Chitosan

PROTASAN Ultrapure Chitosan

chitosan_logo

Chitosan is a biopolymer with a wide range of biomedical applications, including wound healing, tissue engineering and tissue repair, and local delivery of cells, drugs, proteins, genes, and other therapeutics. Currently, nasal drug delivery technology using chitosan to speed the absorption of the API shows great potential, with several applications in clinical trials. Non-viral gene delivery systems using chitosan as a carrier are also the subject of intense research activity.

Chemistry

Chitosan is a linear polysaccharide composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated unit; D) and N-acetyl-D-glucosamine (acetylated unit; A).

Chitosan is produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (crabs, shrimps, etc.). The degree of deacetylation (%DA) can be determined by NMR spectroscopy. Variation in the %DA can lead to varying functionality of chitosans in different applications.

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PROTASAN UP Chitosan Chloride and Chitosan Glutamate Salts are readily soluble in water. The viscosity of a chitosan solution primarily depends on the average molecular weight of the polymer, which can be determined by size exclusion chromatography combined with light scattering detection. The amino group in chitosan has a pKa value of ~6.5, while the charge density is dependent upon the solution pH and the degree of chitosan deacetylation. As a positively charged biopolymer, chitosan is bioadhesive and readily binds to negatively charged surfaces such as mucosal membranes. Chitosan enhances the transport of polar drugs across epithelial surfaces, and is biocompatible and biodegradable.

Lab_Chitosan

Quality

PROTASAN™ UP Chitosans from NovaMatrix® are a series of ultrapurified and well-characterized water-soluble Chitosan Chloride and Glutamate Salts. There are different viscosities and %DA values developed for use in biomedical and pharmaceutical applications.

For our ultrapure sodium alginates, chitosan salts, and sodium hyaluronate, we maintain Drug Master Files with the US FDA.

NovaMatrix® products are intended to be used in applications which require high and consistent quality provided by our manufacturing, Quality Control and Quality Assurance.

NovaMatrix® manufacture PRONOVA™ sodium alginate and PROTASAN™ chitosan salts in our facilities in Norway. We operate according to GMP guidelines; ICH Q7, ICH Q1, ICH Q2 and ISO standards; ISO 9001:2008 and ISO 13485:2003. Furthermore NovaMatrix® holds the Manufacturing License for API’s issued by the Norwegian Medicine Agency (NOMA).

Safety and Toxicology

PROTASAN™ ultrapure chitosan salts are manufactured in accordance with ICH-Q7 GMP guidelines, NS-EN ISO 9001:2000 (Quality Management System) and ISO 13485:2003 (Medical Device Directive) standards. Ultrapure (UP) implies that the products contain low levels of residual endotoxin. The current specification is <100 EU/g for the PROTASAN™ UP G and PROTASAN™ UP CL.

PROTASAN™ chitosans are ideal for a wide variety of pharmaceutical, biomedical, biotechnology and tissue engineering applications. The safety and toxicology profile of the ultrapure PROTASAN™ products are described in Drug Master Files submitted to the US FDA. The table below is meant to serve as a summary over some of the studies performed and the conclusions drawn from them.

Effect of chitosan salts on cell survival of V-79 and 3T3 cells cultured in vitro

Report number : ALG-95-001

  • Animal species : In vitro cell culture 3T3 mouse fibroblasts V79 Chinese hamster
  • Chitosan products : PROTASAN™ UP G 213 (84 mPas), PROTASAN™ UP CL 113 (12 mPas)
  • Concentration : 0-1 mg/ml – 24 hr exposure
  • Conclusion : Little or no effect at up to 1 mg/ml. Reduction in cell survival by 15% (CL) and 35% (G) at 5 mg/ml.

Single dose toxicity study by the intraperitoneal route

Report number : 658/525

  • Animal species : Mouse
  • Chitosan products : PROTASAN™ UP G 213 (84 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/526

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 213 (84 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/527

  • Animal species : Mouse
  • Chitosan products : PROTASAN™ UP CL 113 (12 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/528

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP CL 113 (12 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.

13 week oral (gavage) toxicity study

Report number : 658/539

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 100, 300, 600 mg/kg
  • Conclusion : No treatment related deaths. No differences in body weight nor food consumption. No abnormal clinical signs, normal weight increase.

7 day intranasal tolerance

Report number : 658/540

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 0.5 and 1 mg admin. x 3 per day
  • Conclusion : No mortality during study. Treated animals have some increase in mucus production, no clear dose-effect seen. No abnormal clinical signs, normal weight increase.

Single dose toxicity study by the intravenous route

Report number : 658/538

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 25 mg/kg
  • Conclusion : No mortality was observed. Subdued behaviour up to 30 minutes after injection. Treated animals lost weight between days 1 and 2, thereafter weight gains similar to control.
Report number : 658/543

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 113 (2.8 mPas)
  • Concentration : 20, 50, 100 mg/kg in preliminary, 25 mg/kg in main study
  • Conclusion : No mortality at 20 or 25 mg/kg, 2/2 and 1/2 deaths at 50 and 100 mg/kg, resp. Lower body weight on day 1-2, same as control after.
Report number : 658/557

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP CL 113 (5 mPas)
  • Concentration : 25, 50, 100 mg/kg in preliminary, 50 mg/kg in main study
  • Conclusion : No mortality at 25 and 50 mg/kg in preliminary study. Mortality was 1/10 treated animals in main study at 50 mg/kg.

Sensitizing potential in the guinea pig. Magnusson & Kligman test (GPMT)

Report number : 658/541

  • Animal species : Guinea pig
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 1 mg/kg intradermal, 60 mg/ml topical occlusive induction. 60 mg/ml topical challenge
  • Conclusion : No mortality observed. Body weight not influenced. Induction: moderate irritation at injection site. After challenge no sign of delayed hyper- sensitivity.
Report number : 658/556

  • Animal species : Guinea pig
  • Chitosan products : PROTASAN™ UP CL 113, (5 mPas)
  • Concentration : 0.5 mg/ml intradermal, 60 mg/ml topical occlusive induction. 60 mg/ml topical occlusive challenge
  • Conclusion : No mortality. No influence on body weight. Signs of irritation were noted during induction. No delayed hyper- sensitivity in test animals.

Evaluation of the potential to induce immediate hyper- sensitivity: induced anaphylactic shock

Report number : 658/542

  • Animal species : Guinea pig
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 10 mg/kg subcutaneous induction, 20 mg/kg intra- venous challenge
  • Conclusion : No mortality observed. Body weight similar in all groups. Cyanosis noted in 5 treated animals within 1 hr of challenge, cleared by 4 hr.

Baterial reverse mutation test

Report number : 10396

  • Animal species : Plate incorpora- tion
  • Chitosan products : PROTASAN™ UP G 213 (75 mPas)
  • Concentration : ≤5000 mg
  • Conclusion : No significant increase in numbers of revertants. 5000 μg/plate toxic to TA98 and TA 1537 (± activation).

Acute dermal irritation test

Report number : 507340

  • Animal species : Rabbits
  • Chitosan products : PROTASAN™ UP B 80/20 and 80/500
  • Concentration : 0.5 g
  • Conclusion : No edema or erythema. Non-irritant.

Local lymph node assay (hypersensitivity)

Report number : 507356

  • Animal species : Mouse
  • Alginate products : PROTASAN™ UP B 80/20 and 80/500
  • Concentration : 1, 2.5, 5% – 0.5, 1, 2%
  • Conclusion : No adverse clinical signs. No Simulation Index over 3 = no sensitizing effect.
Report number : ALG-95-001

  • Animal species : In vitro cell culture 3T3 mouse fibroblasts V79 Chinese hamster
  • Chitosan products : PROTASAN™ UP G 213 (84 mPas), PROTASAN™ UP CL 113 (12 mPas)
  • Concentration : 0-1 mg/ml – 24 hr exposure
  • Conclusion : Little or no effect at up to 1 mg/ml. Reduction in cell survival by 15% (CL) and 35% (G) at 5 mg/ml.
Report number : 658/525

  • Animal species : Mouse
  • Chitosan products : PROTASAN™ UP G 213 (84 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/526

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 213 (84 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/527

  • Animal species : Mouse
  • Chitosan products : PROTASAN™ UP CL 113 (12 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/528

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP CL 113 (12 mPas)
  • Concentration : 100, 250, 500 mg/kg
  • Conclusion : No mortality. No abnormal clinical signs, normal weight increase.
Report number : 658/539

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 100, 300, 600 mg/kg
  • Conclusion : No treatment related deaths. No differences in body weight nor food consumption. No abnormal clinical signs, normal weight increase.
Report number : 658/540

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 0.5 and 1 mg admin. x 3 per day
  • Conclusion : No mortality during study. Treated animals have some increase in mucus production, no clear dose-effect seen. No abnormal clinical signs, normal weight increase.
Report number : 658/538

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 25 mg/kg
  • Conclusion : No mortality was observed. Subdued behaviour up to 30 minutes after injection. Treated animals lost weight between days 1 and 2, thereafter weight gains similar to control.
Report number : 658/543

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP G 113 (2.8 mPas)
  • Concentration : 20, 50, 100 mg/kg in preliminary, 25 mg/kg in main study
  • Conclusion : No mortality at 20 or 25 mg/kg, 2/2 and 1/2 deaths at 50 and 100 mg/kg, resp. Lower body weight on day 1-2, same as control after.
Report number : 658/557

  • Animal species : Rat
  • Chitosan products : PROTASAN™ UP CL 113 (5 mPas)
  • Concentration : 25, 50, 100 mg/kg in preliminary, 50 mg/kg in main study
  • Conclusion : No mortality at 25 and 50 mg/kg in preliminary study. Mortality was 1/10 treated animals in main study at 50 mg/kg.
Report number : 658/541

  • Animal species : Guinea pig
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 1 mg/kg intradermal, 60 mg/ml topical occlusive induction. 60 mg/ml topical challenge
  • Conclusion : No mortality observed. Body weight not influenced. Induction: moderate irritation at injection site. After challenge no sign of delayed hyper- sensitivity.
Report number : 658/556

  • Animal species : Guinea pig
  • Chitosan products : PROTASAN™ UP CL 113, (5 mPas)
  • Concentration : 0.5 mg/ml intradermal, 60 mg/ml topical occlusive induction. 60 mg/ml topical occlusive challenge
  • Conclusion : No mortality. No influence on body weight. Signs of irritation were noted during induction. No delayed hyper- sensitivity in test animals.
Report number : 658/542

  • Animal species : Guinea pig
  • Chitosan products : PROTASAN™ UP G 213 (91 mPas)
  • Concentration : 10 mg/kg subcutaneous induction, 20 mg/kg intra- venous challenge
  • Conclusion : No mortality observed. Body weight similar in all groups. Cyanosis noted in 5 treated animals within 1 hr of challenge, cleared by 4 hr.
Report number : 10396

  • Animal species : Plate incorpora- tion
  • Chitosan products : PROTASAN™ UP G 213 (75 mPas)
  • Concentration : ≤5000 mg
  • Conclusion : No significant increase in numbers of revertants. 5000 μg/plate toxic to TA98 and TA 1537 (± activation).
Report number : 507340

  • Animal species : Rabbits
  • Chitosan products : PROTASAN™ UP B 80/20 and 80/500
  • Concentration : 0.5 g
  • Conclusion : No edema or erythema. Non-irritant.
Report number : 507356

  • Animal species : Mouse
  • Alginate products : PROTASAN™ UP B 80/20 and 80/500
  • Concentration : 1, 2.5, 5% – 0.5, 1, 2%
  • Conclusion : No adverse clinical signs. No Simulation Index over 3 = no sensitizing effect.

Regulatory Status
PROTASAN™ chitosan chloride meets the standards set forth in the European Pharmacopoeia (EP 1774). PROTASAN™ chitosan products satisfy ASTM F 2103 for use in tissue engineered medical products (TEMPs). PROTASAN™ chitosan products are manufactured in compliance with current Good Manufacturing Practice and described in a DMF submitted to the US FDA.

Stability and Shelf Life

Chitosan is a polycationic polysaccharide commercially obtained by de-N-acetylation of chitin, which is commonly found in the exoskeleton of crustaceans. It is a linear, binary copolymer, consisting of (1→4)-linked 2-acetamido-2-deoxy-β-D-glucopyranose (GlcNAc, A-unit) and 2-amino-2-deoxy-β-D-glucopyranose (GlcN, D-unit).

chitosan_logo

Figure 1: Representative structure of chitosan, showing monomer identities, and linkage types.

For most applications of chitosans, the degree of deacetylation (fraction or percentage of D-units; %DA) and the molar mass (molecular weight) are important parameters determining the functionality of the chitosan for that specific application. The amino group in chitosan has a pK-value of around 6.5, and the charge density is dependent upon the solution pH and the %DA. Chitosan is, therefore, a polycation, and will normally be soluble under slightly acidic conditions. Aqueous solutions of chitosans are viscous, the observed solution viscosity being dependent on molar mass, %DA, concentration of chitosan, temperature, and potentially also presence of salts and other formulation or environmental parameters. Viscosity measurements are often used as a simple measurable parameter of changes in the molar mass of a polymer, and thereby can be used as a stability indicating parameter. This document reports on the stability, shelf-life and storage conditions for powders of PROTASAN™ UP chitosan salts.

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Depolymerization of polysaccharides will occur in solution and in the solid state due to a variety of mechanisms. Vårum et al. (2001) studied acid hydrolysis of chitosans of different chemical composition in dilute and concentrated HCl by viscometry. One finding of this study was that the specificity of the acid-catalyzed cleavage of different glycosidic linkages was such that A-A and A-D linkages were cleaved about three orders of magnitude faster than D-A and D-D linkages. Holme et al. (2001) showed that acid hydrolysis was the primary depolymerization mechanism of chitosan chlorides in the solid state, with similar specificity as observed by Vårum et al. (2001). Holme et al. (2008) studied kinetics and depolymerization mechanisms of 1% solutions of chitosans, and showed that oxidative-reductive depolymerization (ORD) was the primary mechanism as long as transition metal ions are present in the sample, while acid hydrolysis is the primary mechanism for highly purified samples. Consequently, high purity chitosan has enhanced stability as compared to less purified products, and high %DA and low pH (in solution) is preferred for maximum stability.

NovaMatrix® conducts stability studies according to ICH guidelines for all our product ranges. For PROTASAN™ UP chitosan salts, the parameters monitored are appearance of powder, dry matter content, intrinsic viscosity, apparent viscosity, pH, molar mass, %DA, chloride/glutamate content, ash content, endotoxins, and microbial purity.

Apparent viscosity, intrinsic viscosity, and molar mass, which are all indicative measurements of depolymerization, have been observed to change significantly upon time under certain storage conditions. Figure 2 shows example data for the apparent viscosity (measured in 1% (w/w) chitosan salt solution at 20°C using a Brookfield viscometer at 20 rpm) from stability studies conducted on PROTASAN™ UP CL 213 (chitosan chloride, %DA=86%) and PROTASAN™ UP G 213 (chitosan glutamate, %DA 83%). No significant change in apparent viscosity was apparent after 5 years of storage at 2-8°C/ambient relative humidity (RH), or at -18°C. Higher storage temperatures, such as 25°C/60% RH and 40°C/75% RH, show decreases in apparent viscosity over time. The decrease in apparent viscosity, and therefore molecular weight, is especially marked when the product is stored at an elevated temperature such as 40°C/75% RH.

chistosan-tab

Figure 2: Apparent viscosity values observed during 60 months of storage of PROTASAN™ UP CL 213.

Based upon stability studies, NovaMatrix recommends refrigerated storage (2-8°C) of all PROTASAN™ UP salts. Shelf life of the PROTASAN™ UP salts are as following:
The PROTASAN™ UP CL213 and G213 have a shelf life of 5 years while our other PROTASAN™ UP products ( CL113, G113, CL114, CL214 and G214) have a shelf life of 3 years.

All PROTASAN™ UP chitosan salts are delivered in a closed polyethylene container. During storage, the lid of the container should be kept closed.
Our stability programs show that PROTASAN™ UP chitosan salts can be stored at room temperature (25°C/ 60% RH) for at least 6 months without significant reduction of apparent viscosity. Therefore, we do not consider cool/cold shipment of products to customers a necessity, as transportation and transit times are normally a matter of a few days.
PROTASAN™ UP specifications with respect to apparent viscosity are intended to be broad and suitable for a range of applications. We can offer customer-dedicated specifications if required.

References:

  • Vårum, K.M., Ottøy, M.H., and Smidsrød, O. “Acid hydrolysis of chitosans”, Carbohydr. Polym. 46 (2001), p89-98.
  • Holme, H.K., Foros, H., Pettersen, H.,Dornish, M. and Smidsrød, O. ”Thermal depolymerization of chitosan chloride”, Carbohydr. Polym. 46 (2001), p287-294.
  • Holme, H.K., Davidsen, L., Kristiansen, A. and Smidsrød, O. ”Kinetics and mechanisms of depolymerization of alginate and chitosan in aqueous solution”, Carbohydr. Polym. 73 (2008), p656-664.

Application examples

  • Biomedical applications: wound healing, tissue engineering and tissue repair
  • Local delivery of cells, drugs, proteins, genes, and other therapeutics
  • Nasal drug delivery
  • Non-viral gene delivery systems using chitosan as a carrier
Please select the product to obtain more informations or order

PROTASAN UP CL 113

  • Viscosity  [mPa*s] :  < 20
  • Appr. Mw [kDa] : < 200
  • DA [%] : 75-90
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

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PROTASAN UP CL 114

  • Viscosity  [mPa*s] :  < 20
  • Appr. Mw [kDa] : < 200
  • DA [%] : > 90
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

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PROTASAN UP CL 213

  • Viscosity  [mPa*s] :  20-200
  • Appr. Mw [kDa] : 150-400
  • DA [%] : 75-90
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

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PROTASAN UP CL 214

  • Viscosity  [mPa*s] :  20-200
  • Appr. Mw [kDa] : 150-400
  • DA [%] : > 90
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

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PROTASAN UP G 113

  • Viscosity  [mPa*s] :  < 20
  • Appr. Mw [kDa] : < 200
  • DA [%] : 75-90
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

Purchase the product

PROTASAN UP G 213

  • Viscosity  [mPa*s] :  20-200
  • Appr. Mw [kDa] : 200-600
  • DA [%] : 75-90
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

Purchase the product

PROTASAN UP G 214

  • Viscosity  [mPa*s] :  20-200
  • Appr. Mw [kDa] : 200-600
  • DA [%] : > 90
  • Endotoxins [EU/g] : ≤ 100
  • Total viable count [cfu/g] : ≤ 100

Purchase the product

Product

Viscosity [mPa-s]

Appr. M. [KDa]

DA [%]

Endotoxine [EU/g]

Total viable count [cfu/g]

>20

>200

75-90

≤ 100

≤ 100

>20

150-400

>90

≤ 100

≤ 100

20-200

150-400

75-90

≤ 100

≤ 100

20-200

150-400

>90

≤ 100

≤ 100

>20

<200

75-90

≤ 100

≤ 100

20-200

200-600

75-90

≤ 100

≤ 100

20-200

200-600

>90

≤ 100

≤ 100