ASTM F2064-00
(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
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 F 748 Practice for Selecting Generic Biological Test Meth-
ods for Materials and Devices
1.1 Thisguidecoverstheevaluationofalginatessuitablefor
F 749 Practice for Evaluating Material Extracts by Intracu-
use in biomedical or pharmaceutical applications, or both,
taneous Injection in the Rabbit
including, but not limited to, tissue-engineered medical prod-
F 756 Practice for Assessment of Hemolytic Properties of
ucts (TEMPS).
Materials
1.2 This guide addresses key parameters relevant for the
F 763 Practice for Short-Term Screening of Implant Mate-
functionality, characterization, and purity of alginates.
rials
1.3 As with any material, some characteristics of alginates
F 813 PracticeforDirectContactCellCultureEvaluationof
may be altered by processing techniques (such as molding,
Materials for Medical Devices
extrusion, machining, assembly, sterilization, and so forth)
F 895 Test Method forAgar Diffusion Cell Culture Screen-
required for the production of a specific part or device.
ing for Cytotoxicity
Therefore, properties of fabricated forms of this polymer
F 981 Practice for Assessment of Compatibility of Bioma-
should be evaluated using test methods that are appropriate to
terials for Surgical Implants with Respect to Effect of
ensure safety and efficacy and are not addressed in this guide.
Materials on Muscle and Bone
1.4 This standard does not purport to address all of the
F 1251 Terminology Relating to Polymeric Biomaterials in
safety concerns, if any, associated with its use. It is the
Medical and Surgical Devices
responsibility of the user of this standard to establish appro-
F 1439 Guide for Performance of Lifetime Bioassay for the
priate safety and health practices and determine the applica-
Tumorigenic Potential of Implant Materials
bility of regulatory limitations prior to use.
F 1903 Practice for Testing for Biological Responses to
2. Referenced Documents
Particles In Vitro
F 1904 Practice for Testing the Biological Responses to
2.1 ASTM Standards:
Particles In Vivo
D 2196 Test Methods for Rheological Properties of Non-
F 1905 Practice for Selecting Tests for Determining the
Newtonian Materials by Rotational (Brookfield) Viscom-
Propensity of Materials to Cause Immunotoxicity
eter
F 1906 Practice for Evaluation of Immune Responses in
F 619 Practice for Extraction of Medical Plastics
Biocompatibility Testing Using ELISATests, Lymphocyte
Proliferation, and Cell Migration
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
2.2 USP Document:
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Tissue Characterization.
Current edition approved Nov. 10, 2000. Published February 2001.
Annual Book of ASTM Standards, Vol 06.01.
Annual Book of ASTM Standards, Vol 13.01.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2064–00
4 9
USP Monograph USP 24/NF 19<719> Sodium Alginate 2.8 prEN Documents :
2.3 ISO Documents : prEN 12442-1 Animal tissues and their derivative utilized
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
Williams, DF. The Williams Dictionary of Biomaterials.
ISO/DIS 10993-17—Part 17: Methods for establishment of
Liverpool University Press, 1999.
allowable limits for leachable substances using health-
based risk assessment
3. Terminology
ISO 13408-1: 1998: Aseptic processing of health care
3.1 Definitions of Terms Specific to This Standard: (see also
products—Part 1: General requirements.
Terminology F 1251):
2.4 ICH Documents :
3.1.1 alginate, n—a polysaccharide substance containing
International Conference on Harmonization (ICH) S2B
calcium, magnesium, sodium, and potassium salts obtained
Genotoxicity: A Standard Battery for Genotoxicity Test-
6 from some of the more common species of marine algae.
ing of Pharmaceuticals (July 1997)
Alginate exists in brown algae as the most abundant polysac-
International Conference on Harmonization (ICH) Q1A
charide, mainly occurring in the cell walls and intercellular
ICH HarmonizedTripartiteGuidanceforStabilityTesting
spaces of brown seaweed and kelp. Its main function is to
of New Drug Substances and Products (September 23,
contribute to the strength and flexibility of the seaweed plant.
1994)
Alginate is classified as a hydrocolloid. The most commonly
2.5 FDA Documents :
used alginate is sodium alginate.
FDAGuideline on validation of the Limulus amebocyte test
3.1.2 decomposition, n—structural changes of alginates due
as an end-product endotoxin test for human and animal
to exposure to environmental, chemical or thermal factors,
parenteral drugs, biological products and healthcare prod-
such as temperatures greater than 180°C. Decomposition can
ucts. DHHS, December 1987.
result in deleterious changes to the alginate.
FDA. Interim guidance for human and veterinary drug
3.1.3 degradation, n—change in the chemical structure,
products and Biologicals. Kinetic LAL techniques.
physical properties, or appearance of a material. Degradation
DHHS, July 15, 1991.
of polysaccharides occurs by means of cleavage of the glyco-
2.6 ANSI Documents :
sidic bonds, usually by acid catalyzed hydrolysis. Degradation
ANSI/AAMI/ISO 11737-1: 1995: Sterilization of medical
can also occur thermally. It is important to note that degrada-
devices—microbiological methods—Part 1: Estimation of
tion is not synonymous with decomposition. Degradation is
bioburden on product.
often used as a synonym for depolymerization when referring
ANSI/AAMI/ISO 11737-2: 1998: Sterilization of medical
to polymers.
devices—microbiological methods—Part 2: Tests of ste-
3.1.4 depolymerization, n—reductioninlengthofapolymer
rility performed in the validation of a sterilization process.
chain to form shorter polymeric units. Depolymerization may
2.7 AAMI Documents :
reduce the polymer chain to oligomeric or monomeric units, or
AAMI/ISO 14160—1998: Sterilization of single-use medi-
both. In alginates, hydrolysis of the glycosidic bonds is the
cal devices incorporating materials of animal origin—
primary mechanism.
Validation and routine control of sterilization by liquid
3.1.5 Endotoxin, n—a high-molecular weight lipopolysac-
chemical sterilants.
charide (LPS) complex associated with the cell wall of
AAMI ST67/CDV-2: 1999: Sterilization of medical
gram-negative bacteria that is pyrogenic in humans. Though
devices—requirements for products labeled “sterile”.
endotoxins are pyrogens, not all pyrogens are endotoxins.
AAMI TIR No. 19—1998: Guidance for ANSI/AAMI/ISO
3.1.6 hydrocolloid, n—a water-soluble polymer of colloidal
10993-7: 1995, Biological evaluation of medical
nature when hydrated.
devices—Part 7: Ethylene oxide sterilization residuals.
3.1.7 molecular mass average (molecular weight average),
n—thegivenmolecularweight(Mw)ofanalginatewillalways
represent an average of all of the molecules in the population.
The most common ways to express the Mw are as the number
Available from United States Pharmacopeia and National Formulary, U.S.
¯ ¯
average ~M ! and the weight average ~M !. The two averages
n w
Pharmaceutical Convention, Inc. (USPC), Rockville, MD.
are defined by the following equations:
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
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
7 9
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
8 10
AssociationfortheAdvancementofMedicalInstrumentation1110NorthGlebe Available from Superintendent of Documents, U.S. Government Printing
Rd., Suite 220, Arlington, VA 22201–4795. Office, Washington, DC 20402
F2064–00
903 (m), and 811 (m). The peak designators are: sh: sharp; s:
NM wM NM
(i i i (i i i (i i i
M 5 and M 5 5 (1)
n w
strong; m: medium; w: weak; and b: broad.
N w NM
(i i (i i (i i i
5.2 Physical and chemical characterization of alginate:
where:
5.2.1 The composition and sequential structure of alginate
N = number of molecules having a specific molecular
i
can be a key functional attribute of any alginate. Variations in
weight, M, and
i
the composition or the sequential structure, or both, may, but
w = weight of molecules having a specific molecular
i
not necessarily, cause differences in performance of an alginate
weight M
i
in a particular end use.This information may be determined by
¯ ¯
Inapolydispersemolecularpopulationtherelation M >M
w n
1 13
the following method: High-resolution H and C-nuclear
¯ ¯
is always valid. The coefficient M /M is referred to as the
w n
magnetic resonance spectroscopy (NMR). Sodium alginate
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
guarantee of clinical success in any tissue engineered medical w
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 d
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