1、Designation: F2347 11Standard 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 F2347; the number immediately following the designation indicate
2、s 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 () indicates an editorial change since the last revision or reapproval.INTRODUCTIONHyaluronan, which in this guide will encompass
3、 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 fluid
4、, 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 and
5、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 industrie
6、s.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 hyaluronan
7、.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 testme
8、thods that are appropriate to ensure safety and efficacy andare not addressed in this guide.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns, if any, ass
9、ociated 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 requirements prior to use.2. Referenced Documents2.1 ASTM Standards:2D2196 Test Methods for Rheological Properties of Non-Ne
10、wtonian Materials by Rotational (Brookfield type) Vis-cometerF619 Practice for Extraction of Medical PlasticsF748 Practice for Selecting Generic Biological Test Meth-ods for Materials and DevicesF749 Practice for Evaluating Material Extracts by Intracu-taneous Injection in the RabbitF756 Practice fo
11、r Assessment of Hemolytic Properties ofMaterialsF763 Practice for Short-Term Screening of Implant Materi-alsF813 Practice for Direct Contact Cell Culture Evaluation ofMaterials for Medical DevicesF895 Test Method for Agar Diffusion Cell Culture Screen-ing for CytotoxicityF981 Practice for Assessment
12、 of Compatibility of Biomate-rials for Surgical Implants with Respect to Effect ofMaterials on Muscle and BoneF1251 Terminology Relating to Polymeric Biomaterials inMedical and Surgical DevicesF1439 Guide for Performance of Lifetime Bioassay for the1This guide is under the jurisdiction of ASTM Commi
13、ttee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved March 1, 2011. Published March 2011. Originallyapproved in 2003. Last previous edition approved in 2003 as F2347 03. DOI:10
14、.1520/F2347-11.2For 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 to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor
15、 Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Tumorigenic Potential of Implant MaterialsF1903 Practice for Testing For Biological Responses toParticles In VitroF1904 Practice for Testing the Biological Responses toParticles in vivoF1905 Practice For Selecting Tests for Determi
16、ning thePropensity of Materials to Cause Immunotoxicity3F1906 Practice for Evaluation of Immune Responses InBiocompatibility Testing Using ELISA Tests, LymphocyteProliferation, and Cell Migration32.2 USP Documents:4USP Microbial Limit TestsUSP Sterility TestsUSP Bacterial Endotoxins TestsUSP Heavy M
17、etalsUSP Loss on DryingUSP Sterilization and Sterility Assurance of Com-pendial Articles2.3 EP Documents:5EP Monograph 1472 Sodium HyaluronateEP 2.6.1 Sterility2.4 Other Referenced Documents:ISO 10993 Biological Evaluation of Medical Devices6ISO 10993-1 Biological Evaluation of Medical DevicesPart 1
18、: 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 Identification and Quantification ofPotential Degradation ProductsISO 10993-17 Biological Evaluation of
19、Medical DevicesPart 17: Establishment of Allowable Limits for LeachableSubstancesISO 14160: 1998 Sterilization of Single-Use Medical De-vices Incorporating Materials of Animal OriginValidation and Routine Control of Sterilization by LiquidChemical Sterilants6ISO 11737-1: 1995 Sterilization of Medica
20、l DevicesMicrobiological MethodsPart 1: Estimation of Popula-tion of Microorganisms on Products6ISO 11737-2: 1998 Sterilization of Medical DevicesMicrobiological MethodsPart 2: Tests of Sterility Per-formed in the Validation of a Sterilization Process6ISO 13408-1: 1998 Aseptic Processing of Health C
21、areProductsPart 1: General Requirements6ISO EN 12442-1 Animal Tissues and Their DerivativeUtilized in the Manufacture of Medical DevicesPart 1:Analysis and Management of Risk6ISO EN 12442-3 Animal Tissues and Their DerivativeUtilized in the Manufacture of Medical DevicesPart 3:Validation of the Elim
22、ination and/or inactivation of Virusand Transmissible Agents6ICH S2B A Standard Battery for Genotoxicity Testing ofPharmaceuticals (July 1997)7ICH Q1A Harmonized Tripartite Guidance for StabilityTesting of New Drug Substances and Products (September2001, Revision 1)7FDA Guideline on Validation of th
23、e Limulus AmebocyteTest as an End-Product Endotoxin Test for Human andAnimal Parenteral Drugs, Biological Products and Health-care Products, DHHS, December 19878FDA Interim Guidance for Human and Veterinary DrugProducts and Biologicals, Kinetic LAL Techniques,DHHS, July 15, 19918AAMI TIR No. 7: 1999
24、 Chemical Sterilants and High LevelDisinfectants: A Guide to Selection and Use9AAMI ST67/CDV2: 1999 Sterilization of MedicalDevicesRequirements for Products Labeled “Sterile”921 CFR 312 FDA Title 21, Food and Drugs, InvestigationalNew Drug Applications103. Terminology3.1 Definitions:3.1.1 decomposit
25、ion, nstructural changes of hyaluronandue to exposure to environmental, chemical, or thermal factors.Decomposition may occur at temperatures as low as 121Cduring autoclaving. Decomposition can result in deleteriouschanges to the hyaluronan.3.1.2 degradation, nchange in the chemical structure,physica
26、l 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 decomposition. Degrada-tion is o
27、ften used as a synonym for depolymerization whenreferring to polymers. Degradation (depolymerization) of hy-aluronan may also occur enzymatically by the action ofhyaluronidases.3.1.3 depolymerization, nreduction in length of a polymerchain to form shorter polymeric units. Depolymerization mayreduce
28、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.3.1.4 endotoxin, npyrogenic high molar mass li-popolysaccharide (LPS) complex associated with the cell wallof
29、 gram-negative bacteria.3.1.4.1 DiscussionThough endotoxins are pyrogens, notall pyrogens are endotoxins. Endotoxins are specifically de-tected through a Limulus Amebocyte Lysate (LAL) test.3Withdrawn. The last approved version of this historical standard is referencedon www.astm.org.4Available from
30、 U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852.5Available from European Directorate for the Quality of Medicines (EDQM),Council of Europe, BP 907, 67029 Strasbourg, France.6Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036.
31、7Available from International Conference on Harmonization (ICH) Secretariat,c/o IFPMA, 30 rue de St-Jean, P.O. Box 758, 1211 Geneva 13, Switzerland.8Available from U.S. Food and Drug Administration, 5600 Fishers Lane,Rockville, MD 20857-0001.9Available from Association for the Advancement of Medical
32、 Instrumentation,1110 North Glebe Rd., Suite 220, Arlington, VA 22201-4795.10Available from Standardization Documents Order Desk, DODSSP, Bldg. 4,Section D, 700 Robbins Ave., Philadelphia, PA 19111-5098F2347 1123.1.5 hyaluronan, na polysaccharide with a disacchariderepeating unit composed of D-glucu
33、ronic acid and N-acetyl-D-glucosamine 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.3.1.6 hydrocolloid, na water-soluble polymer of colloidalnature when hydrated.3.1.7 mole
34、cular mass average (molecular 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
35、following equations:Mn5(iNiMi(iNiand Mw5(iwiMi(iwi5(iNiMi2(iNiMiwhere:Ni= number of molecules having a specific molecularweight Mi, andwi= weight of molecules having a specific molecularweight Mi.In a polydisperse molecular population the relation Mw Mnis always valid. The coefficient Mw/ Mnis refer
36、red to as thepolydispersity index, and will typically be in the range 1.2 to3.0 for commercial hyaluronan.3.1.8 non-animal derived, na term describing the absenceof any animal-derived tissue, proteins, or products in themanufacturing process.3.1.9 pyrogen, nany substance that produces fever whenadmi
37、nistered parenterally.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 applicat
38、ion. This guide is 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 t
39、his guide may be 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
40、guide should not 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: identi
41、ty of hyaluronan,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
42、 Hyaluronate Monograph EP Monograph1472.5.1.2 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
43、 and bending and can therefore 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) h
44、aving a sample trough and 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 pea
45、k designators are: sh: sharp; s:strong; m: medium; w: weak; b: broad. A typical FT-IR HATRspectrum is shown in Fig. 1. A reference spectrum can beobtained from the European Pharmacopoeia.115.2 Physical and Chemical Characterization of Hyaluro-nan:5.2.1 The composition and sequential structure of hya
46、luro-nan can be determined 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 hyal
47、uronan is shown below. Hyaluronan 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).125.2.2 Molecular mass (molecular weight
48、) of hyaluronan willdefine 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 polydispers
49、e with respect to molecularweight (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 form viscous solutions in water and is directly proportionalto the average molecular weight of the polymer. The intrinsicviscosity is a characteristic of the polymer under specifiedsolvent and temperature conditions; it is inde
