CN112877313A

CN112877313A - Process for the preparation of thrombin

Info

Publication number: CN112877313A
Application number: CN202110184522.1A
Authority: CN
Inventors: L·韦斯曼; D·塞拉; I·诺尔; 江彩霞; 安宝昌
Original assignee: Guangzhou Bioseal Biotech Co Ltd; Omrix Biopharmaceuticals Ltd
Current assignee: Guangzhou Bioseal Biotech Co Ltd; Omrix Biopharmaceuticals Ltd
Priority date: 2015-02-06
Filing date: 2015-02-06
Publication date: 2021-06-01
Also published as: CN105985940A; CN105985940B

Abstract

The invention relates to the use of a given BaSO₄Reagents as adsorbents for prothrombin, methods for producing thrombin from prothrombin sources, and evaluation of given BaSO₄Reagents for use in a suitable method of preparing thrombin. In at least one embodiment, the method includes causing a given BaSO₄Contacting a sample of the reagent with a source of prothrombin to obtain BaSO₄Adsorbed prothrombin, and subsequent evaluation of BaSO₄Procoagulant activity of the adsorbed prothrombin. In another embodiment, the method comprises the step of assessing procoagulant activity relative to normal mammalian plasma.

Description

Process for the preparation of thrombin

This application is a divisional application of a patent application filed on 2015, 02/06, under application No. 201510064089.2, entitled "method for preparing thrombin".

Technical Field

The present invention relates to the field of thrombin production, and more particularly to the evaluation of a given BaSO₄A method of suitability of a reagent for use as a prothrombin adsorbent in the preparation of thrombin.

Background

Thrombin is a serine protease that promotes blood coagulation 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 multiple proteolytic feedback mechanisms. 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 coagulation factor to stop bleeding from wounds by converting fibrinogen into fibrin. Thrombin is a common component of surgical dressings and has been used in combination with fibrinogen and other coagulation proteins in two-component hemostatic systems such as fibrin glues, adhesives, and sealants.

Thrombin is produced by proteolytic activation of the precursor (zymogen) prothrombin. In order 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 which assembles on a negatively charged phospholipid membrane in the presence of calcium ions.

Thrombin can be produced from prothrombin by contacting a prothrombin source (such as plasma or blood fraction) with a solid adsorbent capable of adsorbing prothrombin from the prothrombin source, for example barium sulfate (BaSO)₄). Fixing deviceThe bulk adsorbent is typically washed with a wash solution to remove contaminants such as unbound proteins, and the prothrombin is subsequently eluted therefrom using an elution solution. After further optional purification and processing steps, the eluted prothrombin may be converted to thrombin by activation using an activating agent, such as calcium ions.

It is reported that when different BaSO are used₄Reagents, such as BaSO from different manufacturers₄Reagents, or even different reagent batches produced by a particular manufacturer, have significant differences in thrombin yield from a given volume of prothrombin source.

It has been suggested in the report that differences in thrombin yield are at least partially attributable to differences in thrombin yield by different BaSO₄Change in the adsorption of prothrombin to the reagent and the adsorption capacity is chosen to be BaSO₄Reagents are key factors for prothrombin adsorption. Surgenor and Neorker (1952) state that certain BaSO₄The reagent was more efficient than others in adsorbing prothrombin from plasma, whereas Voss D. (1965) noted when different BaSO's were used₄When the reagent is used for adsorbing the prothrombin complex, the same amount of BaSO required by the prothrombin complex is adsorbed₄Significant differences were found in the amounts and it is presumed that this was due to differences in their crystal structures. However, in the case of the applicant, different BaSO have been tested₄The morphology of the batches, and no significant differences were determined. In addition, some ions such as Ca²⁺Reportedly contribute to different BaSO₄Different adsorption rates of the batches, and in this case, at different BaSO₄Ca in batch²⁺Nor does it differ significantly.

Thrombin manufacturers have attempted to remove multiple different BaSO from multiple different BaSO by trial and error₄Selecting proper BaSO from the reagent₄Reagents to increase thrombin yield.

Disclosure of Invention

The present invention, in some embodiments thereof, relates to the use of a given BaSO₄Reagents as adsorbents for prothrombin, methods for producing thrombin from prothrombin sources, and methods for evaluating a given BaSO by₄Use of reagents for the preparation ofMethods of thrombin suitability: to give BaSO₄Contacting the reagent with a prothrombin source to obtain BaSO₄Adsorbed prothrombin and, in some embodiments, BaSO is evaluated relative to the procoagulant activity of normal mammalian plasma₄Procoagulant activity of the adsorbed prothrombin.

Contact is used herein in its broadest sense and refers to any type of combined action, such as contacting a prothrombin source with BaSO₄To a close enough proximity to enable a sufficient contact between the BaSO and the substrate₄And the source of prothrombin, a binding interaction will occur. Contacting includes, but is not limited to, mixing, blending and/or adding the sources to the BaSO₄Internally, or mixing BaSO₄Added to the source.

In the present invention, it has surprisingly been found that prothrombin is associated with some BaSO₄Reagent contact triggers conversion to thrombin (i.e., conversion of prothrombin to its intermediates and/or thrombin). This was found to be a premature conversion at the end of the production process which compromises thrombin yield.

In some embodiments of the methods described herein, procoagulant activity occurs upon conversion of prothrombin to its intermediates and/or thrombin. Such intermediates may be formed during the proteolytic conversion of prothrombin to thrombin. Non-limiting examples of intermediates are prethrombin and metathrombin (meizothrombin).

Some embodiments of the methods described herein allow for the identification of BaSO suitable for use as a prothrombin adsorbent₄Reagents, preferably in the presence of a given BaSO₄Reagents are used as adsorbents prior to large scale thrombin production processes.

Specifically, in some embodiments, a given BaSO is used₄Contacting a sample of the reagent with a source of prothrombin to adsorb prothrombin therefrom to obtain BaSO₄Adsorbed prothrombin, and subsequent evaluation of BaSO₄Procoagulant activity of the adsorbed prothrombin. Generally, BaSO₄The lower the procoagulant activity of the adsorbed prothrombin, the given BaSO₄The more suitable the reagent is for use as a prothrombin adsorbent. In some embodiments, a BaSO is given₄Suitability of the reagent as a prothrombin adsorbent in the preparation of thrombin by BaSO₄The procoagulant activity of the adsorbed prothrombin is no greater than that of normal mammalian plasma, e.g., normal human plasma, indicating procoagulant activity.

Normal mammalian plasma such as human plasma is a well known pooled or single donor plasma preparation intended for use as calibration plasma for various coagulation tests.

The normal human plasma may be sterile plasma obtained by: the liquid fractions of whole blood (to which solutions of potassium citrate or sodium citrate or both have been added), for example from eight or more healthy adults, are combined and exposed to ultraviolet light to destroy bacterial and viral contaminants.

The normal human plasma may be Unicalibrator coagulation test calibration plasma 00625.

At least in some cases, the methods described herein eliminate the selection of suitable BaSO by trial and error₄The need for reagents.

Some embodiments of the methods described herein are fast and easy to use, and potentially provide savings in time and/or production costs. Some embodiments of the methods described herein allow for increasing the thrombin yield from a given prothrombin source.

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

According to an aspect of some embodiments described herein, the invention provides for using a given BaSO₄A method for preparing thrombin from a prothrombin source using a reagent as an adsorbent for prothrombin, the method comprising:

a. providing a given BaSO₄Reagents and prothrombin sources;

b. allowing prothrombin from a prothrombin source to be given BaSO₄Under the condition of reagent adsorption, the given BaSO is added₄Contacting a sample of the reagent with a source of prothrombin, thereby obtaining BaSO₄Adsorbed prothrombin; and

c. evaluation of BaSO₄The procoagulant activity of the adsorbed prothrombin,

wherein when c. is evaluated by comparing BaSO₄Given that the procoagulant activity of the adsorbed prothrombin proceeds with that of normal mammalian plasma, BaSO₄Suitability of the reagents for the preparation of thrombin by BaSO₄The procoagulant activity of the adsorbed prothrombin is not greater than that of normal mammalian plasma.

According to a further aspect, the invention provides the use of a given BaSO₄A method for preparing thrombin from a prothrombin source, the method comprising steps a to c above, and further comprising:

d. subjecting the appropriate BaSO to conditions which allow adsorption of prothrombin₄Contacting with a source of prothrombin;

e. using an elution buffer from BaSO₄Eluting a prothrombin-containing fraction from the adsorbed prothrombin;

f. the eluted fraction is subjected to conditions which allow the conversion of prothrombin to thrombin, thereby obtaining thrombin.

In some embodiments, the method comprises collecting the eluted fraction after step "e".

In some embodiments, the method substantially avoids premature conversion of prothrombin to thrombin. By "premature conversion of prothrombin to thrombin" is meant that prothrombin is converted to thrombin at step "d".

Generally, the term "elution" as disclosed herein may be used interchangeably with the term "desorption".

In one embodiment, adsorption of prothrombin to BaSO is allowed in thrombin preparation₄Conditions of (b) include a pH of 7.4-8.6 and/or a BaSO concentration in the range of about 1% -22% (w/v), e.g., about 1%₄。

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.

In one embodiment, the conditions that allow the conversion of prothrombin to thrombin comprise subjecting prothrombin to an activator such as calcium ions.

According to a further aspect of some embodiments described herein, the present invention provides for evaluating a given BaSO₄A method of suitability of a reagent for preparing thrombin from a prothrombin source, the method comprising:

a. providing a given BaSO₄Reagents and prothrombin sources;

b. allowing the adsorption of prothrombin from a prothrombin source to a given BaSO₄Under the condition of reagent, the given BaSO₄Contacting a sample of the reagent with a source of prothrombin, thereby obtaining BaSO₄Adsorbed prothrombin;

c. evaluation of BaSO₄The procoagulant activity of the adsorbed prothrombin,

According to an aspect of some embodiments of the invention, the invention provides for using a given BaSO₄A method for preparing thrombin from a prothrombin source using a reagent as a prothrombin adsorbent, the method comprising:

a. providing a given BaSO₄Reagents and prothrombin sources;

c. evaluation of BaSO₄Procoagulant activity of the adsorbed prothrombin, in some embodiments, given BaSO₄Suitability of the reagent as a prothrombin adsorbent in the preparation of thrombin by BaSO₄The procoagulant activity of the adsorbed prothrombin is not greater than that of normal mammalian plasma. In some embodiments, the evaluation of c₄Procoagulant activity of adsorbed ProthrombinProcoagulant activity of normal mammalian plasma.

According to a further aspect of some embodiments of the present invention, the present invention provides a method for evaluating a given BaSO₄A method of suitability of a reagent for use as a prothrombin adsorbent in the preparation of thrombin from a prothrombin source, the method comprising:

a. providing a given BaSO₄Reagents and prothrombin sources;

c. evaluation of BaSO₄Procoagulant activity of the adsorbed prothrombin. In some embodiments, a BaSO is given₄Suitability of the reagent as a prothrombin adsorbent in the preparation of thrombin by BaSO₄The procoagulant activity of the adsorbed prothrombin is not greater than that of normal mammalian plasma. In some embodiments, the evaluation of c₄The procoagulant activity of the adsorbed prothrombin is performed in conjunction with the procoagulant activity of normal mammalian plasma.

In some embodiments of the methods disclosed herein, assessing procoagulant activity comprises performing a procoagulant assay.

In some embodiments of the methods disclosed herein, the procoagulant assay comprises a functional assay, such as a functional assay or an indicator assay (such as an immunoassay) selected from a coagulation assay (such as a NAPTT assay, an APTT assay, a protease chromogenic assay).

In some embodiments, the eluate obtained has BaSO with NAPTT ratio not less than 0.8₄The reagents are considered suitable for the preparation of thrombin.

In some embodiments, the eluate obtained has a NAPTT ratio of 0.8 or less or BaSO where a clot is immediately visually observed (which occurs after calcium addition and before clotting time can be recorded in the coagulant measurement machine)₄Reagents are considered unsuitable for the preparation of thrombin.

In some embodiments of the methods disclosed herein, the source of prothrombin is selected from plasma (such as oxalate plasma) or a plasma fraction. In some such embodiments, the prothrombin source comprises plasma harvested from a mammal (such as a human, horse, cow, and pig). In some embodiments, the source of prothrombin comprises porcine plasma. In some embodiments, the source of prothrombin is recombinant prothrombin. In some embodiments, the prothrombin source is subjected to a viral inactivation treatment. For example, the source is solvent/detergent (SD) treated plasma.

"Solvent Detergent (SD) virus inactivation treatment" generally refers to a process of inactivating enveloped or lipid-coated viruses by disrupting their lipid envelope. This treatment may 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 tri alkyl phosphate. The solvent-detergent composition for inactivating lipid-coated viruses may be any solvent-detergent composition known in the art, such as TnBP and Triton X-100; polysorbate 80 and sodium cholate, and other combinations.

The solvent (s)/detergent(s) concentrations used may be those commonly used in the art, for example as performed in US5094960A, US 4789545A. The one or more solvents/one or more detergents used may be at a concentration of > 0.1% TnBP and > 0.1% Triton X-100 in combination. The concentration of solvent (s)/detergent(s) used may 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.

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

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

In some embodiments of the methods disclosed herein, a given BaSO is used₄Contacting the sample of reagents with a prothrombin source comprises contacting about 1% to about 22% or about 1% to about 10% (w/v) BaSO₄Reagents are added to a source of prothrombin (e.g., harvested plasma). In some embodiments, about 1% (w/v) BaSO is added₄And (3) a reagent.

In some embodiments, prothrombin from a prothrombin source is allowed to be given BaSO₄The conditions for adsorption of the reagents include a pH of 7.4 to 8.6. In some embodiments, the conditions include, for example, room temperature in the range of 20 ℃ to 25 ℃.

Prothrombin by BaSO₄The adsorption of the reagents may be in batch mode or impregnated with BaSO₄Is carried out in a column.

In one embodiment, the prothrombin is passed through BaSO₄Is carried out in batch mode at room temperature, for example at 25 ℃ for 2 hours at a pH of 7.4 to 8.6.

In some embodiments of the methods described herein, BaSO is evaluated₄The procoagulant activity of the adsorbed prothrombin is adsorption to BaSO₄BaSO in reagent₄Procoagulant activity of the adsorbed prothrombin.

In some embodiments, the methods disclosed herein further comprise, after "b" and before "c", recovering from BaSO₄Elution of BaSO in the reagent₄At least some of the adsorbed prothrombin; and wherein BaSO is evaluated₄The procoagulant activity of the adsorbed prothrombin is eluted BaSO₄Procoagulant activity of at least some of the adsorbed prothrombin. In some such embodiments, from BaSO₄Elution of BaSO in the reagent₄At least some of the adsorbed prothrombin comprises reacting BaSO₄The reagent is contacted with an elution solution having a pH of not less than 6.0 and not more than 7.0.

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 no more than 6.5, no more than 6.6 and even no more than 6.7, or between about pH6.3 and 6.7. In some embodiments, the pH of the elution solution is between 6.3 and 7.4.

In some embodiments, the elution solution comprises a chelating salt. In some embodiments, the chelating salt concentration 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).

In some embodiments, a BaSO is given₄The reagent is a reagent comprising at least 75% (w/w) BaSO (weight/weight)₄E.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, and even at least 88% (w/w) BaSO₄。

Unless defined otherwise, 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 belongs. Furthermore, the descriptions, materials, methods, and examples are illustrative only and are not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

As used herein, the term "given BaSO₄Reagent "refers to a specified batch of BaSO from a specified supplier₄And (3) a reagent. Different given BaSO₄The reagents may thus be reagents provided by different suppliers, or different reagent batches provided by the same supplier.

As used herein, the term "procoagulant activity" refers to the promotion of blood coagulation.

As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" and grammatical variations thereof are to be taken as specifying the stated features, integers, steps or components but not precluding the addition of one or more additional features, integers, steps, components or groups thereof. These terms encompass the terms "consisting of …" and "consisting essentially of …".

As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.

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

Drawings

Some embodiments of the invention are described herein with reference to the accompanying drawings. It will be apparent to those skilled in the art how the description together with the drawings may make embodiments of the invention practical. The drawings are for illustrative purposes and are not intended to show structural details of the embodiments in more detail than is necessary for a fundamental understanding of the invention. For purposes of clarity, some objects depicted in the drawings are not drawn to scale.

In the drawings:

FIG. 1 shows BaSO obtained on a large scale₄Western blot analysis of the eluate of the adsorbed prothrombin sample, said large scale BaSO₄Samples of adsorbed prothrombin were tested using BaSO from different suppliers₄Preparation during thrombin production scale manufacturing of reagents;

FIG. 2 shows Western blot analysis of eluate from porcine plasma using different BaSO from the same supplier₄Preparing reagent batches;

FIG. 3 shows Western blot analysis of eluates from porcine plasma using BaSO from different suppliers₄And (4) preparing a reagent.

Detailed Description

The present invention, in some embodiments thereof, relates to the use of a given BaSO₄Reagents as adsorbents for prothrombin, methods for preparing thrombin from prothrombin sources, and methods for evaluating a given BaSO₄Method of suitability of reagents for the preparation of thrombin: to give BaSO₄Contacting the reagent with a prothrombin source to obtain BaSO₄Adsorbed prothrombin and, in some embodiments, BaSO is evaluated relative to the procoagulant activity of normal mammalian plasma₄Procoagulant activity of the adsorbed prothrombin.

The principles, uses and specific implementations taught herein may be better understood with reference to the accompanying description. Those skilled in the art, having the benefit of this disclosure, will be able to implement the invention without undue effort or experimentation.

Before explaining at least one embodiment in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. The phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Examples of the invention

Materials and methods

Material

Provided are BaSO as shown in Table 1 below₄Reagent:

₄table 1: BaSO reagent

Name of reagent	Suppliers of goods	Batch number
			A	1 (pharmaceutical grade)	1
B1	2 (pharmaceutical grade)	1
			B2	2 (pharmaceutical grade)	2
C	3 (medicine grade)	1
			D1	4 (pharmaceutical grade)	1
D2	4 (white standard grade)	1

Sodium chloride was obtained from Sigma-Aldrich (catalog No. S1679, analytically pure, at least 99.0% by weight).

Trisodium citrate dihydrate was obtained from Sigma-Aldrich (catalog number S1804, analytically pure, at least 99.0% by weight). Oxalate pig blood (including 0.25% (w/w) oxalate solution) was obtained from the slaughterhouse of Kibbutz Lahav (Israel).

BaSO₄The adsorbed prothrombin is obtained during large scale production of thrombin (also referred to herein as large scale samples), wherein BaSO used to adsorb prothrombin₄Selected from BaSO defined in Table 1 above₄One of the reagents A, B1.

Obtained from Sigma-Aldrich (catalog No. P8074, BioXtra, polyethylene glycol sorbitan monooleate).

Tri-n-butyl phosphate (TnBP) was obtained from Merck-Millipore (catalog number 100002, pharmaceutical grade).

Filter 1.2 μm was obtained from Sartorius Stedim India Pvt Ltd (Bangalore, India) (Cat. No.)

2300) And filters 0.45+0.2 μm were obtained from Sartorius Stedim India Pvt Ltd. (catalog No.)

300)。

For gel electrophoresis, a pre-cast polyacrylamide gel was obtained from Bio-Rad Laboratories Inc. (Mini-gel 4-20% from catalog No. 456-1096).

Sheep anti-human thrombin was obtained from Affinity Biologicals Inc. (Canada) (Cat. SAHT-IG)

Anti-sheep IgG alkaline phosphatase conjugate was obtained from Sigma-Aldrich (Cat. No. A-5187).

Example 1: preparation of solvent/detergent (SD) virus inactivated plasma

Oxalate swine blood was delivered on ice. Blood was centrifuged at 5,000g (5440rpm) relative centrifugal force (rcf) and 4 ℃ for 15 minutes. The supernatant containing plasma was collected, divided into aliquots (300-500ml) and stored frozen at-30 ℃ until needed.

For the preparation of SD treated plasma ("SD plasma"), aliquots of the frozen stored plasma were thawed at 37 ℃ 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 and the pH was monitored at 7.4-8.6.

₄Example 2: preparation of laboratory-Scale BaSO-adsorbed Prothrombin eluate from porcine SD plasma

SD plasma as described in example 1 above as a source of Prothrombin with the given BaSO listed in Table 1₄Sample contact of the reagent.

Specifically, for the six BaSO listed in table 1₄Each of the reagents:

(i) mixing BaSO₄(1% w/v) A sample of the reagent was added to one unit of SD plasma.

(ii) SD plasma/BaSO Using magnetic stirrer₄The reagent mixture was stirred at 25 ℃ for 2 hours (pH 7.4-8.6), allowing prothrombin in SD plasma to be bound by BaSO₄And (5) adsorbing the reagent.

(iii) The mixture was centrifuged at 5,000g (5,440rpm) at 4 ℃ for 15 minutes.

(iv) Mixing the supernatant with the precipitate (including BaSO)₄Adsorbed prothrombin) and each precipitate was stored separately at-80 ℃ until needed.

(v) The precipitate was thawed at room temperature (20-25 ℃) for 15 minutes prior to use.

(vi) BaSO the precipitate was mixed at a ratio of 1:1.4₄: the wash buffer (w/w) was resuspended in wash buffer (78mM NaCl pH 6.9-7.1) and mixed using a tube roller for 15 minutes at room temperature.

(vii) The precipitate in the wash buffer was then centrifuged at 5,000g (5,440rpm) for 15 minutes at 4 ℃ and the supernatant was collected and stored at-80 ℃ until needed. This step was repeated twice (i.e. three washes in total).

(viii) After the washing step, the washed precipitate (containing BaSO)₄Adsorbed prothrombin) BaSO at 1:1.4 ratio₄: the elution buffer (w/w) was resuspended in elution buffer (3% sodium citrate pH 6.3-6.7) and mixed for 15 minutes using a tube roller machine.

(ix) The pellet/elution buffer was then centrifuged at 5,000g (5,440rpm) at 4 ℃ for 15 minutes. The prothrombin-containing eluate was collected into a clean container placed on ice and the elution step was repeated four times (i.e., a total of five elution cycles were performed).

(x) The eluates from five elution cycles (containing desorbed prothrombin) were combined into a single eluate, filtered through a 0.2 μm PVDF filter and stored at-80 ℃ in a clean vessel until tested by NAPTT or Western blot.

₄Example 3: preparation of large-scale BaSO-adsorbed prothrombin eluate from porcine SD plasma

Preparation of frozen Prothrombin-adsorbed BaSO from porcine plasma₄Large scale samples of).

BaSO used₄The reagents are a and B1 as defined in table 1.

SD was performed using 1% Tween-80 and 0.3% TnBP for SD treatment. The pH is in the range of 7.4-8.6. The solution was incubated with SD at 24-26 ℃ for 6 hours.

In large scale samples, step v replaces the corresponding step in laboratory scale samples and the samples are processed up to step x.

Example 4: western blot analysis of the prothrombin and thrombin content of the eluate

Each of the eluates obtained as described in examples 2 and 3 above was performed using the Biuret method (each eluate corresponds to the six BaSO of table 1)₄One of the reagents) to calculate the loading volume for the Western blot assay and to verify that residual unbound proteins and other contaminants are removed by the washing steps (vi and vii).

To obtain better resolution of the proteins from each of the eluates, proteins from the eluate samples were separated under reducing conditions using SDS-PAGE gradient micro precast gels (4% -20%). Proteins were transferred from the gel to nitrocellulose membranes. Western blots were performed using sheep anti-human thrombin and secondary antibodies, anti-sheep IgG conjugated to alkaline phosphatase.

Some BaSO was found₄The adsorbed prothrombin sample comprises thrombin (see "results" section below).

Example 5: procoagulant Activity of the eluate

Each of the eight eluates prepared from porcine SD plasma (6 as in example 2 and 2 as in example 3) was evaluated for procoagulant activity using the non-activated partial thromboplastin time (NAPTT) assay, essentially as described in ph. Briefly, and as known to those of ordinary skill in the art, the NAPTT assay involves combining phospholipid (rabbit brain phospholipid) and calcium ion (e.g., CaCl)₂) With addition of the test eluate or adsorbed BaSO₄Adsorbed prothrombin samples and normal human plasma control [ Unicalibrator coagulation test calibration plasma 00625](which inherently contains prothrombin), calcium ions initiate coagulation in the test sample by converting prothrombin to thrombin. Thrombin causes clot formation by converting soluble fibrinogen to insoluble fibrin. Clotting times were measured using a coagulometer (ST ART4 Stago machine).

More specifically, 45 μ l of human plasma was added to each of the four test wells. For control samples, 45. mu.l TBSAlbumin buffer was added to each of two control wells and 45 μ Ι of diluted test sample was added to each of two additional control wells. Then 45 μ l of rabbit brain phospholipid 0.02% 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 ℃ for 1 minute. By mixing 45. mu.l of 0.025M CaCl₂(previous incubation at 37 ℃) reactions were initiated by adding to all wells and the clotting time was measured. The NAPTT ratio is calculated as the clotting time (in seconds) of the test sample divided by the clotting time of the normal human plasma control.

It was found that in the eluate in which active thrombin was identified (example 4), the clotting time was substantially shorter than that of the normal human plasma control. Clotting was found to be immediate in some of the eluates. The clotting time was found to exceed 450 seconds in some eluates.

Found to be due to BaSO₄The eluates of B1 and B2 obtained high yields of thrombin at the end of the process.

The results indicate that the NAPTT assay can be used to identify suitable BaSO for thrombin production₄And (3) a reagent.

₄Example 6: preparation of BaSO-adsorbed Prothrombin from porcine SD plasma

For six BaSO in Table 1₄Repeating steps (i) - (ii) above for each of the reagents.

Evaluation of adsorbed BaSO prepared from porcine SD plasma using the non-activated partial thromboplastin time (NAPTT) assay₄Procoagulant activity of the adsorbed prothrombin sample.

Results

Western blot analysis

Using BaSO₄Western blot analysis of the eluates (example 3) of reagents a and B1 (table 1) is presented in fig. 1.

Lane numbers represent the following:

1-MW ladder

2-human prothrombin Standard

3-use of BaSO₄Large-Scale eluate from reagent A

4-use of BaSO₄Large Scale eluate from reagent B1

5-human prethrombin 2 Standard

6-human alpha Thrombin Standard

In FIG. 1, BaSO is used₄In the large-scale eluate obtained with reagent B1 (lane 4), a band corresponding to prothrombin was clearly visible.

In FIG. 1, BaSO is used₄In the large-scale eluate obtained with reagent A (lane 3), the band corresponding to prothrombin was hardly visible. Other bands corresponding to prothrombin intermediate proteins as well as alpha thrombin are apparent.

Using BaSO₄Western blot analysis of the eluate from porcine SD plasma (example 2) for reagents A, B1 and B2 (table 1) is presented in figure 2.

Lane numbers represent the following:

1-MW ladder

2-human prothrombin Standard

3-use of BaSO₄Eluate obtained from reagent A

4-use of BaSO₄Eluate obtained from reagent B1

5-use of BaSO₄Eluate obtained from reagent B2

6-human alpha Thrombin Standard

The results presented in fig. 2 show that different BaSO supplied by the same supplier were used₄The eluate obtained from the batch (B1-lane 4, B2-lane 5) showed a similar band pattern. Using BaSO₄The eluate obtained with reagent A (lane 3) was run as a reference.

Using BaSO₄Western blot analysis of the eluate from porcine SD plasma (example 2) with reagents A, B2, D2, D1 and C is presented in fig. 3.

Lane numbers represent the following:

1-MW ladder

2-human prothrombin Standard

3-use of BaSO₄Eluate obtained from reagent A

4-use of BaSO₄Eluate obtained from reagent B2

5-use of BaSO₄Eluate obtained from reagent D2

6-use of BaSO₄Eluate obtained from reagent D1

7-use BaSO₄Eluate obtained from reagent C

8-human prethrombin 2 Standard

9-human alpha Thrombin Standard

The results presented in figure 3 show the use of pharmaceutical grade BaSO₄The eluate from porcine SD plasma (C-lane 7, D1-lane 6) showed a similar pattern of bands, including bands corresponding to prothrombin and intermediates. The stripe pattern is similar to the use of BaSO₄Band pattern of the eluate obtained with reagent A (lane 3). Using white standard grade BaSO₄The eluate obtained ("D2" -lane 5) shows a much weaker band corresponding to prothrombin and a more intense band corresponding to the intermediate and alpha thrombin. The eluate obtained using reagent B2 (lane 4) showed the strongest prothrombin band and the weakest band corresponding to the intermediate.

NAPPT assay results

Table 2: NAPTT assay results summarization

Using BaSO₄The results of NAPTT assays for large and laboratory scale eluates obtained with reagents A, B1, B2, C, D1, and D2 are presented in table 2.

The results show that, regardless of the prothrombin source, BaSO with little or no thrombin is shown by Western blot₄Reagents B1 and B2 caused slower clotting than control plasma.

The results also show that the thrombin-like enzyme is related to prothrombinSource independent, BaSO with significant thrombin content as shown by Western blot₄Reagent a showed faster clotting than control plasma.

Involving the use of different BaSO₄Reagent plasma-derived BaSO₄The eluate of the adsorbed prothrombin was analyzed for protein composition using Western blot and for procoagulant activity using NAPTT.

The results of the Western blot analysis (FIG. 1) are shown in the application of BaSO₄Differences in binding patterns between the eluates of samples obtained with reagents a and B (

lanes

3 and 4, respectively). This result suggests that prothrombin in the sample obtained using reagent a has undergone partial conversion to thrombin.

In the NAPTT coagulation assay, BaSO is used when the assay is performed₄The clot was formed immediately upon elution with reagent a, whereas in the eluate obtained with reagent B, no clotting was observed even after 450 seconds. It should be noted that as observed in fact, normal human plasma is expected to produce clots in the NAPTT assay within about 200-.

Various BaSO₄The reagents are used to separate prothrombin from porcine SD plasma and for the production of thrombin. Pharmaceutical grade BaSO₄Unsatisfactory results were obtained with reagents A, C and D1, as lower grade BaSO₄Reagents A and D2 were identical, and some pharmaceutical grades of BaSO₄Reagents B1 and B2 provided excellent results.

The results show the specific BaSO used₄Reagent significantly affects BaSO₄Procoagulant activity of the adsorbed prothrombin and thrombin yield.

Conversion of at least some of the prothrombin to thrombin and/or a thrombin intermediate is also shown. Experimental results show that the procoagulant assay can be used as described herein for the qualitative determination of a given BaSO₄Whether the reagent is suitable for use as a prothrombin adsorbent and produces increased yields of thrombin at the end of the process.

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

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

Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

₄Example 7: prothrombin activation following elution of prothrombin from BaSO reagent

From BaSO₄Prothrombin eluted from a and B1 (as defined in the table below) was activated as follows:

name of reagent	Suppliers of goods	Batch number
			A	1 (pharmaceutical grade)	1
B	2 (pharmaceutical grade)	1

Mixing the eluted prothrombin solution with CaCl₂(6 g/liter) and glycine (10 g/liter) were mixed and the pH was adjusted. Next, the solution was filtered through a 0.22 μ M filter and incubated at 20-25 ℃ for 8 hours followed by incubation at 2-8 ℃ for up to 72 hours. And use of BaSO₄Comparison of Thrombin yield obtained with A as adsorbent, BaSO₄The yield obtained with B1 as adsorbent was about 9 times higher.

Example demonstration of selection of appropriate BaSO Prior to Large Scale Thrombin production₄The reagents are advantageous in order to maximize thrombin yield.

The present application also includes the following items.

1. Using given BaSO₄A method for preparing thrombin from a prothrombin source using a reagent as an adsorbent for prothrombin, said method comprising:

a. providing the given BaSO₄Reagents and the source of prothrombin;

b. allowing prothrombin from said prothrombin source to be bound by said given BaSO₄Allowing the given BaSO to be adsorbed under the condition of reagent adsorption₄Contacting a sample of the reagent with said source of prothrombin, thereby obtaining BaSO₄Adsorbed prothrombin; and

c. evaluating the BaSO₄The procoagulant activity of the adsorbed prothrombin,

wherein when c. is evaluated by comparing the BaSO₄The procoagulant activity of the adsorbed prothrombin is in accordance with the procoagulant activity of normal mammalian plasma, the given BaSO₄Suitability of reagents for the preparation of thrombin by said BaSO₄The procoagulant activity of the adsorbed prothrombin is not greater than the procoagulant activity of the normal mammalian plasma.

2. Using given BaSO₄A method for preparing thrombin from a prothrombin source as an adsorbent for prothrombin, said method comprising steps "a" to "c" as described in item 1, and further comprising:

d. contacting a suitable BaSO4 with a source of prothrombin under conditions that allow adsorption of prothrombin;

e. using an elution buffer from the BaSO₄Eluting a prothrombin-containing fraction from the adsorbed prothrombin;

f. subjecting the eluted fraction to conditions allowing the conversion of prothrombin to thrombin, thereby obtaining thrombin.

3. For evaluating given BaSO₄A method of suitability of a reagent for use as a prothrombin adsorbent in the preparation of thrombin from a prothrombin source, the method comprising:

a. providing the given BaSO₄Reagents and prothrombin sources;

c. evaluating the BaSO₄The procoagulant activity of the adsorbed prothrombin,

wherein when c. is evaluated by comparing the BaSO₄The procoagulant activity of the adsorbed prothrombin is in accordance with the procoagulant activity of normal mammalian plasma, the given BaSO₄Suitability of reagents for use as prothrombin adsorbents in the preparation of thrombin by said BaSO₄The procoagulant activity of the adsorbed prothrombin is not greater than the procoagulant activity of the normal mammalian plasma.

4. The method of any of clauses 1 to 3, wherein assessing procoagulant activity comprises performing a procoagulant assay.

5. The method of clause 4, wherein the procoagulant assay comprises a functional assay.

6. The method of item 5, wherein the functional assay is a coagulation assay.

7. The method of clause 6, wherein the coagulation assay is selected from the group consisting of a NAPTT assay, an APTT assay, and a protease chromogenic assay.

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

9. The method of clause 8, wherein the plasma comprises oxalate plasma.

10. The method of any one of clauses 1-9, wherein the source of prothrombin comprises plasma harvested from a mammal.

11. The method of clause 10, wherein the mammal is selected from the group consisting of human, horse, cow, and pig.

12. The method of any of clauses 1-11, wherein prothrombin from the prothrombin source is allowed to be retained by the given BaSO₄The conditions for reagent adsorption include a pH of 7.4-8.6.

13. The method of any of clauses 9 to 12, wherein the given BaSO is allowed to stand₄Contacting a sample of the reagent with the source of prothrombin comprises contacting about 1% (w/v) BaSO₄Reagents are added to the harvested plasma, which is the source of the prothrombin.

14. The method of any of clauses 1 to 13, wherein the BaSO is evaluated₄The procoagulant activity of the adsorbed prothrombin is adsorption to the BaSO₄The BaSO of the reagent₄Procoagulant activity of the adsorbed prothrombin.

15. The method of any of items 1-13, further comprising:

after "b" and before "c", from the BaSO₄Eluting the BaSO in a reagent₄At least some of the adsorbed prothrombin; and is

Wherein the BaSO is evaluated₄The procoagulant activity of the adsorbed prothrombin is eluted BaSO₄Procoagulant activity of at least some of the adsorbed prothrombin.

16. The method of clause 15, wherein the BaSO is derived from the BaSO₄Elution of the prothrombin-containing fraction from the adsorbed prothrombin involves the use of calcium-chelating salts at a pH of about 6.3 and 7.4.

17. The method of clause 16, wherein the chelating salt comprises sodium citrate.

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

19. The method of any of clauses 1-18, wherein the source of prothrombin comprises porcine plasma.

20. The method of any of clauses 1 to 19, wherein when more than one BaSO is evaluated₄When the reagent is used, proper BaSO is selected₄The reagent is used for adsorbing prothrombin.

Claims

a. providing the given BaSO₄Reagents and the source of prothrombin;

c. evaluating the BaSO₄The procoagulant activity of the adsorbed prothrombin,

2. Using given BaSO₄A method for preparing thrombin from a prothrombin source as an adsorbent for prothrombin, the method comprising steps "a" to "c" of claim 1, and further comprising: