1、Designation: F 2064 00 (Reapproved 2006)e1Standard Guide forCharacterization and Testing of Alginates as StartingMaterials Intended for Use in Biomedical and Tissue-Engineered Medical Products Application1This standard is issued under the fixed designation F 2064; the number immediately following th
2、e 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.e1NOTEMercury warning was editorial
3、ly added in April 2008.INTRODUCTIONAlginate has found uses in a variety of products ranging from simple technical applications such asviscosifiers to advanced biomedical matrices providing controlled drug delivery from immobilizedliving cells. As for most hydrocolloids, the functionality of alginate
4、 is related to its chemical andstructural composition. The aim of this guide is to identify key parameters relevant for thefunctionality and characterization of alginates for the development of new commercial applications ofalginates for the biomedical and pharmaceutical industries.1. Scope1.1 This
5、guide covers the evaluation of alginates suitable foruse in biomedical or pharmaceutical applications, or both,including, but not limited to, tissue-engineered medical prod-ucts (TEMPS).1.2 This guide addresses key parameters relevant for thefunctionality, characterization, and purity of alginates.1
6、.3 As with any material, some characteristics of alginatesmay be altered by processing techniques (such as molding,extrusion, 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 e
7、valuated using test methods that are appropriate toensure safety and efficacy and are not addressed in this guide.1.4 WarningMercury has been designated by EPA andmany state agencies as a hazardous material that can causecentral nervous system, kidney, and liver damage. Mercury, orits vapor, may be
8、hazardous to health and corrosive tomaterials. Caution should be taken when handling mercury andmercury-containing products. See the applicable product Ma-terial Safety Data Sheet (MSDS) for details and EPAs website(http:/www.epa.gov/mercury/faq.htm) for additional informa-tion. Users should be awar
9、e that selling mercury or mercury-containing products, or both, in your state may be prohibited bystate law.1.5 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
10、and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 2196 Test Methods for Rheological Properties of Non-Newtonian Materials by Rotational (Brookfield type) Vis-cometerF 619 Practice for Extraction of Medical Plasti
11、csF 748 Practice 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-
12、rialsF 813 Practice for Direct Contact Cell Culture Evaluation ofMaterials for Medical DevicesF 895 Test Method for Agar Diffusion Cell Culture Screen-ing for CytotoxicityF 981 Practice for Assessment of Compatibility of Bioma-terials for Surgical Implants with Respect to Effect of1This guide is und
13、er the jurisdiction of ASTM Committee 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, 2006. Published April 2006. Originallyapproved in 2000. Last previous edition app
14、roved in 2000 as F 2064 00.2For 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
15、 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Materials 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 P
16、ractice for Testing For Biological Responses toParticles in vitroF 1904 Practice for 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 InBiocompa
17、tibility Testing Using ELISA Tests, LymphocyteProliferation, and Cell Migration2.2 USP Document:USP Monograph USP 24/NF 19 Sodium Alginate32.3 ISO Documents:4ISO 10993 Biological Evaluation of Medical Devices:ISO 10993-1 Biological Evaluation of Medical DevicesPart 1: Evaluation and TestingISO 10993
18、-3 Part 3: Tests for Genotoxicity, Carcinogenicityand Reproductive ToxicityISO 10993-9Part 9: Framework for Identification andQuantification of Potential Degradation ProductsISO 10993-17Part 17: Methods for Establishment ofAllowable Limits for Leachable Substances Using Health-Based Risk AssessmentI
19、SO 13408-1: 1998: Aseptic Processing of Health CareProductsPart 1: General Requirements.2.4 ICH Documents:5International Conference on Harmonization (ICH) S2BGenotoxicity: A Standard Battery for Genotoxicity Test-ing of Pharmaceuticals (July 1997)5International Conference on Harmonization (ICH) Q1AI
20、CH Harmonized Tripartite Guidance for Stability Testingof New Drug Substances and Products (September 23,1994)2.5 FDA Documents:6FDA Guideline on Validation of the Limulus AmebocyteTest as an End-Product Endotoxin Test for Human andAnimal Parenteral Drugs, Biological Products and Health-care Product
21、s. DHHS, December 1987FDA. Interim Guidance for Human and Veterinary DrugProducts and Biologicals. Kinetic LAL techniques.DHHS, July 15, 19912.6 ANSI Documents:4ANSI/AAMI/ISO 11737-1: 1995: Sterilization of MedicalDevicesMicrobiological MethodsPart 1: Estimationof Bioburden on Product.ANSI/AAMI/ISO
22、11737-2: 1998: Sterilization of MedicalDevicesMicrobiological MethodsPart 2: Tests of Ste-rility Performed in the Validation of a Sterilization Process2.7 AAMI Documents:7AAMI/ISO 141601998: Sterilization of Single-UseMedical Devices Incorporating Materials of AnimalOriginValidation and Routine Cont
23、rol of Sterilizationby Liquid Chemical SterilantsAAMI ST67/CDV-2: 1999: Sterilization of MedicalDevicesRequirements for Products Labeled “Sterile”AAMI TIR No. 191998: Guidance for ANSI/AAMI/ISO10993-7: 1995, Biological Evaluation of MedicalDevicesPart 7: Ethylene Oxide Sterilization Residuals2.8 prE
24、N Documents:8prEN 12442-1 Animal Tissues and their Derivative Utilizedin the Manufacture of Medical DevicesPart 1: Analysisand Management of RiskprEN 12442 Part 3:Validation of the Elimination and/orInactivation of Virus and Transmissible Agents2.9 Other Documents:21CFR184.1724 Listing of Specific S
25、ubstances Affirmed asGRASSodium Alginate93. Terminology3.1 Definitions of Terms Specific to This Standard: (see alsoTerminology F 1251):3.1.1 alginate, na polysaccharide substance containingcalcium, magnesium, sodium, and potassium salts obtainedfrom some of the more common species of marine algae.A
26、lginate exists in brown algae as the most abundant polysac-charide, mainly occurring in the cell walls and intercellularspaces of brown seaweed and kelp. Its main function is tocontribute to the strength and flexibility of the seaweed plant.Alginate is classified as a hydrocolloid. The most commonly
27、used alginate is sodium alginate.3.1.2 decomposition, nstructural changes of alginates dueto exposure to environmental, chemical or thermal factors,such as temperatures greater than 180C. Decomposition canresult in deleterious changes to the alginate.3.1.3 degradation, nchange in the chemical struct
28、ure,physical properties, or appearance of a material. Degradationof polysaccharides occurs by means of cleavage of the glyco-sidic bonds, usually by acid catalyzed hydrolysis. Degradationcan also occur thermally. It is important to note that degrada-tion is not synonymous with decomposition. Degrada
29、tion isoften used as a synonym for depolymerization when referringto polymers.3.1.4 depolymerization, nreduction in length of a polymerchain to form shorter polymeric units. Depolymerization mayreduce the polymer chain to oligomeric or monomeric units, orboth. In alginates, hydrolysis of the glycosi
30、dic bonds is theprimary mechanism.3Available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville,MD 20852.4Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036.5Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758,
31、1211 Geneva 13, Switzerland.6Available from U. S. Food and Drug Administration, 5600 Fishers Lane,Rockville MD 20857-0001.7Association for theAdvancement of Medical Instrumentation 1110 North GlebeRd., Suite 220, Arlington, VA 222014795.8Available from European Committee for Standardization CEN, Man
32、agementCentre 36, rue de Stassart B-1050 Brussels, Belgium.9Available from Superintendent of Documents, U.S. Government PrintingOffice, Washington, DC 20402.F 2064 00 (2006)e123.1.5 Endotoxin, na high-molecular weight lipopolysac-charide (LPS) complex associated with the cell wall ofgram-negative ba
33、cteria that is pyrogenic in humans. Thoughendotoxins are pyrogens, not all pyrogens are endotoxins.3.1.6 hydrocolloid, na water-soluble polymer of colloidalnature when hydrated.3.1.7 molecular mass average (molecular weight average),nthe given molecular weight (Mw) of an alginate will alwaysrepresen
34、t an average of all of the molecules 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(iNiand Mw5(iwiMi(iwi5(iNiMi2(iNiMi(1)where:Ni= number of molecules having a specific
35、 molecularweight, Mi, andwi= weight of molecules having a specific molecularweight MiIn a polydisperse molecular population the relation MwMnis always valid. The coefficient Mw/Mnis referred to as thepolydispersity index, and will typically be in the range from 1.5to 3.0 for commercial alginates.3.1
36、.8 pyrogen, nany substance that produces fever whenadministered parenterally.4. Significance and Use4.1 This guide contains a listing of those characterizationparameters that are directly related to the functionality ofalginate. This guide can be used as an aid in the selection andcharacterization o
37、f the appropriate alginate for a particularapplication. This guide is intended to give guidance in themethods and types of testing necessary to properly character-ize, assess, and ensure consistency in the performance of aparticular alginate. It may have use in the regulation of thesedevices by appr
38、opriate authorities.4.2 The alginate covered by this guide may be gelled,extruded, or otherwise formulated into biomedical devices foruse in tissue-engineered medical products or drug deliverydevices for implantation as determined to be appropriate, basedon supporting biocompatibility and physical t
39、est 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 character-ization shou
40、ld be considered. These are: identity of alginate,physical and chemical characterization and testing, impuritiesprofile, and performance-related tests.5. Chemical and Physical Test Methods5.1 Identity of AlginateThe identity of alginates can beestablished by several methods including, but not limite
41、d to thefollowing:5.1.1 Sodium alginate monograph USP 24/NF19.5.1.2 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 bandsrelat
42、ing to bond stretching and bending and can therefore serveas a unique fingerprint of a specific compound. Cast an alginatefilm from a 0.25 % (w/v) solution of sodium alginate by dryingapproximately 500 L of the sample onto a disposable IR cardfor3to4hat60C. Record a background spectrum between4000 a
43、nd 400 cm1using 128 scans at a resolution of 4 cm1.Record the IR spectrum of a dried blank IR card, then recordthe IR spectrum of the sample using 128 scans at a resolutionof4cm1, % transmission mode. Label the peaks. Typicalfrequencies (cm1) for sodium alginate are 3375-3390 (b),1613 (s), 1416 (s),
44、 1320 (w), 1125, 1089, 1031 (s), 948 (m),903 (m), and 811 (m). The peak designators are: sh: sharp; s:strong; m: medium; w: weak; and b: broad.5.2 Physical and chemical characterization of alginate:5.2.1 The composition and sequential structure of alginatecan be a key functional attribute of any alg
45、inate. Variations inthe composition or the sequential structure, or both, may, butnot necessarily, cause differences in performance of an alginatein a particular end use. This information may be determined bythe following method: High-resolution1H and13C-nuclearmagnetic resonance spectroscopy (NMR).
46、 Sodium alginateshould be dissolved in D2O and partially degraded to a degreeof depolymerization of 20 to 30 using mild acid hydrolysisbefore recording proton or carbon NMR spectra (Grasdalen,H., Larsen, B., and Smidsrd, O., Carbohydr. Res., 68, 23-31,1979). Techniques have been developed to determi
47、ne themonad frequencies FG(fraction of guluronate residues) and FM(fraction of mannuronate residues), the four nearest neighbor-ing (diad) frequencies (FGG,FGM,FMG, and FMM) and the eightnext nearest neighboring (triad) frequencies (FGGG,FGGM,FGMM,FGMG,FMGM,FMGG,FMMG, and FMMM). A typical1H-NMR spec
48、trum of alginate is shown as follows. Alginate ischaracterized by calculating parameters such as M/G ratio,G-content, consecutive number of G monomers (that is, G1),and average length of blocks of consecutive G monomers.5.2.2 Molecular mass (molecular weight) of an alginate willdefine certain perfor
49、mance 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 alginates are polydisperse with respect to molecularweight (Mw). Molecular weight may be expressed as thenumber average (MN) or the weight average (MW). Molecularweights may be determined by methods such as, but notlimited, to the following5.2.2.1 Molecular Weight Determination Based on IntrinsicViscosityTh
