WO2010110654A1

WO2010110654A1 - Method for preparation of heparin from mucosa

Info

Publication number: WO2010110654A1
Application number: PCT/NL2010/000055
Authority: WO
Inventors: Jan-Willem Vreeburg; Arie Baauw
Original assignee: Van Hessen Bv
Priority date: 2009-03-24
Filing date: 2010-03-24
Publication date: 2010-09-30

Abstract

This invention relates to a method for the preparation of heparin by enzymatic digestion of raw mucosa, and to protein hydrolysate and heparin thus produced. The method does not require the use of preservatives, and comprises a fast hydrolysis step reducing the viscosity, yielding a mucosa hydrolysate from which the heparin can be isolated with a resin contacting step, as a resin bound heparin. Heparin can be extracted from the resin bound heparin, and high yields as high as 50,000 Units per kg can be obtained.

Description

Title: Method for preparation of heparin from mucosa

Description

Heparin is a negatively charged, highly-sulfated glycosaminoglycan, a carbohydrate, which is highly heterogeneous with respect of molecular weight, physio-chemical properties and biological properties. It is widely used in pharmaceutical compositions and has already been used for a long time in medicine as an anticoagulant and antithrombotic agent. In addition to said use, derivatives of heparin have been developed as (potential) drugs in immunity related conditions such as cancer, transplants and asthma. Heparin is usually derived from animal tissue, more specifically from mucosal tissue which is obtained during the slaughtering of animals. The mucosa tissue contains only a very small fraction of heparin, which can be as low as 0.01 weight percent. The preferred source of heparin for the preparation of human pharmaceuticals is porcine derived mucosal tissue, as porcine heparin most closely resembles human heparin.

Currently, animals are slaughtered in slaughterhouses mainly to produce meat, where also other materials of the slaughtered animals are collected which include the intestine of the animal. The intestine collected can be further processed at a facility especially equipped for intestine processing, which is usually within the slaughterhouse itself. The intestine consists of four distinct tissue layers: mucosa, submucosa (rich in collagenous fibres), circular and longitudinal muscle layers and serosa. The different layers are separated, of which the submucosa layer, is used as a casing material in the meat industry, and the mucosa layer being a source for heparin. In a typical slaughterhouse, where for example pigs are slaughtered, about 5,000 animals are slaughtered everyday. In case of slaughtered pigs, about 0,8 Liter (corresponding to about 0,8 kg) of mucosa is produced per animal, thus, yielding a total volume of approximately 4,000 Liter, or 4 m³, per day. The mucosa is usually stored in very large tanks of up to 80 m³ and collected and stored at the slaughterhouse during several days or even weeks, which has the disadvantage that considerable space and facilities are required for storage. Furthermore, these large volumes of mucosa are picked up by trucks and transported over large distances to plants specialized to facilitate the processing of mucosa, preparing heparin. The transport of such large volumes has a big environmental and logistical impact, especially when taking into account the relatively small fraction of heparin that is contained within the mucosa. Moreover, as it takes considerable time before processing of the mucosa can start, i.e. collection and storage at numerous slaugherhouses and transport to the specialized plant, it is required to preserve the mucosa. To this end, preservatives are added to the mucosa at the slaughterhouses, which also has a negative environmental effect, as it may need to be removed or neutralized before the waste produced during the process can be disposed of safely. The chemical plants have specialized equipment for processing of mucosa, in which mucosa collected from multiple sites is processed in very large volumes that can be as large as 250 m³. The mucosa are subjected to chemical hydrolysis, for instance acid hydrolysis at high temperatures, during which peptide bonds in proteins are broken, after which the obtained mucosa hydrolysate is further processed to extract the heparin.

Attempts to improve the current method have been made, wherein the mucosa was intended to be processed on site. Nevertheless, these alternative procedures still require considerable time, and still require preservatives to be added to the mucosa. Importantly, these procedures are not current practice, as to date mucosa is still collected from slaughterhouses, transported over large distances and processed in chemical plants as described above.

One of the methods for the preparation of heparin from animal tissue on site is described in EP Pat. No. 0996642, which describes a process for the preparation of heparin from mucosa and which involves the enzymatic hydrolysis of mucosa with a proteolytic enzyme. Preservatives are added to the mucosa when the mucosa is collected during the slaughtering process of animals, e.g. sodium metabisulphite, calcium proprionate or phenol. Preservatives can also be added during the lengthy processing of the mucosa tissue. A preincubation step of up to 10 hours is carried out at ambient temperature wherein a proteolytic enzyme for the hydrolysis of the mucosa is added, followed by heating the enzyme mucosa mixture to 50-75⁰C for up to 6 hours, wherein the hydrolysis is continued, after which the mixture is subsequently allowed to slowly cool down overnight. In all of these steps the enzyme is active and prolonged pre-incubation and cooling steps are shown to improve heparin yields. Furthermore, the temperature is raised by addition of large volumes of water with a temperature between 80-100⁰C, resulting in an up to 2-3 fold increase in volume, having the disadvantage that the scale of the process as well as the volume of the waste stream is much increased. A resin capable of binding heparin can be added during the process, allowing to capture the heparin from the reaction mixture, resulting in resin bound heparin. The heparin is retrieved by sieving of the resin bound heparin, and eluting the heparin from the resin. Disadvantages of this method are that hydrolysis is carried out for extremely long periods, with total processing time taking 24 hours or more resulting in only moderate yields, in particular as a function of processing time, i.e., that more or less acceptable yields are obtained after very long processing times. Also, the addition of preservatives to the mucosa is still a prerequisite, having the disadvantage that these additives result in a potentially toxic waste, being a burden to the environment that must be removed or neutralized during the process. Furthermore, the heparin thus obtained may still contain such impurities once the mucosa hydrolysate has been removed therefrom.

Another method describing the hydrolysis of mucosal tissue is described in US Pat. No. 5,607,840. This method, however, is not directed to the preparation of heparin, but to the preparation of a protein hydrolysate. Mammalian mucosa is heated to 55°C and proteolytic enzyme is added, hydrolysis is carried out for up to 60 hours, while the high temperature is maintained. As with the process of EP 0996642, a preservative, such as sodium metabisulphite or calcium propionate is present to prevent deterioration of the mucosa during the time consuming process. The hydrolysate is obtained by removing the negatively charged molecules using a resin capable of binding negatively charged molecules. As this method has the objective to obtain a protein hydrolysate and is not focused on the isolation of heparin, the enzymatic hydrolysis step is designed to be as complete as possible and therefore takes very long. This method was never intended and is not suitable to obtain an efficient procedure for the preparation of heparin. Furthermore, the said method again requires the use of preservatives.

Thus, both these processes have the disadvantage that they require long enzymatic incubation step(s) in order to digest the mucosal tissue and also the total processing time takes at least a day. Average yields in the method of EP '642 were on average 30,000 Units per kg of mucosa. In an attempt to reduce the processing time, a very low yield was obtained of only 18,000 Units per kg, thus emphasizing that only when the processing time is prolonged, attractive yields are obtained (see comparative example). In view of this, a limitation in time was not further considered. In addition, these methods have the disadvantage that they require the use of preservatives in the mucosal tissue or mucosal hydrolysate which introduces undesired potential environmental or subsequent processing effects. The mucosa hydrolysate waste stream contains the preservatives and may be regarded as environmentally hazardous, requiring further processing to allow for safe disposal, leading to increased costs.

The present invention now provides an improved method for the preparation of heparin, allowing on site processing of the mucosa, as it provides a faster and easier way to produce high yields of heparin from raw mucosa as compared to the relatively long procedures known in the art. On the site where the intestines are processed, where raw mucosa is provided, heparin can be isolated from the raw mucosa after a fast hydrolysis step and resin contacting step, as a resin bound heparin. This resin bound heparin contains highly concentrated heparin, as compared to the raw mucosa bulk volume, resulting in a dramatic reduction of volume that needs to be stored and transported to the processing plants. Unexpected high yields as high as 48,460 Units per kg could be obtained (see example 3), possibly at least partly due to the fact that the viscosity was reduced to a value below 25 mPa-s and the complete method can be performed in relatively short processing time.

The invention is thereto characterized in that it comprises a method for the preparation of heparin from raw mucosa, comprising the steps of: a) providing raw mucosa, b) incubating the raw mucosa of step a) under conditions wherein the raw mucosa is hydrolyzed, reducing the viscosity to a value of at most 25 mPa-s, in a time period of 4 hours or less, thereby obtaining a mucosa hydrolysate, c) contacting a resin capable of binding heparin with the mucosa hydrolysate, allowing the heparin from the mucosa hydrolysate to bind with the said resin to obtain resin bound heparin, d) isolating the resin bound heparin of step c) from the hydrolysate, to obtain isolated resin bound heparin.

For example, the viscosity may be reduced in step b) to a viscosity such as as obtained by incubating raw mucosa at a temperature of 50°C at a pH between 8,0 and 9,0 for 0,5 hour using 7 grams of Syder alkaline protease and 1 litre of raw mucosa.

Raw mucosa, is obtained as one of the slaughtering products during the processing of slaughtered animals and can be collected in a container and may be cooled prior to providing raw mucosa in step a). Cooling the raw mucosa has the benefit that it may no longer be required to use preservatives in case the raw mucosa has to be stored for some time before processing of the raw mucosa can commence. Raw mucosa as collected in a slaughterhouse typically may have a temperature of about 44°C, a temperature at which the raw mucosa may deteriorate quickly unless it is preserved. Preferably the raw mucosa is cooled to a temperature as low as possible without freezing the raw mucosa. In a preferred embodiment, the temperature of the raw mucosa is reduced to a temperature between 5- 20⁰C, more preferably between 1-15°C, most preferably between 0-8⁰C. The raw mucosa is preferably substantially free of preservatives, such that the raw mucosa composition and constitution remains practically unaltered, as such preservatives may have a negative effect on the quality and yield of heparin and may introduce undesirable environmental problems. With substantially free of preservatives is meant an amount that is preferably less than 0,2%, more preferably less than 0,1 weight %, even more preferably less than 0,05 weight %, most preferably 0,0001 weight % or less , i.e. in an amount such that the chemicals used do not function as a preservative. The raw mucosa is a mainly water based proteinaceous slurry which is highly viscous. It is the objective of the invention to hydrolyse the raw mucosa in step b) such that viscosity is reduced to a value of at most 25 mPa-s. The viscosity is determined as described in the method of example 10. The viscosity is reduced within 4 hours or less, thus obtaining a mucosa hydrolysate. For example, the viscosity of the mucosa hydrolysate may be a viscosity such as as obtained by incubating raw mucosa at a temperature of 5O⁰C at a pH between 8,0 and 9,0 for 0,5 hour using 7 grams of Syder alkaline protease and 1 liter of raw mucosa. By reducing the viscosity, the heparin within the lysate can be efficiently contacted and isolated with a resin. This fast hydrolysis contrasts to previous methods known in the art in which very long hydrolysis steps were carried out in order to obtain high yields of heparin, and in previous methods the hydrolysis step was not specified by viscosity i.e. where no control of viscosity was suggested. The viscosity can be determined with a rheometer, with a probe within the reactor vessel that allows constant monitoring of viscosity during step b), or alternatively, at regular intervals samples may be taken during step b) of which viscosity is determined. However, once the proper parameters are established (see below), controlling viscosity is no longer necessary.

The skilled person knows how to reduce the viscosity in such a short time as it is known in the art how to select conditions (i.e. establishing proper processing parameters) that may make it possible to perform such a fast hydrolysis step b). For instance, hydrolysis can be carried out chemically or enzymatically. Chemical hydrolysis can be carried out with an acid, such as hydrochloric acid, and optimal pH, pressure and temperature may be selected to carry out such a fast hydrolysis. When an enzymatic hydrolysis is performed, the conditions may be selected as provided by the manufacturer for that enzyme, such that the desired viscosity of the mucosa hydrolysate can be obtained within the set time frame. In addition, it may be contemplated to increase the temperature and also add water to reduce the viscosity. However, the addition of water may have the downside that the reaction volume is increased which may have a negative impact on subsequent processing steps, and the waste volume is increased. Nevertheless, the invention allows the addition of water to at least support the lowering of the viscosity to the envisaged value. Furthermore, the addition of water may have the effect of lowering the buffering capacity of the mucosa, thus requiring less amount of base or acid in case the pH of the mixture needs to be modulated. In addition, by lowering the viscosity to a value of at most 25 mPa-s, filtration steps, i.e. filtering of mucosa hydrolysate before step c), and/or in the isolation of resin bound heparin, step d), are improved, i.e. faster and more efficient. In contrast, in the method of EP Pat No. 0996642, the mucosa hydrolysate can not be filtered before the resin is added, as the hydrolysis and resin contacting step are carried out simultaneously, which has the disadvantage that the dirt remains during the contacting step, which may have a negative effect on the contacting step and subsequent isolation step of the heparin bound resin. Furthermore, the heparin bound resin heparin obtained, may be contaminated with the dirt that may be removed in the current invention.

The addition of the water volume is preferably 1 -3-fold of the raw mucosa volume. From a mucosa hydrolysate thus obtained, i.e. having a reduced viscosity of a value of at most 25 mPa-s, the heparin is released. For example, the viscosity may be reduced to a viscosity such as as obtained by incubating raw mucosa at a temperature of 50⁰C at a pH between 8,0 and 9,0 for 0,5 hour using 7 grams of Syder alkaline protease and 1 liter of raw mucosa. As the heparin present in the mucosa hydrolysate is highly negatively charged, the heparin can be captured using a resin. In particular, a resin capable of binding negatively charged molecules, e.g. an anion-exchange resin, can be used. Alternatively, affinity resins capable of specifically binding heparin may also be used. Anion-exchange resins are commercially available, among others, under the trademarks Dowex, Amberlite, Duolite or Lewatit. Important for the capture of heparin is that the resin is efficiently contacted with the mucosa hydrolysate, allowing the heparin to bind to the resin. The skilled person knows suitable resins, and how to increase the contacting efficiency, which can comprise elevating the temperature and mixing, preferably while adhering to the operating conditions prescribed by the resin manufacturer. In a next step, the resin bound heparin is isolated from the hydrolysate to obtain isolated resin bound heparin. This can be done by convenient means like for instance sedimentation, in which the resin bound heparin sediments to the bottom of a reaction vessel, thus isolating the resin bound heparin from the hydrolysate fraction. Other methods like sieving or centrifugation are also possible and may speed up the processing time and may therefore be preferred. By isolating the resin bound heparin from the mucosa hydrolysate, isolated resin bound heparin is obtained. The isolated resin bound heparin has a weight and/or volume which is about 150-fold reduced as compared to the original raw mucosa that was used as a starting material. The isolated resin bound heparin may be further washed with water to remove hydrolysate remnants and may subsequently be transferred to storage vessels. As the mucosal hydrolysate and heparin are now separated, much of the nutrients for bacterial growth and contaminating bacteria are now removed, and the resin bound heparin is thus highly stabilized and may no longer suffer from decomposition and degradation as compared to the raw mucosa starting material. Nevertheless, a preservative such as sodium bisulphite may be added to the isolated resin bound heparin as a precautionary measure, of which much less is required because of the extreme volume reduction and separation from the hydrolysate, but it is not required. The resin bound heparin can now be produced on site, and can be transported to highly specialized chemical plants, e.g. capable to produce under the conditions suitable and required for medicament production, where it provides excellent starting material that can be used to produce heparin or heparin derived products, and where also the resin may be retrieved and recycled. Furthermore, as the mucosa hydrolysate waste produced on site is not substantially chemically or biologically treated, it may be used as a source for the preparation of biofuel, e.g. for the preparation of biogas in fermentation processes.

In alternative embodiments, preservatives may be added to the raw mucosa in order to maintain the quality of the raw mucosa, i.e. ensure high yields. For example, in one embodiment NaCI, e.g in the form of brine, may be added as a preservative to the raw mucosa. In other embodiments sodium metabisulfite or sodium bisulfite may be used. Suitable amounts of such preservatives are well known in the art. The addition of salt is a much less efficient preservative as compared to reducing preservatives such a bifsulfite. When the raw mucosa is stored overnight, the yields of heparine may already reduce by 50% as compared to the direct processing of raw mucosa or as compared to raw mucosa preserved with sodium bisulfite. or raw mucosa preserved by cooling at a temperature between 1-10⁰C. Thus, when raw mucosa is preserved with NaCI, it is preferred to store the raw mucosa less than 24 hours, more preferably less than 12 hours, most preferably less than 6 hours, in order to ensure acceptable heparin yields.

The advantage of using bisulfite is that the mucosa may be stored for longer periods without significant losses with regard to heparin yield, e.g up to 5 days. However, as mentioned, the downside of the use a strong reducing agents such as sodium bisulfite, is that these will be comprised in the waste stream of the protein hydrolysate of the mucosa hydrolysate from which the resin bound heparin has been separated. The presence of sodium bisulifite or metabisulfite or other preservatives may result in the waste stream being hazardous to the environment, e.g. toxic to human beings, animal life and plants. Moreover, the waste stream, i.e. biomass, may because of the presence of the preservatives not be suitable for the biofuel industry. For example, the preservatives may also be toxic to the microorganisms that ferment the biomass, i.e. the protein hydrolysate waste stream. Thus, it is desirable to remove preservatives to such level that the waste stream becomes suitable for the biofuel industry. Suprisingly, it was found that by using a round separator device (Sweco, Florence, KY, USA, www.sweco.com), in particular the S48S66 model, for filtering the mucosa hydrolysate i.e. seperating dirt and on a later stage resin, the amounts of preservatives were significantly reduced, making the protein hydrolysate a suitable source for the biofuel industry. In contrast, when the preserved mucosa will be filtered using a standard filtration system, the amounts of preservatives may not be reduced. Without being bound by theory, it is the inventors belief that the separator device used also serves as a micro- oxygenation device, i.e. intensifies air and fluid interaction thereby reducing the preservative, e.g. sodium bisulfite or sodium meta bisulfite. Thus, the use of preservatives in this scenario may have the benefit of preserving the mucosa, without the negative effects of the preservative, i.e. without being hazardous to the environment, and the protein hydrolysate is suitable to be used as a source of biomass for the biofuel industry. As according to the invention, the hydrolysis is carried out in a very short time, the resin can be added to the mucosa hydrolysate after a short time, resulting in a total processing time which is much reduced as compared to the previous methods. As the entire process up to the point where the isolated resin bound heparin is obtained can take place on site, where the raw mucosa is produced, it is no longer required to store large volumes of raw mucosa as the heparin is highly concentrated in the resin bound heparin. This has logistical and environmental advantages as no longer very large volumes of mucosa need to be stored and transported but only the condensed resin bound heparin. Furthermore, as the raw mucosa can be processed within a very short time after slaughtering of the animals, and the heparin can be separated from the raw mucosa and/or hydrolysate as soon as possible, the heparin does not suffer or at least significantly suffers less from decomposition caused by bacterial growth or general degradation which has a positive effect on heparin yield and quality, and in contrast to EP'642, high yields of 45,000-50,000 Units per kg of raw mucosa may be obtained within such a short processing time.

A chemical hydrolysis may require high temperatures and/or pressure, and the relative harsh conditions may have an impact on the heparin quality and yield. Preferably the hydrolysis of step b) is carried out by a proteolytic enzyme. Enzymatic proteolysis may be carried out at lower temperatures and milder pH, and as such in general do not affect quality and yield of heparin. The skilled person knows how to select optimal conditions to carry out such a proteolytic hydrolysis step. Parameters such as the amount of water, amount of enzyme, the amount of enzyme, the temperature and/or pH may be selected such that the desired viscosity is obtained within the set time frame. Preferably, the conditions meet the enzyme manufacturer's instructions. The pH of the raw mucosa at ambient temperature is in general between 6 and 7. The pH is preferably adjusted, e.g. with sodium hydroxide, to obtain a desired pH at ambient temperature, e.g. between 8 and 9, which may improve the proteolytic enzyme activity.

In one embodiment, the raw mucosa is obtained by taking the mucosa from one or more slaughtered animal(s), and the time between the moment of slaughtering the said animal(s) and the hydrolysis incubation step b) is less than 72 hours, preferably less than 48 hours, more preferably less than 24 hours, even more preferably less than 18 hours, 12 hours, 8 hours or 6 hours or even less. In principle step b) can be done immediately after the mucosa are separated from the slaughtered animal, so the above time is merely dictated by the logistics of the slaughtering process. It is one of the objectives of the invention to start the process as soon as possible, by reducing the time before the start of the process the heparin quality and yield may be improved as it is exposed to degradation and bacterial growth for a much shorter time. In addition, by reducing the time before the start of the process, the necessary storage volume may also be much reduced.

Without being bound thereto, the present inventors theorized that there is a relation between the viscosity of the mucosa hydrolysate and the yield of heparin. The lower viscosity of the mucosa hydrolysate as compared to the highly viscous raw mucosa is believed to enable the capture of the heparin from the mucosa hydrolysate using a resin capable of binding heparin and the subsequent efficient isolation of resin bound heparin from the mucosa hydrolysate. The lowering of the viscosity may increase the efficiency of the contacting step of the resin with the mucosa hydrolysate and subsequent isolation of resin bound heparin, which may reduce the time required for an efficient contacting and/or isolating step, which may simultaneously result in higher yields of heparin. An upper limit of viscosity indicates the upper limit of viscosity at which the contacting and/or isolating steps may be efficiently carried out. A lower limit of viscosity may for example indicate a viscosity below which the efficiency of the contacting and/or isolating step may not further improve the yield of heparin isolated from resin bound heparin. In certain embodiments, the upper limit of viscosity is preferably up to and thus including 25 mPa-s. In other embodiments, the upper limit of viscosity is preferably up to 22 mPa-s, more preferably up to 19 mPa-s, most preferably up to 16 mPa-s. In certain embodiments, the higher limit of viscosity may for instance be up to 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 mPa s. In certain embodiments, the lower limit of viscosity is preferably from 10 mPa-s. In certain embodiments, the lower limit of viscosity is from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mPa-s. In certain embodiments, the viscosity of the hydrolysate can vary between 10 mPa-s and 25 mPa-s. In other embodiments, the viscosity of the mucosa hydrolysate can vary between a lower limit of viscosity selected from 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 mPa-s, and an upper limit of viscosity selected from 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 mPa-s, as long as the viscosity value of the upper limit of viscosity is higher than the value of the lower limit of viscosity. For instance, when the upper limit of viscosity is 25 mPa s, the lower limit can be selected from 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 mPa s. Furthermore, when the lower limit of viscosity is 10 mPa-s, the upper limit of viscosity can be selected from 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 mPa-s. Thus, the viscosity of the hydrolysate may also vary between 11 mPa-s and 24 mPa-s, between 12 mPa-s and 23 mPa-s, or between 13 mPa-s and 22 mPa-s. Other ranges of viscosity that may preferably be possible are from 10- 15 mPa-s, 10-20 mPa-s, 10-25 mPa-s, 15-20 mPa-s, 15-25 rnPa-s or 20-25 mPa-s. For example, the viscosity may be reduced to a viscosity such as as obtained by incubating raw mucosa at a temperature of 50⁰C at a pH between 8,0 and 9,0 for 0,5 hour using 7 grams of Syder alkaline protease and 1 liter of raw mucosa.

It is to be noted that the viscosity can be measured in many different ways resulting in different viscosity values. The viscosity as indicated herein throughout the description and the claims refers to the viscosity that is determined by the method as described/indicated in example 10 , i.e. viscosity is measured with a Brookfield DV-II+ Pro viscosimeter using an RV-02 spindle, according to the manufacturer's instructions (Brookfield Engineering Laboratories, Inc, Middleboro, MA, USA, 02346, and wherein the temperature of the mucosa hydrolysate is 56 ⁰C and the speed of the spindle 100 rpm.

In a preferred embodiment, the incubation time of step b) of the raw mucosa is 3 hours or less, preferably 2 hours or less. Such short time periods are preferred because according to the invention, the processing of the raw mucosa is as fast as possible. The skilled person knows how to adapt the conditions such that the viscosity is reduced to the desired viscosity within that time frame, and he is aware that there are limits to the minimal time required for efficient hydrolysis, such that the hydrolysis step b) will take at least some time, e.g. 0,5 hour. For example, as shown in example 10, a viscosity below 25 mPa-s may already be obtained within half an hour.

As the quality of the raw mucosa is no longer compromised, as explained above, from for instance bacterial growth or general decomposition, during the short time between raw mucosa collection and the start of the hydrolysis, it is also no longer required to actively preserve the raw mucosa, by e.g. addition of chemicals or applying heat. In addition, because also the processing time of the raw mucosa and mucosa hydrolysate is much reduced in the invention, the quality of the raw mucosa is also maintained during said processing, thus it is also not required to use preservatives during the process. Therefore, in another embodiment, the invention is characterized in that the raw mucosa and the mucosa hydrolysate remain free of sodium metabisulfite, calcium proprionate and/or phenol. Nevertheless, if desired, it is still possible to use preservatives, and the invention does not exclude the use of preservatives. If preservatives are used, much lower amounts may be used, which will reduce the disadvantageous environmental effects of preservatives as previously described. When preservatives are not used, yields may be increased even further when hydrolysis was carried out with an enzyme. This may indicate that preservatives, that have their effect in stopping or slowing down biological processes, also can have a negative effect on the proteolytic hydrolysis step, which is a biological process as well.

In a preferred embodiment , the hydrolysis step is carried out with a proteolytic enzyme and the incubation step b) is carried out at elevated temperature, i.e. above ambient temperature, preferably between 30⁰C and 80 ⁰C, more preferably between 40⁰C and 70 ⁰C, most preferably at a temperature between 5O⁰C and 60⁰C. Although the proteolytic enzyme used in the incubation step b) of the raw mucosa hydrolysis may have activity at ambient temperature, the incubation step is preferably carried out at an elevated temperature in order to increase the activity of the enzyme during the short time period of step b). The skilled person knows which temperature is optimal for the selected enzyme and will preferably select that temperature. When an elevated temperature is used in step b), the said enzyme is added before or when the elevated temperature is reached, and the time between the addition of the enzyme and the time at which the elevated temperature, is reached is 2 hours or less, preferably 1 hour or less. As such, there is no need to pre-incubate the raw mucosa with the proteolytic enzyme.

Any enzyme can be used in the process, provided that the enzyme provides sufficient proteolytic activity at the conditions used, meaning that within the desired time frame under the conditions of step b) the viscosity of the raw mucosa is reduced to a viscosity of at most 25 mPa-s, for example to the viscosities and the viscosity ranges as described above. The viscosity as indicated herein throughout the description and the claims refers to the viscosity that is is determined by the method as described/indicated in example 10, i.e. viscosity is measured with a Brookfield DV-II+ Pro viscosimeter using an RV-02 spindle, according to the manufacturer's instructions (Brookfield Engineering Laboratories, Inc, Middleboro, MA, USA, 02346, and wherein the temperature of the mucosa hydrolysate is 56 °C, and the speed of the spindle is 100 rpm.

Enzymes with proteolytic activity are known in the art and are commercially available, for example proteolytic enzymes isolated from the bacterium Bacillus Subtilis can be used and proteases have also been improved with regard to specificity and stability using techniques of site-directed mutagenesis and/or random mutagenesis. Examples of commercially available enzymes which may be preferably selected are from the group consisting of Syder alkaline protease, Alcalase, Maxatase, Durazym, Maxapem and Purafect, or a combination thereof. Syder alkaline protease, commercially available at Wuxi Syder Bioproducts Ltd, Wuxi City, China, is an alkaline protease extracted from Bacillus Licheniformis and has an enzyme activity of > 100,000 μ/g. 1 μ/g is defined as the amount of enzyme in 1 gram enzyme powder to hydrolyze casein to produce 1 microgram (μg) tyrosine per minute at pH 10,5 and 40⁰C.

The skilled person knows that in order to increase the proteolytic activity, and thus decrease the time to obtain the desired viscosity, he may increase the amount of enzyme used. Nevertheless, increasing the amount of enzyme has the downside that the process becomes less economical from a cost perspective and thus the skilled person may preferably select an optimal amount of enzyme such that the desired viscosity is obtained within set time frame without using excessive amounts of enzyme. Therefore, in another embodiment, the amount of enzyme in the incubation step b) used per liter of raw mucosa is preferably between 1 and 16 grams, more preferably between 4 and 1 1 grams, most preferably between 7 and 8 grams.

In another embodiment, the proteolytic enzyme in the mucosa hydrolysate obtained in step b) is inactivated before or during step c) to obtain an inactivated mucosa hydrolysate. It is preferred to stop the activity of the proteolytic enzyme in the mucosa hydrolysate because it is undesirable to have a potentially active proteolytic enzyme contamination within heparin prepared according to the invention as this may have an impact on medicaments produced therefrom. Inactivation is preferably carried out when the mucosa hydrolysate reaches the desired viscosity, as described above. The skilled person will be aware of the conditions that can be chosen to inactivate the particular proteolytic enzyme or selected enzymes, e.g. most enzymes will not survive a treatment of 85⁰C, whereas heparin, a polysaccharide, can survive such temperatures. Thus, enzyme activity can be in general be inactivated by an inactivation step comprising a heating step. Preferably, the inactivation is effected by raising the temperature to 80⁰C or higher, preferably to between 80°C and 90⁰C, preferably for at least 10 minutes. After the inactivation, the mucosa hydrolysate is allowed to cool down. However, the heparin can also be bound to the resin and optionally be further isolated without denaturation of the enzyme. Alternatively, inactivation can take place during or after step d), i.e. isolating of the resin bound heparin.

A resin capable of binding the heparin present in the mucosa hydrolysate is contacted with the mucosa hydrolysate, resulting in resin bound heparin. The resin can also be added earlier, for instance, during step b), which can have the advantage that the total processing time may be reduced even further. If the method comprises an inactivation step such as described above, the resin is preferably contacted with the mucosa hydrolysate when the temperature is less than 60⁰C in order not to deteriorate the resin. If a method in which resin is added during step b) also comprises heat inactivation of the enzyme, it is preferred to select a resin that can withstand the high temperature of the inactivation step. It is important to note that the addition of the resin can increase the viscosity of the mucosa hydrolysate. This effect on viscosity may not be taken into account when selecting the conditions required to obtain the envisaged viscosity of the mucosa hydrolysate in step b). For instance, from a first process in which resin is added after the envisaged viscosity is reached, exactly the same processing parameters for step b) can be selected for an alternative process wherein during step b) resin is added. This means when one would compare the two processes, at the end of step b) the alternative process in comparison with the first process may have a relatively higher viscosity. Nevertheless, it is preferred to add the resin when the viscosity of the mucosa hydrolysate obtained in step b) has reached the desired viscosity, as a reduced viscosity improves mixing and resin heparin contacting. Thus, because of such an improvement, the amount of resin required in this step may also be reduced. The person skilled in the art may select a suitable contacting time, during which the resin can bind the heparin. Optimal results may be obtained by selecting a contacting time between 10-12 hours, or longer. In a preferred embodiment, the contacting time of step c) of the resin with the mucosa hydrolysate is 10 hours or less, preferably 8 hours or less, more preferably 6 hours or less, most preferably 3 hours or less. By reducing the contacting time, the total processing time is further reduced, and it was found that this reduction maynot have a dramatic negative impact on process yield, which contrasts to the previous methods known in the art, in which very long contacting times of the resin with the mucosa hydrolysate were used to ensure sufficient capture. The contacting step may be at least half an hour, as it requires some time before the hydrolysate is efficiently contacted with the resin, e.g. by mixing. In addition, the temperature of the contacting step c) is preferably between 15-80⁰C, more preferably between 40-60°C. By elevating the temperarature, the interaction of the resin with the heparin is improved, resulting in a more efficient contacting step. In another embodiment, the time between the beginning of step b) and isolating the resin bound heparin in step d) is less than 16 hours, preferably less than 12 hours, more preferably 8 hours or less. Thus, a much shorter time is required as compared to the previous methods known in the art, which require very long processing times of 24 hours and more in order to obtain the desired high yields of heparin. High yields of heparin can now be obtained within a working day or in an overnight process. The skilled person knows how to arrive at such short processing times by selecting conditions as provided by the invention of each step such that the process can be efficiently carried in such a short time.

Preferably the raw mucosa is obtained from slaughtered livestock, preferably from slaughtered pigs. Livestock can include e.g. sheep, cows, pigs, horses and poultry. Most preferred is the raw mucosa obtained from slaughtered pigs, as heparin derived from pigs is preferred for human pharmaceuticals, as it most closely resembles human heparin. Nevertheless other sources of heparin may be used if desired.

In an alternative embodiment, the circular and longitudinal muscle layer and serosa may be also be used as a source of heparin. The circular and longitudinal muscle layer and serosa may be combined to the raw mucosa, subsequently grinded. The grinded mixture thus obtained may be processed according to the methods as described in the invention. An additional yield of approximately 10,000-15,000 IU heparin /pig may be obtained.

In another embodiment, heparin is produced by a method for the preparation of heparin wherein the resin bound heparin is obtained according to the invention and wherein the heparin is released from the resin and isolated therefrom. This may take place on site, but preferably, the heparin is released from the resin in a specialized chemical plant, which may be remotely located from the site where the resin bound heparin is isolated. The heparin can be recovered from the resin bound heparin by elution. It may be desirable to first elute possibly present contaminating bound anions from the resin, e.g. with a water solution that contains a specific salt concentration, which is usually between 0.1-0.5 M sodium chloride, after which the heparin may be eluted at a concentration between 2-4 M sodium chloride. Alternatively, the heparin can be eluted directly with a salt concentration between 2- 4 M sodium chloride, but other elution strategies, know to the skilled person, can also be envisaged. After the heparin is eluted from the resin, the resin may be further processed such that it can be recycled and may be used again in the same process. The eluted heparin can be precipitated with an organic solvent such as methanol and dried, before it is further processed into pharmaceutical compositions. As the use of organic solvents requires special safety and environmental protection measures, such processing preferably takes place at a specialized chemical plant, equipped to deal with such solvents, which may be recovered and reused. The following examples are illustrative for the invention and should not be interpreted as limiting the scope of the invention.

Comparative example

During the slaughtering of pigs, during working hours, intestines of pigs were collected and processed into a submucosa fraction, for use as casing material, and raw mucosa. 300 pigs were slaughtered resulting in 240 Liter of raw mucosa which was stored in a collection tank. To the raw mucosa, 1 % ( w/v) sodium bisulphite was added and to the raw mucosa. The next day, 0,2 kg of proteolytic enzyme, Maxatase, was added. After a pre-incubation of 4 hours, 1 ,8 kg of washed resin was added and NaOH was added and the raw mucosa was continuously mixed until the pH of the raw mucosa was between 8,0 and 9,0. The temperature was increased to 55-65°C for 2 hours, after which the mixture was allowed to cool down overnight. The mucosa hydrolysate was now filtered for half an hour with an 80 mesh filtercloth to remove remaining debris, such as gravel or hairs and such, to obtain a clarified mucosa hydrolysate. The pH was measured and was between 6,8 and 7,0. The resin bound heparin was filtered through a filter cloth, and the mucosa hydrolysate was discarded as sewage. The cloth containing the resin bound heparin was washed with water of 45°C until only clear water came through the cloth from the resin, ensuring that all contaminations were removed. The resin was dried in the filtercloth, by letting the water leak from the resin contained within the cloth. The resin bound heparin was transferred to a container, and a 50 gram/Liter of sodium-bisulphite was added such that a 10 cm layer of fluid covered the resin in the container. The container was stored at 8°C. The heparin was eluted from the washed resin bound heparin, and the eluate was obtained at least 40 hours after slaughtering and contained 30.000-35.000 U/kg mucosa. In an alternative experiment, the pre-incubation was carried out for 2 hours, after which the resin was added and the temperature was maintained at 55-65°C for 3 hours, after which the resin bound heparin was immediately obtained, without slowly cooling down overnight. The heparin yield was only 18.000 U/kg mucosa.

Example 1

During the slaughtering of pigs, during working hours, intestines of pigs were collected and processed into a submucosa fraction, for use as casing material, and raw mucosa. 4.000 pigs were slaughtered resulting in 3.200 Liter of raw mucosa which is stored in a collection tank. The next day, the raw mucosa was transferred to a processing tank. The pH was measured and was between 6,5 and 7,0. NaOH was added while the raw mucosa was continuously mixed until the pH of the raw mucosa was between 8,0 and 9,0. At 6 AM, 22 kg of Syder alkaline protease was added after the temperature of the raw mucosa enzyme mixture was increased to 50⁰C. The temperature was maintained at 50⁰C for 3 hours, after 1 hour, the pH was checked to ensure the pH was between 8,0 and 9,0, if necessary, NaOH or HCI was added to obtain a pH within the desired range. Next, the mucosa hydrolysate was heat inactivated at 82°C for 15 minutes, after which the mucosa hydrolysate was cooled to 60⁰C. The mucosa hydrolysate was now filtered for an hour with an 80 mesh filtercloth to remove remaining debris, such as gravel or hairs and such, to obtain a clarified mucosa hydrolysate. The pH was measured and was between 6,8 and 7,0. At 10:45 AM, 24 kg of washed resin, Amberlite FPA98 Cl (Rohm and Haas Ion Exchange Resins, Philadelphia, PA USA), was added and the resin mucosa hydrolysate mixture was mixed for 10 hours. The resin bound heparin was filtered through a filter cloth, and the mucosa hydrolysate was discarded as sewage. The cloth containing the resin bound heparin was washed with water of 45⁰C until only clear water came through the cloth from the resin, ensuring that all contaminations were removed. The resin was dried in the filtercloth, by letting the water leak from the resin contained within the cloth. The resin bound heparin was transferred to a container, and a 50 gram/Liter of Sodium-Bisulphite was added such that a 10 cm layer of fluid covered the resin in the container. The container was stored at 8°C. The heparin was eluted from the washed resin bound heparin, and the eluate contained 45.000-50.000 U/kg mucosa.

Example 2

During the slaughtering of pigs, during working hours, intestines of pigs were collected and processed into a submucosa fraction, for use as casing material, and raw mucosa. 4.112 Liter of raw mucosa mixed with 4.100 Liter of water of ambient temperature and was stored in a collection tank and preserved with 0.25 weight % Natrium Bisulfite (E222).The next day the raw mucosa was transferred to a processing tank. The pH was measured at 5.9. Next the temperature of the raw mucosa enzyme mixture was increased to 55 °C, when the temperature reached the desired value, NaOH was added until the pH of the raw mucosa was 8.6. The raw mucosa was continuously stirred. At 7:45 AM 38.5 kg of enzyme was added, i.e. Syder alkaline protease, commercially available at Wuxi Syder Bioproducts Ltd, Wuxi City, China. The temperature was maintained at 55°C for 4 hours while the viscosity was monitored. After 1 hour, the pH was checked to ensure the pH was between 7,5 and 8.0, if necessary, NaOH or HCI was added to obtain a pH within the desired range. Within 4 hours the viscosity reached a value of 14.8 mPa-s, as measured as described below in example 10. Next, the mucosa hydrolysate was heat inactivated at 82°C for 15 minutes. After which the heating was turned off. The mucosa hydrolysate was now filtered for an hour and a half to remove debris, 3.9 kg debris remained, which included gravel, hairs and such, obtaining a clarified mucosa hydrolysate. The filtering was done with a micro-oxygenator which exposes the filtrate with maximum amount of air, thus promoting the degeneration of preservatives. We used a Sweco micro-oxygenator, model S48S66 with a 1.200 mm diameter and a 80 mesh filter. After filtering the pH was measured at 7.5. At 3:15 PM, 30.8 kg of resin, was added and the resin mucosa hydrolysate mixture was mixed for 13 hours and 10 minutes. The resin bound heparin was filtered with the same micro-oxygenator as used for the filtering of debris. The mucosa hydrolysate was discarded to the tank for collecting material for the Bio-fuel company. The cloth containing the resin bound heparin was washed with water of 45°C until only clear water came through the cloth from the resin, ensuring that all contaminations were removed. The resin was dried in the filtercloth, by letting the water leak from the resin contained within the cloth. The resin bound heparin was transferred to a container, and 50 gram/Liter of Sodium-Bisulphite was added such that a 10 cm layer of fluid covered the resin in the container. The container was stored at 8°C. The heparin yield was 40.347 IU/kg mucosa.

Example 3

Heparin was prepared according to the method as described in example 2. Four different runs were executed.

Example 4

Heparin is prepared according to the method as described in example 1 , wherein the storage time of the raw mucosa is varied. Also, the method is performed with and without (wo) the use of the preservative of example 2. Results is shown in table 1.

Table 1

Storage of raw mucosa (hrs) Heparin yield^a

0 (wo preservatives) +++++

0 +++++

6 (wo preservatives) ++++

6 +++++

24 (wo preservatives) +++

24 +++++

48 (wo preservatives) ++

48 ++++

72 (wo preservatives) ++

72 +++

168 (wo preservatives) +

168 ++ aHeparin Yield (+ = 0-10,000; ++ = 10,000-20,000; +++= 20,000-30,000; ++++=30,000-40,000; +++++=40,000-50,000, Units of heparin per kg of raw mucosa starting material)

Example 5

Heparin is prepared according to the method as described in example 1 , wherein the temperature of the enzymatic hydrolysis step is varied. Expected results are shown in table

2.

Table 2

Temperature of enzymatic hydrolysis (°C) Heparin yield ambient temperature ++

30 +++

40 ++++

52 +++++

55 +++++

60 ++++

70 +++

80 ++ aHeparin Yield (+ = 0-10,000; ++ = 10,000-20,000; +++= 20,000-30,000; ++++=30,000-40,000; +++++=40,000-50,000, Units of heparin per kg of raw mucosa starting material)

Example 6

Heparin is prepared according to the method as described in example 1 or 2, wherein the amount of enzyme used in the enzymatic hydrolysis step is varied, as well as the duration of the enzyme step. Increasing the amount of enzyme resulted in a higher yield of heparin. Results are shown in table 3.

Table 3 enzyme/kg raw Enzymatic Heparin mucosa hydrolysis yield³

2 1 +

4 1 ++

8 1 ++++

16 1 +++++

2 2 +

4 2 +++

8 2 +++++

16 4 +++++

2 4 +++

4 4 +++++

8 8 +++++

16 8 +++++

2 8 +++++

4 8 +++++ aHeparin Yield (+ = 0-10,000; ++ = 10,000-20,000; +++= 20,000-30,000; ++++=30,000-40,000; +++++=40,000-50,000, Units of heparin per kg of raw mucosa starting material)

Example 7

Heparin is prepared according to the method as described in example 1 , wherein the amount of enzyme used in the enzymatic hydrolysis step is varied, as well as the temperature of the enzyme step. Results are shown in table 4.

Table 4

Grams enzyme/kg Temperature hydrolysis (⁰C) Heparin yield^a mucosa

1 20 +

1 50 +++

7 20 ++

7 50 +++++

50 20 +++++

50 50 +++++ aHeparin Yield (+ = 0-10,000; ++ = 10,000-20,000; +++= 20,000-30,000; ++++=30,000-40,000; +++++=40,000-50,000, Units of heparin per kg of raw mucosa starting material) the enzyme step. Increasing the amount of enzyme resulted in a higher yield of heparin. Results are shown in table 3.

Table 3 enzyme/kg raw Enzymatic Heparin mucosa hydrolysis yield³

2 1 +

4 1 ++

8 1 ++++

16 1 +++++

2 2 +

4 2 +++

8 2 +++++

16 4 +++++

2 4 +++

4 4 +++++

8 8 +++++

16 8 +++++

2 8 +++++

4 8 +++++

Ηeparin Yield (+ = 0-10,000; ++ = 10,000-20,000; +++= 20,000-30,000; ++++=30,000-40,000; +++++=40,000-50,000, Units of heparin per kg of raw mucosa starting material)

Example 7

Table 4

Grams enzyme/kg Temperature hydrolysis (⁰C) Heparin yield^a mucosa

1 20 +

1 50 +++

7 20 ++

7 50 +++++

50 20 +++++

50 50 +++++ aHeparin Yield (+ = 0-10,000; ++ = 10,000-20,000; +++= 20,000-30,000; ++++=30,000-40,000; +++++=40,000-50,000, Units of heparin per kg of raw mucosa starting material) Example 8

Heparin is prepared according to the method as described in example 1 , wherein the contacting time of the resin with the mucosa hydrolysate is varied. Acceptable yields were obtained with reduced contacting times of 8, 6 and 3 hours.

Example 9

Heparin is prepared according to the method as described in example 1 or 2, wherein no enzyme heat inactivation step was carried out. Similar yields were obtained as in examples

1-3.

Example 10

Viscosity was measured using a Brookfield DV-II+ Pro viscosimeter using an RV-02 spindle, according to the manufacturer's instructions (Brookfield Engineering Laboratories, Inc, Middleboro, MA, USA, 02346, http://www.brookfieldengineering.com/ ). Briefly, as described in example 1 , an equal volume of water was added to raw mucosa and the mixture was heated. When the temperature of the mixture reached 55⁰C, enzyme was added (t=0) and at 0.5 hour intervals the viscosity was measured, as well as the temperature and the pH (table 5). The viscosity was determined at a temperature as indicated below. Viscosity is expressed in mPa-s = 10^~3 Pascal -second. Viscosity as used in the description and in the claims refers to the viscosity measured using a Brookfield DV-II+ Pro viscosimeter with an RV-02 spindle, at an 100 rpm, at a temperature of 56°C, wherein preferably the pH of the material measured is 7.5.

Table 5.

Claims

1. Method for the preparation of heparin from raw mucosa, comprising the steps of: a) providing raw mucosa, b) incubating the raw mucosa of step a) under conditions wherein the raw mucosa is hydrolyzed, reducing the viscosity to a value of at most 25 mPa-s, in a time period of 4 hours or less, thereby obtaining a mucosa hydrolysate, c) contacting a resin capable of binding heparin with the mucosa hydrolysate, allowing the heparin from the mucosa hydrolysate to bind with the said resin to obtain resin bound heparin, d) isolating the resin bound heparin of step c) from the hydrolysate, to obtain isolated resin bound heparin

2. Method, according to claim 1 , wherein the hydrolysis of step b) is carried out by a proteolytic enzyme.

3. Method according to claim 1 or claim 2 wherein the raw mucosa of step a) is obtained by taking the mucosa from one or more slaughtered animal(s), the time between the moment of slaughtering the said animal(s) and the incubation step b) being less than 72 hours, preferably less than 48 hours, more preferably less than 24 hours.

4. Method according to any of the preceding claims, wherein in step b) the raw mucosa is hydrolysed, reducing the viscosity to a value of at most 22 mPa s, more preferably 19 mPa-s, most preferably 16 mPa-s..

5. Method according to any of the preceding claims, wherein the incubation time of step b) is 3 hours or less, preferably 2 hours or less.

6. Method according to any of the preceding claims characterized in that the raw mucosa and the mucosa hydrolysate remain free of sodium metabisulfite, calcium proprionate and phenol.

7. Method according to any of the preceding claims, including claim 2, wherein the incubation step b) is carried out at elevated temperature, preferably between 3O⁰C and 80 ⁰C, more preferably between 40⁰C and 70 ⁰C, most preferably at a temperature between 5O⁰C and 60⁰C.

8. Method according to claim 6, wherein in step b) the said enzyme is added before or when the elevated temperature is reached, and that the time between the addition of the enzyme and the time at which the elevated temperature is reached is 2 hours or less, preferably 1 hour or less.

9. Method according to any of the preceding claims, including claim 2, wherein the enzyme is selected from the group consisting of Syder alkaline protease, Alcalase, Maxatase, Durazym, Maxapem and Purafect, or a combination thereof.

10. Method according to any of the preceding claims, including claim 2, wherein the amount of enzyme in the incubation step b) used per Liter of raw mucosa is preferably between 1 and 16 grams, more preferably between 4 and 11 grams, most preferably between 7 and 8 grams.

11. Method according to any of the preceding claims, including claim 2, wherein the proteolytic enzyme in the mucosa hydrolysate obtained in step b) is inactivated before or during step c) to obtain an inactivated mucosa hydrolysate.

12. Method according to claim 11 , wherein the inactivation is effected by raising the temperature to 80°C or higher, preferably to between 80⁰C and 90°C, preferably for at least 10 minutes,

13. Method according to any of the preceding claims wherein the contacting time of step c) of the resin with the mucosa hydrolysate is 10 hours or less, preferably 8 hours or less, more preferably 6 hours or less, most preferably 3 hours or less.

14. Method according to any of the preceding claims wherein the temperature of the contacting step c) is between 15-80°C, preferably between 40-60°C.

15. Method according to any of the preceding claims wherein the time between the beginning of step b) and isolating the resin bound heparin in step d) is less than 16 hours, preferably less than 12 hours, more preferably 8 hours or less.

16. Method according to any of the preceding claims in which the raw mucosa is obtained from slaughtered livestock, preferably from slaughtered pigs.

17. Method for the preparation of heparin wherein the resin bound heparin is obtained according to any of the preceding claims and wherein the heparin is released from the resin and isolated therefrom.

18. Protein hydrolysate prepared according to any of the preceding claims.

19. Heparin prepared according to claim 17.

20. Method according to any of the claims 1-17, wherein the viscosity is measured with a Brookfield DV-I l+ Pro viscosimeter using an RV-02 spindle and wherein the temperature of the mucosa hydrolysate is 56 ⁰C, and the speed of the spindle is 100 rpm.