Thrombin solution and methods of use thereof

Abstract

Provided are methods for lyophilization of an aqueous thrombin solution, thrombin solutions for use in such lyophilization methods, and solid thrombin compositions produced by such methods.

Claims

1. A solid composition comprising: (i) thrombin; (ii) a single saccharide or sugar alcohol, said single saccharide or sugar alcohol comprising one member selected from the group consisting of sucrose, maltose, and mannitol, at a concentration of about 19.5% to about 78%, by total weight of the composition; (iii) sodium acetate at a concentration of about 1% to about 20%, by total weight of the composition; (iv) albumin at a concentration of about 2% to about 53%, by total weight of the total composition; (v) calcium chloride at a concentration of about 2.5% to about 31%, by total weight of the composition; and (vi) sodium chloride at a concentration of about 6% to about 45%, by total weight of the composition, wherein the solid composition has a water content of less than 3%, by total weight of the composition, wherein the composition is a porous monolithic block comprising about 200 to about 2000 IU/cm.sup.3 thrombin.

2. The solid composition of claim 1, wherein said single saccharide or sugar alcohol comprises mannitol.

3. The solid composition of claim 1, comprising about 22% to about 66% mannitol, by total weight of the composition.

4. The solid composition of claim 1, comprising about 1.5% to about 10% sodium acetate, by total weight of the composition.

5. The solid composition of claim 1, comprising about 2.5% to about 43% albumin, by total weight of the composition.

6. The solid composition of claim 1, comprising about 4% to about 17% calcium chloride, by total weight of the composition.

7. The solid composition of claim 1, comprising about 9.5% to about 25% sodium chloride, by total weight of the composition.

8. The solid composition of claim 1, comprising mannitol at a concentration of about 22% to about 66%, by total weight of the composition, sodium acetate at a concentration of about 1.5% to about 10% by total weight of the composition; albumin at a concentration of about 2.5% to about 43%, by total weight of the composition; calcium chloride at a concentration of about 4% to about 17%, by total weight of the composition, and sodium chloride at a concentration of about 9.5% to about 25%, by total weight of the composition.

9. The solid composition of claim 1, having thrombin activity recovery of at least 95%.

10. The solid composition of claim 1, having a water content of not more than about 1.5%, by total weight of the composition, and a thrombin activity recovery of at least 98%.

11. The solid composition of claim 1, being stable for at least 2 years under non-freezing storage conditions.

12. The solid composition of claim 1, being stable for at least 2 years at room temperature.

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 the visual appearance of a cake obtained following an exemplary short lyophilization cycle as described herein.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

(4) The invention, in some embodiments thereof, relates to methods for lyophilization of an aqueous thrombin solution, thrombin solutions for use in such lyophilization methods, and solid thrombin compositions produced by such methods.

(5) 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.

(6) 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.

(7) As mentioned above, known thrombin solutions vary considerably in the number and types of excipients present, and in the concentrations of the individual excipients.

EXAMPLES

(8) Materials and Methods

(9) Thrombin Stock Solution:

(10) The thrombin stock solution used for preparing the aqueous thrombin solutions described in the Examples below comprised about 3,500 IU/ml thrombin, about 200 mM NaCl, with stabilizers 2% mannitol and 0.2% human serum albumin (HSA) (unless indicated otherwise). The stock solution was prepared from Factor II (pro-thrombin) of human plasma that was activated and purified by sequential use of diethylaminoethyl (DEAE) cellulose anion exchange resin and SP cation exchanger resin, essentially as described in U.S. Pat. No. 5,143,838, hereby incorporated by reference.

(11) Lyophilization:

(12) A. Lyophilization of a Thrombin Solution of Height 8 mm within the Lyophilization Vessel:

(13) Lyophilization was carried out on a lyophilizer shelf of a Christ Epsilion 2-8D freeze drier (Christ, Germany) in 8 ml siliconized glass vials (Schott, Germany). Each vial was filled with 2 ml aqueous thrombin solution. The height of the solution in each vial was approximately 8 mm.

(14) Two different short lyophilization cycles were carried out as specified in Table 1 (a total time of 34 hours) and Table 2 (a total time of 30 hours) below. Temperatures given are those of the lyophilizer shelf.

(15) Following lyophilization, a solid thrombin composition having a volume similar to the volume of the aqueous solution prior to lyophilization was obtained.

(16) TABLE-US-00001 TABLE 1 Duration Pressure Temperature Step (hr:min) (μbar) (° C.) Freezing ramp  2 Atm RT to −50° C. Freezing soak  5 Atm −50° C. Primary drying ramp 1:30 Atm to −50° C. to −15° C. 140 μBar Primary drying soak 13 140 μBar −15° C. Secondary drying ramp 1:30 140 μBar to −15° C. to 25° C. 12 μBar Secondary drying soak 11 12 μBar 25° C. Total Time 34

(17) As used herein, the term “soak” with regard to a freezing or drying process refers to maintaining the composition being lyophilized at a constant temperature and pressure for a specified period of time in order to effect freezing or drying, respectively.

(18) As used herein, the term “ramp” with regard to a freezing or drying process refers to a step in which the temperature and pressure of the composition being lyophilized is gradually changed over a specified period of time in order to bring the composition to a specified freezing or drying temperature and pressure respectively.

(19) TABLE-US-00002 TABLE 2 Duration Pressure Temperature Step (hr:min) (μbar) (° C.) Freezing ramp  2 Atm RT to −50° C. Freezing soak 4:40 Atm −50° C. Primary drying ramp 1:20 Atm to −50° C. to −15° C. 140 μBar Primary drying soak 11 140 μBar −15° C. Secondary drying ramp 1:30 140 μBar to −15° C. to 25° C. 12 μBar Secondary drying soak 9:30 12 μBar 25° C. Total Time 30
B. Lyophilization of a Thrombin Solution of Height 17 mm within the Lyophilization Vessel:

(20) Lyophilization was carried out on a lyophilzer shelf of a Christ Epsilion 2-8D freeze drier (Christ, Germany) in LYOGUARD® cups (GORE®). Each vial was filled with 52 ml aqueous thrombin solution. The height of the solution in each vial was approximately 17 mm.

(21) A short lyophilization cycles were carried out as specified in Table 3 (a total time of 68 hours). Temperatures given are those of the lyophilizer shelf.

(22) Following lyophilization, a solid thrombin composition having a volume similar to the volume of the aqueous solution prior to lyophilization was obtained.

(23) TABLE-US-00003 TABLE 3 Duration Pressure Temperature Step (hr:min) (μbar) (° C.) Freezing ramp 2:00 Atm RT to −50° C. Freezing soak 5:40 Atm −50° C. Primary drying ramp 0:50 Atm to −50° C. to −15° C. 120 μBar Primary drying 31:00  120 μBar −15° C. Intermediate drying 0:30 120 μBar −15° C. to 10° C. ramp Intermediate drying 12:00  120 μBar 10° C. Secondary drying ramp 1:00 140 μBar to 10° C. to 25° C. 12 μBar Secondary drying 15:00  12 μBar 25° C. Total Time 68:00 
Quantification of Water Content within the Lyophilized Thrombin Composition:

(24) Water content determination was carried out using the volumetric Karl Fischer Titration method (KFT), which is based on the US Pharmacopoeia assay (USP 27, <921>, P. 2398-2399). Prior to the titration, the water was extracted from the lyophilized composition by adding dried methanol to the vial containing the lyophilized composition and shaking the vial. A sample from the supernatant were removed for the titration.

(25) Determination of Thrombin Activity:

(26) Thrombin activity of aqueous thrombin solutions was determined using a Clotting Time assay by measuring thrombin clotting activity in the different solutions according to the modified European Pharmacopeia Assay (0903/1997) procedure. Briefly, standard solutions of thrombin (4, 6, 8 and 10 IU/ml) or the test solutions were incubated for 2 minutes at 37° C. Then, 40 μl of each test solution or standard solution was mixed with 160 μl fibrinogen solution (0.1%; Enzyme research; cat No FIB1 2800L) and clotting time was measured. A calibration curve of log clotting times vs. log thrombin concentration was plotted using the standards. Thrombin activity in the different test solutions was determined by the clotting time obtained (calculated automatically by a clotting machine (Diagnostica Stago STart Coagulation Analyzer) interpolated from the calibration curve and multiplied by the dilution factor).

(27) Thrombin Activity Recovery Following Reconstitution (% of Initial Activity):

(28) Thrombin activity was measured as described above in the test solutions prior to lyophilization, and in the solid thrombin composition obtained by lyophilization, after reconstitution with purified water to the original volume. The recovered activity was calculated by dividing the activity obtained in the solid thrombin composition following reconstitution by the activity obtained in the thrombin solution before lyophilization and multiplying by 100.

(29) Qualitative and Quantitative Evaluation of Solid Thrombin Compositions Obtained by a Short Lyophilization Process:

(30) Solid thrombin compositions obtained by subjecting different aqueous thrombin solutions to a short lyophilization cycle were evaluated by the following parameters: water content of the solid compositions, thrombin activity recovery following reconstitution of the solid compositions, and structural appearance of the cake (by visual inspection). Typically, a cake having a “good structural appearance” is defined as a cake having a volume similar to that of the aqueous thrombin solution prior to lyophilization, is a monolithic block, has uniform porosity throughout the solid composition, and having no apparent wet areas.

(31) For determining the effect of a short lyophilization process on different thrombin formulations, several aqueous thrombin solutions comprising different ingredients, e.g. different saccharides, salts, and excipients at different concentrations were prepared from the thrombin stock solution described in the “MATERIALS AND METHODS” section, and the solutions were then lyophilized using the short lyophilization cycle, as detailed in Tables 1, 2 or 3.

Example 1: Use of Different Saccharides and Concentrations or a Sugar Alcohol in a Thrombin Solution Subjected to a Short Lyophilization Cycle

(32) Disaccharides (sucrose and maltose), and a sugar alcohol (mannitol) at different concentrations were used in the preparation of aqueous thrombin solutions, and solid thrombin compositions obtained by use of such thrombin solutions in a short lyophilization cycle were studied.

(33) Mannitol was tested at concentrations of 1.6%, 2.1%, 2.6% and 4.6% (w/v) by addition of mannitol to the diluted stock solution as described below; and sucrose and maltose were tested at a concentration of 2% (w/v). [For preparing the thrombin test solutions, the thrombin stock solution described above was diluted 1:3.5 with a solution providing the following final compositions: 0.6% human serum albumin, 20 mM sodium acetate (0.27%), 130 mM NaCl (0.76%) and 0.6% CaCl.sub.2 in water at pH 7.0, and the tested saccharide or sugar alcohol was added to the solution in the concentrations listed above. In the case of mannitol, the solution was supplemented to the listed concentration (following dilution of the stock, the solution comprised about 0.6% mannitol).

(34) Of note, human serum albumin present in the thrombin stock solution was also present in all test solutions as a “background ingredient” in a concentration of approx. 0.06%. Also, the stock solution comprised 0.6% mannitol.

(35) The prepared aqueous thrombin solutions were lyophilized using the short lyophilization cycle as described in Table 2 above, to obtain solid thrombin compositions. The water content of each solid composition, and thrombin activity recovery following reconstitution of the lyophilized composition were measured. The results are presented in Table 4 below.

(36) TABLE-US-00004 TABLE 4 Water content in the Thrombin Activity Saccharide/sugar solid composition Recovery alcohol Tested (%) (%) Sucrose 2% 0.4 96 Maltose 2% 0.6 95 Mannitol 1.6% 0.6 100 Mannitol 2.1% ND 100 Mannitol 2.6% 0.6 98 Mannitol 4.6% 1.7 100

(37) The results showed that each of the tested saccharides and sugar alcohols, when present in the aqueous thrombin solution at a concentration of 2.6% or lower, resulted in solid thrombin compositions having a low water content (0.4-0.6%) following a short lyophilization cycle, and high thrombin activity recovery (95-100%) following reconstitution of the solid composition.

(38) The results also showed that the aqueous thrombin solutions resulted in cakes that maintained their structure without collapse, and which had a good structural appearance as defined above.

(39) In contrast, an aqueous thrombin solution comprising 4.6% mannitol, when subjected to a short lyophilization cycle, resulted in a shrunken cake with a higher water content of 1.7%.

(40) These results show that an aqueous thrombin solution for use in a short lyophilization cycle advantageously comprises about 1.6% to less than about 4.6% saccharides or sugar alcohols in order to obtain a solid and stable (structurally and functionally) thrombin composition.

Example 2: Use of Different Concentrations of Human Serum Albumin in a Thrombin Solution Subjected to a Short Lyophilization Cycle

(41) Different concentrations of HSA were used in the preparation of aqueous thrombin solutions, and solid thrombin compositions obtained by use of such thrombin solutions in a short lyophilization cycle were studied.

(42) HSA was tested at the following concentrations: 0.2%, 0.6%, 3% and 10%.

(43) The solutions were prepared from the thrombin stock solution by 1:3.5 dilution with a solution providing the following final composition: 2.6% mannitol, 20 mM sodium acetate (0.27%), 130 mM NaCl (0.76%) and 0.6% CaCl.sub.2 in water at pH 7.0.

(44) Of note, human serum albumin was present in all test solutions at a concentration of approx. 0.06%, (in addition to the added HSA concentrations listed above).

(45) The prepared aqueous thrombin solutions were lyophilized using the short lyophilization cycle described in Table 2 above, wherein the solution had a height of about 8 mm in the lyophilization vessel, to obtain solid thrombin compositions. The water content of each solid composition, and thrombin activity recovery following reconstitution of the solid composition were measured. The results are presented in Table 5 below.

(46) TABLE-US-00005 TABLE 5 HSA Water content in the Thrombin activity concentration solid composition recovery (%) (%) (%) 0.2 3.0 100 0.6 0.6 98 3 ND 89 10 0.3 28

(47) The results showed that changing the HSA concentration had a significant effect on the water content in the solid thrombin composition and on the thrombin activity recovery following reconstitution of the solid composition.

(48) More specifically, it was shown that a thrombin solution containing 0.6% HSA resulted in a solid composition having both relatively low water content and a high recovery of thrombin activity upon reconstitution. In contrast, thrombin solutions with lower HSA concentrations resulted in solid compositions with increased water content, while solutions with higher HSA concentrations resulted in a lower recovery of thrombin activity upon reconstitution.

(49) Therefore, it is shown that optimal thrombin solutions advantageously comprise HSA at a concentration of higher than about 0.2% to lower than about 3%.

Example 3: Use of Different Salts and Concentrations in a Thrombin Solution Subjected to a Short Lyophilization Cycle

(50) A. Effect of Sodium Chloride (NaCl) Concentration:

(51) Different concentrations of NaCl (90 mM, 120 mM and 150 mM, i.e. 0.5% w/v, 0.7 w/v, and 0.9 w/v, respectively) were used in the preparation of aqueous solutions devoid of thrombin, and solid compositions obtained by use of such solutions in a short lyophilization cycle were studied, wherein the height of the solutions in a lyophilization vessel was about 8 mm.

(52) The solutions contained, in addition to the different concentrations of NaCl, 2% mannitol, 0.6% HSA, 20 mM sodium acetate (0.27%), and 0.6% CaCl.sub.2 at pH 7.0.

(53) The solutions were lyophilized using a short cycle similar to that described in Table 2 above, wherein the height of the solution in the lyophilization vessel was about 8 mm, wherein the primary drying soak was an hour longer than described in Table 2.

(54) Following lyophilization, the water content in the solid composition was tested. The results are presented in Table 6 below.

(55) TABLE-US-00006 TABLE 6 Water content in the solid composition NaCl Concentration (%)  90 mM/0.5% w/v  .sup.  3% 120 mM/0.7% w/v  1.2% 150 mM/0.9% w/v <1.2%

(56) As shown in the Table, the water content was inversely proportional to NaCl concentration up to 150 mM.

(57) In additional similar experiments using thrombin solutions and solutions devoid of thrombin (data not shown), no further decrease in water content was found when NaCl concentration was increased beyond 150 mM.

(58) NaCl concentrations of 300 mM (1.75% w/v) and above had an inhibitory effect on thrombin activity as measured by clotting time assay (data not shown).

(59) With regards to structural appearance of the solid composition, a cake prepared from a solution comprising 90 mM NaCl had a poor appearance, being shrunken compared to the volume of the solution from which it was prepared, and having a granular consistency, with non-uniform porosity throughout the solid composition.

(60) Therefore, it was concluded that advantageously optimal thrombin solutions comprise NaCl at a concentration of at least 120 mM (0.7% w/v) to lower than 300 mM (1.75% w/v).

(61) B. Effect of Partial Replacement of NaCl by Potassium Chloride (KCl)

(62) The potential use of KCl instead of NaCl in the thrombin solution was assessed. Since NaCl is present in the thrombin stock solution used to prepare the test solutions, it cannot be completely removed and thus the solution was supplemented with 65 mM KCl (bringing the solution to a final salt concentration of about 125 mM). Also, an additional thrombin solution was tested, comprising the stock solution supplemented with NaCl, bringing the solution to a final salt concentration of 130 mM).

(63) Solutions supplemented with NaCl or with KCl were prepared by diluting stock solution 1:3.5, to provide a NaCl concentration of 60 mM. A first solution was then supplemented to an NaCl concentration of 130 mM; and a second solution was supplemented with 65 mM KCl.

(64) The stock solution was diluted so as to provide the following final composition: 2.6% mannitol, 20 mM sodium acetate (0.27%), and 0.6% CaCl.sub.2 in water at pH 7.0.

(65) The prepared solutions were lyophilized using the short lyophilization cycle described in Table 2 above, wherein the solution has a height in the lyophilization vessel of about 8 mm, and the water content in the solid compositions and the thrombin activity recovery following reconstitution of the solid composition were measured. The results are presented in Table 7 below.

(66) TABLE-US-00007 TABLE 7 Water content in the Thrombin activity solid composition recovery Salt Tested (%) (%) 130 mM NaCl (0.76% w/v) 0.6 98 65 mM (0.49% w/v) KCl; and 5.2 100 60 mM (0.35% w/v) NaCl

(67) It was found that partial replacement of NaCl with KCl in the solution had a detrimental effect on the water content of the solid thrombin composition obtained following a short lyophilization cycle (5.2% water content of solid composition following lyophilization of a solution containing NaCl plus KCl, as compared to 0.6% following lyophilzation of a solution containing NaCl without KCl). Similar thrombin activity recovery was obtained following reconstitution of a solid composition obtained from lyophilization of a solution comprising 130 mM NaCl and a solution comprising 65 mM KCl and 60 mM NaCl, as shown in Table 7.

(68) Visual inspection of the structural appearance of the solid composition revealed that replacement of NaCl with KCl resulted in a shrunken cake.

(69) It was therefore concluded that advantageously in order to obtain a solid composition with a relatively low water content, it is preferred that KCl is absent from the thrombin solution.

(70) It is further concluded that an optimal aqueous thrombin solution that yields a solid composition with low water content and a high thrombin activity recovery following lyophilization using a short cycle comprises about 1.6% to less than about 4.6% of saccharide or sugar alcohol, at least about 120 mM (0.7% w/v) to lower than about 300 mM (1.75% w/v) sodium chloride, and higher than about 0.2% to lower than about 3% albumin, in addition to about 0.3 to about 1.2% calcium chloride; and about 0.14 to about 0.7% sodium acetate.

Example 4: Use of the Solutions Described Herein and Solutions Similar to Prior Art Solutions in a Short Lyophilization Cycle with Height of Solution in Lyophilization Vessel of 8 mm

(71) Three solutions were prepared: a solution as described herein, and two solution which are similar to prior art solutions, as follows: 1. A thrombin solution as described herein, comprising 2% mannitol, 0.6% human serum albumin, 20 mM sodium acetate (0.27%), 130 mM sodium chloride (0.76%) and 40 mM CaCl.sub.2 (0.6%) in distilled water at pH 7.0. 2. A thrombin solution similar to that disclosed in EP 1766003B1 (Jiang et al.), comprising 3% sucrose, 4% mannitol, 150 mM NaCl (0.9%), 0.1% polyethylene glycol 3350 (PEG3350), 4 mM CaCl.sub.2 (0.04%); and 5 mM (0.08%) histidine in distilled water at pH 6.0. 3. A thrombin solution similar to that disclosed in EP 813598B1 (MacGregor et al.), comprising 40 mM (0.78%) gluconic acid, 20 mM (0.5%) tri-sodium citrate and 150 mM NaCl (0.9%) in distilled water at pH 6.5.
The components of the solutions are further presented in Table 8.

(72) TABLE-US-00008 TABLE 8 Solution 1 Solution 2 Solution 3 Mannitol   2% 4% — Albumin  0.6% — — Sodium acetate 0.27% — — Sodium chloride 0.76% 0.9%   0.9% Calcium chloride  0.6% 4 mM (0.04%) — Sucrose — 3% — PEG — 0.1%   — Histidine — 5 mM (0.08%) — Gluconic acid — — 40 mM (0.78%) Tri- sodium citrate — — 20 mM (0.5%) 

(73) All three thrombin solutions were prepared by diluting the thrombin stock solution described above 1:3.5 with a solution comprising the ingredients listed in each solution 1-3. Mannitol and human serum albumin were present in all three test solutions in a concentration of approx. 0.6% and 0.06%, respectively, as background ingredients

(74) The solutions were lyophilized using the short lyophilization cycles described in Table 1 or Table 2 above (wherein the height of the solution in the lyophilization vessel was about 8 mm). Water content of the solid thrombin composition, and thrombin activity recovery after reconstitution of the solid thrombin composition were measured, as described above.

(75) FIG. 1 shows the visual appearance of a solid thrombin composition obtained following a short lyophilization of the aqueous thrombin solution described herein (solution 1; #1 in FIG. 1); and a solid thrombin composition obtained following a short lyophilization of a solution according to EP 1766003B1 (solution 2; #2 in FIG. 1). The solid composition shown in FIG. 1 was obtained using the cycle as described in Table 1. Similar results were obtained when the cycle as described in Table 2 was used.

(76) Visual inspection of the appearance of the solid thrombin compositions revealed that the solid compositions obtained following lyophilization of the thrombin solution according to the invention and of solution 3 had a “good structural appearance” according to the definition discussed above. However, the cake obtained following use of the thrombin solution according to EP 1766003B1 (solution 2) in the short lyophilization cycle had a granulated structure.

(77) Water content in the three solid thrombin compositions and the thrombin activity recovery of the solid compositions is presented in Table 9 below.

(78) TABLE-US-00009 TABLE 9 Water Content in the Thrombin activity Average solid composition Average recovery thrombin Thrombin (%) water (%) activity Solution Cycle of Cycle of content Cycle of Cycle of recovery Tested Table 1 Table 2 (%) ±SD Table 1 Table 2 (%) ±SD Solution 1 0.8 0.6  0.7 ± 0.14 100 98 99 ± 1.4 Solution 2 3.2 4.1 3.65 ± 0.64 92 96 94 ± 2.8 Solution 3 1.6 3.3 2.45 ± 1.2  93 91 92 ± 1.4

(79) As shown in Table 9, it was found that following the short lyophilization cycle (as described in Tables 1 or 2), the lowest average water content was found in the lyophilized solid composition prepared using the solution described herein (0.7±0.14%).

(80) As further shown in Table 9, it was found that following reconstitution of the short-cycle lyophilized solid compositions, the highest thrombin activity recovery was obtained in the solid compositions prepared using the solution described herein (99±1.4%) according to Tables 1 or 2.

Example 5: Use of the Solutions Described Herein in a Short Lyophilization Cycle with Height of Solution in Lyophilization Vessel of 17 mm

(81) A first thrombin solution as described herein, comprising 2% mannitol, 0.6% human serum albumin, 20 mM sodium acetate (0.27%), 130 mM sodium chloride (0.76%) and 40 mM CaCl.sub.2 (0.6%) in distilled water at pH 7.0; and a second solution, similar to the first solution, but comprising a sodium chloride concentration of lower than 120 mM (0.7%) sodium chloride (lower than 0.76%) were lyophilized. The first solution was lyophilized using the short lyophilization cycles described in Table 3 above; and the second solution (with the lower sodium chloride concentration) was lyophilized in a lyophilization cycle having a duration of 108 hours (wherein the height of each solution in the lyophilization vessel was about 17 mm).

(82) Water content of both solid thrombin compositions, and thrombin activity recovery after reconstitution of both solid thrombin compositions were measured, as described above.

(83) In both solid compositions, water content was 1.5% and the thrombin activity recovery was 100%.

(84) The results show that the thrombin solution according to the invention enabled to shorten the duration of the lyophilization cycle by about 37%. In conclusion, the thrombin solution described herein advantageously enables use of a short lyophilization cycle that yields a solid composition having low water content and the high thrombin activity recovery as compared to known thrombin solutions.

(85) 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 are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

(86) 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.

(87) 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.