Method for the preparation of thrombin

Abstract

The present invention relates to a method for producing thrombin from a thromboplastin source using a given BaSO4 reagent as an adsorbent for prothrombin, and a method for assessing the suitability of a given BaSO4 reagent for the preparation of thrombin. In at least one embodiment, the method comprises contacting a sample of the given BaSO4 reagent with a thromboplastin source to obtain BaSO4-adsorbed thromboplastin, and subsequently assessing the procoagulant activity of the BaSO4-adsorbed thromboplastin. In another embodiment, the method comprises the step of assessing the procoagulant activity relative to normal mammalian plasma.

Description

[0001] This application is a divisional application of patent application No. 201510064089.2, filed on February 06, 2015, with the title "Process for the preparation of thrombin". TECHNICAL FIELD

[0002] The present invention relates to the field of thrombin production, and more specifically, to a method for assessing the suitability of a given BaS04 reagent for use as a prothrombin adsorbent in the preparation of thrombin. BACKGROUND

[0003] Thrombin is a serine protease that promotes blood clotting by catalyzing the conversion of fibrinogen to fibrin. Thrombin is also responsible for activating platelets and indirectly for the regulation of its own production and inhibition through a multiple proteolytic feedback mechanism. Thrombin is also involved in the activation of Factor VIII, Factor V, Factor XI, Factor XIII and Protein C. Thrombin is widely used in clinical applications as a clotting factor to stop bleeding from wounds by converting fibrinogen to fibrin. Thrombin is a common component of surgical dressings and has been combined with fibrinogen and other clotting proteins for use in two-component hemostatic systems such as fibrin glue, adhesives and sealants.

[0004] Thrombin is produced by proteolytic activation of the precursor, prothrombin. To produce thrombin, prothrombin must be cleaved at two sites, generating an intermediate product. The conversion of prothrombin to thrombin in vivo is catalyzed by the prothrombinase complex, which includes activated Factor X and Factor V, and is assembled on a negatively charged phospholipid membrane in the presence of calcium ions.

[0005] Thrombin can be manufactured from prothrombin by contacting a prothrombin source, such as plasma or a blood fraction, with a solid adsorbent capable of adsorbing prothrombin from the prothrombin source, for example barium sulfate (BaS04). The solid adsorbent is typically washed using a wash solution to remove contaminants such as unbound proteins, and then eluted from it using an elution solution. After further optional purification and processing steps, the eluted prothrombin can be converted to thrombin by activation using an activator, for example calcium ions.

[0006] Significant differences in the yield of thrombin from a given volume of a prothrombin source have been reported when using different BaS04 reagents, such as BaS04 reagents from different manufacturers, or even different batches of reagent produced by a particular manufacturer.

[0007] Reports have suggested that differences in thrombin yield can be at least partially attributed to variations in prothrombin adsorption using different BaSO4 reagents, and that adsorption capacity is a key factor in selecting a BaSO4 reagent for prothrombin adsorption. Surgenor and Neortker (1952) stated that certain BaSO4 reagents are more effective than others in adsorbing prothrombin from plasma, while Voss D. (1965) noted significant differences in the amount of BaSO4 required to adsorb the same amount of prothrombin complex when using different BaSO4 reagents, speculating that this was due to differences in their crystal structures. However, in the applicant's case, the morphologies of different batches of BaSO4 were tested, and no significant differences were detected. Additionally, some ions, such as Ca... 2+ Reports have shown that this leads to different adsorption rates for different batches of BaSO4, and in the case of this paper, Ca in different batches of BaSO4... 2+ The existence of [other substances] is not significantly different.

[0008] Thrombin manufacturers attempt to increase thrombin yield by using a trial-and-error method to select the appropriate BaSO4 reagent from a variety of different BaSO4 reagents. Summary of the Invention

[0009] In some embodiments of the present invention, therein relates to a method for producing thrombin from a prothrombin source using a given BaSO4 reagent as an adsorbent for prothrombin, and a method for evaluating the suitability of a given BaSO4 reagent for the preparation of thrombin by contacting the given BaSO4 reagent with a prothrombin source to obtain BaSO4-adsorbed prothrombin, and in some embodiments, evaluating the procoagulant activity of the BaSO4-adsorbed prothrombin relative to the procoagulant activity of normal mammalian plasma.

[0010] Contact is used herein in its broadest sense and refers to any type of combined action, such as bringing the prothrombin source and BaSO4 into sufficiently close proximity such that a binding interaction occurs between the BaSO4 and the prothrombin in the source. Contact includes, but is not limited to, mixing, blending, and / or adding the source to BaSO4, or adding BaSO4 to the source.

[0011] In this invention, it was surprisingly discovered that contact between prothrombin and certain BaSO4 reagents triggers its conversion into thrombin (i.e., prothrombin is converted into its intermediate and / or thrombin). This was found to be a premature conversion that compromises thrombin yield at the end of the production process.

[0012] In some embodiments of the methods described herein, procoagulant activity occurs after prothrombin is converted into its intermediates and / or thrombin. Such intermediates can be formed during the proteolytic conversion of prothrombin to thrombin. Non-limiting examples of intermediates are prethrombin and meisothrombin.

[0013] Some embodiments of the methods described herein allow for the identification of BaSO4 reagents suitable for use as prothrombin adsorbents, preferably before using a given BaSO4 reagent as an adsorbent as part of a large-scale thrombin production process.

[0014] Specifically, in some embodiments, a sample of a given BaSO4 reagent is contacted with a source of prothrombin to adsorb the prothrombin, yielding BaSO4-adsorbed prothrombin, and the procoagulant activity of the BaSO4-adsorbed prothrombin is subsequently evaluated. Generally, the lower the procoagulant activity of the BaSO4-adsorbed prothrombin, the more suitable the given BaSO4 reagent is as a prothrombin adsorbent. In some embodiments, the suitability of a given BaSO4 reagent as a prothrombin adsorbent in the thrombin preparation process is indicated by the procoagulant activity of the BaSO4-adsorbed prothrombin not exceeding that of normal mammalian plasma, such as normal human plasma.

[0015] Normal mammalian plasma, such as human plasma, is a well-known conglomerate or single-donor plasma preparation intended for use as a calibrator for various coagulation tests.

[0016] Normal human plasma can be sterile plasma obtained by combining, for example, the liquid portion of whole blood from eight or more healthy adults (with a solution of potassium citrate or sodium citrate or both added) and exposing it to ultraviolet light to destroy bacterial and viral contaminants.

[0017] Normal human plasma can be Unicalibrator coagulation test calibration plasma 00625.

[0018] At least in some cases, the method described in this paper eliminates the need to select a suitable BaSO4 reagent through trial and error.

[0019] Some embodiments of the methods described herein are rapid and easy to use, and potentially offer savings in time and / or production costs. Some embodiments of the methods described herein allow for increased thrombin yield from a given prothrombin source.

[0020] Aspects and embodiments of the invention are described in the following description and appended claims.

[0021] According to one aspect of some embodiments described herein, the present invention provides a method for preparing thrombin from a prothrombin source using a given BaSO4 reagent as an adsorbent for prothrombin, the method comprising:

[0022] a. Provide a given BaSO4 reagent and source of prothrombin;

[0023] b. Under conditions that allow prothrombin from a prothrombin source to be adsorbed by a given BaSO4 reagent, contact a sample with the given BaSO4 reagent and a source of prothrombin to obtain BaSO4-adsorbed prothrombin; and

[0024] c. Assess the procoagulant activity of prothrombin adsorbed by BaSO4.

[0025] When the assessment of c. is performed by comparing the procoagulant activity of prothrombin adsorbed by BaSO4 with that of normal mammalian plasma, the suitability of a given BaSO4 reagent for the preparation of thrombin is indicated by the procoagulant activity of prothrombin adsorbed by BaSO4 not being greater than that of normal mammalian plasma.

[0026] According to a further aspect, the present invention provides a method for preparing thrombin from a prothrombin source using a given BaSO4 reagent as an adsorbent for prothrombin, the method comprising steps a to c above, and further comprising:

[0027] d. Under conditions that allow for prothrombin adsorption, bring a suitable BaSO4 into contact with a source of prothrombin;

[0028] e. Use elution buffer to elute the prothrombin fraction from the prothrombin adsorbed by BaSO4;

[0029] f. Apply conditions to the eluted fractions that allow prothrombin to be converted into thrombin, thereby obtaining thrombin.

[0030] In some embodiments, the method includes collecting the eluted fractions after step "e".

[0031] In some embodiments, the method substantially avoids premature conversion of prothrombin into thrombin. “Premature conversion of prothrombin into thrombin” means that prothrombin is converted into thrombin in step “d”.

[0032] Generally, the term “elution” as used herein is used interchangeably with the term “desorption”.

[0033] In one embodiment, conditions that allow prothrombin to adsorb onto BaSO4 during thrombin preparation include pH 7.4-8.6 and / or a concentration range of about 1%-22% (w / v), for example, about 1% BaSO4.

[0034] In one embodiment, the elution buffer during thrombin preparation comprises a calcium chelating salt such as sodium citrate at a concentration of 3.0-4.4% (w / v) and / or has a pH between 6.3 and 7.4.

[0035] In one embodiment, the conditions that allow prothrombin to be converted into thrombin include applying an activator such as calcium ions to the prothrombin.

[0036] According to further aspects of some embodiments described herein, the present invention provides a method for evaluating the suitability of a given BaSO4 reagent for the preparation of thrombin from a prothrombin source, the method comprising:

[0037] a. Provide a given BaSO4 reagent and source of prothrombin;

[0038] b. Under conditions that allow prothrombin from a prothrombin source to adsorb onto a given BaSO4 reagent, contact a sample with a given BaSO4 reagent and a prothrombin source to obtain prothrombin adsorbed onto BaSO4.

[0039] c. Assess the procoagulant activity of prothrombin adsorbed by BaSO4.

[0040] When the assessment of c. is performed by comparing the procoagulant activity of prothrombin adsorbed by BaSO4 with that of normal mammalian plasma, the suitability of a given BaSO4 reagent for the preparation of thrombin is indicated by the procoagulant activity of prothrombin adsorbed by BaSO4 not being greater than that of normal mammalian plasma.

[0041] According to one aspect of some embodiments of the present invention, the present invention provides a method for preparing thrombin from a prothrombin source using a given BaSO4 reagent as a prothrombin adsorbent, the method comprising:

[0042] a. Provide a given BaSO4 reagent and source of prothrombin;

[0043] b. Under conditions that allow prothrombin from a prothrombin source to be adsorbed by a given BaSO4 reagent, contact a sample with the given BaSO4 reagent and a source of prothrombin to obtain BaSO4-adsorbed prothrombin; and

[0044] c. Assessing the procoagulant activity of BaSO4-adsorbed prothrombin. In some embodiments, the suitability of a given BaSO4 reagent as a prothrombin adsorbent in the thrombin preparation process is indicated by the procoagulant activity of BaSO4-adsorbed prothrombin not being greater than that of normal mammalian plasma. In some embodiments, the assessment of c. is performed by comparing the procoagulant activity of BaSO4-adsorbed prothrombin with that of normal mammalian plasma.

[0045] According to further aspects of some embodiments of the present invention, the present invention provides a method for evaluating the suitability of a given BaSO4 reagent as a prothrombin adsorbent in the preparation of thrombin from a prothrombin source, the method comprising:

[0046] a. Provide a given BaSO4 reagent and source of prothrombin;

[0047] b. Under conditions that allow prothrombin from a prothrombin source to be adsorbed by a given BaSO4 reagent, contact a sample with the given BaSO4 reagent and a source of prothrombin to obtain BaSO4-adsorbed prothrombin; and

[0048] c. Assess the procoagulant activity of BaSO4-adsorbed prothrombin. In some embodiments, the suitability of a given BaSO4 reagent as a prothrombin adsorbent in the thrombin preparation process is indicated by the procoagulant activity of BaSO4-adsorbed prothrombin not being greater than that of normal mammalian plasma. In some embodiments, the assessment of c. is performed by comparing the procoagulant activity of BaSO4-adsorbed prothrombin with that of normal mammalian plasma.

[0049] In some embodiments of the methods disclosed herein, evaluating coagulant activity includes performing a coagulant assay.

[0050] In some embodiments of the methods disclosed herein, the procoagulant assay includes functional assays, such as those selected from coagulation assays (such as NAPTT assays, APTT assays, protease chromogenic assays) or indicator assays (such as immunoassays).

[0051] In some embodiments, BaSO4 reagents with an eluent having a NAPTT ratio of not less than 0.8 are considered suitable for the preparation of thrombin.

[0052] In some embodiments, BaSO4 reagents with an eluent having a NAPTT ratio of 0.8 or less, or with clots immediately visible to the naked eye (clots occurring after calcium addition and before clotting time can be recorded in a coagulant measuring machine), are considered unsuitable for the preparation of thrombin.

[0053] In some embodiments of the methods disclosed herein, the prothrombin source is selected from plasma (such as oxalate plasma) or plasma fractions. In some such embodiments, the prothrombin source includes plasma harvested from mammals (such as humans, horses, cattle, and pigs). In some embodiments, the prothrombin source includes porcine plasma. In some embodiments, the prothrombin source is recombinant prothrombin. In some embodiments, the prothrombin source is subjected to viral inactivation treatment. For example, the source is solvent / detergent (SD) treated plasma.

[0054] "Solvent-detergent (SD) virus inactivation treatment" generally refers to the process of inactivating enveloped or lipid-coated viruses by disrupting their lipid envelope. This treatment can be carried out by adding a detergent (such as Triton X-45, Triton X-100, or polysorbate 80) and a solvent (such as tri(n-butyl) phosphate (TnBP), di- or trialkyl phosphates). The solvent-detergent combination used to inactivate lipid-coated viruses can be any solvent-detergent combination known in the art, such as TnBP and Triton X-100; polysorbate 80 and sodium cholate, and other combinations.

[0055] The concentrations of one or more solvents / detergents used can be those commonly used in the art, such as those described in US5094960A and US4789545A. The concentrations of one or more solvents / detergents used can be a combination of >0.1% TnBP and >0.1% Triton X-100. The concentrations of one or more solvents / detergents used can be a combination of 1% Triton X-100 and 0.3% TnBP. However, other solvent / detergent combinations and suitable conditions will be apparent to any person skilled in the art.

[0056] In one embodiment, 0.5% Tween-80 and 0.15% TnBP were used for SD treatment. The pH was in the range of 7.4-8.6. The solution was incubated at room temperature for 1 hour.

[0057] In one embodiment, 1% Tween-80 and 0.3% TnBP were used for SD treatment. The pH was in the range of 7.4-8.6. The solution was incubated at room temperature for 6 hours.

[0058] In some embodiments of the methods disclosed herein, contacting a sample of a given BaSO4 reagent with a prothrombin source comprises adding about 1% to about 22% or about 1% to about 10% (w / v) of the BaSO4 reagent to the prothrombin source (e.g., harvested plasma). In some embodiments, about 1% (w / v) of the BaSO4 reagent is added.

[0059] In some embodiments, conditions that allow prothrombin from a prothrombin source to be adsorbed by a given BaSO4 reagent include pH 7.4-8.6. In some embodiments, these conditions include, for example, room temperature in the range of 20°C-25°C.

[0060] Prothrombin can be adsorbed onto a BaSO4 reagent in batch mode or on a column filled with BaSO4.

[0061] In one embodiment, prothrombin is adsorbed onto BaSO4 in batch mode at room temperature, for example at 25°C, and at pH 7.4-8.6 for 2 hours.

[0062] In some embodiments of the methods described herein, the procoagulant activity of prothrombin adsorbed by BaSO4 is evaluated as the procoagulant activity of prothrombin adsorbed by BaSO4 when adsorbed onto the BaSO4 reagent.

[0063] In some embodiments, the method disclosed herein further includes, after “b” and before “c”, eluting at least some of the prothrombin adsorbed by BaSO4 from the BaSO4 reagent; and wherein evaluating the procoagulant activity of the prothrombin adsorbed by BaSO4 is the procoagulant activity of at least some of the prothrombin adsorbed by the eluted BaSO4. In some such embodiments, eluting at least some of the prothrombin adsorbed by BaSO4 from the BaSO4 reagent comprises contacting the BaSO4 reagent with an elution solution having a pH of not less than 6.0 and not greater than 7.0.

[0064] In some embodiments, the pH of the elution solution is not less than 6.1, not less than 6.2, and even not less than 6.3. In some embodiments, the pH of the elution solution is not more than 6.5, not more than 6.6, and even not more than 6.7, or between about pH 6.3 and 6.7. In some embodiments, the pH of the elution solution is between 6.3 and 7.4.

[0065] In some embodiments, the elution solution comprises a chelating salt. In some embodiments, the concentration of the chelating salt in the elution solution is from about 0.2% (w / v) to about 4.4% (w / v) or from about 3.0% (w / v) to about 4.4% (w / v). In some embodiments, the chelating salt comprises sodium citrate. In some embodiments, the concentration of sodium citrate in the elution solution is from about 0.2% (w / v) to about 4.4% (w / v) or from about 3.0% (w / v) to about 4.4% (w / v).

[0066] In some embodiments, a given BaSO4 reagent is a reagent containing at least 75% (w / w) BaSO4 on a weight-to-weight basis, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, and even at least 88% (w / w) BaSO4.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, descriptions, materials, methods, and examples are illustrative only and are not intended to be limiting. Methods and materials similar to or equivalent to those described herein may be used in the practice of this invention.

[0068] As used herein, the term “given BaSO4 reagent” refers to a specified batch of BaSO4 reagent from a specified supplier. Different given BaSO4 reagents may therefore be reagents supplied by different suppliers, or different batches of reagents supplied by the same supplier.

[0069] As used in this article, the term "coagulant activity" refers to the ability to promote blood clotting.

[0070] As used herein, the terms “comprising,” “including,” “having,” and their grammatical variations shall be regarded as specifying the stated feature, integer, step, or component, but do not preclude the addition of one or more additional features, integers, steps, components, or groups thereof. These terms encompass the terms “consisting of” and “substantially consisting of”.

[0071] As used in this article, the indefinite articles “a” and “an” mean “at least one / a kind” or “one or more / a combination of one or more”, unless the context clearly indicates otherwise.

[0072] As used in this article, the term “about” means ±10%. Attached Figure Description

[0073] Some embodiments of the present invention are described herein with reference to the accompanying drawings. The description, together with the drawings, makes it apparent to those skilled in the art how some embodiments of the invention can be practiced. The drawings are for illustrative purposes and do not attempt to show structural details of embodiments more detailed than are necessary for a basic understanding of the invention. For clarity, some objects depicted in the drawings are not drawn to scale.

[0074] In the attached diagram:

[0075] Figure 1 Western blot analysis of the eluents obtained from a large-scale BaSO4-adsorbed prothrombin sample prepared during thrombin production using BaSO4 reagents from different suppliers is shown.

[0076] Figure 2 Western blot analysis of the eluent obtained from porcine plasma, which was prepared using different batches of BaSO4 reagent from the same supplier, is shown.

[0077] Figure 3 Western blot analysis of eluates obtained from porcine plasma, prepared using BaSO4 reagents from different suppliers, is shown. Detailed Implementation

[0078] In some embodiments of the present invention, therein relates to a method for preparing thrombin from a prothrombin source using a given BaSO4 reagent as an adsorbent for prothrombin, and a method for evaluating the suitability of a given BaSO4 reagent for the preparation of thrombin by contacting the given BaSO4 reagent with a prothrombin source to obtain BaSO4-adsorbed prothrombin, and in some embodiments, evaluating the procoagulant activity of the BaSO4-adsorbed prothrombin relative to the procoagulant activity of normal mammalian plasma.

[0079] The principles, uses, and specific implementations taught herein can be better understood by referring to the accompanying specification. After carefully reading the specification, those skilled in the art will be able to implement this invention without excessive effort or experimentation.

[0080] Before detailing at least one embodiment, it should be understood that the invention is not necessarily limited in its application to the details of the construction and arrangement of the components and / or methods shown in the following description. The invention is capable of having other embodiments practiced or implemented in various ways. The wording and terminology used herein are for descriptive purposes and should not be considered limiting.

[0081] Example

[0082] Materials and Methods

[0083] Materials

[0084] The BaSO4 reagents provided are shown in Table 1 below:

[0085] Table 1: BaS04 Reagents

[0086] Reagent Name Supplier Batch Number A 1 (Pharmaceutical grade) 1 B1 2 (Pharmaceutical grade) 1 B2 2 (Pharmaceutical grade) 2 C 3 (Pharmaceutical grade) 1 D1 4 (Pharmaceutical grade) 1 D2 4 (White standard grade) 1

[0087] Sodium chloride was obtained from Sigma-Aldrich (catalog number S1679, analytical grade, at least 99.0% by weight).

[0088] The trisodium citrate dihydrate was obtained from Sigma-Aldrich (catalog number S1804, analytical grade, at least 99.0% by weight). The oxalate-treated pig blood (including a 0.25% (w / w) oxalate solution) was obtained from the Kibbutz Lahav (Israel) slaughterhouse.

[0089] The prothrombin adsorbed by BaSO4 was obtained during the large-scale production of thrombin (also referred to herein as the large-scale sample), wherein the BaSO4 used for adsorbing prothrombin was selected from one of the BaSO4 reagents A and B1 specified in Table 1 above.

[0090] Obtained from Sigma-Aldrich (catalog number P8074, BioXtra, polyethylene glycol dehydrated sorbitan monooleate).

[0091] Tri-n-butyl phosphate (TnBP) is derived from Merck-Millipore (catalog number 100002, pharmaceutical grade).

[0092] The 1.2μm filter is from Sartorius Stedim India Pvt Ltd. (Bangalore, India) (catalog number) 2 300), and the filter 0.45+0.2μm was obtained from Sartorius Stedim India Pvt Ltd. (catalog number) 300).

[0093] For gel electrophoresis, the precast polyacrylamide gel was obtained from Bio-Rad Laboratories Inc. (4-20% microgels were obtained from catalog number: 456-1096).

[0094] Sheep antithrombin is derived from Affinity Biologicals Inc. (Canada) (catalog number SAHT-IG).

[0095] The anti-sheep IgG alkaline phosphatase conjugate was obtained from Sigma-Aldrich (catalog number A-5187).

[0096] Example 1: Preparation of solvent / detergent (SD) virus inactivated plasma

[0097] Deliver oxalate-enriched pig blood on ice. Centrifuge the blood at a relative centrifugal force (rcf) of 5,000 g (5440 rpm) and a temperature of 4 °C for 15 minutes. Collect the supernatant containing plasma, divide it into aliquots (300-500 ml), and store frozen at -30 °C until needed.

[0098] For the preparation of SD-treated plasma (“SD plasma”), aliquots of frozen plasma were thawed at 37°C for approximately 1 hour, then passed through a 1.2 μm filter, followed by a 0.45 ± 0.2 μm filter. At pH 7.4–8.6, 0.5% Tween-80 and 0.15% (v / v) TnBP were added for SD treatment. The solution was stirred at room temperature for 1 hour, with pH monitored at 7.4–8.6.

[0099] Example 2: Preparation of laboratory scale BaS04 adsorbed prothrombin eluate from pig SD plasma

[0100] Contact the SD plasma described in Example 1 above, as a source of prothrombin, with the samples of the given BaSO4 reagents listed in Table 1.

[0101] Specifically, for each of the six BaSO4 reagents listed in Table 1:

[0102] (i) Add a sample of BaSO4 (1% w / v) reagent to one unit of SD plasma.

[0103] (ii) Stir the SD plasma / BaSO4 reagent mixture at 25°C for 2 hours (pH 7.4-8.6) using a magnetic stirrer to allow the prothrombin in the SD plasma to be adsorbed by the BaSO4 reagent.

[0104] (iii) Centrifuge the mixture at 5,000 g (5,440 rpm) at 4 °C for 15 minutes.

[0105] (iv) Separate the supernatant and precipitates (including prothrombin adsorbed by BaSO4) and store each precipitate separately at -80°C until needed.

[0106] (v) Before use, thaw the precipitate at room temperature (20-25°C) for 15 minutes.

[0107] (vi) The precipitate was resuspended in wash buffer (78 mM NaCl pH 6.9-7.1) at a ratio of 1:1.4 BaSO4:wash buffer (w / w) and mixed for 15 minutes at room temperature using a tube roller.

[0108] (vii) The precipitate in the washing buffer is then centrifuged at 5,000 g (5,440 rpm) at 4 °C for 15 minutes, and the supernatant is collected and stored at -80 °C until needed. This step is repeated twice (i.e., a total of three washes are performed).

[0109] (viii) After the washing step, the washed precipitate (containing prothrombin adsorbed by BaSO4) was resuspended in elution buffer (3% sodium citrate, pH 6.3-6.7) at a BaSO4:elution buffer ratio of 1:1.4 (w / w) and mixed for 15 minutes using a roller mixer.

[0110] (ix) The precipitate / elution buffer was then centrifuged at 5,000 g (5,440 rpm) at 4 °C for 15 minutes. The prothrombin-containing eluent was collected in a clean container placed on ice, and the elution step was repeated four times (i.e., a total of five elution cycles were performed).

[0111] (x) The eluents (containing desorbed prothrombin) from five elution cycles were combined into a single eluent, filtered through a 0.2 μm PVDF filter, and stored in a clean container at -80°C until tested by NAPTT or Western blot.

[0112] Example 3: Preparation of large scale BaS04 adsorbed prothrombin eluate from pig SD plasma

[0113] (Preparation of large-scale samples of frozen prothrombin-adsorbed BaSO4 from porcine plasma).

[0114] The BaSO4 reagents used are A and B1 as specified in Table 1.

[0115] SD treatment was performed using 1% Tween-80 and 0.3% TnBP. The pH was maintained in the range of 7.4–8.6. The solution was incubated with SD at 24–26°C for 6 hours.

[0116] In large-scale samples, step v replaces the corresponding step in laboratory-scale samples, and the sample is processed up to step x.

[0117] Example 4: Western blot analysis of prothrombin and thrombin content of eluate

[0118] The total protein measurements in the eluates obtained as described in Examples 2 and 3 above (each eluate corresponding to one of the six BaSO4 reagents in Table 1) were performed using the Biuret method to calculate the loading volume for Western blot assays and to verify the removal of residual unbound proteins and other contaminants by washing steps (vi and vii).

[0119] To obtain better resolution of each protein from the eluent, proteins from the eluent samples were separated under reducing conditions using SDS-PAGE gradient microprecast gels (4%–20%). Proteins were transferred from the gel to nitrocellulose membranes. Western blotting was performed using sheep anti-human thrombin and secondary antibodies, and anti-sheep IgG conjugated with alkaline phosphatase.

[0120] Some BaSO4-adsorbed prothrombin samples were found to contain thrombin (see the "Results" section below).

[0121] Example 5: Coagulation promoting activity of eluate

[0122] The procoagulant activity of each of eight eluates (such as the six in Example 2 and the two in Example 3) prepared from porcine SD plasma was evaluated using a non-activated partial thromboplastin time (NAPTT) assay, essentially as described in Ph. Eur. (2.6.22). Briefly, and as is known to those skilled in the art, the NAPTT assay involves the simultaneous addition of phospholipids (rabbit brain phospholipids) and calcium ions (such as CaCl2) to the eluate being tested or to a sample of prothrombin adsorbed with BaSO4, as well as a normal human plasma control [Unicalibrator coagulation test calibration plasma 00625] (which inherently contains prothrombin). Calcium ions initiate clotting in the test sample by converting prothrombin to thrombin. Thrombin causes clot formation by converting soluble fibrinogen to insoluble fibrin. Clotting time was measured using a coagulation meter (ST ART4 Stago machine).

[0123] More specifically, 45 μl of human plasma was added to each of the four test wells. For the control samples, 45 μl of TBS / albumin buffer was added to each of the two control wells, and 45 μl of diluted test sample was added to each of the two additional control wells. Subsequently, 45 μl of 0.02% rabbit brain phospholipid solution was added to each of the four test wells, and the contents of the wells were mixed by gentle shaking and incubation at 37°C for 1 minute. The reaction was initiated by adding 45 μl of 0.025M CaCl2 (previously incubated at 37°C) to all wells, and clotting time was measured. The NAPTT ratio was calculated as the clotting time (in seconds) of the test sample divided by the clotting time of the normal human plasma control.

[0124] In the eluents in which active thrombin was identified (Example 4), the clotting time was found to be substantially shorter than that of normal human plasma controls. Clotting was found to be immediate in some eluents. Clotting times exceeded 450 seconds in some eluents.

[0125] It was found that the eluents from BaSO4 B1 and B2 yielded a high thrombin yield at the end of the process.

[0126] The results indicate that the NAPTT assay can be used to identify suitable BaSO4 reagents for thrombin production.

[0127] Example 6: Preparation of BaS04 adsorbed prothrombin from pig SD plasma

[0128] Repeat steps (i)-(ii) above for each of the six BaSO4 reagents in Table 1.

[0129] The procoagulant activity of prothrombin samples adsorbed with BaSO4, prepared from porcine SD plasma, was evaluated using the non-activated partial thromboplastin time (NAPTT) assay.

[0130] Results

[0131] Western blot analysis

[0132] Western blot analysis of the eluates (Example 3) using BaSO4 reagents A and B1 (Table 1) is presented in... Figure 1 middle.

[0133] The lane numbers represent the following:

[0134] 1–MW ladder

[0135] 2-Human prothrombin standard

[0136] 3 – Large-scale eluent obtained using BaSO4 reagent A

[0137] 4 – Large-scale eluent obtained using BaSO4 reagent B1

[0138] 5 – Human prethrombin 2 standard

[0139] 6–human α-thrombin standard

[0140] exist Figure 1 In the large-scale eluent (lane 4) obtained using BaSO4 reagent B1, the band corresponding to prothrombin is clearly visible.

[0141] exist Figure 1 In the large-scale eluent (lane 3) obtained using BaSO4 reagent A, the band corresponding to prothrombin is almost invisible. Other bands corresponding to prothrombin intermediate proteins and α-thrombin are clearly visible.

[0142] Western blot analysis of the eluents (Example 2) obtained from porcine SD plasma using BaSO4 reagents A, B1, and B2 (Table 1) is presented in... Figure 2 middle.

[0143] The lane numbers represent the following:

[0144] 1–MW ladder

[0145] 2-Human prothrombin standard

[0146] 3 – Elution obtained using BaSO4 reagent A

[0147] 4 – Elution obtained using BaSO4 reagent B1

[0148] 5 – Eluent obtained using BaSO4 reagent B2

[0149] 6–human α-thrombin standard

[0150] Figure 2 The results presented show that the eluents (lane B1-4, lane B2-5) obtained using different batches of BaSO4 supplied by the same supplier exhibit similar banding patterns. The eluent (lane 3) obtained using BaSO4 reagent A was used as a reference run.

[0151] Western blot analysis of the eluates (Example 2) obtained from porcine SD plasma using BaSO4 reagents A, B2, D2, D1, and C is presented in... Figure 3 middle.

[0152] The lane numbers represent the following:

[0153] 1–MW ladder

[0154] 2-Human prothrombin standard

[0155] 3 – Elution obtained using BaSO4 reagent A

[0156] 4 – Elution obtained using BaSO4 reagent B2

[0157] 5 – Elution obtained using BaSO4 reagent D2

[0158] 6 – Elution obtained using BaSO4 reagent D1

[0159] 7 – Elution obtained using BaSO4 reagent C

[0160] 8 – Human prethrombin 2 standard

[0161] 9–human α-thrombin standard

[0162] Figure 3 The results presented show that the eluent obtained from porcine SD plasma using pharmaceutical-grade BaSO4 (lane C-7, lanes D1-6) exhibits a similar banding pattern, including bands corresponding to prothrombin and intermediates. This banding pattern is similar to that of the eluent obtained using BaSO4 reagent A (lane 3). The eluent obtained using white standard grade BaSO4 ("D2"-lane 5) shows a much weaker band corresponding to prothrombin and stronger bands corresponding to intermediates and α-thrombin. The eluent obtained using reagent B2 (lane 4) shows the strongest prothrombin band and the weakest band corresponding to the intermediate.

[0163] NAPPT assay results

[0164] Table 2: Summary of NAPTT assay results

[0165]

[0166]

[0167] The NAPTT determination results of large-scale and laboratory-scale eluates obtained using BaSO4 reagents A, B1, B2, C, D1, and D2 are presented in Table 2.

[0168] The results showed that, regardless of the source of prothrombin, Western blot analysis revealed that BaSO4 reagents B1 and B2, which contained little or no prothrombin, caused slower clotting than control plasma.

[0169] The results also showed that, regardless of the source of prothrombin, BaSO4 reagent A, which had a significant thrombin content, exhibited faster clotting than control plasma, as indicated by Western blotting.

[0170] The protein composition of eluates of prothrombin adsorbed by BaSO4 from plasma using different BaSO4 reagents was analyzed using Western blotting, and the procoagulant activity was analyzed using NAPTT.

[0171] Western blot analysis results ( Figure 1 The results show the difference in binding patterns between the eluates (lanes 3 and 4, respectively) from samples obtained using BaSO4 reagents A and B. This suggests that prothrombin in the sample obtained using reagent A has undergone partial conversion to thrombin.

[0172] In the NAPTT coagulation test, clots formed immediately when the eluent obtained using BaSO4 reagent A was tested, while no coagulation was observed in the eluent obtained using reagent B, even after 450 seconds. It should be noted that, as actually observed, normal human plasma is expected to form clots within approximately 200–300 seconds in the NAPTT test, as shown in Table 2.

[0173] Various BaSO4 reagents are used to isolate prothrombin from porcine SD plasma and for the production of thrombin. Pharmaceutical-grade BaSO4 reagents A, C, and D1 yielded unsatisfactory results, as did the lower-grade BaSO4 reagents A and D2, while some pharmaceutical-grade BaSO4 reagents B1 and B2 provided excellent results.

[0174] The results showed that the specific BaSO4 reagent used significantly affected the procoagulant activity of BaSO4-adsorbed prothrombin and the thrombin yield.

[0175] It was also shown that at least some of the prothrombin was converted into thrombin and / or thrombin intermediates. Experimental results showed that the procoagulant assay, as described herein, can be used for the qualitative determination of whether a given BaSO4 reagent is suitable as a prothrombin adsorbent and to produce thrombin with increased yield at the end of the process.

[0176] It should be understood that, for clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for simplicity, various features of the invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination or as suitably as in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiments would not function without these elements.

[0177] Although the invention has been described in conjunction with its specific embodiments, it will be apparent to those skilled in the art that many alternative forms, modifications, and variations will be readily apparent. Therefore, it is contemplated that all such alternative forms, modifications, and variations falling within the scope of the appended claims be covered.

[0178] Any references cited or identified in this application should not be construed as an admission that such references are prior art to this invention.

[0179] Example 7: Prothrombin activation after elution of prothrombin from BaS04 reagent

[0180] Prothrombin eluted from BaSO4 A and B1 (as defined in the table below) is activated as follows:

[0181] Reagent Name Supplier Batch Number A 1 (Pharmaceutical grade) 1 B 2 (Pharmaceutical grade) 1

[0182] The eluted prothrombin solution was mixed with CaCl2 (6 g / L) and glycine (10 g / L), and the pH was adjusted. The solution was then filtered through a 0.22 μM filter and incubated at 20–25 °C for 8 hours, followed by incubation at 2–8 °C for up to 72 hours. The yield obtained using BaSO4B1 as an adsorbent was approximately 9 times higher than that obtained using BaSO4A as an adsorbent.

[0183] Examples demonstrate that selecting the appropriate BaSO4 reagent prior to large-scale thrombin production is advantageous in order to maximize thrombin yield.

[0184] This application also includes the following items.

[0185] 1. A method for preparing thrombin from a prothrombin source using a given BaSO4 reagent as an adsorbent for prothrombin, the method comprising:

[0186] a. Provide the given BaSO4 reagent and the source of the prothrombin;

[0187] b. Under conditions allowing prothrombin from the given prothrombin source to be adsorbed by the given BaSO4 reagent, contact a sample of the given BaSO4 reagent with the source of prothrombin to obtain BaSO4-adsorbed prothrombin; and

[0188] c. Evaluate the procoagulant activity of the prothrombin adsorbed by the BaSO4.

[0189] When the assessment of c. is performed by comparing the procoagulant activity of the prothrombin adsorbed by the BaSO4 with that of normal mammalian plasma, the suitability of the given BaSO4 reagent for the preparation of thrombin is indicated by the procoagulant activity of the prothrombin adsorbed by the BaSO4 not being greater than that of the normal mammalian plasma.

[0190] 2. A method for preparing thrombin from a prothrombin source using a given BaSO4 reagent as an adsorbent for prothrombin, the method comprising steps “a” to “c” of item 1, and further comprising:

[0191] d. Under conditions that allow for prothrombin adsorption, bring a suitable BaSO4 into contact with a source of prothrombin;

[0192] e. Using elution buffer, elute the prothrombin fraction from the prothrombin adsorbed by the BaSO4;

[0193] f. Apply conditions to the eluted fraction that allow prothrombin to be converted into thrombin, thereby obtaining thrombin.

[0194] 3. A method for evaluating the suitability of a given BaSO4 reagent as a prothrombin adsorbent in the preparation of thrombin from a prothrombin source, the method comprising:

[0195] a. Provide the given BaSO4 reagent and prothrombin source;

[0196] b. Under conditions allowing prothrombin from the given prothrombin source to be adsorbed by the given BaSO4 reagent, contact a sample of the given BaSO4 reagent with the source of prothrombin to obtain BaSO4-adsorbed prothrombin; and

[0197] c. Evaluate the procoagulant activity of the prothrombin adsorbed by the BaSO4.

[0198] When the evaluation of c. is carried out by comparing the procoagulant activity of the prothrombin adsorbed by the BaSO4 with that of normal mammalian plasma, the suitability of the given BaSO4 reagent as a prothrombin adsorbent in the preparation of thrombin is indicated by the procoagulant activity of the prothrombin adsorbed by the BaSO4 not being greater than that of the normal mammalian plasma.

[0199] 4. The method according to any one of items 1 to 3, wherein assessing the coagulant activity includes performing a coagulant assay.

[0200] 5. The method according to Project 4, wherein the coagulation assay includes a functional assay.

[0201] 6. The method according to Item 5, wherein the functional assay is a coagulation assay.

[0202] 7. The method according to Item 6, wherein the coagulation assay is selected from NAPTT assay, APTT assay and protease chromogenic assay.

[0203] 8. The method according to any one of items 1 to 7, wherein the prothrombin source is selected from plasma or plasma fraction.

[0204] 9. The method according to item 8, wherein the plasma comprises oxalate plasma.

[0205] 10. The method according to any one of items 1 to 9, wherein the source of said prothrombin includes plasma harvested from mammals.

[0206] 11. The method according to item 10, wherein the mammal is selected from humans, horses, cattle and pigs.

[0207] 12. The method according to any one of items 1 to 11, wherein the conditions under which prothrombin from the prothrombin source is allowed to be adsorbed by the given BaSO4 reagent include pH 7.4-8.6.

[0208] 13. The method according to any one of items 9 to 12, wherein contacting the sample of the given BaSO4 reagent with the prothrombin source comprises adding about 1% (w / v) BaSO4 reagent to the harvested plasma, the harvested plasma being the prothrombin source.

[0209] 14. The method according to any one of items 1 to 13, wherein the evaluation of the procoagulant activity of the prothrombin adsorbed by the BaSO4 is the procoagulant activity of the prothrombin adsorbed by the BaSO4 reagent when adsorbed onto the BaSO4 reagent.

[0210] 15. The method according to any one of items 1 to 13, further comprising:

[0211] After "b" and before "c", at least some of the prothrombin adsorbed by BaSO4 is eluted from the BaSO4 reagent; and

[0212] The evaluation of the procoagulant activity of the prothrombin adsorbed by the BaSO4 is the procoagulant activity of at least some of the prothrombin adsorbed by the eluted BaSO4.

[0213] 16. The method according to item 15, wherein eluting the prothrombin fraction from the prothrombin adsorbed by said BaSO4 comprises using a calcium chelate at pH of about 6.3 and 7.4.

[0214] 17. The method according to item 16, wherein the chelate salt comprises sodium citrate.

[0215] 18. The method according to Item 17, wherein the concentration of sodium citrate is from about 3% (w / v) to about 4.4% (w / v).

[0216] 19. The method according to any one of items 1 to 18, wherein the source of said prothrombin includes porcine plasma.

[0217] 20. The method according to any one of items 1 to 19, wherein when more than one BaSO4 reagent is evaluated, a suitable BaSO4 reagent is selected for adsorption of prothrombin.

Claims

1. A method for preparing thrombin from a prothrombin source using a given BaSO4 reagent as an adsorbent for prothrombin, the method comprising: a. Provide the given BaSO4 reagent and the source of the prothrombin; b. Under conditions allowing prothrombin from the given prothrombin source to be adsorbed by the given BaSO4 reagent, contact a sample of the given BaSO4 reagent with the source of prothrombin to obtain BaSO4-adsorbed prothrombin; and c. Evaluate the procoagulant activity of the prothrombin adsorbed by the BaSO4. When the assessment of c. is performed by comparing the procoagulant activity of the prothrombin adsorbed by the BaSO4 with that of normal mammalian plasma, the suitability of the given BaSO4 reagent for the preparation of thrombin is indicated by the procoagulant activity of the prothrombin adsorbed by the BaSO4 not being greater than that of the normal mammalian plasma.

2. A method for preparing thrombin from a prothrombin source using a given BaSO4 reagent as an adsorbent for prothrombin, the method comprising steps "a" to "c" of claim 1, and further comprising: d. Under conditions that allow for prothrombin adsorption, bring a suitable BaSO4 into contact with a source of prothrombin; e. Using elution buffer, elute the prothrombin fraction from the prothrombin adsorbed by the BaSO4; f. Apply conditions to the eluted fraction that allow prothrombin to be converted into thrombin, thereby obtaining thrombin.

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