises microcrystalline cellulose. The depth filtration system includes a layer of microcrystalline cellulose filter aid and a layer of depth filtration media with or without microcrystalline cellulose filter aid. The deep layer filtering medium or the deep layer filtering system disclosed by the invention can be used for filtering samples with larger viscosity, has high filtering efficiency and high relative activity recovery rate, can replace or partially replace the traditional high-speed centrifugation method or filter pressing method to realize solid-liquid separation of liquid to be filtered in a water phase, reduces the requirements on production facility equipment, reduces the production cost, and is suitable for large-scale production and application.

Description

Deep layer filtering medium and its use

Technical Field

The invention relates to the technical field of filtration. In particular, the present invention relates to a depth filtration medium or depth filtration system and its use in filtering a liquid to be filtered in a water phase.

Background

Depth filtration is one of the most widely used filtration techniques in the preparation of plasma protein preparations and is also commonly used for cell culture clarification. Depth filtration media typically have a thickness or depth and an internal structure that is a tortuous labyrinth that retains particles smaller than the pores of the filter media. It is generally believed that depth filtration has multiple retention mechanisms, and that particulate retention achieves size exclusion and adsorption through hydrophobic, ionic and other interactions. Depth filtration filters typically have a gradient density structure of material, generally with larger pores near the top, and fouling mechanisms may include pore blocking, cake formation, and/or pore shrinkage.

The medium for industrial deep filtration is generally made of fiber as a filter plate, generally called as a filter plate, and the fiber is widely used in clarification treatment of wine, oral liquid, medical liquid and the like. The common filter plates are generally divided into two types, one is made of synthetic fiber and plant fiber materials; the other is a filter plate made of asbestos and pulp. The former has higher manufacturing cost, low filtration precision, small flux and easy blockage; the latter is not high in cost, but the filtering precision and flux are not high, and the filtering effect is not good, especially the asbestos product is a material which is limited by the country.

Traditional depth filtration media consist of: (1) cellulose, (2) filter aids, and (3) wet strength resins, wherein the filter aids may be selected from filter aids based on mineral sources such as silica, such as perlite or sand, or may be selected from activated carbon filter aids derived from natural materials such as wood or coconut shells, and the filter plates made therefrom have high ash content. In the filter aids used globally at present, diatomite accounts for 75-80%, for example, diatomite is mostly adopted for the first stage filtration of beer, and microcrystalline cellulose is rarely used as the filter aid of a deep layer filtration medium for the filtration of cell cultures and blood plasma and derivatives (blood plasma fraction components). Microcrystalline cellulose (MCC) is a free-flowing superfine rod-shaped or powdery porous particle which is obtained by hydrolyzing natural cellulose to a limit degree of polymerization (LOOP) through a dilute acid, is white or nearly white, is odorless and tasteless, has a particle size of 20-80 mu m generally, has a limit degree of polymerization (LODP) of 15-375, and has no fiber and extremely high flowability. The microcrystalline cellulose is insoluble in water, dilute acid, organic solvent and grease, is partially dissolved and swollen in dilute alkali solution, and has higher reaction performance in the processes of carboxymethylation, acetylation and esterification. Because of the special properties of low polymerization degree, large specific surface area and the like, the microcrystalline cellulose is widely applied to the industries of medicine, food, cosmetics and light chemical industry. In the pharmaceutical industry, microcrystalline cellulose is commonly used as an adsorbent, a suspending agent, a diluent, a disintegrant, and is particularly widely used in pharmaceutical formulations, mainly as a diluent and a binder in oral tablets and capsules, and can be used not only for wet granulation but also for dry direct compression. Microcrystalline cellulose is very useful in the preparation of tablets due to its lubricating and disintegrating action, and can also be used as a sustained release agent for pharmaceuticals.

The industrially useful depth filter media have a high ash content and are not completely suitable for filtration of samples from the course of isolation and purification of plasma human coagulation Factor VIII (FVIII, Factor VIII) or von Willebrand Factor (vWF), or complexes of FVIII and vWF, which have poor filterability.

At present, filter plates used by blood product enterprises at home and abroad are mainly produced by companies such as American 3M, PALL, EATON and the like, and the common models thereof include SP series filter plates of the 3M company, P series filter plates of the PALL company, BECO series filter plates of the EATON company and the like. In order to solve the problem of filterability of blood samples, the filter plates still have high ash content, such as 49% of the ash content of BECO PR Steril40 filter plates manufactured by EATON corporation, 50P filter plates specially developed by PALL corporation for blood product separation, high-purity filter materials, high wet strength, less fiber shedding, low Al/Fe ion precipitation amount, easy operation, hydrophobic surface of the filter plates, easy protein recovery and high efficiency, but the ash content of the filter plates still reaches 23%.

The poor filterability of plasma or plasma derivatives containing FVIII and/or vWF is related to the very viscous nature of the sample itself, for example, the fluid before and after S/D virus inactivation is extremely viscous and difficult to filter due to the large amount of impurities such as tween80, fibronectin (FN, fibrinection), and lipoprotein. Wherein Tween80 is highly viscous liquid, while the extensive biological activity of plasma fibronectin enables the viscosity of fibronectin-containing liquid to be higher than that of a common protein solution. Plasma fibronectin, a glycoprotein with molecular weight 440-500kDa, exists in plasma as a dimer, has a relatively high sugar content (about 5%) and is soluble and interacts with a variety of extracellular substances such as heparin, fibrinogen, collagen, sialic acid, etc. For example, the aluminum gel precipitate obtained after the plasma cryoprecipitate dissolved solution is adsorbed by aluminum gel is thick paste and is very difficult to separate. On the other hand, poor filterability of human coagulation Factor VIII (FVIII, Factor VIII) or von Willebrand Factor (vWF, von Willebrand Factor) or a sample isolated and purified from a complex of FVIII and vWF may be associated with an excessively large molecular weight of the active ingredient. Human coagulation factor FVIII is a heterodimer consisting of two domains, A1-A2-B, A3-C1-C2, having 2351 amino acid residues, and usually forming a complex with vWF factor. The molecular weight of vWF monomer in human plasma is about 250kDa, and the molecular weight of multimer is 500-20000 kDa, which is the known protein with the largest molecular weight in plasma at present. Therefore, in the preparation process of human plasma FVIII or vWF or the compound of FVIII and vWF, the blockage of a deep layer filtering medium is serious, and the production is discontinuous and the cost is greatly increased due to frequent replacement of a filter, so that centrifugation or membrane filtration is mostly adopted for separating and purifying the FVIII or vWF or the compound of FVIII and vWF, and the deep layer filtration is less adopted. For example, patent CN10534838A discloses a method for preparing highly pure human blood coagulation factor VIII, wherein the process steps are mainly connected with each other by filtering with a 0.45 μm filter element (membrane filtration method); for another example, patent CN107226859A discloses a method for preparing high purity human blood coagulation factor VIII, which only uses DELP deep filter (CUNO DELP deep filter) of CUNO corporation in usa to filter the sample before S/D virus inactivation, and uses filter pressing method instead of high speed centrifugation to improve the separation effect in other process steps. However, as the product instruction discloses, the CUNO DELP deep filter cartridge and the lipids in the plasma and its fraction components generate affinity adsorption to remove lipid impurities, and cannot be further applied to other purification steps, and the filtration rate is limited, so that industrialization is difficult to realize.

The separation and purification of other plasma proteins with high sample viscosity, such as fibrinogen, human coagulation factor IX, human prothrombin complex, etc., and the filtration of cell cultures with high sample viscosity face the same problems, and therefore it is difficult to select an appropriate filter medium. The main purpose of filtration of high viscosity liquids is to remove particulate impurities therefrom. The particles are mostly colloidal particles and semi-colloidal particles with various forms, heterogeneous impurities or solid particles from raw materials, have the particle strength range of approximately 0-100 mu m, are mostly less than 20 mu m, and are very easy to block the gaps of the filter medium. Therefore, it is difficult to select a filter medium suitable for filtering a high-viscosity liquid, and it is necessary to consider the degree of clogging before and after filtration, viscosity, corrosiveness, time stability (since some polymer solutions have thixotropy), heat resistance, limit of filtration rate, charge and potential difference between medium-particles-filtrate, safety conditions, production scale, and the like (international industrial textile and nonwoven fabric seminar in 2003 shanghai and seminar for popularization and application of the second highest technology in the field of industrial textiles, "filtration of high-viscosity liquid", northeast university, sunxin, and the like).

In the separation and purification of the coagulation factor-type protein derived from recombinant or plasma, it is less considered to add a filter aid to improve the filtration effect. The reason is that the common filter aids in the pharmaceutical field are mainly two types, one is perlite and the other is diatomite, and the two types contain certain amount of Ca²⁺、Al、Fe、K⁺And (4) plasma metal ions. Once excessive Al, Fe, Ca²⁺、K⁺With the introduction of purification systems, the coagulation factors are activated to different degrees, and fibrin clots are inevitably formed, which in turn affects the filterability of the intermediate products and the performance and yield of the final preparation (transfusion therapy and blood preparation).

Therefore, there is a real need in the field of pharmaceutical production to further improve the filterability of samples with a relatively high viscosity, and the corresponding deep filtration method or deep filtration medium is to be further studied.

Disclosure of Invention

The present invention aims to solve, at least to some extent, the technical problems of the prior art. Therefore, the invention provides a deep layer filter medium and a deep layer filter system for filtering a sample with higher viscosity, and the deep layer filter medium or the deep layer filter system can effectively improve the filterability of the sample with higher viscosity, further improve the production efficiency, reduce the production cost and is suitable for large-scale production and application.

It should be noted that the present invention has been completed based on the following findings of the inventors:

conventionally, plasma cryoprecipitate gel adsorption precipitation of a plasma source containing human coagulation factor VIII or vWF or a coagulation factor VIII and vWF compound is very difficult to filter due to the characteristic of thick paste, and solid-liquid separation is generally realized by a high-speed centrifugation method or a filter pressing method by using a tubular freezing continuous flow centrifuge in industry, but the centrifugal separation method has obvious defects: firstly, the working environment is poor, the equipment requirement is high, secondly, the shearing force generated by centrifugation is not beneficial to the stability of protein, and the activity loss of the human coagulation factor VIII or vWF is large. In other practical cases, the supernatant obtained after the plasma cold precipitation gel adsorption is subjected to S/D virus inactivation, so as to avoid the blockage of a chromatographic column in a subsequent chromatographic process, and further filtration is also needed to remove solid particles in the supernatant. However, because the sample after S/D virus inactivation contains highly viscous Tween80 and fibronectin with high sugar content, the filtration is also very difficult, and membrane filtration methods are mostly adopted in industry to filter S/D virus inactivated samples, but the filtration efficiency is low, and the filtration cost is high.

The inventor tries to add a microcrystalline cellulose filter aid layer at the liquid inlet end of the filter plate for deep filtration to filter plasma cryoprecipitate gel adsorption precipitation, and compared with the method of singly using the filter plate for deep filtration to filter, the filtration efficiency is obviously improved. For example, a microcrystalline cellulose filter aid layer is added at the liquid inlet end of a Superdur 50P filter plate to filter plasma cryoprecipitate, and aluminum gel is adsorbed and precipitated, so that the filtering efficiency is greatly improved. Further, the inventors have made similar attempts to the sample having poor filterability such as the sample after the plasma cryoprecipitated aluminum gel adsorption centrifugation, the S/D virus inactivated sample and the like and observed a uniform filtration effect.

Accordingly, in a first aspect of the invention, the invention provides a depth filter medium comprising a filter aid comprising microcrystalline cellulose.

Further, the content of the microcrystalline cellulose is not less than 2% of the total mass of the filter aid in percentage by mass.

According to an embodiment of the present invention, the microcrystalline cellulose has a particle size of not more than 500 μm.

According to an embodiment of the present invention, the microcrystalline cellulose has a particle size of not more than 200 μm.

According to the embodiment of the invention, the particle size of the microcrystalline cellulose is 20-100 μm.

According to embodiments of the present invention, the depth filtration media may be a gradient distribution or a hybrid distribution.

Further, the depth filtration media adapted filter may be a filter plate filter, a filter disc filter, a cartridge filter, or a stack filter.

According to an embodiment of the invention, the depth filtration media further comprises a membrane filtration layer.

According to the embodiment of the invention, the filtration pore size of the membrane filtration layer is preferably 0.1-10 μm.

In particular embodiments, the depth filter media layer is optionally selected from one or more of a Supradur P filter plate, a K300 filter plate, a BECOPAD P270 filter plate.

Further, the microcrystalline cellulose may be selected from one or more of alpha-microcrystalline cellulose, modified microcrystalline cellulose, or a derivative of microcrystalline cellulose.

Still further, the alpha-microcrystalline cellulose is crystalline alpha-cellulose prepared from cellulose-containing plant fiber pulp.

In a specific application scenario, the deep layer filtering medium can be used for filtering liquid to be filtered in a water phase, and is particularly suitable for filtering the liquid in the water phase with high viscosity or complex effective components and poor filtering efficiency. In particular, the method can be used for filtering cell cultures or derivatives of cell cultures, and can also be used for separating and purifying plasma proteins from plasma or plasma fraction components or intermediates for separating and purifying the plasma proteins from the plasma or plasma fraction components.

In some embodiments, the depth filter medium described above may be used to filter plasma or plasma fraction fractions containing human factor VIII and/or vWF or intermediates for the isolation and purification of plasma proteins starting therefrom.

In some embodiments, the depth filtration media described above can be used to filter Cohn process fraction fractions as well as NK process fraction fractions.

In other embodiments, the depth filtration medium may be used for plasma cryoprecipitated gel adsorption precipitation or plasma cryoprecipitated gel adsorption precipitation filtration solution comprising human coagulation factor VIII or vWF or a complex of human coagulation factor VIII and vWF or human fibrinogen or human coagulation factor IX or a human prothrombin complex, and a purified intermediate thereof or a virus-inactivated sample thereof.

In a specific embodiment, when the content of the microcrystalline cellulose is increased in percentage by mass of the total mass of the filter aid, the filtration efficiency of the liquid to be filtered is increased.

In a specific embodiment, when the particle size of the microcrystalline cellulose is too small, the filtration efficiency of the liquid to be filtered is improved to a limited extent.

In a second aspect of the invention, a depth filtration system is provided that substantially achieves the above-described depth filtration media filtration results, comprising a microcrystalline cellulose filter aid layer and a depth filtration media layer with or without microcrystalline cellulose.

Preferably, the depth filter media layer is as described in the first aspect of the invention.

Preferably, the content of the microcrystalline cellulose is not less than 10g/m by mass of microcrystalline cellulose contained in the filter material per unit area²。

In a specific application scenario, the deep filtration system can be used for filtering liquid to be filtered in a water phase, and is particularly suitable for filtering the liquid in the water phase with high viscosity or complex effective components and poor filtering efficiency. The aqueous phase liquid sequentially passes through the microcrystalline cellulose filter aid layer and the deep filtration medium layer and/or the membrane filtration layer containing or not containing microcrystalline cellulose, so that the filtration efficiency of the sample difficult to filter and/or the removal of colloidal particles, semi-colloidal particles, heterogeneous impurities or other particulate matters are further improved.

In particular, the method can be used for filtering cell cultures or derivatives of cell cultures, and can also be used for separating and purifying plasma proteins from plasma or plasma fraction components or intermediates for separating and purifying the plasma proteins from the plasma or plasma fraction components.

In some embodiments, the depth filtration system described above may be used to filter plasma or plasma fraction fractions comprising human factor VIII and/or vWF or intermediates for the isolation and purification of plasma proteins starting therefrom.

In other embodiments, the depth filtration system can be used for plasma cryoprecipitated gel adsorption precipitation or plasma cryoprecipitated gel adsorption precipitation filtration solution containing human coagulation factor VIII or vWF or a complex of human coagulation factor VIII and vWF or human fibrinogen or human coagulation factor IX or a human prothrombin complex, and a purified intermediate or a virus-inactivated sample thereof.

In a further aspect of the invention, the invention provides the use of a depth filter medium or depth filtration system as described above for filtering a liquid to be filtered in a water phase.

According to an embodiment of the invention, the liquid to be filtered is derived from a cell culture or a derivative of a cell culture.

According to the embodiment of the invention, the liquid to be filtered is derived from plasma or plasma fraction components, or an intermediate product for separating and purifying plasma proteins by taking the plasma or plasma fraction components as raw materials.

According to an embodiment of the invention, the cell culture or the derivative of a cell culture comprises recombinant coagulation factor VIII and/or vWF.

According to an embodiment of the present invention, the plasma or plasma fraction, or an intermediate product of the plasma or plasma fraction as a raw material for separating and purifying the plasma protein, contains human coagulation factor VIII and/or vWF.

According to an embodiment of the invention, the plasma fraction is derived from a Cohn' S low temperature ethanol reaction system or a NK low temperature ethanol modified reaction system.

In some embodiments, the derivative of the cell culture may be a purified intermediate of a cell culture comprising recombinant factor VIII or recombinant vWF or a complex of recombinant factor VIII and recombinant vWF or a sample after virus inactivation or removal.

In some embodiments, the intermediate product for separating and purifying plasma protein from plasma or plasma fraction components can be a protein purification intermediate product or a virus inactivated or removed intermediate product.

In some embodiments, the plasma fraction may be a plasma cryoprecipitated gel adsorption precipitation or plasma cryoprecipitated gel adsorption precipitation filtration solution comprising human factor VIII or vWF or a complex of human factor VIII and vWF or human fibrinogen or human factor IX or a human prothrombin complex, and purified intermediates thereof or a virus-inactivated sample thereof.

In some embodiments, the plasma cryoprecipitating gel adsorption precipitation is a plasma cryoprecipitating aluminum gel adsorption precipitation.

In some embodiments, the viral inactivation is S/D viral inactivation.

Compared with the prior art, the invention has the beneficial effects that:

1. the deep layer filtering medium or the deep layer filtering system disclosed by the invention can be used for filtering samples with larger viscosity, and the filtering efficiency is high. As disclosed in the examples of the present invention, when the depth filtration media or the depth filtration system disclosed in the present invention is used to filter a biological sample containing recombinant or human coagulation factor VIII or vWF or a complex of coagulation factor VIII and vWF, the filtration efficiency is significantly improved and the filtration amount is generally increased by about 50% or more, as compared to the existing depth filtration media.

2. The deep layer filtering medium or the deep layer filtering system disclosed by the invention filters a sample with higher viscosity, and the relative activity recovery rate is high. Compared with the common filter plate and diatomite deep filtration method, the filter plate and microcrystalline cellulose filtration method disclosed by the embodiment of the invention not only obviously improves the filtration capacity, but also greatly improves the recovery rate of active ingredients such as FVIII (FVIII) and vWF (von Willebrand factor).

3. The deep filtration method disclosed by the invention can replace or partially replace the traditional high-speed centrifugation method or the filter pressing method to realize the solid-liquid separation of the sample, reduce the requirements on production facilities and equipment, and has high filtration efficiency, so the filtration cost is correspondingly reduced.

4. The deep filtration medium disclosed by the invention can be combined with a membrane filtration medium for application, on one hand, the comprehensive application of various filtration principles is favorable for further improving the filtration effect of complex samples such as plasma cold precipitation aluminum gel adsorption precipitation, S/D virus inactivation solution and the like, effectively removing colloidal particles, semi-colloidal particles, heterogeneous impurities or other particulate matters, obtaining a clear solution meeting the requirements, being beneficial to mutual connection among process steps, avoiding the problems of chromatographic column blockage and the like in the subsequent chromatographic process, and improving the feasibility of automatic operation in the large-scale production process. On the other hand, the ingenious combination of a plurality of filtering methods also ensures the inactivation effect of the S/D virus and improves the reliability of the product quality. According to the embodiment of the invention, the human coagulation factor VIII finished product prepared by using the deep layer filtering medium or the method disclosed by the invention is qualified according to the detection result of the main quality index of the finished product verification item of Chinese pharmacopoeia, and the index of visible foreign matters meets the requirements of the pharmacopoeia.

The filter aid disclosed by the invention contains a deep layer filter medium of microcrystalline cellulose, reduces the application of the filter aid from traditional mineral sources such as silicon dioxide and the like, and can reduce the ash content of the deep layer filter medium to a certain extent.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

In one aspect, the present disclosure provides a depth filter media comprising a filter aid, wherein the filter aid comprises microcrystalline cellulose.

Traditional depth filtration media consist of: (1) a fiber-containing material, (2) a filter aid (e.g., siliceous sorbent particles such as diatomaceous earth), and (3) a binder (e.g., a binder resin such as a wet strength resin). According to different requirements of different samples to be filtered on filtering performance, a person skilled in the art can adjust and optimize the content and the proportion of each component of the filtering medium so as to obtain the filtering medium with improved specific performance. The term "wet strength resins" as used herein is known in the art and are water-soluble synthetic polymers having anionic and/or cationic groups for imparting strength to the material when wet, such as polymers based on urea or melamine-formaldehyde, polyaminopolyamide-epichlorohydrin (PAE) polymers and Glyoxalated Polyacrylamides (GPAM).

Adsorbent deep filtration media use a variety of materials to filter fluids, including fiber-containing materials and various adsorbent particulates, and may also include other materials such as glass fibers or synthetic fibers (e.g., polypropylene fibers or polyethylene fibers). Known adsorption-type depth filtration media typically include 30% to 50% fibers. In some cases, the percentage of fibers by weight of the total filter media can be as low as 10%. By maximizing the number of fine adsorbent particles in the depth filter media layer, the performance of the filter can be enhanced. Commonly used fine adsorbent particles include diatomaceous earth, perlite, talc, silica gel, activated carbon, asbestos, molecular sieves, clays, and the like. In industrial applications, siliceous materials such as diatomaceous earth or perlite are most widely used.

According to an embodiment of the present invention, the depth filter medium of the present invention, the filter aid, comprises microcrystalline cellulose, in mass percent.

Further, the inventors have studied and found that the content and particle size of microcrystalline cellulose are different for different liquids to be filtered in order to obtain a desired filterability.

According to an embodiment of the invention, the microcrystalline cellulose content is not less than 2% of the total mass of the filter aid. According to the properties of viscosity, protein content, molecular weight and the like of the liquid to be filtered, the content ratio of the microcrystalline cellulose in the filter aid can be increased so as to obtain expected filterability.

Further, according to embodiments of the present invention, in some embodiments, the microcrystalline cellulose may be present in an amount of 100g/m²In some embodiments, the microcrystalline cellulose may be present in an amount of 1000g/m²In other embodiments, the microcrystalline cellulose may be present in an amount of 2000g/m²。

According to an embodiment of the present invention, the microcrystalline cellulose has a particle size of not more than 500 μm. According to the properties of viscosity, protein content, molecular weight and the like of the liquid to be filtered, microcrystalline cellulose with proper size and particle size is selected to obtain expected filterability.

Further, in some embodiments according to embodiments of the present invention, the microcrystalline cellulose has a particle size of no greater than 200 μm;

further, in some embodiments according to examples of the present invention, the microcrystalline cellulose has a particle size of between 20 μm and 100 μm, inclusive.

Further, the depth filtration media of the present invention may be a gradient distribution or a hybrid distribution. In particular, the depth filtration media-adapted filter of the present invention, including without limitation, may be a filter plate filter, a filter disc filter, a cartridge filter, or a stack filter.

It will be apparent to those skilled in the art, in light of the present examples, that the fiber-containing material, the filter aid comprising microcrystalline cellulose, and the wet strength resin can be made into a process-scale depth filtration medium useful for filtering samples of greater viscosity. Further, alternative processing methods include, but are not limited to: air-laying, melt-pressing, mechanical compression, wet-laying, and the like. Optionally, the compression may be performed by a roller mill to adjust the thickness of the filter media, according to actual development and production needs.

According to the embodiment of the invention, the deep layer filter medium can be further processed or assembled into different types of filters such as filter plates, filter discs, filter elements or filter stacks and the like which are suitable for development or production activities by those skilled in the art according to actual needs. These filter forms are common in the art and may be implemented by those skilled in the art.

According to an embodiment of the invention, the depth filter medium of the invention further comprises a membrane filtration layer.

Furthermore, the filtering pore size of the membrane filtering layer is preferably 0.1-10 μm.

In some embodiments, the membrane filtration layer is disposed at the exit end of the depth filtration media.

In some embodiments, the membrane filtration layer is disposed at the feed end of the depth filtration media.

According to an embodiment of the present invention, the microcrystalline cellulose according to the present invention may be selected from one or more of alpha-microcrystalline cellulose, modified microcrystalline cellulose or a derivative of microcrystalline cellulose.

Further, in some embodiments, the alpha-microcrystalline cellulose is crystalline alpha-cellulose prepared from cellulose-containing plant fiber pulp.

Further, in some embodiments, the microcrystalline cellulose may be selected from silicified microcrystalline cellulose having higher inertness or nano-sized microcrystalline cellulose having better biocompatibility and excellent mechanical properties, which further results in the improved depth filtration media of the present invention.

Further, in some embodiments, the microcrystalline cellulose may be selected from crystalline alpha-cellulose prepared from cellulose-containing plant fiber pulp, which may be used as a pharmaceutical excipient, to further provide the depth filtration media of the present invention.

In another aspect, the present invention provides a depth filtration system capable of substantially achieving the filtration results of the depth filtration media of the present invention, according to an embodiment of the present invention, comprising a layer of microcrystalline cellulose filter aid and a layer of depth filtration media with or without microcrystalline cellulose filter aid.

In some embodiments, the depth filter media layer may be a depth filter media of the present invention.

In some embodiments, the microcrystalline cellulose is present in an amount of not less than 10g/m based on the mass of microcrystalline cellulose per unit area of the filter². For filtration of certain liquids, such as samples containing VIII and/or vWF prior to chromatography, the content of microcrystalline cellulose is as low as 10g/m²A significant increase in filtration efficiency was observed.

In some embodiments, the depth filter media layer is optional in accordance with embodiments of the present invention, including, without limitation, one or more of a Supradur P filter plate, a K300 filter plate, a BECOPAD P270 filter plate.

Further, according to an embodiment of the present invention, the depth filtration system of the present invention further comprises a membrane filtration layer.

In some embodiments, the membrane filtration layer is disposed at the exit end of the depth filtration system.

In some embodiments, the membrane filtration layer is disposed at the feed end of the depth filtration system.

The inventor finds out in practice that the deep filtration system is particularly suitable for filtering viscous plasm-like samples of plasma cryoprecipitate gel adsorption precipitates, and the filtering amount is effectively improved. According to embodiments of the present invention, it is envisioned that the above described depth filtration system is advantageous for the filtration of other high viscosity samples, in particular biological samples comprising recombinant or human factor VIII or vWF or complexes of factor VIII and vWF.

The inventors have also practiced the use of depth filtration media as disclosed herein in combination with membrane filtration media in an attempt to improve filtration. According to the embodiment of the invention, the deep layer filtering medium or the deep layer filtering system disclosed by the invention is combined with the membrane filtering medium for application, so that the filtering effect of complex samples such as plasma cold precipitation aluminum gel adsorption precipitation, S/D virus inactivation solution and the like can be further improved, colloidal particles, semi-colloidal particles, heterogeneous impurities or other particulate matters can be effectively removed, and a clear solution meeting the requirement can be obtained.

The exploration discovers that the method is beneficial to mutual connection among process steps from the viewpoint of optimizing the production process of the medicine, avoids the problems of blockage of a chromatographic column and the like in the subsequent chromatographic process, and improves the feasibility of automatic operation in the large-scale production process; from the aspect of improving the product quality level, the S/D virus inactivation effect is ensured, and the reliability of the product quality is improved. According to the embodiment of the invention, the human coagulation factor VIII finished product prepared by using the deep layer filtering medium or the method disclosed by the invention has high qualification rate according to the full inspection result of the finished product inspection item of Chinese pharmacopoeia, and the visible foreign matter index meets the requirements of the pharmacopoeia and has high qualification rate.

In a further aspect, the present invention provides the use of a depth filter medium or depth filtration system as described above for filtering a liquid to be filtered in a water phase, according to embodiments of the present invention.

According to an embodiment of the invention, the liquid to be filtered may be derived from a cell culture or a derivative of a cell culture.

According to an embodiment of the present invention, the liquid to be filtered may be derived from plasma or plasma fraction components, or an intermediate product for separating and purifying plasma proteins from the plasma or plasma fraction components.

According to embodiments of the present invention, in some embodiments, the derivative of the cell culture may be a purified intermediate of a cell culture comprising recombinant factor VIII or vWF or a complex of factor VIII and vWF or a sample after virus inactivation or removal.

According to embodiments of the present invention, in some embodiments, the plasma or plasma fraction component can be used as a raw material for separating and purifying plasma protein, such as a protein purification intermediate or an intermediate after virus inactivation or removal.

In some embodiments, the plasma fraction may be a plasma Cohn process fraction, according to embodiments of the present invention.

According to embodiments of the invention, in some embodiments, the plasma fraction may be a plasma NK fraction.

In some embodiments, the plasma fraction may be a plasma cryoprecipitated gel adsorption precipitation or plasma cryoprecipitated gel adsorption precipitation filtration solution comprising human coagulation factor VIII or vWF or a complex of coagulation factor VIII and vWF or human fibrinogen or human coagulation factor IX or human prothrombin complex, and purified intermediates thereof or a virus inactivated sample thereof according to embodiments of the present invention.

According to embodiments of the present invention, in some embodiments, the plasma cryoprecipitating gel adsorption precipitation is plasma cryoprecipitating aluminum gel adsorption precipitation.

According to embodiments of the present invention, in some embodiments, the viral inactivation is S/D viral inactivation.

The term "fiber-containing material" as used herein refers to a fiber-containing material that is useful in forming a depth filter material and that typically swells when contacted with an aqueous medium. The depth filter material made from the fiber-containing material may be compressed to a degree that does not compromise the integrity of the filter layer when the filter material is compressed below its original thickness. The maximum compression depends on other components of the filter material, such as the wet strength resin, the amount of filter aid content, and the degree of binding to the fibers. Known depth of adsorption filter media typically include 30% to 50% fibers. In some cases, the percentage of fibers by weight of the total filter media can be as low as 10%.

The microcrystalline cellulose (MCC) is free-flowing superfine short rod-shaped or powdery porous particles which are obtained by hydrolyzing natural cellulose to a limit polymerization degree (LOOP) through a dilute acid, is white or nearly white, is odorless and tasteless, has a particle size of 20-80 mu m generally, has a limit polymerization degree (LODP) of 15-375, and has no fiber and extremely high flowability.

The "microcrystalline cellulose" described herein may, in some embodiments, extend to alpha-microcrystalline cellulose, modified microcrystalline cellulose, or derivatives of microcrystalline cellulose. Such as silicified microcrystalline cellulose with stronger inertia, nano-scale microcrystalline cellulose with better biocompatibility and excellent mechanical property, etc.

The plasma cryoprecipitate gel adsorption precipitation is generally derived from plasma cryoprecipitate of healthy human plasma, and is prepared by dissolving the plasma cryoprecipitate in neutral pH buffer solution or water for injection, and adding Al (OH)₃The gel or A50 gel such as DEAE Sephadex A-50 gel is adsorbed to give a viscous paste. Blood plasma cryoprecipitationThe precipitated gel adsorption precipitate can be further used for preparing FVIII products or vWF products.

The term "S/D virus inactivation method" as used herein refers to a method of disrupting the lipid membrane of enveloped viruses with an organic solvent/detergent mixture (S/D). Once the lipid membrane is disrupted, the virus is no longer able to bind to the infected cells. This method is effective in inactivating lipid-enveloped viruses, but not non-enveloped viruses. Usually, the working solution concentration of S/D is 0.3% of TNBP and 0.78% of Tween-801.0%.

The term "Cohn' S low-temperature ethanol reaction system" used in the present invention refers to a method for fractionating plasma proteins by the Cohn low-temperature ethanol method, which is a method for industrially producing blood products. The classic Cohn low-temperature ethanol method includes a Cohn 6 method, a Cohn 9 method and a Cohn10 method. The corresponding fractionated fractions containing specific kinds and amounts of plasma protein fractions, i.e., "plasma fraction fractions" are FI, FII, FIII, FIV, FV, etc.

The term "NK low-temperature ethanol modified reaction system" used in the present invention is also a method for fractionating plasma proteins in low-temperature ethanol, sometimes referred to as "NK variation method", "NK method", and is mainly different from Cohn original method in that: 1. precipitation of precipitate a, which corresponds to Cohn FII + III, lowers the pH and the concentration of ethanol; 2. from a number corresponding to Cohn S_2,3The method can directly precipitate albumin, and omit FIV separation₁A step (2); 3. referring to the Cohn10 method, instead of dissolving the precipitate and then precipitating, the conglycinin is extracted from the precipitate a. Compared with the Cohn original method, the NK variable method reduces the ethanol consumption by 40 percent, shortens the working volume by 22 percent, shortens the process period by 2 days, improves the recovery rate (the albumin can reach 90 percent, the propylene spheres can reach 89 percent), and has the purity equivalent to that of the original method.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 plasma cryoprecipitating aluminum gel adsorption precipitation, S/D Virus inactivation of FVIII and/or vWF Complex solutions samples

Plasma cryoprecipitated aluminum gel adsorptive precipitation from plasma of healthy persons can be obtained by the following method, referring to patent US 5714590: dissolving the plasma cryoprecipitate with 2 times volume of 2U/mL heparin sodium injection water, adjusting pH of the cryoprecipitate solution to 7.1 with 0.1M acetic acid, and stirring for 30 min. 108g 2% (wt%) Al (OH) are then cryoprecipitated per kg of plasma₃Amount of gel to be added Al (OH)₃Adding the gel into the plasma cryoprecipitate solution, stirring thoroughly, and adjusting pH to 6.5-6.6 with 0.1M acetic acid to obtain suspension, i.e. plasma cryoprecipitate aluminum gel adsorption precipitate.

And further centrifuging plasma cryoprecipitated aluminum gel adsorption precipitation at a high speed, collecting supernatant, adding 80-1.0% (wt%) of Tween and 0.3% (wt%) of TNBP (tributyl phosphate), uniformly stirring, heating to 24-26 ℃, and preserving heat for 6 hours to obtain an S/D virus inactivated sample of the FVIII/vWF compound solution.

Alternatively, FVIII or vWF or a solution of FVIII in complex with vWF can be biosynthesized in transgenic cells or animals by recombinant techniques.

Examples 2-8 were filtered, if not specifically stated, first through microcrystalline cellulose or diatomaceous earth and then through a filter plate.

Example 2 deep filtration plasma Cold precipitation aluminium gel adsorption precipitation

The dosage of 10kg of plasma cryoprecipitate is reduced as in example 1, and the plasma cryoprecipitate aluminum gel adsorption suspension is taken for filtration. Cutting equal-area filter material grouping filtration test: taking commercial filter material 0.1m²The filter aid is 1, microcrystalline cellulose (with the grain diameter of 180-200 mu m) and 2, diatomite, each of which is 200g, and the plasma cold precipitation aluminum gel adsorption precipitation is deeply filtered at the flow rate of about 0.1-0.5 MPa under the condition of 16-26 ℃ until the pressure drop is obviously increased, namely, the filtration cannot be continued.

Commercial filters and specific filtration schemes are as follows:

a: supradur50P filter plate

b: k300 filter plate

c: BECOPAD P270 filter plate

A1: supradur50P filter plate + microcrystalline cellulose

A2: supradur50P filter plate and diatomite

B1: k300 filter plate and microcrystalline cellulose

B2: k300 filter plate and diatomite

C1: BECOPAD P270 filter plate + microcrystalline cellulose

C2: BECOPAD P270 filter plate + diatomite

The several filtration protocols described above filter the plasma cryoprecipitated aluminum gel adsorption precipitate until the pressure drop significantly increases the filtration capacity at which filtration is not possible, and the relative average activity recovery of FVIII/vWF is shown in the table below.

Filtration efficiency is expressed as a percentage (%), filtration efficiency-100% of filtration volume when pressure drop significantly increases without filtration/total volume to be filtered, the same applies below.

The above experimental results show that, in comparison with deep filtration with a filter plate, in a specific case, the addition of a filter aid layer can improve the filtration capacity and the relative average activity recovery rate of FVIII and vWF. The filtration capacity of the filter plate and the microcrystalline cellulose is generally improved by more than 50 percent, and particularly, when a Supradur50P filter plate or a K300 filter plate and the microcrystalline cellulose filtration scheme is selected, the filtration capacity exceeds 85 percent.

The inventor expects that compared with the deep filtration of a filter plate and diatomite, the deep filtration of microcrystalline cellulose is increased, the filtration capacity is obviously improved, and the relative average activity recovery rate of FVIII and vWF is greatly improved.

To further remove particulate matter and obtain a clearer liquid to ensure the S/D virus inactivation effect, the inventors further filtered the filtrate obtained after the deep filtration of the filtration protocol A1 of example 2 through (A1-1)0.45 μm, (A1-2)0.8 μm, (A1-3)1 μm filter element: and carrying out deep filtration on the A1 filtrate at the flow rate of about 0.05-0.3 MPa at the temperature of 16-26 ℃ until the pressure drop is increased, so that the filtration cannot be continued.

The filtration capacity and FVIII/vWF relative average activity recovery of filter plates + microcrystalline cellulose depth filtration series connected with filter elements with different apertures are shown in the following table:

the experiment results show that the deep filtration scheme of the embodiment is connected with filter elements of 0.45 mu m, 0.8 mu m and 1 mu m in series for filtration, the filtration efficiency is not obviously reduced, and the relative average activity recovery rate of FVIII and vWF is not obviously reduced.

EXAMPLE 3 deep filtration of plasma cryoprecipitated aluminum gel precipitated supernatant after high speed centrifugation

10kg of the feed was converted to plasma cryoprecipitate in accordance with example 1, and the plasma cryoprecipitate aluminum gel adsorption suspension was centrifuged to give a supernatant, which was then filtered. Cutting equal-area filter material grouping filtration test: taking commercial filter material 0.1m²The filter aid is 1, microcrystalline cellulose (with the grain diameter of 100-150 mu m) and 2, diatomite, each of which is 100g, and the plasma cold precipitation aluminum gel adsorption precipitation is deeply filtered under the pressure of about 0.1-0.5 MPa at the temperature of 16-26 ℃ until the pressure drop is obviously increased, namely, the filtration cannot be continued.

The filtration protocol was the same as in example 2.

The filtration of plasma cryoprecipitated aluminum gel adsorption precipitates by the several filtration protocols described above until the pressure drop significantly increased the filtration capacity without filtration, and the relative average activity recovery of FVIII/vWF are shown in the following table:

the above experiment results show that when the weight area ratio of the microcrystalline cellulose to the filter material is 1000g/m²The filtration efficiency and the relative average activity recovery of FVIII and vWF of the deep filtration protocol of the present invention are further improved.

In order to further remove particulate matters and obtain a clearer liquid to ensure the S/D virus inactivation effect, the inventor further filters the filtrate obtained after the deep filtration of the filtration scheme A1 in example 3 by (A1-1)0.45 μm, (A1-2)0.8 μm and (A1-3)1 μm filter membranes, namely 'one plate and one membrane', and deeply filters the filtrate A1 at the flow rate of about 0.05-0.3 MPa at the temperature of 16-26 ℃ until the pressure drop is increased, namely the filtration cannot be continued.

The filtration capacity and the relative average activity recovery rate of FVIII/vWF of filter plates and microcrystalline cellulose depth filtration series connected with filter membranes with different apertures are shown in the following table:

the above experiment results show that the depth filtration scheme of this example is serially connected with 0.45 μm, 0.8 μm, 1 μm filtration membranes for filtration, the filtration efficiency is high, and the relative activity recovery rates of FVIII and vWF are not reduced compared with example 2.

EXAMPLE 4 deep filtration of S/D Virus-inactivated samples of FVIII/vWF Complex solutions

Through 0.1m²Supradur50P filter plate (a), and 0.1m²Supradur50P filter plates and 0.1kg of microcrystalline cellulose (a1), and 0.1m²The Suradur 50P filter plate and 0.2kg of microcrystalline cellulose (a2) are used for deep filtration of a sample subjected to S/D virus inactivation of the FVIII/vWF complex solution obtained in example 1 at a flow rate of about 0.05 to 0.3MPa at 16 to 26 ℃ until the pressure drop is increased, and filtration cannot be continued.

Microcrystalline cellulose particle size: 80-150 μm.

The filtration efficiency, FVIII/vWF relative average activity recovery for the 3 depth filtration modes described above are given in the following table:

the above experimental results show that the deep filtration protocol of the present invention is also suitable for filtration of viscous liquids with high detergent content.

After the S/D virus inactivation step, FVIII and/or vWF preparation is typically followed by an ion exchange chromatography purification step. If the chromatographic sample is not fully filtered before being loaded, on one hand, the phenomena of chromatographic medium blockage and chromatographic column tube explosion are easy to occur, and on the other hand, the service life of the chromatographic medium is influenced by frequent regeneration treatment of the chromatographic medium. Therefore, based on the above studies, the inventors further examined the filtration efficiency and FVIII/vWF relative average activity recovery of the filtrate obtained by the deep filtration of the filtration scheme a1 of this example through (a1-1)0.45 μm, (a1-2)0.65 μm, and (a1-3)0.8 μm membrane. The results of the experiment are shown in the following table:

the experiment results show that the deep filtration scheme of the embodiment is connected with the filter membranes of 0.45 mu m, 0.65 mu m and 0.8 mu m in series for filtration, the filtration capacity is large, and the relative average activity recovery rate of FVIII and vWF is not obviously reduced.

EXAMPLE 5 preparation of FVIII from plasma by deep filtration according to the invention

And (3) cold precipitation and dissolution: dissolving the plasma cryoprecipitate with 2 times volume of 2U/mL heparin sodium injection water, adjusting pH of the cryoprecipitate solution to 7.1 with 0.1M acetic acid, and stirring for 30 min.

Adsorption on aluminum gel and depth filtration: 108g 2% (wt%) Al (OH) per kg of plasma cryoprecipitate₃Amount of gel to be added Al (OH)₃Adding the gel into the plasma cryoprecipitate dissolved solution, fully stirring, and then adjusting the pH to 6.0-6.6 by using 0.1M acetic acid to obtain plasma cryoprecipitate aluminum gel adsorption precipitate. Then filtering the mixture by a Superdur 50P filter plate and a microcrystalline cellulose series 0.8 mu m filter membrane at 16-26 ℃, and collecting clear filtrate.

S/D virus inactivation and deep filtration: adding Tween80 to 1.0 wt% and TNBP (tributyl phosphate) to 0.3 wt%, stirring, heating to 24-26 deg.C, and holding for 6 hr. Then filtering the mixture by a Superdur 50P filter plate and a microcrystalline cellulose series 0.45 mu m filter membrane at 16-26 ℃, and collecting clear filtrate.

Anion exchange chromatography: the clear filtrate was subjected to anion exchange chromatography with Fractogel TMAE 650M column gel height no higher than the column diameter. The column packed with the anionic gel was previously well equilibrated with an equilibration buffer (120mM sodium chloride, 10mM NaCitrato.5H)₂O, 120mM glycine, 1mM CaCl 2H₂O, pH 6.5-7.5) and then loaded. Sample loadingAfter completion, the column was washed with equilibration buffer and then with washing buffer (120-₂O, 120mM glycine, 1mM CaCl 2H₂O, pH 6.9-7.0), eluting with elution buffer (400mM sodium chloride, 20mM naci 5H2O, 200mM glycine, 1.0mM ca 2H2O, pH 6.8-7.4), and collecting the purified FVIII solution.

And (3) further filtering the anion chromatography purified eluent by a Suradur 50P filter plate and microcrystalline cellulose series-connected 0.45 mu m filter membrane at the temperature of 16-26 ℃, and performing ultrafiltration and aseptic filtration.

The ultrafiltration dialysate contains: 20mM sodium citrate, 10g/L albumin, 35g/L arginine hydrochloride; the pH was 7.2.

Sterilizing, filtering, preparing according to the specification of the finished product, and adding 200mM glycine as a stabilizing agent. Subpackaging, freeze-drying, sealing and taking out of the cabinet, and performing dry heat inactivation (100 ℃ is multiplied by 30min or 80 ℃ is multiplied by 72h) on the freeze-dried product to obtain the finished product FVIII.

EXAMPLE 6 preparation of FVIII from plasma by deep filtration according to the invention

And (3) cold precipitation and dissolution: dissolving plasma cryoprecipitate with 3 times volume of 2U/mL heparin sodium injection water, adjusting pH of the cryoprecipitate solution to 7.1 with 0.1M acetic acid, and stirring for 30 min.

Adsorption on aluminum gel and depth filtration: 108g 2% (wt%) Al (OH) per kg of plasma cryoprecipitate₃Amount of gel to be added Al (OH)₃Adding the gel into the plasma cryoprecipitate dissolved solution, stirring thoroughly, adjusting pH to 6.0-6.6 with 0.1M acetic acid to obtain plasma cryoprecipitate aluminum gel adsorption precipitate, centrifuging at high speed, and collecting supernatant. And filtering the centrifugal supernatant through a Supradur50P filter plate and a microcrystalline cellulose series 0.65-micron filter membrane at 16-26 ℃, and collecting clear filtrate.

S/D virus inactivation and deep filtration: adding Tween80 to 1.0 wt% and TNBP (tributyl phosphate) to 0.3 wt%, stirring, heating to 24-26 deg.C, and holding for 6 hr. Then filtering the mixture by a Superdur 50P filter plate and a microcrystalline cellulose series 0.8 mu m filter membrane at 16-26 ℃, and collecting clear filtrate.

Anion exchange chromatography: the same as in example 5.

Purifying the eluent by anion chromatography, and further performing ultrafiltration, sterilization and filtration.

The ultrafiltration dialysate contains: 10mM sodium citrate, 10g/L glycine; the pH was 7.2.

Preparing the FVIII product according to the finished product before subpackaging, sterilizing, filtering, subpackaging, freeze-drying, sealing and taking out of a cabinet, and performing dry heat inactivation (100 ℃ is multiplied by 30min or 80 ℃ is multiplied by 72h) on the freeze-dried product to obtain the FVIII finished product.

EXAMPLE 7 preparation of FVIII from plasma by the deep filtration method of the invention

Adsorption on aluminum gel and depth filtration: 108g 2% (wt%) Al (OH) per kg of plasma cryoprecipitate₃Adding Al (OH) to the gel₃Adding the gel into the plasma cryoprecipitate dissolved solution, stirring thoroughly, adjusting pH to 6.0-6.6 with 0.1M acetic acid to obtain plasma cryoprecipitate aluminum gel adsorption precipitate, centrifuging at high speed, and collecting supernatant. And centrifuging the supernatant, filtering the supernatant by a P270 filter plate and a microcrystalline cellulose series 1.0 mu m filter membrane at 16-26 ℃, and collecting clear filtrate.

Anion exchange chromatography: the clear filtrate was applied to a CaptoQ column, which was previously fully equilibrated with equilibration buffer (buffer composition 0.02M Tris-HCl, 0.1M NaCl, 1.5% (wt%) glycine, 0.005M CaCl)₂pH6.80-7.40); after the end of the column, the column was washed with equilibration buffer and then with washing buffer (0.02M Tris-HCl, 0.25M NaCl, 1.5% (wt%) glycine, 0.005M CaCl₂pH6.80-7.40), and then eluted with an elution buffer (0.02M Tris-HCl, 2.0M NaCl, 1.5% (wt%) glycine, 0.00l CaCl₂Ph6.50-6.60) and collecting the purified FVIII solution.

The ultrafiltration dialysate contains: 50mM sodium chloride, 10mM sodium citrate, 1mM calcium chloride, 1.5% (wt%) glycine; the pH was 7.0.

Sterilizing, filtering, and making into final product. Subpackaging, lyophilizing, sealing, taking out, and performing dry heat inactivation (100 deg.C for 30min or 80 deg.C for 72h) to obtain FVIII product.

EXAMPLE 8 preparation of FVIII from plasma by deep filtration according to the invention

And (3) cold precipitation and dissolution: dissolving plasma cryoprecipitate with 5 times volume of 2U/mL heparin sodium injection water, adjusting pH of the cryoprecipitate solution to 7.1 with 0.1M hydrochloric acid, and stirring for 30 min.

Adsorption on aluminum gel and depth filtration: 108g 2% (wt%) Al (OH) per kg of plasma cryoprecipitate₃Amount of gel to be added Al (OH)₃Adding the gel into the plasma cryoprecipitate dissolved solution, stirring thoroughly, adjusting pH to 6.0-6.6 with 0.1M acetic acid to obtain plasma cryoprecipitate aluminum gel adsorption precipitate, centrifuging at high speed, and collecting supernatant. And centrifuging the supernatant, filtering the supernatant by a K300 filter plate and a microcrystalline cellulose series 1.0 mu m filter membrane at 16-26 ℃, and collecting clear filtrate.

S/D virus inactivation and deep filtration: adding Tween80 to 1.0 wt% and TNBP (tributyl phosphate) to 0.3 wt%, stirring, heating to 24-26 deg.C, and holding for 6 hr. Then filtering the mixture by a K700 filter plate and a microcrystalline cellulose series 0.8 mu m filter membrane at 16-26 ℃, and collecting clear filtrate.

Anion exchange chromatography: and (3) feeding the clear filtrate into a Q-Sepharose-FF ion exchange column, washing the column by using a washing solution until the column becomes a base line after the sample is loaded, eluting by using an eluent, and collecting a purified FVIII solution.

The preparation method of the equilibrium liquid comprises the following steps: adding 2.5g of tris (hydroxymethyl) aminomethane, 1.0g of calcium chloride and 17.5g of sodium chloride, adding 1L of water for injection, and adjusting the pH value to 6.5-7.5; the preparation method of the cleaning solution comprises the following steps: adding 2.5g of tris (hydroxymethyl) aminomethane, 1.0g of calcium chloride and 19.5g of sodium chloride, adding 1L of water for injection, and adjusting the pH value to 6.5-7.5; the preparation method of the eluent comprises the following steps: 2.5g of tris (hydroxymethyl) aminomethane, 1.0g of calcium chloride and 58.5g of sodium chloride, and adding water for injection to 1L, and adjusting the pH value to 6.5-7.

The ultrafiltration dialysate comprises: 120mM sodium chloride, 20mM sodium citrate, 1mM calcium chloride, 10g/L histidine; the pH was 7.2.

Sterilizing, filtering, preparing according to the specification of the finished product, and adding 100mM glycine as a stabilizing agent. Subpackaging, lyophilizing, sealing, taking out, and performing dry heat inactivation (100 deg.C for 30min or 80 deg.C for 72h) to obtain FVIII product.

According to the regulation of Chinese pharmacopoeia, the main quality indexes of the human blood coagulation factor VIII finished products prepared in the examples 5 to 8 are detected, and the detection of visible foreign matters is particularly concerned. The main quality index detection results are as follows:

the filtration schemes used in examples 2-8 can achieve substantially similar technical results with depth filtration media having filter aids comprising microcrystalline cellulose, such as the depth filtration media described in example 9 or example 10.

Example 9

The invention relates to a deep layer filtering medium which comprises the following raw materials in parts by mass:

fiber: 0.5 portion

α -microcrystalline cellulose: 0.1 part, particle size 60 μm:

silicon-containing filter aid: 0.2 part

Polyaminopolyamide-epichlorohydrin polymer: 0.05 part

Filter discs with a thickness of 0.1cm to 1.0cm were made by wet-laid methods known in the art and further assembled into filter plate depth filters.

Further, it can be referred to the method disclosed in CN102489075B to make filter sheets with gradient distribution, and further assemble into a filter plate depth filter.

Example 10

The deep layer filtering medium comprises the following raw materials in parts by mass:

fiber: 0.5 portion

α -microcrystalline cellulose: 0.006 part, particle size 500 μm:

silicon-containing filter aid: 0.294 portion

Polyaminopolyamide-epichlorohydrin polymer: 0.05 part of

The depth filters prepared in examples 9-10 were used to filter poor filterable plasma or plasma fraction fractions rich in thromboxane VIII or vWF or a complex thereof, and were fast in filtration flow rate, less prone to clogging, and superior to the filtration effect of commercially available depth filter plates without microcrystalline cellulose.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A depth filter medium comprising a filter aid, wherein the filter aid comprises microcrystalline cellulose.

2. The depth filter medium of claim 1, wherein the microcrystalline cellulose is present in an amount of no less than 2% by mass of the total mass of the filter aid.

3. The depth filtration media of claim 1, wherein the microcrystalline cellulose has a particle size of no more than 500 μ ι η;

further, the microcrystalline cellulose has a particle size of not higher than 200 μm;

further, the particle size of the microcrystalline cellulose is 20-100 μm.

4. The depth filter media of claim 3, wherein the depth filter media is a gradient or a hybrid;

5. The depth filter media of claim 1, further comprising a membrane filtration layer;

preferably, the filtering pore size of the membrane filtering layer is preferably 0.1-10 μm.

6. The depth filtration media of claim 1, wherein the microcrystalline cellulose may be selected from one or more of alpha-microcrystalline cellulose, modified microcrystalline cellulose, or a derivative of microcrystalline cellulose;

further, the alpha-microcrystalline cellulose is crystalline alpha-cellulose prepared from cellulose-containing plant fiber pulp.

7. A depth filtration system comprising a layer of microcrystalline cellulose filter aid and a depth filtration media layer with or without microcrystalline cellulose filter aid;

preferably, the depth filtration media layer is as in any one of claims 1-6;

8. The depth filtration system of claim 7, further comprising a membrane filtration layer;

9. Use of a depth filtration medium according to any one of claims 1 to 6 or a depth filtration system according to any one of claims 7 or 8 for filtering a liquid to be filtered in a water phase.

10. The use according to claim 9, wherein the liquid to be filtered is derived from a cell culture or a derivative of a cell culture,

or the liquid to be filtered comes from plasma or plasma fraction components, or an intermediate product for separating and purifying plasma proteins by taking the plasma or plasma fraction components as raw materials;

further, the cell culture or the derivative of the cell culture contains recombinant coagulation factor VIII and/or vWF, and the plasma or plasma fraction component contains or takes the plasma or plasma fraction component as an intermediate product for separating and purifying the plasma protein and contains human coagulation factor VIII and/or vWF;

further, the plasma fraction is derived from a Cohn' S low temperature ethanol reaction system or a NK low temperature ethanol modification reaction system.