FR2600897A1 - Proteolytic and absorbent dressing and process for its preparation - Google Patents

Abstract

A new proteolytic and absorbent dressing and process for its preparation. The dressing according to the invention is characterised in that it comprises a support based on a starch derivative onto which trypsin is grafted by means of a chemical bond.

Description

 The present invention relates to a proteolytic and absorbent dressing for wounds and its method of preparation.

 If, for millennia, man has been interested in wound healing and if today many works are still devoted to this subject, it turns out that each of the methods of care already advocated has gaps and / or shortcomings as to the effectiveness obtained.

 One of the first methods of care of antiquity was the use of honey dressing for burns, frostbite, small wounds and ulcers. The oxygenated water produced by the action of glucose oxidase honey stimulates the formation of granulation tissue at the beginning of treatment and sometimes reduces local pain.

 Sugar is also again widely used. When applied to a wound, it dissolves and creates an environment where water activity is low, which inhibits bacterial growth.

Thus, after surgical debridement, the application of sugar on infected wounds causes a change in the odor of the latter and a disappearance of secretions in 72 to 96 hours.

 In fact, the coating according to the present invention provides all the advantages of the above methods, namely, granulation tissue formation, pain reduction, deodorization and inhibition of bacterial growth.

 But in addition, the dressing according to the present invention aims to promote the healing of the lesion much more than did traditional materials.

 Indeed, said proteolytic and wound absorbent dressing is characterized in that it comprises a support based on a starch derivative on which is grafted, via a chemical bond, trypsin.

 The term "dressing" will designate throughout the description an object of any shape and constitution, intended to be applied to a wound.

 The dressing according to the present invention allows the lysis of proteins, or proteolysis. It is indeed a matter of rapidly degrading the devitalised and necrotic tissues by means of trypsin, a pancreatic enzyme known to accelerate the natural autolysis of proteins.

 This enzymatic wound debridement will be particularly useful as a complement to the surgical treatment of necrotic and inert lesions, mainly burns, whose healing is facilitated by rapid degradation of necrotic tissue.

 According to the present invention, the trypsin is grafted by a chemical bond on a starch derivative whose high absorption capacity makes it possible to rapidly remove the exudates or excess of secretion of the wound.

Starch is a polymer, or better, a polycondensed glucose, in which the glucopyranose alpha radicals ("boat-shaped" rings) form chains where they are united by the reducing function and the alcoholic oxhydryl at position 4. It there is no other mode of binding in straight-chain linear starch, whereas a second type is found in the so-called branched starch, where the branches are made by an alpha glucosidic bond from the oxhydryl of the primary alcohol in position 6:

 Preferably, according to the present invention, the chemical bond is carried out on the carbon atoms 2 and 3 of the glucopyranosic moieties, namely the carbon atoms carrying the hydroxy functions.

dans lequel
G représente le glutaraldéhyde,
T représente la trypsine, et
n est un nombre entier compris entre 1 et 4, de préférence égal
à 2.In particular, the bond can be established on each of said carbon atoms 2 and 3, via the grouping

in which
G represents glutaraldehyde,
T represents trypsin, and
n is an integer between 1 and 4, preferably equal
to 2.

 The support preferably used according to the present invention is the starch-acrylonitrile copolymer whose cyano groups are partially hydrolyzed to amide and sodium (or potassium) carboxylate. This starch copolymer, when hydrolysed and evaporated to dryness, has an absorption capacity which is particularly effective with distilled water. It is in the form of a light brown powder, very hygroscopic, whose structure is defined by its method of production.

 The number of base units and bonds of this copolymer remains undetermined.

dans laquelle
n, m, x et z sont des facteurs indépendants les uns des autres et varient de O à l'infini.It can be represented by the following formula I

in which
n, m, x and z are independent of each other and vary from 0 to infinity.

The copolymer can be synthesized by various techniques known to those skilled in the art. In particular, the copolymerization can be carried out by reacting starch, such as corn starch, with acrylonitrile in water in the presence of a catalyst, such as a salt of series, which makes it possible to generate free radicals. Polyacrylonitrile chains are formed at these free radicals. It is then sufficient to saponify the copolymer thus formed by hydrolysis in an alkali such as potassium hydroxide or sodium hydroxide and to dry it under vacuum or by any other suitable method known to those skilled in the art. following reaction scheme illustrates a mode of preparation of the starch-acrylonitrile copolymer

The absorbent and proteolytic dressing according to the present invention is prepared by the following successive steps
a) the OH functions carried by the carbon atoms 2 are oxidized
and 3 glucopyranosic moieties of the starch derivative
With the help of a salt of metaperiodic acid, it will be preferable
sodium metaperiodate
b) rinsed with the pyrophosphate buffer and recovered by
filtration the product from step a), it will be preferable
of vacuum filtration on a frit of porosity
four
c) the oxidized functions are aminated during step a) using
of an alkylenediamine in aqueous solution, it will be preferred
ence of ethylenediamine
d) rinsed with the pyrophosphate buffer and recovered by
filtration the product from step c), the filtration will be
preferably a vacuum filtration on a porosity frit
adequate
e) the amino functions are activated during step c) by
putting in contact with a solution of glutaraldehyde in
pyrophosphate buffer, this activation will be preferred
at a slightly basic pH
f) the excess glutaraldehyde is removed by repeated washing of the
product from step e) with the pyrophosphate buffer,
will preferably renew these washes until - de-sm-Hw
giant gives a negative reaction to an aldehyde detection test
g) the functions activated during step e) are grafted with a
trypsin solution in pyrophosphate buffer, this grafting
preferably at a slightly basic pH, and
h) the non-grafted trypsin is removed by repeated washing of the
product from step (g) with pyrophosphate buffer then
sodium chloride.

Trypsin can have various origins. In the following detailed examples, SIGMA trypsin type IX is used which is extracted from porcine pancreas and contains 1-5% calcium acetate. It is preferably according to the present invention, dissolved in 0.05 M sodium pyrophosphate buffer at pH 8.6. The preferred immobilization method according to the present invention (FIG. 1) is the following
A sample of 500 mg of support is oxidized with 100 ml of sodium metaperiodate (MERCK) and then aminated with ethylene diamine (FLUKA), dissolved in water. The volume occupied by the support being much lower in the pyrophosphate buffer than in distilled water, the buffer was used to rinse the support between each step. The support is recovered by filtration under vacuum on a frit with adequate porosity.

 The activated support is contacted with 100 ml of glutaraldehyde (25% MERCK) in 0.05 M sodium pyrophosphate buffer, pH 8.6.

 The excess glutaraldehyde is removed by repeated washing with 100 ml of the same buffer, until the supernatant gives a negative reaction to the Schiff reagent (aldehyde detection) test.

 The activated support is then either re-rinsed with pyrophosphate buffer to serve as a control, or contacted with 100 ml of trypsin solution in 0.05 M pyrophosphate buffer (PROLABO) pH 8.6.

 The unbound trypsin is removed by repeated washing with the same pyrophosphate buffer and then 1M sodium chloride (PROLABO).

 The dressing thus obtained is in the form of dry particles. Preferably, according to the present invention, it is partially moistened until a gel is obtained.

 The dressings may be applied as such to the wound, in the form of powder or gel. They may also include another application material such as gauze, cotton, tape or plaster.

 The present invention will be better understood on reading the following examples and with the aid of the appended figures.

 Figure 1 shows the dressing preparation method. In this figure: a) corresponds to the oxidation by NaIO4, c) represents the amination by H2N- (CH2) 2 -NH2, e) represents the activation by glutaraldehyde (G) of formula: OHC- (CH2) 3-CHO and g) corresponds to the trypsin binding step).

FIG. 2 represents the evolution of the activity per gram of support and of the immobilization yield, as a function of the quantity of trypsin introduced, under the following conditions
Sodium metaperiodate: 2 mM, 10 min. at 20 C;
Ethylenediamine: 5 mM, 10 min. at 20 C
Glutaraldehyde: 2.5%, 6 h to 20 C
Trypsin: 12 h to 40C.

FIG. 3 represents the evolution of the activity of immobilized trypsin per gram of dry support, as a function of the concentration of ethylenediamine, at various temperatures, under the following conditions
Sodium metaperiodate: 5 mM, 10 min. at 20 C;
Ethylenediamine: 10 min .;
Glutaraldehyde: 2.5%, 6 hrs. 20 C
Trypsin: 19900 U / g support, 12 h to 4 C.

FIG. 4 represents the influence of the duration of contact on the activity per gram of dry support, for different initial amounts of trypsin, under the following conditions
Sodium metaperiodate: 5 mM, 10 min. at 200C
Ethylenediamine: 100 mM, 10 min. at 20 C
Glutaraldehyde: 2.5%, 1 hour at 200C
Trypsin: +4 C.

FIG. 5 represents the evolution of the immobilization yield as a function of the initial quantity of trypsin, for different contact times, under the following conditions
Sodium metaperiodate: 5mM, 10 min. at 20 C
Ethylenediamine: 100 mM, 10 min. at 20 C
Glutaraldehyde: 2.5%, 1 hr at 20 ° C
Trypsin: +4 C.

Methods used
A / Measurement of enzymatic activity
The enzymatic activity is then measured at pH-stat using a synthetic trypsin substrate: N-Benzoyl-L-Arginine
Ethyl Ester (BAEE) (LASKOWSKI, 1955, WALSH and WILCOX, 1970).

 Trypsin SIGMA type IX corresponds. at 14900 U from B.A.E.E.E.

per mg of protein. 1 unit B.A.E.E.E.E. causes an absorbance change at 253 nm of 0.001 min at pH 7.6; 25 ° C in 3.2 ml.

 B.A.E.E.E.E. (SIGMA and BOEHRINGER). is in solution in 50 mM Tris-HCl buffer and 20 mM CaCl 2; pH 8.0. The concentration is 100 mM.

* Principle of measurement
The enzymatic activity is determined by measuring the initial rate of the reaction which corresponds to the amount of protons released.

 indeed, trypsin, like all proteases, hydrolyzes the peptide bonds of the substrate (here the BAEE) and releases protons in the reaction medium. The resulting decrease in pH is compensated by the continuous addition of a precise volume of previously titrated sodium hydroxide. The pH is thus kept constant throughout the hydrolysis.

 The volume of soda added allows access to the concentration of protons released. The equipment used is a TACUSSEL pH-stat consisting of a pin81 pH meter, a TTS titrator and an electronic burette. The temperature of the reaction mixture is ensured by a thermostatic bath.

* Determination of enzymatic activity
The enzymatic activity is determined by introducing the support on which the trypsin is immobilized in a thermostated cell provided with a stirring system and into which the electrode of the pH meter is immersed. The reaction is bubbled with nitrogen in order to avoid carbon dioxide interference. When the thermal equilibrium is reached, 10 ml of BAEE also thermostated and whose pH has been adjusted to that of the set point (8.0). The volume of soda poured is measured for ten minutes by a recorder connected to the automatic burette.

* Calculation
Unit: unit = 1 pmol of NaOH poured / min.

slope at origin = #y (ml.min-1)
NN: normality of soda
AA: enzymatic activity expressed in moles of
soda poured - per minute
A - x N x 0 03 (pmol.min -1) = U #x
mm: dry mass of support on which activity A
has been measured.This mass is obtained by
washing and lyophilization of the measured sample
A5: immobilized enzymatic activity per gram
dry support
A = A (pmoles.min ~ l .g = Ug l sec)
m
B / Capital Performance Calculation
The capital return R is the ratio

A: Immobilized enzyme activity (U / g) per gram of dry support
M: Total mass of support at the end of manipulation (g) A1: Activity introduced: number of units contained in the volume of
enzymatic solution brought into contact with activated support
AL: Recovered activity: number of units collected during
different washes.

C1 Debugging the immobilization protocol
The dressing development protocol was realized
following a factorial design plan that allows to vary the
experimental factors acting on the reactions and to estimate the significance
effect on the intended response.

Appearance and behavior of the support
The final batch of support is in the aspect of
particles of irregular granulometry of light color. Wet, they
do not form a homogeneous gel. The hydrated particles remain
individual, regardless of size.

The absorption capacities of the support are large and vary
important according to the nature of the liquid absorbed. This
change in absorbed volume appears to be due to an effect of salts.

Once lyophilized, the support is quickly hydrated and
absorbed.

Before starting the optimization of the various stages of the
immobilization process, the development of the experimental protocol
has been deepened. In particular, the evolution of the desorption of
trypsin was studied, as well as the influence of the concentration of
zyme introduced on activity and performance.

These tests were carried out by choosing concentrations
and arbitrary but identical contact times from one manipulation to the other, in order to be able to compare the results.

* Influence of washes on desorption of non-trypsin
immobilized
The purpose of this study is to determine the minimum number
washing after bringing the activated support into contact with the trypsin,
so that all ltenzs me not immobilized by covalent bond be
eliminated.

 For this, the enzymatic activity was measured after various washings, on the support and in the "washing supernatant" in order to calculate the immobilization efficiency.

 These measurements were made during two fixed assets, one of which served as a witness. For this tem- perature manipulation, the glu taraldehyde was replaced by 0.05 M pyrophosphate buffer, pH 8.6.

The results obtained are shown in the table below.
TABLE l
Evolution of the enzymatic activity measured on the support as a function of the number of washes.

<Tb>

<SEP> Activity <SEP> (U / g <SEP> support <SEP> sec)
<tb><SEP> Step <SEP> Witness <SEP> Test
<tb><SEP> Withdrawal <SEP> Enzyme <SEP> 1961 <SEP> 2404
<tb><SEP> 1 <SEP> wash <SEP> pyrophosphate <SEP> 487 <SEP> 652
<tb><SEP> 2nd <SEP> wash <SEP> pyrophosphate <SEP> 94 <SEP> 185
<tb><SEP> 1 <SEP> wash <SEP> NaCI <SEP> 21 <SEP>;<SEP> 140
<tb><SEP> 2nd <SEP> wash <SEP> NaCI <SEP> 25149 <SEP>
<tb><SEP> 3rd <SEP> wash <SEP> NaCl <SEP> 20 <SEP> 125
<tb> Immobilization efficiency <SEP><SEP> 0.1 <SEP>% <SEP> 4.3 <SEP>%
<Tb>
As the results obtained during the two manipulations clearly show, the trypsin which is not immobilized by covalent bonding is rapidly desorbed during the washes.

The measured activity evolved substantially more after two washes with 0.05 M pyrophosphate buffer, pH 8.6 and one wash at
NaCl 1M.

 For the control manipulation, only 20 U / g dry support remains. This corresponds to the trypsin included in the support gel. This value is sufficiently low, compared to the activity measured during immobilization, to be neglected.

 After the support has been washed twice with pyrophosphate buffer, a single 1M NaCl wash is sufficient to desorb the non-immobilized trypsin.

* Influence of the amount of trypsin
Figure 2 shows that the variation of 990 to 20,000 in the number of trypsin units contacted with one gram of support is accompanied by a continuous - and significant - increase in activity per gram of dry support.

 Beyond this value, the phenomenon is less marked and for an initial concentration of enzyme five times higher (99 000 U / g), the activity is multiplied by a factor of 1.5.

 The immobilization efficiency, on the other hand, decreases when the concentration of trypsin increases.

The amount of enzyme used is set at 19800 U / g of support. This value is a good compromise between the amount of trypsin to be used and the activity per gram of dry support.

a) Relative importance and possible dependence of the different
steps
In the experimental design used, the factors are three in number: oxidation, amination and activation. The attachment of the enzyme is not taken into account because this step is independent of the activation of the support.

 The experience matrix allowed the calculation of the effects of the three aforementioned factors: main effects, first-order interaction effects and second-order interaction effects.

 Analysis of these effects concluded that the three steps: oxidation, amination and activation were dependent.

 This effect is particularly pronounced for oxidation and amination, which should therefore be optimized together.

 Glutaraldehyde activation is the most important step in the preparation of the support.

 During the optimization of the various stages, it is necessary to take into account the physicochemical behavior of the support, which must retain its gel properties.

b) Optimization of oxidation and amination
The experiment matrix made it possible to highlight the influence exerted by the three factors studied on the oxidation and amination steps. The most pronounced effect is due to the concentrations of reagents whose increase causes an increase in the activity of the immobilized enzyme.

 The duration and the contact temperature exert effects of the same order of magnitude. As the values of these factors increase, immobilized activity decreases.

 The factors studied are dependent, in particular the reagent concentrations and the duration of contact.

 To achieve maximum activity, temperatures should be low, contact times short and reagent concentrations as high as possible.

 The preceding manipulations make it possible to fix the values of certain parameters of the oxidation and amination steps.

 Contact times, which must be brief, are set at 10 minutes.

 The concentrations of sodium metaperiodate and ethylenediamine should be high.

 That of metaperiodate should be 5 mM because, all the other conditions being identical, for lower values, the activity is less and for higher concentrations, the support is degraded.

 Only the concentration of ethylenediamine remains to be determined, knowing that the optimum value is between 5 and 500 mM. Indeed, a higher concentration causes a loss of the gel properties of the support. Capital expenditures were made for concentrations of 5, 50,100, 250 and 500 mM.

 The temperature that gives the highest activities is + 40C. However, the manipulations were performed in duplicate, one series with oxidation and amination at + 40C and another with these steps at + 20 ° C, to better understand the effect of temperature.

 The conditions of the various fixed assets are presented in Table 2 below and the results in Figure 3.

Table 2
Conditions of immobilization

<tb><SEP> Step <SEP> Duration <SEP> Concentration
<tb><SEP> reagent
<tb> Oxidation <SEP> 10 <SEP> min. <SEP> 5 <SEP> mM
<tb> Amination <SEP> 10 <SEP> min. <SEP> 5-50-100-250-500mM
<tb> Activation <SEP> (20 "C) <SEP> 6 <SEP> h <SEP> 2.5 <SEP> 96 <SEP>
<tb> Immobilization <SEP> (4 "C) <SEP> 15 <SEP> h <SEP> 1 <SEP> no <SEP> mg / l
<Tb>
The activity per gram of dry support follows the same evolution as a function of the concentration of ethylenediamine, when the oxidation and the amination are carried out at +400C and +20 ° C. The curves show a fairly sharp break with the concentration 100 mM.

 Below this value, the activity increases strongly with the concentration of ethylenediamine, while beyond that, the increase is much slower.

 This break in the curve corresponds to a visual observation. Indeed, in tests where the concentration of ethylenediamine is greater than 100 mM, a yellow-orange color of the support occurs on contact with glutaraldehyde.

This coloration intensifies as the concentration increases.

 This effect is particularly marked for tests where oxidation and amination were performed at 200C. At this temperature, the amine support with ethylenediamine 250 and 500 mM has virtually lost its gel appearance.

 Oxidation and amination at + 40C lead to higher activity than when performed at 20 ° C (negative temperature effect), which decreases when the concentration of ethylenediamine becomes greater than 100. mu.

 This series of manipulations indicates that the optimum concentration of ethylenediamine is 100 mM.

 It remains to determine the oxidation and amination temperature.

When the concentration of ethylenediamine is 100 mM,
The activity is essentially the same (3 G of difference), at 20 ° C or 40C.

Conclusion
Successive steps made it possible to determine the optimal oxidation and amination conditions. These conditions are as follows: for 550 mg of support in a volume of 100 ml.

Oxidation
Sodium metaperiodate: 5 mM
Duration: 10 min.

Temperature: 200C
amination
Ethylenediamine: 100 mM
Duration: 10 min.

Temperature: 20 "C c) Optimization of glutaraldehyde activation
The experiment matrix shows that, in the glutaraldehyde activation step, the most important factor is the concentration, the effect of which is much greater than that of the duration and temperature of contact. These last two are of the same order of magnitude and the parameters they characterize are dependent.

 This series of manipulations makes it possible to know that the activation with glutaraldehyde can be carried out at 200C. The positive effect of the contact temperature is small and goes in the direction of a simplification of the manipulations.

 As a result, the concentration of glutaraldehyde must be high and the contact times rather long, for. that the activity of the immobilized enzyme is maximal.

 The activation temperature is thus set at 20 ° C. The concentration and the duration of contact remain to be determined, the first being between 0.5% and 6%, the second between 10 minutes and 6 hours. at acceptable limits given the experimental constraints and the mechanical behavior of the support.

 The tests were therefore performed for glutaraldehyde concentrations of 0.596; 1.25%; 2,596 and 596. In each case the contact times were: 10 min, 1 h, 3 h and 6 h.

 Trypsin is still used in the proportion of 19 900 U / g of support, the immobilization being carried out at +4 ° C. The activities obtained per gram of support in these various cases are shown in FIG.

 As the duration of contact increases, the maximum activity increases and is reached for lower and lower glutaraldehyde concentrations.

 For a contact time of 6 h, only the decreasing part of the curve, after the maximum activity was reached, could be described under the chosen conditions.

The values used for glutaraldehyde concentration
and the contact time are: 2.5% for 3 hours. Indeed, for
these values, the activity is high (2000 U / g of support) and is low
modified by small variations in glutaraldehyde concentration
hyde. The conditions 1.25 96 for 3 hours make it possible to obtain a
activity (2900 U / g of support) but as soon as
deviates a little bit from the concentration 1.25%, this activity decreases rapidly.

Conclusion
The conditions giving the best results chosen for the following manipulations are as follows
Glutaraldehyde: Concentration: 2.5%
Duration of contact: 1 hour
Contact temperature: 20 "C d) Optimization of immobilization of trypsin
The experimental design revealed the strong influence of the initial amount of trypsin on the activity.

 It also indicates that a low temperature is preferable.

 In the following tests, the immobilization of the trypsin will therefore be carried out at +4 ° C for a rather long time, the amount of trypsin contacted with one gram of support being high.

 During the development of the experimental immobilization protocol, the influence of the initial amount of trypsin per gram of support on the activity was demonstrated. Under conditions which had not yet been opltimized, activity reached a plateau when the amount of trypsin reached 19 900 U / g. Since the various steps have been optimized, the study has been repeated, but only for three quantities of enzyme. The chosen values: 9 900, 19 800 and 39 600 U / g of support are centered on the quantity determined during the preliminary study.

 In each case, the contact times are: 2h, 4h and 12h.

 During these various tests, the immobilization yields were also determined. The results obtained are shown in FIG.

 When we follow the evolution of the activity as a function of the contact time, it appears that for two quantities of trypsin out of three, the activity passes through a maximum and then decreases. This phenomenon is very low for 9 900 U / g of support. In the other two cases, the maximum activity reached is identical (1623 U / g sec and 1695 U / g) while the quantity of trypsin is multiplied by two (19 900 U introduced / g and 39 600 U introduced / g) .

Moreover, we find the phenomenon highlighted in the experimental plan: when the contact time becomes very large,
The activity decreases. In this series of tests, it was only 12 hours at most, and the effect is already noticeable.

 FIG. 7 shows that the immobilization efficiency also passes through maxima (25% and 28%) when the amount of trypsin introduced is 19 900 U / g of support, for 2 hours or 4 hours. For 12 hours of contact, the yield is much lower, regardless of the initial amount of trypsin.

Conclusion
From these two series of manipulations, it appears that the most favorable conditions for the immobilization of trypsin on the support are
Trypsin: Quantity introduced: 19 900 U / g of support
Duration of contact: 4 h
Contact temperature: +4 "C.

The best results were obtained under the following conditions: for 500 mg of support in a volume of 100 ml
Oxidation
Sodium metaperiodate 5 mM in aqueous solution for 10 min. at 20 "C.

amination
Ethylenediamine: 100 mM in aqueous solution for 10 min. at 20 C.

activation
Glutaraldehyde: 2.5% in solution in 0.05 M sodium pyrophosphate buffer, pH 8.6 for 1 h at 20 ° C.

immobilization
Trypsin introduced in the proportion of 19 800 U / g of support, for 4 hours at +4 ° C. The enzyme is in solution in 0.05 M sodium pyrophosphate buffer, pH 8.6.

 The activity of trypsin immobilized under these conditions is about 1600 U / g of dry support, with a yield of 26%.

Claims (11)

 1. Proteolytic and wound absorbent dressing, characterized in that it comprises a support based on a starch derivative on which is grafted, via a chemical bond, trypsin.  2. Dressing according to claim 1, characterized in that the chemical bond is established at carbon atoms 2 and 3 of glucopyranosic fragments of the starch derivative.  3. Dressing according to claim 2, characterized in that the carbon atoms 2 and 3 each carry a group of formula

 in which  G represents glutaraldehyde  T represents trypsin, and  n is an integer from 1 to 4.  4. Dressing according to claim 3, characterized in that n is equal to 2.  5. Dressing according to one of claims 1 to 4, characterized in that the carrier is a starch-acrylonitrile copolymer whose cyano groups are partially hydrolyzed to amide and sodium carboxylate.  6. Process for preparing the dressing according to one of claims I to 5, characterized in that it comprises the following steps  a) the OH functions carried by the carbon atoms 2 are oxidized  and 3 glucopyranosic moieties of the starch derivative  using a salt of metaperiodic acid  b) rinsed with the pyrophosphate buffer and recovered by  filtration the product resulting from step a)  c) the oxidized functions are aminated during step a) using  an alkylenediamine in aqueous solution;  d) rinsed with the pyrophosphate buffer and recovered by  filtration the product from step c)  e) the amino functions are activated during step c) by  putting in contact with a solution of glutaraldehyde in  pyrophosphate buffer  f) the excess glutaraldehyde is removed by repeated washing of the  product from step e) with the pyrophosphate buffer  g) the functions activated during step e) are grafted into  contacting with a solution of trypsin in  pyrophosphate buffer, and  h) the unbound trypsin is removed by repeated washing of the product  from step g) with pyrophosphate buffer, then chlo  sodium chloride.  7. Method according to claim 6, characterized in that for 500 mg of support is grafted with a quantity of trypsin of between 10,000 and 30,000 units / of support.  The process according to claim 7, characterized in that step a) is carried out with an aqueous solution of 5mM sodium metaperiodate for about 10 minutes at about 20 ° C.  9. Process according to claim 7, characterized in that step c) is carried out using an aqueous solution of 100 mM ethylenediamine for about 10 minutes at about 200C.  10. Process according to claim 7, characterized in that step e) is carried out using a solution of 2.5% glutaraldehyde in 0.15 M sodium pyrophosphate buffer at a pH of about 8.6 for about 1 hour and at an approximate temperature of 20 ° C.  11. Process according to claim 7, characterized in that step g) is carried out by introducing a trypsin solution in 0.05 M sodium pyrophosphate buffer at a pH of approximately 8.6 at 19.900 units / g of support for about 4 hours at a temperature of approximately 4 ° C.