USO09527929B2

(12) United States Patent (10) Patent No.: US 9,527,929 B2

Francois et al. (45) Date of Patent: Dec. 27, 2016

(54) OPTIMIZED CHITOSAN REACETYLATION 8,318,913 B2 11/2012 Bristow

8,507,563 B2 8/2013 Berger et al.
w 8,703,924 B2 4/2014 Andersson
(71) Applicant: Sofradim Production, Trévoux (FR) 8,899,277 B2 12/2014 Chiu et al.
8,940,881 B2 1/2015 Ifuku et al.
(72) Inventors: Sebastien Francois, Jassans-Riottier 8,945,609 B2 2/2015 Schuetz et al.
(FR); Sebastien Ladet, Caluire & Cuire 9,078,949 B2 7/2015 Gislason et al.
(FR): Muriel Guerin, Anse (FR) 2001 OO24655 A1 9, 2001 Schneider et al.
2002fOO18732 A1 2/2002 Hung et al.
2002fOO68151 A1 6/2002 Kim et al.
(73) Assignee: Sofradim Production, Trevoux (FR) 2002/0177577 A1 1 1/2002 Hung et al.
2003.0034304 A1 2/2003 Huang et al.
(*) Notice: Subject to any disclaimer, the term of this 2004/0242537 A1 12, 2004 Oh I
patent is extended or adjusted under 35 2005/0042265 A1 2/2005 Guillot et al.
U.S.C. 154(b) by 16 days. 2005.0053663 A1 3/2005 Struszczyk et al.
2009.0075383 A1 3/2009 Buschmann et al.
2009/O197789 A1 8, 2009 Brooker et al.
(21) Appl. No.: 14/596,672 2010, O190704 A1 7, 2010 Shimada
2013, O184356 A1 7/2013 Andersson
(22) Filed: Jan. 14, 2015 2014/0275291 A1 9, 2014 McGrath et al.
2014/0314824 A1 10/2014 Kim et al.
(65) Prior Publication Data 2015,0065454 A1 3/2015 Dupasquier et al.
2015,0174153 A1 6, 2015 Nothias et al.
US 2015/021078O A1 Jul. 30, 2015
FOREIGN PATENT DOCUMENTS
(30) Foreign Application Priority Data
EP 382210 A1 8, 1990
Jan. 30, 2014 (FR) ...................................... 14 50716 EP 512849 A1 11F1992
EP T78286 A2 6, 1997
WO 9004608 A1 5, 1990
(51) Int. Cl. WO 962O730 A1 T 1996
C8B 37/08 (2006.01) WO 97O3708 A1 2, 1997
C7H I/08 (2006.01) WO 98O8877 A1 3, 1998
(52) U.S. Cl. WO 9938893 A1 8, 1999
WO O3O37935 A1 5, 2003
CPC ............... C08B 37/003 (2013.01); C07H I/08 WO 2005O19272 A1 3, 2005
(2013.01) WO 2007OOTO14 A2 1, 2007
(58) Field of Classification Search WO WO 2008063503 A2 * 5/2008 ...... CO8E3 37,0003
None WO 201OO61454 A1 6, 2010
See application file for complete search history. WO 2012080772 A1 6, 2012
WO 2015032984 A1 3, 2015
WO 2015.09.2289 A1 6, 2015
(56) References Cited
U.S. PATENT DOCUMENTS OTHER PUBLICATIONS

4,159,932 A 7, 1979 Peniston et al. Taghizadeh et al., “Preparation, characterization, and Swelling
4, 195,175 A 3, 1980 Peniston et al. behavior of N-acetylated and deacetylated chitosons', www.
4,301,067 A 11/1981 Koshugi Sciencedirect.com, 8 pages.
4,308,377 A 12/1981 Koshugi
4,356,236 A 10/1982 Koshugi * cited by examiner
4,373,096 A 2/1983 Koshugi
4,378,017 A 3/1983 Kosugi et al.
4,401,807 A 8/1983 Koshugi Primary Examiner — Layla Berry
4,879,340 A 11/1989 Moriguchi et al.
4,996,307 A 2f1991 Itoi et al. (57) ABSTRACT
5,620,706 A 4/1997 Dumitriu et al.
5,897,821 A 4/1999 Kawasaki The present invention relates to a method for the preparation
5,902,798 A 5, 1999 Gouda et al. of chitosan of a degree of acetylation DA, from chitosan B
6,130,321 A 10, 2000 Johnson et al. having a degree of acetylation DA, lower than DA, com
6,156,330 A 12/2000 Tsukada et al. prising at least one step of purification and at least one step
6,251,959 B1 6/2001 Kawahara et al.
6,333,399 B1 12/2001 Teslenko et al. of acetylation of said chitosan B, said purification step
6,444,797 B1 9, 2002 Son et al. comprising at least one step of solubilization of said chitosan
6,589,999 B2 7/2003 Gurny et al. B in an acidic aqueous medium and at least one step of
6,867,287 B2 3/2005 Carlucci et al. precipitation of said thus solubilized chitosan,
6,887,564 B2 5/2005 Gagliardini et al. wherein said acetylation step is carried out in a solution on
7,189,326 B2 3/2007 Domard et al.
7,544,785 B2 6, 2009 Cowan et al. said solubilized chitosan before said precipitation step.
8,153,612 B2 4/2012 Ben-Shalom et al.
8,236,781 B2 8/2012 Laugier et al. 19 Claims, No Drawings
 US 9,527,929 B2
1. 2
OPTIMIZED CHITOSAN REACETYLATION controlled drug release systems. The pharmaceutical and
medical sectors also focus on the coagulant, cicatrizing and
CROSS-REFERENCE TO RELATED controlled salting out properties of therapeutic agents of the
APPLICATIONS chitosan. Chitosan also has antibacterial and antifungal
properties.
This application claims priority of French Patent Appli The effectiveness of the chitosan in medical applications,
cation Serial No. 14/50716 filed Jan. 30, 2014, the disclosure and in particular its biodegradability, its enzymatic degra
of the above-identified application us hereby incorporated dation rate or biodegradation kinetics, and its mechanical
by reference in their entirety. properties, including its Swelling capacity in aqueous
10 medium, are based on the one hand, to its molecular weight
BACKGROUND and, on the other hand, to its degree of acetylation. The latter
must hence be able to be attained and characterized rigor
The present invention relates to a method for the prepa ously and particularly in a reproducible manner.
ration of chitosan having a determined degree of acetylation, The extraction of chitin from exoskeletons of arthropods
in particular a degree of acetylation beyond 40% from 15 involves steps of protein and lipid hydrolysis, of depigmen
chitosan B of lower degree of acetylation, with a good tation and of demineralization. Usually, the hydrolysis of
reproducibility and good precision. proteins and lipids is carried out in presence of Soda, the
Chitosan is a polysaccharide obtained from the deacety demineralization requiring the use of hydrochloric acid. As
lation of chitin. regards to endoskeletons of cephalopods, the latter being
Chitin with cellulose is the most widespread natural devoid of minerals, the extraction of chitin requires only one
polysaccharide on earth. Chitin is for example present in the step of hydrolysis of protein.
exoskeletons of arthropods, in the endoskeletons of cepha Once the chitin is extracted, the chitosan is obtained by a
lopods as well as in the fungi. deacetylation step, which consists in hydrolyzing acetamide
Chitin is a biopolymer constituted of the repetition of groups. This reaction is generally carried out at high tem
units B(1-4)N-acetyl-2-amino-2-deoxy-D-glucose. Chitosan 25 perature in an alkaline Solution, for example a solution of
is obtained by extensive deacetylation of chitin. Thus, it is soda (NaOH) at 48% in water, at 90° C.
composed of units f(1-4)N-acetyl-2-amino-2-deoxy-D-glu Due to its crystalline structure, chitin B, which is extracted
cose and units 2-amino-2-deoxy-D-glucose. The degree of from endoskeletons of cephalopods, has a better hydrophilia
acetylation (DA) of a chitosan is the percentage of units and a greater reactivity to deacetylation. Thus, due, on the
B(1-4)N-acetyl-2-amino-2-deoxy-D-glucose with respect to 30 one hand, to its crystalline structure, and, on the other hand,
the total number of units B(1-4)N-acetyl-2-amino-2-deoxy to a less aggressive corrosive step during the extraction of
D-glucose and 2-amino-2-deoxy-D-glucose constituting this chitin B, chitosan B, particularly obtained from endoskel
chitosan. Thus, from the chemical point of view, chitin and etons of cephalopods, also has a better reactivity and is
chitosan are distinguished from each other Substantially by particularly interesting for medical applications. Chitosan B
their degrees of acetylation which give them different prop 35 from chitin B may also attain molecular weights which are
erties: in particular, as opposed to chitin which is insoluble impossible to attain with chitosan C. from chitin C.
in the majority of known solvents, chitosan is soluble in Chitosan B obtained after deacetylation of chitin B has a
weakly acidic aqueous solutions and may accordingly be relatively low acetylation rate, generally in the range of 1 to
easily treated and/or transformed. Chitosan, hence, is dis 10%, giving it a very good solubility in diluted acidic media.
tinguished from chitin first by its capacity to be soluble in an 40 However, according to its desired usage, this chitosan B of
aqueous medium. The degree of acetylation of chitosan may low degree of acetylation should for example be reacety
vary from 0 to around 55%. lated, that is to say, its degree of acetylation must be
Furthermore, chitin is mainly found under two different increased again, for example to give it specific properties.
crystalline forms: chitin C. and chitin B. Chitin C. is substan Thus, in the field of biodegradable implants which serve
tially extracted from exoskeletons of arthropods such as 45 as reinforcement for abdominal walls, compounds which
lobster, crab, shrimp, whereas chitin B is extracted from the give the implant both mechanical properties ensuring the
endoskeleton of cephalopods such as Squid. function, even temporary, of reinforcement and Support, and
Chitin C. has polysaccharide chains disposed in a antipar a controlled biodegradation, are needed. Thus, according to
allel manner between planes of Successive chains, thus, the kinetics of biodegradation required for the implant,
resulting in numerous hydrogen bonds. Such a structure 50 chitosan should have a degree of acetylation of 20, 30, 40,
gives chitin C. an important stiffness and a low reactivity or even 50% or 55%.
with respect to deacetylation. Chitin C. is not soluble in the Moreover, for Such medical and/or pharmaceutical appli
aqueous solvents and Swells only very slightly in aqueous cations, chitosan B must of course be purified and perfectly
medium. characterized: in particular, it must have very low rates of
Chitin B, on the contrary, has polysaccharide chains all 55 bacteria, endotoxins, contaminants, such as heavy metals
parallel to each other; the hydrogen bonds are less numerous and insoluble particles; it must not include any risk of viral
therein, this gives chitin Babetter reactivity and hydrophily. contamination; its physico-chemical parameters, such as its
Chitin B, thus, has the capacity of highly Swelling in water. molecular weight and its polydispersity index must be
As seen above, due to its solubility in acidic aqueous controlled.
medium, chitosan may be easily treated or transformed. 60 Methods for the purification of chitosan B exist. However,
Furthermore, chitosan has properties, such as biodegradabil it has been observed that the acetylation of a starting
ity, bioresorbability, biocompatibility, non-toxicity, chitosan B having a low degree of acetylation and which is
mechanical properties, which make it particularly interesting purified was hardly reliable and hardly reproducible from
for medical applications. Thus, chitosan may be incorpo the moment degrees of acetylation higher than or equal to
rated in medical devices as a constituent of implants, for 65 40% were sought to be attained.
example in the form of Sponges or films for reinforcements Indeed, the acetylation of a starting chitosan B with a low
of the abdominal wall, or even as excipient or Support for degree of acetylation, for example ranging from 1 to 10%,
 US 9,527,929 B2
3 4
is generally carried out by means of acetic anhydride, which reacetylated chitosan higher than or equal to 40%, for
is reacted with the quantity of chitosan B sought to be example close to 50%, said method being reliable and
acetylated in Stoichiometric proportions, method for which reproducible.
one can be based on a statistical model which allows The present invention is about a method for the prepara
determining a priori the required quantity of acetic anhy- 5 tion of chitosan of a degree of acetylation DA, from
dride as follows: the number (n-NH2) of amine functions chitosan B having a degree of acetylation DA, lower than
to be acetylated depends on the initial degree of acetylation DA, comprising at least one step of purification and at least
of the chitosan (DA) and the degree of acetylation sought to one step of acetylation of said chitosan B, said purification
be attained, (DA). Determining this quantity is hence car step comprising at least one step of solubilization of said
ried out using the following formula: 10 chitosan B in an acidic aqueous medium and at least one step
of precipitation of said thus solubilized chitosan,
(no-NH2)=(no-NH2)-(no-NH2), wherein said acetylation step is carried out in Solution on
Where (n-NH), is the number of free amine functions said solubilized chitosan before said precipitation step.
of chitosan before acetylation, that is to say, DA, and 15 Thus, in the method according to the invention, the
(n-NH), is the number of free amine functions of chitosan purification
global step.
and the acetylation are carried out in one single
The method according to the invention allows
of the degree of acetylation DA, sought to be attained, obtaining purified chitosans having a high degree of acety
With
lation (DA), for example ranging from 40% to 55%, for
(no-NH2), nix (1-DA) example of around 50%, in a reliable and reproducible
2O manner. In particular, in the method of the invention, the

and absolute value of the average A as mentioned above is for

example lower than or equal to 2%, and its standard devia
tion O is lower than or equal to 2%.
(n C-NH2), nix (1 -DA) Thus, thanks to the method according to the invention, it
DA, is the initial degree of acetylation of the chitosan B, 25 is possible to obtain purified chitosan and having a high DA
DA, is the final degree of acetylation sought to be attained, inpharmaceutical
a precise manner, and thus, use it in medical and/or
applications. Thus, it is possible to carry out
And
implants having a totally predictable and controlled kinetics
of degradation.
mchito X (1 -% water) 30 In addition, as will become apparent from the following
fitti F
Mo description, the method according to the invention allows
getting free from numerous time-consuming washing and
drying steps, steps which are generally found in the known
where -% represents the moisture content of the initial methods where the chitosan is purified before being acety
chitosan B. The considered mass Mo is that of a unit of the 35 lated.
polymer of the initial chitosan B. The chitosan B of initial degree of acetylation DA, con
The mass of acetic anhydride (m) to be added is stituting the starting material of the method according to the
obtained by the following relationship: invention may be obtained by deacetylation of the chitin B
in the following manner: chitin B, for example extracted
from endoskeleton of ground squid, as described above by
where Maa is the molar mass of the acetic anhydride, in hydrolyzing proteins, is washed in water then centrifuged. It
other words 102.09 gmol'. is then dried.
Such a method generally allows producing a succession of The thus, ground chitin undergoes a step of deproteini
homogenous lots of reacetylated chitosan with a control of zation at room temperature (around 20-25° C.): to do this,
the degradation kinetics of the reacetylated chitosan, the 45 chitin in powder form is mixed with a solution of NaOH 1N
chitosan being reacetylated homogenously. for 24 h.
In order to determine the reproducibility and precision of The chitin is then washed in successive water baths.
such a method, for a determined number N of tests, it is The thus washed chitin is deacetylated in order to obtain
measured the value D of the difference between the value of chitosan: during this step, the washed chitin is introduced in
the final desired or theoretical degree of acetylation, namely 50 a bath of NaOH 50% (in weight) at 90° C. for 20 min and
DA, and the value of the real or measured degree of then washed. This step is repeated a second time.
acetylation on the end product, in other words the reacety The obtained product is dried in an oven: it consists of
lated chitosan, for example by nuclear magnetic resonance chitosan of low degree of acetylation.
spectroscopy (NMR), namely, DA. Then the average A of Chitosan B having a low degree of acetylation is obtained,
the percentage of differences D is calculated with respect to 55 in other words a degree of acetylation ranging from about
the theoretical target, and its standard deviation (a) with 1% to about 10%, for example ranging from 2 to 6%.
respect to the number N of carried out tests. The following publications also describe methods for the
Thus, it has been noted that in the case where the deacetylation of chitin B in order to obtain chitosan of low
theoretical degree of acetylation was 40% or more for the degree of acetylation: “Lamarque, G., C. Viton, and A.
reacetylation of purified chitosan, the average A and its 60 Domard, New Route of Deacetylation of C- and B-Chitins by
standard deviation O, determined as aforementioned, Means of Freeze-Pump Out-Thaw Cycles. Biomacromol
attained too high values, making the method hardly reliable ecules, 2005. 6(3): p. 1380-1388.”, “Lamarque, G., C. Viton,
and non reproducible. and A. Domard, Comparative Study of the First Heteroge
Hence, it would be desirable to be able to have a method neous Deacetylation of C- and B-Chitins in a Multistep
which allows both purifying and acetylating a chitosan 3 of 65 Process. Biomacromolecules, 2004. 5(3):p. 992-1001.’,
low initial degree of acetylation, said method allowing “Lamarque, G., C. Viton, and A. Domard, Comparative
attaining a real degree of acetylation measured on the Study of the Second and Third Heterogeneous Deacety
 US 9,527,929 B2
5 6
lations of C- and B-Chitins in a Multistep Process. Biomac to remove the insoluble particles and contaminants that are
romolecules, 2004. 5(5):p. 1899-1907.”, “Tolaimate, A., et potentially present in the acidic aqueous solution of chito
al., Contribution to the preparation of chitins and chitosans san. The filtration of the chitosan solution obtained in the
with controlled physico-chemical properties. Polymer, 2003. previous step may for example be carried out on porous
44(26): p. 7939-7952.” membranes such as millipore cartridges of porosity 1.0/0.5
The degree of acetylation of chitosan may be character um sold under brand name “OpticapR XL by the Millipore
ized for example by one of the following methods: Fourier company.
transform infrared spectroscopy (FTIR), UV spectrometry, The Solution of chitosan is for example poured into a
nuclear magnetic resonance (NMR). filtering tank suited to the worked volume and is put under
In the present application, the degrees of acetylation DAi, 10 pressure with compressed air, the outlet of the tank being
DAr and DAfare characterized by Nuclear Magnetic Reso connected to the porous filtering membrane. If necessary, in
nance (NMR) spectroscopy according to the protocol order to accelerate filtration if necessary, the solution of
described in the publication of Lavertu M. et al <A chitosan may be diluted in demineralized water.
validated "H NMR method for the determination of the Once the solution is filtered as described above, we
degree of deacetylation of chitosand Journal of Pharma 15 proceed to the acetylation step.
ceutical and Biomedical Analysis, 32 (2003) 1149-1158. The acetylation step comprises for example the addition
In a form of implementation of the method according to of acetic anhydride in Stoichiometric proportion with respect
the invention, DA, ranges from around 1% to around 10%, to the quantity of chitosan to be acetylated in order to obtain
for example from 2 to 6%. said chitosan of degree of acetylation DA.
The method according to the invention comprises at least The quantity of chitosan to be acetylated, in other words
one step of purification and at least one step of acetylation the number (n-NH) of amine functions to be acetylated,
of said chitosan B. depends on the degree of acetylation of the solubilized
The purification step of the method according to the chitosan (DA) and the degree of acetylation sought to be
invention comprises a first step of solubilization of the attained (DA). Determination of this quantity is hence done
chitosan B in an acidic aqueous medium. For example, the 25 using the following formula:
solubilization may be carried out using acetic acid. The
chitosan B may for example be solubilized in a solution of
demineralized water by adding a stoichiometric quantity of Where (n-NH), is the number of free amine functions
acetic acid. The molar mass of acetic acid is of 60.052 g/mol. of the solubilized chitosan before acetylation and (n-
The determination of the quantity of acetic acid to be added 30 NH), is the number of free amine functions of chitosan of
is carried out by calculating the number of amine functions degree of acetylation DA, sought to be attained,
that are potentially protonable along the chain of chitosan. With
The number of amine functions that are potentially pro (no-NH2), -nix(1-DA)
tonable is obtained by the following formula:
35 and

n(NH2) = m(chito San) X (1 - DAi) X (1 - %ate.) (nc-NH2)-nix(1 -DA)

M (O) DA, is the initial degree of acetylation of the chitosan,
Ris the final degree of acetylation sought to be attained,
40
Wherein -% represents the moisture content of starting
chitosan and m(chitosan) the dry mass used.
M(O) is the molar mass of chitosan and is calculated based mchito X (1 -% water)
on the following formula: fitti F
Mo
M(0)=203xDA+161x (1-DA) 45
Then, the mass of acetic acid (100%) to be added is The considered mass Mo is that of a unit of the initial
determined using the following formula: chitosan polymer.
The mass of acetic anhydride (m) to be added is
obtained by the following relationship:
where M(CH3COOH) is the molar mass of the acetic 50
acid, namely 60.052 g/mol.
Solubilization is done by stirring until obtaining a homog where Maa is the molar mass of the acetic anhydride, in
enous solution. For example, the concentration in chitosan other words 102.09 gmol
may be set at 0.5% (in weight) in acidic aqueous solution in Before adding the acetic anhydride to the solubilized
order to preferably obtain a rather low viscosity in the final 55 chitosan, a co-solvent may be added, for example 1.2-
Solution. Propanediol to the solution of chitosan. The addition of such
In other implementations of the method according to the a co-solvent allows homogenizing the reaction.
invention, Solubilization may be carried out using hydro Then, the acetic anhydride is added in stoichiometric
chloric acid, glutamic acid, lactic acid or a mixture thereof. proportion, as determined above. The set is preferably mixed
According to the method of the invention, the step of 60 under stirring.
acetylation of the chitosan is carried out in Solution on said Once the acetylation step carried out, the purification of
solubilized chitosan, before any precipitation step. This the chitosan is continued with a precipitation step in order to
allows the chitosan to have very good reactivity with respect obtain a precipitate of chitosan.
to the acetylation. In a form of implementation of the method according to
In a form of implementation of the method according to 65 the invention, the precipitation step comprises adjusting at a
the invention, a filtration step is carried out after said pH ranging from 9.5 to 11 the chitosan obtained after the
solubilization step and before said acetylation step, in order acetylation step.
 US 9,527,929 B2
7 8
The precipitation step may include a viral deactivation in Soda 1N, and extensive deacetylation in a solution of soda
step. The viral deactivation step is a step which allows 48% (in weight) in order to obtain a chitosan B of initial
hydrolyzing the components related to the viruses, prions degree of acetylation DA, of around 3%. The molar mass of
and residual toxins. chitosan is of 162.26 g/mol.
Thus, in a form of implementation of the method accord- 5 In the present example, the degrees of acetylation DA, and
ing to the invention, the step of precipitation comprises: DA, are characterized by the Nuclear Magnetic Resonance
A first precipitation at a pH ranging from 9.5 to 12 of the spectroscopy (NMR) according to the protocol described in
chitosan obtained after the acetylation step, the publication of Lavertu M. etal <A validated "H NMR
A viral deactivation step with a molar concentration of method for the determination of the degree of deacetylation
NaOH strictly higher than 1N of the precipitate of chitosan 10 of chitosand Journal of Pharmaceutical and Biomedical
obtained at the end of said first precipitation,
A second precipitation at a pH ranging from 9.5 to 11 of Analysis, 32 (2003) 1149-1158.
We proceeded to the purification and the acetylation of
the chitosan obtained after said viral deactivation step.
For example, in a mode of implementation of the method chitosan B as follows:
according to the invention, a first precipitation of the chi liters In a first step, 55.38 g of chitosan were solubilized in 5
tosan obtained after the acetylation step is carried out in 15 of demineralized water to which were added acetic
basic medium. For example, a solution of soda 5N is acid in Stoichiometric quantity, namely 18.88 ml of acetic
prepared. Before the precipitation, the solution of reacety acid. The solubilization is carried out under stirring at a
lated chitosan, in other words obtained after the acetylation temperature of around 50° C. for around 2 ha.0, until
step, is preferably filtered. The solution is then taken to a pH obtaining a homogenous Solution.
ranging from 9.5 to 12 to carry out the precipitation. The The solution is then filtered on a millipore cartridge of
precipitate of chitosan is recovered by filtration. porosity 1.0/0.5 um sold under brand name “Opticap(R) XL
In a form of implementation of the method according to by the Millipore company.
the invention, a viral deactivation step is carried out on the Then 400 ml of 1.2-Propanediol were added to the solu
precipitate at the end of this first precipitation. For example, tion of chitosan.
to this end, the precipitate recovered by filtration is plunged 25 The quantity of acetic anhydride to be added, namely the
under mechanical stirring in a quantity of soda which allows Stoichiometric quantity, has been determined using the fol
attaining a molar concentration of NaOH higher than 1N, for lowing formulae for a desired final degree of acetylation DA,
example a pH higher than or equal to 12. For example, the of around 50%:
viral deactivation step comprises a solubilization of the
precipitate of chitosan obtained at the end of said first 30
precipitation in a solution of sodium hydroxide. The increase
of the degree of acetylation of the chitosan with respect to ofWhere the
(n-NH), is the number of free amine functions
solubilized chitosan before acetylation and (n-
its initial degree of acetylation promotes the solubilization of
the chitosan during this viral deactivation step. Thus, at the NH), is the number of free amine functions of the chitosan
end of the viral deactivation step, the reacetylated chitosan 35 ofWithdegree of acetylation DA, desired to be attained,
may again be in Solubilized form.
In a form of implementation of the method of the inven (non-NH2);-nix(1-DA)
tion, a second precipitation is then carried out, this time on
the solution of reacetylated chitosan obtained at the end of and
the viral deactivation step. The solution of chitosan being
highly basic, for example with a molar concentration of 40 (nc-NH2)-nix(1 -DA)
NaOH higher than 1N after the viral deactivation step, the DA, is the initial degree of acetylation of the chitosan,
precipitation may be carried out by means of addition of
glacial acetic acid until obtaining a pH ranging from 9.5 to DA, is the final degree of acetylation desired to be
11. The obtained precipitate of chitosan is recovered, which attained,
is the purified chitosan. This chitosan thus has a degree of 45 And
acetylation DA.
In a form of implementation of the method of the inven mchito X (1 -% water)
tion, a washing step is carried out on the precipitate of n = --
Mo
chitosan obtained at the end of the precipitation step, in other
words on the precipitate obtained from the second precipi 50
tation when the precipitation step includes a viral deactiva The considered mass Mo is that of a unit of the polymer
tion step as described above. This washing step may for of initial chitosan.
example be carried out with sterile water up to a neutral pH, The mass of acetic anhydride (m) to be added is
then for example with ethanol until obtaining a conductivity obtained by the following relationship:
lower than 1 LS/cm. 55
In a form of implementation of the method of the inven
tion, the washed precipitate of purified chitosan of degree of where Maa is the molar mass of the acetic anhydride, in
acetylation DA is dried, for example under laminar flow. other words 102.09 gmol
The invention and its advantages will become more 14.791 g of acetic anhydride were added to proceed to the
apparent with the examples below. 60 acetylation.
Then the purification of chitosan was carried out using a
EXAMPLE 1. solution of soda 5N. 100 ml of soda is poured to take the
solution of chitosan to a pH of 11.56.
According to the Invention A viral deactivation step is carried out on the precipitate
65 obtained at the end of this first precipitation. To this end, the
It is provided chitosan B obtained as follows: grinding of precipitate recovered by filtration is plunged by mechanical
squid pen, then deproteinization of the powder of squid pen stirring in a quantity of Soda which allows attaining a molar
 US 9,527,929 B2
10
concentration of NaOH strictly higher than 1N, for example However, the purification and the acetylation of the
here a pH of around 12.66, for around 1 hour. chitosan B were carried out in two separate steps, the
The Solution is precipitated again by adding acetic acid purification in a first step, then the acetylation in a second
until obtaining a pH of around 9.53. step.
The obtained precipitate is then washed with sterile water 1) Purification:
until obtaining a washing water with neutral pH, then with In a first step, 110.4 g of chitosan were solubilized in 20
ethanol until obtaining a conductivity for the washing water liters of demineralized water to which were added the acetic
lower than 1 LS/cm. The precipitate of purified chitosan is acid in Stoichiometric quantity, namely 40 ml of acetic acid.
dried in a vacuum oven for around 96 h. 41.6 g of dry The solution is then filtered on a millipore cartridge of
reacetylated and purified chitosan is recovered. 10 porosity 1.0/0.5 um sold under brand name “Opticap(R) XL
We proceeded to nineteen tests. by the Millipore company.
For each test, the real degree of acetylation DA obtained Then, we proceeded to the purification of the chitosan
has been measured by Nuclear Magnetic Resonance spec using a solution of soda 5N. 150 ml of soda is poured to take
troscopy according to the aforementioned method. the solution of chitosan to a pH of 11.79.
For each test, the value D was then determined from the 15 A viral deactivation step is carried out on the precipitate
difference between the value of the final desired or theoreti from this first precipitation. To this end, the precipitate
cal degree of acetylation, namely DA, and the value of the recovered by filtration is plunged by mechanical stirring in
real or measured degree of acetylation on the end product, a quantity of Soda which allows reaching a molar concen
in other words the reacetylated chitosan, for example by tration of NaOH of 2N.
nuclear magnetic resonance (NMR), namely DA, The obtained precipitate is then washed with sterile water
Values DADA, and Dare gathered in the following table until obtaining a washing water with neutral pH, then with
I: ethanol until obtaining a conductivity for the washing water
lower than 1 LS/cm.
TABLE I 2) Reacetylation:
25 34.2 g of chitosan obtained in step 1) are solubilized in
differences between values of theoretical 3 liters of demineralized water to which were added acetic
DA (DA) and measured DA (DA acid in Stoichiometric quantity, namely 10.9 g.
Tests DAf DAir D = DAf- DAir Then, 2500 ml of 1.2-Propanediol is added to the solution
of chitosan.
1 46.18% 44.00% 2.18%
2 SO.05% 49.00% 1.05%
30 9.1 g of acetic anhydride is added to proceed to the
3 SO.04% 49.00% 1.04% acetylation.
4 SO.12% SO.00% O.12% The solution was then precipitated using a solution of
5
6
SO.04%
SO.02%
48.00%
SO.00%
2.04%
O.02%
soda 5N. 71.6 ml of soda is poured to take the solution of
7 49.96% 48.00% 1.96% chitosan to a pH of 11.6.
8 SO.O.3% S1.00% -O.97% 35 The obtained precipitate is then washed with sterile water
9 SO.02% 49.00% 1.02% until obtaining a washing water with neutral pH, then with
10
11
52.92%
49.66%
S1.00%
45.00%
1.92%
4.66%
ethanol until obtaining a conductivity for the washing water
12 SO.00% SO.00% O.00% lower than 1 LS/cm. The purified precipitate of chitosan is
13 SO.00% SO.00% O.00% dried by lyophilization. 35.5 g of dry reacetylated and
14 SO.00% 48.00% 2.00% 40 purified chitosan is recovered.
15
16
S.O.39%
S.O.38%
49.00%
43.00%
1.39%
7.38%
We proceeded to 58 tests.
17 50.37% 49.00% 1.37% For each test, the real degree of acetylation obtained DA,
18 50.37% 49.00% 1.37% has been measured by Nuclear Magnetic Resonance accord
19 50.37% 49.00% 1.37% ing to the protocol described in Example 1.
Average SO.05% 48.47% 1.57% 45 For each test, we then determined the value D of the
Standard deviation 1.15% 2.20% 1.84% difference between the value of the final desired or theoreti
cal degree of acetylation, namely DA, and the value of the
The average of the differences D is calculated on the set real or measured degree of acetylation on the end product,
of the 19 carried out tests. Statistical analyses are carried out in other words the reacetylated chitosan, for example by
with the “Minitab(R) 16.2 software of Microsoft. Thus, the 50 nuclear magnetic resonance (NMR), namely DA,
average of the differences is equal to 1.57%+1.84%. We then calculated the average of the differences D on the
Thus, the reproducibility and precision of the method set of 58 carried out tests. The statistical analyses are carried
according to the invention is excellent and allows obtaining out with the “Minitab(R) 16.1' software of Microsoft. Thus,
chitosan having a degree of acetylation higher than 40%, for the average of the differences D is equal to -2.65%+2.84%.
example here approximately equal to 50%, in a very reliable, 55 Thus, the average of the differences D is high and does not
reproducible and precise manner. allow carrying out the purification and the acetylation of the
chitosan B in a reliable, precise and reproducible manner.
EXAMPLE 2
The invention claimed is:
Comparative 60 1. A method for the preparation of chitosan of a degree of
acetylation DA, from chitosan f having a degree of acety
It is provided the same chitosan B as in Example 1, lation DA, lower than DA, comprising at least one step of
obtained in the same manner and with the same degree of purification and at least one step of acetylation of said
acetylation DA, of 3%. chitosan B, said purification step comprising at least one step
The added quantities of acetic anhydride have been deter 65 of Solubilization of said chitosan B in an acidic aqueous
mined using the same formula as in Example 1 for each final medium and at least one step of precipitation of said thus
desired degree of acetylation DA namely around 50%. solubilized chitosan, said acetylation step being carried out
 US 9,527,929 B2
11 12
in a solution on said solubilized chitosan before said pre 9. The method according to claim 6, wherein a washing
cipitation step, wherein the precipitation step includes a viral step is carried out on the precipitate of chitosan obtained at
deactivation step. the end of said precipitation step.
2. The method according to claim 1, wherein DA, ranges 10. The method according to claim 1, wherein the solu
from about 1% to about 10%. bilization in acidic aqueous medium is carried out using
acetic acid.
3. The method according to claim 1, wherein a filtration 11. The method according to claim 1, wherein the acety
step is carried out after said solubilization step and before lation step comprises the addition of acetic anhydride in
said acetylation step, in order to remove the insoluble Stoichiometric proportion with respect to the quantity of
particles and contaminants that are potentially present in the 10 chitosan f3 to be acetylated in order to obtain said chitosan
acidic aqueous solution of chitosan. of degree of acetylation DA.
4. The method according claim 1, wherein the precipita 12. The method according to claim 1, wherein the viral
tion step comprises adjusting at a pH ranging from 9.5 to 11 deactivation step comprises a solubilization of a precipitate
the chitosan obtained after the acetylation step. of chitosan obtained at the end of a first precipitation in a
5. The method according to claim 1, wherein the precipi 15
solution of sodium hydroxide.
tation step comprises: 13. The method according to claim 5, wherein the viral
a first precipitation at a pH ranging from 9.5 to 12 of the deactivation step comprises a solubilization of the precipi
chitosan obtained after the acetylation step, tate of chitosan obtained at the end of said first precipitation
a viral deactivation step with a molar concentration of in a solution of sodium hydroxide.
NaOH higher than 1N of the precipitate of chitosan 14. The method according to claim 6, wherein the viral
obtained at the end of said first precipitation which deactivation step comprises a solubilization of the precipi
forms a chitosan solution, tate of chitosan obtained at the end of said first precipitation
a second precipitation at a pH ranging from 9.5 to 11 of in a solution of sodium hydroxide.
the chitosan obtained at the end of said viral deactiva 15. The method according to claim 1, wherein DA, ranges
tion step. from 2 to 6%.
25
6. The method according to claim 4, wherein the precipi 16. The method according to claim 5, wherein the solu
tation step comprises: bilization in acidic aqueous medium is carried out using
a first precipitation at a pH ranging from 9.5 to 12 of the acetic acid.
chitosan obtained after the acetylation step, 17. The method according to claim 6, wherein the solu
a viral deactivation step with a molar concentration of 30
bilization in acidic aqueous medium is carried out using
acetic acid.
NaOH higher than 1N of the precipitate of chitosan 18. The method according to claim 5, wherein the acety
obtained at the end of said first precipitation which lation step comprises the addition of acetic anhydride in
forms a chitosan solution, Stoichiometric proportion with respect to the quantity of
a second precipitation at a pH ranging from 9.5 to 11 of chitosan f3 to be acetylated in order to obtain said chitosan
the chitosan obtained at the end of said viral deactiva 35
tion step. of degree of acetylation DA.
7. The method according to claim 1, wherein a washing 19. The method according to claim 6, wherein the acety
Step is carried out on the precipitate of chitosan obtained at lation step comprises the addition of acetic anhydride in
the end of said precipitation step. Stoichiometric proportion with respect to the quantity of
8. The method according to claim 5, wherein a washing 40
chitosan B to be acetylated in order to obtain said chitosan
Step is carried out on the precipitate of chitosan obtained at of degree of acetylation DA.
the end of said precipitation step.