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WO2004050700A1 - Procede de preparation de solutions de thrombine concentrees et utilisation desdites solutions dans de la colle de fibrine - Google Patents

Procede de preparation de solutions de thrombine concentrees et utilisation desdites solutions dans de la colle de fibrine Download PDF

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Publication number
WO2004050700A1
WO2004050700A1 PCT/SE2003/001548 SE0301548W WO2004050700A1 WO 2004050700 A1 WO2004050700 A1 WO 2004050700A1 SE 0301548 W SE0301548 W SE 0301548W WO 2004050700 A1 WO2004050700 A1 WO 2004050700A1
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Prior art keywords
thrombin
solution
process according
concentration
activation
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PCT/SE2003/001548
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English (en)
Inventor
Stefan Winge
Peter Aizawa
Göran Karlsson
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Biovitrum Ab
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Priority to AU2003267906A priority Critical patent/AU2003267906A1/en
Publication of WO2004050700A1 publication Critical patent/WO2004050700A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/106—Fibrin; Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)

Definitions

  • Thrombin cleaves four peptide bonds in the central region of fibrinogen to release four fibrinopeptides .
  • a fibrin molecule devoid of these fibrinopeptides is called fibrin monomer. Fibrin monomers spontaneously assemble into the ordered fibrous arrays called fibrin. (Stryer, L. (1987) Biochemistry 3rd ed.)
  • fibrin monomers spontaneously assemble into the ordered fibrous arrays called fibrin.
  • thrombin preparation which is obtained from prothrombin which is, after activation to thrombin, purified by hydrophobic interaction chromatography (HIC) .
  • the adsorbent employed in the HIC is a gel with coupled phenyl radicals or other ligands with a similar hydrophobicity .
  • cation exchange chromatography Before or after the HIC it is also possible in addition to carry out cation exchange chromatography. The chromatographies in this case may be carried out as "positive" (binding of thrombin) or as "negative” (binding of the impurities) chromatography.
  • the thrombin solution obtained after chromatographic purification can then be subjected to virus inactivation or virus reduction such as filtration. Virus inactivation or reduction can also take place before the chromatographic purification.
  • a thrombin preparation which contains as stabiliser a noncovalently binding inhibitor, and to which further stabilisers can be added for stabilisation, is also described.
  • the abovementioned known production process is very synoptic and thus has a great potential for improvements, It is partly focused on the HIC and its combination with cation exchange chromatography. This combination is laborious and does not result in a satisfactory thrombin concentration. Additionally, the hydrophobicity of the phenyl radicals used as adsorbent during the HIC is too high to give an optimal result.
  • the abovementioned process is further focused on stabilisation of the final thrombin preparation. It does not, however, provide for the stabilisation necessary in connection with heat treatment methods for virus inactivation. Such methods are essential when preparing a high quality thrombin solutio .
  • An object of the present invention is to provide an improved process for the preparation of a concentrated thrombin solution.
  • a second object of the invention is to provide a process for the preparation of a concentrated thrombin solution which utilises synergy effects between its steps.
  • a further object of the present invention is to enable the production of an improved fibrin glue.
  • the invention provides a process for the preparation of a concentrated thrombin solution from an initial thrombin solution obtainable by activation of a prothrombin solution, which process comprises subjecting such an initial thrombin solution to the following consecutive steps:
  • the invention provides a thrombin solution having a thrombin concentration higher than about 100,000 IU/ l and the use of a thrombin solution in a component for a fibrin glue.
  • a pathogen inactivating method is included in the process to ensure a high virus safety of the biological material.
  • Heat treatment is a well known and efficient such method.
  • stabilisers must normally be added to a protein solution to protect the proteins from heat denaturation during heat treatment.
  • the thrombin solution may contain some heat denatured proteins after being subjected to the heat treatment. Possibly contaminating proteins and inactive thrombin molecules are present in the solution as well. Said stabilisers and protein impurities are removed during the hydrophobic interaction chromatography step.
  • an aliphatic adsorbent facilitates suitably weak bindings to the target protein to allow desorption of said protein in its native state. It is highly desirable to keep the thrombin in its native state throughout the process.
  • the combination of the heat treatment step and the subsequent hydrophobic interaction chromatography step offers additional advantages related to synergy effects:
  • the heat treatment step involves the use of salts as stabilisers.
  • the invention provides subsequent purifying by hydrophobic interaction chromatography, which is a process able to bind thrombin also at high salt levels.
  • a corresponding binding of thrombin at high salt levels can not be achieved by e.g. ion exchange chromatography on a gel with immobilised diethylaminoethyl (DEAE) groups.
  • a thrombin solution having a thrombin concentration higher than about 100,000 IU/ml allows the use of the solution in different applications without causing dilution of other components.
  • thrombin solution is used as a component in a fibrin glue .
  • optional steps can be present. Such optional steps improve the performance of the process by their specific actions as well as by introducing interaction advantages among steps.
  • the following description serves to further clarify the functions and features of each step, whether essential or optional, as well as interactions among steps.
  • certain optional steps can be omitted and/or present in a different order. Also described is a possible way of obtaining the initial thrombin solution by activation of prothrombin to thrombin. The thrombin solution according to the invention and its use are finally described.
  • the activation process of prothrombin to thrombin is, in vivo, a very complex system including several cofactors and inhibitors. It is essential to provide necessary cofactors as not all factors involved in the enzymatic cascade are present in plasma. Examples of such factors important for in vivo activation, not present in plasma, are calcium, phospholipids and tissue factor. Further, in commercial plasma fractionation some cofactors are purified separately and not available in the fraction used for preparation of thrombin. One way of obtaining the initial thrombin solution involves an activation process, which is optimised for in vi tro use. Thus, inhibitors are removed and the only cofactor that is needed to convert prothrombin to thrombin is factor X.
  • the initial thrombin solution of the present invention is obtainable by activation of a prothrombin solution.
  • a prothrombin solution that is essentially free from FVII, FIX, FVIII, calcium and/or phospholipids.
  • the initial thrombin solution is obtainable by activation of a prothrombin solution, said activation comprising adding a salt, preferably sodium citrate, in the concentration range of from 5 to 50 %wt, preferably from 20 to 40 %wt, most preferably around 28 %wt, to the prothrombin solution.
  • a salt preferably sodium citrate
  • the initial thrombin solution is obtainable by activation of a prothrombin solution at a temperature in the range of from 5 to 50 °C, preferably in the range of from 25 to 50 °C and more preferably around 44 °C.
  • Pathogen inactivating methods are included among the steps of the process of the present invention to ensure a high virus safety of the biological material.
  • Pasteurisation is a well-known virus inactivation method useful for protein solutions. It is performed at a temperature higher than 50 °C for a period of more than 1 hour, preferably at 60 °C for 10 h.
  • the heat treatment is performed at a pH in the range of from 6.5 to 7.5, preferably about 7.0.
  • stabilisers must normally be added to the protein solution to protect the proteins from heat denaturation during heat treatment. The choice of suitable stabilisers for a thrombin solution is discussed in Example 2 below.
  • a preferred stabiliser is sucrose, preferably at a concentration of from 25 to 75 %wt, more preferably around 50 %wt .
  • Other preferred stabilisers are the amino acids alanine (preferably at around 0.25 %wt) , arginine (preferably at around 1.5 %wt) , glycine (preferably at around 0.5 %wt) and lysine (preferably at around 1.3 %wt) .
  • a further preferred stabiliser is NaCl, preferably at around 0.4 %wt .
  • the said stabilisers may be present solely or in any combination, also in combination with other stabilising substances. Most preferred is a combination of all said stabilisers at their respective preferred concentrations.
  • the thrombin solution may contain some heat denatured proteins after being subjected to the heat treatment. Possibly, contaminating proteins and inactive thrombin molecules are present in the solution as well. The amount of said stabilisers and protein impurities is reduced during the hydrophobic interaction chromatography step.
  • HIC hydrophobic interaction chromatography
  • the adsorbent is a C 4 -C 8 alkyl gel.
  • the use of a hydrophobic ligand with a short carbon chain is advantageous.
  • a gel having phenyl or long carbon chain (more than about 8 carbon atoms) ligands may cause too strong bindings to the target enzyme (thrombin) and thus make it difficult to desorb in a native state.
  • the adsorbent is a butyl gel.
  • the hydrophobic interaction chromatography is preferably performed so as to bind thrombin to the gel.
  • Impurities are bound to the gel together with thrombin and are preferably washed away with a first sodium citrate solution before elution of thrombin.
  • the first sodium citrate solution preferably has a concentration in the interval of from 0.2 to 1.0 M, more preferably from 0.3 to 0.6 M.
  • more than one washing may take place. It is then desirable to perform the first washing at a concentration in the upper part of said range and further washings at lower concentrations.
  • thrombin is eluted from the gel by means of lowering the ionic strength.
  • the eluent is water or a second sodium citrate solution of a lower concentration than the first sodium citrate solution, preferably lower than 0.2 M, more preferably about 0.17 M.
  • Temperature and pH are important process parameters during the hydrophobic interaction chromatography. It is suitably performed at a pH in the interval of from 6.5 to 7.5, preferably about 7.0.
  • the hydrophobic interaction chromatography is suitably performed at a temperature in the interval of from 20 to 30 °C, preferably about 25 °C.
  • Virus filtration In addition to the abovementioned heat treatment step for virus inactivation, there are legal requirements for further virus reduction of biological material administered to humans.
  • the combination of different virus inactivation and reduction steps improves the biological safety of the product obtainable by the process of the present invention.
  • a suitable method for further virus reduction of the thrombin solution is virus filtration.
  • the thrombin solution is preferably filtered through a filter having a cut-off of about 15-20 nm, corresponding to a molecular weight of about 70,000. Two or more filters in series may optionally be used.
  • the number of viable viruses is preferably reduced at least 10 4 times by the described virus filtration.
  • the thrombin solution is optionally subjected to a further process step for formulation.
  • the formulation is preferably performed by concentration and desalting of the solution. Concentration and desalting can be performed simultaneously by ultrafiltration of the solution against a buffer solution. Stabilisers are then transferred from the buffer solution to the thrombin solution.
  • suitable stabilisers for long term storage is discussed in Preparatory example 4 below.
  • the buffer solution comprises sucrose, preferably from 2 to 10 %wt; NaCl, preferably from 0.5 to 1.5 % wt; arginine, preferably from 2 to 4 %wt; lysine, preferably from 1 to 4 %wt; and/or glycine, preferably from 0.5 to 1.5 %wt .
  • the buffer solution preferably has a pH of about 7.0.
  • the formulation is performed so as to achieve a thrombin solution of a concentration higher than about 100,000 IU/ml. It is also performed so as to achieve a thrombin solution comprising sucrose, preferably from 2 to 10 %wt; NaCl, preferably from 0.5 to 1.5 % wt; arginine, preferably from 2 to 4 %wt; lysine, preferably from 1 to 4 %wt; and/or glycine, preferably from 0.5 to 1.5 %wt .
  • the obtained thrombin solution can be further stabilised by freezing, preferably at a temperature lower than -70 °C. It will then be stable for at least one year. Further stabilisation can also be achieved by freeze-drying.
  • the purity of the thrombin solution achieved after the disclosed purification method is sufficiently high for in vivo purposes. However, for e.g. standard or stability purposes it may be advantageous to reach an even higher purity.
  • This can be achieved by adding an anion-exchange gel to the thrombin solution, stirring for a suitable period of time (about 1 hour) , and removing the gel by filtration or centrifugation. It is also possible to process the thrombin solution through a column with such a gel or through a positively charged filter. This additional procedure is preferably performed at a stage where the pH and ionic strength of the thrombin solution are at their physiological values, suitably after formulation of the thrombin solution but before final concentration of the same. The purity will typically be > 95 % after such a procedure.
  • the thrombin solution of the present invention is characterised in that it has a thrombin content higher than about 100,000 IU/ml. Such a high thrombin concentration is desirable in several applications as it decreases the dilution of other components being present in the application. Such a high concentration may also facilitate stable long term storage.
  • the thrombin solution of the present invention preferably has a purity higher than about 1000 IU/mg. High purity is a prerequisite for safe medical and surgical applications as well as for stable storage conditions .
  • the thrombin solution of the invention is stable for more than 12 months frozen.
  • Fibrin glues are applied to e.g. tissue wounds and consist substantially of a thrombin component and a fibrinogen component.
  • the components are mixed upon use and an adhesive, fibrin, is formed.
  • a highly viscous glue is desired to prevent the glue from draining off its point of application.
  • the thrombin solution according to the present invention is thus particularly useful for use in a component for a fibrin glue, as its high concentration prevents unnecessary dilution of the glue.
  • the thrombin component is preferably a composition further comprising hyaluronic acid.
  • Hyaluronic acid is a non-toxic substance enhancing the viscosity of the glue.
  • Hyaluronic acid is known to have positive effects on wound healing.
  • Such a composition may comprise from 50 to 5000, preferably around 500, IU thrombin/ml of hyaluronic acid.
  • the composition of the inventive thrombin solution and hyaluronic acid is stable for at least 2 months at 3 °C.
  • the thrombin solution according to the invention can also be used as a glue component or to prevent blood loss on its own, using fibrinogen from a sieving wound.
  • One such application is to apply the thrombin solution to a plaster.
  • Figures 1-3 show the effects of pH and temperature on the activation of prothrombin according to results from Preparatory example 1.
  • Figure 4 shows the effect of temperature on the activation of prothrombin according to results from Preparatory example 1.
  • Figure 5 shows the effect of salt concentration on the activation of prothrombin according to results from Preparatory example 1.
  • prothrombin (7.29 IU/ml) and FX (4.57 IU/ml) was subjected to activation of prothrombin to thrombin under different conditions.
  • prothrombin in 1561.7 g of a solution of prothrombin and factor X was activated to thrombin by the following procedure. 624.7 g of trisodium citrate was added to the solution and pH was adjusted to 8.0 by addition of 22.7 ml of 0.5 M sodium hydroxide. The solution was incubated under continuous stirring for 18 hours at 44 °C. Comparative example 1: Self activation of prothrombin
  • Starting material for this example was a solution of prothrombin and FX with the following properties: concentrated prothrombin/FX fraction with an absorbance at 280 nm of 200, pH 6.0, buffer 0.05 M sodium citrate, prothrombin concentration 600 IU/ml and factor X concentration 300 IU/ml.
  • the solution was stirred at 22-23 °C for 91 h, whereafter precipitated (milky looking solution) calcium citrate was removed using centrifugation (2 h, 3000 r/min) and filtration (pore sizes 5 ⁇ m, 1.2 ⁇ m and 0.8 ⁇ m in series) .
  • the obtained clear solution contained 34,390 IU/ml thrombin (thrombin recovery 24 %) and had a thrombin activity of 734 IU/mg.
  • Self activation and sodium citrate activation of prothrombin are compared in Table 1. Sodium citrate activation is preferable as it is faster and gives a higher recovery of thrombin.
  • thrombin can be successfully stabilised with different substances during heat treatment.
  • Thrombin recovery > 100 % may be due to activation of prothrombin residues present in the thrombin solution or to measurement inaccuracy.
  • Example 1 Heat treatment and hydrophobic interaction chromatography
  • Stabilisers were added in the following amounts: 0.24 %wt alanin, 1.52 %wt arginine, 0.54 %wt glycine, 0.42 %wt sodium chloride,
  • the heat treated thrombin solution was diluted (1 part of protein solution and 3 parts of aqueous buffer) to reach a salt content of 155 mg-of trisodium citrate and 3.6 mg of disodium phosphate per gram of solution (pH was adjusted to 7.0 using citric acid) .
  • the gel (equilibrated with the same salt solution) was loaded with 12 mg protein/ml gel at a flow of 2 column volumes/h.
  • the column was thereafter washed with 2 column volumes of equilibration buffer and 5 column volumes of an aqueous wash buffer which included 108 mg of trisodium citrate and 3.6 mg of disodium phosphate per gram of solution (pH was adjusted to 7.0 using citric acid).
  • the fraction which did not bind to the column and the wash fractions were discarded.
  • the bound thrombin fraction was eluted with an aqueous buffer containing 50 mg of trisodium citrate and 3.6 mg of disodium phosphate per gram of solution (pH was adjusted to 7.0 using citric acid) .
  • the fractions were analysed according to thrombin recovery, thrombin activity and protein recovery. As can be seen in Table 3, a specific activity of more than 1500 IU/mg and a thrombin recovery of about 50 % is obtained with this purification method.
  • a thrombin solution containing 7785 IU thrombin/ml and 18,1 mg protein/ml, and having a purity of 430 IU/mg was used as initial material. Portions of the initial material were adjusted to different pH (6.0, 6.5, 7.0, 7.5 and 8.0) and polyethylene glycol (PEG-4000) was added to different concentrations (10, 15 and 20 %wt) . The resulting solutions were stirred for 1 h at 25 °C and thereafter filtered through a 0.8 ⁇ m filter. The clear filtrates were analysed regarding protein content and thrombin activity. As can be seen in Table 4, the maximum activity achieved was slightly above 600 IU/mg, with no decrease in thrombin recovery. Table 4
  • the column was thereafter washed with 2 column volumes of equilibration buffer and the thrombin fraction was eluted with 5 column volumes of a solution of 0.02 M sodium phosphate and 0.05 M sodium citrate, pH 7.5.
  • the fraction was analysed according to thrombin recovery, thrombin activity and protein recovery. As can be seen in Table 5, a thrombin activity of more than 1100 IU/mg and a recovery of 100 %, is obtained with this purification method.
  • Example 2 Virus filtration
  • suitable stabilisers To ensure a stable purified thrombin drug, suitable stabilisers must be added to the final product. To samples of a protein solution comprising 252 IU thrombin/ml and 1.36 mg protein/ml, having a purity of 540 IU thrombin/mg protein, in a buffer solution of 0.05 M trisodium citrate at pH 7.0, different candidates of stabilisers, according to Tables 8 and 9, were added. The samples were then incubated at 37 °C, to accelerate the denaturation of the protein, (Table 7) or 2-8 °C (Table 8) and subsequently analysed for biological activity of thrombin after different periods of time. Finally the thrombin recovery was calculated. As can be seen in Tables 7-8, some of the stabilisers have better effects than others.
  • Thrombin recovery > 100 % may be due to activation of prothrombin residues present in the thrombin solution or to measurement inaccuracy.
  • Human blood plasma comprises 1 IU prothrombin/ml. In vivo conversion of 1 IU prothrombin theoretically results in 238 IU thrombin. The purification method used to obtain the concentrate presented in Table 9, has a recovery of 45 IU thrombin from 1 ml of human blood plasma. Analysis methods
  • thrombin standard and samples are diluted with buffer and added to a standard amount of fibrinogen. Since the concentration of fibrinogen is high and constant, the rate of clot formation is primarily determined by the thrombin concentration in the sample. The clotting time is inversely proportional to the thrombin concentration. The thrombin "clot" concentration analysis only detects active thrombin. The unit used for thrombin "clot" concentration is IU/ml.
  • Factor Ila hydrolyses the chromogenic substrate S-2238 and thereby liberates the chromoforic group para- nitroaniline (pNa) .
  • the hydrolysis is stopped with acid and the yellow color intensity, which is proportional to the factor Ila activity of the sample, is read photometrically at 405 nm against a reagent blank.
  • the thrombin concentration (substrate) analysis detects total thrombin (active and denatured forms) .
  • the unit used for thrombin concentration is IU/ml.
  • the ratio of thrombin "clot" concentration to thrombin concentration (substrate) reflects the amount of active thrombin compared to the total amount.
  • a ratio ⁇ 1 indicates that denatured thrombin molecules are present.
  • Thrombin recovery is a comparison of the total amount of proteins after and before an operation performed on a protein solution. Based on the analyses of thrombin concentration (substrate) (see above), the thrombin recovery is calculated as
  • Total protein concentration was measured as absorbance in a 1 cm cuvette at 280 nm against a diluent in a spectrophotometer from Amersham Pharmacia Biotech, model Ultraspec II, alternatively Ultraspec 2000. If necessary, the samples were diluted with saline to obtain an absorbance between 0.1 and 1.0. An absorptivity of 1.0 ml cm -1 mg -1 was assumed for all samples. The unit used for protein concentration is thus mg/ml .
  • Protein recovery is a comparision of the total amount of proteins after and before an operation performed on a protein solution. Based on the analysis of protein concentration (see above), the protein recovery is calculated as
  • thrombin activity (thrombin concentration) / (protein concentration) .
  • Virus inactivation Purification processes for pharmaceutical products derived from human or animal sources must contain virus reduction steps to ensure a safe product. This is stated by regulative authorities and specific procedures for the validation of such reduction steps are recommended.
  • Model viruses e.g. HIV, parvoviruses, hepatitis viruses
  • a reduction factor is determined for each model virus and each inactivation step.

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Abstract

Un des aspects de la présente invention concerne un procédé de préparation d'une solution de thrombine concentrée à partir d'une solution de thrombine initiale pouvant être obtenue par activation d'une solution de prothrombine. Ledit procédé consiste à soumettre ladite solution de thrombine initiale aux étapes consécutives suivantes : (a) traitement thermique et (b) chromatographie d'interaction hydrophobe sur un adsorbant aliphatique. Un autre aspect de la présente invention concerne une solution de thrombine ayant une concentration de thrombine supérieure à environ 100 000 IU/ml. Un autre aspect encore concerne l'utilisation d'une solution de thrombine dans un constituant pour une colle de fibrine.
PCT/SE2003/001548 2002-12-02 2003-10-06 Procede de preparation de solutions de thrombine concentrees et utilisation desdites solutions dans de la colle de fibrine WO2004050700A1 (fr)

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AU2003267906A AU2003267906A1 (en) 2002-12-02 2003-10-06 Process for preparating concentrated thrombin solutions and use in fibrin glue

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5866122A (en) * 1996-03-20 1999-02-02 Immuno Aktiengesellschaft Pharmaceutical preparation for treating blood coagulation disorders
US20010033837A1 (en) * 2000-03-18 2001-10-25 Aventis Behring Gmbh. Thrombin preparations and process for their production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5866122A (en) * 1996-03-20 1999-02-02 Immuno Aktiengesellschaft Pharmaceutical preparation for treating blood coagulation disorders
US20010033837A1 (en) * 2000-03-18 2001-10-25 Aventis Behring Gmbh. Thrombin preparations and process for their production

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHIMICA ET BIOPHYSICA ACTA, vol. 427, no. 2, April 1976 (1976-04-01), pages 575 - 585 *
DATABASE MEDLINE [online] HATTON M. W. ET AL.: "The affinity of human, rabbit and bovine thrombins for sepharose-lysine and other conjugates", accession no. NCBI Database accession no. 1268219 *

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