Kit for purification of plasmin

Details Kit for purification of plasmin Kit for purification of plasmin

2000-02-16

2001-02-06

Kim, John (Department: 1723)

Chemical apparatus and process disinfecting, deodorizing, preser

Analyzer, structured indicator, or manipulative laboratory...

Sample mechanical transport means in or for automated...

C210S198200, C210S200000, C210S201000, C422S105000, C435S217000, C530S413000, C530S416000, C436S177000, C436S178000

Reexamination Certificate

active

06183692

ABSTRACT:

I. Field of the Invention
The present invention relates to a method for the rapid purification of a blood component and, in particular, plasminogen which is then converted to plasmin.
II. Description of the Prior Art
There are many situations in which it is desirable to purify a particular blood component, such as plasminogen, from human blood. The separated blood component may then be broken down into further subcomponents, such as plasmin.
For example, our prior U.S. Pat. No. 5,304,118 which issued on Apr. 19, 1994, describes a method of injecting plasmin into a human eye in order to induce a posterior vitreous detachment. The plasmin is produced by adding an enzyme to plasminogen which is extracted from human blood.
For non-life-threatening surgical procedures, such as a vitrectomy utilizing plasmin injection, the risk of infection, such as AIDS infection, from unidentified whole blood sources is simply unacceptable. Consequently, it has been the previously known practice to extract blood from the patient in such non-life-threatening surgical procedures and then to extract the blood component, e.g. plasminogen, from the patient's own blood. Thus, even if such blood contains a viral infection, the reinjection of the blood component or subcomponent into the individual patient causes no harm.
In order to extract blood components such as plasminogen from blood, it has been the previous practice to utilize column chromatographic separation procedures. These types of columns are gravity fed and are open at the inlet. The flow rate through the column is limited by the force of gravity as well as the column dimensions. U.S. Pat. No. 3,943,245 to Silverstein discloses a method for purifying plasminogen from human and non-human mammalian plasma by modified affinity chromatography using Sepharose-L-lysine. The Sepharose-L-lysine is placed in a column which requires low flow rates through the column to prevent collapse of the column. U.S. Pat. No. 5,371,007 to Linnau et al. discloses a method of producing lys-plasminogen using a lysine-polyacrylamide gel as the matrix for affinity chromatography. However, both the method disclosed in Silverstein and the method disclosed in Linnau et al. utilized structurally weak materials for the affinity media which when utilized under high pressures collapse or create excessive back pressure. Furthermore, the Linnau et al. patent specifically seeks to provide a method for the large-scale isolation of plasminogen. While such procedures are effective, they are time consuming to perform, oftentimes requiring a full day or even more of laboratory time and require the use of pooled plasma. Fisher, Linnau et al., and Silverstein suggest an additional time consuming step by requiring that the plasma be extracted by stirring for two hours at 4° C. in phosphate buffer prior to the affinity chromatography. Castellino,
Methods of Enzy.,
Vol. 80, 265-377, 1984, calls for the removal of E-ACA from plasminogen by extensive dialysis against water at 4° C. This would require at least four additional hours and the use of a refrigerator. Linnau et al. suggest that for large scaled separations, the E-ACA could be removed by gel filtration or ion exchange chromatography. This would consist of packed columns containing weak column material which would require gravity feed, low pressure and a slow flow rate and thereby increasing separation time.
SUMMARY OF THE PRESENT INVENTION
The present invention overcomes the previously known disadvantages of the previously known blood component separation methods by providing a rapid separation and purification of a blood component utilizing the patient's own blood and which can be accomplished within a few minutes.
In brief, blood is first drawn from the patient and the blood plasma is separated from the cellular blood elements using conventional methods, such as centrifugation. After centrifugation, the blood plasma containing the blood component is retained and the cellular blood elements are discarded.
An affinity cartridge of the type which binds with the desired blood component is prepared prior to the time of use. L-lysine (or other protein binding component) is covalently attached to silica which has been epoxy activated. Silica is a material which can withstand very high pressure and still maintain its shape. This matrix yields low back pressure and high flow rates. Three mls of the lysine-silica is loaded into a cartridge and the lid is put on. The lid has a female luer-locked inlet to which a syringe can be attached. Liquids can be pushed through the cartridge with the use of a syringe at a rate which is up to ten times of that which could be achieved with the use of a gravity fed column.
A syringe containing equilibration buffer is attached to the cartridge and the buffer is pushed through it to pre-wet the cartridge. The syringe is discarded and replaced with a syringe containing plasma (normally 10 ml). The plasma is passed through the cartridge such that the desired blood component (PLGN) binds with the affinity cartridge and is removed from the plasma.
Thereafter, an affinity cartridge of the type which binds with the desired blood component is first washed with an equilibration buffer in order to activate the cartridge. The separated blood plasma is then passed through the affinity cartridge such that the desired blood component binds with the affinity cartridge and is effectively removed from the plasma. The plasma is then discarded.
Thereafter, an equilibration buffer is passed through the affinity cartridge to wash and remove any unbound proteins or the like that may be contained within the affinity cartridge. Upon completion of the washing step, only the bound blood component remains within the affinity cartridge.
The blood component is then eluted from the affinity cartridge by injecting an elution buffer containing a releasing agent. The releasing agent is selected such that the releasing agent frees or unbinds the blood component from the affinity cartridge. If desired, the releasing component (E-ACA) can be removed from the blood component by attaching an ion exchanging solid-phase extraction device to the outlet “out” of the affinity cartridge, prior to the blood component elution step. These devices consists of female luer inlets and male luer outlets. They contain strong supports which will not collapse when passing materials through them with an attached syringe. They do have limited capacity and could not be used for large scale separation purposes.
The elution buffer, releasing agent and blood component solution is then passed through an ion exchange cartridge. The ion exchange cartridge effectively removes the releasing agent and the eluted blood component is then collected within a sterile tube.
A known amount of an enzyme is then added to the eluted blood component to obtain the desired subcomponent, such as plasmin. The blood component or subcomponent is then used, optionally after further filtering, as desired in the surgical procedure.


REFERENCES:
patent: 3943245 (1976-03-01), Silverstein
patent: 4604358 (1986-08-01), Fisher et al.
patent: 4724207 (1988-02-01), Hou et al.
patent: 5304118 (1994-04-01), Trese et al.
patent: 5371007 (1994-12-01), Linnau et al.
Castellino and Powell (1981) Human Plasminogen, Methods in Enzymology, vol. 80, pp. 365-378.

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