Chitosan production process

Abstract

The present disclosure provides a method of producing chitosan, the method comprising the steps of: a. obtaining exuviae from a group of crustacean animals, preferably belonging to one or more of the following infraorders: achelata, polychelida and astacidea; 5 b. demineralizing and deproteinizing the exuviae to provide chitin; and c. deacetylating the chitin to provide chitosan. Further provided herein are the chitosan obtained from this method and its applications.

Description

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[0003] Chitosan Production Process

[0004] FIELD OF THE INVENTION

[0005] The present disclosure relates to a method of producing chitosan, chitosan produced according to this method and its uses and applications.

[0006] BACKGROUND OF THE INVENTION

[0007] Chitosan is a linear polysaccharide composed of randomly distributed p-(1 -^4)-linked D-glu-cosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is frequently obtained from chitin, which in turn can be obtained from the shell remains of crustaceans. In many processes, the shell remains are first demineralized, then deproteinized and decolorized to obtain the chitin. The chitin can be converted to chitosan by deacetylation, which can be performed under alkaline conditions or enzymatically. The resulting chitosan typically has a broad distribution of molecular weights.

[0008] Numerous different processes for producing chitosan are known from the prior art. However, the different processes suffer from significant disadvantages.

[0009] One major disadvantage is that many of the known processes display poor reproducibility. For example, high batch-to-batch deviations are observed in key quality parameters such as molecular weight and the degree of deacetylation of chitosan, as well as its solubility, its molecular weight distribution, its protein content, its ash content and color, among several other quality parameters. The lack of reproducibility constitutes a severe limitation because several industrial applications of chitosan require high reproducibility and low batch-to-batch deviations, most notably biomedical applications of chitosan. Therefore, many chitosan production processes are not suitable to supply adequate chitosan for biomedical applications or other applications. Especially for biomedical applications, the regulatory requirements areP25951 PCOO February 2026

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[0011] high and require the chitosan to fulfill stringent quality control requirements with high fidelity and predictability.

[0012] Another challenge in the field of chitosan production is that it is difficult to produce chitosan which has both a high molecular weight and a high degree of deacetylation. Indeed, these two targets often compete with each other: to achieve high degrees of deacetylation, harsh reaction conditions may be chosen for the deacetylation step, but this leads to increasing chain shortening and thus a reduction in molecular weight. Conversely, deacetylation conditions that are rather mild in order to avoid undesired reductions in molecular weight are often insufficient to achieve high degrees of deacetylation. Although enzymatic deacetylation systems are known, they also face the problem of balancing high molecular weight and high deacetylation degrees, and they also suffer from further limitations, including high production costs and / or complicated and therefore expensive reaction processes.

[0013] A particular problem arises when considering the combined requirement to produce chitosan having a high molecular weight and a high degree of deacetylation in a highly reproducible fashion. The multidimensional challenges in the synthesis of chitosan are difficult to overcome with the known processes.

[0014] Besides molecular weight and high degrees of deacetylation, further desired product properties of the chitosan include a high solubility of the chitosan, colorlessness, a low ash content and a low protein content. Other desirable properties may also include low metal concentrations (especially low heavy metal concentrations, but also low calcium concentrations), low toxin concentrations, low viscosity in solution, good film-forming properties and no unpleasant odor. High storage stability, low production costs, a low environmental impact and sustainability may also be desirable. Finally, if the raw material is ultimately obtained from crustacean animals, the health and well-being of the animals is also a concern.

[0015] For many applications, it is necessary to meet high quality standards consistently and repro-ducibly in a range of different properties, including molecular weight, deacetylation degree,P25951 PCOO February 2026

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[0017] a low ash content, low toxin levels, solubility and the ability to form a colorless solution upon solubilization. Being able to realize these quality features consistently and with high reproducibility has so far remained an elusive goal.

[0018] In conclusion, there is a need to further develop the state of the art in the field of chitosan production.

[0019] SUMMARY OF THE DISCLOSURE

[0020] It is the general object of the present disclosure to advance the state of the art in the field of chitosan production, and preferably to address at least some of the disadvantages of the prior art, such as the ones discussed above, fully or at least partly. In advantageous embodiments, the present disclosure provides a method of producing chitosan that allows to produce the chitosan in a highly reproducible fashion. In particular, the method allows to ensure that key quality parameters of the chitosan are met with high predictability and reproducibility and with minimal deviations across batches and / or across time. The key quality parameters generally include a range of different properties, such as one or more of the following: a controlled (typically high) molecular weight, a controlled (typically high) degree of deacetylation, a low residual protein content, a low ash content, a low level of toxins such as endotoxins, a high solubility, preferably forming a colorless and clear solution, and a low metal content (particularly a low calcium content and / or a low heavy metal content). It is of particular interest, at least in some embodiments, to provide chitosan having high molecular weight and a high deacetylation degree in a reproducible fashion.

[0021] The general object is achieved by the subject-matter of the independent claims. Further favorable embodiments follow from the dependent claims and the overall disclosure.

[0022] In a first aspect, the present disclosure provides a method of producing chitosan. The method comprises the steps of:P25951 PCOO February 2026

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[0024] a. obtaining exuviae from a group of crustacean animals;

[0025] b. demineralizing and deproteinizing the exuviae to provide chitin; and

[0026] c. deacetylating the chitin to provide chitosan.

[0027] Several different embodiments of this general method are described herein and will be described hereinafter. In a particularly advantageous embodiment, the group of crustacean animals belong to one or more of the following infraorders: achelata, polychelida and astacidea, preferably achelata. Choosing these infraorders was found to lead to high-quality chitosan. It was also found to ensure high reproducibility and minimal deviations across batches and / or across time. In particular, minimal deviations across batches and across time were observed with respect to the degree of deacetylation, the molecular weight, the solubility (i.e. the chitosan was consistently and reliably soluble across batches and time) and the colorless nature of the chitosan upon solubilization. Additionally, further quality criteria are met such as a low protein content, low heavy metal content and low endotoxin levels. More generally, the method described herein allows to produce chitosan that is suitable for biomedical applications.

[0028] Obtaining the Exuviae

[0029] In the method disclosed herein, exuviae from the groups of crustacean animals are used to ultimately produce chitosan. As the skilled person knows, exuviae are the remains of an exoskeleton and related structures that are left after the crustaceans have molted. Depending on the application, the exuviae can be obtained in different ways.

[0030] In some embodiments, the exuviae are obtained by molting of live crustacean animals (i.e. of the live crustacean animals of the group of crustacean animals). In other words, in some embodiments, the exuviae are obtained by the natural molting process rather than by an unnatural (e.g. manual) process of stripping or otherwise removing the exoskeleton from theP25951 PCOO February 2026

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[0032] flesh of the (typically dead) crustacean animals. Surprisingly, it was found that using exuviae obtained by molting of live crustacean animals ensures that the chitosan can be manufactured with high reproducibility and with minimal deviations across batches. In particular, it was found that the different batches of chitosan which were produced from different batches of exuviae consistently display similarly high molecular weights, similarly high degrees of deacetylation, are consistently soluble and consistently form colorless solutions upon solubilization. Without wishing to be bound to a theory, it is possible that the natural molting process ensures that the resulting exuviae display a high degree of homogeneity with respect to their chemical composition. A further advantage of using exuviae from molting of live crustacean animals is that the crustacean animals are unaffected by the method, thereby providing sustainable and animal-friendly access to chitosan. Furthermore, the live animals can molt several times, thereby providing access to more chitosan over time. Finally, by relying on the natural molting process, the resulting exuviae are purer and are free of remaining flesh that would result from manual removal of the exoskeleton from the animals. Thus, in some embodiments, the obtained exuviae are essentially free from flesh.

[0033] In some embodiments, the step of providing the exuviae includes collecting the exuviae during a post-molt period.

[0034] The unexpected advantages of high reproducibility and consistently achieving key quality parameters come at the cost of a range of practical disadvantages, including reliance on a natural process that is less predictable, less plannable and more challenging to scale up, as well as the need to collect the exuviae from the locations where the animals molted, which tend to be in locations that are difficult to access because crustaceans tend to molt in secluded or difficult-to-access locations because they offer protection during the period of molting during which they are particularly vulnerable to prey.

[0035] It was found to be particularly advantageous to use exuviae which were obtained by molting of live crustacean animals belonging to the infraorders: achelata, polychelida and astacidea, preferably achelata, more preferably to the family Palinuridae, more preferably to the genusP25951 PCOO February 2026

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[0037] Palinurus. Using molts from these animals ensured high reproducibility and consistently high-quality chitosan (high molecular weight, high deacetylation degree, solubility and formation of a colorless solution). Using molts from these infraorders is particularly counterintuitive because these animals tend to molt less frequently than other crustaceans. For example, spiny lobsters tend to molt once every couple of months, which limits supply of these molts.

[0038] Surprisingly, it was found to be particularly advantageous to use substrate structures such as those described in WO 2024 / 033292 A1 to aid the crustacean animals during molting. Therefore, in some embodiments, the exuviae used in the process were obtained by molting of live crustacean animals in the presence of a substrate structure. Preferably, the crustacean animals molt on the substrate structure. Consequently, the molts may in some embodiments of the process described herein be collected from the substrate structure.

[0039] The substrate structure may for example comprise:

[0040] - at least two support gratings (2) arranged parallel to each other; and

[0041] - at least two interspacing units (3) arranged between the two support gratings (2) for spacing the two support gratings (2) at a distance of at least 20 mm, such that at least two floors (4) are formed, which provide support for the crustacean animals.

[0042] Further preferred embodiments of the substrate structure are described in further detail in WO 2024 / 033292 A1 , which is incorporated herein in its entirety, in particular with respect to the features describing different embodiments of the substrate structure. Therefore, the embodiments of the substrate structure described in WO 2024 / 033292 A1 are also preferred embodiments of the substrate structure as used in the process described herein. It is understood that should there be discrepancies between any the terminology or any definitions used in WO 2024 / 033292 A1 and the present disclosure, the present disclosure prevails.P25951 PCOO February 2026

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[0044] Depending on the application, the crustacean animals may be reared to achieve better results. For example, in some embodiments, the method further comprises the step of rearing the group of crustacean animals before the step of obtaining their exuviae. By rearing the crustacean animals, chitosan can be manufactured with high reproducibility and consistently high quality across batches. It is believed that the high reproducibility can be achieved at least in part because of controlled rearing conditions to the animals.

[0045] As used herein, rearing typically involves raising or upbringing of the crustacean animals. This may e.g. involve raising or upbringing of juveniles, pre-adults and / or adults. In a typical embodiment, rearing does not include reproduction, e.g. sexual reproduction, of the crustacean animals. In other words, rearing typically does not include sexual crossing of the whole genomes of two crustacean animals.

[0046] Depending on the application, the rearing process may be performed in different ways. In some embodiments, the crustacean animals are reared for at least two months before obtaining their exuviae, preferably for at least six months. These embodiments may be used to ensure that the resulting chitosan is of high quality.

[0047] In some embodiments, the crustacean animals are reared in a semi-closed or closed aquaculture system. Using semi-closed or closed aquaculture systems rather than open aquaculture systems allows more control over the rearing conditions, which in turn can be used to increase reproducibility. As used herein, semi-closed and closed aquaculture systems are land-based, as opposed to open aquaculture systems, which refer to an enclosure in natural water, such as an enclosure in an ocean or in the sea. In a semi-closed or closed aquaculture system, the crustacean animals are not kept in the sea or in an ocean. Semi-closed and closed aquaculture systems typically include water recirculation. The water recirculation may include water treatment, such as water purification and / or filtration. In a semi-closed aquaculture system, water is recirculated with the open sea. In a closed aquaculture system, the water cycle is closed.P25951 PCOO February 2026

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[0049] In some embodiments, the crustacean animals are reared under controlled conditions. For example, in some embodiments, at least two, preferably at least three, more preferably at least four, of the following water parameters are monitored and maintained within the respective ranges:

[0050] - temperature: from 16 °C to 22 °C;

[0051] - pH level from 7.8 to 8.2;

[0052] - oxygen concentration: from 5 ppm to 7 ppm;

[0053] - calcium concentration: from 400 ppm to 420 ppm;

[0054] - magnesium concentration: from 1200 ppm to 1400 ppm;

[0055] - ammonium concentration: less than 0.1 ppm;

[0056] - salinity: from 28 per thousand to 37 per thousand.

[0057] The parameters described in the preceding list are water parameters, e.g. water parameters of the semi-closed or closed aquaculture system. Thus, it is understood that they refer to the water in which the crustacean animals are reared. The indicated parameter ranges were found to be particularly advantageous to maximize the amount of exuviae obtained over time, while still ensuring that the final chitosan product is of consistently high quality (particularly regarding consistently high molecular weight, consistently high degree of deacetylation, consistently high solubility and formation of a consistently colorless solution upon dissolution).

[0058] One water parameter which was found to be particularly advantageous to control is the temperature, which is advantageously monitored and maintained to be from 16 °C to 22 °C. TheP25951 PCOO February 2026

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[0060] temperature was found to be particularly advantageous for the infraorders achelata, poly-chelida and astacidea. In addition to this temperature range, it is also particularly advantageous to also monitor and maintain the pH level to be from 7.8 to 8.2 and / or to monitor and maintain the salinity to be from 28 per thousand to 37 per thousand.

[0061] In some embodiments, the group of crustacean animals comprises less than 10’000 crustacean animals, preferably less than 1’000 crustacean animals, more preferably less than 100 crustacean animals, even more preferably from 2 to 50 crustacean animals. Choosing a group of this size ensures high reproducibility and consistently in the quality of the resulting chitosan, while still being large enough to ensure high output of exuviae and to ensure longterm survival of the group.

[0062] It was also found to be advantageous to control a nutrition fed to the crustacean animals. For example, feeding the crustacean animals essentially the same nutrition each day can be beneficial, e.g. to ensure a homogenous chemical composition of the exuviae. In other words, the nutrition fed to the crustacean animals during the rearing may preferably be kept essentially constant.

[0063] Further preferred rearing conditions are described in patent application WO 2024 / 115526 A1 , which is incorporated herein by reference in its entirety. The reference extends, in particular, to the rearing conditions. If any definitions or specifications set forth in the present disclosure conflict with any definitions or specifications provided in WO 2024 / 115526 A1 , the definitions and specifications of the present disclosure shall prevail.

[0064] Depending on the application, different parts of the exoskeleton may be used as exuviae. For example, a cephalothorax portion and / or an abdomen portion and / or an appendages portion may be used. In typical embodiments, the exuviae each comprise a cephalothorax portion. In some embodiments, the exuviae additionally comprise an abdomen portion and / or an appendages portion.P25951 PCOO February 2026

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[0066] Depending on the application, the exuviae may have different physical or chemical characteristics. In some embodiments, it was found to be advantageous if the obtained exuviae have a protein content of less than 20 wt.-%, preferably less than 15 wt.-%, more preferably less than 10 wt.-%, even more preferably from 5 wt.-% to 9 wt.-%. Alternatively or in combination, the obtained exuviae may have a chitin content of at least 10 wt-%, preferably from 14 wt-% to 17 wt-%. Alternatively or in combination, the obtained exuviae may have a water content from 55 wt-% to 65 wt-%. Alternatively or in combination, the obtained exuviae may have a mineral content from 36 wt-% to 39 wt-%. It is understood that these ranges refer to the exuviae before the step of demineralization and deproteinization.

[0067] Crustacean Animals

[0068] Depending on the application, different crustacean animals may be used in the method disclosed herein.

[0069] In some embodiments, the group of crustacean animals belong to the phylum Anthropoda, preferably to the class Malacostraca, more preferably to the order Decapoda, even more preferably to the suborder Pleocyemata, even more preferably to the clade Reptantia.

[0070] In some embodiments, the group of crustacean animals belong to one of the following infraorders: achelata, polychelida or astacidea. Preferably, the group of crustacean animals belong to the infraorder achelata.

[0071] Within this infraorder, the crustacean animals can belong to different families. For example, in some embodiments, the group of crustacean animals belong to the family Palinuridae, preferably to the genus Palinurus.

[0072] In some embodiments, the group of crustacean animals belong to one or more of the following species: Palinurus elephas, Palinurus japonicas, Palinurus homarus, Palinurus strimp-soni, Palinurus guttatus, Palinurus versicolor, Palinurus omatus, Palinurus Jasus, PalinurusP25951 PCOO February 2026

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[0074] Justitia, Palinurus Linuparus, Palinurus Nupalirus. In a preferred embodiment, the group of crustacean animals belong to the species Palinurus elephas.

[0075] It has been found that the chitosan obtained by the method disclosed herein displays particularly advantageous properties if it is obtained from crustacean animals belonging to the infraorders achelata, polychelida and astacidea. In particular, certain quality characteristics of the chitosan (including e.g. molecular weight and high degree of deacetylation, as well as high solubility and formation of a colorless solution upon solublization) were achieved with high reproducibility and consistency across batched and across time. The high reproducibility was particularly pronounced for crustacean animals belonging to the family Palinuridae, particularly to the genus Palinurus and, within this genus, in particular for the species Palinurus elephas.

[0076] In further embodiments, at least some of the crustacean animals of the group of crustacean animals may be shrimps. For example, the crustacean animals may optionally be shrimps. In some embodiments, the crustacean animals belong to the suborder dendrobranchiata and / or pleocyemata.

[0077] Regardless of the specific species (or genus or family or order or class or phylum) to which the crustacean animals belong, it was found to be particularly advantageous that all individual animals of the group of crustacean animals belong to the species, genus, gamily, suborder or order. Thus, in some embodiments, all individual animals of the group of crustacean animals belong to the same order, preferably to the same suborder, more preferably to the same taxonomic family, even preferably to the same genus, even more preferably to the same species. Most preferably, all individual animals of the group of crustacean animals belong to the same genus (particularly to the genus Palinurus), preferably to the same species (particularly to the species Palinurus elephas). By using a group in which all individual animals belong to the same order, suborder, family, genus or species, the properties of the chitosan obtained from different batches display a high degree of consistency. In particular, the different batches consistently and reproducibly display a constant and consistently high molecularP25951 PCOO February 2026

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[0079] weight, a constant and consistently high degree of deacetylation, a constant and consistently high solubility, and consistent formation of a colorless solution upon dissolution. The consistency in chemical properties across batches was particularly pronounced if all individual animals belong to the same genus, preferably to the same species.

[0080] Demineralization, Deproteinization and Decolorization

[0081] The method described herein typically comprises at least a demineralizing step, a deproteinizing step and a deacetylating step. Optionally, the method may additionally comprise a decolorization step.

[0082] Depending on the application, these steps may be carried out in different orders. For example, in some embodiments, the steps of demineralizing, deproteinizing and optionally also decolorization are carried out before the deacetylation. The demineralization, deproteinization and the optional decolorization in turn may also be carried out in different orders. For example, the exuviae may optionally be demineralized before they are deproteinized, or the other way around. The decolorization may for example be performed after the demineralization. In preferred embodiments, the exuviae are subsequently demineralized, deproteinized and decolorized to provide the chitin.

[0083] In some embodiments, the obtained exuviae are ground, milled, crushed, chopped or otherwise reduced in size before demineralization and deproteinization. For example, it was found to be particularly advantageous to crush or ground the exuviae to an average size of less than 150 micrometers.

[0084] It was found to be particularly advantageous to pre-dry the obtained exuviae before they are ground, milled, crushed, chopped or otherwise reduced in size. For example, the obtained exuviae may be pre-dried at more than 35 °C, e.g. at 40 °C to 60 °C, such as 50 °C, for at least 2 hours, preferably for at least 5 hours, before the pre-dried exuviae are ground, milled, crushed, chopped or otherwise reduced in size. In preferred embodiments, the exuviae areP25951 PCOO February 2026

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[0086] pre-dried at 40 °C to 60 °C for at least 2 hours, preferably for at least 5 hours, more preferably for at least 10 hours. The pre-drying is advantageous because it ensures that the subsequent grounding, milling, crushing, chopping or otherwise reducing in size proceeds more efficiently and more smoothly. In a particularly preferred embodiments, the obtained exuviae are predried to a water content of less than or equal to 20 wt.-% before the pre-dried exuviae are ground, milled, crushed, chopped or otherwise reduced in size.

[0087] The demineralization step serves to remove calcium (e.g. in the form of calcium oxide or other calcium-containing minerals) from the exuviae. As such, it is understood that calcium, as used herein, includes calcium in different forms, e.g. calcium carbonate. Depending on the application, the demineralization may be performed in different ways. It has been found to be particularly advantageous for efficient and quantitative calcium removal to use a pH of 2.0 to 3.0, particularly 2.3 to 2.7. Thus, in some embodiments, the exuviae are demineralized at a pH in the range from 2.0 to 3.0, preferably from 2.3 to 2.7. In particular, it was found that no traces of calcite were left after demineralization when using a pH in this range.

[0088] Preferably, the exuviae are treated with a mineral acid such as HCI during the demineralization. In some embodiments, the mineral acid is added to the exuviae. This is advantageous for proper heat and foam management during the reaction. Preferably, the mineral acid is added to a suspension of the (preferably ground) exuviae in water.

[0089] Furthermore, it was found to be particularly advantageous to wash the demineralized exuviae obtained after treatment of the exuviae with acid (e.g. at a pH of 2.3 to 2.7) multiple times until all calcium salts (particularly CaCh formed during the reaction) have been washed out.

[0090] The deproteinization step serves to remove proteins from the exuviae. Depending on the application, it may be performed in different ways. For example, in some embodiments, the step of deproteinizing the exuviae comprises treatment of the exuviae with a base (e.g. NaOH, KOH or a mixture thereof). This base may for example have a concentration of at least 1 M, e.g. at least 2 M. Irrespective of the concentration, the exuviae may be treated withP25951 PCOO February 2026

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[0092] the base at a temperature of at least 40 °C, such as from 40 °C to 70 °C, for at least 3 h, preferably from 3 h to 10 h. Once again, it is understood that the deproteinization is preferably carried out after the demineralization step, but may in principle also be carried out before the demineralization step.

[0093] The decolorization step serves to decolorize the exuviae or the chitin, e.g. by removing colored impurities from the product. For example, after demineralization and deproteinization the exuviae, the resulting material is typically yellow, orange or red as a result of colored impurities. By contrast, chitin and chitosan in their purest form are essentially colorless, off-white or at most yellowish white. More specifically, chitin tends to be yellowish white, and chitosan tends to be off-white or essentially colorless. Different colored impurities may be present in the product. For example, the colored impurities may comprise or consist of pigments, such as astaxanthin.

[0094] It has been found that treatment of the chitin with an alcohol (e.g. ethanol) at a temperature of at least 40 °C for at least 1 hour is particularly suitable for efficient decolorization without causing any other chemical harm to the material. Thus, in some embodiments, the step of decolorizing the chitin before deacetylation, wherein the decolorization comprises treatment of the chitin with a C(1-4) alcohol, preferably ethanol, at a temperature of at least 40 °C, preferably from 40 °C to 65 °C, for a period of at least 1 hour, preferably from 1 hour to 3 hours. It is preferred to carry out the decolorization step described in the previous sentence on the chitin, after demineralization and after deproteinization. However, it is also possible to perform the decolorization step on the exuviae, e.g. after demineralization and before deproteinization.

[0095] C(1 -4) alcohol, as used herein, refers to a linear or branched alkane having 1 , 2, 3 or 4 carbon atoms and being substituted with at least one hydroxyl group, preferably with exactly one hydroxyl group.P25951 PCOO February 2026

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[0097] In some embodiments, the decolorization comprises at least two (e.g. exactly two) ethanol cleaning operations in which the chitin is treated with ethanol, preferably for 1 to 3 hours, e.g. for 2 hours.

[0098] Deacetylation

[0099] The deacetylation step serves to deacetylate chitin to provide the chitosan. Depending on the application, this step may be carried out in different ways. A typical challenge encountered in deacetylation of chitin is to choose conditions which are harsh enough to ensure efficient deacetylation, but which are also mild enough to avoid deleterious side-reactions, which may e.g. result in reduced molecular weight.

[0100] To strike this balance in an efficient and reproducible manner, it has been found that it is particularly advantageous to treat the chitin with concentrated NaOH for a defined period of time. Specifically, in some embodiments, the deacetylation of the chitin comprises treatment of the chitin with aqueous NaOH at a temperature from 95 °C to 120 °C, wherein the aqueous NaOH has a concentration of at least 15 M, preferably at least 17.5 M, more preferably at least 18.5 M. Preferably, the chitin is treated with aqueous NaOH having a concentration of at least 17.5 M, preferably at least 18.5 M, at a temperature from 100 °C to 120 °C, preferably from 105 °C to 115 °C. These reaction conditions have been found to be particularly advantageous for providing chitosan from crustacean animals belonging to the infraorders ache-lata, polychelida and astacidea, preferably acelata, in particular the family Palinuridae, e.g. the genus Palinurus.

[0101] Regarding the reaction time of the deacetylation, the deacetylation reaction is typically monitored and stopped as soon as a desired degree of deacetylation has been reached. In present disclosure, it has been found to be particularly advantageous to expose the chitin to concentrated NaOH at more than 95 °C for less than 4 hours. Thus, in some embodiments, during the deacetylation of the chitin, a maximum overall duration during which the chitin is treated with the NaOH at a temperature of more than 95 °C, particularly more than 100 °C,P25951 PCOO February 2026

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[0103] does not exceed 7 hours, preferably does not exceed 5 hours, more preferably does not exceed 4 hours. In particular, in some embodiments, during the deacetylation of the chitin, a maximum overall duration during which the chitin is treated with the NaOH (e.g. having a concentration of at least 17.5 M, such as at least 18.5 M) at a temperature of more than 100 °C, does not exceed 7 hours, preferably does not exceed 5 hours, more preferably does not exceed 4 hours. In a specific exemplary embodiment, during the deacetylation of the chitin, a maximum overall duration during which the chitin is treated with the NaOH having a concentration of at least 17.5 M at a temperature of more than 100 °C, does not exceed 7 hours, preferably does not exceed 5 hours, more preferably does not exceed 4 hours. In a more specific exemplary embodiment, during the deacetylation of the chitin, a maximum overall duration during which the chitin is treated with the NaOH having a concentration of at least 18.5 M at a temperature of 105 °C or more, does not exceed 7 hours, preferably does not exceed 5 hours, more preferably does not exceed 4 hours.

[0104] Depending on the application, the deacetylation may be carried out in a single step or in multiple steps. It has been found to be particularly advantageous to perform the deacetylation in at least two steps, preferably in two, three or four steps, more preferably in two steps.

[0105] In some embodiments, the deacetylation comprises:

[0106] - A first deacetylation step comprising treatment of the chitin with aqueous NaOH at a temperature from 95 °C to 120 °C for a period from 1.2 hours to 2.8 hours, preferably from 1.7 hours to 2.3 hours, wherein the aqueous NaOH of the first deacetylation step of the first deacetylation step has a concentration of at least 15 M, preferably at least 17.5 M, more preferably at least 18.5 M, to provide a partially deacetylated intermediate product;

[0107] - A second deacetylation step comprising treatment of the partially deacetylated intermediate product with aqueous NaOH at a temperature from 95 °C to 120 °C for a period from 1 .2 hours to 2.8 hours, preferably from 1 .7 hours to 2.3 hours, whereinP25951 PCOO February 2026

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[0109] the aqueous NaOH of the second deacetylation step has a concentration of at least 15 M, preferably at least 17.5 M, more preferably at least 18.5 M, to provide the chitosan.

[0110] Preferably, the first deacetylation step comprises treatment of the chitin with aqueous NaOH at a temperature from 100 °C to 120 °C for a period from 1.2 hours to 2.8 hours, preferably from 1.7 hours to 2.3 hours, wherein the aqueous NaOH of the first deacetylation step has a concentration of 18.5 M, to provide a partially deacetylated intermediate product. Alternatively or in combination, preferably, the second deacetylation step comprises treatment of the partially deacetylated intermediate product with aqueous NaOH at a temperature from 100 °C to 120 °C for a period from 1.2 hours to 2.8 hours, preferably from 1.7 hours to 2.3 hours, wherein the aqueous NaOH of the second deacetylation step has a concentration of at least 18.5 M, to provide the chitosan.

[0111] In a preferred embodiment, the deacetylation step consists of the two deacetylation steps described above.

[0112] The embodiments described in the previous paragraphs in the context of the deacetylation were found to be particularly advantageous to allow manufacturing the chitosan with high reproducibility and with minimal deviations across batches and / or across time. In particular, minimal deviations across batches and across time were observed with respect to the degree of deacetylation, the molecular weight, the solubility (i.e. the chitosan was consistently and reliably soluble across batches and time) and the colorless nature of the chitosan upon solubilization.

[0113] In particular, it was found that, surprisingly, the use of a base such as NOH with a relatively high concentration, e.g. at least 17.5 M, preferably at least 18.5 M, leads to a colorless and transparent product, while lower concentrations were found to lead to non-transparent, cloudy products. In particular, it was found that treatment with NaOH having a concentrationP25951 PCOO February 2026

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[0115] of at least 17.5 M, preferably at least 18.5 M, at a temperature of at least 100 °C, preferably from 100 °C to 120 °C, leads to formation of product having high transparency.

[0116] In some embodiments, the deacetylation comprises treatment of the chitin with NaOH, KOH or a mixture thereof in the presence if a C(1-6) alcohol, preferably isopropanol, methanol or ethanol, even more preferably isopropanol. In some embodiments, for example, the deacetylation comprises treatment of the chitin with NaOH having a concentration of at least 12 M, preferably at least 15 M, in the presence of isopropanol, methanol or ethanol, preferably isopropanol. In some embodiments of the embodiments described in the previous sentence, the NaOH has a concentration of less than 17 M. It was found that, surprisingly, the additional of isopropanol aids the deacetylation and may also allow the use of slightly less concentrated base, thereby allowing milder reaction conditions, ultimately leading to higher molecular weight product.

[0117] Depending on the application, a raw product of the deacetylation may be worked up and purified by filtration. In some cases, filter clogging may be observed, potentially due to formation of aggregate sediments. It was found that avoiding the filter residue to run dry is helpful to avoid renewed filter clogging. Thus, in some embodiments, a raw product obtained from the deacetylation is filtered, wherein a filter cake obtained during filtering is not allowed to run dry.

[0118] Irrespective of the risk of running dry of the filter cake, it was also found that first adding an alcohol such as ethanol to the filter cake before adding water may also be helpful to avoid future filter clogging. Thus, in some embodiments, an alcohol such as ethanol is added to a filter cake obtained from filtering a raw product obtained from the deacetylation.

[0119] Further Embodiments

[0120] One advantage of this method is that it allows manufacturing the chitosan with high reproducibility and with minimal deviations across batches and / or across time.P25951 PCOO February 2026

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[0122] Thus, in some embodiments, the exuviae used in the method for chitosan production were obtained from the same or different individual animals by molting at two different time points. For example, the exuviae use in the method may comprise a first batch obtained from molting of a first group of crustacean animals at a first time point, and a second batch obtained from molting of a second group of crustacean animals at a second time point.

[0123] In some embodiments, the first time point and the second time point are at least 1 month, preferably at least 2 months, apart. In some embodiments, the first time point and the second time point may even be at least 5 months, e.g. at least 8 months, apart. Alternatively or in combination, in some embodiments, the first group of crustacean animals and the second group of crustacean animals may share at least some individual animals belonging to the first group and to the second group. However, in further embodiments, the first group of crustacean animals and the second group of crustacean animals are distinct from each other, i.e. do not comprise crustacean animals belonging to the first group and to the second group.

[0124] Chitosan Produced According to the Method Disclosed Herein and Further Aspects

[0125] In a second aspect, the present disclosure provides the chitosan produced according to any of the embodiments of the method of the first aspect described herein.

[0126] One advantage of the chitosan is that the chitosan consistently and reproducibly realizes certain quality characteristics. The quality characteristics include, among others, a range of different properties, such as one or more of the following: a controlled (typically high) molecular weight, a controlled (typically high) degree of deacetylation, a low residual protein content, a low ash content, a low level of toxins such as endotoxins, a high solubility, preferably forming a colorless and clear solution, and a low metal content (particularly a low calcium content and / or a low heavy metal content). In particular, it is possible to obtain chitosan having high molecular weight and a high deacetylation degree in a reproducible fashion.P25951 PCOO February 2026

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[0128] In some embodiments, the chitosan has a degree of deacetylation of at least 65%, preferably at least 75%, more preferably from 80% to 100%.

[0129] Alternatively or in combination, in some embodiments, the chitosan has a molecular weight of at least 300 kDa, such as from 300 kDa to 500 kDa.

[0130] In some embodiments, the chitosan has:

[0131] Either a degree of deacetylation of at least 90%, preferably from 94% to 100%, and a molecular weight of at least 300 kDa, preferably from 300 kDa to 400 kDa;

[0132] - Or a degree of deacetylation of at least 76%, preferably from 76% to 86%, and a molecular weight of at least 350 kDa, preferably from 350 kDa to 450 kDa.

[0133] Unless otherwise specified, molecular weights indicated herein generally refers to weight average molecular weights (Mw).

[0134] Depending on the application, the chitosan may have a molecular weight distribution of less than 2.2, preferably less than 2.0, more preferably from 1.2 to 2.0. The molecular weight distribution, as used herein, may e.g. be determined by the polydispersity index, i.e. the ratio of weight-average molecular weight (Mw) to number-average molecular weight (Mn).

[0135] Alternatively or in combination, the chitosan may have an ash content of less than 2 wt.-%, preferably less than 1 wt.-%.

[0136] Alternatively or in combination, the chitosan may have a residual protein content of less than 2 wt.-%, preferably less than 1 wt.-%, even more preferably less than 0.5 wt.-%.P25951 PCOO February 2026

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[0138] Alternatively or in combination, the chitosan may be soluble in 0.06 M aqueous HCI in an amount of at least 100 mg chitosan per 10 mL of aqueous HCI, preferably forming a clear colorless solution.

[0139] In a third aspect, the present disclosure provides the use of the chitosan of the second aspect for use in one or more of the following:

[0140] - Wound healing;

[0141] - As inhaling agents for treating lung infections, wherein preferably the chitosan is present in the inhaling agent in the form of nanoparticles;

[0142] - As hydrogel implant for regeneration of spinal cord tissue after injury;

[0143] - As nanoparticles, e.g. for drug delivery.

[0144] In a further aspect, the present disclosure provides the chitosan of any of the embodiments of the second aspect disclosed herein, for use in the treatment of a wound and / or in the treatment of lung infections and / or for regeneration of spinal cord tissue after injury. In the case of spinal cord tissue regeneration, preferably, a patient is treated with the chitosan in the form of a hydrogel implant. In the case of treatment of lung infections, preferably, a patient is treated with an inhaling agent comprising the chitosan, wherein the chitosan may e.g. be present in the inhaling agent in the form of nanoparticles.

[0145] In a fourth aspect, the present disclosure provides the use of exuviae from crustaceans for producing chitin and / or chitosan. Preferred embodiments with respect to the exuviae and / or to the crustaceans are disclosed herein in the context of the first aspect and also apply to this fourth aspect, i.e. they are also embodiments of this fourth aspect.P25951 PCOO February 2026

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[0147] It is to be understood that both the foregoing general description and the following detailed description present embodiments, and are intended to provide an overview or framework for understanding the nature and character of the disclosure. The description of preferred embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the disclosure.

[0148] BRIEF DESCRIPTION OF THE FIGURES

[0149] The disclosure described herein will be more fully understood from the detailed description given herein below and the accompanying drawings which should not be considered limiting to the invention described in the appended claims. The drawings show:

[0150] Fig. 1 Shows the comparison of an X-ray powder diffraction analysis of a sample obtained after deproteination with reference chitosan;

[0151] Fig. 2 Shows the comparison of an X-ray powder diffraction analysis of a sample obtained after the first respectively second deacetylation with reference chitosan;

[0152] Fig. 3 Fig. 3 shows the results of a dissolution test.

[0153] DESCRIPTION OF AN EXEMPLARY EMBODIMENT

[0154] In the following, an exemplary process for manufacturing chitosan is described.

[0155] 1 : Obtaining the exuviae

[0156] The exuviae were obtained by collecting the molts of spiny lobsters of the species Palinurus elephas. The lobsters were allowed to molt naturally and the molts were collected afterwards.P25951 PCOO February 2026

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[0158] The molts were cryo-milled, thereby giving a beige powder. The powder was dried in an oven at 50 °C under partial vacuum overnight. A total of 400 g (dry mass) of exuviae was used in the following process.

[0159] Step 2: Demineralization

[0160] The starting material (cryo-milled and dried exuviae) was poured in a 4 L of distillated water (10:1 ratio). A brown suspension was formed (pH = 9.8). 400 mL of hydrochloric acid (34%) were then dosed. Foam appears at circa pH = 6.5. This reverse addition was found useful for proper foam and heat management.

[0161] The pH was then adjusted with HCI to reach the target22.3 < pH < 2.7. The reaction medium was stirred for 1 h in this window.

[0162] The resulting suspension was filtered on a sintered under vacuum. No difficulty was observed during this step. The cake obtained was washed twelve times with 800 mL of water at 40±5°C (i.e. 20-25°C). The last wash was monitored at pH = 3.6.

[0163] Results:

[0164]

[0165] An X ray powder diffraction analysis was performed on the dried sample and compared to a chitin sample reference (DA >90%). The profile obtained was compliant with pure chitin. No trace of calcium carbonate was identified, confirming the good elimination via the acid treat-ment.P25951 PCOO February 2026

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[0167] Step 3: Deproteination

[0168] Humid powder demineralized was introduced in a sodium hydroxide 2.5M solution, ratio 10:1, at room temperature. A brown suspension was formed. Reaction medium was heated at 50±3°C and maintained under stirring for 7h. No significant aspect evolution was observed during the treatment. Then the suspension was cooled down to 20-25°C and filtered on sintered filter under vacuum. The cake was washed six times with water (1 vol).

[0169]

[0170] X-ray powder diffraction (XRPD) analysis was performed on a dried sample. As shown in Fig. 1, the profile of the dried sample obtained (labelled as “sample from experimental ex-ample” in Fig. 1) is similar to and matches with the profile of reference chitin (labelled as “chitin reference” in Fig. 1).

[0171] Step 4: Depigmentation

[0172] The wet powder deproteinated was suspended in ethanol (99%) (ratio 20:1 ). The suspension formed was stirred for 2h at 50±3°C. Then the suspension was cooled down to 20-25°C and filtered on a sintered under vacuum (ca. 4 min of filtration on diameter = 12 cm sintered). Wet cake was washed four times with water (1 vol).

[0173] These operations were repeated two times.

[0174] Results:P25951 PCOO February 2026

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[0176]

[0177] Step 5: Deacetylation

[0178] I) First deacetylation

[0179] The wet powder depigmented is suspended in sodium hydroxide 50% (i.e. 1230 mL) at 20-25°C. Then the suspension is heated to 105°-110°C and maintained at this temperature for 2h. Suspension is then cooled down at 20-25°C and filtered. The cake obtained was washed twice with 800 mL of water.

[0180] 315 g of wet cake were obtained. The solid was then engaged in a second deacetylation, according to the same process.

[0181] ii) Second deacetylation

[0182] The humid cake obtained (=365 mL) was washed 8 times with 730 ml of water. In order to improve the effectiveness of the washes (i.e. discoloration and purge of impurities), the 4th wash was carried out in re-slurry. The product was mechanically stirred in suspension in the washing water on the sintered for 5 min. The other washes were operated by displacement (i.e. piston). The cake was drained after the last wash. The cake was then dried in oven at 50°C under partial vacuum.

[0183] Results:P25951 PCOO February 2026

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[0185]

[0186] Global yield of the process: ca. 11w% (on the dry)

[0187] Samples of both deacetylation steps were dried in an oven at 50°C under partial vacuum overnight, and analyzed by X ray powder diffraction, as shown in Fig.2. The profile observed for the dried sample (labelled as “sample from experimental example after first deacetylation” respectively “sample from experimental example after second deacetylation”) matched with the profile of a reference sample of chitosan (labelled in Fig. 2 as “chitosan reference”).

[0188] Analysis of the chitosan product and of some intermediates

[0189] The water content and ash content of some intermediates and of the chitosan product ob-tained after the first and second deacetylation steps are summarized in the table below. It is understood that in this and the following tables, “demi” refers to the product obtained from the demineralization, “deprot” refers to the product obtained from the deproteination, “deal” refers to the product obtained from the first deacetylation step, and “dea2” refers to the product obtained from the second deacetylation step.

[0190]

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[0193] Furthermore, the degree of acetylation was determined by1H NMR. The results are summarized in the table below.

[0194]

[0195] Furthermore, the molar mass distribution was determined. The results are summarized in the table below.

[0196]

[0197] Optimization of the deacetylation conditions

[0198] Three different deacetylation protocols were tested (tests i-iii), involving different starting materials to be subjected to the deacetylation, and different deacetylation conditions. The three tests are summarized in the following table:

[0199]

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[0202]

[0203] Apart from the differences shown in the table, the samples were treated under otherwise identical conditions. The results observed for the three tests are also summarized in the table (fourth and fifth column): good conversion (as monitored by XRPD) was observed only for tests ii) and iii), and a dissolution test was only passed for test iii).

[0204] The results of the dissolution tests are also illustrated in Fig. 3, which shows:

[0205] - On the left side the flask resulting from test i);

[0206] - In the middle the flask resulting from test iii); and

[0207] - On the right side the flask resulting from test ii).

[0208] As shown in Fig. 3, the conditions of test iii) yield a colorless and transparent production solution, whereas the products obtained from tests i) and ii) are cloudy.

Claims

P25951 PCOO February 202629 / 33PATENT CLAIMS1 . Method of producing chitosan, the method comprising the steps of:a. obtaining exuviae from a group of crustacean animals, preferably belonging to one or more of the following infraorders: achelata, polychelida and astacidea;b. demineralizing and deproteinizing the exuviae to provide chitin; andc. deacetylating the chitin to provide chitosan.

2. The method according to claim 1 , wherein the group of crustacean animals belong to the infraorder achelata, preferably to the family Palinuridae, more preferably to the genus Palinurus.

3. The method according to any one of the previous claims, wherein the exuviae are obtained by molting of live crustacean animals.

4. The method according to any one of the previous claims, further comprising the step of rearing the group of crustacean animals before the step of obtaining their exuviae, wherein the crustacean animals are preferably reared in a semi-closed or closed aquaculture system.

5. The method according to claim 4, wherein during rearing of the group of crustacean animals, at least two, preferably at least three, more preferably at least four, of the following water parameters are monitored and maintained within the respective ranges:a. temperature: from 16 °C to 22 °C;P25951 PCOO February 202630 / 33b. pH level: from 7.8 to 8.2;c. oxygen concentration: from 5 ppm to 7 ppm;d. calcium concentration: from 400 ppm to 420 ppm;e. magnesium concentration: from 1200 ppm to 1400 ppm;f. ammonium concentration: less than 0.1 ppm;g. salinity: from 28 per thousand to 37 per thousand.

6. The method according to any one of the previous claims, wherein the exuviae are demineralized at a pH in the range from 2.0 to 3.0, preferably from 2.3 to 2.7.

7. The method according to any one of the previous claims, wherein the deacetylation of the chitin comprises treatment of the chitin with aqueous NaOH at a temperature from 95 °C to 120 °C, wherein the aqueous NaOH has a concentration of at least 15 M, preferably at least 17.5 M, more preferably at least 18.5 M.

8. The method according to claim 7, wherein during the deacetylation of the chitin, a maximum overall duration during which the chitin is treated with the NaOH at a temperature of more than 95 °C, particularly more than 100 °C, does not exceed 7 hours, preferably does not exceed 5 hours, more preferably does not exceed 4 hours.

9. The method according to any one of claims 7 or 8, wherein the deacetylation of the chitin comprises:a. A first deacetylation step comprising treatment of the chitin with aqueous NaOH at a temperature from 95 °C to 120 °C for a period from 1.2 hours toP25951 PCOO February 202631 / 332.8 hours, preferably from 1.7 hours to 2.3 hours, wherein the aqueous NaOH of the first deacetylation step has a concentration of at least 15 M, preferably at least 17.5 M, more preferably at least 18.5 M, to provide a partially deacetylated intermediate product;b. A second deacetylation step comprising treatment of the partially deacetylated intermediate product with aqueous NaOH at a temperature from 95 °C to 120 °C for a period from 1.2 hours to 2.8 hours, preferably from 1.7 hours to 2.3 hours, wherein the aqueous NaOH of the second deacetylation step has a concentration of at least 15 M, preferably at least 17.5 M, more preferably at least 18.5 M, to provide the chitosan.

10. The method according to any one of the previous claims, further comprising the step of decolorizing the chitin before deacetylation, wherein the decolorization comprises treatment of the chitin with a C(1-4) alcohol, preferably ethanol, at a temperature of at least 40 °C, preferably from 40 °C to 65 °C, for a period of at least 1 hour, preferably from 1 hour to 3 hours.

11. The method according to any one of the previous claims, wherein the crustacean animals belong to one or more of the following species: Palinurus elephas, Palinurus japonicas, Palinurus homarus, Palinurus strimpsoni, Palinurus guttatus, Palinurus versicolor, Palinurus omatus, Palinurus Jasus, Palinurus Justitia, Palinurus Linuparus, and Palinurus Nupalirus, preferably Palinurus elephas.

12. The method according to any one of the previous claims, wherein all individual animals of the group of crustacean animals belong to the same order, preferably to the same suborder, more preferably to the same taxonomic family, even preferably to the same genus, even more preferably to the same species.P25951 PCOO February 202632 / 3313. The method according to any one of the previous claims, wherein the group of crustacean animals comprises less than 10’000 crustacean animals, preferably less than 1 ’000 crustacean animals, more preferably less than 100 crustacean animals, even more preferably from 2 to 50 crustacean animals.

14. Chitosan produced according to the method of any one of the previous claims.

15. The chitosan according to claim 14, wherein the chitosan has a degree of deacetylation of at least 65%, preferably at least 75%, more preferably from 80% to 100%.

16. The chitosan according to claim 15, wherein the chitosan has:a. Either a degree of deacetylation of at least 90%, preferably from 94% to 100%, and a molecular weight of at least 300 kDa, preferably from 300 kDa to 400 kDa;b. Or a degree of deacetylation of at least 76%, preferably from 76% to 86%, and a molecular weight of at least 350 kDa, preferably from 350 kDa to 450 kDa.

17. The chitosan according to any one of claims 14-16, wherein the chitosan has a molecular weight distribution of less than 2.2, preferably less than 2.0, more preferably from 1 .2 to 2.0.

18. The chitosan according to any one of claims 13-17, wherein the chitosan is soluble in 0.06 M aqueous HCI in an amount of at least 100 mg chitosan per 10 mL of the aqueous HCI, preferably forming a clear colorless solution.

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