1、Designation: F 2103 01 (Reapproved 2007)e1Standard Guide forCharacterization and Testing of Chitosan Salts as StartingMaterials Intended for Use in Biomedical and Tissue-Engineered Medical Product Applications1This standard is issued under the fixed designation F 2103; the number immediately followi
2、ng the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.e1NOTEFormatting and grammar w
3、ere corrected editorially throughout in April 2007.INTRODUCTIONBiopolymers from marine sources have been studied and used in commercial applications andproduct development for a number of years. Chitosan, a linear polysaccharide consisting ofglucosamine and N-acetyl glucosamine derived mainly from c
4、rustacean shells, has been used in manytechnical applications such as water purification (as a flocculant), in cosmetics, and recently as aproposed fat-binding weight control product. In solution, the cationic nature of chitosan gives thispolymer a mucoadhesive property. Chitosan salts can be used a
5、s a matrix or scaffold material as wellas in non-parenteral delivery systems for challenging drugs. Chitosan salts have been shown toincrease the transport of polar drugs across the nasal epithelial surface. The purpose of this guide isto identify key parameters relevant for the functionality and ch
6、aracterization of chitosan salts for thedevelopment of new commercial applications of chitosan salts for the biomedical and pharmaceuticalindustries.1. Scope1.1 This guide covers the evaluation of chitosan saltssuitable for use in biomedical or pharmaceutical applications,or both, including, but not
7、 limited to, tissue-engineered medi-cal products (TEMPS).1.2 This guide addresses key parameters relevant for thefunctionality, characterization, and purity of chitosan salts.1.3 As with any material, some characteristics of chitosanmay be altered by processing techniques (such as molding,extrusion,
8、 machining, assembly, sterilization, and so forth)required for the production of a specific part or device.Therefore, properties of fabricated forms of this polymershould be evaluated using test methods that are appropriate toensure safety and efficacy.1.4 This standard does not purport to address a
9、ll of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 2196 Test Methods
10、 for Rheological Properties of Non-Newtonian Materials by Rotational (Brookfield type) Vis-cometerF 619 Practice for Extraction of Medical PlasticsF 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and DevicesF 749 Practice for Evaluating Material Extracts by Intracu-taneous
11、 Injection in the RabbitF 756 Practice for Assessment of Hemolytic Properties ofMaterialsF 763 Practice for Short-Term Screening of Implant Mate-rialsF 813 Practice for Direct Contact Cell Culture Evaluation ofMaterials for Medical DevicesF 895 Test Method for Agar Diffusion Cell Culture Screen-ing
12、for CytotoxicityF 981 Practice for Assessment of Compatibility of Bioma-terials for Surgical Implants with Respect to Effect of1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomateria
13、ls and Biomolecules for TEMPs.Current edition approved Feb. 1, 2007. Published February 2007. Originallyapproved in 2001. Last previous edition approved in 2001 as F 2103 01.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For
14、Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Materials on Muscle and BoneF 1251 Terminology Relating to Polymeric Bio
15、materials inMedical and Surgical DevicesF 1439 Guide for Performance of Lifetime Bioassay for theTumorigenic Potential of Implant MaterialsF 1903 Practice for Testing For Biological Responses toParticles in vitroF 1904 Practice for Testing the Biological Responses toParticles in vivoF 1905 Practice
16、For Selecting Tests for Determining thePropensity of Materials to Cause ImmunotoxicityF 1906 Practice for Evaluation of Immune Responses InBiocompatibility Testing Using ELISA Tests, LymphocyteProliferation, and Cell Migration2.2 Ph. Eur. Document:Ph. Eur. Monograph Chitosan Chloride, Nov. 200032.3
17、ISO Documents:ISO 10993 Biological Evaluation of Medical Devices4ISO 10993-1 Biological Evaluation of Medical DevicesPart 1: Evaluation and Testing4ISO 10993-3Part 3: Tests for Genotoxicity, Carcinogenic-ity and Reproductive Toxicity4ISO 10993-9Part 9: Framework for Identification andQuantification
18、of Potential Degradation Products4ISO 10993-17Part 17: Methods for Establishment ofAllowable Limits for Leachable Substances Using Health-Based Risk Assessment4ISO 13408-1: 1998: Aseptic Processing of Health CareProductsPart 1: General Requirements42.4 ICH Documents:International Conference on Harmo
19、nization (1997) Guid-ance for Industry M3 Nonclinical Safety Studies for theConduct of Human Clinical Trials for Pharmaceuticals 62FR 629225International Conference on Harmonization (1996) Guide-line for Industry S2A Specific Aspects of RegulatoryGenotoxicity Tests for Pharmaceuticals 61 FR 181995In
20、ternational Conference on Harmonization (1997) Guid-ance for Industry S2B Genotoxicity: A Standard Batteryfor Genotoxicity Testing of Pharmaceuticals 62 FR624725International Conference on Harmonization (1994) Guide-line for Industry S5A Detection of Toxicity to Reproduc-tion for Medicinal Products
21、59 FR 487465International Conference on Harmonization (1996) Guid-ance for Industry S5B Detection of Toxicity to Reproduc-tion for Medicinal Products: Addendum on Toxicity toMale Fertility 61 FR 153605International Conference on Harmonization (1996) Guide-line for Industry S1A The Need for Long-term
22、 RodentCarcinogenicity Studies of Pharmaceuticals 61 FR 81535International Conference on Harmonization (1998) Guid-ance for Industry S1B Testing for Carcinogenicity ofPharmaceuticals 63 FR 89835International Conference on Harmonization (1995) Guide-line for Industry S1C Dose Selection for Carcinogen
23、icityStudies of Pharmaceuticals 60 FR 112785International Conference on Harmonization (1997) S1CRGuidance for Industry Addendum to Dose Selection forCarcinogenicity Studies of Pharmaceuticals: Addition of aLimit Dose and Related Notes 62 FR 642595International Conference on Harmonization (ICH) Q1A I
24、CHHarmonized Tripartite Guidance for Stability Testing ofNew Drug Substances and Products (September 23,1994)52.5 FDA Documents:FDA Guideline on Validation of the Limulus AmebocyteTest as an End-Product Endotoxin Test for Human andAnimal Parenteral Drugs, Biological Products and Health-care Products
25、 DHHS, December 19876FDA Interim Guidance for Human and Veterinary DrugProducts and Biologicals. Kinetic LAL Techniques-DHHS, July 15, 199162.6 ANSI Documents:ANSI/AAMI/ISO 11737-1: 1995 Sterilization of MedicalDevicesMicrobiological MethodsPart 1: Estimationof Bioburden on Product4ANSI/AAMI/ISO 117
26、37-2: 1998 Sterilization of MedicalDevicesMicrobiological MethodsPart 2: Tests of Ste-rility Performed in the Validation of a Sterilization Pro-cess42.7 AAMI Documents:AAMI TIR No. 191998: Guidance for ANSI/AAMI/ISO109937: 1995, Biological Evaluation of MedicalDevicesPart 7: Ethylene Oxide Steriliza
27、tion Residuals7AAMI/ISO 141601998: Sterilization of Single-UseMedical Devices Incorporating Materials of AnimalOriginValidation and Routine Control of Sterilizationby Liquid Chemical Sterilants7AAMI ST67/CDV-2: 1999: Sterilization of MedicalDevicesRequirements for Products Labeled “Sterile”72.8 EN D
28、ocuments:EN 12442-1 Animal Tissues and Their Derivative Utilizedin the Manufacture of Medical DevicesPart 1: Analysisand Management of Risk8EN 12442-Part 3: Validation of the Elimination and/orInactivation of Virus and Transmissible Agents83. Terminology3.1 Definitions:3.1.1 chitosan, na linear poly
29、saccharide consisting ofb(14) linked 2-acetamido-2-deoxy-D-glucopyranose(GlcNAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN).3Available from EDQM, Publications and Services European Pharmacopoeia,BP 907 226, avenue de Colmar, F-67029 Strasbourg Cedex 1, France.4Available from American National Standa
30、rds Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.5Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, PO Box 758,1211 Geneva 13, Switzerland.6Available from Food and Drug Administration (FDA), 5600 Fishers Ln.,Rockville, MD 20857, http:/www.fda.gov.7As
31、sociation for the Advancement of Medical Instrumentation, 111 N. GlebeRd., Suite 220, Arlington, VA 222014795.8Available from European Committee for Standardization, CEN ManagementCentre, 36 rue de Stassart, B-1050 Brussels, Belgium.F 2103 01 (2007)e123.1.1.1 DiscussionChitosan is a polysaccharide d
32、erivedby N-deacetylation of chitin.3.1.2 decomposition, nstructural changes of chitosans as aresult of exposure to environmental, chemical, or thermalfactors, such as temperatures greater than 200C.3.1.2.1 DiscussionDecomposition can result in deleteri-ous changes to the chitosan.3.1.3 degradation,
33、nchange in the chemical structure,physical properties, or appearance of a material.3.1.3.1 DiscussionDegradation of polysaccharides occursby means of cleavage of the glycosidic bonds, usually by acidcatalyzed hydrolysis. Degradation can also occur thermally.Note that degradation is not synonymous wi
34、th decomposition.Degradation is often used as a synonym for depolymerizationwhen referring to polymers.3.1.4 degree of deacetylation, nthe fraction or percentageof glucosamine units (deacetylated monomers) in a chitosanpolymer molecule.3.1.5 depolymerization, nreduction in length of a polymerchain t
35、o form shorter polymeric units.3.1.5.1 DiscussionDepolymerization may reduce thepolymer chain to oligomeric or monomeric units, or both. Inchitosan, hydrolysis of the glycosidic bonds is the primarymechanism.3.1.6 endotoxin, na high-molecular-weight lipopolysac-charide (LPS) complex associated with
36、the cell wall ofgram-negative bacteria that is pyrogenic in humans.3.1.6.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endotoxins.3.1.7 molecular mass average (molecular weight average),nthe given molecular weight (Mw) of a chitosan will alwaysrepresent an average of all of the mol
37、ecules in the population.The most common ways to express the Mw are as the numberaverage (Mn) and the weight average (Mw). The two averagesare defined by the following equations:Mn5(iNiMi(iNiandMw5(iWiMi(iWi5(iNiMi2(iNiMiwhere:Ni= number of molecules having a specific molecularweight Miandwi= weight
38、 of molecules having a specific molecularweight Mi. In a polydisperse molecular population therelation Mw Mnis always valid. The coefficient Mw/Mnis referred to as the polydispersity index, and willtypically be in the range 1.5 to 3.0 for commercialchitosans.3.1.8 pyrogen, nany substance that produc
39、es fever whenadministered parenterally.4. Significance and Use4.1 This guide contains a listing of those characterizationparameters that are directly related to the functionality ofchitosan. This guide can be used as an aid in the selection andcharacterization of the appropriate chitosan or chitosan
40、 salt fora particular application. This standard is intended to giveguidance in the methods and types of testing necessary toproperly characterize, assess, and ensure consistency in theperformance of a particular chitosan. It may have use in theregulation of devices containing chitosan by appropriat
41、e au-thorities.4.2 The chitosan salts covered by this guide may be gelled,extruded, or otherwise formulated into biomedical devices foruse as tissue-engineered medical products or drug deliverydevices for implantation as determined to be appropriate, basedon supporting biocompatibility and physical
42、test data. Recom-mendations in this guide should not be interpreted as aguarantee of clinical success in any tissue-engineered medicalproduct or drug delivery application.4.3 To ensure that the material supplied satisfies require-ments for use in TEMPs, several general areas of characteriza-tion sho
43、uld be considered. These include identity of chitosan,physical and chemical characterization and testing, impuritiesprofile, and performance-related tests.5. Chemical and Physical Test Methods5.1 Identity of ChitosanThe identity of chitosan andchitosan salts can be established by several methods inc
44、luding,but not limited to the following:5.1.1 Chitosan chloride monograph Ph. Eur.5.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)Almost all organic chemical compounds absorb infrared radia-tion at frequencies characteristic for the functional groups inthe compound. A FT-IR spectrum will show a
45、bsorption bandsrelating to bond stretching and bending and can therefore serveas a unique fingerprint of a specific compound. Cast a chitosanfilm from a 0.25 % (w/v) solution of chitosan (in 1 % aceticacid) or chitosan salt (dissolved in water) by drying approxi-mately 500 L of the sample onto a dis
46、posable IR card9for 3to4hat60C. Record a background spectrum between 4000and 400 cm-1 using 128 scans at a resolution of 4 cm-1. Recordthe IR spectrum of a dried blank IR card, then record the IRspectrum of the sample using 128 scans at a resolution of 4cm-1, percent transmission mode. Label the pea
47、ks. Typicalfrequencies (cm-1) for chitosan are as follows:Chitosan Base(as Acetate)Chitosan Chloride Chitosan Glutamate3362b 3344b 1555b1556 1605 13961406 1513 11541153 1379 1085s1083s 11541086sThe peak designators are: sh: sharp; s: strong; m: medium;w: weak; and b: broad.5.2 Physical and Chemical
48、Characterization of Chitosan:5.2.1 The composition and sequential structure of chitosancan be a key functional attribute of any chitosan or chitosansalt. Variations in the composition or the sequential structure,9No suitable commercially available IR cards are available for the IR analysis ofchitosa
49、n glutamate salt. Alternative methods are under investigation.F 2103 01 (2007)e13or both, may, but not necessarily will, cause differences inperformance of a chitosan in a particular end use. Thisinformation may be determined by the following method:High-resolution1H- and13C-nuclear magnetic resonance spec-troscopy (NMR).5.2.2 The degree of deacetylation of chitosan can be estab-lished using a number of techniques including, but not limitedto, the following:5.2.2.1 High-resolution1H- and13C-Nuclear MagneticResonance Spectroscopy (NM
