WO2010137043A1

WO2010137043A1 - Haemostatic biomaterial from waste fractions of human plasma fractionation process

Info

Publication number: WO2010137043A1
Application number: PCT/IT2009/000230
Authority: WO
Inventors: Raniero D'ascoli; Leonardo Pajewski; Francesco Veglio'
Original assignee: Baxter Manufacturing S.P.A.
Priority date: 2009-05-27
Filing date: 2009-05-27
Publication date: 2010-12-02

Abstract

The present invention relates to a biocompatible biomaterial, in particular a biocompatible sponge, deriving from protein pastes obtained as by-products of human plasma fractionation, optionally processed in a scaffold of other biomaterials such as collagen, hyaluronic acid, etc. The technology of the invention enables to recycle one or more protein fractions obtained as by-products, which are currently disposed of as industrial waste, thus increasing manufacturing costs. The described method enables to obtain a biomaterial with haemostatic properties. Other possible uses relate to its use as a scaffold for cell cultures in tissue engineering applications.

Description

"Haemostatic biomaterial from waste fractions of human plasma fractionation process"

DESCRIPTION Field of the invention

The present invention relates to a biocompatible biomaterial, in particular a biocompatible sponge, deriving from protein pastes obtained as by-products of human plasma fractionation, if necessary processed in a scaffold of other biomaterials such as collagen, hyaluronic acid, etc. The invention further relates to a method for obtaining the biocompatible material and to the use thereof as a biocompatible material and as a support for cell cultures in tissue engineering applications.

State of the art

E.J. Cohn, L. E. Strong (J. Am. Chem. Soc. 68 (1946) 459) and other researchers studied the separation and the properties of plasma proteins by defining a method, known as Cohn' s method, for their separation into fractions.

A variant of Conn's process, which is currently used for plasma protein fractionation, is based on a system of five variables whose limits are shown in Table 1. Table 1

By suitably varying pH, alcohol concentration, ionic force, protein concentration, the protein fractions shown in Table 2 are separated. ' Table 2

V Albumin, α-and β-globulins

At present, protein pastes obtained from a variant of Conn's fractionation process, e.g. FRACTION I, FRACTION III and FRACTION IV 1-4, are disposed of as industrial waste.

This involves quite high costs for manufacturers of plasma proteins, which must provide for the disposal of processing by-products.

A way to reduce these costs consists in recycling protein fractions by turning them into useful products which can be marketed and thus become a source of profit (and not of costs) for the company. The method of the invention, disclosed in the present patent application, enables to produce a biomaterial that is useful for different aims and therefore marketable.

In the specific field it is known about haemostatic biomaterials obtained from proteins of animal origin, in particular of bovine origin, such as Spongostan sponge (manufactured by Ferrosan) .

It is further known about haemostatic pads based on regenerated cellulose or based on collagen (see e.g. US 6,649,162, US 6,454,787, US 7,186,684). It is also known about biomaterials obtained from plasma components (see e.g. US 7,009,039, - A -

US 7,166,709, US 6,762,336, US 6,548,729), but no publication mentions haemostatic biomaterials that can be obtained by processing waste fractions from human plasma fractionation process, in particular from modified Conn' s process as disclosed in detail in the present patent application.

The haemostatic biomaterial of the present invention is therefore an advantageous alternative to haemostatic pads of animal origin and to other known or commercial sponges.

As a matter of fact, a haemostatic product deriving from human plasma proteins is advantageous in that it has a better interaction with body fluids favored by the porous structure and by its biocompatibility, it accelerates platelet activation (thus reducing blood coagulation times) and improves absorption and metabo- lization of the material itself. Moreover, manufacturing this product involves no costs for raw materials since it is obtained from the transformation of a waste protein paste. The recycling of waste material, which would normally be disposed of according to existing regulations, involves lower disposal costs and enables the company to turn a cost into a material to be sold. These advantages add to better performances for the haemostatic biomaterial of the invention with respect to those present on the market, as shown in the comparative examples disclosed in the experimental part. Description of the invention

The invention is described below in detail also with reference to the accompanying figures, wherein:

- Figure 1 shows a block diagram of the plasma protein fractionation process known in the field;

- Figure 2 shows a block diagram of the process for manufacturing the haemostatic material from plasma protein waste fractions according to the invention;

- Figure 3 shows the typical mechanical behavior of the new material, which, submitted to a compression test, is deformable and soft and wholly recovers the high deformations it has undergone;

- Figure 4 shows the average load variation expressed in kPas, corresponding to a material deformation of 80%, after treatment with glutaraldehyde and gamma irradiation with a dose of 25 kGy;

- Figure 5 shows the modes of in-vitro biocompatibili- ty test by cultivating human fibroblasts on the new biomaterial immersed in the culture medium;

- Figure 6 shows the results of a comparison between the haemostatic properties of the material according to the invention, with respect to the haemostatic ma- terials most frequently used in surgery;

- Figure 7 shows growth curves for fibroblasts in cell cultures prepared for verifying the biocompatibility of the pads of material according to the invention; the biomaterial is biocompatible irrespective of the reticulation executed with glutaraldehyde;

- Figure 8 shows the colonization of the biomaterial surface by adhering fibroblasts; left: pad obtained with addition of polyethylene glycol, right: pad obtained with addition of polyethylene glycol and reticulated with glutaraldehyde;

- Figure 9 shows samples of haemostatic devices used in in-vivo haemostaticity tests;

- Figure 10 shows the diagram of comparative in-vivo haemostaticity test;

- Figure 11 shows average haemorrhage duration in rats not treated with heparin as a function of different haemostatic materials used;

- Figure 12 shows average haemorrhage duration in rats treated with heparin as a function of different haemostatic materials used;

- Figure 13 shows haemostatic collagen pads modified by addition of waste fraction Fraction I; on the left a collagen felt; on the right a collagen felt fragment imbibed with Fraction I solution and dried; - Figure 14 shows sponges obtained from Fraction I with addition of collagen.

The present invention relates to a method for preparing a biomaterial from at least one protein fraction obtained as a by-product of human plasma fractionation process, in particular of modified Conn' s process, disclosed in detail below.

Preferably, the biomaterial is obtained from waste fractions obtained as a protein by-product from modified^* Cohn's process for processing plasma: FRACTION I, FRACTION III or FRACTION IV 1-4.

The most preferred protein fraction as a starting product for preparing the biomaterial of the invention is FRACTION I.

In another embodiment, the biomaterial according to the invention is obtained from whole, untreated plasma .

In another embodiment, the biomaterial of the invention is obtained by adding to a biomaterial scaffold, e.g. a collagen or hyaluronic acid scaffold, at least one waste fraction obtained from modified Conn' s human plasma fractionation process. In this embodiment, the waste fraction that are preferably used are FRACTION I, FRACTION III OR FRACTION IV 1-4. FRACTION I is the most preferred. The biomaterial obtained with the single steps covered by the present invention has shown haemostatic properties, i.e. it can reduce and/or stop blood flow from surface or internal injuries.

The biomaterial has proved particularly useful as a haemostatic material' for use in surgery when haemostatic pads that are effectively able to reduce, control and stop bleeding are needed.

The haemostatic biomaterial according to the present invention is preferably a haemostatic sponge. The biomaterial according to the invention has also proved useful as a scaffold for cell cultures in tissue engineering applications.

Cohn' s human plasma fractionation process, with which the protein waste fractions that are preferably used as starting products for the biomaterial according to the inventive process are obtained, is known in the field and will be described below in detail with reference to Figure 1.

Plasma, obtained from blood by plasmapheresis or by separation from whole blood, is subjected to a fractionation treatment using modified Cohn' s process, which has already been used for a long time by the Applicant for protein separation. Plasma, stored at a temperature of < -20⁰C, is sent to the first blood serum processing line (Mass-Capture) where it is thawed.

Back to its liquid state, plasma is subjected to a first centrifugation giving a precipitate (Cryo paste) . -From the precipitated paste factor VIII and a supernatant (Cryo Supernatant) are obtained, the latter undergoing a purification treatment with ion exchange resins for recovering factors IX, VII and the complex AT III contained therein.

The solution obtained after Mass Capture process (Cohn's plasma), free of coagulation factors, is subjected to a fractionation process for recovering albumin and γ-globulin.

The first process step involves the addition of ethyl alcohol up to a concentration of 5 to 10% (v/v) , pH adjustment around 7 by adding hydrochloric acid and soda, and an ageing of the suspension thus obtained for about 2 hours at a temperature of -1 to 1°C. This step is followed by a centrifugation giving a cake (FRACTION I), which is an industrial waste, and a supernatant (SUPERNATANT I) which continues processing and undergoes another centrifugation, after adjusting alcohol concentration to values of 20 to 30% (v/v) . This second centrifugation gives a cake (FRACTION II + III) , which undergoes in its turn further processing operations for obtaining immuno-globulins, in particular immuno-gamma globulin IgG, and a supernatant (SUPERNATANT II + III) .

The supernatant II + III, by precipitation with alcohol and pH adjustment, gives a precipitate (FRACTION IV-I, 4) containing β-globulins and α-globulins, and a supernatant (SUPERNATANT IV-I, 4) subjected to a filtration operation with a press filter, enhanced by ce- lite addition.

This process step gives a cake (FRACTION IV-I, 4 + CE- LITE) and a filtrate.

The filtrate is subjected to a subsequent treatment for adjusting pH to values around 5.30-5.40, and sent to a further filtration, still with press filter, so as to obtain a supernatant (RAW SUPERNATANT V) , which is then subjected to distillation for recovering etha- nol, and a precipitate (RAW FRACTION V) from which albumin is extracted.

RAW FRACTION V is re-suspended in physiological solution, subjected to pH adjustment up to values of 4.4- 4.8 with hydrochloric acid and soda, adjusted to an alcohol concentration of 8 to 15% and then filtered. Filtration, together with the addition of celite, gives a cake, which is disposed of, and a filtrate, which is subjected to the same operations as the pre- vious one, suitably modifying experimental parameters. Filtration is executed in a press filter and allows to separate a supernatant, which is subjected to distillation for ethanol recovery, and a solid portion, rich in albumin, which is known as PURIFIED FRACTION V. This fraction is further treated for obtaining the end product (albumin) , first by clarification filtration and then by washing.

FRACTION I is more preferably used in the present invention as a starting product for preparing a bioma- terial, in particular a haemostatic sponge. FRACTION I comprises 20 to 40% of proteins dispersed in an aqueous phase.

The proteins included in FRACTION I are albumin, α- globulins, β-globulins, γ-globulins and fibrinogen. The temperature of the process step in which FRACTION I is obtained is preferably of -1 to 1°C. After obtaining the material, the latter is kept at a temperature of -18 to -20⁰C.

Waste protein fractions from a plasma fractionation process, in particular FRACTION I, FRACTION III or FRACTION IV 1-4 (preferably FRACTION I) of modified Conn' s fractionation process, are subjected to the recycling process of the present invention (disclosed below with reference to Figure 2) for obtaining a bio- material which is preferably a haemostatic sponge. A waste protein fraction of paste, preferably FRACTION I, including plasma proteins, water and alcohol, is mixed with a basic solution, preferably a sodium bicarbonate solution, so as to keep pH at a value of 7 to 9, preferably of 7.2 to 8.2.

The paste and the basic solution are mixed in a solid/liquid ratio of 1 to 4, preferably of 1 to 3. The protein paste is then shredded to a small particle size (few millimeters) so as to simplify protein dissolution process with the solvent.

If necessary, the protein paste can be homogenized before dissolution.

Dissolution is obtained by stirring the system proteins/solvent for a time of 1 to 4 hours, preferably of 1.5 to 3 hours (time necessary for complete re- suspension of protein paste) .

At the end of dissolution a colloidal suspension is obtained, to which polyethylene glycol (PEG) with a molecular weight of 400 and 6,000 Da can be added if necessary.

Polyethylene glycol can coordinate water molecule so as to reduce evaporation during the drying step, and it is added in an amount of 20 to 60%, preferably of 30 to 50%, of the protein content of the colloidal so- lution .

Polyethylene glycol interacts with water molecules and gives the solid structure a higher deformability without breaks, as shown in Figure 3.

In an alternative embodiment, at least one biomateri- al, e.g. collagen and/or hyaluronic acid, is further added.

In a preferred embodiment, said at least one bioma- terial is used as a gel or powder.

Preferably, collagen or hyaluronic acid in powder form obtained by grinding collagen or hyaluronic acid felts is used. The biomaterial powder (preferably collagen and/or hyaluronic acid) is added with physiological solution up to balance with water. Then, after removing the supernatant, the biomaterial gel particles thus obtained are added to the solution of fraction I, preferably in an amount of 20 to 70% m/m. After adding if necessary polyethylene glycol and/or a biomaterial (preferably collagen and/or hyaluronic acid) to the colloidal solution, the latter is turned into foam by intense mechanical stirring, so as to increase the air-exposed surface and enable adsorption of the proteins at the interface liquid/gas. Mechanical stirring is executed for 5 to 15 minutes, preferably for 6 to 10 minutes, and anyhow until a clearly stable foam is obtained. The temperature of the solution is of 20 to 30⁰C, preferably 25°C. The foam thus obtained can be added with a solution of a reticulating agent which is able to bind two amine groups of the protein, acting as intermolecular cross- linker. The addition of a reticulation agent enhances the mechanical properties of the material. The reticulating agent is preferably added while the foam is still under stirring, short before the drying step. Thus, foam is homogenized.

The reticulating agent used is an aldehyde, preferably ^• glutaraldehyde .

The amount of reticulating agent to be added to the foam is of 5 to -40% v/v, preferably 10 to 20% v/v. The reticulating agent is advantageously used in aqueous solutions at concentrations of 0.00001 to 0.00005% of reticulating agent.

The addition of this compound should be executed short before the drying step so as to avoid premature formation of intermolecular links. The drying step occurs at a temperature of 60 to 98⁰C, preferably of 70 to 95°C, for a time ranging from 10 to 40 hours, preferably from 15 to 25 hours. It is thus possible to remove both the alcohol, which volatilizes at lower temperatures, and water, which requires higher temperatures and times to be completely eliminated.

In a preferred embodiment, before the drying step, a pre-heating (or stabilization) step is carried out, preferably in a microwave oven.

This is needed to avoid foam collapse as a result of viscosity lowering with increasing temperature, and subsequent separation of draining liquid.

Irradiation with microwaves has two effects: it flattens temperature profile inside the foam; it causes a rapid thermal denaturation of proteins, turning the liquid foam into a meta-stable gelatinous foam.

Pre-heating is executed for the time required to rapidly reach a temperature of about 80⁰C inside the foam to be dried.

After drying the end product is obtained, i.e. the biomaterial which in the preferred case is a haemostatic sponge.

The biomaterial thus obtained is advantageously packaged with a hermetic polymeric package (preferably made of polyethylene) and subjected to sterilization with radiation.

The biomaterial is advantageously irradiated with a dose of gamma or beta sterilizing radiations in the range from 10 to 40 kGy, preferably from 20 to 30 kGy. This treatment ensures the reduction of. bacterial charge and of most viruses, which are also very sensitive to the thermal treatment in an oven, which the biomaterial is subjected to even before the treatment with radiations.

An alternative embodiment makes use of a scaffold of at least one biomaterial, preferably collagen and/or hyaluronic acid, which is added with at least one waste fraction from a human plasma fractionation process, in particular from modified Conn' s process. Thus, a solid scaffold of at least one biomaterial imbibed with at least one waste fraction from a human plasma fractionation process is obtained. The fraction which is preferably used is FRACTION I, FRACTION III or FRACTION IV 1-4, preferably FRACTION I. The scaffold thus imbibed can be further added with polyethylene glycol.

The scaffold thus treated is dried (as described above) so as to obtain a biomaterial comprising hyaluronic acid and/or collagen and a protein waste fraction of the human plasma fractionation prpcess. The drying step can follow a pre-heating step and be executed 'before packaging and irradiation, as described above. The biomaterial obtained from the process of the in- vention can stop bleeding caused by injuries, i.e. it has haemostatic properties.

The biomaterial according to the invention has also proved useful as a scaffold for cell cultures in tissue engineering applications.

A preferred object of the present invention is therefore a haemostatic biomaterial, in particular a haemostatic sponge, obtained from FRACTION I, from other waste fractions of the human plasma fractionation process or from whole, untreated plasma, if necessary mixed with a biomaterial, preferably collagen and/or hyaluronic acid. Experimental part

Example 1. Raw material to be used (see Figure 1) . All waste fractions from plasma fractionation process can be used, including whole, untreated plasma. Some of these require a few preliminary operations such as microfiltration in order to remove solid celite particles from Fraction 4. The best results were achieved using Fraction 1 and the following descriptions refer to such raw material.

Example 2. Dissolution of proteins constituting Fraction 1 (see Figure 2) .

In order to obtain the colloidal solution or suspension of proteins it is necessary to use an aqueous solvent characterized by a suitable pH and ionic force. It was observed that solubility expressed as percentage of dissolved proteins varies as a function of solvent pH as shown below. Ratio solvent/paste 1.5 (m/m) . Temperature 25°C. System stirring time 1 hour.

Dissolution pH - yield %

5.0 21.3

6.0 24.7

7.0 27.1

8.0 83.6

^■ 9.0 72.7

For the following operation a solution of 2% sodium bicarbonate (m/v) in distilled water was chosen as solvent. It was observed that the dissolution yield expressed as percentage of dissolved proteins after 3 hours of system stirring varies as a function of the ratio r of the solvent volume to the mass of the protein paste (v/m) as shown below. Temperature 25°C. r (ml/g) Dissolution yield %

1.5 41.7

3.0 57.9

5.0 72.9

10.0 100.0

The solution of the proteins constituting Fraction 1 was prepared using as solvent the 2% solution of sodium bicarbonate NaHCO₃. Dissolution was carried out at 25⁰C stirring the system for a time t = 3 hours. A ratio solvent/protein paste r = 2.5 ml/g was chosen. At the end of the dissolution process, the colloidal protein suspension was added with polyethylene glycol (PEG) with a molecular weight of 400 Da. The amount of added PEG expressed in ml corresponds to 40% of the mass of dissolved protein, i.e. 11 ml for 100 ml of solution for a Fraction 1 paste containing 28% of proteins .

Example 3. Homogenization of the system proteins/solvent (see Figure 2) .

Considering that the dissolution process occurs at the interface paste-solvent and a relevant factor is the specific surface of the paste, it was thought to carry out a homogenization step before actual dissolution, all the other experimental conditions being unchanged. The following table shows a comparison between the dissolution yield obtained with dissolution only of gross fragments of protein paste and the yield obtained with an additional homogenization step. The results were obtained by determining the dry weight of the solution. Test conditions: temperature T = 25°C. Solvent: H₂O + 2% NaHCO₃. Dissolution time = 3h. Stirring speed = 500 rpm. r = ratio of solvent volume to protein paste mass.

The solution of proteins constituting Fraction 1 was prepared using as solvent the 2% solution of sodium bicarbonate NaHCO₃ and with a ratio solvent/protein¹ paste r = 2.5 ml/g. In order to help dissolution, the system paste/solvent was homogenized until a sub-

__ J i i J — _ . tained. Dissolution was carried out at 25°C stirring the system for a time t = 3 hours.

Example 4. Transformation of the protein solution into foam (see Figure 2) .

After adding polyethylene glycol to the colloidal solution, the latter is turned into foam by an intense mechanical stirring. Mechanical stirring was carried out for a time t = 7 minutes until a clearly stable foam was obtained. Solution temperature T = 25°C. As representative quantity of the foaming ability of the colloidal solution, obtained after subjecting the protein paste to homogenization and dissolution, percentage overrun was monitored, i.e. foam volume increase versus initial solution volume. The following values were obtained for different ratios of solvent volume to protein paste mass.

Ratio solvent/protein paste (ml/g) Overrun %

2.0 295

2.5 339

3.0 391

4.0 505

Since foams are a meta-stable system and tend to drain liquid, the drainage of the liquid phase from the foams obtained with different ratios solvent/orotein paste was monitored. As reference quantity for determining the best ratio solvent/paste in order to ensure lower protein losses from the finished sponge, the percentage of proteins left in the dry foam at the end of heat treatment was chosen. It was observed that the drainage phenomenon increases with higher ratios solvent/protein paste, as shown in the following table.

Ratio solvent/protein % of proteins in paste (w/w) dry foam

2 . 0 71 . 41

2 . 5 51 . 20

3 . 0 49 . 87 4_^1O 32 . 29

Experimental data obtained demonstrate that the foam resists the more the heat treatment the more the starting colloidal solution is concentrated. The phenomenon of liquid phase drainage shows an inverse development with respect to foam yield, i.e. drainage increases with an increasing foam volume with respect to initial solution volume.

As the best compromise between these two phenomena, a ratio solvent/protein paste of 2.5 ml/g was chosen. Example 5. Addition of glutaraldehyde (see Figure 2) . This reticulating agent can be added to dissolved pro- of the finished foam. While testing the mechanical properties of the finished pads with the compression test, pads both made of weakly reticulated proteins and not were examined. Also biocompatibility tests with in-vitro cell cultures were carried out both on reticulated and non-reticulated pads. The glutaralde- hyde solution was added to the foam at the end of the formation thereof (after 7 minutes stirring) in an amount of 10% v/v of the initial protein solution volume. The concentration of the aqueous glutaraldehyde solution added is of 0.000025% v/v.

Example 6. Transfer of foam into moulds (see Figure 2) -

After a stable foam was formed and, if necessary, glutaraldehyde was added, the protein foam was placed into moulds in which the drying process was to be carried out. Moulds used were shaped as parallelepipeds with the following side ratios: 1:2.5:5 and their walls were perforated in order to ease steam discharge during the drying process.

Example 7. Microwave pre-heating (see Figure 2) . In order to reduce the phenomena of liquid phase drainage from the foam during the drying step, the foam contained in the moulds was rapidly pre-heated. Preheating was carried out using microwaves with a double goal: flattening the temperature profile within the foam and causing a rapid thermal denaturation of proteins, turning the liquid foam into a meta-stable gelatinous foam. Microwave pre-heating was carried out until a temperature of 80⁰C was reached at the center of the foam mass.

Example 8. Oven drying (see Figure 2) .

After microwave pre-heating the moulds containing moist foam were transferred to a temperature- controlled, ventilated oven. Drying was carried out at a temperature of 95⁰C for a time of 12 hours. The aim of this treatment, beyond water discharge, is viral deactivation of strains potentially present in human plasma.

Example 9. Fad packaging (see Figure 2) . Pads were cut from the masse of dried foam and then placed into hermetic polyethylene bags, closed by heat sealing .

Example 10. Final sterilization (see Figure 2). The pads closed inside the heat-sealed blisters were subjected to sterilization of a dose of 2'5 kGy of gamma radiation.

Example 11. Mechanical compression tests (see Figure 3) . Mechanical compression tests were performed on 2x2x2 cubes cut from the sponge. Tests aimed both at determining the behavior of the material subjected to deformations, and at verifying the influence of reticulation with glutaraldehyde and of gamma radiation on the mechanical properties of the material. Tests were performed following a factorial pattern with two factors on two levels. Factor A = reticulation with glu;- taraldehyde (the first level without addition of glutaraldehyde or in short -GA, and the second level with glutaraldehyde added as indicated in Example 5 and referred to as +GA) . The effect of radiation, referred to as factor B, was investigated on two levels (the first level without sterilization, referred to as -γ, and the second level with a dose 25 kGy of applied gamma radiation, referred to as +γ) . Tests were carried out twice using dynamometer ZWICK BZ2.5/TN1S at a deformation speed of 4 mm/minute. Compression tests were performed until a relative deformation of 80% was achieved.

The most relevant information that can be inferred from these tests is the high deformability of all tested samples, the preservation of their integrity despite the high deformations applied, and the recovery of initial size at the end of the compression test. From a qualitative point of view, all the pro- files stress/deformation obtained from the tests are as indicated in Figure 3.

In order to emphasize the effect of reticulation with glutaraldehyde and of gamma sterilization on the mechanical properties of the pads, only load values corresponding to the maximum deformation of 80% are listed. Other properties such as elastic modulus, strength and yield point show a similar relation with reticulation and radiation treatment.

Load values corresponding to 80% deformation The analysis of variance of the results enables to state that the main effects of reticulation and radiation are estimated at a level of significance p>99%.

Figure 4 shows the average development of load corresponding to a deformation of 80% as a function of the treatment with glutaraldehyde and with radiation. Example 12. In-vitro biocompatibility tests (see Figure 5) .

In order to verify the biocompatibility of the material obtained with the procedures described in the examples above, cell cultures of human fibroblasts were carried out in presence of fragments of pads treated with glutaraldehyde (tests referred to as +GA) und not treated with glutaraldehyde (referred to as -GA) . The pads used for the tests were sterilized with a dose of 25 kGy of gamma radiation. The cell line used for experimentation consists of human fibroblasts (intestinal submucosa) proliferating spontaneously in presence

-; +- =κi ,1 -1-1 -,^i becco's Modified Minimum Essential Medium) with the addition of 10% FCS (fetal calf serum) , antibiotics (Streptomycin, Penicillin) , Amphoterycin B (as anti- mycotic agent) and L-Glutamine as essential amino acid. The cells were inoculated in Petri dishes (type Nunclon 174926) equipped with a suitable grid on the bottom in^' order to ease cell count, in an amount corresponding to an inoculation density of 2xlO⁴ cells/cm³ and incubated for 3, 7, 14 and 21 days at 37⁰C in controlled humidity atmosphere containing 5% CO₂. On the pre-established times dyeing (May- Grumvald/Giemsa' s method) and cell count were executed on the cell single layer adhering to the plate bottom. Counting results are shown below.

Figure 7 summarizes the results of these counts as growth curves which show the biocompatibility of the pads obtained both without reticulation with glutaral- dehyde and with materials reticulated with the latter. After 21 days from the inoculation, cells adhering to the pad surface were found, which demonstrates not only the biocompatibility of the material, but also that this can be used as scaffold for tissue engineering applications .

Example 13. In-vivo control of haemostatic properties of the new biomaterial (see Figure 9) .

Since there are no reference guidelines for the control of haemostatic properties of a medical device, the performance of four haemostatic pads were compared with each other: the new pad obtained from waste fractions of human plasma fractionation, and common haemostatic devices used in surgery: cotton gauze, regenerated cellulose gauze and bovine gelatin sponge. Tests were performed on male adult Wistar rats weighing 250-300 g, supplied by Harlan-Nossan, measuring the time from injury incision to haemorrhage stop. Before starting the tests, all the animals were anesthetized with 2 ml i.p. of Hydrate Chloralium (3.6 g/100 ml in physiological solution) . Tests were carried out on groups of 4 animals on which the pad of cotton gauze was applied on the left and the haemostatic pad (the new pad, the regenerated cellulose gauze and the bovine gelatin sponge) on the right, both applied on the injury of small vein, medium vein and liver incision, for a total of 6 tests on each animal as schematically shown in Figure 10. The results of these measures (72 in total) were subjected to an analysis of variance, which showed that the level of significance of the influence of the main factor "haemostatic material" is p>95%.

A second group of rats was pre-treated with a heparin dose (2000 IU/kg) . In heparin tests the gauze was not taken into account, since it is not suitable for stopping bleeding injuries in such extreme conditions. The test pattern is shown in Figure 10, where the new bio- material was applied on the left side of the animal in the same place as the cotton gauze, whereas on the right size commercial haemostatic pads were applied (regenerated cellulose gauze and bovine gelatin sponge) , still _.in groups of 4 animals for a total of 48 assays. Also these measures were subjected to an analysis of variance, which showed that the level of significance of the influence of the main factor "haemostatic material" is p>95%. In both groups of animals it was determined on a sta- tistical basis that the average haemorrhage time is halved by using the new biomaterial in the same place as haemostatic devices available on the market. Example 14. Modification of a haemostatic pad comprising collagen by addition of waste proteins from human plasma processing (see Figure 13) .

A haemostatic collagen pad available on the market was used. The pad was imbibed with the solution of Fraction 1 obtained as described in examples 2 and 3. Then the material was subjected to processing as described in examples 7, 8, 9 and 10.

Example 15. Haemostatic pad obtained from powder collagen added to waste proteins from human plasma processing (see Figure 14) .

A collagen in powder form was used, obtained by grinding felts as referred to in example 14. The powders were covered with physiological solution (0,9% NaCl in H₂O) and balanced with water. Then, after removing the supernatant, the collagen gel particles were added to the solution of fraction 1 obtained as described in examples 2 and 3, in an amount of 50% m/m. The collagen particle suspension was subjected to processing as described in examples 4, 5, 6, 7, 8, 9 and 10.

Claims

1. A process for preparing a biomaterial comprising the following steps: a) mixing a waste protein paste obtained from a human plasma process or ex vivo whole plasma with a basic aqueous solution; b) if necessary, homogenizing the mixture protein paste/basic solution; c) dissolving the homogenized mixture by mechanical stirring so as to obtain a colloidal suspension; d) optionally, adding to the colloidal suspension polyethylene glycol and/or at least one biomaterial; e) turning the solution of step d) into foam by intense mechanical stirring; f) optionally, adding a reticulating agent; g) subjecting the foam thus obtained to drying so as to obtain a sponge-like biomaterial.

2. The process according to claim 1, wherein said sponge-like biomaterial is a haemostatic sponge.

3. The process according to claim 1 or 2, wherein said at least one biomaterial is collagen and/or hyaluronic acid.

4. The process according to claim 1, 2 or 3, wherein waste protein paste is a protein by-product of modified Conn' s human plasma fractionation process.

5. The process according to any one of the claims 1 to 4, wherein said waste protein paste is waste FRACTION I, FRACTION III or FRACTION IV 1-4 of modified Cohn' s human plasma fractionation process.

6. The process according to any one of the claims 1 to 5, wherein said waste protein paste comprises 10 to 40% of proteins dispersed in water.

7. The process according to claim 6, wherein said proteins are: albumin, α-globulins, β-globulins, γ- globulins and fibrinogen.

8. The process according to any one of the claims 1 to 7, wherein said basic solution is sodium bicarbonate and pH is kept at a value ranging from 7 to 9.

9. The process according to any one of the claims 1 to 8, wherein said homogenization is carried out for a time ranging from 20 to 60 seconds, preferably from 25 to 40 seconds.

10. The process according to any one of the claims 1 to 9, wherein said dissolution is carried out for a time ranging from 1 to 4 hours, preferably from 1.5 to 3 hours.

11. The process according to any one of the claims 1 to 10, wherein said polyethylene glycol is added in an amount of 20 to 60%, preferably of 30 to 50% of the protein content of the colloidal solution.

12. The process according to any one of the claims 1 to 11, wherein said transformation of the liquid into foam is carried out for a time ranging from 5 to 15 minutes, preferably from 6 to 10 minutes.

13. The process according to any one of the claims 1 to 12, wherein said reticulating agent is added in an amount of 5 to 40% v/v, preferably of 10 to 20% v/v.

14. The process according to any one of the claims 1 to 13, wherein said reticulating agent is added during the step of transformation of the solution into foam.

15. The process according to any one of the claims 1 to 14, wherein before the drying step a pre-heating step is performed, preferably in a microwave oven.

16. The process according to claim 15, wherein said pre-heating is carried out for the time required to reach inside the foam a temperature of 60 to 95⁰C, preferably of 80⁰C.

17. The process according to any one of the claims 1 to 16, wherein said drying occurs at a temperature of 60 to 90⁰C, preferably of 70 to 80°C, for a time ranging from 1 to 4 hours, preferably from 1.5 to 3 hours; temperature is then increased to values ranging from 85 to 98⁰C for a time ranging from 10 to 40 hours, preferably from 15 to 25 hours.

18. The process according to any one of the claims 1 to 17, wherein after the drying step the haemostatic biomaterial is subjected to irradiation with gamma or beta radiation.

19. The process according to any one of the claims 1 to 18, wherein a scaffold of at least one biomaterial, preferably collagen and/or hyaluronic acid, is soaked with at least a waste protein paste and, optionally, also with polyethylene glycol.

20. The process according to claim 19, wherein said soaked scaffold is subjected to the drying step according to claim 1 and, optionally, to the pre-heating and irradiations steps according to claims 15 and 18.

21. A biomaterial obtained from a waste protein paste of a human plasma fractionation process.

22. The biomaterial according to claim 21, comprising a biomaterial, preferably collagen and /or hyaluronic acid.

23. The biomaterial according to claim 21 or 22, wherein said waste protein paste is obtained from a human plasma fractionation process comprising the fol- lowing steps: i) subjecting human plasma to centrifugation, thus obtaining a precipitate (Cryo precipitate) and a solution referred to as Conn' s plasma; ii) adding to Conn' s plasma ethyl alcohol at a concen- tration of 5 to 10% v/v; iii) centrifuging the solution of step ii) obtaining said waste protein paste and a supernatant .__

24. The biomaterial according to any one of the claims 21 to 23, obtainable by the process according to any one of the claims 1 to 20.

25. The biomaterial according to any one of the claims 21 to 24 for use as a medicament for slowing and/or stopping blood haemorrhages deriving from external injuries or from internal injuries due to surgery.

26. The biomaterial according to any one of the claims 21 to 24 for use as a scaffold for cell culture and for tissue engineering applications.