WO2021134180A1 - Method for cryopreserving blood coagulation factor viii intermediate product - Google Patents

Abstract

The present invention relates to the field of blood products, and disclosed is a method for cryopreserving a blood coagulation factor VIII (FVIII) intermediate product. The blood coagulation FVIII intermediate product is taken from an FVIII concentrated solution or an FVIII chromatographic eluent before stock solution preparation or before ultrafiltration concentration or after ultrafiltration concentration in a preparation process of a blood coagulation FVIII preparation, and a combination or transformation thereof. Further disclosed in the present invention are use of the cryopreservation method in preparation of the blood coagulation FVIII preparation, reduction of impurity precipitation of a blood coagulation FVIII product, and improvement of long-term stability of the blood coagulation FVIII product, etc. The FVIII intermediate product is cryopreserved by means of the method of the present invention, so that it is ensured that the activity of FVIII is maintained or even improved, and the FVIII intermediate product can be prepared to a final product satisfying Chinese pharmacopoeia requirements by means of subsequent processes. The method for cryopreserving the blood coagulation FVIII intermediate product disclosed in the present invention improves the flexibility of an FVIII production process, and is favorable for productivity expansion and large-scale production of FVIII preparations.

Description

A method for freezing storage of intermediate product of coagulation factor Ⅷ Technical field

The invention belongs to the technical field of blood products, and specifically relates to a method for freezing an intermediate product of coagulation factor VIII, and a preparation process of a coagulation factor VIII preparation containing a method for freezing an intermediate product of coagulation factor VIII.

Background technique

Coagulation factor Ⅷ preparation (hereinafter referred to as FⅧ preparation) is a special blood preparation for preventing and treating bleeding in patients with hemophilia A. Infusion of coagulation factor Ⅷ preparations in patients with hemophilia A can directly supplement the FⅧ level in their plasma, thereby treating or preventing bleeding and improving symptoms. Hemophilia A is a sex-associated genetic disease, and the incidence of hemophilia A in the population is about 1/5000. The clinical manifestations of joints, muscles, internal organs and deep tissues are spontaneous or difficult to stop bleeding after minor trauma. It often starts in childhood. Repeated joint bleeding leads to gradual joint dysfunction and disability in children. Due to defects in the coagulation mechanism, patients with hemophilia A need to infuse enough FⅧ preparations for bleeding after injury. Although the FⅧ preparation has a significant hemostatic effect, it cannot last in the body, and re-infusion is required for re-bleeding. If the preventive therapy of concentrated FⅧ is used for patients with regular infusion, even patients with severe hemophilia can live almost the same life as normal people. Therefore, although the total number of patients with hemophilia A is small, the demand for FⅧ preparations is very large.

Coagulation factor replacement therapy is still the most effective hemostatic measure for acute bleeding in hemophilia. The principle of treatment is early, adequate, and full course of treatment. The dosage and duration of alternative treatment should consider the location of bleeding and the severity of bleeding. Acute bleeding should be treated as needed: genetically recombined FⅧ preparations or virus-inactivated blood-derived FⅧ preparations can be selected. Cryoprecipitate or fresh frozen plasma can be used for those who are unconditional. However, there is a risk of transfusion-transmitted viral infections. Children with hemophilia should try their best Avoid use.

The problem encountered in clinical use of FⅧ recombinant products is: high-dose repeated use of genetically recombined high-purity FⅧ, inhibitors, namely FⅧ inhibitory antibodies, have a higher risk of production (SFDA.EMEA warns of the risk of recombinant human coagulation factor Ⅷ producing antibodies. Pharmacovigilance Express, 2006, 3(3): 187), and FⅧ inhibitory antibodies are difficult to clear automatically, which in turn causes uncontrolled bleeding, increased hospitalization rates, and joint damage, which ultimately leads to increased morbidity and mortality. Compared with the high-purity FⅧ of genetic recombination, the degree of FⅧ inhibitory antibodies produced after repeated use of blood-derived FⅧ is lower, especially for children with hemophilia A. Therefore, for patients with hemophilia A who need long-term infusion of FⅧ, blood-derived FⅧ inhibitory antibodies are safer, cheaper, and have a lighter medical burden. Therefore, there is a need to further develop and optimize the process route of human FⅧ products derived from plasma to meet the availability of preventive/therapeutic medications and the selectivity of clinical medications for patients with hemophilia A.

Acid precipitation combined with column chromatography purification is one of the classic methods for preparing blood-derived FⅧ. Its principle is: FⅧ has low temperature insolubility, and about 50% of FⅧ in plasma is precipitated with cryoprecipitation. Plasma cryoprecipitation is used as The raw material dissolving solution absorbs vitamin K-dependent coagulation factors through aluminum gel, breaking the coagulation chain, so that the manufacturing process FⅧ is not activated and loses activity. According to the characteristics of the amino acid residues of the fibrinogen (Fg) protein molecule, it is slightly acidic. The pH condition precipitates and removes Fg, while most of FⅧ remains in the supernatant. Due to the limitation of the processing capacity of the front-end purification process steps, the large-scale production of FⅧ preparations is difficult. The reality faced by blood product companies is that compared with intravenous human immunoglobulin, human albumin and other products, blood product companies have a limited amount of FⅧ finished products that can be obtained for each batch of plasma dosage. Compared with aliquoted freeze-dried The processing capacity of other process steps, the processing capacity of the front-end purification process steps such as the process steps before the sterilization and filtration of the stock solution is much smaller. Therefore, the production scale of FⅧ preparations is limited to a certain extent by the preparation scale of cryoprecipitation of raw plasma and the preparation scale of intermediate products before sterilization and filtration of the stock solution. However, blood product companies cannot simply increase the slurry dosage to solve the above problems. The reason is that the production line of FⅧ is developed based on the production lines of important blood products such as human immunoglobulin and human albumin. Therefore, the amount of FⅧ products that can be obtained for each batch of blood product companies is limited; for blood product companies Said that after the production line is completed, the volume of slurry for each batch is fixed, and the production line cannot be changed at will without the approval of the drug regulatory authority. Usually in order to expand production capacity, a parallel approach is used to obtain greater production capacity within the same time, but this puts higher and greater requirements on capital, organization and infrastructure. To sum up, regardless of the production scale of blood product companies, the contradiction between the front-end and back-end production scale of the FⅧ preparation production process always exists.

Therefore, on the one hand, in order to meet the needs of patients with hemophilia A for the availability of preventive/therapeutic drugs and the selectivity of clinical drugs, on the other hand, in order to solve the problem of blood product companies' difficulty in expanding the production capacity of FⅧ products, development FⅧ product production process that is conducive to large-scale production and can be flexibly applied.

Summary of the invention

In order to solve the above technical problems, the present invention provides a freezing storage method of the intermediate product of coagulation factor VIII, which helps to flexibly realize the large-scale production of FVIII preparations.

The present invention is realized by adopting the following scheme:

On the one hand, the present invention discloses a method for cryopreservation of an intermediate product of coagulation factor Ⅷ. The intermediate product of coagulation factor VIII is optionally selected from the FⅧ before the preparation of the original solution or before the ultrafiltration and concentration or after the ultrafiltration and concentration in the preparation process of the coagulation factor VIII preparation. Concentrate or FⅧ chromatography eluent and their combination or deformation.

When the "FⅧ intermediate product" in the present invention specifically refers to FⅧ chromatographic purification eluent. Further, FⅧ heparin affinity chromatography purification eluent or FⅧ anion chromatography purification eluent can be selected.

When the "FⅧ intermediate product" in the present invention refers to the FⅧ concentrate before the preparation of the stock solution, it is preferably the FⅧ concentrate before the sterilization and filtration before the preparation of the stock solution; preferably the FⅧ concentrate before the nanomembrane filtration before the preparation of the stock solution; FⅧ concentrate before ultrafiltration before preparation of stock solution.

In one embodiment, the modification of the "FVIII intermediate product" of the present invention may be the FVIII concentrate after the FVIII purification eluate is dialyzed and/or the concentration of the buffer is adjusted. Furthermore, the “FⅧ intermediate product” referred to in the present invention can also be the FⅧ ultrafiltration concentrate obtained by ultrafiltration of the above FⅧ concentrate. Further, the "FⅧ intermediate product" referred to in the present invention can also be the sample solution before the original solution is prepared after the concentration of the FⅧ ultrafiltration concentrate is adjusted by the buffer.

In one embodiment, the modification of the "FⅧ intermediate product" of the present invention may be the FⅧ concentrated liquid after the FⅧ chromatographic purification eluate is virus-inactivated.

In one embodiment, the modification of the "FⅧ intermediate product" of the present invention may be a chromatographic purification eluent added with an FⅧ active protective agent.

Further, the freezing rate of the intermediate product of coagulation factor VIII is not less than 1°C/min, and is stored under the condition of -15°C to -196°C.

Further, the freezing rate is 1°C/min to 10°C/min.

Furthermore, the freezing rate is 1°C/min to 3°C/min.

Furthermore, the freezing rate is 1.5°C/min to 3°C/min.

In a specific embodiment, the freezing rate is 1.5°C/min.

Further, the intermediate product of coagulation factor VIII is stored at -20°C to -80°C.

Furthermore, the intermediate product of coagulation factor VIII is stored at -20°C to -55°C.

Furthermore, the intermediate product of coagulation factor VIII is stored in liquid nitrogen or dry ice.

The specific activity of the intermediate preparation of coagulation factor VIII is: ≥1IU/mg or ≥10IU/mg or ≥50IU/mg or ≥70IU/mg or ≥100IU/mg or ≥200IU/mg or ≥1000IU/mg.

The pH of the solution of the intermediate product of the coagulation factor VIII preparation is 6.5-7.5.

On the other hand, the present invention provides a preparation process of a coagulation factor VIII preparation containing the freezing method of the above-mentioned intermediate product of the coagulation factor VIII preparation.

In another aspect, the present invention provides a method for reducing the precipitation of impurities in blood coagulation factor VIII products. The method includes freezing the intermediate product of coagulation factor VIII according to the above freezing method during the preparation of the coagulation factor VIII product.

In another aspect, the present invention provides a method for improving the long-term stability of blood coagulation factor VIII products. The method includes freezing the intermediate product of coagulation factor VIII according to the above freezing method during the preparation process of the blood coagulation factor VIII product.

In another aspect, the present invention provides a product prepared according to the above method.

Further, the product is an FⅧ preparation or a complex of FⅧ and vWF.

The beneficial technical effects achieved by the present invention:

In the present invention, the FⅧ intermediate product solution is frozen at a freezing rate of not less than 1°C/min, and frozen for a long time under the condition of -15°C to -196°C. The test results confirm that the FⅧ intermediate product is frozen and stored by the method disclosed in the present invention, which can ensure that the FⅧ activity remains unchanged while the specific activity is increased. Further, the FⅧ intermediate product freezing storage method of the present invention can improve the stability of the FⅧ intermediate product to a certain extent.

The frozen blood coagulation factor Ⅷ preparation intermediate product solution can be further used as the raw material for the preparation of the FⅧ follow-up production process and the finished product to prepare qualified FⅧ finished products that meet the requirements of the 2015 Chinese Pharmacopoeia. The results of the investigation of the stability of the FⅧ finished product prove that the above-mentioned FⅧ finished product is kept stable in 48 months under the condition of 2～8℃.

In summary, under the premise of ensuring that the FⅧ activity recovery rate and specific activity recovery rate are not reduced, and without sacrificing the long-term storage stability of the FⅧ preparation, the method for freezing storage of the intermediate product solution of the blood coagulation factor Ⅷ preparation disclosed in the present invention Increased the flexibility of the FⅧ production process, which is conducive to the expansion of production capacity and large-scale production of FⅧ preparations.

Description of the drawings

Figure 1 is a flow chart of the preparation process of FⅧ finished product, and the shaded area is the process step that can be used to prepare the "factor Ⅷ intermediate product" in the present invention.

Detailed ways

In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes the present invention in detail with reference to specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not used to limit the present invention.

As is generally known in the industry by those skilled in the art, the current industrialized production of blood source FⅧ products or the further development and optimization of the process are continuous and uninterrupted. Because the content of FⅧ in serum is very small (0.05-0.1mg/L) and it is extremely unstable in vitro, the activity loss of FⅧ preparation or purified intermediate product solution is very serious at room temperature. Therefore, in the industrial production scale, the production of FⅧ preparation is from raw materials. From the beginning of plasma cryoprecipitate preparation to the end of virus inactivation, all processes must be carried out continuously and uninterrupted, even at the laboratory level, scientists often freeze protein samples at each purification stage at any time for the convenience of experiments. Moreover, the process design of storing different batches of purified intermediate products and combining a considerable amount of different batches of purified intermediate products for subsequent production of FⅧ preparations is not recognized by the drug regulatory authority, because the freezing and re-thawing of intermediate products will cause FⅧ activity Reduce (Stability of von Willebrand factor and factor VIII in canine cryoprecipitate under various conditions of storage. Stokol T et al. Res Vet Sci. (1995)), which directly affects the potency and quality of the finished product. The possible reason for this adverse consequence is: the protein solution will form small ice crystals and a relatively large ice-liquid interface surface area during the freezing process, which will increase the exposure of protein molecules to the ice-liquid interface, thereby increasing protein denaturation The risk of inactivation; and in the thawing process, the recrystallization of the protein solution further exposes the protein molecules exposed to the ice-liquid interface to large surface tension or mechanical shearing force, thereby exacerbating the damage (Effect of freezing and thawing rates on denaturation). of proteins in aqueous solutions, Biotechnology and Bioengineering, VOL.82, NO.6, JUNE 20, 2003).

Furthermore, in the production process of FⅧ products disclosed or recorded in patent literature, other scientific literature and reference books in the field (such as "Blood Transfusion Therapy and Blood Preparations" edited by Liu Junxiang), there is no freezing of FⅧ intermediate products. Description or introduction of this step. For example, the domestic blood products company Taibang discloses a preparation method of FⅧ in patent CN108218981A. The specific method is as follows: plasma cryoprecipitate is used as raw material, dissolved and then subjected to acid precipitation, DEAE Sephadex A-50 gel adsorption, S/D virus Inactivation, TOYOPEARL DEAE-650M gel column chromatography, ultrafiltration, semi-finished products and finished products are prepared to obtain FⅧ finished products. For another example, the preparation method of FⅧ disclosed by Hualan Biological in patent CN107880112A is as follows: using plasma cryoprecipitate as raw material, dissolving with tromethamine solution and then precipitating with polyethylene glycol, S/D virus inactivation, ToyopearlDEAE 650M gel Column chromatography, ultrafiltration, semi-finished products and finished products are prepared to obtain FⅧ finished products. The preparation method of FⅧ disclosed by Wuhan Blood Products Co., Ltd. in patent CN107337727A is as follows: plasma cryoprecipitate is used as raw material, PEG precipitation and virus inactivation are carried out after dissolution to obtain the original stock solution, anion exchange chromatography to obtain the refined stock solution, ultrafiltration and dialysis Obtain purified liquid, freeze-dry, dry heat inactivation and packaging to obtain FⅧ finished product. The production processes of FⅧ preparations disclosed by the above-mentioned blood product companies do not include the step of freezing the FⅧ intermediate products.

The inventor has discovered through long-term mass production practice that under the production scale, through special control of the freezing storage conditions of FⅧ intermediate products, the unexpected technical effect of FⅧ activity recovery rate and specific activity recovery rate can be achieved without loss. At the same time, the inventor unexpectedly observed that after the FⅧ intermediate product solution was stored at room temperature, the degree of precipitation of foreign matter was reduced after cryopreservation. The FⅧ intermediate product solution was further tested for the FⅧ:C activity after being stored at room temperature for 24 hours. The size found that the loss of FⅧ activity was slowed down, and the room temperature stability was significantly improved.

The freezing storage method of FⅧ intermediate product disclosed in the present invention has a freezing rate of not less than 1°C/min, preferably 1°C/min～10°C/min, more preferably 1°C/min～3°C/min, more preferably 1.5 ℃/min～3℃/min. When the FⅧ intermediate product is completely frozen, the freezing temperature should at least keep the FⅧ intermediate product in a completely frozen state. The FⅧ intermediate product disclosed in the present invention is stored under the conditions of -15°C to -196°C after being frozen under the above conditions, preferably -20°C to -80°C, more preferably -20°C to -55°C.

In some specific embodiments, the FVIII intermediate product is frozen and stored using liquid nitrogen or dry ice.

In some embodiments, the frozen storage time of FVIII intermediate product can be 0-24 months or longer.

In some cases, the specific activity of FⅧ intermediate product (IU/mg) is ≥1IU/mg, in other cases, the specific activity of FⅧ intermediate product (IU/mg) is ≥10IU/mg or ≥50IU/mg or ≥70IU /mg or ≥100IU/mg or ≥200IU/mg or ≥1000IU/mg.

In some embodiments, the pH of the solution of the FVIII preparation intermediate is 6.5-7.5. In some more specific embodiments, the buffer of the FVIII intermediate product may be a buffer containing one or more amino acids, and the buffer may be sodium citrate buffer, phosphate buffer, Tris buffer, and the like. Because the isoelectric point of FⅧ and von Willebrand factor complex is 5.5-6.0, when the pH is 6.5-7.5, FⅧ and von Willebrand factor complex are negatively charged, which is suitable for further purification of FⅧ by anion chromatography; at the same time; The partial neutral conditions with a pH of 6.5-7.5 help protect the activity of FⅧ.

Further, the freezing storage method of FVIII intermediate product disclosed in the present invention has a freezing effect that is less affected by other pre-process steps. After the frozen FⅧ intermediate product is thawed, it is filtered, semi-finished product preparation, sterilization, sub-packaging, freeze-drying, dry heat inactivation and other process steps to prepare qualified FⅧ preparations that meet the requirements of the 2015 Chinese Pharmacopoeia.

The "FVIII preparation" referred to in the present invention can also be referred to as "anti-hemophilia globulin preparation".

The "preparation process of FⅧ preparation" referred to in the present invention refers to the entire production process of FⅧ preparation. The freezing storage method of the FⅧ intermediate product to be highlighted in the embodiment of the present invention can be a link in the overall production process of the FⅧ preparation.

In the prior art, the FⅧ production process starts with cryoprecipitation of plasma, and is prepared by multiple steps of gel adsorption, virus inactivation, and chromatography to prepare the FⅧ intermediate product. The FⅧ intermediate product is further formulated and packaged to be freeze-dried or other viruses. The inactivation process is the finished product of FⅧ preparation. It usually includes several specific steps: preparing plasma cryoprecipitate, dissolving cryoprecipitate to obtain dissolving liquid; adjusting pH for acid precipitation of dissolving liquid, and filtering out the precipitated part to obtain supernatant liquid; subjecting supernatant liquid to gel adsorption ; The flow-through liquid after gel adsorption is subjected to the process steps of virus inactivation, chromatography, ultrafiltration, sterilization, and the final product FⅧ is obtained through the steps of batching, sub-packaging, freeze-drying, dry heat virus inactivation, etc. .

In order to realize the present invention, a basic FⅧ preparation process route is: preparing plasma cryoprecipitate; dissolving plasma cryoprecipitate to obtain a dissolving solution; pre-precipitating the dissolving solution by adjusting pH, and filtering the precipitated part to obtain the supernatant ; Adsorb the supernatant through a balanced gel; the supernatant obtained after the gel is adsorbed is subjected to the process steps of virus inactivation, chromatography, ultrafiltration, sterilization, etc., and is batched, sub-packaged, frozen Dry, dry heat virus inactivation and other steps finally make Ⅷ finished product.

In order to realize the present invention, an optional basic FⅧ preparation process route is: preparing plasma cryoprecipitate; dissolving the plasma cryoprecipitate to obtain a dissolving solution; adsorbing the dissolving solution through a balanced gel; The supernatant is subjected to the process steps of virus inactivation, chromatography, ultrafiltration, sterilization, and the steps of batching, sub-packaging, lyophilization, and dry heat virus inactivation are finally made into a finished product Ⅷ.

In order to realize the present invention, an optional basic FⅧ preparation process route is: preparing plasma cryoprecipitate; Tris buffer solution dissolving cryoprecipitate, after polyethylene glycol precipitation, 0.3% tributyl phosphate and 1% Tween 80 treatment The lipid enveloped virus was inactivated for 6 hours, and then purified by DEAE 650M chromatography. Most of the miscellaneous protein, tributyl phosphate and Tween80 flow through the flow-through fluid directly, and the eluate is the intermediate product of human coagulation factor Ⅷ (reference document "Human coagulation factor Ⅷ original solution production process scale-up feasibility study" published).

Unless otherwise specified, as shown in Figure 1, the "FⅧ intermediate product" referred to in the present invention refers to the FⅧ concentrate before the preparation of the stock solution or before or after ultrafiltration and concentration, or the FⅧ chromatographic purification eluent.

When the "FⅧ intermediate product" referred to in the present invention specifically refers to the FⅧ chromatography purification eluent, the FⅧ heparin affinity chromatography purification eluent or the FⅧ anion chromatography purification eluent can be selected. Preferably, the eluent is purified by FVIII anion chromatography. Because the isoelectric point of the complex of FⅧ and von Willebrand factor is 5.5-6.0, when the pH is 6.5-7.5, the complex is negatively charged; meanwhile, neutral conditions can help protect the activity of FⅧ, so anion chromatography is preferred Purify FⅧ. Common optional anion chromatography media include but are not limited to: DEAE Sepharose A-50, TOYOPEARL DEAE-650M, DEAE 650, Q-sepharose-FF, DEAE-SAPHROSEFF, DEAE-FractogelTSK650M, DEAE SepharoseTSK650M, DEAESepharoseFF, CaptoDEAE, QsepharoseFF, CaptoQ and QSepharoseHP etc. If necessary, an anion exchange chromatography membrane can be used instead of an anion exchange chromatography matrix. Commercially available anion exchange membranes include, but are not limited to, SartobindTM Q from Sartorius, MustangTM Q from Pall Technologies, and InterceptTM Q from Millipore.

When the "FⅧ intermediate product" referred to in the present invention specifically refers to the FⅧ concentrate before the preparation of the stock solution, it is preferably the FⅧ concentrate before the sterilization and filtration before the preparation of the stock solution, and preferably the FⅧ concentrate before the nanomembrane filtration before the preparation of the stock solution.

In one embodiment, the FⅧ intermediate product can be prepared by plasma cryoprecipitation, followed by Al(OH) ₃ gel adsorption, centrifugation to remove insoluble matter, and the supernatant is purified by DEAE Sephadex A-50 gel adsorption. FⅧ chromatographic purification eluent.

In one embodiment, the FⅧ intermediate product can be prepared by plasma cryoprecipitation, followed by acid precipitation, DEAE Sephadex A-50 gel adsorption, centrifugation to remove incompatible materials, and the supernatant can be purified by TOYOPEARL DEAE-650M gel adsorption. The obtained FVIII chromatographic purification eluent.

In one embodiment, the FⅧ intermediate product can be prepared by plasma cryoprecipitation and then adsorbed with Al(OH)3 gel, centrifuged to remove incompatible materials, the supernatant is inactivated by S/D virus, DEAE Sephadex A- 50 FⅧ chromatographic purification eluent obtained by gel adsorption purification.

In one embodiment, the FⅧ intermediate product can be prepared by plasma cryoprecipitation and then dissolved in a neutral pH buffer and then used in Al(OH) ₃ gel (W/V) for adsorption, centrifugation to remove incompatible materials, The supernatant was treated by S/D, and then purified by anion gel (Fractogel TMAE 650). The eluate was heated at 63°C for 10 hours under the protection of sugar and amino acids, and then subjected to the second anion gel column chromatography to remove the stabilizer. FⅧ chromatographic purification eluent.

In one embodiment, the FVIII intermediate product may be a sample solution prepared by cryoprecipitating plasma and then using Al(OH)3 gel adsorption, glycine precipitation, NaCl precipitation, and buffer concentration adjustment before preparing the original solution.

In one embodiment, the FⅧ intermediate product can be prepared by plasma cryoprecipitation and then fractionated with PEG, heparin affinity gel (such as Heparin

6 FF) FⅧ chromatography purification eluent obtained by adsorption purification.

Further, the "FVIII intermediate product" referred to in the present invention can also be the FVIII concentrate obtained by dialysis and/or buffer adjustment of the FVIII chromatographic purification eluent. Furthermore, the “FⅧ intermediate product” referred to in the present invention can also be the FⅧ ultrafiltration concentrate obtained by ultrafiltration of the above FⅧ concentrate. Further, the "FⅧ intermediate product" referred to in the present invention can also be the sample solution before the original solution is prepared after the concentration of the FⅧ ultrafiltration concentrate is adjusted by the buffer.

A kind of "modification of FⅧ intermediate product" referred to in the present invention can be FⅧ concentrated liquid after FⅧ chromatographic purification eluate has been inactivated by virus. It can also be the chromatographic purification eluent added with FVIII active protective agent. The FⅧ activity protection agent can be selected from carbohydrates and amino acids.

The "FⅧ intermediate product" in this embodiment can also be the FⅧ concentrate or FⅧ chromatography purification wash obtained in the preparation of FⅧ preparations before the preparation of the original solution or before the ultrafiltration and concentration or after the ultrafiltration and concentration. Deliquation is not exhaustive here.

The following describes the freezing storage method of FⅧ intermediate products in combination with specific examples.

Example 1

The inventors first investigated the change of FⅧ:C activity recovery rate before and after FⅧ chromatographic purification eluent liquid nitrogen quick freezing and low temperature (-196℃ to -15℃) storage.

Preparation of FⅧ Chromatography Purification Eluent: Using plasma cryoprecipitate as raw material, after dissolving, the FⅧ layer is prepared by acid precipitation, DEAESephadex A-50 gel adsorption, S/D virus inactivation, TOYOPEARL DEAE-650M gel column chromatography Analyze the purified eluate. Detect the FⅧ:C activity size of the eluate from FⅧ chromatography purification.

The prepared FⅧ chromatographic purification eluate was divided into multiple parallel samples, frozen in liquid nitrogen, and stored at -196℃, -80℃, -60℃, -40℃, -20℃, -15℃12 Months later, the room temperature water bath melted, and the activity of FⅧ:C was detected, and the activity recovery rate was calculated.

The test results of FⅧ:C activity recovery rate are shown in Table 1: FⅧ chromatographic purification eluent liquid nitrogen is frozen and stored at low temperature (-196°C to -15°C) for 12 months without loss of activity.

Table 1 Example 1 FⅧ purification eluate before and after cryopreservation FⅧ:C activity recovery rate

The inventors thawed the above 6 frozen FⅧ purified eluate samples in a room temperature water bath and ultrafiltered them. After placing them at room temperature for 2 hours, there was unexpectedly no visible foreign matter precipitation (the visible foreign matter inspection method refers to the 2015 edition of the Chinese Pharmacopoeia). "get on). Previously, the inventors repeatedly observed that if the FⅧ chromatographic purification eluate sample without cryopreservation is ultra-filtered, if it is placed at room temperature for 2 hours, the precipitation of visible foreign matter can be clearly observed with the naked eye.

Furthermore, the inventor kept the above-mentioned sample at room temperature for 24 hours to detect the activity of FⅧ:C to investigate the change of the recovery rate of FⅧ:C activity.

The test results are shown in Table 2: Compared with the samples that have not been frozen and stored in low temperature with liquid nitrogen, after the FⅧ purified eluate is stored at low temperature, the activity loss of 24 hours at room temperature is significantly reduced and the stability is improved.

Table 2 Example 1 Activity recovery rate of FⅧ purified eluate after cryopreservation at room temperature for 24 hours

Further, the inventors investigated the effects of different freezing conditions on the activity recovery rate of the FⅧ purified eluent/FⅧ ultrafiltration concentrate, the stability at room temperature, and the influence on the quality of the final FⅧ product.

Example 2

FⅧ Chromatography purification eluent preparation: Using plasma cryoprecipitate as raw material, after dissolving, it is prepared by acid precipitation, DEAE Sephadex A-50 gel adsorption, S/D virus inactivation, TOYOPEARL DEAE-650M gel column chromatography to obtain FⅧ Chromatographic purification of the eluent. The protein content and FⅧ:C activity of FⅧ chromatographic purification eluate were detected, and the specific activity of FⅧ chromatographic purification eluate was calculated to be (IU/mg)=257IU/mg.

FⅧ Chromatographic Purification Eluent Freezing: Freeze at a rate of 1°C/min, and store it at -30°C to -45°C for 12 months.

FⅧ Finished product preparation: Thaw the frozen FⅧ chromatographic purification eluate in a water bath at room temperature, ultrafiltration, sterile filtration of the thawed FⅧ chromatographic purification eluate. After sterilization and filtration, it is prepared according to the specifications of the finished product, and 100mM glycine is added as a stabilizer. After packaging, freeze-drying, sealing and out of the cabinet, the freeze-dried product is inactivated by dry heat (100℃×30min or 80℃×72h) and then packaged to obtain the FⅧ finished product.

Ultrafiltration dialysate: 50mmol/L sodium chloride, 20mmol/L sodium citrate, 1mmol/L calcium chloride, 7g/L albumin, 35g/L arginine hydrochloride; pH 7.0.

Example 3

FⅧ chromatography purification eluent preparation: using plasma cryoprecipitate as raw material, dissolving with tromethamine solution and then precipitating with polyethylene glycol, S/D virus inactivation, ToyopearlDEAE 650M gel column chromatography to prepare FⅧ chromatography Purify the eluent. The protein content and FⅧ:C activity of FⅧ chromatographic purification eluate were detected, and the specific activity of FⅧ chromatographic purification eluate was calculated to be (IU/mg)=151IU/mg.

FⅧ Chromatographic Purification Eluent Freezing: Freeze at a rate of 10°C/min, and store it at -60°C to -45°C for 12 months.

FⅧ Finished product preparation: Thaw the frozen FⅧ chromatographic purification eluate in a water bath at room temperature, ultrafiltration, sterile filtration of the thawed FⅧ chromatographic purification eluate. After sterilization and filtration, it is prepared according to the specifications of the finished product, and 10mM glycine is added as a stabilizer. After packaging, freeze-drying, sealing and out of the cabinet, the freeze-dried product is inactivated by dry heat (100℃×30min or 80℃×72h) and then packaged to obtain the FⅧ finished product.

Ultrafiltration dialysate: 50mmol/L sodium chloride, 6mmol/L sodium citrate, 1mmol/L calcium chloride, 6.7g/L albumin, 35g/L arginine hydrochloride; pH 6.5.

Example 4

Preparation of FⅧ Chromatography Purification Eluent: Using plasma cryoprecipitate as raw material, dissolving it, it is prepared by PEG precipitation, S/D virus inactivation, and ToyopearlDEAE 650M gel column chromatography to obtain FⅧ chromatographic purification eluent. The protein content and FⅧ:C activity of FⅧ chromatographic purification eluate were detected, and the specific activity of FⅧ chromatographic purification eluate was calculated to be (IU/mg)=85IU/mg.

FⅧ Chromatographic Purification Eluent Freezing: Freeze at a rate of 1.5°C/min, and freeze on dry ice for 12 months.

FⅧ Finished product preparation: Thaw the frozen FⅧ chromatographic purification eluate in a water bath at room temperature, ultrafiltration, sterile filtration of the thawed FⅧ chromatographic purification eluate. After sterilization and filtration, it is prepared according to the specifications of the finished product, and 20mM glycine is added as a stabilizer. After packaging, freeze-drying, sealing and out of the cabinet, the freeze-dried product is inactivated by dry heat (100℃×30min or 80℃×72h) and then packaged to obtain the FⅧ finished product.

The ultrafiltration dialysate is: 50mmol/L sodium chloride, 20mmol/L sodium citrate, 1mmol/L calcium chloride, 7g/L albumin, 35g/L arginine hydrochloride; pH 7.2.

Example 5

FⅧ Chromatographic Purification Eluent Preparation: After dissolving the plasma cryoprecipitate as the raw material, it is prepared by DEAE Sephadex A-50 gel adsorption, S/D virus inactivation, TOYOPEARL DEAE-650M gel column chromatography to obtain FⅧ chromatographic purification Eluent. The protein content and FⅧ:C activity of the FⅧ chromatographic purification eluate were detected, and the specific activity of the FⅧ chromatographic purification eluate was calculated to be (IU/mg)=54IU/mg.

FⅧ Chromatography Purification Eluent Freezing: Freeze at a rate of 3°C/min, and after freezing at -20°C to -30°C for 12 months, melt the FⅧ chromatography high-purity eluate in a water bath at room temperature.

FⅧ Finished product preparation: Thaw the frozen FⅧ chromatographic purification eluate in a water bath at room temperature, ultrafiltration, sterile filtration of the thawed FⅧ chromatographic purification eluate. After sterilization and filtration, it is prepared according to the specifications of the finished product, and 200mM glycine is added as a stabilizer. After packaging, freeze-drying, sealing and out of the cabinet, the freeze-dried product is inactivated by dry heat (100℃×30min or 80℃×72h) and then packaged to obtain the FⅧ finished product.

Ultrafiltration dialysate: 50mmol/L sodium chloride, 20mmol/L sodium citrate, 1mmol/L calcium chloride, 7g/L albumin, 35g/L arginine hydrochloride; pH 7.0.

Examples 6-9

The cryopreserved samples used in Examples 6 to 9 were taken from the ultrafiltration concentrates of the purified eluates in Examples 1 to 4FⅧ. The preparation method of the ultrafiltration concentrate is as follows: the FⅧ purified eluate is dialyzed and concentrated through an ultrafiltration membrane with a cut-off molecular weight of 10-30KD to obtain an FⅧ ultrafiltration concentrate.

The freezing conditions are the same as those in Examples 2-5, and the steps of dialysis concentration, sterilization, semi-finished product preparation, packaging, freeze-drying, and dry heat inactivation after batching are the same as those in Examples 2-5.

The FⅧ intermediate product FⅧ:C value after ultrafiltration and concentration is as follows:

Example 6 Cryopreservation conditions: cryopreservation fluid FⅧ: C 111IU/mL.

Example 7 Cryopreservation conditions: Cryopreservation fluid FⅧ: C 98IU/mL.

Example 8 Cryopreservation conditions: cryopreservation fluid FⅧ: C 73IU/mL.

Example 9 Cryopreservation conditions: cryopreservation fluid FⅧ: C 52IU/mL.

Comparative example 1

The difference between Comparative Example 1 and Example 2 is that the FⅧ chromatographic purification eluent prepared by TOYOPEARL DEAE-650M gel column chromatography directly enters the FⅧ finished product step, excluding the FⅧ chromatography purification eluent freezing step.

Other processes FⅧ purification process and finished product preparation process refer to Example 2 to prepare FⅧ finished product.

The following are the comparisons of various indexes of the finished products before and after the freezing storage of the intermediate products in Examples 2-9 FⅧ and the prepared products.

1. The effect of different freezing conditions on the activity recovery rate of FⅧ purified eluate/FⅧ ultrafiltration concentrate

The protein concentration and FⅧ:C activity of FⅧ purified eluate/FⅧ ultrafiltration concentrated liquid before and after cryopreservation in Examples 2-9 were tested, and the activity recovery rate and specific activity recovery rate were calculated. The test results are shown in Table 3.

The results showed that:

The intermediate product of coagulation factor VIII of Examples 2-9 was frozen at a rate of 1°C/min and above, and after being stored at -20°C to -60°C for 12 months, the activity recovery rate did not decrease, and the FⅧ:C activity was able to maintain.

The intermediate product of coagulation factor VIII of Examples 2-9 was frozen at a rate of 1°C/min and above, and after being stored at -20°C to -60°C for 12 months, the specific activity recovery rate did not decrease, and the ratio of FⅧ:C Activity is improved.

The above test results show that the FⅧ intermediate product freezing storage method of the present invention has basically no effect on the FⅧ:C activity recovery rate and the specific activity recovery rate, and can ensure that the FⅧ activity is maintained while the specific activity is increased.

Table 3 Example 2-9 FⅧ purification eluate/FⅧ ultrafiltration concentrate before and after the activity recovery rate and specific activity recovery rate

2. The influence of different freezing conditions on the stability of FⅧ purified eluent/FⅧ ultrafiltration concentrate at room temperature

The FⅧ purified eluent/FⅧ ultrafiltration concentrated solution sample after freezing treatment in Examples 2-9 was thawed in a water bath at room temperature and ultrafiltered, and its FⅧ:C activity was tested after being placed at room temperature for 24 hours. At the same time, the FⅧ purified eluate/FⅧ ultrafiltration concentrated solution of Examples 2-9 that were not cryopreserved was ultrafiltered, and the FⅧ:C activity was tested after being placed at room temperature for 24 hours. Compare the two test results, see Table 4 for details.

The test results show that after the FⅧ purified eluent/FⅧ ultrafiltration concentrate is frozen and stored in the methods of Examples 2-9, the activity loss of 24 hours at room temperature is significantly reduced, and the storage stability at room temperature is improved.

Table 4 Example 2-9 Low-temperature cryopreservation treatment affects the activity recovery rate of FⅧ purified eluate stored at room temperature

3. The effect of different freezing conditions on the quality of the final FⅧ product obtained from FⅧ purified eluent/FⅧ ultrafiltration concentrate

According to the provisions of the 2015 edition of the Chinese Pharmacopoeia, all indicators of the FⅧ finished products prepared in Examples 2-9 were fully inspected, with special attention to the detection of visible foreign bodies. The test results are shown in Table 5.

The test results show that:

The FⅧ finished products prepared in Examples 2 to 9 meet the requirements of the 2015 edition of the Chinese Pharmacopoeia for FⅧ preparations, especially the detection results of visible foreign bodies comply with the regulations of the Chinese Pharmacopoeia. The FⅧ finished product obtained in Examples 2-9 and the finished product obtained in Comparative Example 1 have no difference in quality inspection results.

The above test results further prove that the FⅧ purified eluate/FⅧ ultrafiltration concentrate processed by the freezing storage method of the present invention can be used as the raw material for the preparation of the FⅧ follow-up production process and the finished product.

Table 5 Test results of main quality indicators of FⅧ finished products prepared in Examples 2-9

4. Investigation on the long-term stability of FⅧ finished products prepared in Examples 2-9

The FⅧ finished products prepared in Examples 2-9 were refrigerated at 2-8°C for 48 months, and all indicators of the FⅧ finished products were fully inspected according to the provisions of the 2015 Chinese Pharmacopoeia. The test results are shown in Table 6.

The test results show that the FⅧ finished product prepared with the frozen FⅧ purified eluent/FⅧ ultrafiltration concentrate is stored under the condition of 2～8℃, and its performance is stable within 48 months. The qualified rate of foreign matter in FⅧ finished products obtained in Examples 2-9 is better than that of Comparative Example 1.

The FⅧ finished product obtained in Examples 2-9 and the finished product obtained in Comparative Example 1 have no difference in quality inspection results.

Table 6 Long-term storage stability of FⅧ finished products prepared in Examples 2-9

The above test results prove:

The FⅧ intermediate product is frozen and stored by the method disclosed in the present invention, and the FⅧ activity is maintained and the specific activity is increased. At the same time, the FⅧ intermediate product freezing storage method of the present invention can improve the room temperature storage stability of the FⅧ intermediate product to a certain extent.

The intermediate product of coagulation factor Ⅷ preparation frozen by the freezing storage process disclosed in the present invention can be used to prepare qualified FⅧ finished products. The stability investigation data proves that the FⅧ finished product prepared from the frozen FⅧ intermediate product is kept stable under the condition of 2～8℃ and has stable performance within 48 months.

The above content is only the preferred embodiments of the present invention. For those of ordinary skill in the art, in accordance with the ideas of the present invention, there will be changes in the specific embodiments and the scope of application. The content of this specification should not be understood. To limit the present invention.

Claims (10)

1. A method for freezing an intermediate product of coagulation factor Ⅷ, characterized in that the intermediate product of coagulation factor Ⅷ is optionally the FⅧ concentrate before the preparation of the original solution or before the ultrafiltration concentration or after the ultrafiltration concentration during the preparation process of the coagulation factor VIII preparation. FⅧ chromatography eluate and their combination or deformation are frozen.
2. The freezing storage method of the intermediate product of coagulation factor VIII according to claim 1, characterized in that the freezing rate of the intermediate product of coagulation factor VIII is not less than 1°C/min, and the temperature is between -15°C and -196°C. Next store.
3. The freezing storage method of the intermediate product of coagulation factor VIII according to claim 2, wherein the freezing rate is 1°C/min-10°C/min;

   Preferably, the freezing rate is 1°C/min to 3°C/min;

   Preferably, the freezing rate is 1.5°C/min to 3°C/min.
4. The cryopreservation method of the intermediate product of coagulation factor VIII according to claim 2, wherein the intermediate product of coagulation factor VIII is stored at -20°C to -80°C;

   Preferably, the intermediate product of coagulation factor VIII is stored at -20°C to -55°C;

   Preferably, the intermediate product of coagulation factor VIII is stored using liquid nitrogen or dry ice.
5. The cryopreservation method of the intermediate product of coagulation factor VIII according to any one of claims 1 to 4, wherein the specific activity of the intermediate product of coagulation factor VIII is: ≥1IU/mg or ≥10IU/mg or ≥50IU/ mg or ≥70IU/mg or ≥100IU/mg or ≥200IU/mg or ≥1000IU/mg.
6. The cryopreservation method of the intermediate product of coagulation factor VIII according to any one of claims 1 to 4, wherein the pH of the solution of the intermediate product of coagulation factor VIII is 6.5-7.5.
7. A preparation process of a coagulation factor VIII preparation comprising the freezing storage method of the coagulation factor VIII intermediate product according to any one of claims 1 to 6.
8. A method for reducing the precipitation of impurities in blood coagulation factor VIII products, which is characterized in that the intermediate product of coagulation factor VIII is frozen according to the freezing storage method of any one of claims 1 to 6 during the preparation process of the coagulation factor VIII products.
9. A method for improving the long-term stability of coagulation factor VIII products, characterized in that, during the preparation process of coagulation factor VIII products, the intermediate product of coagulation factor VIII is cryopreserved according to the cryopreservation method of any one of claims 1 to 6.
10. A product prepared by the method of claims 1-9.

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