Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2000-01-03
2001-05-15
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C536S021000
Reexamination Certificate
active
06232093
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the preparation of heparin. More particularly, it relates to a simplified process for the removal of proteins present in an animal tissue source of heparin
BACKGROUND OF THE INVENTION
Heparin is a very complicated glycosaminoglycan composed of alternating sequences of differently sulfated residues of uronic acid and &agr;-D-glucosamine linked by &agr; and &bgr; (1-4) bonds. Due to the complexity of its primary structure, heparin is a polydisperse heteropolysaccharide which is strongly heterogeneous in terms of molecular weight, physico-chemical properties and biological activities.
Heparin has been used for already a long time as an anticoagulant and antithrombotic agent in the treatment and prevention of venous thrombosis. It is present in a variety of animal tissues and may be obtained by isolation therefrom. Currently a large part of the heparin used for these purposes is isolated from porcine intestinal mucosa. The isolation process involves hydrolysis of the mucosa followed by extraction of the heparin.
U.S. Pat. No. 5,607,840, hereby included by reference, describes a process for hydrolysis of mucosa tissue. The method involves hydrolysis of an aqueous mixture containing mammalian mucosa with a proteolytic enzyme at a temperature of about 55° C., adsorption of polyanions to an anion exchange resin and subsequent recovery of the anions from the resin and the protein hydrolysate from the digested aqueous solution. In order to stabilize the raw mucosa material and to prevent bacterial growth, salts in the form of an oxygen scavenger or bacteriocides are introduced into the solution.
The heparin content in the mucosa-containing aqueous medium is very low and consequently large amounts of mucosa tissue have to be processed. Therefore, for economic reasons, the hydrolysis process is carried out in reaction vessels of more than 50 m
3
. During the reaction time the temperature is kept at a constant level for more than 24 hours and the mixture is stirred vigorously using advanced equipment.
Usually heparin extraction plants are not located at a short distance from the slaughterhouses where the mucosa is collected and transportation over long distances is required. Especially in remote areas this results in a delivery of low quality material at the production plant and an increase of costs.
SUMMARY OF THE INVENTION
The present invention provides a new and easy way of isolating heparin from mucosa tissue. It has been found that protein in an aqueous mixture comprising mammalian mucosa tissue can also be digested with a proteolytic enzyme at a temperature between 50-75° C. for up to approximately 6 hours. A simple “pre-hydrolysis” step at ambient temperature can optionally be included. This was shown to improve the efficiency of the proteolysis significantly. After the raise in temperature hydrolysis can be continued preferably in the presence of polyanion adsorbent material at a temperature below 50° C. The digestion can be carried out in containers with intermediate capacity.
DETAILED DESCRIPTION OF THE INVENTION
Thus, the process according to the invention comprises the step of raising the temperature of the mucosa to approximately 50-75° C. and incubating the mucosa within that temperature range for up to approximately 6 hours in the presence of a proteolytic enzyme.
The process has the advantage that it can be carried out at a relatively small scale. The process is very well suited to be carried out at places where the raw mucosa material is produced and thus no transportation of raw material is needed. Furthermore, no special skills are required.
According to the present invention also one or both of the following incubation steps can be included in the method:
a) incubating the mixture in a container for up to approximately 8 hours at ambient temperature prior to the temperature raise
b) further incubating after the raise in temperature whilst the solution is allowed to cool down to ambient temperature.
The incubation time at 50-75° C. preferably is less than 4 hours. Preferably the incubation time is more than 1 hour. The temperature is raised preferably to approximately 60-70° C.
The temperature can easily be raised by direct heating of the container containing the aqueous mucosa solution. For example, a temperature of about 60-70° C. can be reached rapidly with an ordinary gas flame but also e.g. by the addition of steam. The thus obtained temperature can be maintained by moderate heating for several hours. Alternatively, external addition of heat can be stopped as soon as the desired temperature is reached allowing the mixture in the container to cool down gradually. Because of the gradual temperature decrease, the mixture will retain its temperature above 50° C. for several hours. Thus, initially protein hydrolysis occurs at a temperature above 50° C. After removal of the external heat source, temperature decreases and hydrolysis continues at a lower temperature. There is no limitation to the extent of incubation time at the decreased temperature, however, incubation times of less than 24 hours are to be preferred, more preferably the incubation is carried out over night. Continuation of a prolonged incubation at ambient temperature is not a prerequisite but it was shown to have influence on the heparin yield. Similarly the “pre incubation” step at ambient temperature was shown to improve heparin yields.
The reaction vessel can e.g. be a steel container but no specific requirements are needed.
Alternatively, the temperature can be raised by the addition of 1-3 volumes of an aqueous solution with a temperature between 80 and 100° C. Preferably water is used but also dilute salt solutions can be used. Preferably, the temperature of the water is the boiling temperature. Due to the volume enlargement the heat capacity increases. Therefore the temperature can be maintained at the desired temperature for a sufficiently long period of time whereas simultaneously the cooling down process progresses slowly. An additional advantage of the addition of water is that the viscosity of the solution is reduced resulting in a better sievability of the adsorbent-heparin complex which is important in the extraction of heparin. As no direct heating is required containers from different materials e.g. plastic can be used.
A convenient way of carrying out the procedure according to the invention is to collect the mucosa during a working day and to start with hydrolysis by the addition of proteolytic enzyme during storage of the raw material in a container. At the end of the day the temperature can be increased to 50-75° C., optionally kept at a constant level for a few hours, and then further incubated over night whilst the temperature decreases gradually to ambient temperature.
It is to be preferred that during the hydrolysis process the mixture is stirred in order to obtain a homogeneous solution and to have a good contact between the adsorbent and the heparin-containing solution.
The mucosa-containing solution can be obtained by dispersing animal tissues in water. Tissues can be obtained from e.g. beef, dog, hog and sheep. The tissues have endothelial or mucosal components. Heparin is present e.g. in lung, intestine, skin and liver from a variety of animals. Preferably, intestinal mucosa from porcine origin is used.
A great variety of enzymes may be employed in the process provided that they possess a proteolytic activity. Suitable enzymes are well known in the art, e.g. microbial protease's, trypsin, chymotrypsin. A suitable enzyme for use in alkaline hydrolysis is commercially available under the trademark maxatase®. The enzyme to substrate ratio usually is approximately 0.02-0.2%. The preservative normally present in the mucosa tissue solution can also be added in the process according to the present invention. Preferably as a preservative sodium metabisulfite is used but also other preservatives such as e.g. calcium propionate or phenol will suffice. The amount of bisulfite to be added is approximately 0.5-5 kg/100 liters o
Sanders Adrianus Lambertus Maria
Van Houdenhoven Francois Egbert Abraham
Van Zuthpen Petrus Johannes Josephus
Akzo Nobel N.V.
Blackstone William M.
Patten Patricia
Weber Jon P.
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