ASTM F2064-00(2006)
(Guide)Standard Guide for Characterization and Testing of Alginates as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Products Application
Standard Guide for Characterization and Testing of Alginates as Starting Materials Intended for Use in Biomedical and Tissue-Engineered Medical Products Application
SCOPE
1.1 This guide covers the evaluation of alginates suitable for use in biomedical or pharmaceutical applications, or both, including, but not limited to, tissue-engineered medical products (TEMPS).
1.2 This guide addresses key parameters relevant for the functionality, characterization, and purity of alginates.
1.3 As with any material, some characteristics of alginates may 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 polymer should be evaluated using test methods that are appropriate to ensure safety and efficacy and are not addressed in this guide.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:F2064–00 (Reapproved 2006)
Standard Guide for
Characterization and Testing of Alginates as Starting
Materials Intended for Use in Biomedical and Tissue-
Engineered Medical Products Application
This standard is issued under the fixed designation F 2064; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Alginate has found uses in a variety of products ranging from simple technical applications such as
viscosifiers to advanced biomedical matrices providing controlled drug delivery from immobilized
living cells. As for most hydrocolloids, the functionality of alginate is related to its chemical and
structural composition. The aim of this guide is to identify key parameters relevant for the
functionality and characterization of alginates for the development of new commercial applications of
alginates for the biomedical and pharmaceutical industries.
1. Scope Newtonian Materials by Rotational (Brookfield type) Vis-
cometer
1.1 Thisguidecoverstheevaluationofalginatessuitablefor
F 619 Practice for Extraction of Medical Plastics
use in biomedical or pharmaceutical applications, or both,
F 748 Practice for Selecting Generic Biological Test Meth-
including, but not limited to, tissue-engineered medical prod-
ods for Materials and Devices
ucts (TEMPS).
F 749 Practice for Evaluating Material Extracts by Intracu-
1.2 This guide addresses key parameters relevant for the
taneous Injection in the Rabbit
functionality, characterization, and purity of alginates.
F 756 Practice for Assessment of Hemolytic Properties of
1.3 As with any material, some characteristics of alginates
Materials
may be altered by processing techniques (such as molding,
F 763 Practice for Short-Term Screening of Implant Mate-
extrusion, machining, assembly, sterilization, and so forth)
rials
required for the production of a specific part or device.
F 813 PracticeforDirectContactCellCultureEvaluationof
Therefore, properties of fabricated forms of this polymer
Materials for Medical Devices
should be evaluated using test methods that are appropriate to
F 895 Test Method forAgar Diffusion Cell Culture Screen-
ensure safety and efficacy and are not addressed in this guide.
ing for Cytotoxicity
1.4 This standard does not purport to address all of the
F 981 Practice for Assessment of Compatibility of Bioma-
safety concerns, if any, associated with its use. It is the
terials for Surgical Implants with Respect to Effect of
responsibility of the user of this standard to establish appro-
Materials on Muscle and Bone
priate safety and health practices and determine the applica-
F 1251 Terminology Relating to Polymeric Biomaterials in
bility of regulatory limitations prior to use.
Medical and Surgical Devices
2. Referenced Documents F 1439 Guide for Performance of Lifetime Bioassay for the
Tumorigenic Potential of Implant Materials
2.1 ASTM Standards:
F 1903 Practice for Testing For Biological Responses to
D 2196 Test Methods for Rheological Properties of Non-
Particles in vitro
F 1904 Practice for Testing the Biological Responses to
Particles in vivo
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
F 1905 Practice For Selecting Tests for Determining the
Surgical Materials and Devices and is the direct responsibility of Subcommittee
Propensity of Materials to Cause Immunotoxicity
F04.42 on Biomaterials and Biomolecules for TEMPs.
F 1906 Practice for Evaluation of Immune Responses In
Current edition approved March 1, 2006. Published April 2006. Originally
approved in 2000. Last previous edition approved in 2000 as F 2064 – 00.
Biocompatibility Testing Using ELISATests, Lymphocyte
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Proliferation, and Cell Migration
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2064–00 (2006)
2.2 USP Document: 2.8 prEN Documents:
USP Monograph USP 24/NF 19<719> Sodium Alginate prEN 12442-1 Animal Tissues and their Derivative Utilized
2.3 ISO Documents: in the Manufacture of Medical Devices—Part 1:Analysis
ISO 10993 Biological Evaluation of Medical Devices: and Management of Risk
ISO 10993-1 Biological Evaluation of Medical Devices— prEN –12442 Part 3:Validation of the Elimination and/or
Part 1: Evaluation and Testing Inactivation of Virus and Transmissible Agents
ISO10993-3 Part3:TestsforGenotoxicity,Carcinogenicity 2.9 Other Documents:
and Reproductive Toxicity 21CFR184.1724 Listing of Specific SubstancesAffirmed as
ISO 10993-9—Part 9: Framework for Identification and GRAS–Sodium Alginate
Quantification of Potential Degradation Products
3. Terminology
ISO 10993-17—Part 17: Methods for Establishment of
AllowableLimitsforLeachableSubstancesUsingHealth- 3.1 Definitions of Terms Specific to This Standard: (see also
Based Risk Assessment Terminology F 1251):
ISO 13408-1: 1998: Aseptic Processing of Health Care 3.1.1 alginate, n—a polysaccharide substance containing
Products—Part 1: General Requirements. calcium, magnesium, sodium, and potassium salts obtained
2.4 ICH Documents:
from some of the more common species of marine algae.
International Conference on Harmonization (ICH) S2B Alginate exists in brown algae as the most abundant polysac-
Genotoxicity: A Standard Battery for Genotoxicity Test-
charide, mainly occurring in the cell walls and intercellular
ing of Pharmaceuticals (July 1997) spaces of brown seaweed and kelp. Its main function is to
International Conference on Harmonization (ICH) Q1A
contribute to the strength and flexibility of the seaweed plant.
ICH HarmonizedTripartiteGuidanceforStabilityTesting Alginate is classified as a hydrocolloid. The most commonly
of New Drug Substances and Products (September 23,
used alginate is sodium alginate.
1994) 3.1.2 decomposition, n—structural changes of alginates due
2.5 FDA Documents:
to exposure to environmental, chemical or thermal factors,
FDA Guideline on Validation of the Limulus Amebocyte such as temperatures greater than 180°C. Decomposition can
Test as an End-Product Endotoxin Test for Human and
result in deleterious changes to the alginate.
AnimalParenteralDrugs,BiologicalProductsandHealth- 3.1.3 degradation, n—change in the chemical structure,
care Products. DHHS, December 1987 physical properties, or appearance of a material. Degradation
FDA. Interim Guidance for Human and Veterinary Drug of polysaccharides occurs by means of cleavage of the glyco-
Products and Biologicals. Kinetic LAL techniques. sidic bonds, usually by acid catalyzed hydrolysis. Degradation
DHHS, July 15, 1991 can also occur thermally. It is important to note that degrada-
2.6 ANSI Documents: tion is not synonymous with decomposition. Degradation is
ANSI/AAMI/ISO 11737-1: 1995: Sterilization of Medical often used as a synonym for depolymerization when referring
Devices—Microbiological Methods—Part 1: Estimation to polymers.
of Bioburden on Product. 3.1.4 depolymerization, n—reductioninlengthofapolymer
ANSI/AAMI/ISO 11737-2: 1998: Sterilization of Medical chain to form shorter polymeric units. Depolymerization may
Devices—Microbiological Methods—Part 2:Tests of Ste- reduce the polymer chain to oligomeric or monomeric units, or
rilityPerformedintheValidationofaSterilizationProcess both. In alginates, hydrolysis of the glycosidic bonds is the
2.7 AAMI Documents: primary mechanism.
AAMI/ISO 14160—1998: Sterilization of Single-Use 3.1.5 Endotoxin, n—a high-molecular weight lipopolysac-
Medical Devices Incorporating Materials of Animal charide (LPS) complex associated with the cell wall of
Origin—Validation and Routine Control of Sterilization gram-negative bacteria that is pyrogenic in humans. Though
by Liquid Chemical Sterilants endotoxins are pyrogens, not all pyrogens are endotoxins.
AAMI ST67/CDV-2: 1999: Sterilization of Medical 3.1.6 hydrocolloid, n—a water-soluble polymer of colloidal
Devices—Requirements for Products Labeled “Sterile” nature when hydrated.
AAMI TIR No. 19—1998: Guidance for ANSI/AAMI/ISO 3.1.7 molecular mass average (molecular weight average),
n—thegivenmolecularweight(Mw)ofanalginatewillalways
10993-7: 1995, Biological Evaluation of Medical
Devices—Part 7: Ethylene Oxide Sterilization Residuals represent an average of all of the molecules in the population.
The most common ways to express the Mw are as the number
¯ ¯
average ~M ! and the weight average ~M !. The two averages
n w
are defined by the following equations:
Available from U.S. Pharmacopeia (USP), 12601Twinbrook Pkwy., Rockville,
MD 20852. NM wM NM
( ( (
i i i i i i i i i
4 M 5 and M 5 5 (1)
n w
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
N w NM
( ( (
i i i i i i i
4th Floor, New York, NY 10036.
Available from ICH Secretariat, c/o IFPMA, 30 rue de St-Jean, P.O. Box 758,
1211 Geneva 13, Switzerland.
6 8
Available from U. S. Food and Drug Administration, 5600 Fishers Lane, Available from European Committee for Standardization CEN, Management
Rockville MD 20857-0001. Centre 36, rue de Stassart B-1050 Brussels, Belgium.
7 9
AssociationfortheAdvancementofMedicalInstrumentation1110NorthGlebe Available from Superintendent of Documents, U.S. Government Printing
Rd., Suite 220, Arlington, VA 22201–4795. Office, Washington, DC 20402.
F2064–00 (2006)
5.2.1 The composition and sequential structure of alginate
where:
can be a key functional attribute of any alginate. Variations in
N = number of molecules having a specific molecular
i
the composition or the sequential structure, or both, may, but
weight, M, and
i
w = weight of molecules having a specific molecular notnecessarily,causedifferencesinperformanceofanalginate
i
weight M in a particular end use.This information may be determined by
i
1 13
¯ ¯
Inapolydispersemolecularpopulationtherelation M >M the following method: High-resolution H and C-nuclear
w n
¯ ¯
is always valid. The coefficient M /M is referred to as the magnetic resonance spectroscopy (NMR). Sodium alginate
w n
polydispersityindex,andwilltypicallybeintherangefrom1.5 should be dissolved in D O and partially degraded to a degree
to 3.0 for commercial alginates. of depolymerization of 20 to 30 using mild acid hydrolysis
3.1.8 pyrogen, n—any substance that produces fever when
before recording proton or carbon NMR spectra (Grasdalen,
administered parenterally. H., Larsen, B., and Smidsrød, O., Carbohydr. Res., 68, 23-31,
1979). Techniques have been developed to determine the
4. Significance and Use
monad frequencies F (fraction of guluronate residues) and F
G M
4.1 This guide contains a listing of those characterization
(fraction of mannuronate residues), the four nearest neighbor-
parameters that are directly related to the functionality of
ing(diad)frequencies(F ,F ,F ,andF )andtheeight
GG GM MG MM
alginate. This guide can be used as an aid in the selection and
next nearest neighboring (triad) frequencies (F ,F ,
GGG GGM
characterization of the appropriate alginate for a particular
F ,F ,F ,F ,F , and F ). A typical H-
GMM GMG MGM MGG MMG MMM
application. This guide is intended to give guidance in the
NMR spectrum of alginate is shown as follows. Alginate is
methods and types of testing necessary to properly character-
characterized by calculating parameters such as M/G ratio,
ize, assess, and ensure consistency in the performance of a
G-content, consecutive number of G monomers (that is, G>1),
particular alginate. It may have use in the regulation of these
and average length of blocks of consecutive G monomers.
devices by appropriate authorities.
5.2.2 Molecular mass (molecular weight) of an alginate will
4.2 The alginate covered by this guide may be gelled,
define certain performance characteristics such as viscosity or
extruded, or otherwise formulated into biomedical devices for
gel strength, or both.As such and depending on the sensitivity
use in tissue-engineered medical products or drug delivery
of a particular end use to these variations, determination of
devicesforimplantationasdeterminedtobeappropriate,based
molecular mass directly or indirectly may be necessary. Com-
on supporting biocompatibility and physical test data. Recom-
mercial alginates are polydisperse with respect to molecular
mendations in this guide should not be interpreted as a
weight (M ). Molecular weight may be expressed as the
w
guarantee of clinical success in any tissue engineered medical
number average (M ) or the weight average (M ). Molecular
N W
product or drug delivery application.
weights may be determined by methods such as, but not
4.3 To ensure that the material supplied satisfies require-
limited, to the following
ments for use in TEMPS, several general areas of character-
5.2.2.1 Molecular Weight Determination Based on Intrinsic
ization should be considered. These are: identity of alginate,
Viscosity—The intrinsic viscosity describes a polymer’s ability
physical and chemical characterization and testing, impurities
to form viscous solutions in water and is directly proportional
profile, and performance-related tests.
to the average molecular weight of the polymer. The intrinsic
5. Chemical and Physical Test Methods viscosity is a characteristic of the polymer under specified
solvent and temperature conditions; it is independent of con-
5.1 Identity of Alginate—The identity of alginates can be
centration. The intrinsic viscosity (h) is directly related to the
establishedbyseveralmethodsincluding,butnotlimitedtothe
molecular weight of a polymer through the Mark-Houwink-
following:
a
Sakurada (MHS) equation: [h]=KM , where K is a constant,
5.1.1 Sodium alginate monograph USP 24/NF19.
M is the viscosity derived average molecular weight, and a is
5.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)—
an empirical constant describing the conformation of the
Almost all organic chemical compounds absorb infrared radia-
polymer. For alginate, the exponent (a) is close to unity at an
tion at frequencies characteristic for the functional groups in
ionic strength of 0.1 (for example, 0.1 M NaCl). By measuring
the compound. A FT-IR spectrum will show absorption bands
the intrinsic viscosity, the viscosity average molecular weight
relating to bond stretching and bending and can therefore serve
can be determined if K and a are accurately known for the
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