Method for preparation of thrombin

Abstract

The present invention is directed to methods for the production of thrombin from a source of prothrombin using a given BaSO.sub.4 reagent as an adsorbent of prothrombin as well as methods for evaluating the suitability of a given BaSO.sub.4 reagent for use in preparation of thrombin. In at least one embodiment, the method includes contacting a sample of the given BaSO.sub.4 reagent with a source of prothrombin to obtain BaSO.sub.4-adsorbed prothrombin, and subsequently evaluating the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin. In another embodiment, the method includes the step of evaluating relative to the pro-coagulant activity of normal mammalian plasma.

Claims

1. A method for the preparation of thrombin from a source of prothrombin, the method comprising: a. providing one or more BaSO.sub.4 reagents and a source of prothrombin; b. contacting the one or more BaSO.sub.4 reagents and the source of prothrombin under conditions allowing adsorption of prothrombin with the one or more BaSO.sub.4 reagents, thereby obtaining BaSO.sub.4-adsorbed prothrombin; c. testing the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin, and testing same pro-coagulant activity of normal mammalian plasma source; d. evaluating the one or more BaSO.sub.4 reagents as suitable BaSO.sub.4 adsorbing reagent and selecting a suitable reagent based on said evaluation; wherein the evaluation is carried out by comparing the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin to the same pro-coagulant activity of a normal mammalian plasma source, the selection of suitable BaSO.sub.4 reagent for use in preparing thrombin is based on the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin being not greater than the pro-coagulant activity of normal mammalian plasma source; e. contacting the suitable BaSO.sub.4 with a source of prothrombin under conditions allowing prothrombin adsorption; f. eluting a prothrombin-containing fraction from the BaSO.sub.4-adsorbed prothrombin by contacting the adsorbed prothrombin obtained in step e. with an elution buffer; and g. subjecting the eluted fraction to conditions which allow conversion of prothrombin into thrombin, thereby obtaining thrombin.

2. A method for evaluating the suitability of one or more BaSO.sub.4 reagents for use as a prothrombin adsorbent in preparing thrombin from a source of prothrombin, the method comprising: a. providing one or more BaSO.sub.4 reagents and a source of prothrombin; b. contacting the one or more BaSO.sub.4 reagents and the source of prothrombin under conditions allowing adsorption of prothrombin to the one or more BaSO.sub.4 reagents, thereby obtaining BaSO.sub.4-adsorbed prothrombin; c. testing the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin and testing same pro-coagulant activity of normal mammalian plasma source; and; d evaluating the one or more BaSO.sub.4 reagents as suitable BaSO.sub.4 adsorbing reagents and selecting those suitable reagents based on said evaluation; wherein the evaluation is carried out by comparing the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin to the same pro-coagulant activity of a normal mammalian plasma source, the selection of suitable one or more BaSO.sub.4 reagents for use as a prothrombin adsorbant in method of preparing thrombin is based on the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin being not greater than the pro-coagulant activity of normal mammalian plasma source.

3. The method of claim 2, wherein testing of the pro-coagulant activity comprises performing a pro-coagulant assay.

4. The method of claim 3, wherein the pro-coagulant assay comprises a functional assay.

5. The method of claim 4, wherein the functional assay is a clotting assay.

6. The method of claim 5, wherein the clotting assay is selected from the group consisting of an Non-Activated Partial Thromboplastin Time (NAPTT) assay, an Activated Partial Thromboplastin Time (APTT) assay and a protease chromogenic assay.

7. The method of claim 2, wherein the source of prothrombin is selected from the group consisting of blood plasma and a plasma fraction.

8. The method of claim 7, wherein the blood plasma or plasma fraction comprises oxalated plasma.

9. The method of claim 2, wherein the source of prothrombin comprises plasma harvested from a mammal.

10. The method of claim 9, wherein the mammal is selected from the group consisting of a human, an equine, a bovine and a porcine.

11. The method of any of claim 2, wherein the conditions allowing adsorption of prothrombin from the source of prothrombin by the BaSO.sub.4 one or more reagents comprise pH 7.4-8.6.

12. The method of claim 2, wherein contacting the sample of the one or more BaSO.sub.4 reagents and the source of prothrombin comprises adding about 1% (w/v) BaSO.sub.4 reagent to the harvested plasma that is the source of prothrombin.

13. The method of claim 2, wherein testing of the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin is performed while the prothrombin is adsorbed to the one or more BaSO.sub.4 reagents.

14. The method of claim 2, further comprising: subsequently to b and prior to c, eluting at least some of the BaSO.sub.4-adsorbed prothrombin from the BaSO.sub.4 reagent; and wherein testing of the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin is of at least some of the eluted BaSO.sub.4-adsorbed prothrombin.

15. The method of claim 14, wherein eluting a prothrombin-containing fraction from the BaSO.sub.4-adsorbed prothrombin comprises use of a calcium chelating salt at a pH of about 6.3 and 7.4.

16. The method of claim 15, wherein the chelating salt comprises sodium citrate.

17. The method of claim 16, wherein the concentration of sodium citrate is from about 3% (w/v) to about 4.4% (w/v).

18. The method of claim 2, wherein the source of prothrombin comprises porcine plasma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Some embodiments of the invention are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments of the invention may be practiced. The figures are for the purpose of illustrative discussion and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the invention. For the sake of clarity, some objects depicted in the figures are not to scale.

(2) In the Figures:

(3) FIG. 1 shows a Western blot analysis of eluants obtained from full scale BaSO.sub.4-adsorbed prothrombin samples prepared during production-scale manufacturing of thrombin using BaSO.sub.4 reagents from different suppliers;

(4) FIG. 2 shows a Western blot analysis of eluants obtained from porcine plasma, prepared using different lots of BaSO.sub.4 reagent from the same supplier;

(5) FIG. 3 shows a Western blot analysis of eluants obtained from porcine plasma, prepared using BaSO.sub.4 reagents from different suppliers

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

(6) The invention, in some embodiments thereof, relates to method for the preparation of thrombin from a source of prothrombin using a given BaSO.sub.4 reagent as an adsorbent of prothrombin as well as methods for evaluating the suitability of a given BaSO.sub.4 reagent for use in preparation of thrombin by contacting the given BaSO.sub.4 reagent with a source of prothrombin to obtain BaSO.sub.4-adsorbed prothrombin, and evaluating the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin, in some embodiments, relative to the pro-coagulant activity of normal mammalian plasma.

(7) The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description. Upon perusal of the description, one skilled in the art is able to implement the invention without undue effort or experimentation.

(8) 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 or of being practiced or carried out in various ways. The phraseology and terminology employed herein are for descriptive purpose and should not be regarded as limiting.

EXAMPLES

Materials and Methods

(9) Materials

(10) Provided were BaSO.sub.4 reagents as shown below in Table 1:

(11) TABLE-US-00001 TABLE 1 BaSO.sub.4 reagents Reagent designation Supplier Lot no. 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 std. grade) 1

(12) Sodium Chloride was obtained from Sigma-Aldrich (Cat #S1679, analytical grade, at least 99.0% by weight).

(13) Trisodium Citrate dihydrate was obtained from Sigma-Aldrich Cat #S1804, analytical grade, at least 99.0% by weight). Oxalated porcine blood (including 0.25% (w/w) oxalate solution) was obtained from the abbatoir of Kibbutz Lahav, Israel.

(14) BaSO.sub.4-adsorbed prothrombin was obtained during full scale production of thrombin (also referred to herein as full scale samples), wherein the BaSO.sub.4 used to adsorb prothrombin was selected from one of the BaSO.sub.4-reagents A, B1 defined above in Table 1.

(15) Tween-80 was obtained from Sigma-Aldrich (Cat #P8074, BioXtra, Polyethylene glycol sorbitan monooleate).

(16) Tri-n-butyl phosphate (TnBP) was obtained from Merck-Millipore (Cat #100002, Pharmaceutical Grade).

(17) Filter 1.2 m was obtained from Sartorius Stedim India Pvt Ltd., Bangalore, India (Cat #Sartopore 2 300) and filter 0.45+0.2 m was obtained from Sartorius Stedim India Pvt Ltd (Cat #Sartopore 300).

(18) For gel electrophoresis, precast polyacrylamide gels were obtained from Bio-Rad Laboratories Inc. (Mini-gel 4-20% was obtained from Cat. #: 456-1096).

(19) Sheep anti Human Thrombin was obtained from Affinity Biologicals Inc., Canada, (Cat #SAHT-IG)

(20) Anti Sheep IgG alkaline phosphatase conjugate was obtained from Sigma-Aldrich (Cat #A-5187).

Example 1: Preparation of Solvent/Detergent (SD) Viral Inactivated Plasma

(21) Oxalated porcine blood was delivered on ice. The blood was centrifuged at relative centrifugal force (rcf) of 5,000 g (5440 rpm) and temperature of 4 C. for 15 minutes. The supernatant, comprising plasma, was collected, divided into aliquots (300-500 ml) and stored frozen at 30 C. until required.

(22) For preparation of SD treated plasma (SD plasma), an aliquot of frozen stored plasma was thawed at 37 C. for approximately 1 hour, then passed through a 1.2 m filter, then through a 0.45+0.2 m filter. 0.5% Tween-80 and 0.15% (v/v) TnBP were added for the SD treatment, at pH 7.4-8.6. The solution was stirred at room temperature for 1 hour and the pH was monitored at 7.4-8.6.

Example 2: Preparation of Lab Scale Eluates of BaSO4-Adsorbed Prothrombin from Porcine SD Plasma

(23) The SD plasma described above in Example 1 as a source of prothrombin was contacted with samples of the given BaSO.sub.4 reagents listed in Table 1.

(24) Specifically, for each one of the six BaSO.sub.4 reagents listed in Table 1: (i) a sample of BaSO.sub.4 (1% w/v) reagent was added to a unit of SD plasma. (ii) The SD plasma/BaSO.sub.4 reagent mixture was stirred using a magnetic stirrer at 25 C. for 2 hours (pH 7.4-8.6), allowing adsorption of prothrombin in the SD plasma by the BaSO.sub.4 reagent. (iii) The mixture was centrifuged at 5,000 g (5,440 rpm) at 4 C. for 15 minutes. (iv) The supernatant and sediment (including BaSO.sub.4-adsorbed prothrombin) were separated and each sediment was stored separately at 80 C. until required. (v) Prior to use, the sediment was thawed for 15 minutes at room temperature (20-25 C.). (vi) The sediment was resuspended in washing buffer (78 mM NaCl pH 6.9-7.1) at 1:1.4 ratio BaSO.sub.4:washing buffer (w/w) and mixed using a tube roller for 15 minutes at room temperature. (vii) The sediment in washing buffer was then centrifuged at 5,000 g (5,440 rpm) at 4 C. for 15 minutes and the supernatant collected and stored at 80 C. until required. The step was repeated twice (i.e. a total of three washes were carried out). (viii) Following the washing steps, the washed sediment (including BaSO.sub.4-adsorbed prothrombin) was resuspended in elution buffer (3% sodium citrate pH 6.3-6.7) at 1:1.4 ratio BaSO.sub.4:elution buffer (w/w) and mixed using a tube roller for 15 minutes. (ix) The sediment/elution buffer was then centrifuged at 5,000 g (5,440 rpm) at 4 C. 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 (containing desorbed prothrombin) from the five elution cycles were combined into a single eluate, filtered through a 0.2 m PVDF filter and stored in a clean container at 80 C. until tested by NAPTT or Western Blot.

Example 3: Preparation of Full Scale Eluates of BaSO4-Adsorbed Prothrombin from Porcine SD Plasma

(25) Full scale samples of frozen prothrombin-adsorbed BaSO.sub.4 from porcine plasma were prepared).

(26) BaSO.sub.4 reagents used were A and B1 as defined in Table 1.

(27) SD was carried out using 1% Tween-80 and 0.3% TnBP for the SD treatment. The pH was in the range of 7.4-8.6. The solution was incubated with SD for 6 hour at 24-26 C.

(28) In the full scale samples, step v took the place of the corresponding step in lab-scale samples and the sample was processed up to step x.

Example 4: Western Blot Analysis of Prothrombin and Thrombin Content of Eluates

(29) Total protein measurement in each of the eluates (each eluate corresponding to one of the six BaSO.sub.4 reagents of Table 1) obtained as described above in Examples 2 and 3 was performed using the Biuret method in order to calculate the loading volume for Western blot assay and to verify that residual unbound proteins and other contaminants were removed by the washing steps (vi and vii).

(30) In order to obtain a better resolution of the proteins from each one of the eluates, proteins from the eluate samples were separated using SDS-PAGE gradient mini precast gel (4%-20%) under reducing conditions. The proteins were transferred from the gel onto a nitrocellulose membrane. Western blot was performed using Sheep anti Human Thrombin and a secondary antibody, Anti Sheep IgG conjugated to alkaline phosphatase.

(31) It was found that some BaSO.sub.4-adsorbed prothrombin samples included thrombin (see Results section below).

Example 5: Pro-Coagulation Activity of Eluates

(32) Pro-coagulation activity of each one of the eight eluates prepared from porcine SD plasma (6 as in Example 2 and 2 as in Example 3) were evaluated using the Non-Activated Partial Thromboplastin Time (NAPTT) assay, essentially as described in Ph. Eur. (2.6.22). Briefly and as known to a person having ordinary skill in the art, the NAPTT assay comprises concurrently adding phospholipids (Rabbit Brain Cephalin) and calcium ions (as CaCl.sub.2) to a tested eluate or adsorbed BaSO.sub.4-adsorbed prothrombin sample and to a normal human plasma control [Unicalibrator calibration Plasma for Coagulation Tests 00625] (that inherently contains prothrombin), the calcium ions initiate coagulation in a test sample by conversion of prothrombin to thrombin. The thrombin causes clot formation by conversion of soluble fibrinogen to insoluble fibrin. Clotting time is measured using a Coagulometer (ST ART4 Stago machine).

(33) More specifically, 45 l of human plasma were added to each of four test wells. For the control samples, 45 l of TBS/albumin buffer were added to each of two control wells, and 45 l of diluted test sample were added to each of two additional control wells. 45 l of Rabbit Brain Cephalin 0.02% solution were then added to each of the four test wells, followed by mixing the content of the wells by gentle shaking and incubation for 1 minute at 37 C. The reaction was started by addition of 45 l of 0.025 M CaCl.sub.2 (previously incubated at 37 C.) into all wells and the clotting time was measured. The NAPTT ratio was calculated as the clotting time (in seconds) of a tested sample divided by the clotting time of the normal human plasma control.

(34) It was found that in eluates in which active thrombin was identified (Example 4), the clotting time was substantially shorter than that of the normal human plasma control. It was found that in some eluates the clotting was immediate. It was found that in some eluates clotting time was above 450 seconds.

(35) It was found that eluates resulting from BaSO.sub.4 B1 and B2 gave a high yield of thrombin at the end of the process.

(36) The results indicate that the NAPTT assay can be used for identifying a suitable BaSO.sub.4 reagent for thrombin production.

Example 6: Preparation of BaSO4-Adsorbed Prothrombin from Porcine SD Plasma

(37) Steps (i)-(ii) above are repeated for each one of the six BaSO.sub.4 reagents in Table 1.

(38) Pro-coagulation activity of a sample of adsorbed BaSO.sub.4-adsorbed prothrombin prepared from porcine SD plasma is evaluated using the Non-Activated Partial Thromboplastin Time (NAPTT) assay.

(39) Results

(40) Western Blot Analysis

(41) Western blot analysis of the eluates (Example 3), using BaSO.sub.4 reagents A and B1 (Table 1) are presented in FIG. 1.

(42) Lane numbers represent the following:

(43) 1MW ladder

(44) 2Human Prothrombin std.

(45) 3Full scale eluate obtained using BaSO.sub.4 reagent A

(46) 4Full scale eluate obtained using BaSO.sub.4 reagent B1

(47) 5Human Prethrombin 2 std.

(48) 6Human alpha Thrombin std

(49) In FIG. 1, in the full scale eluate obtained using BaSO.sub.4 reagent B1 (lane 4), a band corresponding to prothrombin is clearly visible.

(50) In FIG. 1, in the full scale eluate obtained using BaSO.sub.4 reagent A (lane 3), a band corresponding to prothrombin is barely visible. Other bands corresponding to prothrombin intermediate proteins as well as alpha thrombin are apparent.

(51) Western blot analysis of eluates obtained from porcine SD plasma (Example 2) using BaSO.sub.4 reagents A, B1 and B2 (Table 1) are presented in FIG. 2.

(52) Lane numbers represent the following:

(53) 1MW ladder

(54) 2Human Prothrombin std.

(55) 3Eluate obtained using BaSO.sub.4 reagent A

(56) 4Eluate obtained using BaSO.sub.4 reagent B1

(57) 5Eluate obtained using BaSO.sub.4 reagent B2

(58) 6Human alpha Thrombin std.

(59) The results presented in FIG. 2 show that eluates obtained using different lots of BaSO.sub.4 supplied by the same manufacturer (B1lane 4, B2lane 5) displayed similar banding patterns. The eluate obtained using BaSO.sub.4 reagent A (lane 3) was run as a reference.

(60) Western blot analysis of eluates obtained from porcine SD plasma (Example 2) using BaSO.sub.4 reagents A, B2, D2, D1 and C are presented in FIG. 3.

(61) Lane numbers represent the following:

(62) 1MW ladder

(63) 2Human Prothrombin std.

(64) 3Eluate obtained using BaSO.sub.4 reagent A

(65) 4Eluate obtained using BaSO.sub.4 reagent B2

(66) 5Eluate obtained using BaSO.sub.4 reagent D2

(67) 6Eluate obtained using BaSO.sub.4 reagent D1

(68) 7Eluate obtained using BaSO.sub.4 reagent C

(69) 8Human prethrombin 2 std.

(70) 9Human alpha Thrombin std.

(71) The results presented in FIG. 3 show that eluates obtained from porcine SD plasma using pharmaceutical grade BaSO.sub.4 (Clane 7, D1lane 6) showed similar banding pattern, including bands corresponding with prothrombin and intermediates. This banding pattern was similar to that of eluates obtained using BaSO.sub.4 reagent A (lane 3). The eluate obtained with white std grade BaSO.sub.4 (D2lane 5) showed a much weaker band corresponding to prothrombin and more intense bands corresponding to intermediates and alpha thrombin. The eluate obtained using reagent B2 (lane 4) showed the most intense prothrombin band and the faintest bands corresponding to intermediates.

(72) NAPPT Assay Results

(73) TABLE-US-00002 TABLE 2 Summary of NAPTT assay results Prothrombin BaSO.sub.4 Clotting time Clotting time of eluates used reagent of control (sec) test sample (sec) Full scale A 308 Immediate Full scale B1 291 >450 Lab scale A 290 Immediate Lab scale B1 325 >450 Lab scale B2 276 >450 Lab scale C 283 Immediate Lab scale D1 286 Immediate Lab scale D2 267 Immediate

(74) Results of the NAPTT assay for full scale and lab-scale eluates obtained using BaSO.sub.4 reagents A, B1, B2, C, D1 and D2 are presented in Table 2.

(75) The results show that irrespective of the prothrombin source, BaSO.sub.4 reagents B1 and B2 that were shown to have little or no thrombin by Western Blot caused slower clotting than the control plasma.

(76) The results also show that irrespective of the prothrombin source, BaSO.sub.4 reagents A were shown to have a significant thrombin content by Western Blot faster clotting than the control plasma.

(77) Eluates including BaSO.sub.4-adsorbed prothrombin obtained from blood plasma using different BaSO.sub.4 reagents were analyzed for protein composition using Western Blot and pro-coagulant activity using NAPTT.

(78) The Western blot analysis results (FIG. 1) show differences in banding patterns between eluates from samples obtained using BaSO.sub.4 reagents A and B (lanes 3 and 4, respectively). This result suggests that prothrombin in the sample obtained using reagent A had undergone partial conversion to thrombin.

(79) In an NAPTT clotting assay, a clot was formed immediately when testing the eluate obtained using BaSO.sub.4 reagent A, whereas in the eluate obtained using reagent B, no clotting was observed even after 450 seconds. It should be noted that normal human plasma is expected to produce a clot in the NAPTT assay within about 200-300 seconds, as indeed was observed as seen in Table 2.

(80) Various different BaSO.sub.4 reagents were used to isolate prothrombin from porcine SD plasma and for producing thrombin. Pharmaceutical grade BaSO.sub.4 reagents A, C and D1 gave unsatisfactory results like lower-grade BaSO.sub.4 reagents A and D2, while some pharmaceutical-grade BaSO.sub.4 reagents B1 and B2 provide superior results.

(81) The results showed that the specific BaSO.sub.4 reagent used significantly affects the pro-coagulant activity of the BaSO.sub.4-adsorbed prothrombin and the yield of thrombin.

(82) It is also shown that at least some of the prothrombin is converted to thrombin and/or thrombin intermediates. The experimental results show that a procoagulation assay can be used as described herein to qualitatively determine whether a given BaSO.sub.4 reagent is suitable for use as a prothrombin adsorbent and production of increased yields of thrombin at the end of the process.

(83) 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 sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

(84) Although 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 appended claims.

(85) 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 invention.

Example 7: Activation of Prothrombin after Elution of the Prothrombin from the BaSO4 Reagent

(86) Prothrombin eluted from BaSO.sub.4 A and B1 (as defined in the Table below) was activated as follows:

(87) TABLE-US-00003 Reagent designation Supplier Lot no. A 1 (pharmaceutical grade) 1 B 2 (pharmaceutical grade) 1

(88) The eluted prothrombin solutions were mixed with CaCl.sub.2 (6 g/liter) and Glycine (10 g/liter) and the pH (was adjusted. Next, the solutions were filtered through 0.22 M Milter and incubated at 20-25 C. for 8 hours followed by incubation at 2-8 C. for up to 72 hours. The thrombin yield obtained using BaSO.sub.4 B1 as an adsorbent was about 9 times higher as compared to the yield obtained using BaSO.sub.4 A as an adsorbent.

(89) The example demonstrates that it is of advantage to select a suitable BaSO.sub.4 reagent prior to full scale thrombin manufacture in order to maximize thrombin yields.