CA2304396A1 - A pharmaceutical preparation comprising vitamin k-dependent single factors - Google Patents
A pharmaceutical preparation comprising vitamin k-dependent single factors Download PDFInfo
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- CA2304396A1 CA2304396A1 CA002304396A CA2304396A CA2304396A1 CA 2304396 A1 CA2304396 A1 CA 2304396A1 CA 002304396 A CA002304396 A CA 002304396A CA 2304396 A CA2304396 A CA 2304396A CA 2304396 A1 CA2304396 A1 CA 2304396A1
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- preparation
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4833—Thrombin (3.4.21.5)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a pharmaceutical substance separated from a prothrombin complex, containing at least two different blood factors which are highly purified and vitamin k-dependent.
Description
MAR-15-00 06:09 +43 1 512 98 05 P.02 R-871 Job-375 15/09 '00 12:09 FAX +4a 1 512 98 05 PAT. ATT. VIENNA l~ 002/04B
A Pharmaceutical >Qreparation Comprising Vitamin K-Dependent Single Factors The invention relates to a pharmaceutical preparation comprising ,critamin K-dependent single factors.
Vitamin KJdependent proteins are characterized in that they essentially requix-e vitamin K for their biosynthesis. Thus, e.g., prothrombin ifactor Iz) formed under the influence of vitamin K antagon~.st,s, in contrast to normal prothrombin can not bind Ca2+.
Normal prothrombin contains y-carboxyglutamate at the N-terminal eza,d, i.e. a second carboxyl group at the glutamate residue. For, in the course of biogenesis of funct~.onal prothrombin, the first ten glutamate residues in the amino-terminal region of the protein are carboxyJ.ated to ~-carboxyglutamate by a vitamin K-dependent enzyme system. Tb~.s y-carboxyglutamate group is a very strong chela.ting agent for calcium .ions_ Via these bound Ga2+ ionss, prothrdmbin is bound on phospholipid membranes which are derived from cell membranes, e.g. from blood platelets, to thus obtain the correct topo7.ogy for initiation of blQOd coagulation.
It is not only prothrombin which has 'y-carboxy-glutamate xesidues, but also the coagulation factors vzl, IX and X are carboxylated on specific glutamate MAR-15-00 06:09 +43 1 512 98 05 P.03 R-971 Job-375 15/0 '00 12:10 FAX +43 1 512 98 05 PAT. ATT. SIENNA C~00a/046 residues to thus form a high affinity relative to calcium ions. Yet also further proteins involved in the coagulation cascade, such as protein C, protein S and protein Z, rEquire vitamin K for their biosynthesis.
Vitamin K-dependent single factors, in particular factors II, VTI, IX and X, knave similar physical-chemical properties, such as, e_g_, similar mol weights, pIs, eleetrophoretid mobility, etc_ and therefore as a rule are recovered togethex as prothrombin Complex (othex' designations factor IX
complex or PPSB-complex). On account of the similar protein characteristic, it is difficult to prepare the factors individually. The production of prpthrombin complex preparations with a simultaneous isolation of all the factors contained therefore has always been preferred in the prior art over the production of blood factor concentrates when producing pharmaceutical preparations (cf. Brummelhuis in: Methods of Plasma Protezn Fractionation, ed_ Curling, 1980, Academic Pross, pp_ 117-128)_ Yet it h8S also been shown that the prothrombin complex factors, on account of their different stabilities or half-li~cres, respectively, can never be obtained in a physiological ratio (always 1 U
of the protein) (cf_ Mullez et al_, Kranlmenhausphar-mazie 13 (11), (1992), 528-531; Kohler et al_, Thrombosis Research 60 (1990), pp. 63-70).
Tn EP-A 0 700 684, a prothrombin compleac MAR-15-00 06:09 +43 1 512 9B 05 P.04 R-971 Job-375 15/03 '00 12:10 FAX +43 1 512 98 05 PAT. ATT. VIENNA 0 004/046 concentrate together with at least one further blood-coagulation promoting component as an antidote for blood anticoagulants has been described.
There is the risk that prothrombin complex concentrates contain activated coagulation factors because they have been purified from complex protein mixtures, which activated coagulation factors in most instances are serine proteases. yet particularly patients should not be coagulated, since the formation of thromboses may be or is, respectively, fatal. even if only traces of these activated coagulation factors, in particular thrombin, are present in such a preparation, proteo~ytic inactivation of singe factors will occur which, depending on the individual stability of the single factors, may have severe consequences. On the whole, thus, prothrombin complex concentrates tend to instabilities and are not suitable for an extended storage. These degradation reactions, in particular due to thrombin, occur even in the solid state, which means that also drying or lyophilisation, respectively, of the preparations will not result in a reliable storage stability.
Moreover, prothrombin complex concentrates are ~xtramely unfl~xibl~ as regards the relatives ratios of, the invididual factors contained. There are hardly any possibilities to irifluence these relative ratios in the course of purifying the prothrombin complex, which is MAR-15-00 06:09 +43 1 512 98 05 P.05 R-971 Job-375 15/Oa '00 12:10 FAX +43 1 512 98 05 PAT. ATT. VIENNA 1005/046 particularly disadvantageous if a prothrombin complex in which certain factors are enriched is desired-thus, it zs an object of the present invention to provide pharmaceutical combination preparations of vitamin K-dependent proteins which are, or can be, respectively, precisely defined as regards their composition, which have a high stability, in particular during extended storage, and which are highly flexible as regards variation of their composition.
According to the invention, this object is achieved by a pharmaceutical separated prothrombin complex preparation comprising at least two chrvmatographiaally purified vitamin K-dependent single factors as active substances_ Above all, the preparation according to the invention shall contain highly purified factor IX in combination with at least one further highly purified vitamin K-dependent single faCtor_ In Contrast to the prior art in which the prothrombin complex has always been purified as a complex and has not been prepared by combination of single factors and, moreover, even at its best is present only up to an intermediary, i_e_ moderate, purity, with the present invention a preparation is provided which contains the individual blood factors in highly purified form, which arE freed from interfering contaminatipns, i~ particular of a thrombin activity.
zn particular, the single factars td be combined MAR-15-00 06:09 +43 1 512 9B 05 P.O6 R-971 Job-375 15/03 '00 12:10 FAg +43 1 512 98 05 PAT. ATT. VIENNA f~]006/046 r according to the invention are purified by chromato-graphic purification methods, such as ion e~rchange chromatography, hydrophobic chromatography, affinity chromatography and/or molecular exclusion chromato-graphy, from plasma or recombinant cells. Trx this manner, specific activities of at least 50% of the thEOretical purity, preferably at least ~0~, in particular at least ~0%, up to the theoretical purity can be attained far most vitamin K-dependent single factors in each case _ .F~ccordingly, it is also preferred to use factors which are substantially free (s 5%) of denaturing products.
In the pharmaceutical production of blood coagulation protein preparations, as a rule it is differentiated between three different degrees of purity: low, intermediary, azxd high.
Table 1 In -vivo Half-'Life Theoretical PuriWtr Factor II 60 h 0 _ 0. U/mg Factor vII 2 .- 2_5 h 2000 (1667 - 2500) Factor IX 18 - 29 h 250 (200 - 333) Factor X 40 h 118 ( 0.00 - 143 In particular when determining the factor II
activity, the results repeatedly are falsified because -MAR-15-00 06:09 +43 1 512 98 05 P.07 R-971 Job-375 15/03 '00 12:11 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~007/046 x of the presence of traces of factor zza, since even traces of factor IIa will interfere with the concentration determination of factor II such that values even by a multiple higher than the theoretical purity can be detearznined. With factor VII, the ~tralues therefore are somewhat lower relative to the other factors, since factor vzz is a very labile protein which is e~ctxemely rapidly converted to factor VITa_ Therefore, even a factor VII preparation which has more them Zoo of the theoretical purity is considered as highly purified.
since the preparation according to the invention is composed of siz~gle factor preparations which are strictly defined particularly as regards their activities and their degrees of purity, respectively, also the ratio of the single factors to each other can be optimally adjusted. 'thus, also problems occurring in the prior art in the course of further processing of the total protein complex concentrates, a_g. by activity losses duriz~.g virus inactivatioza., but also by processes which occur in the course of the pu~cification procedure, can be avoided from the very beginning, since after combin~.ng the highly puxzfied singJ.e factors to the preparation according to the ir~vention, preferably no further processing steps will be carried out_ This means that the preparation according to the MAR-15-00 06:09 +43 1 512 98 05 P.08 R-971 Job-3T5 15/Oa '00 12:11 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~ 008/04B
invention on the whole has the advantage of being standardizable. In this manner it is ensured that the respective individual factors in the added GOriCC~ritra.t10i1 are Go~J,tained in the preparation +/- 10ro deviation.
According to the invention, preferred single factors are selected from the group consisting of factor Iz, factor VI1, Factor lx, factor x, protein C, protein S and protein Z. Preferred production methods for highly purified preparations of these proteins can be found e.g. in EP 0 796 623 (factors TT and X), A 594/97 (factor VII), EP 0 496 725 (factor rX), EP 0 533 20~ (protein C) and EF 0 406 216 (protein S) _ In the px'eparation according to the. invezztion, preferably at least the factor TT, VTT, >;X and X, starting from highly purified single factors, are combined, wherein,, optionally, also the highly purified single factors protein C, protein S and/or protein Z
are admixed, so as to be able to provide a prothrombin complex which is as physiological as possible, i.e. a prothrombin complex whose composition corresponds tv the physiological one - yet without the interfering accomparyring proteins which are included during the purification of the prothrombzn complex from plasma, such as, e.g_, thrombin.
The single factors may be purified from plasma, in particular human plasma, or be prepared by recombinant MAR-15-00 06:09 +43 1 512 98 05 P.09 8-971 Job-3Z5 15/03 '00 12:11 FAX +43 1 512 98 05 PAT. ATT. VIENNA 1009/046 techr~ology_ Since in the pxeparatzon by recomb~.nant DNA
technology a separation of the structurally and physically-chemically very similar factors is not necessary, the preparation according to the invention preferably is combined of highly purified recombinant single factors. The factor may also be tranagenically prepared; it may be a derivative, in particular a peptide, and/or a fragment.
'fhe vitamin K-dependent single factors, factor II, factor VII, factor IX, J=actor x, protein S aza,d C, have been cloned and sequenced, and their production has, e.g., been described in Falkner et al., Tk~rombosi.s and Haemostasis GS (2) (1992), pp. 119 to 124, for vitamin K-dependent proteins.
AcGOrdizxg to the invention, the relatives ratios of the highly purified single factors are easily adjustable in any desired relation to each other within the preparation,, e.g. in that these ratios correspond to the natural ratios in blood, i.e. in that approximately p.er unit of the one factor, one unit each of the other factor is present_ A preferred preparation according to the present invention therefoz~e contains the highly purified single factors, factor II, Factor vzl, factor IX az~.d factox x at xelative ratios, based on international units, of (0.5 to 2) . (0_5 to 2) (0.5 to 2) . (0.5 to 2). If also protein C, protein S
and/or protein Z are present in the preparation, also _ g -MAR-15-00 06:09 +43 1 512 98 05 P.10 R-971 Job-375 15/03 '00 12:11 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~010/048 these single factors preferably have relative ratios of f rom 0 . 5 . >~v 2 .
On the other hand, it is also possible according to the invention to adjust the individual factors, due to their relative stabilities, in particular at the ratios of their relative half-lives, i_e. that more is provided of a less stable factor, and, correspondingly, less of the stable factor. In this instance, also the intended period of time of application or action, respectively, may additionally be taken into consideration, i_e_ the longer this time period, the higher also the relative ratios have to be considered.
Also recombinant proteins of, e_g., changed half-lives, may then be standardized accordingZy_ Also further factors, such as, e.g., the in vivo recovery, may be included in a standardization of the factors.
A preparation which is preferred in this respect therefore comprises the single factors, factor zI, factor VI2, factor TX and factor X at relative ratios, based on international units, of (0.5 to 2) _ (5 to 35) . (0.5 to 7) . (0.5 to 5), since the half-lives of prothrombin are 60 hours, of factor VII 2 hours, of factor IX 2o hours and of factor X 40 hours. A
preferred preparation with these factors therefore comprises the single factors, factor IT, factor VII, factor IX, factor X, protein C and protein S at relative ratios, based an international units, of (O.S
MAR-15-00 D6:09 +43 1 512 98 05 P.11 R-971 Job-375 15/0 '00 12:11 FAX +4~ 1 512 98 05 PAT. ATT. VIENNA I~ 011/046 to 2) _ (5 to as) _ (o.s to ~) . (o.~ to s) _ (1 to i5) _ (1 to 15) .
Since factor II by far is the most stable one of these factors and, moreover, also in its activated form thrombin carries the highest stability risk, a preparatlon which does not contain prothrombin is preferred_ Factor VII mostly is considered as rather unstable, and therefore in the preparation accord~.ng to the invention it is preferably provided to an increased extent, a_g_ in l0~fold concentration (based on international units). A particiularly preferred preparation thus contains single factor VIZ arid slz~.gle factor II at a ratio of greater 10 _ 1_ With the present preparation, preferably a prothrornbin complex or a partzal prothrombin complex is provided from highly purified single factors. lrr. any event, ~.t is preferred that the single factors in the preparation do not form a complex. This may be shown, a.g., by analytical. ion exchange chromatography on Q--Sepharose (Pharmacia), wherein the single factors during elution with a salt gradient can be discretely eluted. In contrast thereto are the complexes as they occur in prothrombinase or in pro-prothrombinase_ Prothrombsnase is an enzyme, substrate comple~c wrhich forms on a phospholipid surface and enables the activation of prothrombin_ Pxothrornbinase by definition consists of factor II (prothrombin), activated factor X
- io -MAR-15-00 06:09 +43 1 512 98 05 P-12 R-971 Job-375 15/Oa '00 12:12 FAX +43 1 512 98 05 PAT. ATT. VIENNA 1012/046 (factor Xa) cofactors V and/or Va, respectively, phospholipids and calcium ions_ In vivo, these factors are present as a transient complex for the activation of prothrombin and the formation of thrombin. A
corresponding pro-prothrombinase is defined as a complex of factors which are present at least partially modified or activated, respectively, for the formation of a prothrombinase. Pro-prothrombinase therefore is to be understood as a precursor of prothrombinase and as a complex in which one or more components are present in their precursors, as zymogens, or as proforms and which is formed on the basis of affizaities of the components to each other.
Por stability xeasons, it is advantageous to avoid any presence of activated coagu~.ation factors in the preparation accord~.ng to the invention_ A preferred embodiment therefore is characterized in that the preparation does not comprise any activated coagulation factors, in particular does not comprise any factor IIa, TXa, Xa and, optionally, VITa.
Preferred preparations according to the iwcrention comprise less than 0.1 U of factor VITT:C or factor Vzz :l.~g/mg of protein and/or less than 0 . 1 tT of factor zza/unzt of prothrombin and/or less than o.1 U of factor Xa/unit of factor X.
To maintain the excellent stability of the preparation according to the invention as long as MAR-15-00 06:09 +43 1 512 98 05 P.13 R-9T1 Job-3T5 15/03 '00 12:12 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~ 013/048 possible, it is advantageous to provide the preparation according to the invention in lyophilized form. zn this manner it is possible to store the preparation according to the invezxtion for an almost unlimited period of time and nevertheless reconstitute it as a lyophilisatE within a short period of time to a ready-to-use solution.
According to a further preferred embodiment, the preparation according to the invention further comprises magnesW m tons. These ions act cvmpet~.tzvely to ca~.cium ions and can displace the calcium ions primarily in the complete or partial prothrombin complex. zn this manner, a premature thrombin formation in a solution of the preparation according to the invention is prevented to an even higher extent and tha latter thus is stabilized so much that even in an agueous solution it will remain stable for many hours_ It has been shown that the pharmaceutical preparation according.to the invention can even be provided as a stable infusion solution, primarily if it is ensured that it does not contain any free calcium ions. The content of free calcium ions can easily be determined by the known ion titration or by other analytical methods_ To complex the caloium ions, e_g_ a pharmaceutically acceptable chelatir~g agent, preferably EDTA, and related substances, such as citrate, are suitable.
MAR-15-00 06:08 +43 1 512 98 05 P.14 8-971 Job-375 15/09 '00 12:12 FAX +49 1 512 98 05 PAT. ATT. VIENNA f~014/046 Preferably, the preparation according to the invention furthermore comprises antithrombin III in those amounts in which hitherto it has been used in stabilizz~r~.g manner in prothromb3n complex concentrates, optionally together with heparin. Although this measure does not seem absolutely necessary because of the hzgh degree of purity of the single factors, it may be considered advantageous for pharmaceutical reasons or also for requirements of pharmacopoieas or other rules with a view Go the prothrombzn complex concentrates of the prior art_ In another preferred embodiment, the preparation therefore is free of albumin and/or stabilizers, such as in particular antithrombin zII and/or heparin. In particular, the preparation according to the izzvention is also free from phosphol.~pids _ According to a preferred embodiment, the pharmaceutical preparation according to the invention is freed from infectious v~.xttses or other infectious agents as a consequence of a treatment for virus inactivatzon_ This treatment far virus .inactivation preferably is ensured by t.wo z.ndependent virus inactivation or vsrus depletion methods.
preferably, this s.nactivation t~reatmezxt is ensured by a tenside and/or beat treatment, e_g. by a heat treatment in the solid state, in particular a vapor treatment according to EP-o X.59 31.1, EP,O 519 901 ox MAR-15-00 06:09 +43 1 512 98 05 P.15 R-971 Job-375 15/03 '00 12:12 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~ 015/048 EP-0 674 531.
Fuxther treatments for virus inactivation also comprise the treatment w~.th chemical or chemical/physical methods, e.g. with chaotrvpiG
substances according to WO 94/13329, T7E 44 34 538 or EP-0 131 740 (solvent) yr photoinactivation.
Nanofiltration or the antibody-intensif~.ed nazlof~.ltration (w0 9740851) also constitute a preferred method for virus,depletion within the scope of the present invention.
Preferably, the preparation according to the invention further comprises pharmaceutically acceptable buffer substances or stabilizers, e.g_ the substance~a already provided for prothrombin complex concentrates in the pharmacopoeias.
A particularly preferred variant for ensuring the freedom from viruses of the product according to the invention consists in that it is composed of highly purified vitamin ic-dependent single factors which in turn have already been virus-inactivated and optionally already have been freed from denaturing pxoducts being formed and from stabilizers, so that they can be present in virus-inactivated and nevertheless precisely defined form as regards their activities.
Since above all in thermal virus inactivation methods, partial inactivation processes of the prothrombin factors may occur which, depending on the MAR-15-00 06:09 +43 1 512 98 05 P.i6 R-971 Job-375 15/03 '00 12:13 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 018/046 stability of the single factor, may lead to different yields and specific activities after the thex-mal treatment of prothrombin complex preparations, one embodiment of the method according to the invention also allows for adjusting the ratios in the mixture of single factors such that even after a thermal treatment, the factors will be present at the desired ratios to each other. E~'or instance, if it is known that the thermal treatment of prothrombin does not result in a loss of activity, whereas the factors, factor X and factor IX also present in the combination preparation will be inactivated by 20% each, a mixed, thermally virus-inactivated "complex" can be adjusted by combining the factors z>:, IX and X at activity ratios of 1 . 1.25 . 1.25. After this adjustment of the ratios, virus inactivation may then be carried out, and from this a preparation will directly result which Comprises these factors at the x'atio of 1 _ 1 . 1.
Preferalaly, factor X a.s used in its a-form and/or (3-form_ A subject matter of the present l.nvention is also a diagnostic preparation which according to the iz~.vention, is composed of the highly purified vitamin Tt-dependent single factors. Also for di~gr~.osis, the advantages of definition, variability of concentration ratios and stability are of particular advantage.
'rhe pharmaceutical preparation according iLo the ~,5 MAR-15-00 06:09 +43 1 512 96 05 P.17 R-971 Job-375 15/03 '00 12:13 FAQ +43 1 512 98 05 PAT. ATT. VIENNA 0 017/046 invention may, of course, be used for all other previous zz~,di_cat~-ozzs of the pxothrombin complex _ Thus, a subject matter of the present invention is also the use df the preparation according to tk~e invention for producing a preparation for the treatment of acquired or inherited blood coagulation disorders, for the treatment of-severe hemorrhages, for the prophylaxis of hemorrhages, in particular if inherited blood coagulation disorders are present, for substitution therapy and for the treatmez~.t of hemophilia B.
.Also for liver dysfunctions, the preparation according to the irmrention proves to be indicated.
Where administered to a patient, the administration regimen has to be taken as a basis for dosage, wherein, however, the more precise definitiQrx of the preparations of the invention is advantageous.
The present invention will be explained in more detail by way of the ~olZowing examples to which, however, it shall not be restra.cted.
E x a. m p 1 a s E x a m p 1 a 1 . Productioh of the single factor preparatioas T.~.>Qroduction of single factor pr~paratioas of plasmatic ~aGtor X and plasmatic factor II:
A lyophilized prothrombin complex factor preparation which contained factors II, TX, X as well MAR-15-00 06:09 +43 1 512 98 05 P.19 R-971 Job-375 15/03 '00 12:15 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 018/098 as protein C and protein S was prepared according to the method of Brummelhuis, H_G._J_, Preparation of the Prothrombiri complc~x_ In: Methods of Plasma Protein Fractionation, Gurl.zxlg, J.M. ed_ , 117--128, Academic Press, New York, (1980), and heat-treated for virus inactivation according to EP 159 311. Accordingly, the lyophilisate (1,QOQ U of factor X/g, 1,200 U of factor zz/g) was dissolved in distilled water so that the latter contained 50,000 a of factor X/1, and adjusted to pH 7_0. After additon of 12~ (v/v) Tween~' 80, i.t was stirred for 1 hour at room temperature. Subsequently, zt was diluted Z.5 with a 2o mM Tris-HCl buffer, pH
7.0, and the prothrombir~ complex protein fraction was adsorbed on ca~.cium phosphate (Ca3 (PO4 ) a ) at a concentrat~.on of 30 g of Ca3 (1704 ) z per 7. of prothrombin complex solution by stirring for 1 h at room temperature. Subsequently, the solid phase was separated by centrifugation, 20 min at 5,x00 rpm, and the precipitate was washed twice wzth 20 mM Tris-HC1 buffer, pH 7_0, aontaW ing 10~ ammonium sulfate, by resuspension and renewed cen.trifugation_ A third washing was carried out in. an analogous manner with 20 mM Tris-HC1 buffer, pH 7.0, containing 150 mM NaC~..
Elution of the protrirombln complex fraction eras effected with Z M sodium phosphate solution, pH 7.0, wherein 25 m1 of this solution per g of calcium phosphate were si=.ix~red for 1 hour at room temperature, MAR-15-00 06:09 +43 1 512 98 05 P-19 R-971 Job-375 15/03 '00 12:13 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~019/046 and subsequently the remaining precipitate was separated by centrif,igatian as above_ subsequently, the supernatant was subjected to an ammonium sulfate prec~.pitation with 366 g of ammonium sulfate per 1 for 15 hours at 4~C under stirring. The precipitate conta~.ning the prothrombin complex fract~.on was separated by centrifugation as above. The precipitate was taken up in a 25 mM trisodium citrate dihydrate buffer containing 100 mM NaCl, 1 mM benzamidzne hydrochloride, pH 6.0, and rebuffered on a column fil7.ed with Sephadex~ G-25 at 4°~ with a linear flow of Z cm/min against 25 mrZ tra..sodium citrate dihydrate buffer containing 100 mM NaCl az~d 1 mM benzamidine hydrochloride, pH 6.0, so as to separate the ammonium sulfate. In doing so, th.e UV absorption at 280 nm and the electx~.c conductivity were measuxed in the eluate flow. The protein-containing fractions were combined and subsequently subjected to an ion exchange chromatography over DEAF-Sepharose FF~ (Pharmacies). The fractions were appl~.ed on a column (inner diameter _ gel bed heigk-i.t = 1 . 1 . 3 ) with a gel volume of 8 _ 2 1, 0.55 g of pxotein/1 of gel, at a l~.near flow of 0.36 cm/min. Chromatography was carried out at 22°c_ Before the prc5telr~s wire applied, the coluziui had been equilibrated with a 25 mrn trisodium citrate dehydrate buffer, containing 100 mM Na~~., 1 mM benzamida.ne hydrochloride, pH 6Ø Elution of the proteizt fractions _ 18 _ MAR-15-00 06:09 +43 1 512 98 05 P.20 R-971 Job-375 15/09 '00 12:14 FAX +49 1 512 98 05 PAT. ATT. VIENNA f~020/048 was carried out in several steps with a buffer 1 (25 ml~
trisodium citrate dehydrate, 1 mM benzamidine hydrochloride, 245 mM NaCl, pH 6.0), buffer 2 (25 mM
trisodium citrate dehydrate, 1 mM benzamidiz~.e hydrochloride, 270 mM NaCl, pH 6.0) and a buffer 3 {25 mM trisodium citrate dehydrate, 1 mM benzamidine hydrochloride, 400 mM NaCl, pH 6.0). Elution with buffer 1 was carried out with 2.4 column volumes, and en doing so, inert protein was separated. Elution was carried out with 5_6 column volumes in buffer 2, and here the fractions were collected which were analyzed for their contents of factor TT, factor X, protein C
and factor TX. The factor X-containing fractions which were free from factors II, IX and protein C were com~.,ined. This highly purified factor x preparation had a specific activity of 60 U/mg of protein.
By elution with buffer 3 (1.9 column volumes), factor TI was desorbed, whereizz, again, the fractions were collected and assayed.for their contents of factor X, factor zX and factor II. The factor II-containing fractions were pooled_ loth factor II and also the factor x-containing pool optionally could be subjected to an additional treatment for inactivation of pathogerW a impuxa.tiea by the additicazx of 1 M KSCN and irxcubation at 22°C for sevexal hours.
~rhe thus recovered factor II pool was adjusted to 1.8 M NaCl by the addition of sodium chloride, and the MAR-15-00 06:08 t43 1 512 98 05 P.21 R-9T1 Job-375 15/03 '00 12:14 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 021/046 pH was corrected to pfl '7Ø Subsequently, this solution was adsorbed on a gel, Phenylsepharose Thigh Performances (Pharmacia), by hydrophobic interaction, whereby 3 g of protein/1 of gel being bovnd_ In a column with a ratio of inner diameter . gel bed height - ~. . 1.9, the protein fraction was adsorbed at a linear flow of 0.25 cm/min, and subsequently was freed fz~om inert protein by washing with a buffer (25 mM
Tris-I-ICI, 3 M NaCI, pH 7.4). By gradient elution with 21_5 column volumes of 3 M - 0.9 M NaCI with simultaneous collection of fractions, factor II was eluted from the column_ Those fractions which contained factor II activity were pooled and yet were free from factors x and IX. Subsequently, the collected factor II-fractions were concentrated 10 times by ultra/diafiltration via an ultrafil.tration membrane having a cut-off of 30 kD, and rebuffered against a buffer containing 4 g of trisodium citrate dihydrate/l, 8 g of macl/1, pH 7Ø A thus prepared factor II-preparation had a specific activity of 6.9 U/mg o~
protein_ betermination of the factor II activity was carried out with the 1 step method, based on the thromboplastin time, by using a factor 11 deficiency plasma aga~.nst the International Facta~ ~I Standard, employing the reagent combination fxom BAXTER, Vienna_ zn the coagulation analyses, other coagulation factors were detectable in traces or were not detectable any MAR-15-00 06:09 +43 1 512 96 05 P.22 R-9T1 Job-375 15/03 '00 12:15 FAX +43 1 512 98 05 PAT. ATT. VIENNA 1022/046 longer (factor VII ~ 0_OOQ02 U/U factor II, factor IX
0.0002 U/U Factor II, factor X 0_004 ~LT/U factor II, protein C 0.003 L7/U' factor II, and factor VZT~T ~ 0.0002 U/U factor IT) _ As an alternative production method for a highly purified factor II, also a method was used in which fzrst factor IX was separated from a lyophilized prothrombin complex factor preparation by hydrophobic chromatography, subsequently factor z1 was isolated, and the latter was highly purified by chromatography on hydroxyl apatite.
The prothrombin complex factor preparation was dissolved as described above azxd incubated with a detergent for 1 h at room temperature. Subsequently, a factor II, IX an X-containing fraction was isolated by ion exchange chromatography on DEAF Sepharose FF~
(Pharmacia). From this, subsequently the factor Ix-containing fraction was removed by interaction with Butyl-TOyopaarl~ (Toso F3aas) . ~rhe adsorption supernatant subsequently was purified on Phenyl-sepharose I-Iigh Performance~ (Pharmacia) by a fuz~thex hydrophobic interaction chromatography, wherein approximately ~ g of protein/1 of gel could be adsorbed. In a column havzng a ratio of in.nar dlamete~
. gel bed height = 1 . 1.9, the protein fraction was adsorbed at a linear flow of o.25 cm/min, subsequently the inert protein was removed by washing with 20 mM
MAR-15-00 06:09 +43 1 512 98 05 P.23 R-971 Job-375 15/03 '00 12:15 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 023/048 Tris-I~Cl, ~ rZ I~IaCl, pII ~.4, and finally the factor II--containing fraction was isolated by step-wise elution, wh~.ck~ Exaction de sorbed from the gel at 1.9 1K maCl at decreasing conductivity. The factor 11-containisag fraction then was directly adsorbed on Ceramik-Hydroxylapatit~ (BioRad). This was carried out on a column with a ratzo of inner diameter . gel bed height - 1 . ~.8. Elution was effected at a linear flow of 3 cm/min. By elution with a salt gradient, factor II
could be desorbed from the columrx. The factor II-containing fractions were collected and concentrated by ul.tra/diafiltration via polysulfone membranes having a cut-cuff of 30 kD, until the factor Ilmconcentration was 50-100 U/rn1_ A thus-prepared factor II preparation had a specific activ~.ty of at least 7 U/mg protein. Other coagulation factors, in particular factor TX and faotor vIII, were merely detectable in traces or not detectable at all. By selecting a suitable diafiltration buffer, the factor TT preparat~.vn was transferred into a pharmaceutica~.ly acceptable buffer (e_g_ 4 g trisodium citrate dihydrate/l, 8 g NaC1/1, pH 7.0) .
1.2 Etecovery of plasmatic factor vzz:
30 ml of fresh frozen human citrated plasma were thawed at 0 - +4~G, and the cryoprecipitate incurred was separated by centrifugation at +2~C. The "cryosupernatant" resulting therefrom was admixed with MAR-15-00 06:09 +43 1 512 98 05 P.24 8-971 Job-3Z5 15/03 '00 12:15 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~024/046 2 IU of heparin/ml. Subsequently, the proteins of the prothrombin complex were adsorbed with I7EAE-Sephadex°
A-50 (Pharmacia? at a concentration of 0.5 mg/ml_ The gel--protein complex was separated from the solution and washed respectively with buffer 1 (4 g/1 Na3 citrate _ 2H2 O, 7 g/1 NaCl, 9 g/1 Naz HPp4 .2Ha O, 500 TU
heparin/1, pH 7.5) and subsequently with buffer 2 (4 g/1 Na3citrate.2H20/1, 7 g/1 NaCl, 500 ZU hepax~in/1, pH 7.5) .
In this manner, the prothrombin complex fraction comprising the coagulation factors prothrombin, small amounts of factor VII, factor ZX and factor X was separated. The main portion of coagulation factor VII
remaining in the supernatant after adsorption on DEAE-SephadexQ° A50 was then recovered by adsorptiorx_on aluminum hydroxide_ Thereto, 10 ml of a 2% alumin~um-hydrogel suspension were admixed per 1 1 of aupernataz~.t after the prothrombin complex has been separated, and it was r~t~.rxed at 4°C for 30 minutes. subsequently, the aluminum hydroxide-protein complex was separated by means of cen~.rifugation at 5000 rpm far IO minutes at approximately 4°C in a Sor~crall RG3B Rotor H6000A. The supernatant was discarded, and the precipitate was suspended with 3_50 of the volume of the prothrombin Complex supernatant used for adsorption in a solution of 4 g Na3c~.tra><e.2H20/1 and 7 g NaCl/1, pH 7.5, and stirred for 30 minutes. zn this manner the inert MAR-15-00 06:09 +43 1 512 98 05 P.25 R-971 Job-375 15/03 '00 12:16 FAX +43 1 512 98 05 PAT. ATT. VIENNA 0 025/046 protein was desorbed from alumxrmm hydroxide. Factor VII remaining on the aluminum hydroxide was pelletized by renewed centrifugation as described above. The supernatant was discarded, and the precipitate was used for further processizag. fox desorption of the protein fraction, the aluminum hydroxide factor VII complex was stirred for 30 minutes with 1 vol.-~ of the prothrombin complex supernatant of an 0.3 mol/1 phosphate buffer used for adsorption, pH 8 . 6 ( 53 . 4 g Nay HPO,~ _ 2H2 O/1 ware adjusted to pH 8.6 with a solution of 41.1 g NaH2 Pp4 Hz Cj/1) containiza.g 3.~ 'fWEEN'~80 _ For pathogen inactivation, subsequently TWEEN~-so detergent was admixed to a final concentratioz~ of 15%, Followed by stirring for ~ 1~ at 4.0°G.
After the solution had been cooled to 2~°G, one aliquot of 2p ml was rebuffered by means of column chromatography via Sephadex~ G-50 (Pharmacia) against a solution Qf 4 g Na3citrate.2Ha0, pH 7.4, and diluted to the ZO-fold with the same solution. 'fhezi, the entire solution was applied onto a column which was packed with heparin sepharose CL6B {Pharmacia), having a diameter of Z6 mm and a height of In cm. ~rhe flow rate was 1 ml/min. Subsequently, it was eluted with the 10-fold column volume raith a salt gradient of from O-1 M
Natal in a solution of 4 g Na3citrate.2HZp/l, pH 7.4.
The protein fractzon was quantitated by measuring Uv absorption at 280 nm. Simultaneously, fractions were MAR-15-00 06:09 +43 1 512 96 05 P.26 R-971 Job-375 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA C~02B/046 collected, and factor vIZ was determined therein with a chromogenic factor VII assay (Immunochrom Faktor v1Z:10, BAXTER AG, Vienna). The fractions of the eluted protein which contained factor VII were collected and combined. The elution volume of the factor VzI fraction was 25 m~_ In the protein fraction, the protein content was determined according to the method of Bradford, M.M. (Anal. Biochem. 72: 248-254, 1976), and factor VII
as described above. Likewise, factor VIIa was quantitated according to the method of uS 5,72,850 (Ser_ No. 683582)_ Frpm this zt could be calculated that the eluting factor VII fraction had a specific activity of 98 units/mg of protein, while the factor VZIa content was below one unit/ms. Accordingly, a highly purified factor VII preparation without any substantial activation of factor VII could be recovered_ The yield at the chromatography was more than 50~.
1.3 Plasmatic factor I8.
150 ml of prothrombin complex were pre-purified by means of dextrane sulfate (Miletich et al_, Analytical Biochemistry 105, 304 (1980))_ 20 ml of eluate (912 I.U. of factor IX, 160 I.U. of factor X) were applied on a column with 20 ml of an agarose polymer with octyl groups (Octyl-Sepharose-CL-4B (Pharmacia, Sweden)) with a flow rate of 360 ml/h. The column previously had been equilibrated with 80 ml of buffer A. After washing of MAR-15-00 06:09 +43 1 512 96 05 P.27 R-871 Job-375 15/03 '00 12:16 FAX +43 1 512 98 05 PAT. ATT. VIENNA 01027/048 the loaded gel with 120 ml of buffer A, the factor TX-containing fraction. was eluted with 80 ml of a 250 mmol l~TaC1 solution _ The yield of factor IX was approximately 5-4~ of the starting activ~.ty. The specific activ~.ty was 186 1.U.
of factor IX/mg of protein. Factor X was only present in traces any more.
l.ø Recovery of plasmatic Qroteizx C
Highly pure protein C was recovered from a crude protein C fraction which was prepared from commercially obtainable prothrombin Complex concentrate.
Purification was carried out by affinity chromatography by zneaz~.s of moza,oclvnal ant~.bvdies. Monoclonal anti-protein C antibody was prepared as follows:
BALB/C mice were immunized by intraperitoriea3.
inject~.oza. with 100 ~Cg of human protein C at two-week intervals. After six weeks, once again 50 Acg of the human protein C were injected, and 3 days afterwards fusion was carried out- The rnyelvma ce~.J. line (P3-X-63-AGS-6~3, 2 _ 5 ~x i0' cells) was ma.xed rwith 1 . 7 x 108 spleen cells from a mouse, and fusion was effected according to a modified Kohler ~ Milstein method by using PEG 1500 (Kohler G_, Milstein C., Nature 256 (I975) . ~k95-497) .
Positive clones, tested by means of an ELZSA, were subcloned twice. Ascites productiozz was effected by injection pf S x 10d hybridoma cells per BALB/C mouse MAR-15-00 06:09 +43 1 512 9B 05 P.26 8-971 Job-375 15/03 '00 12:17 FAX +43 1 512 98 05 PAT. ATT. VIENNA I~028/046 two weeks a~tex~ Pristan treatment.
The immunoglobulin ra~as purified from ascites by ammonium sulfate px~ecip~.tation and subsequent chromatography by means of QAE-Sephadex and, thereafter, chromatography on Sephadex 6200. To reduce the risk of transmitting murine viruses, the ant~.body ~c,ras additionally subjected to a virus inactivation step prior to immobilisation. The thus obtained monoclonal antibodies against protein C were coupled to CNBR-Sepharose 48 fPharmacia). To purify the protein C by means of affinity chromatography, the follow ng buffers were used:
as adsorption buffer: 20 mM Tris, 2 mM EDTA, 0.25 M
NaCl and 5 mM benzamidine;
as washing buffer, 20 mM Tris, 1 M lVaCl, 2 mM
benzamidine, 2 mM EDTA was used; the pH was as elution buffer, 3 M NaSCN, 20 mM Tris, 1 M NaCl, 0.5 mM benzamidine, 2 mM EDTA was used.
h further suitable monoclonal antibody for purifying protein C has been described ~,n US 5,336,610.
This antibody binds to the act~.vation peptide of protein C and thus is suitable to selectively purify the zymogen protein C from the activated form_ In de~.ail: The prothrombin eomple~r concentrate was dissolved in adsorpt~.orz buffer, wherein approximately g. of the prothrombin complex conGentSeate were used for a 20 rnl monoclonal antibody column. Subsequently, MAR-15-00 06:09 +43 1 512 98 05 P.29 R-971 Job-3T5 15/03 '00 12:17 FAX +43 1 512 98 05 PAT. ATT. VIENNA 01029/048 the dissolved prothrombin complex concentrate was filtered, centrifuged for 15 min at 20,000 rpm, and Sterile-filtered through a 0.8 ~.em filter. The sterile-filtered and dissolved prothrombin complex concentrate was applied to a co~.umn at a flow rate of 10 ml/h. Then the column was washed with a washing b2lffer so as to be free from protein, and finally the bound protein C was eluted with the elution buffer at a flow rate of ml/h, and the fractions were collected. The eluted protein C was dialyzed against 3 buffer (0.2 M Tris, 0.15 M glycizle, and 2 mM EDTA, pH 8.3). The protein C
content was determind with respect to antigen by means of the method according to Laurell and with respect to activity after Protac activation_ The protein C el.uate obtained was finished to a pharmaceutically applicable preparation in the following manner:
At first, the eluate was subjected to an ultrafiltration and a di.a~i~tration step- For diafiltration, a buffer having a pH of 7.4 was used, which contained 3.50 mM I~5aG1 and 15 mM trisodium citrate . 2H2 O _ The filtrate obtained was freeze--dried and virus inactivated by a one-hour vappr treatment at 80°C + 5°G and X375 + 35 mbar.
The lyophilized, virus-inactivated material then was dissolved in a sterile isotonic rlacl so7.ut~.vzl, and possib~.y present antibodies or serum amylvid P, I~AR-15-00 06:09 +43 1 512 98 05 P.30 R-971 Job-375 15/03 '00 12:17 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 030/046 z'espeCtivply, were removed by means pf ipn e~GChange chromatography on c~-Sephaxose. The purified solution was concentrated by a further ultrafiltration and diafiltration step_ 'thereafter, 10 g albumin, 150 mM
NaCl and 15 mM trisodium citrate were added. The pH of the solution was 7.5. Murine zmmunoglobulin as well as the factors 1=I, VII, IX arid X could not be detected.
Subsequently, the solution was sterile-filtered, filled into containers and lyophilized. The specific activity was 14 units of protein C/mg_ As the activity assay, an amidolytic assay was used wherein protein C was activated by means of Prptac (Pentapharm).
1.5 Purification of plasmatic protein S
Human protein S was prepared from factor Ix concentrate (Frothrombin complex STIM-3, BAXTER AG, Vienna) by means of QAE-Sephadex and Blue Sepharose CL-~8 chromatography (Pharmacia) izz the following mariner:
The lyophilized concentrate (100 g) was dissvJ.ved in 200 ml of sterile, ion-free water and dialyzed against a buffer consisting of 0.01 M 2-(N-morpholinpethane-sulfonic acid), pH 6.0; 0.18 M Nacl, mM EDTA, 2 mM benzamidine-HCl and 0.02 NaN3 (starting buffer). Then the dialyzed material was applied to a QAE-Sephadex coJ.umn (8 x 19 cm) and eqlzilibrated with the buffer men~i,pned. As washing solution, 1.5 1 of buffer (starting buffer) were used.
Protein S was eluted at 1.10 ml/h with a linear NaCl MAR-15-00 06:09 +43 1 512 98 05 P.31 R-971 Job-375 15/03 '00 12:17 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~031/046 gradient Consisting of 1.2 1 Of starting buffer and 1.2 1 of a further buffer which differs from the first buffer by the addition of 0.5 M NaCl. The pxote~.z~ S
fractions were examined for protein s by means of Fast Flow SDS PAGE (PharmaCia) arid antigen daterm~uatic~n (LaureJ.l) . ~xote~.r~ S-containing fractions were pooled and finally dialyzed against a buffer. This buffer contained 50 mM Tris-HCl, pH 7.4, 150 ml~ NaCl, 2 mM
EDTA, 1 mM benzamidine-HC1 and 0_2~ NaN3. After dialysis, the protein S pool was applied onto a Blue Sepharose column CL-6B (2_5 cm x 10.5 cm) and equilibrated with the starting buffer.
wash~.z~.g was carried out with 500 ml of starting buffer at a flow rate of 15 ml/h. Thus, protein S could be eluted in the void volume, whereas prothz~vmbln adsorbed to the column. Again, the protein S rich fractions were determined by means of SDS~PAGE Fast Flow System (Pharmacia) and according to Laurell (Scand_ J_ Clin_ Invest. {Suppl_) 29 (1977) 21 (Suppl_ 124) ) .
On a reduced SDS-PAGE, the thus prepared protein S
had a morphology character~.stic of protein S, namely two close bands (doublet) with a moleGUlar weight of appro~cimately 96, oop azzd 76, 000, respectsvel~r. The protein concentration was determined spectrophoto-metrically by means of an extinction coefficiezxt Qf 0.1 at 280 nm for human protein S and was confirmed by the MAR-15-00 06:09 +43 1 512 96 05 P.32 8-971 Job-375 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~032/046 Method accordiza.g to LOwlzY (Lowry O , et al . , Protein measurement with the Folin phenol reagent, ~_ I3io1_ Chem. 193 (1951) 265).
The thus prepared, pre-purified protein S was used for preparing sheep antiserum against protein S by carrying out four immunizing injections. 100 ug of protein s had been applied subcutaneously with Freund~s adjuvant in the first two injections and incomplete adjuvant being employed in the following boosters.
After further boosters, the antiserum was tested by means of double immunodiffusion and showed a precipitation with purified protein S and with normal plasma.
The TgG fraction from 450 ml antiserum was obtained by alcohol precipitation and subsequent adsorption on Sephadex A 50 in TRTS-HCl buffer, pH 6_8_ From 454 ml of antiserum, 1.14 g of anti-protein s-zgG were present in the supernatant. The IgG fraction was coupled to 450 ml of Sepharose CL-4B, with 5.7 mg protein/m7.
Sepharose being used. The coupling efficiency was 76~.
The anti-protein S column was equilibrated with glycine-HCl, pH 3, and adsorption buffer, pI-~ ~.5.
The adsorption buffer was comprised of 20 m1K TRIS, 2 rr~ EDTA, O_25 M NaCl, 2 mM benzamidine, O_02~
Tween~ 20 and 0.02 Nal~T3, pH 7.4_ Tha washing buffer solution had the following contents: 20 mM TRTS, 2 mM
EDTA, 3._0 M IrTaCl, 0.5 mM benzamidine, 0.01% Tween~ 20;
MAR-15-00 06:09 +43 1 512 98 05 P.33 R-971 Job-375 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA I~ 033/048 0.02 NaN~, pH 7.4.
The elution buffer had a composition like the washing buffer solution, except that 0.05 Tween~ 20 and, additionally, 243.3 g of NaSCN, pH 7.4 (a 3 M
rhodanid solution) were used.
The dialysis buffer solution contayned 20 mM Tris, 0.15 mM glycine, 1 mM EI7TA, 2 mM benzamidine, pH B.3.
For further purification o~ the protein S fraction prepared from prothrombin complex concentrate, 100 g of the fraction were dissolved in 1 1 of adsorption buffer and dialyzed over night against an adsorption buffer solution. Aftex application of the sample onto the column, the column was washed with washing buffer, approx~.mately 5 1, to be free from protein, subsequently elution was carried out with 3 M NaSCN in the elution bufTer solutzon_ The eluate was dialyzed immediately until SCN was below the detection limit;
the eluate had a concentration of 500 ug/ml protein S_ zt was f x~ee f rom G~ -birxding prote in .
Monoclonal anti-protein S antibodies were prepared as follows:
BALB/C mice were immunized at two-week intervals by intraperitoneal injection of 100 N,g of the protein S
produced_ After six weeks, once again 50 E.tg of the human protein S were injected, and 3 days afterwards fus~.oz~, was carried out. The myeloma cell line (P3-X-63-.A.G8-653, 1.5 x 10' cells) was mixed with 1.7 x 108 MAR-15-00 06:09 X43 1 512 98 05 P.34 R-9T1 Job-3T5 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~034/046 spleen cells from a mouse, and fusion was effected according to a modified Icohler & Milstein method by using PEG 1500 (KShler G., Milstein C., Nature 256 (3.975) , 495-497) .
Positive clones, tested by means of an ELISA, were subcloned twice. Ascites production was effected Iay injection of 5 x 106 hybridoma cells per BALB/C mouse two weeks after Pristan treatment.
The immunoglobulin was purified from ascites by anunoza.~.um sulfate precipitation and subsequent chromatography by means of QAE-Sephadex and, thereafter, chromatography on sephadex 6200.
The IgG fraction obtained from ascites and pre-purified an protein A-Sepharose was coupled to Sepharase CL-~B. The affinity chromatographic purification o~ protein S which had been recovered from the prothrombin complex concentrate was effected under the conditions descxzbed for polyclonal protein S
antibodies. The concentration of the eluate on protein 8 was 600 ~.i.g/ml. ~t was free from C4-bind~.ng protein.
'fhe protein S preparations highly purified according to th.e method of polyclonai or monoclonal affinity chromatography were subjected to an SDS-PAGE
(gradient gel. 8 to 12%), and ~rere designated as more than 95~ pure according to Coomassie stainixa,g (Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970), -MAR-15-00 06:09 +43 1 512 98 05 P.35 R-971 Job-375 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~035/046 6$0) _ Subsequently, the eluates were subjected to an ultra~iltration and a diafiltxation step. Far diafiltration, a buffer having a pH of 7_4 was used which contained 150 mM NaCl and 15 mM txisodium citrate.2H~0_ The filtrates obtairmd were freeze-dried and virus-inactivated by a one-hour vapor treatment at 80°C ~ 5°C and 1375 + 35 mbar (to remove possibly present viral contaminations of polyclonal or monoclonal antibody)_ Then the lyophilized virus-inactivated material was dissolved in. a sterile isotonic NaCl solution and possibly present antibodies and serum amyloid P, respectively, were removed by ion exchange crrx~omatography on Q-Sepharose_ The purified solution was concentrated by a further ultrafiltration and diafiltration step. Thereafter, 10 g of albumin, 150 mM
NaCl and 15 mM trisodium citrate were added per liter to the solution obtained. The pH of the sdluta..o~c~, was 7.5. zt contained 3000 ~.g/ml protein. S. This content of protein S corresponds to a 500--fold enrichment as compared to plasma. Mur,iz~e ~.mmunoglobulin as well as the factors lZ, VII, 1X and X could not be detected.
Sub~equez~.tl.y, the solution was sterile-filtered, filled into containers and 3.yophi~.ized _ E x a m p 1 a ~ _ phax~0.aCeutiCal pr~paratioa of highly .purified vitams.n x--depe~.deat protei~2es:
.. 34 -MAR-15-00 06:09 +43 1 512 98 05 P.36 R-971 Job-375 15/03 '00 12:19 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~ 036/046 The highly purified single factors prothrombin, factor VII, factor 1X, factor X, pxotein C and protein S were combined such that a solution containing 25,000 units/1 of each of the single factors was foz~med in aqueous solutiozx of 4 g Na3 citrate _ 2Hz O and 8 g rlaCl/1. The solution was sterile-filtered via a nylon filter having a retention rate of 0_2 um and subsequently filled at 20 ml each in steri7~a.zed glass bottles having a filling volume of 50 ml_ It was freeze-dried under sterile conditions, whereafter the bottles were closed under sterile conditions.
8 x a m p 1 a 3 . Phaxmaeeutioai preparation o~
the prothromb.im complex:
As described in the previous example, a highly purified prothrombzn complex comprising the factors prothrombin, factor VII, factor ZX and factor X, yet without protel.n C and protein S, was prepared and filled into containers under sterile conditions.
l~ccordirzg to international recommendations, the solution was admixed with 7.0,000 international units of heparin/1 before filling into containers.
E x a m p 1 a 4 . pha.rmaceutiaal preparation of 'v'x.tamin x-dependent proteins for continu~aus substitution and ma~.ntaizaxn,g of coaetant plasma levels:
After substitution with a vitamin K-dependent protein concentrate in which plasma levels as constant as possible of the factors of the prothrombin complex MAR-15-00 06:29 +43 1 512 9B 05 P.02 R-972 Job-376 15/03 '00 12:29 FAX +43 1 512 98 05 PAT. ATT. VIENNA 0 037/048 ax'e to be attained, due to the different half-lives of the indi~cridual factors it is necessary to carry out the substitution rates of these prate~.ns ~.z~. a non-equivalent ratio. Accordingly, a pharmaceutical preparation as descxibed above was pxpduced which contained prothrombin, factor vII, factor 1X, factor X, protein C and protein S at a ratio of 1:30:3:1.5:6:6.
a m p 1 a 5 . Phariuaaeutical preparation of a partial prratl~xomb3ss complex:
bn account of the different a.rl va.vo half-lives of the factors of the prothrombin complex, in particular because of the long half-life of prothrombin of 2-5 days as compared to the short half--life of factor VTI
of 4-7 h, and of the factor IX of less than 24 h, in the continuous substitution with prothrombin complex preparations it happens that when adjusting a normal plasma concentration of 1 unit of factor VTZ/ml, already after 24 h the plasma concentration has decreased again to below the concentration of at least 20% required for a functioning haemostasis, whereas, a_g., the prothrombin p~.asma level still is appro~c~.mately normal, i . a . is at 1 unit/ml . A z~epeated substitution with a pxothrombin complex concentrate of the same composition as in the primary substif.ur.ion will have the result that the prothrombin plasma concentration in the patient will be increased to above the normal range so as to attain ozzce more equivalent MAR-15-00 06:29 +43 1 512 9B 05 P.03 R-972 Job-376 15/03 '00 12:29 FAX +43 1 512 98 05 PAT. ATT. VIENNA 0 038/048 Factor VTI or factor IX plasma concexitrations_ Raising the plasma prothrombin concentration to above the standard range, however, means an-increased risk of thrombosis for the patient treated, since Poort et al_ (Blood 88:3698-3703, X996) have found that a plasma level of prothrombin raised by as little as 30%
involves a significantly increased risk of venous thromboses. This problem can be avoided by using combined prothrombin cample~c preparations for secozz~dary substitutions.
Such a preparation is produces as fv~.lows: ,mss described in the previous examples, a prothrombin complex concentrate comprising factor VII, 2X and X at a ratio of 30:3:1.5, yet without prothrombin, is prepared and provided as a pharmaceutical formulation.
The Gontaizaexs of this preparation intended for administration each contained 500 international units of the coagulation factors vzz, IX and X in a dissolution volume of 10 ml after reconstitution of the lyophili.~ed powder-E x a, nay p 1 a 6 . Quality chealc of the 8arothrombin complex preparations:
The izidividual coagulation factors in the final aonta~.rxers of the prothrombin complex preparations were assayed for their content of prothrombin, factor 'V2I, factor Ix, factor x, protein C and protein S by means of ~-step coagulation tests according to standard MAR-15-00 06:29 +43 1 512 9B 05 P.04 R-9~2 Job-376 15/03 '00 12:30 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 039/048 methods, by using deficient plasmas and coagulation reagents from BAXTER, Vienna_ Details to the methods used can be taken from the paper by Muller, H_G. and Bonilc K. (Kranlcenhauspharma-zie 13:528-531, 1992)_ To determine the content of activated prothrombin complex factors, the chromogenic substrates S-2238, s-2222, S-244, S-2366 and S-2251 (Chromogenix) were used. The determinations were carried out by using the buffer conditions indicated by the producer. With the chromogenic substz~ates, in each case the activated factors thrombin, factor VIIa, factor IXa, factor Xa, activated px'otein C which developed proteolytic activities against the peptide substrates used, could be determined. After evaluation of the photometric analysis of the turnover of the chromogenic substrate it could be determined that for all the substrates the content in the previously described preparatioz~.
comprising vitamin K dependent proteins or selected factors of tha prothrombin complex was less than mU/ml in each ca6e_
A Pharmaceutical >Qreparation Comprising Vitamin K-Dependent Single Factors The invention relates to a pharmaceutical preparation comprising ,critamin K-dependent single factors.
Vitamin KJdependent proteins are characterized in that they essentially requix-e vitamin K for their biosynthesis. Thus, e.g., prothrombin ifactor Iz) formed under the influence of vitamin K antagon~.st,s, in contrast to normal prothrombin can not bind Ca2+.
Normal prothrombin contains y-carboxyglutamate at the N-terminal eza,d, i.e. a second carboxyl group at the glutamate residue. For, in the course of biogenesis of funct~.onal prothrombin, the first ten glutamate residues in the amino-terminal region of the protein are carboxyJ.ated to ~-carboxyglutamate by a vitamin K-dependent enzyme system. Tb~.s y-carboxyglutamate group is a very strong chela.ting agent for calcium .ions_ Via these bound Ga2+ ionss, prothrdmbin is bound on phospholipid membranes which are derived from cell membranes, e.g. from blood platelets, to thus obtain the correct topo7.ogy for initiation of blQOd coagulation.
It is not only prothrombin which has 'y-carboxy-glutamate xesidues, but also the coagulation factors vzl, IX and X are carboxylated on specific glutamate MAR-15-00 06:09 +43 1 512 98 05 P.03 R-971 Job-375 15/0 '00 12:10 FAX +43 1 512 98 05 PAT. ATT. SIENNA C~00a/046 residues to thus form a high affinity relative to calcium ions. Yet also further proteins involved in the coagulation cascade, such as protein C, protein S and protein Z, rEquire vitamin K for their biosynthesis.
Vitamin K-dependent single factors, in particular factors II, VTI, IX and X, knave similar physical-chemical properties, such as, e_g_, similar mol weights, pIs, eleetrophoretid mobility, etc_ and therefore as a rule are recovered togethex as prothrombin Complex (othex' designations factor IX
complex or PPSB-complex). On account of the similar protein characteristic, it is difficult to prepare the factors individually. The production of prpthrombin complex preparations with a simultaneous isolation of all the factors contained therefore has always been preferred in the prior art over the production of blood factor concentrates when producing pharmaceutical preparations (cf. Brummelhuis in: Methods of Plasma Protezn Fractionation, ed_ Curling, 1980, Academic Pross, pp_ 117-128)_ Yet it h8S also been shown that the prothrombin complex factors, on account of their different stabilities or half-li~cres, respectively, can never be obtained in a physiological ratio (always 1 U
of the protein) (cf_ Mullez et al_, Kranlmenhausphar-mazie 13 (11), (1992), 528-531; Kohler et al_, Thrombosis Research 60 (1990), pp. 63-70).
Tn EP-A 0 700 684, a prothrombin compleac MAR-15-00 06:09 +43 1 512 9B 05 P.04 R-971 Job-375 15/03 '00 12:10 FAX +43 1 512 98 05 PAT. ATT. VIENNA 0 004/046 concentrate together with at least one further blood-coagulation promoting component as an antidote for blood anticoagulants has been described.
There is the risk that prothrombin complex concentrates contain activated coagulation factors because they have been purified from complex protein mixtures, which activated coagulation factors in most instances are serine proteases. yet particularly patients should not be coagulated, since the formation of thromboses may be or is, respectively, fatal. even if only traces of these activated coagulation factors, in particular thrombin, are present in such a preparation, proteo~ytic inactivation of singe factors will occur which, depending on the individual stability of the single factors, may have severe consequences. On the whole, thus, prothrombin complex concentrates tend to instabilities and are not suitable for an extended storage. These degradation reactions, in particular due to thrombin, occur even in the solid state, which means that also drying or lyophilisation, respectively, of the preparations will not result in a reliable storage stability.
Moreover, prothrombin complex concentrates are ~xtramely unfl~xibl~ as regards the relatives ratios of, the invididual factors contained. There are hardly any possibilities to irifluence these relative ratios in the course of purifying the prothrombin complex, which is MAR-15-00 06:09 +43 1 512 98 05 P.05 R-971 Job-375 15/Oa '00 12:10 FAX +43 1 512 98 05 PAT. ATT. VIENNA 1005/046 particularly disadvantageous if a prothrombin complex in which certain factors are enriched is desired-thus, it zs an object of the present invention to provide pharmaceutical combination preparations of vitamin K-dependent proteins which are, or can be, respectively, precisely defined as regards their composition, which have a high stability, in particular during extended storage, and which are highly flexible as regards variation of their composition.
According to the invention, this object is achieved by a pharmaceutical separated prothrombin complex preparation comprising at least two chrvmatographiaally purified vitamin K-dependent single factors as active substances_ Above all, the preparation according to the invention shall contain highly purified factor IX in combination with at least one further highly purified vitamin K-dependent single faCtor_ In Contrast to the prior art in which the prothrombin complex has always been purified as a complex and has not been prepared by combination of single factors and, moreover, even at its best is present only up to an intermediary, i_e_ moderate, purity, with the present invention a preparation is provided which contains the individual blood factors in highly purified form, which arE freed from interfering contaminatipns, i~ particular of a thrombin activity.
zn particular, the single factars td be combined MAR-15-00 06:09 +43 1 512 9B 05 P.O6 R-971 Job-375 15/03 '00 12:10 FAg +43 1 512 98 05 PAT. ATT. VIENNA f~]006/046 r according to the invention are purified by chromato-graphic purification methods, such as ion e~rchange chromatography, hydrophobic chromatography, affinity chromatography and/or molecular exclusion chromato-graphy, from plasma or recombinant cells. Trx this manner, specific activities of at least 50% of the thEOretical purity, preferably at least ~0~, in particular at least ~0%, up to the theoretical purity can be attained far most vitamin K-dependent single factors in each case _ .F~ccordingly, it is also preferred to use factors which are substantially free (s 5%) of denaturing products.
In the pharmaceutical production of blood coagulation protein preparations, as a rule it is differentiated between three different degrees of purity: low, intermediary, azxd high.
Table 1 In -vivo Half-'Life Theoretical PuriWtr Factor II 60 h 0 _ 0. U/mg Factor vII 2 .- 2_5 h 2000 (1667 - 2500) Factor IX 18 - 29 h 250 (200 - 333) Factor X 40 h 118 ( 0.00 - 143 In particular when determining the factor II
activity, the results repeatedly are falsified because -MAR-15-00 06:09 +43 1 512 98 05 P.07 R-971 Job-375 15/03 '00 12:11 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~007/046 x of the presence of traces of factor zza, since even traces of factor IIa will interfere with the concentration determination of factor II such that values even by a multiple higher than the theoretical purity can be detearznined. With factor VII, the ~tralues therefore are somewhat lower relative to the other factors, since factor vzz is a very labile protein which is e~ctxemely rapidly converted to factor VITa_ Therefore, even a factor VII preparation which has more them Zoo of the theoretical purity is considered as highly purified.
since the preparation according to the invention is composed of siz~gle factor preparations which are strictly defined particularly as regards their activities and their degrees of purity, respectively, also the ratio of the single factors to each other can be optimally adjusted. 'thus, also problems occurring in the prior art in the course of further processing of the total protein complex concentrates, a_g. by activity losses duriz~.g virus inactivatioza., but also by processes which occur in the course of the pu~cification procedure, can be avoided from the very beginning, since after combin~.ng the highly puxzfied singJ.e factors to the preparation according to the ir~vention, preferably no further processing steps will be carried out_ This means that the preparation according to the MAR-15-00 06:09 +43 1 512 98 05 P.08 R-971 Job-3T5 15/Oa '00 12:11 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~ 008/04B
invention on the whole has the advantage of being standardizable. In this manner it is ensured that the respective individual factors in the added GOriCC~ritra.t10i1 are Go~J,tained in the preparation +/- 10ro deviation.
According to the invention, preferred single factors are selected from the group consisting of factor Iz, factor VI1, Factor lx, factor x, protein C, protein S and protein Z. Preferred production methods for highly purified preparations of these proteins can be found e.g. in EP 0 796 623 (factors TT and X), A 594/97 (factor VII), EP 0 496 725 (factor rX), EP 0 533 20~ (protein C) and EF 0 406 216 (protein S) _ In the px'eparation according to the. invezztion, preferably at least the factor TT, VTT, >;X and X, starting from highly purified single factors, are combined, wherein,, optionally, also the highly purified single factors protein C, protein S and/or protein Z
are admixed, so as to be able to provide a prothrombin complex which is as physiological as possible, i.e. a prothrombin complex whose composition corresponds tv the physiological one - yet without the interfering accomparyring proteins which are included during the purification of the prothrombzn complex from plasma, such as, e.g_, thrombin.
The single factors may be purified from plasma, in particular human plasma, or be prepared by recombinant MAR-15-00 06:09 +43 1 512 98 05 P.09 8-971 Job-3Z5 15/03 '00 12:11 FAX +43 1 512 98 05 PAT. ATT. VIENNA 1009/046 techr~ology_ Since in the pxeparatzon by recomb~.nant DNA
technology a separation of the structurally and physically-chemically very similar factors is not necessary, the preparation according to the invention preferably is combined of highly purified recombinant single factors. The factor may also be tranagenically prepared; it may be a derivative, in particular a peptide, and/or a fragment.
'fhe vitamin K-dependent single factors, factor II, factor VII, factor IX, J=actor x, protein S aza,d C, have been cloned and sequenced, and their production has, e.g., been described in Falkner et al., Tk~rombosi.s and Haemostasis GS (2) (1992), pp. 119 to 124, for vitamin K-dependent proteins.
AcGOrdizxg to the invention, the relatives ratios of the highly purified single factors are easily adjustable in any desired relation to each other within the preparation,, e.g. in that these ratios correspond to the natural ratios in blood, i.e. in that approximately p.er unit of the one factor, one unit each of the other factor is present_ A preferred preparation according to the present invention therefoz~e contains the highly purified single factors, factor II, Factor vzl, factor IX az~.d factox x at xelative ratios, based on international units, of (0.5 to 2) . (0_5 to 2) (0.5 to 2) . (0.5 to 2). If also protein C, protein S
and/or protein Z are present in the preparation, also _ g -MAR-15-00 06:09 +43 1 512 98 05 P.10 R-971 Job-375 15/03 '00 12:11 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~010/048 these single factors preferably have relative ratios of f rom 0 . 5 . >~v 2 .
On the other hand, it is also possible according to the invention to adjust the individual factors, due to their relative stabilities, in particular at the ratios of their relative half-lives, i_e. that more is provided of a less stable factor, and, correspondingly, less of the stable factor. In this instance, also the intended period of time of application or action, respectively, may additionally be taken into consideration, i_e_ the longer this time period, the higher also the relative ratios have to be considered.
Also recombinant proteins of, e_g., changed half-lives, may then be standardized accordingZy_ Also further factors, such as, e.g., the in vivo recovery, may be included in a standardization of the factors.
A preparation which is preferred in this respect therefore comprises the single factors, factor zI, factor VI2, factor TX and factor X at relative ratios, based on international units, of (0.5 to 2) _ (5 to 35) . (0.5 to 7) . (0.5 to 5), since the half-lives of prothrombin are 60 hours, of factor VII 2 hours, of factor IX 2o hours and of factor X 40 hours. A
preferred preparation with these factors therefore comprises the single factors, factor IT, factor VII, factor IX, factor X, protein C and protein S at relative ratios, based an international units, of (O.S
MAR-15-00 D6:09 +43 1 512 98 05 P.11 R-971 Job-375 15/0 '00 12:11 FAX +4~ 1 512 98 05 PAT. ATT. VIENNA I~ 011/046 to 2) _ (5 to as) _ (o.s to ~) . (o.~ to s) _ (1 to i5) _ (1 to 15) .
Since factor II by far is the most stable one of these factors and, moreover, also in its activated form thrombin carries the highest stability risk, a preparatlon which does not contain prothrombin is preferred_ Factor VII mostly is considered as rather unstable, and therefore in the preparation accord~.ng to the invention it is preferably provided to an increased extent, a_g_ in l0~fold concentration (based on international units). A particiularly preferred preparation thus contains single factor VIZ arid slz~.gle factor II at a ratio of greater 10 _ 1_ With the present preparation, preferably a prothrornbin complex or a partzal prothrombin complex is provided from highly purified single factors. lrr. any event, ~.t is preferred that the single factors in the preparation do not form a complex. This may be shown, a.g., by analytical. ion exchange chromatography on Q--Sepharose (Pharmacia), wherein the single factors during elution with a salt gradient can be discretely eluted. In contrast thereto are the complexes as they occur in prothrombinase or in pro-prothrombinase_ Prothrombsnase is an enzyme, substrate comple~c wrhich forms on a phospholipid surface and enables the activation of prothrombin_ Pxothrornbinase by definition consists of factor II (prothrombin), activated factor X
- io -MAR-15-00 06:09 +43 1 512 98 05 P-12 R-971 Job-375 15/Oa '00 12:12 FAX +43 1 512 98 05 PAT. ATT. VIENNA 1012/046 (factor Xa) cofactors V and/or Va, respectively, phospholipids and calcium ions_ In vivo, these factors are present as a transient complex for the activation of prothrombin and the formation of thrombin. A
corresponding pro-prothrombinase is defined as a complex of factors which are present at least partially modified or activated, respectively, for the formation of a prothrombinase. Pro-prothrombinase therefore is to be understood as a precursor of prothrombinase and as a complex in which one or more components are present in their precursors, as zymogens, or as proforms and which is formed on the basis of affizaities of the components to each other.
Por stability xeasons, it is advantageous to avoid any presence of activated coagu~.ation factors in the preparation accord~.ng to the invention_ A preferred embodiment therefore is characterized in that the preparation does not comprise any activated coagulation factors, in particular does not comprise any factor IIa, TXa, Xa and, optionally, VITa.
Preferred preparations according to the iwcrention comprise less than 0.1 U of factor VITT:C or factor Vzz :l.~g/mg of protein and/or less than 0 . 1 tT of factor zza/unzt of prothrombin and/or less than o.1 U of factor Xa/unit of factor X.
To maintain the excellent stability of the preparation according to the invention as long as MAR-15-00 06:09 +43 1 512 98 05 P.13 R-9T1 Job-3T5 15/03 '00 12:12 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~ 013/048 possible, it is advantageous to provide the preparation according to the invention in lyophilized form. zn this manner it is possible to store the preparation according to the invezxtion for an almost unlimited period of time and nevertheless reconstitute it as a lyophilisatE within a short period of time to a ready-to-use solution.
According to a further preferred embodiment, the preparation according to the invention further comprises magnesW m tons. These ions act cvmpet~.tzvely to ca~.cium ions and can displace the calcium ions primarily in the complete or partial prothrombin complex. zn this manner, a premature thrombin formation in a solution of the preparation according to the invention is prevented to an even higher extent and tha latter thus is stabilized so much that even in an agueous solution it will remain stable for many hours_ It has been shown that the pharmaceutical preparation according.to the invention can even be provided as a stable infusion solution, primarily if it is ensured that it does not contain any free calcium ions. The content of free calcium ions can easily be determined by the known ion titration or by other analytical methods_ To complex the caloium ions, e_g_ a pharmaceutically acceptable chelatir~g agent, preferably EDTA, and related substances, such as citrate, are suitable.
MAR-15-00 06:08 +43 1 512 98 05 P.14 8-971 Job-375 15/09 '00 12:12 FAX +49 1 512 98 05 PAT. ATT. VIENNA f~014/046 Preferably, the preparation according to the invention furthermore comprises antithrombin III in those amounts in which hitherto it has been used in stabilizz~r~.g manner in prothromb3n complex concentrates, optionally together with heparin. Although this measure does not seem absolutely necessary because of the hzgh degree of purity of the single factors, it may be considered advantageous for pharmaceutical reasons or also for requirements of pharmacopoieas or other rules with a view Go the prothrombzn complex concentrates of the prior art_ In another preferred embodiment, the preparation therefore is free of albumin and/or stabilizers, such as in particular antithrombin zII and/or heparin. In particular, the preparation according to the izzvention is also free from phosphol.~pids _ According to a preferred embodiment, the pharmaceutical preparation according to the invention is freed from infectious v~.xttses or other infectious agents as a consequence of a treatment for virus inactivatzon_ This treatment far virus .inactivation preferably is ensured by t.wo z.ndependent virus inactivation or vsrus depletion methods.
preferably, this s.nactivation t~reatmezxt is ensured by a tenside and/or beat treatment, e_g. by a heat treatment in the solid state, in particular a vapor treatment according to EP-o X.59 31.1, EP,O 519 901 ox MAR-15-00 06:09 +43 1 512 98 05 P.15 R-971 Job-375 15/03 '00 12:12 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~ 015/048 EP-0 674 531.
Fuxther treatments for virus inactivation also comprise the treatment w~.th chemical or chemical/physical methods, e.g. with chaotrvpiG
substances according to WO 94/13329, T7E 44 34 538 or EP-0 131 740 (solvent) yr photoinactivation.
Nanofiltration or the antibody-intensif~.ed nazlof~.ltration (w0 9740851) also constitute a preferred method for virus,depletion within the scope of the present invention.
Preferably, the preparation according to the invention further comprises pharmaceutically acceptable buffer substances or stabilizers, e.g_ the substance~a already provided for prothrombin complex concentrates in the pharmacopoeias.
A particularly preferred variant for ensuring the freedom from viruses of the product according to the invention consists in that it is composed of highly purified vitamin ic-dependent single factors which in turn have already been virus-inactivated and optionally already have been freed from denaturing pxoducts being formed and from stabilizers, so that they can be present in virus-inactivated and nevertheless precisely defined form as regards their activities.
Since above all in thermal virus inactivation methods, partial inactivation processes of the prothrombin factors may occur which, depending on the MAR-15-00 06:09 +43 1 512 98 05 P.i6 R-971 Job-375 15/03 '00 12:13 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 018/046 stability of the single factor, may lead to different yields and specific activities after the thex-mal treatment of prothrombin complex preparations, one embodiment of the method according to the invention also allows for adjusting the ratios in the mixture of single factors such that even after a thermal treatment, the factors will be present at the desired ratios to each other. E~'or instance, if it is known that the thermal treatment of prothrombin does not result in a loss of activity, whereas the factors, factor X and factor IX also present in the combination preparation will be inactivated by 20% each, a mixed, thermally virus-inactivated "complex" can be adjusted by combining the factors z>:, IX and X at activity ratios of 1 . 1.25 . 1.25. After this adjustment of the ratios, virus inactivation may then be carried out, and from this a preparation will directly result which Comprises these factors at the x'atio of 1 _ 1 . 1.
Preferalaly, factor X a.s used in its a-form and/or (3-form_ A subject matter of the present l.nvention is also a diagnostic preparation which according to the iz~.vention, is composed of the highly purified vitamin Tt-dependent single factors. Also for di~gr~.osis, the advantages of definition, variability of concentration ratios and stability are of particular advantage.
'rhe pharmaceutical preparation according iLo the ~,5 MAR-15-00 06:09 +43 1 512 96 05 P.17 R-971 Job-375 15/03 '00 12:13 FAQ +43 1 512 98 05 PAT. ATT. VIENNA 0 017/046 invention may, of course, be used for all other previous zz~,di_cat~-ozzs of the pxothrombin complex _ Thus, a subject matter of the present invention is also the use df the preparation according to tk~e invention for producing a preparation for the treatment of acquired or inherited blood coagulation disorders, for the treatment of-severe hemorrhages, for the prophylaxis of hemorrhages, in particular if inherited blood coagulation disorders are present, for substitution therapy and for the treatmez~.t of hemophilia B.
.Also for liver dysfunctions, the preparation according to the irmrention proves to be indicated.
Where administered to a patient, the administration regimen has to be taken as a basis for dosage, wherein, however, the more precise definitiQrx of the preparations of the invention is advantageous.
The present invention will be explained in more detail by way of the ~olZowing examples to which, however, it shall not be restra.cted.
E x a. m p 1 a s E x a m p 1 a 1 . Productioh of the single factor preparatioas T.~.>Qroduction of single factor pr~paratioas of plasmatic ~aGtor X and plasmatic factor II:
A lyophilized prothrombin complex factor preparation which contained factors II, TX, X as well MAR-15-00 06:09 +43 1 512 98 05 P.19 R-971 Job-375 15/03 '00 12:15 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 018/098 as protein C and protein S was prepared according to the method of Brummelhuis, H_G._J_, Preparation of the Prothrombiri complc~x_ In: Methods of Plasma Protein Fractionation, Gurl.zxlg, J.M. ed_ , 117--128, Academic Press, New York, (1980), and heat-treated for virus inactivation according to EP 159 311. Accordingly, the lyophilisate (1,QOQ U of factor X/g, 1,200 U of factor zz/g) was dissolved in distilled water so that the latter contained 50,000 a of factor X/1, and adjusted to pH 7_0. After additon of 12~ (v/v) Tween~' 80, i.t was stirred for 1 hour at room temperature. Subsequently, zt was diluted Z.5 with a 2o mM Tris-HCl buffer, pH
7.0, and the prothrombir~ complex protein fraction was adsorbed on ca~.cium phosphate (Ca3 (PO4 ) a ) at a concentrat~.on of 30 g of Ca3 (1704 ) z per 7. of prothrombin complex solution by stirring for 1 h at room temperature. Subsequently, the solid phase was separated by centrifugation, 20 min at 5,x00 rpm, and the precipitate was washed twice wzth 20 mM Tris-HC1 buffer, pH 7_0, aontaW ing 10~ ammonium sulfate, by resuspension and renewed cen.trifugation_ A third washing was carried out in. an analogous manner with 20 mM Tris-HC1 buffer, pH 7.0, containing 150 mM NaC~..
Elution of the protrirombln complex fraction eras effected with Z M sodium phosphate solution, pH 7.0, wherein 25 m1 of this solution per g of calcium phosphate were si=.ix~red for 1 hour at room temperature, MAR-15-00 06:09 +43 1 512 98 05 P-19 R-971 Job-375 15/03 '00 12:13 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~019/046 and subsequently the remaining precipitate was separated by centrif,igatian as above_ subsequently, the supernatant was subjected to an ammonium sulfate prec~.pitation with 366 g of ammonium sulfate per 1 for 15 hours at 4~C under stirring. The precipitate conta~.ning the prothrombin complex fract~.on was separated by centrifugation as above. The precipitate was taken up in a 25 mM trisodium citrate dihydrate buffer containing 100 mM NaCl, 1 mM benzamidzne hydrochloride, pH 6.0, and rebuffered on a column fil7.ed with Sephadex~ G-25 at 4°~ with a linear flow of Z cm/min against 25 mrZ tra..sodium citrate dihydrate buffer containing 100 mM NaCl az~d 1 mM benzamidine hydrochloride, pH 6.0, so as to separate the ammonium sulfate. In doing so, th.e UV absorption at 280 nm and the electx~.c conductivity were measuxed in the eluate flow. The protein-containing fractions were combined and subsequently subjected to an ion exchange chromatography over DEAF-Sepharose FF~ (Pharmacies). The fractions were appl~.ed on a column (inner diameter _ gel bed heigk-i.t = 1 . 1 . 3 ) with a gel volume of 8 _ 2 1, 0.55 g of pxotein/1 of gel, at a l~.near flow of 0.36 cm/min. Chromatography was carried out at 22°c_ Before the prc5telr~s wire applied, the coluziui had been equilibrated with a 25 mrn trisodium citrate dehydrate buffer, containing 100 mM Na~~., 1 mM benzamida.ne hydrochloride, pH 6Ø Elution of the proteizt fractions _ 18 _ MAR-15-00 06:09 +43 1 512 98 05 P.20 R-971 Job-375 15/09 '00 12:14 FAX +49 1 512 98 05 PAT. ATT. VIENNA f~020/048 was carried out in several steps with a buffer 1 (25 ml~
trisodium citrate dehydrate, 1 mM benzamidine hydrochloride, 245 mM NaCl, pH 6.0), buffer 2 (25 mM
trisodium citrate dehydrate, 1 mM benzamidiz~.e hydrochloride, 270 mM NaCl, pH 6.0) and a buffer 3 {25 mM trisodium citrate dehydrate, 1 mM benzamidine hydrochloride, 400 mM NaCl, pH 6.0). Elution with buffer 1 was carried out with 2.4 column volumes, and en doing so, inert protein was separated. Elution was carried out with 5_6 column volumes in buffer 2, and here the fractions were collected which were analyzed for their contents of factor TT, factor X, protein C
and factor TX. The factor X-containing fractions which were free from factors II, IX and protein C were com~.,ined. This highly purified factor x preparation had a specific activity of 60 U/mg of protein.
By elution with buffer 3 (1.9 column volumes), factor TI was desorbed, whereizz, again, the fractions were collected and assayed.for their contents of factor X, factor zX and factor II. The factor II-containing fractions were pooled_ loth factor II and also the factor x-containing pool optionally could be subjected to an additional treatment for inactivation of pathogerW a impuxa.tiea by the additicazx of 1 M KSCN and irxcubation at 22°C for sevexal hours.
~rhe thus recovered factor II pool was adjusted to 1.8 M NaCl by the addition of sodium chloride, and the MAR-15-00 06:08 t43 1 512 98 05 P.21 R-9T1 Job-375 15/03 '00 12:14 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 021/046 pH was corrected to pfl '7Ø Subsequently, this solution was adsorbed on a gel, Phenylsepharose Thigh Performances (Pharmacia), by hydrophobic interaction, whereby 3 g of protein/1 of gel being bovnd_ In a column with a ratio of inner diameter . gel bed height - ~. . 1.9, the protein fraction was adsorbed at a linear flow of 0.25 cm/min, and subsequently was freed fz~om inert protein by washing with a buffer (25 mM
Tris-I-ICI, 3 M NaCI, pH 7.4). By gradient elution with 21_5 column volumes of 3 M - 0.9 M NaCI with simultaneous collection of fractions, factor II was eluted from the column_ Those fractions which contained factor II activity were pooled and yet were free from factors x and IX. Subsequently, the collected factor II-fractions were concentrated 10 times by ultra/diafiltration via an ultrafil.tration membrane having a cut-off of 30 kD, and rebuffered against a buffer containing 4 g of trisodium citrate dihydrate/l, 8 g of macl/1, pH 7Ø A thus prepared factor II-preparation had a specific activity of 6.9 U/mg o~
protein_ betermination of the factor II activity was carried out with the 1 step method, based on the thromboplastin time, by using a factor 11 deficiency plasma aga~.nst the International Facta~ ~I Standard, employing the reagent combination fxom BAXTER, Vienna_ zn the coagulation analyses, other coagulation factors were detectable in traces or were not detectable any MAR-15-00 06:09 +43 1 512 96 05 P.22 R-9T1 Job-375 15/03 '00 12:15 FAX +43 1 512 98 05 PAT. ATT. VIENNA 1022/046 longer (factor VII ~ 0_OOQ02 U/U factor II, factor IX
0.0002 U/U Factor II, factor X 0_004 ~LT/U factor II, protein C 0.003 L7/U' factor II, and factor VZT~T ~ 0.0002 U/U factor IT) _ As an alternative production method for a highly purified factor II, also a method was used in which fzrst factor IX was separated from a lyophilized prothrombin complex factor preparation by hydrophobic chromatography, subsequently factor z1 was isolated, and the latter was highly purified by chromatography on hydroxyl apatite.
The prothrombin complex factor preparation was dissolved as described above azxd incubated with a detergent for 1 h at room temperature. Subsequently, a factor II, IX an X-containing fraction was isolated by ion exchange chromatography on DEAF Sepharose FF~
(Pharmacia). From this, subsequently the factor Ix-containing fraction was removed by interaction with Butyl-TOyopaarl~ (Toso F3aas) . ~rhe adsorption supernatant subsequently was purified on Phenyl-sepharose I-Iigh Performance~ (Pharmacia) by a fuz~thex hydrophobic interaction chromatography, wherein approximately ~ g of protein/1 of gel could be adsorbed. In a column havzng a ratio of in.nar dlamete~
. gel bed height = 1 . 1.9, the protein fraction was adsorbed at a linear flow of o.25 cm/min, subsequently the inert protein was removed by washing with 20 mM
MAR-15-00 06:09 +43 1 512 98 05 P.23 R-971 Job-375 15/03 '00 12:15 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 023/048 Tris-I~Cl, ~ rZ I~IaCl, pII ~.4, and finally the factor II--containing fraction was isolated by step-wise elution, wh~.ck~ Exaction de sorbed from the gel at 1.9 1K maCl at decreasing conductivity. The factor 11-containisag fraction then was directly adsorbed on Ceramik-Hydroxylapatit~ (BioRad). This was carried out on a column with a ratzo of inner diameter . gel bed height - 1 . ~.8. Elution was effected at a linear flow of 3 cm/min. By elution with a salt gradient, factor II
could be desorbed from the columrx. The factor II-containing fractions were collected and concentrated by ul.tra/diafiltration via polysulfone membranes having a cut-cuff of 30 kD, until the factor Ilmconcentration was 50-100 U/rn1_ A thus-prepared factor II preparation had a specific activ~.ty of at least 7 U/mg protein. Other coagulation factors, in particular factor TX and faotor vIII, were merely detectable in traces or not detectable at all. By selecting a suitable diafiltration buffer, the factor TT preparat~.vn was transferred into a pharmaceutica~.ly acceptable buffer (e_g_ 4 g trisodium citrate dihydrate/l, 8 g NaC1/1, pH 7.0) .
1.2 Etecovery of plasmatic factor vzz:
30 ml of fresh frozen human citrated plasma were thawed at 0 - +4~G, and the cryoprecipitate incurred was separated by centrifugation at +2~C. The "cryosupernatant" resulting therefrom was admixed with MAR-15-00 06:09 +43 1 512 98 05 P.24 8-971 Job-3Z5 15/03 '00 12:15 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~024/046 2 IU of heparin/ml. Subsequently, the proteins of the prothrombin complex were adsorbed with I7EAE-Sephadex°
A-50 (Pharmacia? at a concentration of 0.5 mg/ml_ The gel--protein complex was separated from the solution and washed respectively with buffer 1 (4 g/1 Na3 citrate _ 2H2 O, 7 g/1 NaCl, 9 g/1 Naz HPp4 .2Ha O, 500 TU
heparin/1, pH 7.5) and subsequently with buffer 2 (4 g/1 Na3citrate.2H20/1, 7 g/1 NaCl, 500 ZU hepax~in/1, pH 7.5) .
In this manner, the prothrombin complex fraction comprising the coagulation factors prothrombin, small amounts of factor VII, factor ZX and factor X was separated. The main portion of coagulation factor VII
remaining in the supernatant after adsorption on DEAE-SephadexQ° A50 was then recovered by adsorptiorx_on aluminum hydroxide_ Thereto, 10 ml of a 2% alumin~um-hydrogel suspension were admixed per 1 1 of aupernataz~.t after the prothrombin complex has been separated, and it was r~t~.rxed at 4°C for 30 minutes. subsequently, the aluminum hydroxide-protein complex was separated by means of cen~.rifugation at 5000 rpm far IO minutes at approximately 4°C in a Sor~crall RG3B Rotor H6000A. The supernatant was discarded, and the precipitate was suspended with 3_50 of the volume of the prothrombin Complex supernatant used for adsorption in a solution of 4 g Na3c~.tra><e.2H20/1 and 7 g NaCl/1, pH 7.5, and stirred for 30 minutes. zn this manner the inert MAR-15-00 06:09 +43 1 512 98 05 P.25 R-971 Job-375 15/03 '00 12:16 FAX +43 1 512 98 05 PAT. ATT. VIENNA 0 025/046 protein was desorbed from alumxrmm hydroxide. Factor VII remaining on the aluminum hydroxide was pelletized by renewed centrifugation as described above. The supernatant was discarded, and the precipitate was used for further processizag. fox desorption of the protein fraction, the aluminum hydroxide factor VII complex was stirred for 30 minutes with 1 vol.-~ of the prothrombin complex supernatant of an 0.3 mol/1 phosphate buffer used for adsorption, pH 8 . 6 ( 53 . 4 g Nay HPO,~ _ 2H2 O/1 ware adjusted to pH 8.6 with a solution of 41.1 g NaH2 Pp4 Hz Cj/1) containiza.g 3.~ 'fWEEN'~80 _ For pathogen inactivation, subsequently TWEEN~-so detergent was admixed to a final concentratioz~ of 15%, Followed by stirring for ~ 1~ at 4.0°G.
After the solution had been cooled to 2~°G, one aliquot of 2p ml was rebuffered by means of column chromatography via Sephadex~ G-50 (Pharmacia) against a solution Qf 4 g Na3citrate.2Ha0, pH 7.4, and diluted to the ZO-fold with the same solution. 'fhezi, the entire solution was applied onto a column which was packed with heparin sepharose CL6B {Pharmacia), having a diameter of Z6 mm and a height of In cm. ~rhe flow rate was 1 ml/min. Subsequently, it was eluted with the 10-fold column volume raith a salt gradient of from O-1 M
Natal in a solution of 4 g Na3citrate.2HZp/l, pH 7.4.
The protein fractzon was quantitated by measuring Uv absorption at 280 nm. Simultaneously, fractions were MAR-15-00 06:09 +43 1 512 96 05 P.26 R-971 Job-375 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA C~02B/046 collected, and factor vIZ was determined therein with a chromogenic factor VII assay (Immunochrom Faktor v1Z:10, BAXTER AG, Vienna). The fractions of the eluted protein which contained factor VII were collected and combined. The elution volume of the factor VzI fraction was 25 m~_ In the protein fraction, the protein content was determined according to the method of Bradford, M.M. (Anal. Biochem. 72: 248-254, 1976), and factor VII
as described above. Likewise, factor VIIa was quantitated according to the method of uS 5,72,850 (Ser_ No. 683582)_ Frpm this zt could be calculated that the eluting factor VII fraction had a specific activity of 98 units/mg of protein, while the factor VZIa content was below one unit/ms. Accordingly, a highly purified factor VII preparation without any substantial activation of factor VII could be recovered_ The yield at the chromatography was more than 50~.
1.3 Plasmatic factor I8.
150 ml of prothrombin complex were pre-purified by means of dextrane sulfate (Miletich et al_, Analytical Biochemistry 105, 304 (1980))_ 20 ml of eluate (912 I.U. of factor IX, 160 I.U. of factor X) were applied on a column with 20 ml of an agarose polymer with octyl groups (Octyl-Sepharose-CL-4B (Pharmacia, Sweden)) with a flow rate of 360 ml/h. The column previously had been equilibrated with 80 ml of buffer A. After washing of MAR-15-00 06:09 +43 1 512 96 05 P.27 R-871 Job-375 15/03 '00 12:16 FAX +43 1 512 98 05 PAT. ATT. VIENNA 01027/048 the loaded gel with 120 ml of buffer A, the factor TX-containing fraction. was eluted with 80 ml of a 250 mmol l~TaC1 solution _ The yield of factor IX was approximately 5-4~ of the starting activ~.ty. The specific activ~.ty was 186 1.U.
of factor IX/mg of protein. Factor X was only present in traces any more.
l.ø Recovery of plasmatic Qroteizx C
Highly pure protein C was recovered from a crude protein C fraction which was prepared from commercially obtainable prothrombin Complex concentrate.
Purification was carried out by affinity chromatography by zneaz~.s of moza,oclvnal ant~.bvdies. Monoclonal anti-protein C antibody was prepared as follows:
BALB/C mice were immunized by intraperitoriea3.
inject~.oza. with 100 ~Cg of human protein C at two-week intervals. After six weeks, once again 50 Acg of the human protein C were injected, and 3 days afterwards fusion was carried out- The rnyelvma ce~.J. line (P3-X-63-AGS-6~3, 2 _ 5 ~x i0' cells) was ma.xed rwith 1 . 7 x 108 spleen cells from a mouse, and fusion was effected according to a modified Kohler ~ Milstein method by using PEG 1500 (Kohler G_, Milstein C., Nature 256 (I975) . ~k95-497) .
Positive clones, tested by means of an ELZSA, were subcloned twice. Ascites productiozz was effected by injection pf S x 10d hybridoma cells per BALB/C mouse MAR-15-00 06:09 +43 1 512 9B 05 P.26 8-971 Job-375 15/03 '00 12:17 FAX +43 1 512 98 05 PAT. ATT. VIENNA I~028/046 two weeks a~tex~ Pristan treatment.
The immunoglobulin ra~as purified from ascites by ammonium sulfate px~ecip~.tation and subsequent chromatography by means of QAE-Sephadex and, thereafter, chromatography on Sephadex 6200. To reduce the risk of transmitting murine viruses, the ant~.body ~c,ras additionally subjected to a virus inactivation step prior to immobilisation. The thus obtained monoclonal antibodies against protein C were coupled to CNBR-Sepharose 48 fPharmacia). To purify the protein C by means of affinity chromatography, the follow ng buffers were used:
as adsorption buffer: 20 mM Tris, 2 mM EDTA, 0.25 M
NaCl and 5 mM benzamidine;
as washing buffer, 20 mM Tris, 1 M lVaCl, 2 mM
benzamidine, 2 mM EDTA was used; the pH was as elution buffer, 3 M NaSCN, 20 mM Tris, 1 M NaCl, 0.5 mM benzamidine, 2 mM EDTA was used.
h further suitable monoclonal antibody for purifying protein C has been described ~,n US 5,336,610.
This antibody binds to the act~.vation peptide of protein C and thus is suitable to selectively purify the zymogen protein C from the activated form_ In de~.ail: The prothrombin eomple~r concentrate was dissolved in adsorpt~.orz buffer, wherein approximately g. of the prothrombin complex conGentSeate were used for a 20 rnl monoclonal antibody column. Subsequently, MAR-15-00 06:09 +43 1 512 98 05 P.29 R-971 Job-3T5 15/03 '00 12:17 FAX +43 1 512 98 05 PAT. ATT. VIENNA 01029/048 the dissolved prothrombin complex concentrate was filtered, centrifuged for 15 min at 20,000 rpm, and Sterile-filtered through a 0.8 ~.em filter. The sterile-filtered and dissolved prothrombin complex concentrate was applied to a co~.umn at a flow rate of 10 ml/h. Then the column was washed with a washing b2lffer so as to be free from protein, and finally the bound protein C was eluted with the elution buffer at a flow rate of ml/h, and the fractions were collected. The eluted protein C was dialyzed against 3 buffer (0.2 M Tris, 0.15 M glycizle, and 2 mM EDTA, pH 8.3). The protein C
content was determind with respect to antigen by means of the method according to Laurell and with respect to activity after Protac activation_ The protein C el.uate obtained was finished to a pharmaceutically applicable preparation in the following manner:
At first, the eluate was subjected to an ultrafiltration and a di.a~i~tration step- For diafiltration, a buffer having a pH of 7.4 was used, which contained 3.50 mM I~5aG1 and 15 mM trisodium citrate . 2H2 O _ The filtrate obtained was freeze--dried and virus inactivated by a one-hour vappr treatment at 80°C + 5°G and X375 + 35 mbar.
The lyophilized, virus-inactivated material then was dissolved in a sterile isotonic rlacl so7.ut~.vzl, and possib~.y present antibodies or serum amylvid P, I~AR-15-00 06:09 +43 1 512 98 05 P.30 R-971 Job-375 15/03 '00 12:17 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 030/046 z'espeCtivply, were removed by means pf ipn e~GChange chromatography on c~-Sephaxose. The purified solution was concentrated by a further ultrafiltration and diafiltration step_ 'thereafter, 10 g albumin, 150 mM
NaCl and 15 mM trisodium citrate were added. The pH of the solution was 7.5. Murine zmmunoglobulin as well as the factors 1=I, VII, IX arid X could not be detected.
Subsequently, the solution was sterile-filtered, filled into containers and lyophilized. The specific activity was 14 units of protein C/mg_ As the activity assay, an amidolytic assay was used wherein protein C was activated by means of Prptac (Pentapharm).
1.5 Purification of plasmatic protein S
Human protein S was prepared from factor Ix concentrate (Frothrombin complex STIM-3, BAXTER AG, Vienna) by means of QAE-Sephadex and Blue Sepharose CL-~8 chromatography (Pharmacia) izz the following mariner:
The lyophilized concentrate (100 g) was dissvJ.ved in 200 ml of sterile, ion-free water and dialyzed against a buffer consisting of 0.01 M 2-(N-morpholinpethane-sulfonic acid), pH 6.0; 0.18 M Nacl, mM EDTA, 2 mM benzamidine-HCl and 0.02 NaN3 (starting buffer). Then the dialyzed material was applied to a QAE-Sephadex coJ.umn (8 x 19 cm) and eqlzilibrated with the buffer men~i,pned. As washing solution, 1.5 1 of buffer (starting buffer) were used.
Protein S was eluted at 1.10 ml/h with a linear NaCl MAR-15-00 06:09 +43 1 512 98 05 P.31 R-971 Job-375 15/03 '00 12:17 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~031/046 gradient Consisting of 1.2 1 Of starting buffer and 1.2 1 of a further buffer which differs from the first buffer by the addition of 0.5 M NaCl. The pxote~.z~ S
fractions were examined for protein s by means of Fast Flow SDS PAGE (PharmaCia) arid antigen daterm~uatic~n (LaureJ.l) . ~xote~.r~ S-containing fractions were pooled and finally dialyzed against a buffer. This buffer contained 50 mM Tris-HCl, pH 7.4, 150 ml~ NaCl, 2 mM
EDTA, 1 mM benzamidine-HC1 and 0_2~ NaN3. After dialysis, the protein S pool was applied onto a Blue Sepharose column CL-6B (2_5 cm x 10.5 cm) and equilibrated with the starting buffer.
wash~.z~.g was carried out with 500 ml of starting buffer at a flow rate of 15 ml/h. Thus, protein S could be eluted in the void volume, whereas prothz~vmbln adsorbed to the column. Again, the protein S rich fractions were determined by means of SDS~PAGE Fast Flow System (Pharmacia) and according to Laurell (Scand_ J_ Clin_ Invest. {Suppl_) 29 (1977) 21 (Suppl_ 124) ) .
On a reduced SDS-PAGE, the thus prepared protein S
had a morphology character~.stic of protein S, namely two close bands (doublet) with a moleGUlar weight of appro~cimately 96, oop azzd 76, 000, respectsvel~r. The protein concentration was determined spectrophoto-metrically by means of an extinction coefficiezxt Qf 0.1 at 280 nm for human protein S and was confirmed by the MAR-15-00 06:09 +43 1 512 96 05 P.32 8-971 Job-375 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~032/046 Method accordiza.g to LOwlzY (Lowry O , et al . , Protein measurement with the Folin phenol reagent, ~_ I3io1_ Chem. 193 (1951) 265).
The thus prepared, pre-purified protein S was used for preparing sheep antiserum against protein S by carrying out four immunizing injections. 100 ug of protein s had been applied subcutaneously with Freund~s adjuvant in the first two injections and incomplete adjuvant being employed in the following boosters.
After further boosters, the antiserum was tested by means of double immunodiffusion and showed a precipitation with purified protein S and with normal plasma.
The TgG fraction from 450 ml antiserum was obtained by alcohol precipitation and subsequent adsorption on Sephadex A 50 in TRTS-HCl buffer, pH 6_8_ From 454 ml of antiserum, 1.14 g of anti-protein s-zgG were present in the supernatant. The IgG fraction was coupled to 450 ml of Sepharose CL-4B, with 5.7 mg protein/m7.
Sepharose being used. The coupling efficiency was 76~.
The anti-protein S column was equilibrated with glycine-HCl, pH 3, and adsorption buffer, pI-~ ~.5.
The adsorption buffer was comprised of 20 m1K TRIS, 2 rr~ EDTA, O_25 M NaCl, 2 mM benzamidine, O_02~
Tween~ 20 and 0.02 Nal~T3, pH 7.4_ Tha washing buffer solution had the following contents: 20 mM TRTS, 2 mM
EDTA, 3._0 M IrTaCl, 0.5 mM benzamidine, 0.01% Tween~ 20;
MAR-15-00 06:09 +43 1 512 98 05 P.33 R-971 Job-375 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA I~ 033/048 0.02 NaN~, pH 7.4.
The elution buffer had a composition like the washing buffer solution, except that 0.05 Tween~ 20 and, additionally, 243.3 g of NaSCN, pH 7.4 (a 3 M
rhodanid solution) were used.
The dialysis buffer solution contayned 20 mM Tris, 0.15 mM glycine, 1 mM EI7TA, 2 mM benzamidine, pH B.3.
For further purification o~ the protein S fraction prepared from prothrombin complex concentrate, 100 g of the fraction were dissolved in 1 1 of adsorption buffer and dialyzed over night against an adsorption buffer solution. Aftex application of the sample onto the column, the column was washed with washing buffer, approx~.mately 5 1, to be free from protein, subsequently elution was carried out with 3 M NaSCN in the elution bufTer solutzon_ The eluate was dialyzed immediately until SCN was below the detection limit;
the eluate had a concentration of 500 ug/ml protein S_ zt was f x~ee f rom G~ -birxding prote in .
Monoclonal anti-protein S antibodies were prepared as follows:
BALB/C mice were immunized at two-week intervals by intraperitoneal injection of 100 N,g of the protein S
produced_ After six weeks, once again 50 E.tg of the human protein S were injected, and 3 days afterwards fus~.oz~, was carried out. The myeloma cell line (P3-X-63-.A.G8-653, 1.5 x 10' cells) was mixed with 1.7 x 108 MAR-15-00 06:09 X43 1 512 98 05 P.34 R-9T1 Job-3T5 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~034/046 spleen cells from a mouse, and fusion was effected according to a modified Icohler & Milstein method by using PEG 1500 (KShler G., Milstein C., Nature 256 (3.975) , 495-497) .
Positive clones, tested by means of an ELISA, were subcloned twice. Ascites production was effected Iay injection of 5 x 106 hybridoma cells per BALB/C mouse two weeks after Pristan treatment.
The immunoglobulin was purified from ascites by anunoza.~.um sulfate precipitation and subsequent chromatography by means of QAE-Sephadex and, thereafter, chromatography on sephadex 6200.
The IgG fraction obtained from ascites and pre-purified an protein A-Sepharose was coupled to Sepharase CL-~B. The affinity chromatographic purification o~ protein S which had been recovered from the prothrombin complex concentrate was effected under the conditions descxzbed for polyclonal protein S
antibodies. The concentration of the eluate on protein 8 was 600 ~.i.g/ml. ~t was free from C4-bind~.ng protein.
'fhe protein S preparations highly purified according to th.e method of polyclonai or monoclonal affinity chromatography were subjected to an SDS-PAGE
(gradient gel. 8 to 12%), and ~rere designated as more than 95~ pure according to Coomassie stainixa,g (Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970), -MAR-15-00 06:09 +43 1 512 98 05 P.35 R-971 Job-375 15/03 '00 12:18 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~035/046 6$0) _ Subsequently, the eluates were subjected to an ultra~iltration and a diafiltxation step. Far diafiltration, a buffer having a pH of 7_4 was used which contained 150 mM NaCl and 15 mM txisodium citrate.2H~0_ The filtrates obtairmd were freeze-dried and virus-inactivated by a one-hour vapor treatment at 80°C ~ 5°C and 1375 + 35 mbar (to remove possibly present viral contaminations of polyclonal or monoclonal antibody)_ Then the lyophilized virus-inactivated material was dissolved in. a sterile isotonic NaCl solution and possibly present antibodies and serum amyloid P, respectively, were removed by ion exchange crrx~omatography on Q-Sepharose_ The purified solution was concentrated by a further ultrafiltration and diafiltration step. Thereafter, 10 g of albumin, 150 mM
NaCl and 15 mM trisodium citrate were added per liter to the solution obtained. The pH of the sdluta..o~c~, was 7.5. zt contained 3000 ~.g/ml protein. S. This content of protein S corresponds to a 500--fold enrichment as compared to plasma. Mur,iz~e ~.mmunoglobulin as well as the factors lZ, VII, 1X and X could not be detected.
Sub~equez~.tl.y, the solution was sterile-filtered, filled into containers and 3.yophi~.ized _ E x a m p 1 a ~ _ phax~0.aCeutiCal pr~paratioa of highly .purified vitams.n x--depe~.deat protei~2es:
.. 34 -MAR-15-00 06:09 +43 1 512 98 05 P.36 R-971 Job-375 15/03 '00 12:19 FAX +43 1 512 98 05 PAT. ATT. VIENNA f~ 036/046 The highly purified single factors prothrombin, factor VII, factor 1X, factor X, pxotein C and protein S were combined such that a solution containing 25,000 units/1 of each of the single factors was foz~med in aqueous solutiozx of 4 g Na3 citrate _ 2Hz O and 8 g rlaCl/1. The solution was sterile-filtered via a nylon filter having a retention rate of 0_2 um and subsequently filled at 20 ml each in steri7~a.zed glass bottles having a filling volume of 50 ml_ It was freeze-dried under sterile conditions, whereafter the bottles were closed under sterile conditions.
8 x a m p 1 a 3 . Phaxmaeeutioai preparation o~
the prothromb.im complex:
As described in the previous example, a highly purified prothrombzn complex comprising the factors prothrombin, factor VII, factor ZX and factor X, yet without protel.n C and protein S, was prepared and filled into containers under sterile conditions.
l~ccordirzg to international recommendations, the solution was admixed with 7.0,000 international units of heparin/1 before filling into containers.
E x a m p 1 a 4 . pha.rmaceutiaal preparation of 'v'x.tamin x-dependent proteins for continu~aus substitution and ma~.ntaizaxn,g of coaetant plasma levels:
After substitution with a vitamin K-dependent protein concentrate in which plasma levels as constant as possible of the factors of the prothrombin complex MAR-15-00 06:29 +43 1 512 9B 05 P.02 R-972 Job-376 15/03 '00 12:29 FAX +43 1 512 98 05 PAT. ATT. VIENNA 0 037/048 ax'e to be attained, due to the different half-lives of the indi~cridual factors it is necessary to carry out the substitution rates of these prate~.ns ~.z~. a non-equivalent ratio. Accordingly, a pharmaceutical preparation as descxibed above was pxpduced which contained prothrombin, factor vII, factor 1X, factor X, protein C and protein S at a ratio of 1:30:3:1.5:6:6.
a m p 1 a 5 . Phariuaaeutical preparation of a partial prratl~xomb3ss complex:
bn account of the different a.rl va.vo half-lives of the factors of the prothrombin complex, in particular because of the long half-life of prothrombin of 2-5 days as compared to the short half--life of factor VTI
of 4-7 h, and of the factor IX of less than 24 h, in the continuous substitution with prothrombin complex preparations it happens that when adjusting a normal plasma concentration of 1 unit of factor VTZ/ml, already after 24 h the plasma concentration has decreased again to below the concentration of at least 20% required for a functioning haemostasis, whereas, a_g., the prothrombin p~.asma level still is appro~c~.mately normal, i . a . is at 1 unit/ml . A z~epeated substitution with a pxothrombin complex concentrate of the same composition as in the primary substif.ur.ion will have the result that the prothrombin plasma concentration in the patient will be increased to above the normal range so as to attain ozzce more equivalent MAR-15-00 06:29 +43 1 512 9B 05 P.03 R-972 Job-376 15/03 '00 12:29 FAX +43 1 512 98 05 PAT. ATT. VIENNA 0 038/048 Factor VTI or factor IX plasma concexitrations_ Raising the plasma prothrombin concentration to above the standard range, however, means an-increased risk of thrombosis for the patient treated, since Poort et al_ (Blood 88:3698-3703, X996) have found that a plasma level of prothrombin raised by as little as 30%
involves a significantly increased risk of venous thromboses. This problem can be avoided by using combined prothrombin cample~c preparations for secozz~dary substitutions.
Such a preparation is produces as fv~.lows: ,mss described in the previous examples, a prothrombin complex concentrate comprising factor VII, 2X and X at a ratio of 30:3:1.5, yet without prothrombin, is prepared and provided as a pharmaceutical formulation.
The Gontaizaexs of this preparation intended for administration each contained 500 international units of the coagulation factors vzz, IX and X in a dissolution volume of 10 ml after reconstitution of the lyophili.~ed powder-E x a, nay p 1 a 6 . Quality chealc of the 8arothrombin complex preparations:
The izidividual coagulation factors in the final aonta~.rxers of the prothrombin complex preparations were assayed for their content of prothrombin, factor 'V2I, factor Ix, factor x, protein C and protein S by means of ~-step coagulation tests according to standard MAR-15-00 06:29 +43 1 512 9B 05 P.04 R-9~2 Job-376 15/03 '00 12:30 FAX +43 1 512 98 05 PAT. ATT. VIENNA ~ 039/048 methods, by using deficient plasmas and coagulation reagents from BAXTER, Vienna_ Details to the methods used can be taken from the paper by Muller, H_G. and Bonilc K. (Kranlcenhauspharma-zie 13:528-531, 1992)_ To determine the content of activated prothrombin complex factors, the chromogenic substrates S-2238, s-2222, S-244, S-2366 and S-2251 (Chromogenix) were used. The determinations were carried out by using the buffer conditions indicated by the producer. With the chromogenic substz~ates, in each case the activated factors thrombin, factor VIIa, factor IXa, factor Xa, activated px'otein C which developed proteolytic activities against the peptide substrates used, could be determined. After evaluation of the photometric analysis of the turnover of the chromogenic substrate it could be determined that for all the substrates the content in the previously described preparatioz~.
comprising vitamin K dependent proteins or selected factors of tha prothrombin complex was less than mU/ml in each ca6e_
Claims (34)
1. A pharmaceutical, separated prothrombin complex preparation, characterized in that it comprises at least 2 chromatographically purified vitamin K-dependent single factors as the active substances.
2. A preparation according to claim 1, characterized in that the single factors are selected from the group of factor II, factor VII, factor IX, factor X, protein C, protein S and protein Z.
3. A preparation according to claim 1 or 2, characterized in that it comprises at least the factors II, VII, IX and X.
4. A preparation according to any one of claims 1 to 3, characterized in that it comprises the single factors protein C and protein S.
5. A preparation according to any one of claims 1 to 4, characterized in that at least one of the single factors is a recombinant factor, a transgenically prepared factor, a derivative, in particular a peptide, and/or a fragment.
6. A preparation according to any one of claims 1 to 5, characterized in that it comprises the single factors contained at a ratio which substantially corresponds to the ratio of these factors in blood.
7. A preparation according to any one of claims 1 to 6, characterized in that it comprises the single factors factor II, factor VII, factor IX and factor X
at relative ratios, based on international units, of (0.5 to 2) : (0.5 to 2) : (0.5 to 2) : (0.5 to 2) .
at relative ratios, based on international units, of (0.5 to 2) : (0.5 to 2) : (0.5 to 2) : (0.5 to 2) .
8. A preparation according to any one of claims 1 to 7, characterized in that it comprises the individual factors factor II, factor VII, factor IX, factor X, protein C and protein S at relative ratios, based on international units, of (0.5 to 2) : (0.5 to 2) : (0.5 to 2) : (0.5 to 2) : (0.5 to 2) : (0.5 to 2) .
9. A preparation according to any one of claims 1 to 5, characterized in that it comprises the single factors contained at ratios which correspond to the relative half-lives of the single factors.
10. A preparation acording to claim 9, characterized in that it comprises the single factors factor II, factor VII, factor IX and factor X at relative ratios, based on international units, of (0.5 to 2) : (5 to 35) : (0.5 to 7) : (0.5 to 5) .
11. A preparation according to any one of claims 9 or 10, characterized in that it comprises the single factors factor II, factor VII, factor IX, factor X, protein C and protein S at relative ratios, based on international units, of (0.5 to 2) : (5 to 35) : (0.5 to 7) : (0.5 to 5) : (1 to 15) : (1 to 15).
12. A preparation according to claim 1, 2, 4, 5, 6 or 9, characterized in that it comprises factor VII and factor II at a ratio of greater than 10 : 1.
13. A preparation according to any one of claims 1 to 12, characterized in that the single factors in the preparation do not form a complex.
14. A preparation according to any one of claims 1 to 13, characterized in that it comprises a partial prothrombin complex.
15. A preparation according to any one of claims 1 to 14, characterized in that it does not contain an activated coagulation factor selected from IIa, IXa, Xa and, optionally, VIIa.
16. A preparation according to any one of claims 1 to 15, characterized in that it contains less than 0.1 U
of factor VIII:C or factor VIII:Ag/mg of protein.
of factor VIII:C or factor VIII:Ag/mg of protein.
17. A preparation acording to any one of claims 1 to 16, characterized in that it contains less than 0.1 U
of factor IIa/U of prothrombin.
of factor IIa/U of prothrombin.
18. A preparation according to any one of claims 1 to 17, characterized in that it contains less than 0.1 U
of factor Xa/U of factor X.
of factor Xa/U of factor X.
19. A preparation according to any one of claims 1 to 18, characterized in that it is present in lyophilized form.
20. A preparation according to any one of claims 1 to 19, characterized in that it comprises magnesium ions.
21. A preparation according to any one of claims 1 to 20, characterized in that it does not contain free calcium ions.
22. A preparation according to claim 21, characterized in that it comprises a chelating agent for complexing free calcium ions.
23. A preparation according to any one of claims 1 to 22, characterized in that it further comprises antithrombin III in stabilizing amounts, optionally together with heparin.
24. A preparation according to any one of claims 1 to 23, characterized in that it is free from infectious viruses on account of a virus inactivation or virus depletion treatment.
25. A preparation according to claim 24, characterized in that its freedom from infectious viruses is ensured by two independent virus inactivation or virus depletion methods.
26. A preparation according to any one of claims 1 to 25, characterized in that it further comprises pharmaceutically acceptable buffer substances or stabilizers.
27. A preparation according to any one of claims 1 to 26, characterized in that it comprises highly purified vitamin K-dependent single factors which are virus-inactivated.
28. A preparation according to any one of claims 1 to 27, characterized in that it comprises the single factor X in its .alpha.-form and/or in its .beta.-form.
29. A diagnostical preparation, comprising a preparation according to any one of claims 1 to 28.
30. The use of a preparation according to any one of claims 1 to 28 for producing a preparation for treating acquired or inherited blood coagulation disorders.
31. The use of a preparation according to any one of claims 1 to 28 for producing a preparation for treating severe hemorrhages.
32. The use of a preparation according to any one of claims 1 to 28 for producing a preparation for the prophylaxis of hemorrhages, in particular in case of inherited blood coagulation disorders.
33. The use of a preparation according to any one of claims 1 to 28 for producing a preparation for substitution therapy.
34. The use of a preparation according to any one of claims 1 to 28 for producing a preparation for treating hemophilia B.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT0159197A AT409334B (en) | 1997-09-19 | 1997-09-19 | PHARMACEUTICAL PREPARATION CONTAINING VITAMIN K-DEPENDENT INDIVIDUAL FACTORS |
ATA1591/97 | 1997-09-19 | ||
PCT/AT1998/000224 WO1999015196A1 (en) | 1997-09-19 | 1998-09-17 | Pharmaceutical substance containing various vitamin k-dependent factors |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2304396A1 true CA2304396A1 (en) | 1999-04-01 |
Family
ID=3516739
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002304396A Abandoned CA2304396A1 (en) | 1997-09-19 | 1998-09-17 | A pharmaceutical preparation comprising vitamin k-dependent single factors |
Country Status (10)
Country | Link |
---|---|
EP (1) | EP1015020A1 (en) |
JP (1) | JP2001517636A (en) |
AT (1) | AT409334B (en) |
AU (1) | AU743102B2 (en) |
BR (1) | BR9812222A (en) |
CA (1) | CA2304396A1 (en) |
HU (1) | HUP0003681A1 (en) |
NO (1) | NO20001415L (en) |
SK (1) | SK4022000A3 (en) |
WO (1) | WO1999015196A1 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7790852B2 (en) | 2003-06-25 | 2010-09-07 | Novo Nordisk Health Care A/G | Liquid composition of factor VII polypeptides |
US7897734B2 (en) | 2003-03-26 | 2011-03-01 | Novo Nordisk Healthcare Ag | Method for the production of proteins |
US8022031B2 (en) | 2001-12-21 | 2011-09-20 | Novo Nordisk Health Care A/G | Liquid composition of factor VII polypeptides |
US8026214B2 (en) | 2003-08-14 | 2011-09-27 | Novo Nordisk Health Care Ag | Liquid, aqueous pharmaceutical compositions of factor VII polypeptides |
US8299029B2 (en) | 2002-06-21 | 2012-10-30 | Novo Nordisk Health Care Ag | Stabilised solid compositions of factor VII polypeptides |
US8536127B2 (en) | 2003-05-23 | 2013-09-17 | Novo Nordisk Healthcare Ag | Protein stabilization in solution |
US8658597B2 (en) | 2003-12-19 | 2014-02-25 | Novo Nordisk Healthcare Ag | Stabilised compositions of factor VII polypeptides |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005528351A (en) * | 2002-03-08 | 2005-09-22 | イーライ・リリー・アンド・カンパニー | Activated protein C formulation |
WO2006088741A2 (en) * | 2005-02-16 | 2006-08-24 | The Board Of Trustees Of The University Of Illinois | Procoagulants based on metal-chelating lipids |
WO2006096345A2 (en) | 2005-03-04 | 2006-09-14 | The Board Of Trustees Of The University Of Illinois | Coagulation and fibrinolytic cascades modulator |
US8821861B2 (en) | 2007-10-05 | 2014-09-02 | The Board Of Trustees Of The University Of Illinois | Fibrin sealant |
WO2009061697A1 (en) | 2007-11-09 | 2009-05-14 | The Board Of Trustees Of The University Of Illinois | Anticoagulant antagonist and hemophilia procoagulant |
EP2624859B1 (en) | 2010-10-06 | 2017-03-01 | Medimmune Limited | Factor ii alone or in combination with further factors for treatment of impaired haemostasis associated with dilutional coagulopathy |
PL3579857T3 (en) * | 2017-02-09 | 2022-07-25 | Csl Behring Gmbh | A blood coagulation factor replacement product for use in the treatment or prophylaxis of bleedings |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8729822D0 (en) * | 1987-12-22 | 1988-02-03 | Central Blood Lab Authority | Chemical process |
IT1262899B (en) * | 1992-03-27 | 1996-07-22 | Sclavo Spa | PROCESS FOR THE INSULATION OF FACTOR IX, FACTOR X AND FACTOR II HIGHLY PURIFIED FROM THE PROTROMBINIC COMPLEX OR HUMAN PLASMA |
DE4342132C1 (en) * | 1993-12-10 | 1994-11-03 | Octapharma Ag | Process for the preparation of virus-inactivated vitamin K-dependent plasma components, and protein C and protein S-containing compositions by membrane chromatography |
DE4430205A1 (en) * | 1994-08-26 | 1996-02-29 | Behringwerke Ag | Compositions suitable as antidotes for blood anticoagulants and their use |
DE59712322D1 (en) * | 1996-03-20 | 2005-06-30 | Baxter Ag | Pharmaceutical preparation for the treatment of blood clotting disorders |
-
1997
- 1997-09-19 AT AT0159197A patent/AT409334B/en not_active IP Right Cessation
-
1998
- 1998-09-17 CA CA002304396A patent/CA2304396A1/en not_active Abandoned
- 1998-09-17 HU HU0003681A patent/HUP0003681A1/en unknown
- 1998-09-17 WO PCT/AT1998/000224 patent/WO1999015196A1/en not_active Application Discontinuation
- 1998-09-17 EP EP98944886A patent/EP1015020A1/en not_active Withdrawn
- 1998-09-17 BR BR9812222-3A patent/BR9812222A/en not_active Application Discontinuation
- 1998-09-17 JP JP2000512565A patent/JP2001517636A/en active Pending
- 1998-09-17 AU AU92449/98A patent/AU743102B2/en not_active Ceased
- 1998-09-17 SK SK402-2000A patent/SK4022000A3/en unknown
-
2000
- 2000-03-17 NO NO20001415A patent/NO20001415L/en not_active Application Discontinuation
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8022031B2 (en) | 2001-12-21 | 2011-09-20 | Novo Nordisk Health Care A/G | Liquid composition of factor VII polypeptides |
US8461116B2 (en) | 2001-12-21 | 2013-06-11 | Novo Nordisk Healthcare Ag | Liquid composition of factor VII polypeptides |
US8729022B2 (en) | 2002-06-21 | 2014-05-20 | Novo Nordisk Healthcare Ag | Stabilised solid compositions of factor VII polypeptides |
US8299029B2 (en) | 2002-06-21 | 2012-10-30 | Novo Nordisk Health Care Ag | Stabilised solid compositions of factor VII polypeptides |
US7897734B2 (en) | 2003-03-26 | 2011-03-01 | Novo Nordisk Healthcare Ag | Method for the production of proteins |
US8084587B2 (en) | 2003-03-26 | 2011-12-27 | Novo Nordisk Health Care Ag | Method for the production of proteins |
US8921530B2 (en) | 2003-03-26 | 2014-12-30 | Novo Nordisk Healthcare Ag | Method for the production of proteins |
US8530630B2 (en) | 2003-03-26 | 2013-09-10 | Novo Nordisk Healthcare A/G | Method for the production of proteins |
US8536127B2 (en) | 2003-05-23 | 2013-09-17 | Novo Nordisk Healthcare Ag | Protein stabilization in solution |
US7790852B2 (en) | 2003-06-25 | 2010-09-07 | Novo Nordisk Health Care A/G | Liquid composition of factor VII polypeptides |
US8026214B2 (en) | 2003-08-14 | 2011-09-27 | Novo Nordisk Health Care Ag | Liquid, aqueous pharmaceutical compositions of factor VII polypeptides |
US8318904B2 (en) | 2003-08-14 | 2012-11-27 | Novo Nordisk Health Care Ag | Liquid, aqueous pharmaceutical compositions of factor VII polypeptides |
US8658597B2 (en) | 2003-12-19 | 2014-02-25 | Novo Nordisk Healthcare Ag | Stabilised compositions of factor VII polypeptides |
Also Published As
Publication number | Publication date |
---|---|
ATA159197A (en) | 2001-12-15 |
AU9244998A (en) | 1999-04-12 |
WO1999015196A1 (en) | 1999-04-01 |
AU743102B2 (en) | 2002-01-17 |
AT409334B (en) | 2002-07-25 |
SK4022000A3 (en) | 2000-09-12 |
JP2001517636A (en) | 2001-10-09 |
NO20001415L (en) | 2000-05-19 |
BR9812222A (en) | 2000-07-18 |
HUP0003681A1 (en) | 2001-02-28 |
NO20001415D0 (en) | 2000-03-17 |
EP1015020A1 (en) | 2000-07-05 |
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Legal Events
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FZDE | Discontinued |