Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2000-07-21
2003-06-24
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C530S383000, C530S389300, C530S413000, C435S214000
Reexamination Certificate
active
06582603
ABSTRACT:
REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/JP98/04812 filed Oct. 23, 1998.
TECHNICAL FIELD
This invention relates to an affinity adsorbent possessing as a ligand a thrombin or thrombin analogue deprived of nucleophilicity (nucleophilic activity) without causing stereostructure to the linkage with a substrate, affinity chromatography formed by using the affinity adsorbent, and further a method for the refinement of a novel thrombin substrate characterized by comprising therein a step of working the affinity chromatography. More particularly, this invention relates to a method for the refinement of a thrombin substrate by using as a ligand for affinity chromatography a thrombin or thrombin analogue deprived of nucleophilicity (nucleophilic activity) without causing stereostructure to the linkage with such a substrate as anhydrothrombin. Further, this invention relates to a method for the elimination of a thrombin substrate contained as an extraneous matter in some other substance subjected to refinement by utilizing the specific adsorbing-desorbing activity of the affinity chromatography.
BACKGROUND ART
The blood coagulation factor VIII (FVIII) participates in the endogenous mechanism of the first phase of blood coagulation from the start of blood coagulation until before the formation of thrombin in the mechanism of blood coagulation. Since the disease due to the deterioration of this FVIII activity is called hemophilia A, the FVIII factor is referred to as antihemophilic globulin or antihemophilic factor A.
The hemophilia A is a recessive inheritance of sex chromosome and is sorted by the degree of deterioration of factor activity into three classes, light illness, medium illness, and serious illness. The patients of light illness rarely suffer spontaneous hemorrhage and, even when they do, they barely conceive of this symptom by personally experiencing difficulty in attaining hemostasis in connection with injury, surgical operation, or extraction of teeth. The patients of medium to serious illness suffer spontaneous hemorrhage as a main symptom and, owing to the obstruction to the endogenous coagulation, sustain this hemorrhage in the depths of tissue. Particularly, the intra-articular hemorrhage characterizes this disease and, when suffered to occur repeatedly, induces arthritis, compels the joints to develop contractual deformation and eventually manifest inevitable functional obstruction, and shows discernible signs of intramuscular hemorrhage, intracranial hemorrhage, and renal hemorrhage.
Though the gene therapy or the transplantation therapy is conceivable for the cure of a patient of hemophilia, it is still on the stage of undergoing a clinical study at present. It has not yet been put to use on patients of hemophilia. In this state of things, it is necessary to effect the cure by replenishing the patient with the FVIII, a factor depleted on the occurrence of a hemorrhage, in an effort to stop the hemorrhage or make this replenishment preparatorily in expectation of the hemorrhage.
As the FVIII preparation for the cure of a patient of hemophilia (by heightening the level in the blood of the depleted FVIII up to the level fit for hemostasis and preventing the blood flow), the cryoprecipitate (allowing the presence of FVIII at a high concentration), a precipitate which occurs when the Chon I fraction in a frozen state is gradually dissolved at a low temperature, is pharmaceutically prepared and used because the concentration of FVIII in the human blood plasma is so low as to fall in the range of 0.1 to 0.2 mg/lit. Since the transfusion of this preparation has the possibility of exposing the patent to infection with hepatitis B or hepatitis C or AIDS (acquired immunodeficiency syndrome), such heated preparations of very high safety as the dry concentrated human coagulant factor VIII preparations which has been dried by heating at 60° C. for 10 hours, and the dry concentrated human coagulant factor VIII which has been dried by heating at 65° C. for 95 hours, are now in use. Besides, the dry concentrated human coagulant factor VIII which has been produced by subjecting the donor's blood plasma as a raw material to a heat treatment or a treatment with an organic solvent/surfactant (as the treatment with TNBP/Octoxynol 9) thereby inactivating viruses possibly entrained by the plasma and separating the resultant coagulant in a refined state by the use of an anti-FVIII monoclonal antibody and the recombinant preparation which is produced, as disclosed in JP-A-07-278,197 (U.S. Pat. No. 2,513,993), by refining a product obtained by the gene recombination technique as by affinity chromatography using a monoclonal antibody to the VIII:C factor active C-terminal subunit have been similarly finding utility.
Even the preparations which have been endowed with heightened safety by the inactivation of viruses due to the heat treatment or the treatment with an organic solvent/surfactant cannot allow perfect denial of the possibility of exposing the patients who have used the preparation to such virus infectious as non-A or non-B type hepatitis. In the case of the heated preparations, since they contain fibrinogen, their administration has the possibility of excessively increasing the fibrinogen concentration in the patients' blood. Then, in the case of the recombinant preparation separated in a refined state by the use of the aforementioned monoclonal antibody, (1) the preparation has the possibility of including a heterogenous animal protein because the monoclonal antibody more often than not uses the heterogenous animal protein and (2) even from the viewpoint of cost, the preparation eventually obtained becomes inevitably expensive.
The coagulant factor XIII (hereinafter referred to simply as “FXIII”) participates in the formation of a stable fibrin polymer which constitutes itself the final stage of the mechanism of blood coagulation. Hence, it is called a fibrin stabilizing factor. The FXIII normally occurs in an inactive state in the blood plasma. When the blood plasma forms thrombin in consequence of hemorrhage, the FXIII is activated with thrombin and calcium ion and consequently enabled to stabilize the fibrin. In the case of a patient who has been depleted slightly or seriously of the FXIII, therefore, the FXIII which displays a normal value during the coagulation of blood manifests such phenomena as secondary hemorrhage because the fibrin clots consequently formed are not stable. Clinically, therefore, the FXIII preparations are widely used for promoting the cure of wounds.
The FXIII conventionally used domestically has originated in the placenta. Owing to the charge with the complaints {circle around (1)} that the state of inclusion of the FXIII differs in the placenta and in the blood plasma, {circle around (2)} that the FXIII has the possibility of including such an impurity as transforms ultimately into an immunogen, and {circle around (3)} that the relevant process of inactivation of virus has a problem (JP-A-01-250,326), it has altered the origin to the blood plasma. Since the blood plasma contains the FXIII only in a minute amount, however, the FXIII preparation has relied on the imported blood plasma for its source.
The FXIII preparation is extracted from the bloodplasma, the blood platelets, and the placenta, separated in a refined state, and pharmaceutically prepared by pasteurization (a treatment of liquid heating at 60° C. for 10 hours) which inactivates various viruses (retroviruses such as hepatitis B virus and HIV-1). It is otherwise obtained by producing a recombinant FXIII (including an analogue thereof which has a blood coagulating activity) in the yeast cells by the technique of gene recombination and then, for the purpose of pharmaceutical preparation, separating the produced recombinant FXIII in a refined state from the supernatant covering the ruptured yeast cells. The main methods which are available for the manufacture of the FXIII in the refined state from the blood plasma, the blood platelets, the placenta, or the reco
Hosokawa Kazuya
Nagata Masanori
Suzuki Toyoaki
Fujimori Kogyo Co. Ltd.
Nissim, Esq. Stuart H.
Therkorn Ernest G.
Woodbridge & Associates PC
Woodbridge, Esq Richard C.
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