1、Designation: F 2064 00 (Reapproved 2006)Standard 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 the
2、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.INTRODUCTIONAlginate has found uses i
3、n 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 is related to its chemical andstructural composition. The a
4、im 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 guide covers the evaluation of alginates suitable foruse in
5、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.1.3 As with any material, some characteristics of alginatesma
6、y 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 evaluated using test methods that are appropriate toensure sa
7、fety and efficacy and are 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 practices and determine the applica-bility
8、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 PlasticsF 748 Practice for Selecting Generic Biological Test
9、 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 Practice for Direct Contact Cell Culture Ev
10、aluation 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 ofMaterials on Muscle and BoneF 1251 Terminology Relating to Polymeric Bio
11、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
12、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 Migration1This guide is under the jurisdiction of ASTM Committee F04 on Medical and
13、Surgical 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 approved in 2000 as F 2064 00.2For referenced ASTM standard
14、s, 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 Drive, PO Box C700, West Conshohocken, PA 1
15、9428-2959, United States.2.2 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 10993-3 Part 3: Tests for Genotoxicity, Carcinogenicityand
16、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 AssessmentISO 13408-1: 1998: Aseptic Processing of Health CarePro
17、ductsPart 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) Q1AICH Harmonized Tripartite Guidance for Stability Testin
18、gof 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 Products. DHHS, December 1987FDA. Interim Guidance for Human
19、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 11737-2: 1998: Sterilization of MedicalDevicesMicrobio
20、logical 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 Control of Sterilizationby Liquid Chemical SterilantsAAMI
21、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 prEN Documents:8prEN 12442-1 Animal Tissues and their Der
22、ivative 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 Substances Affirmed asGRASSodium Alginate93. Terminolog
23、y3.1 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.Alginate exists in brown algae as the most abundant pol
24、ysac-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 commonlyused alginate is sodium alginate.3.1.2 decomposition,
25、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 structure,physical properties, or appearance of a material.
26、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. Degradation isoften used as a synonym for depolymerization wh
27、en 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 glycosidic bonds is theprimary mechanism.3.1.5 Endotoxin, na
28、high-molecular weight lipopolysac-charide (LPS) complex associated with the cell wall ofgram-negative bacteria that is pyrogenic in humans. Thoughendotoxins are pyrogens, not all pyrogens are endotoxins.3.1.6 hydrocolloid, na water-soluble polymer of colloidalnature when hydrated.3.1.7 molecular mas
29、s average (molecular weight average),nthe given molecular weight (Mw) of an alginate will alwaysrepresent 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
30、equations:Mn5(iNiMi(iNiand Mw5(iwiMi(iwi5(iNiMi2(iNiMi(1)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
31、 St-Jean, P.O. Box 758,1211 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 S
32、tandardization CEN, ManagementCentre 36, rue de Stassart B-1050 Brussels, Belgium.9Available from Superintendent of Documents, U.S. Government PrintingOffice, Washington, DC 20402.F 2064 00 (2006)2where:Ni= number of molecules having a specific molecularweight, Mi, andwi= weight of molecules having
33、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.3.1.8 pyrogen, nany substance that produces fever whenadmin
34、istered 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 of the appropriate alginate for a particularapplication.
35、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 appropriate authorities.4.2 The alginate covered by this gui
36、de 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 test data. Recom-mendations in this guide should not be i
37、nterpreted 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 should be considered. These are: identity of alginate,physic
38、al 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 limited to thefollowing:5.1.1 Sodium alginate monograph USP 24
39、/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 bandsrelating to bond stretching and bending and can therefore ser
40、veas 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 and 400 cm1using 128 scans at a resolution of 4 cm1.Recor
41、d 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), 1320 (w), 1125, 1089, 1031 (s), 948 (m),903 (m), and 81
42、1 (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 alginate. Variations inthe composition or the sequential st
43、ructure, 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). Sodium alginateshould be dissolved in D2O and partially
44、 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 determine themonad frequencies FG(fraction of guluronate residu
45、es) 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 spectrum of alginate is shown as follows. Alginate ischaract
46、erized 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 performance characteristics such as viscosity orgel strength,
47、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 (M
48、N) 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 IntrinsicViscosityThe intrinsic viscosity describes a polymers abilityto form viscous solutions in water and is directly proportionalt
49、o the average molecular weight of the polymer. The intrinsicviscosity 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 Mark-Houwink-Sakurada (MHS) equation: h=KMa, where K is a constant,M is the viscosity derived average molecular weight, and a isan empirical constant describing the conformation of thepolymer. For alginate, the exponent (a) is close to unity at anionic s
