1、Designation: F 2347 03Standard Guide forCharacterization and Testing of Hyaluronan as StartingMaterials Intended for Use in Biomedical and TissueEngineered Medical Product Applications1This standard is issued under the fixed designation F 2347; the number immediately following the designation indica
2、tes 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.INTRODUCTIONHyaluronan, which in this guide will encomp
3、ass hyaluronic acid, hyaluronate, and its salt forms, isthe simplest of the glycosaminoglycans. Hyaluronan is soluble in water and forms highly viscoussolutions. Hyaluronan is found in ubiquitously in the body as part of the extracellular matrix of tissues,with high concentrations in the synovial fl
4、uid, vitreous humor, and skin, as well as in cartilage.Hyaluronan has found uses in a variety of products ranging from viscosupplements (treatment ofosteoarthritis), adhesion prevention (prevention of post-surgical adhesions), viscoelastics (ocularprotection), and dermal implants (lip augmentation a
5、nd wrinkle removal). New applications, such asscaffolds for tissue engineering, are emerging. The aim of this guide is to identify key parametersrelevant to the characterization of hyaluronan for the development of new commercial applications ofhyaluronan for the biomedical and pharmaceutical indust
6、ries.1. Scope1.1 This guide covers the evaluation of hyaluronan suitablefor use in biomedical or pharmaceutical applications, or both,including, but not limited to, Tissue Engineered MedicalProducts (TEMPs).1.2 This guide addresses key parameters relevant to thecharacterization and purity of hyaluro
7、nan.1.3 As with any material, some characteristics of hyaluro-nan may be altered by processing techniques, such as cross-linking and sterilization, required for the production of aspecific formulation or device. Therefore, properties of fabri-cated forms of this polymer should be evaluated using tes
8、tmethods that are appropriate to ensure safety and efficacy andare not addressed in this guide.1.4 This standard does not purport to address all 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 pr
9、actices and determine the applica-bility of regulatory requirements prior to use.2. Referenced Documents2.1 ASTM Standards:2D 2196 Test Methods for Rheological Properties of Non-Newtonian Materials by Rotational (Brookfield) Viscom-eterF 619 Practice for Extraction of Medical PlasticsF 748 Practice
10、for Selecting Generic Biological Test Meth-ods for Materials and DevicesF 749 Practice for Evaluating Material Extracts by Intracu-taneous Injection in the RabbitF 756 Practice for Assessment of Hemolytic Properties ofMaterialsF 763 Practice for Short-Term Screening of Implant Mate-rialsF 813 Practi
11、ce for Direct Contact Cell Culture Evaluation ofMaterials for Medical DevicesF 895 Test Method for Agar Diffusion Cell Culture Screen-ing for Cytotoxicity1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of Subco
12、mmitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Nov. 1, 2003. Published December 2003.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer
13、 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.F 981 Practice for Assessment of Compatibility of Bioma-terials (Nonporous) for Surgical Implants with Respect toEffect of Mat
14、erials on Muscle and BoneF 1251 Terminology Relating to Polymeric Biomaterials 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
15、 Testing the Biological Responses toParticles in vivoF 1905 Practice 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 U
16、SP Documents:3USP Microbial Limit TestsUSP Sterility TestsUSP Bacterial Endotoxins TestsUSP Heavy MetalsUSP Loss on DryingUSP Sterilization and Sterility Assurance of Com-pendial Articles2.3 EP Documents:4EP Monograph 1472 Sodium HyaluronateEP 2.6.1 Sterility2.4 Other Referenced Documents:ISO 10993
17、Biological Evaluation of Medical Devices5ISO 10993-1 Biological Evaluation of Medical DevicesPart 1: Evaluation and TestingISO 10993-7 Biological Evaluation of Medical DevicesPart 7: Ethylene Oxide Sterilization ResidualsISO 10993-9 Biological Evaluation of Medical DevicesPart 9: Framework for Ident
18、ification and Quantification ofPotential Degradation ProductsISO 10993-17 Biological Evaluation of MedicalDevicesPart 17: Establishment of Allowable Limits forLeachable SubstancesISO 14160-1998 Sterilization of Single-Use Medical De-vices Incorporating Materials of Animal OriginValidation and Routin
19、e Control of Sterilization by LiquidChemical Sterilants5ISO 11737-1: 1995 Sterilization of Medical DevicesMicrobiological MethodsPart 1: Estimation of Popula-tion of Microorganisms on Products5ISO 11737-2: 1998 Sterilization of Medical DevicesMicrobiological MethodsPart 2: Tests of Sterility Per-for
20、med in the Validation of a Sterilization Process5ISO 13408-1: 1998 Aseptic Processing of Health CareProductsPart 1: General Requirements5ISO EN 12442-1 Animal Tissues and Their DerivativeUtilized in the Manufacture of Medical DevicesPart 1:Analysis and Management of Risk5ISO EN 12442-3 Animal Tissue
21、s and Their DerivativeUtilized in the Manufacture of Medical DevicesPart 3:Validation of the Elimination and/or inactivation of Virusand Transmissible Agents5International Conference on Harmonization (ICH) S2BGenotoxicity A Standard Battery for Genotoxicity Testingof Pharmaceuticals (July 1997)6Inte
22、rnational Conference on Harmonization (ICH) Q1AICH Harmonized Tripartite Guidance for Stability Testingof New Drug Substances and Products (September 2001,Revision 1)6FDA Guideline on Validation of the Limulus AmebocyteTest as an End-Product Endotoxin Test for Human andAnimal Parenteral Drugs, Biolo
23、gical Products and Health-care Products, DHHS, December 19877FDA Interim Guidance for Human and Veterinary DrugProducts and Biologicals, Kinetic LAL Techniques,DHHS, July 15, 19917AAMI TIR No. 7: 1999 Chemical Sterilants and High LevelDisinfectants: A Guide to Selection and Use8AAMI ST67/CDV-2: 1999
24、 Sterilization of MedicalDevicesRequirements for Products Labeled “Sterile”821 CFR 312 FDA Title 21, Food and Drugs, InvestigationalNew Drug Applications93. Terminology3.1 Definitions:3.1.1 hyaluronan, na polysaccharide with a disacchariderepeating unit composed of D-glucuronic acid and N-acetyl-D-g
25、lucosamine in b-(13) linkage. Each disaccharide unit isattached to the next by b-(14) bonds. Hyaluronan is a linearpolymer. Other common names are hyaluronic acid and sodiumhyaluronate.3.1.2 hydrocolloid, na water-soluble polymer of colloidalnature when hydrated.3.1.3 molecular mass average (molecul
26、ar weight average),nthe given molecular weight (Mw) of hyaluronan willalways represent an average of all of the molecules in thepopulation. The most common ways to express the Mw are asthe number average (Mn) and the weight average (Mw). Thetwo averages are defined by the following equations:Mn5(iNi
27、Mi(iNiand Mw5(iwiMi(iwi5(iNiMi2(iNiMiwhere:Ni= number of molecules having a specific molecularweight Mi, and3Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852.4Available from European Directorate for the Quality of Medicines (EDQM),Council of Europe, BP 907, 67029 Str
28、asbourg, France.5Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036.6Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758,1211 Geneva 13, Switzerland.7Available from U.S. Food and Drug Administration, 5600 Fishers Lane,Rock
29、ville, MD 20857-0001.8Available from Association for the Advancement of Medical Instrumentation,1110 North Glebe Rd., Suite 220, Arlington, VA 22201-4795.9Available from Standardization Documents Order Desk, DODSSP, Bldg. 4,Section D, 700 Robbins Ave., Philadelphia, PA 19111-5098F2347032wi= weight o
30、f molecules having a specific molecularweight Mi.In a polydisperse molecular population the relation Mw Mnis always valid. The coefficient Mw/ Mnis referred to as thepolydispersity index, and will typically be in the range 1.2 to3.0 for commercial hyaluronan.3.1.4 depolymerization, nreduction in len
31、gth of a polymerchain to form shorter polymeric units. Depolymerization mayreduce the polymer chain to smaller molecular weight poly-mers, oligomeric, or monomeric units, or combination thereof.In hyaluronan, acid hydrolysis of the glycosidic bonds is theprimary mechanism.3.1.5 degradation, nchange
32、in the chemical structure,physical properties or appearance of a material. Degradation ofpolysaccharides occurs via cleavage of the glycosidic bonds,usually by acid catalyzed hydrolysis. Degradation can alsooccur thermally and by alkali. It is important to note thatdegradation is not synonymous with
33、 decomposition. Degrada-tion is often used as a synonym for depolymerization whenreferring to polymers. Degradation (depolymerization) of hy-aluronan may also occur enzymatically by the action ofhyaluronidases.3.1.6 decomposition, nstructural changes of hyaluronandue to exposure to environmental, ch
34、emical, or thermal factors.Decomposition may occur at temperatures as low as 121Cduring autoclaving. Decomposition can result in deleteriouschanges to the hyaluronan.3.1.7 pyrogen, nany substance that produces fever whenadministered parenterally.3.1.8 endotoxin, na high molecular weight lipopolysac-
35、charide (LPS) complex associated with the cell wall ofgram-negative bacteria that is pyrogenic in humans. Thoughendotoxins are pyrogens, not all pyrogens are endotoxins.3.1.9 non-animal derived, na term describing the absenceof any animal-derived tissue, proteins, or products in themanufacturing pro
36、cess.4. Significance and Use4.1 This guide contains a listing of those characterizationparameters that are directly related to the functionality ofhyaluronan. This guide can be used as an aid in the selectionand characterization of the appropriate hyaluronan for aparticular application. This guide i
37、s intended to give guidancein the methods and types of testing necessary to properlycharacterize, assess, and ensure consistency in the performanceof a particular hyaluronan. It may have use in the regulation ofthese devices by appropriate authorities.4.2 The hyaluronan covered by this guide may be
38、gelled,cross-linked, extruded, or otherwise formulated into biomedi-cal devices for use in tissue engineered medical products ordrug delivery devices for implantation as determined to beappropriate, based on supporting biocompatibility and physicaltest data. Recommendations in this guide should not
39、beinterpreted as a guarantee of clinical success in any tissueengineered medical product 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 should be considered. These are: identity of hyaluronan,
40、physical and chemical characterization and testing, impuritiesprofile, and performance-related tests.5. Chemical and Physical Test Methods5.1 Identity of HyaluronanThe identity of hyaluronan canbe established by several methods including, but not limited tothe following:5.1.1 Sodium Hyaluronate Mono
41、graph EP 1472.5.1.2 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 absorption bandsrelating to bond stretching and bending and can there
42、fore serveas a unique fingerprint of a specific compound. Direct FT-IRanalysis of hyaluronan powder is perhaps the easiest techniqueto perform. One method utilizes a horizontal attenuated totalreflectance (HATR) accessory with a zinc-selenium (ZnSe)crystal (or equivalent) having a sample trough and
43、a pressureplate. Record background and sample spectra between 4000and 600 cm-1at an appropriate resolution. Label the peaks.Typical frequencies (cm-1) for hyaluronan (sodium salt) are3275-3390 (b), 1615 (s), 1405 (m), 1377 (m), 1150, 1077, 1045(s), 946 (m), 893 (w). The peak designators are: sh: sha
44、rp; s:strong; m: medium; w: weak; b: broad. A typical FT-IR HATRspectrum is shown in Fig. 1. A reference spectrum can beobtained form the European Pharmacopoeia.105.2 Physical and Chemical Characterization of Hyaluro-nan:5.2.1 The composition and sequential structure of hyaluro-nan can be determined
45、 by the following method: High-resolution1H- and13C-nuclear magnetic resonance spectros-copy (NMR). Hyaluronan should be dissolved in D2O. If theresulting solution is viscous, viscosity may be reduced bychemical or enzymatic depolymerization. A typical1H-NMRspectrum of hyaluronan is shown below. Hya
46、luronan is char-acterized by calculating parameters such as glucuronic acid:N-acetylglucosamine ratio. Some literature references to thedetermination of composition and structure of hyaluronan aregiven in the References section (1-4).115.2.2 Molecular mass (molecular weight) of hyaluronan willdefine
47、 certain performance characteristics such as viscosity orgel strength, or both. As such and depending on the sensitivityof a particular end use to these variations, determination ofmolecular mass directly or indirectly may be necessary. Com-mercial hyaluronan is polydisperse with respect to molecula
48、rweight (Mw). Mwmay be expressed as the number average(MN) or the weight average (MW). Molecular weights may bedetermined by methods such as, but not limited to the follow-ing:5.2.2.1 Molecular Weight Determination Based on IntrinsicViscosityThe intrinsic viscosity describes a polymers abilityto for
49、m viscous solutions in water and is directly proportionalto the average molecular weight of the polymer. The intrinsic10EDQM, European Pharmacopoeia, Council of Europe, B.P. 907, F-67029Strasbourg France; www.pheur.org11The boldface numbers in parentheses refer to the list of references at the endof this standard.F2347033viscosity is a characteristic of the polymer under specifiedsolvent and temperature conditions; it is independent of con-centration. The intrinsic viscosity (h) is directly related to themolecular weight of a polymer through the Mar
