Purification of fibrinogen

0001The present invention relates to a process for the purification of fibrinogen, characterized in that it contains one or more process steps in which one or more contaminating proteins are depleted by negative chromatography and / or negative adsorption, in each case using hydrophobic gel. Furthermore, the invention relates to the fibrinogen obtained by the process according to the invention, which is distinguished by improved stability, and to the production and use of pharmaceutical preparations which contain this fibrinogen.

0002Fibrinogen plays a key role in blood clotting. Blood vessels are almost always damaged and bleeding occurs during injuries or operations. The blood clots in the area of ​​smaller wounds and the bleeding stops. The coagulation system protects the body against high blood losses. During blood coagulation, the soluble fibrinogen contained in the blood plasma is converted into the fibrous, insoluble fibrin in the presence of thrombin. If there is no fibrinogen, blood coagulation does not work properly. The deficiency can be compensated for by administering fibrinogen isolated from, for example, human blood plasma. Because of its importance for hemostasis and wound healing, fibrinogen is of great importance in clinical use.

0003Due to the high clinical importance of fibrinogen, there are many references in the literature that deal with different methods for the purification of this important protein. Fibrinogen is mainly purified from human, less often from animal plasma. It is also possible to purify recombinant fibrinogen, e.g. B. from cell culture after recombinant expression, or from the milk of transgenic animals. Human plasma contains a complex mixture of more than 100 proteins, with fibrinogen accounting for approximately 2% of the total amount of protein. The cleaning and isolation of fibrinogen therefore usually requires several steps and the possible combinations of these individual process steps are diverse.

0004Precipitation is traditionally an important part of the purification of fibrinogen from human plasma. Known precipitation methods use amino acids such as glycine or alanine, see for example the<patcit id="pcit0001" dnum="EP0383234A"><text>EP 0 383 234</text></patcit>, the <patcit id="pcit0002" dnum="WO0148016A"><text>WO 01/48016</text></patcit> or <nplcit id="ncit0001" npl-type="s"><text>Jakobsen &amp; Kierulf (Thrombosis Research 3 (1973) 145-159</text></nplcit>), Ammonium sulfate, see for example the <patcit id="pcit0003" dnum="US5773033A"><text>US 5,773,033</text></patcit>, <patcit id="pcit0004" dnum="US6037457A"><text>US 6,037,457</text></patcit> or <nplcit id="ncit0002" npl-type="s"><text>Takeda (Journal of Clinical Investigation 45 (1966) 103-111</text></nplcit>), Polymers such as polyethylene glycol (PEG), see for example the <patcit id="pcit0005" dnum="WO9525748A"><text>WO 95/25748</text></patcit> or <nplcit id="ncit0003" npl-type="s"><text>Vila et al. (Thrombosis Research 39 (1985) 651-656</text></nplcit>), Ethanol, see for example <patcit id="pcit0006" dnum="EP0408029A"><text>EP 0 408 029</text></patcit>where fibrinogen is precipitated with 5-10% ethanol and separated from other plasma proteins, or the <patcit id="pcit0007" dnum="US5099003A"><text>US 5,099,003</text></patcit> or <nplcit id="ncit0004" npl-type="s"><text>Blombäck &amp; Blombäck (Arkiv För Kemi 10 (1956) 415-443</text></nplcit>), Sulfated polysaccharides (SPS, eg heparin), see for example <patcit id="pcit0008" dnum="WO9937680A"><text>WO 99/37680</text></patcit> and <patcit id="pcit0009" dnum="US4210580A"><text>US 4,210,580</text></patcit> and low ionic strength solutions, see for example <patcit id="pcit0010" dnum="US4188318A"><text>US 4,188,318</text></patcit> and <patcit id="pcit0011" dnum="DE2636757"><text>DE 26 36 757</text></patcit>.

0005However, the purity of fibrinogen obtained solely on the basis of precipitations is not yet sufficient for some applications, so that various additional adsorption and chromatography steps for purifying fibrinogen are described in the prior art, additionally or alternatively.

0006Anion exchange chromatography is frequently used in the field of ion exchange chromatography. In this connection, reference should be made to the<patcit id="pcit0012" dnum="EP0555135A"><text>EP 0 555 135</text></patcit>, in which fibrinogen is bound to an anion exchanger column in a main process step, while eg albumin and inactivators do not bind. Fibrinogen is then eluted from the column. In the<patcit id="pcit0013" dnum="WO9305067A"><text>WO 93/05067</text></patcit> the binding of fibrinogen to anion exchangers is used to remove detergents added for virus inactivation. The<patcit id="pcit0014" dnum="WO0148016A"><text>WO 01/48016</text></patcit> describes the binding of fibrinogen to ion exchange material, preferably using the ω-amino acids which delay fibrinogen degradation, such as, for example, ε-aminocaproic acid (EACA) in the application and / or washing buffer. Such a process step enables in particular an efficient depletion of the contaminating plasminogen from solutions containing fibrinogen.

0007The cation exchanger chromatography is used, for example, in the purification of fibrinogen from the milk of transgenic animals described in the <patcit id="pcit0015" dnum="WO0017234A"><text>WO 00/17234</text></patcit>. In this case too, conditions are selected which result in the binding of fibrinogen to the column material and thus enable the separation of, for example, casein.

0008The property of binding fibrinogen to cation exchangers is described in the <patcit id="pcit0016" dnum="WO9101808A"><text>WO 91/01808</text></patcit> exploited to selectively fibrinogen, lipoproteins and urea from liquids such. B. to remove blood.

0009In the <patcit id="pcit0017" dnum="WO8912065A"><text>WO 89/12065</text></patcit> fibrinogen is purified in one process step through a heparin-Sepharose column. The conditions are chosen so that fibrinogen is adsorbed on the column material and above all albumins, immunoglobulins and virus inactivating substances can be separated.

0010The possibility of purifying fibrinogen via hydrophobic interactions is also described. In the<patcit id="pcit0018" dnum="WO0017239A"><text>WO 00/17239</text></patcit> the purification of fibrinogen from milk of transgenic animals is achieved in one process step by binding to a hydrophobic column in the presence of salts with subsequent elution. The possibility of using a hydrophobic column for fibrinogen from human plasma is also mentioned.

0011Various affinity chromatographies are also described, which have different ligands which are able to selectively bind fibrinogen. In this context, it should be noted, for example, that<patcit id="pcit0019" dnum="US6037457A"><text>US 6,037,457</text></patcit> or the <patcit id="pcit0020" dnum="WO9905176A"><text>WO 99/05176</text></patcit>that describe antibodies that specifically bind fibrinogen or fibrinogen peptides and can be used, among other things, to purify fibrinogen. The<patcit id="pcit0021" dnum="EP0789030A"><text>EP 0 789 030</text></patcit>, <patcit id="pcit0022" dnum="US5723579A"><text>US 5,723,579</text></patcit> and <patcit id="pcit0023" dnum="US5783663A"><text>US 5,783,663</text></patcit> describe peptides that bind fibrinogen and can accordingly be used as ligands in affinity columns for the purification of fibrinogen. Immobilized ristocetin was also used to bind fibrinogen and this was again eluted with 8 M urea (<nplcit id="ncit0005" npl-type="s"><text>Suzuki et al., Thrombosis Research 18 (1980) 707-715</text></nplcit>). In the<patcit id="pcit0024" dnum="WO9920655A"><text>WO 99/20655</text></patcit> finally, inactivated thrombin is used as a ligand for thrombin substrates, which also includes fibrinogen. In the<patcit id="pcit0025" dnum="WO9012803A"><text>WO 90/12803</text></patcit> describes the so-called IMAC (Immobilized Metal Affinity Chromatography) for the purification of certain proteins. Human fibrinogen is also listed as a possible protein that binds to the metal chelate matrix. Fibrinogen also binds to protamine agarose and can then be eluted in acid pH, see for example<patcit id="pcit0026" dnum="US6037457A"><text>US 6,037,457</text></patcit> or <nplcit id="ncit0006" npl-type="s"><text>Dempfle &amp; Heene (Thrombosis Research 46 (1987) 19-27</text></nplcit>).

0012All of these processes or process steps listed have in common that the conditions are selected such that fibrinogen binds to the chromatography or adsorption material and then has to be detached again from the gel material.

0013Processes in which fibrinogen passes through the column have hitherto only been described for processes in which the separation of the starting material into different “useful fractions” is desired or in which fibrinogen is an impurity.

0014In addition to fibrinogen, plasma or cryoprecipitate also contains large amounts of other clinically meaningful plasma proteins, so that it is also used as a starting material for fractionating and obtaining factor VIII (F VIII), fibronectin and Willebrand factor. For this reason, chromatographic process steps are also used which are able to separate all four or at least two of the main proteins. Anion exchangers are used for this under conditions that do not bind fibrinogen, but for example F VIII, from Willebrand factor and fibronectin, which are then selectively eluted by different ionic strengths. Please refer to the<patcit id="pcit0027" dnum="WO8912065A"><text>WO 89/12065</text></patcit>, the <patcit id="pcit0028" dnum="EP0359593A"><text>EP 0 359 593</text></patcit> or the <patcit id="pcit0029" dnum="US5252709A"><text>US 5,252,709</text></patcit>. In the<patcit id="pcit0030" dnum="EP0383234A"><text>EP 0 383 234</text></patcit> conditions are selected for the anion exchanger in which only F VIII binds, whereas Willebrand factor, fibronectin and fibrinogen pass through the column or remain in the supernatant (batch process). The<patcit id="pcit0031" dnum="EP0408029A"><text>EP 0 408 029</text></patcit> and <patcit id="pcit0032" dnum="US5138034A"><text>US 5,138,034</text></patcit> Finally, include a fractionation process in which a freeze / thaw treatment first separates F VIII and fibonectin by precipitation, factor IX (F IX) is adsorbed on a subsequent anion exchange column and the fibrinogen is precipitated from the passage of the column over ethanol. All of these cases involve the separation of the clinically important main proteins, which can then be added to further purification steps individually before they are used for pharmaceutical purposes. A similar process step with an anion exchange column is also used as a possible intermediate step in fibrinogen purification for removing F VIII in the<patcit id="pcit0033" dnum="EP0555135A"><text>EP 0 555 135</text></patcit>. In the<patcit id="pcit0034" dnum="WO9602571A"><text>WO 96/02571</text></patcit> or. <patcit id="pcit0035" dnum="US5834420A"><text>US 5,834,420</text></patcit> is used in an embodiment for the purification of fibrinogen, ion exchange material, similar to that in the <patcit id="pcit0036" dnum="WO8912065A"><text>WO 89/12065</text></patcit>, used to remove F VIII and Willebrand factor. Proteins such as F VIII and von Willebrand factor have no influence on the stability of fibrinogen. This process step is often also used in the production of F VIII concentrates in order to remove contaminants such as, for example, immunoglobulins, fibronectin and fibrinogen as completely as possible. In this context, reference is made, for example, to the<patcit id="pcit0037" dnum="US5043428A"><text>US 5,043,428</text></patcit> and <patcit id="pcit0038" dnum="EP0173242A"><text>EP 0 173 242</text></patcit>. In the<patcit id="pcit0039" dnum="US4210580A"><text>US 4,210,580</text></patcit> describes a process for the purification of fibronectin, in which fibrinogen can be separated off after the heparin precipitation by washing a subsequent anion exchange column. Also the<patcit id="pcit0040" dnum="US5099003A"><text>US 5,099,003</text></patcit> uses an anion exchanger, but in this case to remove factors II, VII, IX and X, which would otherwise cause the fibrinogen-containing solution to gel and clot after virus inactivation treatment with β-propiolactone and UV radiation. In the<patcit id="pcit0041" dnum="DE2902158"><text>DE 29 02 158</text></patcit>The process for the production of fibrinogen, prothrombin complex, antithrombin III and a solution of storage-stable serum proteins describes, among other things, a variant is described in which the prothrombin complex (F II, F VII, F IX and FX) is first obtained by adsorption on anion exchangers and then fibrinogen is isolated from the run by adsorption with colloidal silica. However, since this cleaning sequence often already leads to fibrinolysis, which has a disadvantageous effect on fibrinogen recovery, the variant is preferred in which fibrinogen is first separated by adsorption on silica and then the prothrombin complex is isolated with the aid of anion exchangers. In this case, the use of an anion exchanger has a rather adverse effect on the quality of the fibrinogen to be obtained from the run at the same time.

0015In the <patcit id="pcit0042" dnum="WO9951724A"><text>WO 99/51724</text></patcit> finally, a "negative" chromatography process is described for the purification of heterologous proteins, including fibrinogen, from the milk of transgenic animals. In this case, however, hydroxyapatite is used as the gel material to bind contaminating milk proteins.

0016Also described are special negative affinity chromatography, such as immobilized gelatin, which is able to deplete fibronectin through specific binding (see e.g. <nplcit id="ncit0007" npl-type="s"><text>Vuento &amp; Vaheri, Biochem. J. (1979) 183, 331-337</text></nplcit>). However, fibronectin is a structural protein that has no effect on the stability of fibrinogen.

0017It is also described that lysine or analogous compounds can bind plasminogen in a very specific manner and that this ability can be exploited for the purification of plasminogen by means of affinity chromatography with L-lysine substituted Sepharose (<nplcit id="ncit0008" npl-type="s"><text>Deutsch &amp; Mertz, Science 170 (1970) 1095-1096</text></nplcit>; <nplcit id="ncit0009" npl-type="s"><text>Matsuda et al., Thrombosis Research 1 (1972) 619-630</text></nplcit>). This ability of lysine ligands is used in some fibrinogen purification processes as a process step to deplete contaminating plasminogen. In this regard, reference is made, for example, to the<patcit id="pcit0043" dnum="WO9525748A"><text>WO 95/25748</text></patcit> or <patcit id="pcit0044" dnum="WO9305067A"><text>WO 93/05067</text></patcit>. In this case, however, only one protein, the plasminogen, is depleted very specifically, since the lysine binds very specifically to a specific epitope (Kringel domains) of the plasminogen.

0018In the <patcit id="pcit0045" dnum="WO0127623A"><text>WO 01/27623</text></patcit> describes a method of how isoagglutinins (blood group antibodies) can be removed from blood or blood components such as fibrinogen. In this case, antigens (eg oligosaccharides) are used as ligands of an affinity column that can specifically bind isoagglutinins. Removal of isoagglutinins is advantageous to avoid complications if the blood groups of the donor blood or the donor blood components differ from the blood group of the recipient. Isoagglutinins have no influence on the stability of fibrinogen against proteolysis.

0019They also apply <patcit id="pcit0046" dnum="EP0366946A"><text>EP 0 366 946</text></patcit> or. <patcit id="pcit0047" dnum="US5094960A"><text>US 5,094,960</text></patcit>which describe a method for removing lipid-soluble process chemicals from biological material, such as plasma, cryoprecipitate, coagulation factors and fibrinogen. A hydrophobic chromatography column is used, which adsorbs the process chemicals but not the proteins of the biological material under the selected conditions. This procedure is only about the removal of chemical substances, e.g. were added to inactivate viruses. Proteins, such as coagulation factors, which also include fibrinogen-degrading factors such as factor XI (F XI) (<nplcit id="ncit0010" npl-type="s"><text>Scott et al., Arch Biochem Biophys 249 (1986) 480-488</text></nplcit>), should not bind under the chosen conditions and should not be depleted in this patent. Similarly, in the<patcit id="pcit0048" dnum="DE2903131"><text>DE 29 03 131</text></patcit> only ions can be exchanged through the use of ion exchange material. This patent describes a process for the simultaneous purification of four products (antihemophilic globulin A, prothrombin complex, solution of storage-stable serum proteins and fibrinogen), a plasma from which calcium ions have been removed or additionally citrate ions when using citrate plasma is required as the starting material have been removed. Cation exchangers replace the calcium ions for sodium or hydrogen ions and anion exchangers the citrate ions for chloride or hydroxide ions. Fibrinogen, inter alia, can then be isolated from the ion exchange plasma thus obtained by adsorption on silica. The cation exchanger used in this case, however, only serves to exchange the calcium ions in order to avoid the onset of a coagulation reaction; this step does not purify.

0020Adsorption is also used in the purification of plasma proteins. So in the<patcit id="pcit0049" dnum="DE2903131"><text>DE 29 03 131</text></patcit> a tricalcium phosphate (TCP) adsorption was used to purify the prothrombin complex. In the<patcit id="pcit0050" dnum="WO9525748A"><text>WO 95/25748</text></patcit> it is used to remove the prothombin complex as a process step for the purification of fibrinogen. Also worth mentioning are the adsorption using aluminum hydroxide (Al (OH)₃), which binds factors of the prothrombin complex and can also play a role, for example, in the removal of lipids. Al (OH) is used₃ for example in the <patcit id="pcit0051" dnum="EP0383234A"><text>EP 0 383 234</text></patcit> or <patcit id="pcit0052" dnum="WO0148016A"><text>WO 01/48016</text></patcit>. A possible use of barium sulfate (BaSO₄).

0021All of the described methods for negative chromatography / adsorption are, with the exception of <patcit id="pcit0053" dnum="EP0366946A"><text>EP 0 366 946</text></patcit> or. <patcit id="pcit0054" dnum="US5094960A"><text>US 5,094,960</text></patcit>, in which only process chemicals are removed, as well as the <patcit id="pcit0055" dnum="DE2903131"><text>DE 29 03 131</text></patcit>, in which only calcium and citrate ions are removed, together that gel materials other than those described for process step (s) according to the invention are used. In addition, the particular advantage of the method step according to the invention is that by efficiently depleting one or more contaminating proteins, in particular fibrinogen-degrading proteins or their precursors, a fibrinogen preparation can be obtained, the stability of which is significantly increased in solution. For technical production, it is of particular interest to develop a cleaning process that is not only economically feasible from an industrial point of view, but also leads to a fibrinogen that remains largely stable when stored in solution for a long time.

0022Fibrinogen is a very large protein with a complex structure. It is a glycoprotein of approx. 340 kDa consisting of two symmetrical halves. Two alpha (Aα) □, beta (Bβ) and gamma (y) chains form an elongated molecular structure (approx. 47 nm) that forms three domains (one central E domain and two identical D domains). This complex structure is essential for the efficient formation of a stable fibrin matrix. The alpha chain in particular is affected by the beginning of proteolytic degradation by fibrinogen-degrading proteases. Such damage to the alpha chain causes a delayed start of coagulation after exposure to thrombin and suggests that proteolysis of the alpha chain (s) can influence fibrin polymerization. The three-dimensional structure of the fibrin clot is also affected by a damaged alpha chain. The result is a finer fibrin network with thinner fibrils and less mechanical stability. The C-terminal ends of the alpha chains contain amino acid sequences at which bonds between neighboring fibrin molecules, catalyzed by activated F XIII, are established. Missing cross-links reduce the stability of the fibrin matrix. All of this shows that fibrinogen with severely damaged alpha chains has a reduced quality and is not desirable in pharmaceutical use, for example for fibrin adhesives. The same applies to the proteolytic degradation of the other fibrinogen chains, which usually takes place with slower kinetics. The advantage of the method steps according to the invention lies in the depletion of fibrinogen-degrading proteins with the result of an increased stability of the fibrinogen in aqueous solution.

0023Processes which bring about the greatest possible removal of fibrinogen-degrading proteins or their proenzymes are therefore particularly advantageous since the stability and efficiency of the fibrinogen solution obtained is decisively improved even when stored in liquid form for a long time. Storage in liquid form is of particular advantage for fibrinogen, since the active substance can be used immediately on the patient and thus the time required for the reconstitution of lyophilized preparations or the thawing and heating of frozen preparations is unnecessary. However, even when stored as a lyophilisate or in the frozen state, it is advantageous if the reconstituted or thawed fibrinogen is stable for even longer. This can be seen, for example, in situations where e.g. B. As a precautionary material for operations was reconstituted, but then the use was not necessary for medical reasons. This material has to be discarded with only short-term stability and could no longer be used at a later point in time. When using fibrin glue, it is particularly advantageous if fibrinogen is in liquid form. The commercially available adhesives are usually two components. One component comprises fibrinogen, often together with factor XIII and a fibrinolysis inhibitor such as aprotinin, and the other component comprises thrombin, often together with calcium ions. Reconstituting to make the glue ready for use takes a relatively long time, especially since fibrinogen is present in high concentrations.

0024Another great advantage of the fibrinogen, which was obtained by the method according to the invention, is the possibility of liquid storage for a certain time even at room temperature, which can, for example, improve the application properties in emergency situations. Even on longer shipping routes, where low temperatures may not always be guaranteed, it is advantageous if the stability must also be guaranteed at room temperature during this phase. Stable storage of fibrinogen in solution thus facilitates the manufacture, use, transport and administration to the patient in many ways. Because of the advantageously increased stability of the fibrinogen, which is produced by the process according to the invention, it is also possible in many pharmaceutical preparations to add fibrinolysis or fibrinogenolysis inhibitors, which under certain circumstances can lead to undesirable side effects or which are avoided to reduce potential risks are supposed to do without. In summary, despite the numerous cleaning methods described, there remains a need for improved methods which enable economical, large-scale production of stable fibrinogen solutions.

0025The present invention is therefore based on the object of specifying a method for purifying fibrinogen which provides a fibrinogen of high stability with a good yield. Surprisingly, cleaning processes have been found which can also be carried out economically on an industrial scale and lead to a fibrinogen, the stability of which is significantly increased in solution.

0026The invention therefore relates to a process for the purification of fibrinogen, characterized in that it contains one or more process steps in which one or more contaminating proteins are depleted by negative chromatography (s) and / or negative adsorption (s) using hydrophobic gel be carried out, the negative chromatography and / or the negative adsorption at a pH between 5.5 and 9. The invention furthermore relates to a fibrinogen preparation which is obtainable by the process steps described and which is distinguished by improved stability after storage at room temperature and / or by a reduced content of fibrinogen-degrading proteases or their proenzymes.

0027The proportion of low-molecular fibrinogen degradation fragments when stored at 30 ° C. is taken as a measure of the fibrinogen stability. These degradation fragments are determined by size exclusion chromatography as peaks with a smaller molecular weight than the main fibrinogen peak (peak 4 or ≤ peak 4). A fibrinogen preparation is considered stable in the sense of the invention if the absolute proportion of fibrinogen degradation fragments (peak 4 or ≤ peak 4) after storage at 30 ° C. after one month in the liquid state is below 3%, preferably below 2%, 5%.

0028Purification in the sense of the present invention can be a chromatographic purification process or a batch adsorption process.

0029Processes have proven successful in which a fibrinogen preparation of at least 50% (w / w), preferably at least 70% (w / w), purity is first produced by one or more precipitation steps, which purification is then carried out by negative chromatography (s) and / or negative adsorption (s) is further purified using hydrophobic gel and optionally by additional described methods.

0030The methods according to the prior art fundamentally differ from the method step of negative chromatography or negative adsorption according to the invention, in which the conditions are chosen such that fibrinogen does not bind or only binds to a minor extent and thus passes through the column with chromatographic separation. The predominant proportion of fibrinogen is thus in the passage or in the supernatant during adsorption. The advantage of the process step according to the invention is, inter alia, that conditions can be selected which do not impair the structure and functionality of the fibrinogen and, moreover, lead to high yields. Since essentially only secondary components have to be bound and separated off in the process step according to the invention, in particular after prior purification by precipitation, an economical, large-scale implementation is possible due to small column dimensions and simple technical equipment. In contrast, the chromatographies / adsorptions with binding of fibrinogen described so far have some disadvantages. Since fibrinogen is bound, large column dimensions are required which, due to the large amounts of gel material and due to the more complex technical equipment, necessitate a more expensive process. For large-scale production, elutions via salt gradients, for example, can also be a bottleneck. In some cases, harsh chemical conditions that can impair the functionality of fibrinogen must also be used in order to elute the bound fibrinogen again. In some cases, the cleanings described are also associated with high losses of fibrinogen or cannot be used economically for the large-scale purification of fibrinogen, for. B. due to expensive or not commercially available gel materials.

0031The following embodiments have proven to be advantageous:

0032Method as described above, wherein the hydrophobic gel contains alkyl groups as functional groups.

0033Method as described above, wherein the hydrophobic gel contains phenyl groups or derivatized phenyl groups as functional groups.

0034Method as described above, wherein the hydrophobic gel contains propyl, butyl, pentyl, hexyl or octyl groups as functional groups.

0035The method as described above, wherein as a hydrophobic gel Macro Prep Methyl, Fractogel EMD Propyl 650, Fractogel EMD Butyl 650, Fractogel TSK Butyl 650, Macro Prep t Butyl, Butyl Cellufine, Butyl Sepharose 4 Fast Flow, Butyl S-Sepharose 6 Fast Flow, HIC Fractogel Pentyl, Hexyl S-Sepharose 6 Fast Flow, Octyl Sepharose CL 4B, HIC-Fractogel HW 65 propyl tentacle, Fractogel HW 65 butyl tentacle, Fractogel TA 650, Phenyl Sepharose HP, Phenyl Sepharose Fast Flow, Phenylalanine Sepharose, Thiopropyl Sepharose 6B and / or Pyridyl S-Sepharose 6 Fast Flow is used.

0036Process as described above, wherein a pH between 6 and 9 is used during the process step with negative chromatography and / or negative adsorption.

0037Process as described above, the process step being carried out with negative chromatography and / or negative adsorption at a temperature of 0-30 ° C.

0038Process as described above, wherein the yield of fibrinogen in the course of the negative chromatography or in the supernatant of the negative adsorption is ≥ 50%, preferably ≥ 70%.

0039Process as described above, the process step involving negative adsorption or chromatography being carried out in the presence of substances which weaken the binding of plasminogen to fibrinogen.

0040Method as described above, wherein a mixture obtained from blood, milk from transgenic animals or a fermentation supernatant or a fraction produced therefrom is used as the starting material.

0041Process as described above, wherein human plasma, a plasma fraction or cryoprecipitate is used as the starting material.

0042Process as described above, wherein one or more process steps include aluminum hydroxide treatment.

0043Process as described above, which includes one or more process steps in which fibrinogen is precipitated.

0044Method as described above, wherein one or more precipitations with glycine or other amino acids are contained.

0045Process as described above, which includes one or more process steps in which plasminogen is removed via gel material with lysine or lysine analogues as a functional group.

0046Method as described above, wherein one or more method steps for the depletion and / or inactivation of infectious particles are included.

0047Method as described above, wherein one method step is pasteurization and / or UV radiation and / or nanofiltration.

0048Method as described above, wherein one or more ultrafiltrations and / or dialyses are contained.

0049Method as described above, using filter materials with a molecular weight cut-off of 100 to 500 kDa.

0050Method as described above, wherein one or more sterile filtrations are contained.

0051Process as described above, combining the following process steps: production of a plasma fraction, adsorption on aluminum hydroxide, inactivation of infectious particles such as viruses, precipitation, further purification and / or inactivation steps, negative chromatography and / or negative adsorption, ultrafiltration, sterile filtration.

0052Process as described above, the further purification step being a process step for separating plasminogen.

0053Method as described above, the sequence and / or the number of individual method steps being changed.

0054Process as described above, wherein one of the process steps according to the invention is used and the plasma proteins to be separated are eluted from the hydrophobic gel after carrying out the negative chromatography and / or negative adsorption.

0055Method as described above for the isolation of F XII, plasminogen, t-PA and / or F XI.

0056Fibrinogen preparation which can be obtained by one of the processes described above.

0057F XII, plasminogen, t-PA and / or F XI, which were prepared by one of the methods described above.

0058Production of a pharmaceutical preparation, characterized in that it contains a fibrinogen according to one of the processes described above.

0059Pharmaceutical preparation, characterized in that it contains a fibrinogen according to one of the processes described above, that the F XI content is ≤ 1 ng per OD_280-320 lies, that the content of plasminogen is ≤ 5 ng per OD280-320, that the t-PA content is ≤ 0.02 ng per OD280-320 and that the proportion of fibrinogen breakdown fragments when stored at 30 ° C after one month in the liquid state is below 3%.

0060Pharmaceutical fibrinogen preparation, characterized in that the content of F XI is preferably 0.2 ng per OD_280-320 lies.

0061Pharmaceutical fibrinogen preparation, characterized in that the content of F XII is ≤ 20, preferably ≤ 10 ng per OD_280-320 lies.

0062Pharmaceutical fibrinogen preparation, characterized in that the content of t-PA is preferably ≤ 0.01 ng per OD_280-320 lies.

0063Pharmaceutical preparation as described above, the absolute proportion of fibrinogen degradation fragments when stored at 30 ° C. after one month in the liquid state preferably being below 2.5%. Use of a pharmaceutical preparation according to one of the processes described above.

0064Use of a pharmaceutical preparation according to one of the methods described above as a preparation for the treatment of fibrinogen deficiency states.

0065Use of a pharmaceutical preparation according to one of the methods described above as a component of a fibrin glue.

0066Use of a pharmaceutical preparation according to one of the processes described above as a component for producing a fibrin matrix.

0067Use of a pharmaceutical preparation according to one of the methods described above as a component of a diagnostic agent.

0068Fibrinogen preparation according to one of the methods described above as a component of a multi-component adhesive. Fibrinogen preparation according to one of the processes described above, containing the formulation component arginine as a component of a multi-component adhesive. Fibrinogen preparation according to one of the processes described above, containing the formulation constituents NaCl, Na₃-Citrate, ArgxHCl and CaCl₂ as a component of a multi-component adhesive. Fibrinogen preparation according to one of the methods described above, containing the formulation components NaCl (0-500 mM), Na₃-Citrate (0-50 mM), Arg or Arg x HCl (0.05-2.0 mol / l) and CaCl₂ (0.1-5 mM) or mixtures thereof as a component of a multi-component adhesive. Fibrin glue, characterized in that it contains a fibrinogen component according to one of the methods described above, an F XIII and a thrombin component as separate components.

0069Fibrin glue, characterized in that it contains a fibrinogen component according to one of the processes described above, to which F XIII has been added, and a thrombin component as separate components.

0070Further embodiments relate to the subject matter of the claims and further features and advantages of the invention emerge from the description of the preferred embodiments, the examples and figures.

0071A brief description of the figures follows:<ul id="ul0001" list-style="none"><li><figref idref="f0001 f0002">Figure 1</figref> shows the examination of fibrinogen-containing solutions for fibrinogen degradation fragments by means of SEC-HPLC. It is shown that the use of negative hydrophobic interaction chromatography can significantly reduce the proportion of fibrinogen degradation fragments when stored for two months at 30 ° C and thus increase the stability of fibrinogen in solution.</li><li><figref idref="f0001">Figure 1a</figref>: Fibrinogen-containing solution, which was additionally purified using hydrophobic interaction chromatography in negative chromatography mode, before starting storage.</li><li><figref idref="f0001">Figure 1b</figref>: Fibrinogen-containing solution, which was additionally purified using hydrophobic interaction chromatography in negative chromatography mode, after 2 months' storage at 30 ° C.</li><li><figref idref="f0002">Figure 1c</figref>: Starting solution containing fibrinogen, without additional purification via hydrophobic interaction chromatography, after 2 months' storage at 30 ° C.</li></ul>

0072The term fibrinogen is preferably to be understood as human fibrinogen which can be purified, for example, from a mixture obtained from human blood and containing fibrinogen. The term “mixture obtained from blood” means, for example, whole blood, blood plasma, plasma fractions or plasma precipitates. Fibrinogen from human plasma, cryoprecipitate or Cohn fraction 1 is particularly preferred. Fibrinogen can be isolated from pooled plasma donations as well as from individual donations. Human fibrinogen can also be obtained from the milk of transgenic animals, see for example<patcit id="pcit0056" dnum="US5639940A"><text>US 5,639,940</text></patcit>. Also see fibrinogen, which is obtained by recombinant expression from cell culture, for example<patcit id="pcit0057" dnum="US6037457A"><text>US 6,037,457</text></patcit>is included. It is thus possible to isolate fibrinogen from the corresponding fermentation supernatants or fractions made from them. However, fibrinogen is also isolated from a mixture obtained from animal blood and contains fibrinogen, preferably from animals such as mammals (eg pigs, horses, cattle, goats, sheep and dogs).

0073Contaminating proteins in the sense of the invention are to be understood in principle as all proteins which occur in the plasma in addition to fibrinogen or occur in the milk of transgenic animals or in the cell culture supernatant. It is particularly preferably fibrinogen-degrading proteins which can proteolytically degrade fibrinogen or precursors of the fibrinogen-degrading proteins (proenzymes) which have to be activated beforehand for proteolytic degradation of fibrinogen, or activators of fibrinogen-degrading proteases. A proteolytic process produces fibrinogen degradation fragments that are smaller than fibrinogen and can be of low molecular weight. The alpha and / or beta and / or gamma chains of fibrinogen can be affected by proteolytic degradation. Possible fibrinogen-degrading or degradation-supporting proteins or their precursors, which can appear as possible contaminants in the purification of fibrinogen from plasma, are for example plasmin, F XIa, kallikrein, factor VII activating protease (FSAP), F Xlla, plasminogen activators such as t-PA and u-PA, thrombin, metalloproteases (MMP's) or the corresponding precursors such as plasminogen, F XI, precallikrein, sc-FSAP, F XII, single-chain plasminogen activators such as sct-PA and scu-PA, prothrombin and Pro-MMP's. In the following, a distinction is no longer made between the activated forms and the respective preliminary stages, but the designation of the non-activated proenzymes is used as a representative for both forms.

0074The process according to the invention preferably enables the depletion of fibrinogen-degrading proteases or their proenzymes and activators by the process step (s) according to the invention. These can be, for example, plasminogen, F XI, precallikrein, F XII, Pro-MMP's and / or plasminogen activators sct-PA and scu-PA. The depletion factor (AF) is above 1, preferably above 2. The proportion of F XII can particularly preferably be ≤ 20 ng per OD_280-320 be minimized. The proportion of F Xl can be ≤ 1 ng per OD_280-320' particularly preferably to ≤ 0.2 ng per OD_280-320 be minimized. The proportion of plasminogen can be particularly preferred up to values ​​of 5 5 ng per OD_280-320 be reduced. The process step according to the invention particularly preferably increases the stability of fibrinogen in solution and in particular the proteolytic stability of the fibrinogen chains. The degradation fragments resulting from the proteolysis of fibrinogen by fibrinogen-degrading proteins can be detected by different methods. The separation into SDS polyacrylamide gels under reducing or non-reducing conditions, HPLC methods such as SEC (Size Exclusion Chromatography) HPLC or immunological methods are mentioned here as examples. The activity of remaining fibrinogen can continue according to common methods such as<nplcit id="ncit0011" npl-type="s"><text>Clauss (Acta-Haematol. 17 (1957) 237-246</text></nplcit>) described, determined. The depletion of fibrinogen-degrading proteins can, for example, be specifically demonstrated with the aid of antibodies, such as in the known ELISA (enzyme-linked immunosorbent assay) or RIA (radioimmunoassay) method. The antibodies are, for example, antibodies which are directed, inter alia, against the named or other known fibrinogen-degrading proteases. However, the depletion can also be demonstrated nonspecifically by storing the fibrinogen solution in the liquid state and determining the degree of a possible degradation reaction after storage in comparison to a likewise stored, non-depleted control.

0075Chromatography in the sense of this invention is to be understood as a separation method in which a solution containing fibrinogen is passed over a stationary phase with the aid of a liquid stream, and components of the mixture separate. The stationary phase is preferably the filling material of a chromatographic column. The filling material, hereinafter also called gel material, consists of a solid carrier material, preferably of approximately the same size, porous or non-porous particles, on which there are covalently bonded functional groups which determine the separation mode. The carrier material can be, for example, biopolymers such as agarose, cellulose and dextran (preferably Sepharose and Sephadex), or synthetic polymers such as e.g. B. Methacrylates, polyvinylbenzene, polystyrene and polyacrylamide or inorganic polymers, such as. As silica or porous glass beads act. The solution in which the fibrinogen to be purified is dissolved with contaminating proteins, as well as possible washing buffers and equilibration buffers are to be regarded as mobile phases. Negative chromatography in the sense of this invention is to be understood to mean that the stationary and the mobile phase are to be selected such that fibrinogen interacts as weakly or as little as possible with the stationary phase, while one or more contaminating proteins have stronger interactions with the stationary phase enter as fibrinogen. The fibrinogen thus predominantly passes through the column and is predominantly in the passage (> 50%), while one or more contaminating proteins are predominantly bound to the stationary phase. The column is pretreated and equilibrated prior to loading with the fibrinogen-containing solution in accordance with the requirements of the gel material. The equilibration buffer preferably corresponds to the solution in which fibrinogen and the contaminating proteins are dissolved. In order to minimize losses of fibrinogen, fibrinogen still remaining in the mobile phase of the column can be washed out, the volume of the washing buffer preferably corresponding approximately to that of the column volume. The wash buffer preferably corresponds to the solution in which the fibrinogen and the contaminating proteins are dissolved. The wash buffer can optionally be combined with the run. The contaminating proteins bound to the stationary phase can be eluted with appropriate solutions and the stationary phase can be used several times for a negative chromatographic purification step by regeneration to save costs. If regeneration is not possible or is not economical, the used gel material is discarded. The proteins eluted from the stationary phase can also be used as starting material for the isolation of these protein components if required. Protein concentrates can be obtained by optionally further purification or process steps. In this way, e.g. B. plasminogen, F XII, F XI and other concentrates can be produced.

0076In negative chromatography in the sense of this invention, basically any material can be used for the stationary phase which, under the selected conditions, in particular the choice of the mobile phase, is able to interact more strongly with one or more contaminating proteins than with the fibrinogen . For the process step according to the invention, gel materials (stationary phases), which belong to the group of hydrophobic gels, are included. There is a wide range of commercially available column materials or pre-packed columns, e.g. from Amersham, Bio-Rad, Biosepra, Merck, Perseptive Biosystems, Pharmacia, Prometic, Toso Haas, which are to be regarded as included. Some were tested as examples among the listed examples. The mobile phases have to be adapted to the respective gel materials. In general, however, pH values ​​in the range between 5 and 9 are preferred. Buffer systems must be selected accordingly that buffer this area well. Examples would be citrate, phosphate and Tris buffer.

0077The negative chromatography step is preferably carried out at a temperature between 2 and 30 ° C.

0078A hydrophobic chromatography is characterized in that the non-polar surface regions of a protein interact with hydrophobic functional groups (ligands) of the stationary phase at mostly higher salt concentrations. The hydrophobic gel as the stationary phase is preferably a synthetic polymer, silicate or a biopolymer such as Sepharose, the surfaces of which are modified as a functional group by hydrophobic ligands. The hydrophobic ligands are preferably alkyl groups with 1 to more than 24 carbon atoms (C), which can be linear or branched, or aromatic ligands. For example C1 (methyl), C3 (propyl), C4 (butyl), C5 (pentyl), C6 (hexyl) and C8 (octyl) groups are possible, particularly preferred are propyl, butyl, pentyl, hexyl and octyl -Groups. The alkyl groups can be derivatized, such as thiopropyl groups. Preferably, phenyl groups or aromatic compounds containing a phenyl derivative, such as. B. phenylalanine groups can be used. Phenyl groups can, for example, also be linked to alkyl chains. For example, pyridyl groups or their derivatives can also be used. Hydrophobic materials are well known to the person skilled in the art and are commercially available, for example from the companies Amersham, Bio-Rad, Biosepra, Merck, Perseptive Biosystems, Pharmacia, Prometic and Toso Haas. Particularly preferred as the stationary phase are, for example Macro Prep Methyl HIC Support (Bio-Rad), Fractogel EMD Propyl 650 (Merck), Fractogel EMD Butyl 650 (Merck), Fractogel TSK Butyl 650 (Merck), Macro Prep t Butyl HIC Support (Bio-Rad), Butyl Cellufine ( Amicon), Butyl Sepharose 4 Fast Flow (Pharmacia), Butyl S-Sepharose 6 Fast Flow (prototype, Pharmacia), HIC-Fractogel Pentyl (Merck), Hexyl S-Sepharose 6 Fast Flow (prototype, Pharmacia), Octyl Sepharose CL 4B (Pharmacia), Fractogel HW 65 propyl tentacle (Merck), Fractogel HW 65 butyl tentacle (Merck), Fractogel TA 650 (Merck), Phenyl Sepharose High Performance (Pharmacia), Phenyl Sepharose Fast Flow (Pharmacia), Phenylalanin Sepharose (Pharmacia), Thiopropyl Sepharose 6B (Pharmacia) or Pyridyl S-Sepharose 6 Fast Flow (Pharmacia).

0079The hydrophobic interaction of the proteins with the stationary phase is influenced not only by the functional groups (e.g. increasing chain length of alkyl groups increases the hydrophobic character), but also very strongly by the ionic strength and pH of the solution that forms the mobile phase. Depending on the stationary phase used, the mobile phase should be selected so that one or more contaminating proteins enter into hydrophobic interactions with the stationary phase, while fibrinogen preferably passes through the column to more than 50%. Possible mobile phases and adaptations to the respective stationary phase are known to the person skilled in the art. The pH should preferably be in the range above about 5 and up to about 9. Known salts can be used for hydrophobic interaction chromatography, particularly preferred are, for example, NaCl, Na₂SO₄ and (NH₄)₂SO₄. Depending on the stationary phase, the salt concentration is preferably in the range from 0.01 to 2 M.

0080The pH of the mobile phase is preferably in the range from about 5 to about 9.

0081For the purposes of this invention, negative adsorption is understood to mean that a solution containing fibrinogen is mixed in a suitable container with an adsorption material which, under the selected conditions, does not adsorb fibrinogen or only adsorbs it in small amounts, but adsorbs one or more contaminating proteins. Fibrinogen remains mostly in the solution. This process is also known as a batch or batch process. The conditions of the adsorption should be chosen so that the contaminating proteins have enough time and opportunity to bind to the adsorption material. For example, a mixture e.g. B. by careful stirring at suitable temperatures. Suitable temperatures are, for example, between 2 and 30 ° C. In principle, any material can be used as the adsorption material which, under the selected conditions, is able to bind one or more contaminating proteins and does not bind fibrinogen or only binds to a small extent. Included materials are gel materials that have already been listed under chromatography and thus belong to the group of hydrophobic gels. Preferred solutions correspond to preferred mobile phases in chromatography. The adsorption material with the bound, contaminating proteins can be separated from the fibrinogen-containing solution by methods known to the person skilled in the art. The methods of filtration, centrifugation and / or sedimentation should preferably be mentioned here. The adsorption material can preferably be regenerated by elution of the contaminants and re-buffers, so that it can be used several times. If necessary, the proteins eluted from the adsorption material can also be used as a starting basis for the isolation of these protein components. Protein concentrates can be obtained in this way by optionally further purification or process steps. In this way, for example, plasminogen, F XII, F XI and other concentrates can be produced.

0082Batch format negative adsorption is a technique that can be used in addition to or instead of negative chromatography.

0083The process steps according to the invention generally enable a step yield of ≥ 50% fibrinogen in the run or in the supernatant, preferably of ≥ 70%.

0084In a further embodiment, substances which weaken the binding of plasminogen to fibrinogen are added during the process according to the invention and in particular during the process steps according to the invention. It is known that fibrinogen tends to bind other proteins such as plasminogen. This repeatedly leads to partial co-purification of contaminants such as plasminogen. By weakening substances, co-purifications can be minimized. These substances preferably include ω-amino acids such as ε-aminocaproic acid, tranexamic acid, PAMBA (p-aminomethylbenzoic acid), lysine and other lysine analogues.

0085It is known that the enrichment and isolation of fibrinogen generally requires several process steps and that there are numerous possible combinations of these individual purification steps. In addition to one or more process step (s) according to the invention, the process according to the invention can therefore contain additional process steps which are basically to be found in the prior art for the purification of fibrinogen and are encompassed thereby. The state of the art for the purification of fibrinogen has already been set out essentially and the correspondingly cited patents and publications are hereby included. Process steps which do not involve chromatography or adsorption, in which fibrinogen binds to a large extent and which would therefore require elution with all of its disadvantages, are preferred.

0086In a preferred embodiment, the process according to the invention contains one or more process steps in which aluminum hydroxide (Al (OH)₃) is added to the fibrinogen-containing solution to be cleaned. The Al (OH)₃ primarily adsorbs the factors of the prothrombin complex and can be removed, for example, by centrifugation and / or filtration.

0087In a preferred embodiment, the process contains one or more process steps in which the fibrinogen is precipitated. The addition of glycine or other amino acids for precipitation is particularly preferred. If the glycine concentration is sufficiently high, this leads to a direct precipitation of fibrinogen (single-stage glycine precipitation). However, alternatively or additionally, a two-stage glycine precipitation is possible, in which the glycine concentration is selected in a first step such that fibrinogen essentially remains in the supernatant and precipitated proteins are separated off by, for example, centrifugation, and only in a second step is the main amount of the fibrinogen increased the glycine concentration, as also shown in the examples, is precipitated. As mentioned above, other amino acids can be used instead of the amino acid glycine. Examples include alanine, glutamine or glutamic acid. However, other known precipitation agents (such as sodium chloride, ammonium sulfate or polyethylene glycol) can also be used here.

0088In a further preferred embodiment, the process contains one or more process steps in which plasminogen is depleted in a gel material with lysine ligands or analog ligands. Examples here would be Lysine-Sepharose, Lys-Hyper D, Lys-Fractogel, Aminohexyl-Sepharose or the like. This leads to an advantageous depletion of plasminogen, which can additionally have an advantageous effect on the stability of fibrinogen in liquid storage. In addition, depletion of plasminogen is also advantageous if the fibrinogen concentrate is to be used as a component of a fibrin glue.

0089In a further preferred embodiment, the method comprises one or more method steps in which potentially existing infectious particles such as viruses are inactivated or depleted as far as possible. If the fibrinogen is isolated from human plasma, this is a particularly preferred component of the method according to the invention. Inactivation or depletion of infectious particles can be carried out according to techniques known to the person skilled in the art, which are hereby included. These are processes such as pasteurization, heating in the dry state, nanofiltration, chemical additives (for example detergents), radiation or combinations thereof.

0090In a further preferred embodiment, the process contains one or more process steps which include ultrafiltration and / or dialysis. These methods are used to advantage in order to buffer the fibrinogen-containing solution, ie to change components of the solution or to concentrate the protein solution. This is particularly preferred in preparation for a cleaning step or at the end of the method according to the invention in order to choose formulation components which are suitable for storage and use as a pharmaceutical preparation.

0091Ultrafiltration also offers the possibility of depleting additional contaminating proteins by choosing filters that allow contaminating proteins to pass through but retain fibrinogen, such as in the <patcit id="pcit0058" dnum="WO9317776A"><text>WO 93/17776</text></patcit> described. In the course of this invention it was possible to show that if suitable filters with a molecular weight limit (cut off) of, for example, 300 kDa are selected, depletion of fibrinogen-degrading proteins is also possible. In a particularly preferred embodiment, filters with a molecular weight cut-off of 50 to 500 kDa are used for the ultrafiltration.

0092In a further preferred embodiment, the method contains sterile filtration. This is particularly useful at the end of the process for producing a pharmaceutical preparation.

0093In a preferred embodiment, several of the above-mentioned method steps are combined with one or more method steps according to the invention to form a method according to the invention. The order of individual process steps can be varied. Individual process steps, such as precipitation, can also be used several times. In a particularly preferred embodiment, a combination contains, in addition to one or more process steps according to the invention, at least the steps of Al (OH)₃ Adsorption, one and / or two-stage glycine precipitation, as well as pasteurization. These process steps are advantageously combined with an affinity column with lysine ligands. After cleaning is complete, the solution components are replaced by a solution that is suitable for storage. Possible methods such as dialysis, ultrafiltration, etc. are known to the person skilled in the art and are hereby encompassed. Sterile filtration can be connected to the cleaning. In a particularly preferred embodiment, the cleaning method according to the invention consists of the following method steps:<ul id="ul0002" list-style="dash" compact="compact"><li>Preparation of a plasma fraction</li><li>Adsorption on aluminum hydroxide</li><li>Inactivating infectious particles such as viruses</li><li>Precipitation</li><li>Further cleaning and / or inactivation steps</li><li>negative chromatography (s) and / or negative adsorption (s)</li><li>Ultrafiltration / Diafiltation</li><li>Sterile filtration</li></ul>

0094In a particularly preferred embodiment, the further purification steps include an affinity column with lysine ligands, such as, for example, Lys-Sepharose. Both the number and the sequence of individual process steps can be varied. For example, one or more negative adsorptions or chromatography steps directly after the Al (OH)₃ Adsorption take place. This would have the advantage that possible fibrinogen-degrading proteins are depleted at an early point in time, which proteins can therefore no longer adversely affect the stability of the fibrinogen in the course of further purification.

0095When using the particularly preferred embodiments, fibrinogen degradation can be significantly reduced when fibrinogen is stored in liquid form for 1 month at 30 ° C. in the absence of added fibrinolysis inhibitors, ie significantly less low-molecular degradation fragments are generated in this storage time. An analysis by means of SEC-HPLC shows that the increase in the proportion of low-molecular breakdown fragments compared to the remaining peaks when using the particularly preferred embodiments, which additionally use an affinity column with lysine ligands, is below 2.5%, in particular is less than 1.5% -2%. The subject of this application is therefore also a fibrinogen concentrate or a corresponding pharmaceutical preparation with fibrinogen which, after liquid storage at 30 ° C. for 1 month, has less than 2.5-3% fibrinogen degradation fragments, determined by means of SEC-HPLC, or by less than approx. 1.5-2.5% of degradation fragments based on the total peak area increases.

0096Another object of the invention is fibrinogen which has been purified by the process according to the invention. The fibrinogen obtained in this way or a corresponding pharmaceutical preparation only contains ≤ 0.2 ng per OD_280-320 at F XI and / or ≤ 5 ng per OD_280-320 of plasminogen and / or ≤ 0.02 ng per OD_280-320 of t-PA and the proportion of low-molecular fibrinogen degradation fragments after storage at 30 ° C for one month in the liquid state is below 3% and preferably only ≤ 20 ng per OD_280-320 to F XII.

0097The contaminating proteins bound during the process step according to the invention for the purification of fibrinogen by means of negative chromatography or adsorption on hydrophobic gels can serve as a starting basis for the isolation of these protein components. Appropriate protein concentrates can be obtained by elution of the proteins from the corresponding gel material and, if appropriate, further purification or process steps. In this way, e.g. B. Plasminogen, t-PA, F XII or F XI concentrates can be produced. This application accordingly includes a method for isolating one or more plasma proteins and producing corresponding pharmaceutical preparations using a gel material which binds one or more plasma proteins, does not bind fibrinogen under the chosen conditions and is suitable for hydrophobic interaction chromatography.

0098The application also relates to the production of a pharmaceutical preparation which contains fibrinogen which has been purified by the process according to the invention. Possible pharmaceutical preparations are known to the person skilled in the art and the additives depend on the intended use. For intravenous administration of fibrinogen, other formulation components known to the person skilled in the art can be advised than for use as fibrin glue. Certain additives should also be taken into account in the case of planned interim storage as lyophilisate, in the liquid or in the frozen state. Known lyophilization aids for proteins are for example saccharides such as sucrose, mannose, galactose and glucose, sugar alcohols such as mannitol and sorbitol or amino acids. Possible formulation components for the storage forms mentioned are monovalent metal salts (for example sodium or potassium chloride), divalent metal salts (for example magnesium or calcium chloride), amino acids (for example glycine, arginine, aspartic acid, glutamic acid, histidine, lysine, isoleucine), carbohydrates (for example glucose, Sucrose, trehalose, lactose, cellulose, sorbitol, mannitol and glycosaminoglycans), Detergents (e.g. poloxamers or polysorbates), chaotropic agents (e.g. urea and guanidine or derivatives thereof), inhibitors such as aprotinin, alpha-2-antiplasmin, alpha-2-macroglobulin, alpha-1-antiplasmin, C1-inhibitor, antithrombin, plasminogen activator inhibitors (PAI), thrombin-activatable fibrinolysis inhibitor (TAFI) and lysine analogues such as epsilonaminocaproic acid, plasma proteins (e.g. F XIII), antioxidants (e.g. ascorbic acid), Buffer substances (for example amino acids such as arginine, buffer systems such as citrate, phosphate, acetate, succinate, tris (hydroxymethyl) aminomethane (Tris), glycylglycine, carbonate and bicarbonate) or mixtures thereof. The pH is preferably between about 5 and about 8.

0099The following additives, for example, are suitable for preparing the fibrinogen solution as a component of a liquid fibrin adhesive: NaCl (0-400 mM), Na₃-Citrate (0-50 mM), L-Arg x HCl (0.1 - 1 M), CaCl₂ (0-10 mM), and possibly other stabilizing agents such as amino acids, carbohydrates or detergents.

0100Another object of the invention is the use of fibrinogen or a pharmaceutical preparation containing fibrinogen which has been purified by a process which contains one or more of the process steps according to the invention. Possible applications are known to the person skilled in the art and the fibrinogen produced by the process according to the invention can be used for all known uses of fibrinogen. The medical application preferably relates to humans, but applications are also included in veterinary medicine. In general, the fibrinogen preparation according to the invention is suitable for the therapy of fibrinogen deficiency states. These deficiency states can occur, for example, in the case of large-area wounds, heavy bleeding, large-area burns, pathological activation of blood coagulation (consumption coagulopathy also called DIC (disseminated intravascular coagulation)), medication or severe liver diseases (e.g. in the case of synthesis disorders due to liver parenchymal damage) . In addition to the described hypofibrinogenemia (reduced fibrinogen in the blood) or Afibrinogenaemia (missing or highly reduced fibrinogen in the blood) is also rarely congenital afibrinogenaemia or hypofibrinogenaemia, which can be caused by a lack of or reduced fibrinogen synthesis in the liver.

0101In the case of hypofibrinogenemia or afibrinogenemia, the fibrinogen preparation according to the invention is preferably injected intravenously into the patient in order to compensate for corresponding deficiency states in fibrinogen. Dosages are based on the level of the defect that occurs.

0102Fibrinogen is of great importance in fibrin therapy as an important component of so-called fibrin glue. A fibrin glue simulates the last step of blood coagulation by combining fibrinogen with thrombin and auxiliary substances such as calcium and F XIII to form stabilized fibrin.

0103There are many possible uses for fibrin glue in medicine, which are widely known (see e.g. <nplcit id="ncit0012" npl-type="s"><text>Sierra, Journal of Biomaterials Applications 7 (1993) 309-352</text></nplcit>; <nplcit id="ncit0013" npl-type="s"><text>Martinowitz &amp; Spotnitz, Thrombosis and Haemostasis 78 (1997) 661-666</text></nplcit>; <nplcit id="ncit0014" npl-type="s"><text>Radosevich et al. Vox Sanguinis 72 (1997) 133-143</text></nplcit>). Hemostasis, wound closure, sealing of sutures and wound healing are important to mention. Local intraoperative hemostasis is particularly important on parenchymatous organs and in the cardiovascular area. Even heavy bleeding after liver or spleen injuries can be stopped. Fibrin glue is also used to close and fix skin wounds (including skin grafts) and to seal seams (e.g. on the duodenal stump). The use in the sealing of a plastic replacement of the dura and for cavity sealing as well as for gluing the pleural sheets for the palliative treatment of pleural effusions is also mentioned as an example. For gluing, the fibrin glue can also be used advantageously in connective tissues such as bones, cartilage and tendons. A fibrinogen component free of synthetic fibrinolysis inhibitors has e.g. B. Its advantages, particularly when applied for dura sealing, since substances such as tranexamic acid have been shown to be neurotoxic (Lit: <nplcit id="ncit0015" npl-type="s"><text>MG Schlag, R. Hopf, U. Zifko and H. Redl; Acta Neurochir 144: 63-69 (2002</text></nplcit>)). Fibrin glue can also be used to prevent postoperative adhesions.

0104Fibrinogen can also be used as a component for making a fibrin matrix. Such a carrier material can be used for the slow release of active substances, such as growth factors (e.g. also together with osteoinductive proteins as a matrix for bone and / or cartilage regeneration), antibiotics, cytostatics, anti-inflammatory additives and / or wound healing additives. The carrier can also consist of a mixture of fibrin with other materials.

0105A fibrin matrix also has wide-ranging uses in biotechnology, such as, for example, as a carrier material and culture medium for cells and tissues in tissue engineering or for covering implants such as biosensors.

0106The fibrinogen according to the invention can also be used as a component of a diagnostic agent.

0107The invention is further illustrated by the following examples, which, however, are not intended to have any restrictive effect.

Example 1:

0108This example shows that hydrophobic gels are able to purify a solution containing fibrinogen by means of negative chromatography in such a way that the stability of the fibrinogen with respect to the starting material is increased.

0109For the production of fibrinogen starting material as in <patcit id="pcit0059" dnum="EP0103196A"><text>EP 0 103 196</text></patcit> described a pasteurized fibrinogen concentrate prepared and fractionally precipitated by adding glycine.

0110The fibrinogen-rich precipitate was first in a suitable aqueous solvent (50 mM NaCl; 20 mM tri-sodium citrate dihydrate, 0.05% NaN₃ pH 7.3) and served as the starting material for the further purification steps using negative chromatography.

0111The chromatography columns (Ø 0.7 cm) used for this purification were each filled with 1.0 ml of the respective gel material. The columns were equilibrated in a buffer.<tables id="tabl0001" num="0001"><table frame="none"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="98mm" /><tbody><row><entry>Buffer:</entry><entry>1000 mM NaCl, 50 mM sodium phosphate pH 6.5 for hydrophobic gels</entry></row></tbody></tgroup></table></tables>

011230-40 ml of fibrinogen-containing solution were dialyzed against the buffer (see Table 1) and with the buffer to an OD_280-320 diluted by 10. 15 ml of this fibrinogen solution were applied per column and the columns were washed with 1.0 ml of the buffer. The column run and the wash solution were combined. First the optical density at 280 and 320 nm (OD_280-320) measured in order to determine the yield (in%) compared to the starting material. An aliquot of this material was retained for analysis to determine the 0 value of the subsequent storage trial. The column runs combined with the respective washing solutions and the starting material were against a buffer containing 100 mM NaCl, 20 mM Na₃-Citrate, 5% L-Arg pH 7.2 dialyzed and 1 ml each of which was mixed with sodium azide (0.05% w / v final concentration). These differently purified fibrinogen preparations and the starting material were stored as a control at + 30 ° C for storage times of up to 3 months. The stability of fibrinogen was determined after each storage period by means of SEC-HPLC. This is size exclusion chromatography (SEC), which separates the proteins and cleavage products according to their molecular weight. Small molecular fragments, which are the result of a proteolytic breakdown of fibrinogen, can be found as a new peak (fragment peak 4) as well as possibly other new peaks with an increased retention time. The area of ​​the fragment peaks (≤ peak 4) was determined and calculated as a percentage of all peaks. The value was adjusted for the small proportion before the start of storage (zero value) and the values ​​determined from this are shown in Table 1 as a result. They reflect the increase in degradation fragments during storage under accelerated conditions.

0113An appropriate HPLC system with an SEC column (TSK gel G 4000 SWXL, 7.5 x 300 mm from TOSO HAAS) was used to carry out the analysis. The proteins and protein fragments were separated after appropriate storage times at a flow rate of 0.5 ml / min in a suitable running buffer at 20-25 ° C. The protein or protein fragment peaks were detected at 280 nm.

0114The <figref idref="f0001 f0002">Figure 1</figref> shows exemplary separation processes of fibrinogen using SEC HPLC. It shows the separation of a solution containing fibrinogen, which was purified with the aid of lysine-Sepharose chromatography and a negative HIC before the start of storage (t = 0, zero value,<figref idref="f0001">Figure 1 a)</figref> and after 2 months storage at 30 ° C (t = 2 months, <figref idref="f0001">Figure 1 b)</figref>.

0115<figref idref="f0002">Figure 1c</figref> on the other hand shows the separation of a solution containing fibrinogen without additional purification via negative HIC after storage for 2 months at 30 ° C (t = 2 months). The zero value (t = 0) for this fibrinogen-containing solution is not shown, since it is comparable to a separation result<figref idref="f0001">Figure 1a</figref> shows.

0116The main peak with a retention time of approx. 15-16 min corresponds to fibrinogen. The peak with a retention time of approx. 24 min and peaks with longer retention times are the degradation fragments of fibrinogen. Of the<figref idref="f0001">Figure 1b</figref> it can be clearly seen that by using the hydrophobic gel, the area under the peaks corresponding to the degradation fragments after two months of storage at 30 ° C compared to the control material without HIC cleaning, which was also stored for 2 months at 30 ° C (<figref idref="f0002">Figure 1 c)</figref>, is significantly reduced. The lower occurrence of degradation fragments is evidence of the higher stability of fibrinogen after cleaning via a negative HIC.<tables id="tabl0002" num="0002"><img file="EP1568709B1_D0001.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP1568709B1_D0002.tif" /></tables>

0117Table 1 shows that the proportion of degradation fragments with increased retention time after purification of the fibrinogen used on one of the listed gel materials increases significantly less than in the control (starting material). The proven increased stability of fibrinogen compared to the control (starting material before negative chromatography) can be clearly determined after 1 month, but then increases with longer storage times. After longer storage times of 3 months, but usually after 2 months, it is also possible to discriminate the various good column materials. Even improvements over the affinity chromatography using lysine-Sepharose 4B known in the prior art, which depletes plasminogen, can still be achieved. Very good yield results are also consistently achieved, as a rule over 70%. The example further illustrates that with a large number of different hydrophobic gels, a substantial stabilization of fibrinogen and thus a reduction in the formation of degradation fragments can be achieved, so that it can be assumed that generally support materials from the group of the chromatographic separation principles mentioned achieve improved stability results to let.

Example 2:

0118In this example, the buffer conditions of the mobile phases were varied for the hydrophobic gel Phenyl Sepharose HP used in Example 1. The improved stability of fibrinogen was again determined by the reduced formation of degradation fragments in the course of a storage test. In addition, it was checked whether fibrinogen-degrading proteins or their inactive precursors (proenzymes) are depleted.

0119Fibrinogen was used as the starting material, which in addition to the analogous purification from Example 1 after being taken up in a suitable aqueous solvent (50 mM NaCl; 20 mM tri-sodium citrate, 0.05% NaN₃ pH 7.4, buffer AM in table 2) and preferably after dialysis against the solvent, was still purified over lysine-Sepharose.

0120Chromatography columns (column body Q3 = 0.7 cm, h = 2.5 cm from Qiagen) were used, each of which was filled with 1.0 ml of the respective gel material. The gel material was equilibrated with the appropriate buffers.

Equilibration buffer:

0121<tables id="tabl0004" num="0004"><table frame="none"><tgroup cols="4" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="10mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><colspec colnum="3" colname="col3" colwidth="31mm" /><colspec colnum="4" colname="col4" colwidth="32mm" /><tbody><row><entry>3a:</entry><entry>50 mM NaCl,</entry><entry>20 mM Na₃Citrate,</entry><entry>0.05% NaN₃ pH 6.5</entry></row><row><entry>3b:</entry><entry>50 mM NaCl,</entry><entry>20 mM Na₃Citrate,</entry><entry>0.05% NaN₃ pH 7.5</entry></row><row><entry>3c:</entry><entry>500 mM NaCl,</entry><entry>20 mM Na₃Citrate,</entry><entry>0.05% NaN₃ pH 7.5</entry></row><row><entry>3d:</entry><entry>1000 mM NaCl,</entry><entry>20 mM Na₃Citrate,</entry><entry>0.05% NaN₃ pH 7.5</entry></row></tbody></tgroup></table></tables>

0122The fibrinogen-containing application solution was optionally adjusted to the above buffer conditions by adding NaCl and adjusting the pH and a solution volume corresponding to a protein amount of 150 or 300 OD_280-320 per ml of gel (see table 2) at free drip rate. The columns were washed with 1 ml each of the corresponding buffer. The washing solution was combined with the respective run and against 50 mM NaCl, 20 mM Na₃-Citrate, 0.05% NaN₃ pH 7.4 dialyzed overnight at 4 ° C. The storage took place at 30 ° C for different storage times (0 - 2 months (Mo)).

0123The analysis using SEC-HPLC was carried out as described in Example 1.

0124In addition, the amount of plasminogen, factor XI and factor XII was determined using ELISA measurements. In a sandwich ELISA, plasminogen was first bound to rabbit polyclonal antibodies (IgG preparation from Dade Behring), which were immobilized as capture antibodies on a microtiter plate. The detection was carried out using the same polyclonal antibody preparation, but which was labeled with peroxidase. F XII was determined using the F XII ELISA kit from Kordia Life Sciences (Netherlands) according to the manufacturer's instructions. F XI was also quantified using the F XI ELISA kit from Kordia Life Sciences (Netherlands) according to the manufacturer's instructions.

0125The depletion factor (AF) is the quotient of the amount of the particular protein (eg plasminogen, F XI or F XII) per OD_280-320 in the starting material and the amount of the specific protein per OD_280-320 in the fibrinogen-containing solution after the respective negative chromatography.

0126The results are shown in Table 2.<tables id="tabl0005" num="0005"><table frame="all"><title>Table 2</title><tgroup cols="9"><colspec colnum="1" colname="col1" colwidth="31mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><colspec colnum="5" colname="col5" colwidth="15mm" /><colspec colnum="6" colname="col6" colwidth="15mm" /><colspec colnum="7" colname="col7" colwidth="25mm" /><colspec colnum="8" colname="col8" colwidth="14mm" /><colspec colnum="9" colname="col9" colwidth="15mm" /><thead><row><entry align="center" valign="middle"><b>Gel material</b></entry><entry align="center" valign="middle"><b>buffer</b></entry><entry align="center" valign="middle"><b>Loading the column OD_280-320</b></entry><entry align="center" valign="middle"><b>Yield (%)</b></entry><entry align="center" valign="middle"><b>δ Peak 4 after 1 Mo / 30 ° C</b></entry><entry align="center" valign="middle"><b>δ Peak 4 after 2 Mo / 30 ° C</b></entry><entry align="center" valign="middle"><b>AF plasminogen</b></entry><entry align="center" valign="middle"><b>AF F XII</b></entry><entry align="center" valign="middle"><b>AF F XI</b></entry></row></thead><tbody><row><entry morerows="4" valign="middle">Phenyl Sepharose High Performance</entry><entry align="center" valign="middle">3a</entry><entry align="center" valign="middle">150</entry><entry align="center" valign="middle">87</entry><entry align="center" valign="middle">1,32</entry><entry align="center" valign="middle">3,03</entry><entry align="center" valign="middle">2,4</entry><entry align="center" valign="middle">1,8</entry><entry align="center" valign="middle">1,3</entry></row><row><entry align="center" valign="middle">3b</entry><entry align="center" valign="middle">150</entry><entry align="center" valign="middle">91</entry><entry align="center" valign="middle">1,30</entry><entry align="center" valign="middle">3,10</entry><entry align="center" valign="middle">2,0</entry><entry align="center" valign="middle">1,1</entry><entry align="center" valign="middle">1,3</entry></row><row><entry align="center" valign="middle">3c</entry><entry align="center" valign="middle">150</entry><entry align="center" valign="middle">76</entry><entry align="center" valign="middle">1,17</entry><entry align="center" valign="middle">2,81</entry><entry align="center" valign="middle">1,7</entry><entry align="center" valign="middle">0,9</entry><entry align="center" valign="middle">1,7</entry></row><row><entry align="center" valign="middle">3d</entry><entry align="center" valign="middle">150</entry><entry align="center" valign="middle">66</entry><entry align="center" valign="middle">1,09</entry><entry align="center" valign="middle">2,69</entry><entry align="center" valign="middle">1,8</entry><entry align="center" valign="middle">0,9</entry><entry align="center" valign="middle">≥ 2, 2</entry></row><row><entry align="center" valign="middle">3b</entry><entry align="center" valign="middle">300</entry><entry align="center" valign="middle">96</entry><entry align="center" valign="middle">1,43</entry><entry align="center" valign="middle">3,27</entry><entry align="center" valign="middle">2,0</entry><entry align="center" valign="middle">1,2</entry><entry align="center" valign="middle">1,5</entry></row><row><entry valign="middle">Source material</entry><entry align="center" valign="middle">AT THE</entry><entry align="center" valign="middle">-</entry><entry align="center" valign="middle">-</entry><entry align="center" valign="middle">1,85</entry><entry align="center" valign="middle">4,33</entry><entry align="center" valign="middle">-</entry><entry align="center" valign="middle">-</entry><entry align="center" valign="middle">-</entry></row></tbody></tgroup><tgroup cols="9" rowsep="0"><colspec colnum="1" colname="col1" colwidth="31mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><colspec colnum="4" colname="col4" colwidth="20mm" /><colspec colnum="5" colname="col5" colwidth="15mm" /><colspec colnum="6" colname="col6" colwidth="15mm" /><colspec colnum="7" colname="col7" colwidth="25mm" /><colspec colnum="8" colname="col8" colwidth="14mm" /><colspec colnum="9" colname="col9" colwidth="15mm" /><tbody><row><entry namest="col1" nameend="col9" align="justify">AF: depletion factor</entry></row></tbody></tgroup></table></tables>

0127Table 2 shows, by way of example, that the negative chromatographies used, with appropriate selection of the separation parameters, allow plasminogen, F XII and F XI - even if to different degrees - to be depleted.

0128Hydrophobic gels show a dependence on the mobile phase, especially on the salt concentration of the buffer. In the case of the Phenyl Sepharose HP, for example, a depletion of plasminogen and increased stability of the fibrinogen, with a good yield of fibrinogen, can be achieved over a wide range of NaCl. All selected conditions lead to increased stability compared to the starting material, so that basically several conditions can be used advantageously. Other hydrophobic gels and / or other buffer conditions can be optimized in a similar way.

0129The table also shows that the different gels in this example contribute to different levels of depletion of plasminogen, F XII and F XI.

Example 3:

0130In this example, further hydrophobic gels or conditions for negative chromatography were tested.

0131The starting material was obtained using the same purification scheme as described in Example 2. As already described in Example 2, the columns were equilibrated with the respective buffer listed below.<ul id="ul0003" list-style="none" compact="compact"><li>3a: 1000 mM NaCl, 20 mM Na₃-Citrate, 0.05% NaN₃ pH 7.5</li><li>3b: 2000mM NaCl, 20mM Na₃-Citrate, 0.05% NaN₃ pH 7.5</li></ul>

0132The starting material was dialyzed against the appropriate buffer. The chromatography columns were each with an OD_280-320 loading of 150 (corresponding to 15 ml) per ml of gel at a free drip rate. The first 0.5 ml of column runs were discarded. The columns were washed with 1 ml of the respective buffer. The washing solution was combined with the respective run and against 50 mM NaCl, 20 mM Na₃-Citrate, 0.05% NaN₃ pH 7.4 dialyzed overnight at 4 ° C. The storage took place at 30 ° C for a storage time of 2 months.

0133The analysis using SEC-HPLC was carried out as described in Example 1 and the depletion factors (AF) for plasminogen, F XII and F XI were carried out as described in Example 2.<tables id="tabl0006" num="0006"><table frame="all"><title>Table 3:</title><tgroup cols="9"><colspec colnum="1" colname="col1" colwidth="31mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><colspec colnum="4" colname="col4" colwidth="24mm" /><colspec colnum="5" colname="col5" colwidth="20mm" /><colspec colnum="6" colname="col6" colwidth="25mm" /><colspec colnum="7" colname="col7" colwidth="10mm" /><colspec colnum="8" colname="col8" colwidth="11mm" /><colspec colnum="9" colname="col9" colwidth="13mm" /><thead><row valign="top"><entry align="center"><b>Gel material</b></entry><entry align="center"><b>buffer</b></entry><entry align="center"><b>Loading the column OD_280-320</b></entry><entry align="center"><b>Gel volume (ml)</b></entry><entry align="center"><b>Yield (%)</b></entry><entry align="center"><b>AF plasminogen</b></entry><entry align="center"><b>AF F XII</b></entry><entry align="center"><b>AF F XI</b></entry><entry align="center"><b>δ Peak 4 after 2Mo / 30 ° C</b></entry></row></thead><tbody><row valign="middle"><entry>Macro</entry><entry align="center">3a</entry><entry align="center">150</entry><entry align="center">1,0</entry><entry align="center">100</entry><entry align="center">0,8</entry><entry align="center">1,1</entry><entry align="center">1,4</entry><entry align="center">2,11</entry></row><row valign="middle"><entry>Prep t Butyl HIC Support</entry><entry align="center">3b</entry><entry align="center">150</entry><entry align="center">1,0</entry><entry align="center">89</entry><entry align="center">1,1</entry><entry align="center">1,3</entry><entry align="center">1,6</entry><entry align="center">2,08</entry></row><row valign="middle"><entry>Fractogel EMD Butyl 650 (S)</entry><entry align="center">3a</entry><entry align="center">150</entry><entry align="center">1,0</entry><entry align="center">91</entry><entry align="center">1,4</entry><entry align="center">1,5</entry><entry align="center">1,8</entry><entry align="center">1,63</entry></row><row valign="middle"><entry>Source material</entry><entry align="center">AT THE</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">2,32</entry></row></tbody></tgroup></table></tables>

0134Table 3 shows that the other gels and conditions tested are also suitable for reducing the formation of degradation fragments. Increasing the application quantity (loading the column) leads to an improved yield.

Example 4:

0135Some gel materials which were found to be suitable on the basis of Examples 1 to 3 were tested for their suitability on a larger scale. Chromatography columns with a gel volume of approx. 500 ml were used and the fibrinogen-containing solution was pumped through the columns.

0136Cryoprecipitate was used to obtain a fibrinogen-containing solution <patcit id="pcit0060" dnum="EP0103196A"><text>EP 0 103 196</text></patcit> worked up to the pasteurized fibrinogen solution.

0137The now pasteurized fibrinogen solution was mixed with three times the volume of dilution solution (3.5 g / l NaCl; 5.88 g / l tri-sodium citrate dihydrate in water, pH 7.5). 90 g of glycine per liter of diluted solution were added with stirring. The resulting precipitate was separated by centrifugation or filtration and discarded.

0138The supernatant was optionally brought to 200 mM L-Lys x HCl or EACA by adding solid L-Lys x HCl or EACA. A further 75 g of glycine were added per liter. The fibrinogen-rich precipitate was obtained by centrifugation and stored at -25 ° C until further processing.

0139For the further purification and depletion of traces of plasminogen, the fibrinogen-rich precipitate was dissolved and preferably after dialysis against buffer solution (20 mM tri-sodium citrate, 50 mM NaCl pH 7.4, optionally containing 0.05% NaN₃ as a preservative), pumped through a chromatography column with a matrix which carries L-lysyl residues as ligands. The run was used for the subsequent steps.

0140To separate further contaminants that affect the stability of fibrinogen, the fibrinogen-containing solution was pumped through various second chromatography columns, if necessary with a previous change in the buffer composition:<ul id="ul0004" list-style="none"><li>A: The fibrinogen-containing solution was brought to a final concentration of 1 M NaCl by adding crystalline NaCl and over a column (Ø 6 cm, volume approx. 500 ml) with a hydrophobic matrix which carries phenyl groups as ligands (Phenyl-Sepharose HP) pumped and the run collected. The column was washed with 1 SV buffer solution (20 mM tri-sodium citrate, 1 M NaCl pH 7.4, optionally containing 0.05% NaN₃ washed as a preservative).</li><li>B: The fibrinogen-containing solution was brought to a final concentration of 1 M NaCl by adding crystalline NaCl and over a column (Ø 7 cm, volume approx. 577 ml) with a hydrophobic matrix which carries butyl groups as ligands (Macro Prep t Butyl HIC Resin) pumped and the run collected. The column was washed with 1 SV buffer solution (20 mM tri-sodium citrate dihydrate, 1 M NaCl pH 7.4, optionally containing 0.05% NaN₃ washed as a preservative).</li></ul>

0141To produce fibrinogen preparations, the fibrinogen-containing runs were combined with the respective washing solutions and, first, using a suitable ultrafiltration method, depending on the use, to a protein concentration of approximately OD_280-320nm = 2 - 200, preferably approx. 20 - 160, brought and then dialyzed against solutions which contained the following formulation components: NaCl, Na₃-Citrate x 2H₂O, L-Arg x HCl, optional CaCl₂.

0142After final concentration and sterile filtration, fibrinogen preparations were obtained, which were tested for the content of fibrinogen degradation fragments using SEC-HPLC (see Example 1) after storage for 1 month at 30 ° C. (see Table 4).<tables id="tabl0007" num="0007"><table frame="all"><title>Table 4:</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="60mm" /><colspec colnum="2" colname="col2" colwidth="62mm" /><colspec colnum="3" colname="col3" colwidth="45mm" /><thead><row><entry valign="top"><b>Gel material</b></entry><entry align="center" valign="top"><b>buffer</b></entry><entry align="center" valign="top"><b>δ (s peak 4) after 1 Mo / 30 ° C</b></entry></row></thead><tbody><row><entry>Phenyl Sepharose High Performance</entry><entry>20 mM Na₃Citrate, 100mM NaCl, 50g / L L-ArgxHCl, 2.5mM CaCl₂ pH 7.2</entry><entry align="center">1,2</entry></row><row><entry>Macroprep-t Butyl HIC Support</entry><entry>20 mM Na₃Citrate, 100mM NaCl, 50g / L L-ArgxHCl, 2.5mM CaCl₂ pH 7.2</entry><entry align="center">1,1</entry></row><row><entry>Without additional negative chromatography</entry><entry>20 mM Na₃Citrate, 100mM NaCl, 50g / L L-ArgxHCl, 2.5mM CaCl₂ pH 7.2</entry><entry align="center">2,6</entry></row></tbody></tgroup></table></tables>

0143The results show that even on a larger scale, the use of negative chromatographies can reduce the difference between the degradation fragments (δ (≤ Peak 4)) that occur after accelerated storage for less than 2% and the stability of the fibrinogen in solution when stored at 30 ° C is significantly increased.

Example 5:

0144In this example, two negative batch adsorbents were combined using a dye gel and a hydrophobic gel.

0145To obtain a solution containing fibrinogen, cryoprecipitate was used as the starting material as in <patcit id="pcit0061" dnum="EP0103196A"><text>EP 0 103 196</text></patcit> described twice with Al (OH)₃ Suspension adsorbed.

0146To reduce / remove contaminating proteins such as fibrinogen-degrading proteins in particular, further adsorption was carried out in batch format. For this purpose, the Blue Sepharose 6 FF was used, a gel material that carries a derivatized anthraquinone dye as ligand. 0.5 g of nutsch-moist gel was added to each 10 g of solution containing fibrinogen. (Stirring time: 90 min) The dye gel was then removed by centrifugation (20 min at 25 ° C. and 1500 g).

0147The fibrinogen-containing supernatant was subjected to a further negative adsorption. Phenyl Sepharose HP was used for this. The gel material was also used in a ratio of 0.5 g per 10 g of fibrinogen-containing solution (stirring time: 90 min). Following adsorption, the gel material with the bound contaminating proteins was removed by centrifugation.

0148The subsequent pasteurization and glycine precipitation was carried out accordingly <patcit id="pcit0062" dnum="EP0103196A"><text>EP 0 103 196</text></patcit> with the exception that the precipitation was carried out in the presence of 200 mM Lys.

0149For further purification and plasminogen removal, the fibrinogen-rich precipitate was first dissolved in a suitable aqueous solvent, filtered and preferably after dialysis against buffer solution (20 mM trisodium citrate dihydrate, 50 mM NaCl, pH 7.4, optionally containing 0.05% NaN₃ as a preservative) and setting an optical density of approx. 10 via a chromatography column with a gel material which carries L-lysyl residues as ligand.

0150To produce fibrinogen preparations, the fibrinogen-containing solution was first adjusted to a protein concentration of approximately OD using suitable ultrafiltration processes, depending on the use_280-320nm = 2 - 200, preferably approx. 20 - 160, brought and then dialyzed against solutions containing the formulation constituents mentioned in Example 4.

0151Sterile filtration gave fibrinogen preparations which were tested for stability in accordance with Example 1 using SEC-HPLC. After storage for 1 month at 30 ° C and after deduction of the zero value, there was a share of degradation fragments of &lt;1.0%, measured using peak 4 and smaller peaks, i.e. significantly less than in the case of corresponding refurbishments.

0152This example showed that negative adsorption in batch format can also be used in order to achieve the highest possible stability of the fibrinogen in solution. It was also shown that several negative adsorptions can be combined. Furthermore, it can be seen that process steps with negative adsorption and / or negative chromatographies can be meaningfully integrated at different points in the process for the purification of fibrinogen. In this example, in contrast to the previous examples, the negative adsorption is used very early in the cleaning process, directly after the aluminum hydroxide treatment. Pasteurization, precipitation with glycine and removal of further plasminogen by lysine-Sepharose took place only afterwards.

Example 6:

0153In this example it was investigated to what extent fibrinogen-degrading proteins can be depleted with the help of ultrafiltration by selecting suitable pore sizes.

0154To obtain a fibrinogen-containing solution, the procedure according to Example 1 was followed up to and including the production of a pasteurized, precipitated precipitate.

0155For the further purification and depletion of fibrinogen-degrading proteins, the fibrinogen-rich precipitate was first dissolved in a suitable aqueous solvent and, using ultrafiltration membranes with a cut-off of 300 kDa, intensively against buffer solution (20 mM tri-sodium citrate dihydrate, 50 mM NaCl pH 7, 4, optionally containing 0.05% NaN₃ as a preservative) diafiltered.

0156The solution for plasminogen removal was then pumped through a chromatography column with a gel material which carries L-lysyl residues as ligands.

0157To prepare fibrinogen preparations, the fibrinogen-containing solution was first reduced to a protein concentration of approx. OD using suitable ultrafiltration processes and membranes (cut off = 300 kDa), depending on the use_280-320nm = 2 - 200, preferably approx. 20 - 160, brought and then dialyzed against solutions containing suitable formulation components.

0158After final concentration and sterile filtration, fibrinogen preparations were obtained, which were tested for the content of fibrinogen degradation fragments with the aid of SEC-HPLC (see Example 1) before the start of storage and after 1 month of storage at 30 ° C. Afterwards, after deduction of the zero value, there was a reduced proportion of degradation fragments compared to a control with conventional ultrafiltration.

0159This showed that additional fibrinogen-degrading proteins can be depleted by ultrafiltration with a cut-off of 300 kDa and that a more stable fibrinogen concentrate can be produced.

Example 7:

0160A fibrinogen precipitate was prepared as described in Example 1 and additionally purified by means of negative chromatography on Lys-Sepharose. After adding 1 mol of sodium chloride per liter of fibrinogen solution, this was added to a chromatography column filled with butyl Sepharose and rinsed with buffer. The passage of the column was concentrated, dialyzed and tested for the content of t-PA, plasminogen and F XI. As a control, a comparison was carried out in which the negative chromatography on butyl-Sepharose was not carried out. As a result it could be shown that the additional HIC significantly reduced the concentration of t-PA, plasminogen and F XI and that the stability after storage at 30 ° C was increased (the proportion of fibrinogen fragments was at 30 ° C after 1 month about 20% lower when the HIC was performed).<tables id="tabl0008" num="0008"><table frame="all"><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="32mm" /><colspec colnum="2" colname="col2" colwidth="33mm" /><colspec colnum="3" colname="col3" colwidth="34mm" /><colspec colnum="4" colname="col4" colwidth="34mm" /><colspec colnum="5" colname="col5" colwidth="35mm" /><thead><row><entry valign="top" /><entry valign="top">t-PA (ng / OD280-320)</entry><entry valign="top">Plasminogen (ng / OD280-320)</entry><entry valign="top">F XI (ng / OD280-320)</entry><entry valign="top">Fibrinogen fragments after 1 month at 30 ° C (processing without HIC = 100%)</entry></row></thead><tbody><row><entry>Refurbishment without HIC</entry><entry>0,046</entry><entry>2,47</entry><entry>0,069</entry><entry>100 %</entry></row><row><entry>Refurbishment including HIC</entry><entry>0,003</entry><entry>2,04</entry><entry>0,038</entry><entry>79 %</entry></row></tbody></tgroup></table></tables>

Example 8:

0161A fibrinogen precipitate is prepared as described in Example 1 and purified by means of negative chromatography on Lys-Sepharose. Further cleaning takes place using HIC. The fibrinogen solution is transferred to the following formulation buffers by diafiltration and ultrafiltration and, after sterile filtration, is stored at 30 ° C. in an accelerated manner:<dl id="dl0001" compact="compact"><dt>1.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 50 g / l L-Arg x HCl pH 7.2</dd><dt>2.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 70 g / l L-Arg x HCl pH 7.2</dd><dt>3.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 100 g / l L-Arg x HCl pH 7.2</dd><dt>4.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 50 g / l L-Arg x HCl, 2.5 mM CaCl₂ pH 7.2</dd><dt>5.</dt><dd>20 mM Na₃-Citrate, 100mM NaCl, 70g / L L-Arg x HCl, 2.5mM CaCl₂ pH 7.2</dd><dt>6.</dt><dd>20 mM Na₃-Citrate, 100mM NaCl, 100g / L L-Arg x HCl, 2.5mM CaCl₂ pH 7.2</dd><dt>7.</dt><dd>4th mM Na₃-Citrate, 100 mM NaCl, 50 g / l L-Arg x HCl, 0.5 mM CaCl₂ pH 7.2</dd><dt>8.</dt><dd>12th mM Na₃-Citrate, 100 mM NaCl, 50 g / l L-Arg x HCl, 1.5 mM CaCl₂ pH 7.2</dd><dt>9.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 50 g / l L-Arg x HCl, 2.5 mM CaCl₂ pH 7.2</dd><dt>10.</dt><dd>20 mM Na₃Citrate, 200 mM NaCl, 50 g / l L-Arg x HCl, 2.5 mM Cacl₂ pH 7.2</dd><dt>11.</dt><dd>20 mM Na₃-Citrate, 100mM NaCl, 70g / L L-Arg x HCl, 2.5mM CaCl₂ pH 6.8</dd><dt>12.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl, 1% L-His, 2.5 mM CaCl₂ pH 7.2</dd><dt>13.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl, 2% L-His, 2.5 mM CaCl₂ pH 7.2</dd><dt>14.</dt><dd>4th mM Na₃Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl, 0.5 mM CaCl₂ pH 7.2</dd><dt>15.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl, 30 mM aminobenzoic acid, 2.5 mM CaCl₂ pH 7.2</dd><dt>16.</dt><dd>4th mM Na₃-Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl pH 7.2</dd><dt>17.</dt><dd>4th mM Na₃-Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl, 2% L-His pH 7.2</dd><dt>18.</dt><dd>4th mM Na₃-Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl, 2% L-His pH 6.4</dd><dt>19.</dt><dd>4th mM Na₃-Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl, 2% L-His, 0.5 mM CaCl₂ pH 7.2</dd><dt>20.</dt><dd>20 mM Na₃-Citrate, 100 mM NaCl, 60 g / l L-Arg x HCl, 2.5 mM CaCl₂ pH 7.2</dd></dl>

0162The stability of the formulations listed is very good and fragment formation is observed only to a very small extent (fewer than 2% fragments (δ (≤ peak 4)) are produced during storage for one month at 30 ° C. The solutions are suitable to serve as a component of a fibrin adhesive that can be stored in the liquid state and consist of two or more components.