ASTM F2212-20

Standard Guide for Characterization of Type I Collagen as Starting Material for Surgical Implants and Substrates for Tissue Engineered Medical Products (TEMPs)

SIGNIFICANCE AND USE
4.1 The objective of this guide is to provide guidance in the characterization of Type I collagen as a starting material for surgical implants and substrates for tissue engineered medical products (TEMPs). This guide contains a listing of physical and chemical parameters that are directly related to the function of collagen. This guide can be used as an aid in the selection and characterization of the appropriate collagen starting material for the specific use. Not all tests or parameters are applicable to all uses of collagen.  
4.2 The collagen covered by this guide may be used in a broad range of applications, forms, or medical products, for example (but not limited to) medical devices, tissue engineered medical products (TEMPs) or cell, drug, or DNA delivery devices for implantation. The use of collagen in a practical application should be based, among other factors, on biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success in any tissue engineered medical product or drug delivery application.  
4.3 The following general areas should be considered when determining if the collagen supplied satisfies requirements for use in TEMPs. These are source of collagen, chemical and physical characterization and testing, and impurities profile.  
4.4 The following documents or other appropriate guidances from appropriate regulatory bodies relating to the production, regulation, and regulatory approval of TEMPs products should be considered when determining if the collagen supplied satisfies requirements for use in TEMPs:    
FDA CFR:  
21 CFR 3: Product Jurisdiction:  
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=3    
21 CFR 58: Good Laboratory Practice for Nonclinical Laboratory Studies:  
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=58    
FDA/CDRH CFR and Guidances:  
21 CFR Part 803: Medical Device ...
SCOPE
1.1 This guide for characterizing collagen-containing biomaterials is intended to provide characteristics, properties, and test methods for use by producers, manufacturers, and researchers to more clearly identify the specific collagen materials used. With greater than 20 types of collagen and the different properties of each, a single document would be cumbersome. This guide will focus on the characterization of Type I collagen, which is the most abundant collagen in mammals, especially in skin and bone. Collagen isolated from these sources may contain other types of collagen, for example, Type III and Type V. This guide does not provide specific parameters for any collagen product or mix of products or the acceptability of those products for the intended use. The collagen may be from any source including, but not limited to, animal or cadaveric sources, human cell culture, or recombinant sources. The biological, immunological, or toxicological properties of the collagen may vary, depending on the source material. The properties of the collagen prepared from each of the above sources must be thoroughly investigated, as the changes in the collagen properties as a function of source materials is not thoroughly understood. This guide is intended to focus on purified Type I collagen as a starting material for surgical implants and substrates for tissue engineered medical products (TEMPs); some methods may not be applicable for gelatin or tissue implants. This guide may serve as a template for characterization of other types of collagen.  
1.2 The biological response to collagen in soft tissue has been well documented by a history of clinical use (1, 2)2 and laboratory studies (3-6). Biocompatibility and appropriateness of use for a specific application(s) is the responsibility of the product manufacturer.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this...

General Information

• Status: Published
• Publication Date: 30-Nov-2020
• ICS: 11.100.99 - Other standards related to laboratory medicine
• Technical Committee: F04 - Medical and Surgical Materials and Devices
• Drafting Committee: F04.42 - Biomaterials and Biomolecules for TEMPs

Relations

• Refers: ASTM F749-20 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Feb-2020
• Refers: ASTM F2148-18 - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
• Effective Date: 01-Dec-2018
• Refers: ASTM F1903-18 - Standard Practice for Testing for Cellular Responses to Particles <emph type="bdit" >in vitro</emph>
• Effective Date: 01-Oct-2018
• Refers: ASTM F1439-03(2018) - Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
• Effective Date: 01-Feb-2018
• Refers: ASTM F720-17 - Standard Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
• Effective Date: 01-Sep-2017
• Refers: ASTM F756-17 - Standard Practice for Assessment of Hemolytic Properties of Materials
• Effective Date: 01-Mar-2017
• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• Refers: ASTM F895-11(2016) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Apr-2016
• Refers: ASTM F756-13 - Standard Practice for Assessment of Hemolytic Properties of Materials
• Effective Date: 01-Dec-2013
• Refers: ASTM F1439-03(2013) - Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
• Effective Date: 01-Oct-2013
• Refers: ASTM F720-13 - Standard Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
• Effective Date: 01-Jun-2013
• Refers: ASTM F2148-13 - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
• Effective Date: 01-Jun-2013
• Refers: ASTM F749-13 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Mar-2013
• Refers: ASTM F813-07(2012) - Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
• Effective Date: 01-Oct-2012
• Refers: ASTM F2148-07(2012) - Standard Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
• Effective Date: 01-Oct-2012

Overview

ASTM F2212-20 is the standard guide developed by ASTM International for the characterization of Type I collagen as a starting material for surgical implants and substrates used in tissue engineered medical products (TEMPs). This guide outlines parameters and methods for evaluating the chemical, physical, and biological properties of Type I collagen, which is crucial for use in a wide array of biomedical applications such as medical devices, drug delivery systems, and tissue engineering scaffolds. The document serves as a comprehensive reference for producers, manufacturers, and researchers to ensure that collagen starting materials meet appropriate safety, quality, and functional benchmarks.

Key Topics

Scope and Purpose

• Provides a framework for the characterization of Type I collagen from various sources, including animal, human, or recombinant origins.
• Details physical and chemical parameters directly linked to the collagen's intended function in medical products and implants.
• Offers guidance for selecting and evaluating collagen for specific applications, not prescribing universal requirements.

Characterization Areas

• Source Assessment: Evaluation of the collagen's tissue and organism origin, noting its potential impact on biocompatibility and product safety.
• Physical and Chemical Characterization: Includes assays and analytical methods such as SDS-PAGE, amino acid analysis, peptide mapping, and electron microscopy.
• Impurity Profiling: Identification and quantification of impurities like endotoxins, heavy metals, residual crosslinkers, and other protein types.
• Crosslinking and Stability: Addresses both the natural stability of collagen and additional crosslinking processes, which can alter resistance to degradation and affect mechanical properties.
• Microbiological Safety: Ensures sterility and absence of contaminating microorganisms, vital for implantable materials.
• Regulatory Compliance: Recommends consideration of relevant FDA, ISO, and pharmacopeia guidelines during material evaluation.

Testing Considerations

• Not all characterization methods are required for every application; selection depends on the form and intended use of collagen.
• Emphasizes validated, widely accepted protein chemistry methods for accuracy and reproducibility.
• Biocompatibility and appropriateness for use in humans remain the responsibility of the manufacturer.

Applications

Type I collagen is the most prevalent form in mammals and is extensively employed in various biomedical applications, making its careful characterization essential. Key applications include:

• Surgical Implants: Used as graft materials, wound dressings, and reconstructive devices.
• Tissue Engineered Medical Products (TEMPs): Forms scaffolds or substrates for cell growth and tissue regeneration.
• Biomaterial Carriers: Collagen serves as a matrix for the delivery of cells, drugs, DNA, or other therapeutic agents.
• Medical Devices: Found in hemostatic sponges, dermal equivalents, and injectable formulations for soft tissue augmentation.

The use of well-characterized collagen contributes not only to product efficacy but also to regulatory compliance, patient safety, and predictable clinical outcomes.

Related Standards

For comprehensive assessment and regulatory alignment, manufacturers and researchers should consult the following relevant standards and guidelines:

• ASTM Standards: F619, F748, F813, F895, F981, F1439, F2148
• ISO Standards: ISO 10993 series (Biological Evaluation of Medical Devices), ISO 13408-1, ISO 14971, ISO 22442 parts 1-3
• US and European Pharmacopeia
• FDA Regulations: 21 CFR Parts 820, 58, 312, 610, 1271; FDA Guidance for Industry on Pyrogen and Endotoxin Testing
• AAMI Standards: ANSI/AAMI/ISO 11737, ANSI/AAMI ST67
• ICH Guidelines: ICH M3(R2), S1A/B/C, S2A/B, S5A/B

These standards address aspects such as biocompatibility, sterilization, purity, safety, risk management, and quality systems for medical products utilizing collagen.

---

By following ASTM F2212-20 for the characterization of Type I collagen, organizations ensure robust material assessment, support regulatory submissions, and ultimately enhance the safety and performance of collagen-based surgical implants and tissue engineered products.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F2212 − 20
Standard Guide for
Characterization of Type I Collagen as Starting Material for
Surgical Implants and Substrates for Tissue Engineered
Medical Products (TEMPs)
This standard is issued under the fixed designation F2212; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Collagen-based medical products are becoming more prevalent, especially in the area of soft tissue
augmentation. The use of collagen in surgery dates back to the late 1800s, with the use of catgut
sutures, human cadaveric skin, and fascia. More recently, collagen has been used in hemostatic
sponges, dermal equivalents, injectables for soft tissue augmentation, as a matrix for cell-based
products, and as a vehicle for drug delivery. It is because of the versatility of collagen in medical
applications that specific characterizations should be performed as a way to compare materials.
1. Scope gelatin or tissue implants. This guide may serve as a template
for characterization of other types of collagen.
1.1 This guide for characterizing collagen-containing bio-
materials is intended to provide characteristics, properties, and 1.2 The biological response to collagen in soft tissue has
test methods for use by producers, manufacturers, and re- been well documented by a history of clinical use (1, 2) and
searchers to more clearly identify the specific collagen mate- laboratory studies (3-6). Biocompatibility and appropriateness
rials used. With greater than 20 types of collagen and the of use for a specific application(s) is the responsibility of the
different properties of each, a single document would be product manufacturer.
cumbersome. This guide will focus on the characterization of
1.3 The values stated in SI units are to be regarded as
Type I collagen, which is the most abundant collagen in
standard. No other units of measurement are included in this
mammals, especially in skin and bone. Collagen isolated from
standard.
thesesourcesmaycontainothertypesofcollagen,forexample,
1.4 Warning—Mercury has been designated by EPA and
Type III and Type V. This guide does not provide specific
many state agencies as a hazardous material that can cause
parameters for any collagen product or mix of products or the
central nervous system, kidney, and liver damage. Mercury, or
acceptability of those products for the intended use. The
its vapor, may be hazardous to health and corrosive to
collagen may be from any source including, but not limited to,
materials.Cautionshouldbetakenwhenhandlingmercuryand
animal or cadaveric sources, human cell culture, or recombi-
mercury-containing products. See the applicable product Ma-
nant sources. The biological, immunological, or toxicological
terial Safety Data Sheet (MSDS) for details and EPA’s website
properties of the collagen may vary, depending on the source
(http://www.epa.gov/mercury/faq.htm) for additional informa-
material. The properties of the collagen prepared from each of
tion. Users should be aware that selling mercury or mercury-
the above sources must be thoroughly investigated, as the
containingproducts,orboth,inyourstatemaybeprohibitedby
changes in the collagen properties as a function of source
state law.
materials is not thoroughly understood. This guide is intended
1.5 The following precautionary caveat pertains only to the
to focus on purified Type I collagen as a starting material for
surgical implants and substrates for tissue engineered medical test method portion, Section 5, of this guide. This standard
does not purport to address all of the safety concerns, if any,
products (TEMPs); some methods may not be applicable for
associated with its use. It is the responsibility of the user of this
standard to establish appropriate safety, health, and environ-
mental practices and determine the applicability of regulatory
This guide is under the jurisdiction ofASTM Committee F04 on Medical and
limitations prior to use.
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs.
Current edition approved Dec. 1, 2020. Published January 2021. Originally
approved in 2002. Last previous edition approved in 2019 as F2212–19. DOI: Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
10.1520/F2212-20. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2212 − 20
1.6 This international standard was developed in accor- ISO 13408–1Aseptic Processing of Health Care Products—
dance with internationally recognized principles on standard- Part 1: General Requirements
ization established in the Decision on Principles for the ISO 14971Medical Devices—Application of Risk Manage-
Development of International Standards, Guides and Recom- ment to Medical Devices
mendations issued by the World Trade Organization Technical ISO 22442–1Animal Tissues and their Derivatives Utilized
Barriers to Trade (TBT) Committee. in the Manufacture of Medical Devices—Part 1:Applica-
tion of Risk Management
2. Referenced Documents
ISO 22442–2Animal Tissues and their Derivatives Utilized
in the Manufacture of Medical Devices—Part 2: Controls
2.1 ASTM Standards:
on Sourcing, Collection, and Handling
F619Practice for Extraction of Materials Used in Medical
ISO 22442–3Animal Tissues and their Derivatives Utilized
Devices
in the Manufacture of Medical Devices—Part 3: Valida-
F720PracticeforTestingGuineaPigsforContactAllergens:
tion of the Elimination and/or Inactivation of Viruses and
Guinea Pig Maximization Test
Transmissible Spongiform Encephalopathy (TSE) Agents
F748PracticeforSelectingGenericBiologicalTestMethods
for Materials and Devices
2.3 U. S. and European Pharmacopeia Documents:
F749Practice for Evaluating Material Extracts by Intracuta- European Pharmacopeia 5.0
neous Injection in the Rabbit
United States Pharmacopeia (USP), Edition XXX (30)
F756Practice for Assessment of Hemolytic Properties of
USP 30/NF 19Viral Safety Evaluation of Biotechnology
Materials
Products Derived from Cell Lines of Human or Animal
F763Practice for Short-Term Screening of Implant Materi-
Origin
als 6
2.4 Code of Federal Regulations:
F813Practice for Direct Contact Cell Culture Evaluation of
9 CFR 113Standard Requirements
Materials for Medical Devices
21 CFR 312Investigational New Drug Application
F895TestMethodforAgarDiffusionCellCultureScreening
21 CFR Part 820 Quality System Regulation
for Cytotoxicity
21 CFR Parts 207, 807, and 1271Human Cells,Tissues and
F981Practice for Assessment of Compatibility of Biomate-
Cellular and Tissue-Based Products, Establishment Reg-
rials for Surgical Implants with Respect to Effect of
istration and Listing
Materials on Muscle and Insertion into Bone
21 CFR Part 1271, Part CSuitability Determination for
F1439Guide for Performance of Lifetime Bioassay for the
Donors of Human Cell and Tissue-based Products, Pro-
Tumorigenic Potential of Implant Materials
posed Rule
F1903Practice for Testing for Cellular Responses to Par-
CFR610.13(b) Rabbit Pyrogen Assay
ticles in vitro
Current Good Tissue Practice for Manufacturers of Human
F1904Practice for Testing the Biological Responses to
Cellular and Tissue-Based Products,Inspection and En-
Particles in vivo
forcement. Proposed Rule. Federal Register/Vol. 66, No.
F2148Practice for Evaluation of Delayed Contact Hyper-
5/January 8, 2001/Proposed Rules, pp. 1552-1559
sensitivity Using the Murine Local Lymph Node Assay
Guidance for Screening and Testing of Donors of Human
(LLNA)
Tissue Intended for Transplantation, Availability. Federal
2.2 ISO Standards:
Register/Vol. 62, No. 145/July 29, 1997/Notices
ISO 10993–1Biological Evaluation of Medical Devices—
Guidance for Industry and for FDA Reviewers,Medical
Part 1: Evaluation andTesting within a Risk Management
Devices Containing Materials Derived from Animal
Process
Sources(Exceptfor In VitroDiagnosticDevices),Novem-
ISO 10993–9Framework for Identification and Quantifica-
ber 6, 1998, U.S. Department of Health and Human
tion of Potential Degradation Products
Services, Food and Drug Administration, Center for De-
ISO 10993–10Biological Evaluation of Medical Devices—
vices and Radiological Health
Part 10: Tests for Irritation and Skin Sensitization
Federal Register Vol. 43,No. 141, Friday, July 21, 1978
ISO 10993–17 Establishment of Allowable Limits for
Federal Register, Vol. 66,No. 13, Jan 19, 2001/Rules and
Leachable Substances Using Health-Based Risk Assess-
Regulations, p. 5447
ment
Federal Register, Vol. 72, No. 8, Jan. 12, 2007, pp.
1581–1619, Proposed Rule: Use of Materials Derived
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook
the ASTM website. Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
4 6
Available from International Organization for Standardization (ISO), ISO AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
Geneva, Switzerland, http://www.iso.org. www.access.gpo.gov.
F2212 − 20
from Cattle in Medical Products Intended for Use in U.S. Food and Drug Administration (FDA) Center for
Humans and Drugs Intended for Use in Ruminants Biologics Evaluation and Research (CBER), 1993Points
to Consider in the Characterization of Cell Lines Used to
2.5 ICH Documents:
Produce Biologicals
ICH M3(R2)Guidance for Industry M3 Nonclinical Safety
U.S. Food and Drug Administration (FDA) Center for
Studies for the Conduct of Human Clinical Trials and
Biologics Evaluation and Research (CBER), 1997Points
Marketing Authorizations for Pharmaceuticals 62 FR
toConsiderintheManufactureandTestingofMonoclonal
62922 (2009)
Antibody Products for Human Use, 94D-0259
ICH S1AGuideline for Industry S1A The Need for Long-
term Rodent Carcinogenicity Studies of Pharmaceuticals.
2.7 AAMI Documents:
61 FR 8153 (1996)
AAMI TIR 19:1998Guidance forANSI/AAMI/ISO 10993-
ICH S1BGuidance for Industry S1B Testing for Carcinoge-
7:1995, Biological Evaluation of Medical Devices—Part
nicity of Pharmaceuticals. 63 FR 8983 (1998)
7: Ethylene Oxide Sterilization Residuals
ICH S1CGuideline for Industry S1C Dose Selection for
ANSI/AAMI/ISO 11737-1:2018Sterilization of Healthcare
Carcinogenicity Studies of Pharmaceuticals. 60 FR 11278
Products—Microbiological Methods—Part 1: Determina-
(1995)
tion of a Population of Microorganisms on Products
ICH S1C(R)Guidance for Industry Addendum to Dose
ANSI/AAMI/ISO 11737-2:2009Sterilization of Medical
Selection for Carcinogenicity Studies of Pharmaceuticals:
Devices—MicrobiologicalMethods—Part2:TestsofSte-
AdditionofaLimitDoseandRelatedNotes.62FR64259
rility Performed in the Definition, Validation and Mainte-
(1997)
nance of a Sterilization Process
ICH S2AGuideline for Industry S2A Specific Aspects of
ANSI/AAMI/ISO 14160:2011/(R) 2016 Sterilization of
RegulatoryGenotoxicityTestsforPharmaceuticals.61FR
Health Care Products—Liquid Chemical Sterilizing
18199 (1996)
Agents for Single-Use Medical Devices UtilizingAnimal
ICH S2BGuidance for Industry S2B Genotoxicity: A Stan-
Tissues and Their Derivatives—Requirements for
dard Battery for Genotoxicity Testing of Pharmaceuticals
Characterization, Development, Validation and Routine
62 FR 62472 (1997)
Control of a Sterilization Process for Medical Devices
ICH S5AGuideline for Industry S5A Detection of Toxicity
ANSI/AAMI ST67:2011/(R) 2017Sterilization of Health
to Reproduction for Medicinal Products. 59 FR 48746
CareProducts—RequirementsandGuidanceforSelecting
(1994)
a Sterility Assurance Level (SAL) for Products Labeled
ICH S5BGuidance for Industry S5B Detection of Toxicity
“Sterile”
to Reproduction for Medicinal Products: Addendum on
2.8 Other References:
Toxicity to Male Fertility. 61 FR 15360 (1996)
DraftGuidanceforPreclinicalandClinicalInvestigationsof
ICH Q1A(R2)HarmonizedTripartite Guideline for Stability
UrethralBulkingAgentsUsedintheTreatmentofUrinary
Testing of New Drug Substances and Products (February
Incontinence, November 29, 1995. (ODE/DRARD/
6, 2003)
ULDB), Document No. 850
2.6 FDA Documents:
Council Directive 93/42/EEC,with Respect to Medical De-
FDAGuidance for Industry, Pyrogen and Endotoxins Test- 11
vices Using Tissues of Animal Origin
ing: Questions andAnswers, June 2015, U.S. Department
CommissionDirective2003/32/EC,withRespecttoMedical
of Health and Human Services, Food and Drug
Devices Manufactured Using Tissues of Animal Origin
Administration,CenterforDrugEvaluationandResearch,
EMEA/410/01-rev.2,Committee for Proprietary Medical
Center for Biologics Evaluation and Research, Center for
Products, Note for Guidance on Minimizing the Risk of
VeterinaryMedicine,CenterforDevicesandRadiological
TransmittingAnimal Spongiform EncephalopathyAgents
Health, Office of Regulatory Affairs
via Human and Veterinary Medical Products
FDAInterim Guidance for Human and Veterinary Drug
The European Agency for the Evaluation of Medicinal
Products and Biologicals, Kinetic LAL Techniques,
Products, (EMEA),Committee for Proprietary Medicinal
DHHS, July 15, 1991
Products(CPMP)GuidanceDocumentforDecisionTrees
U.S. Food and Drug Administration (FDA) and Committee
for the Selection of Sterilisation Methods (CPMP/QWP/
for Proprietary Medicinal Products (CPMP), 1998Inter-
054/98 corr 2000) and Annex to Note for Guidance on
national Conference on Harmonization (ICH), Quality of
Biotechnological Products: Viral Safety Evaluation of
Biotechnology Products Derived from Cell Lines of Hu-
Available from Association for the Advancement of Medical Instrumentation
man or Animal Origin, Consensus Guideline ICH Viral
(AAMI), 4301 N. Fairfax Dr., Suite 301, Arlington, VA 22203-1633, http://
Safety Document: Step 5
www.aami.org.
Available from U.S. Food and Drug Administration (FDA), 10903 New
Hampshire Ave., Silver Spring, MD 20993, http://www.fda.gov.
7 11
Available from International Conference on Harmonisation of Technical Available from Office for Official Publications of the European
Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH Communities—European Law, 2, rue Mercier, L-2985, Luxembourg, http://eur-
Secretariat, 9, chemin des Mines, P.O. Box 195, 1211 Geneva 20, Switzerland, lex.europa.eu/en/’index.htm.
http://www.ich.org. Available from European Medicines Agency (EMEA), 7 Westferry Circus,
Available from U.S. Food and Drug Administration (FDA), 10903 New Canary Wharf, London E14 4HB, U.K., http://www.eudora.org/emea.html, and
Hampshire Ave., Silver Spring, MD 20993, http://www.fda.gov. http://www.emea.europa.eu/pdfs/human/bwp/TSE%20NFG%20410-rev2.pdf.
F2212 − 20
Development Pharmaceutics (CPMP/QWP/155/96) 3.1.10 suspension, n—the dispersion of a solid through a
liquidwithaparticlesizelargeenoughtobedetectedbypurely
optical means.
3. Terminology
3.1 Definitions:
4. Significance and Use
3.1.1 adventitious agent, n—an unintentionally introduced
4.1 The objective of this guide is to provide guidance in the
microbiological or other infectious contaminant.
characterization of Type I collagen as a starting material for
3.1.1.1 Discussion—In the production of TEMPs, these
surgical implants and substrates for tissue engineered medical
agents may be unintentionally introduced into the process
products (TEMPs). This guide contains a listing of physical
stream or the final product, or both.
and chemical parameters that are directly related to the
3.1.2 biocompatibility, n—a material may be considered
function of collagen. This guide can be used as an aid in the
biocompatibleifthematerialperformswithanappropriatehost
selection and characterization of the appropriate collagen
response in a specific application (7).
startingmaterialforthespecificuse.Notalltestsorparameters
3.1.3 collagen, n—a family of at least 20 genetically differ-
are applicable to all uses of collagen.
ent secreted proteins that serve a predominantly structural
4.2 The collagen covered by this guide may be used in a
function and possess a unique triple helical structure configu-
broad range of applications, forms, or medical products, for
ration of three polypeptide units known as alpha chains.
example(butnotlimitedto)medicaldevices,tissueengineered
3.1.4 degradation, n—change in chemical, physical, or mo-
medical products (TEMPs) or cell, drug, or DNA delivery
lecularstructureorappearance(thatis,grossmorphology)ofa
devices for implantation. The use of collagen in a practical
material.
application should be based, among other factors, on biocom-
patibility and physical test data. Recommendations in this
3.1.5 endotoxin,n—pyrogenichighmolarmasslipopolysac-
guide should not be interpreted as a guarantee of clinical
charide (LPS) complex associated with the cell wall of
success in any tissue engineered medical product or drug
gram-negative bacteria.
delivery application.
3.1.5.1 Discussion—Though endotoxins are pyrogens, not
all pyrogens are endotoxins. Endotoxins are specifically de-
4.3 The following general areas should be considered when
tected through a Limulus amebocyte lysate (LAL) test.
determining if the collagen supplied satisfies requirements for
3.1.6 medical product, n—any diagnostic or therapeutic use in TEMPs. These are source of collagen, chemical and
treatment that may be regulated as a device, biologic, drug, or physical characterization and testing, and impurities profile.
combination product.
4.4 The following documents or other appropriate guid-
3.1.7 microorganism, n—bacteria, fungi, yeast, mold,
ances from appropriate regulatory bodies relating to the
viruses, and other infectious agents.
production, regulation, and regulatory approval of TEMPs
products should be considered when determining if the colla-
3.1.7.1 Discussion—However,itshouldbenotedthatnotall
gen supplied satisfies requirements for use in TEMPs:
microorganisms are infectious or pathogenic.
FDA CFR:
3.1.8 solubility, n—a measure of the extent to which the
21 CFR 3: Product Jurisdiction:
material can be dissolved.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=3
3.1.8.1 Discussion—Any colloidal system without obvious
21 CFR 58: Good Laboratory Practice for Nonclinical Laboratory Studies:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
phase separation can be considered soluble. In the context of
CFRSearch.cfm?CFRPart=58
collagen, solubility refers to the dissociation of the fibrillar
aggregates of collagen molecules into a solution. Native Type
FDA/CDRH CFR and Guidances:
21 CFR Part 803: Medical Device Reporting:
I collagen, which is soluble in dilute acids, but not soluble at
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
physiological conditions, is termed “insoluble” or “acid
CFRSearch.cfm?CFRPart=803
soluble,” while simple aggregates of non-fibrillar collagen
21 CFR 812: Investigational Device Exemptions:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
soluble in neutral salt solutions are termed “neutral salt
CFRSearch.cfm?CFRPart=812
soluble.” Post translational surface charge modifications may
21 CFR 814: Premarket Approval of Medical Devices :
alter the solubility of collagen in neutral pH condition.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=814
3.1.9 sterilization, n—the destruction or removal of all
21 CFR 820: Quality System Regulation:
microorganisms in or about an object (for example, by chemi-
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
cal agents, electron beam, gamma irradiation, or filtration). CFRSearch.cfm?CFRPart=820
Design Control Guidance for Medical Device Manufacturers:
3.1.9.1 Discussion—If the medical product collagen http://www.fda.gov/cdrh/comp/designgd.pdf
Preproduction Quality Assurance Planning Recommendations for
permits,terminalsterilizationispreferentialtoasepticprocess-
Medical Device Manufacturers (FDA 90-4236):
ing.
http://www.fda.gov/cdrh/manual/appende.html
The Review and Inspection of Premarket Approval Applications under the
Bioresearch Monitoring Program—Draft Guidance for Industry and FDA
Staff:
http://www.fda.gov/cdrh/comp/guidance/1602.pdf
Available from European Medicines Agency (EMEA), 7 Westferry Circus,
Canary Wharf, London E14 4HB, U.K., http://www.eudora.org/emea.html, and
FDA/CDRH Search Engines:
http://www.emea.europa.eu/pdfs/human/qwp/005498en.pdf.
F2212 − 20
acids must be within the range of published data for highly
CDRH Guidance Search Engine:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfggp/search.cfm
purified collagen preparations, generally in the acid soluble
CDRH Premarket Approval (PMA) Search Engine:
form). Amino acid analysis is routinely performed on hydro-
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMA/pma.cfm
lyzed collagens by reverse phase High Performance Liquid
CDRH 510(k) Search Engine:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.cfm
Chromatography(HPLC).Thismethodcanbeusedtoquantify
CDRH Recognized STANDARDS Search Engine :
hydroxyproline, tyrosine, tryptophan, and cysteine. There are
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/
other methods available for amino acid analysis.
search.cfm
5.4 Purity of soluble collagen can be analyzed by SDS-
FDA/CBER CFR and Guidances:
PAGE, either on the collagen directly or after digestion of the
21 CFR 312: Investigational New Drug Application :
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
collagen with purified bacterial collagenase to detect any
CFRSearch.cfm?CFRPart=312
remaining proteins.
21 CFR 314: Applications for FDA Approval to Market a New Drug:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
5.5 Elastin Assay—Elastin can be a component of the
CFRSearch.cfm?CFRPart=31
impuritiesinaninsolublecollagenpreparation.Onemethodto
21 CFR 610: General Biological Products Standards:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/ assay for elastin, although other methods are available, in-
CFRSearch.cfm?CFRPart=610
volves the detection of desmosine (15). These impurities can
21 CFR 1271: Human Cells, Tissues and Cellular and Tissue-Based
be detected by Western blots, enzyme-linked immunosorbent
Products:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/ assays (ELISAs), and other types of assays.
CFRSearch.cfm?CFRPart=1271
5.6 Peptide mapping is one possible method to identify
Cellular & Gene Therapy Guidances and Other Publications:
http://www.fda.gov/cber/genetherapy/gtpubs.htm
Type I collagen. The most commonly used peptide mapping
Human Tissue Guidances and Other Publications:
method utilizes Cyanogen Bromide (CNBr) digestion. The
http://www.fda.gov/cber/tissue/docs.htm
digest can be analyzed by SDS-PAGE or HPLC.
CBER Product Approval Information:
http://www.fda.gov/cber/efoi/approve.htm
5.7 Impurities Profile—The term impurity relates to the
21 CFR 600, 601 BLA Regulations:
http://www.access.gpo.gov/nara/cfr/waisidx_07/21cfrv7_
presence of extraneous substances and materials in the colla-
07.html
gen. These impurities can be detected by Western blots,
21 CFR 210, 211 GMP Regulations:
ELISAs, gas chromatography/mass spectrometry (GC-MS),
http://www.access.gpo.gov/nara/cfr/waisidx_07/21cfr210_
07.html
andothertypesofassays.Ifthereisaconcernforthepresence
of processing aids or other impurities associated with the
5. Chemical and Physical Characterizations
collagen,theyshouldbeaddressedwiththesupplier.Themajor
5.1 General Comments on Chemical and Physical Charac-
impurities of concern include, but are not limited to the
terization of Collagen—These methods are suggested assays;
following: endotoxins, glycosaminoglycans, elastin, lipids,
however,othervalidatedassaymethodsmaybeused.Selection
improperlyalignedcollagenmolecules,hostcellcontaminants,
of assay systems will vary, depending on the configuration of
cell culture contaminants, heavy metals, bioburden, viruses,
the collagen (that is, soluble or insoluble). The user should
transmissible spongiform encephalopathy (TSE) agents, cross-
ensure that the method selected is reliable and commonly
linking and enzymatic agents, and components used in extrac-
accepted in protein chemistry. A review of collagen materials
tionorsolubilization(forexample,acids,surfactants,solvents,
may be found in Li, 2000 (8), while a review of the collagen
and so forth). Type III collagen may also be associated with
familyofproteinsmaybefoundinRefs (9-14).Whenselecting
TypeIcollagen.Whileitspresencemayhavenoadverseeffect
anappropriatetestmethod,theusershouldnotethatimpurities
on product quality, levels should be evaluated and controlled
in highly purified collagen are low or lower than 1 to 2 %, so
for lot-to-lot consistency. The inclusion of urea can be used to
sensitivetestmethodsneedtobeutilized.Forsolublecollagen,
resolve Type I and Type II collagen alpha 1 chains (16).
the following represents a non-inclusive list of assay systems
Assessment of collagens other than Type I and III is discussed
available: Sodium Dodecyl Sulfate Polyacrylamide Gel Elec-
in 5.19. At a minimum, any protein impurity of greater than
trophoresis (SDS-PAGE); peptide mapping; and physico-
1% in the final collagen preparation should be identified and
chemical analysis. A similar list for insoluble collagen may
quantified.
include, but not be limited to, assay methods for hexosamine
5.8 Crosslinking Reactions with Collagen—Collagen is a
(that is, detection of glycoproteins), lipid, total sugar, des-
very stable protein due to its triple-helical structure, imparting
mosine (that is, elastin), and amino acid composition (that is,
resistance to most proteolytic enzymes. It is still sensitive to
collagen composition profile; non-collagenous amino acids).
collagenase, however. The stability can be enhanced by cross-
Additionally, methods such as transmission electron micros-
linking the molecule by physical or chemical means. Both
copy may be helpful in characterizing the collagen fibers or
inter- and intrachain crosslinking can occur due to the propen-
collagen superstructure.
sity of collagen fibers to naturally crosslink. Crosslinking
5.2 The concentration of collagen should be expressed in
agents and methods include aldehydes, carbodiimides,
mass/volumeormass/mass.Colorometricassaysoraminoacid
epoxides, diisocyanates, non-enzymatic glycosylation, dehy-
analysis for hydroxyproline are commonly used methods to
drothermal treatment (DHT), radiation (for example, gamma,
measure collagen content.
electron beam), and ultraviolet light. For chemical
5.3 Amino acid analysis will provide information on the crosslinking, excess crosslinker should be removed and quan-
composition of the amino acids of collagen (that is, the amino titatedbeforeoratthefinalproductstage.Acrosslinkermaybe
F2212 − 20
cytotoxicormutagenic,orboth,andanycomponentinthefinal GuidanceforIndustry,PyrogenandEndotoxinsTesting:Ques-
product needs to be quantitated. There are several methods tions and Answers for information pertaining to the rabbit
available,includingliquidchromatography/massspectrophom- pyrogen assay.
etry (LC/MS), GC/MS, or other assays. Any crosslinker used
5.10 Heavy Metal Content by the USP Method—This test is
that has the potential of reacting with DNAshould be consid-
provided to demonstrate that the content of heavy metal
ered a mutagen. A mutagenic assay should at least be per-
impurities does not exceed a limit in the individual product
formed on the final product in that case. A cytotoxicity
specification. This method is based on <231> Heavy Metals,
assessment will also provide a measure of acceptable cross-
1st and 6th Supplement USP-NF. Substances that typically
linker levels. Physical crosslinking may result in unwanted
respond to this test are lead, mercury, bismuth, arsenic,
changestothestructureofthecollagenmoleculeandshouldbe
antimony, tin, cadmium, silver, copper, and molybdenum.
assessed with qualification assays appropriate to the clinical
Under the specified test conditions, the limit is determined by
indication under consideration. Direct measurement of colla-
a concomitant visual comparison of metals that are colored by
gen crosslinking can be performed by looking at the altered
sulfide ion with a control prepared from a Standard Lead
amino acid composition and using methods appropriate for the
Solution.Additionalheavymetalcontaminantsmaybepresent
crosslinker. One method, for example (other methods exist), to
due to processing. If necessary, the user may detect these
measure degree of crosslinking when lysine residues are
contaminantsbyvariousmethodsthatmayinclude,butarenot
involved is to detect free lysines and hydroxylysines by
limited to, spectrographic, chromatographic, and flame atomic
labelingthe ε-aminoacidgroupswith2.4.6trinitrobenzenesul-
absorption techniques.
fonic acid (TNBS), where the TNBS-labeled amino acids
5.11 Microbiological Safety—Bacteria, viruses, and fungi
absorb at 345 nm with a molar absorptivity of 1.46 × 10
arealsocontaminantsthatcanariseinabiologicalsample.The
L/mole × cm. Amino acid composition can also be examined
user will validate sterilization and characterize its effect on the
by analysis of sodium borohydride-treated collagen. The ther-
product. The presence of bacteria may also contribute to the
mal denaturation characteristics can also be measured by
presenceofendotoxins.ThefollowingMicrobiologicalTestsin
Differential Scanning Calorimetry (DSC) (17). The thermal
USP 30 are of particular relevance: Microbial Limit Tests
denaturation characteristics can sometimes be correlated with
<61>, SterilityTests <71>, Sterilization and sterility assurance
the crosslink density.The % water uptake (% swell), using the
of compendial articles <12211>, the Biological Tests and
equation (W –W )/W , where W = dry weight and W =
W D W D W
Assays: Bacterial Endotoxins Tests <85>, and viral validation
wet weight, is also an indirect measure of collagen crosslink-
studies <1050>. The user should also consider other relevant
ing. The tensile strength can be altered by crosslinking.
standards, such as, but not limited to, Association for the
Measurements using a universal testing machine (UTM) or a
Advancement of Medical Instrumentation (AAMI) standards
rheometer will note a change in properties after crosslinking.
and international standards, of which the following are ex-
Collagen crosslinking imparts a resistance to the proteolytic
amples: ANSI/AAMI/ISO 11737-1:2018; ANSI/AAMI/ISO
enzyme collagenase. Collagenase is the one enzyme that will
11737-2:2009; and ISO 13408–1. The collagen is first dis-
digesttriple-strandedcollagen.Whencollageniscrosslinked,it
solved in a sterile, aqueous solution, then filtered using sterile
is more resistant to breakdown and extensive crosslinking will
techniques through a 0.45 µm membrane filter. The filters are
afford the greatest resistance to collagenase.
subsequently incubated on Tryptic Soy Agar to determine the
5.9 Endotoxin Content—Endotoxin contamination is diffi-
presence of bacteria, and on Sabouraud Dextrose Agar to
cult to prevent because it is ubiquitous in nature, stable, and determinethepresenceofyeastandmold.Ifcollagenproducts
small enough to pass through sterilizing filters (0.22 µm).
are intended to serve as a barrier to microorganisms, this
Endotoxin tests for collagen include the gel clot, endpoint
function will need to be validated with specific experiments.
assay, and the kinetic assay (Food and Drug Administration
5.12 Carbohydrate analysis of collagens can be carried out
GuidanceforIndustry,PyrogenandEndotoxinsTesting:Ques-
by classical gas-liquid chromatographic methods or spectro-
tionsandAnswers).Thegelclottestisthesimplestandeasiest
photometric methods. Novel sources of collagen may result in
oftheLimulusamebocytelysate(LAL)testmethods,although
a different glycosylation pattern or sugars that differ from
much less sensitive than the kinetic assay. The quantitative
human collagen, or both. A potential risk of autoimmune
kinetic assay, which measures the amount of time required to
disease may be present. If a novel source of collagen is used
reach a predetermined optical density, is the most sensitive.
and the carbohydrate pattern is unknown, a risk assessment
Each new lot of reagents should meet acceptance criteria
should be performed. This should include an analysis of the
established by appropriate qualification or validation studies
sugars present on the collagen. If necessary, the gylcosylation
(for investigational or licensed/cleared products, respectively).
properties of the collagen should be examined and an assess-
Theendotoxinlevelincollagenwillultimatelybecriticaltoits
ment of the autoimmunity potential performed.
useinbiomedicalapplicationswherethereareregulatorylimits
totheamountofendotoxinthatcanbeimplantedintohumans. 5.13 Trypsin susceptibility will detect that portion of colla-
Relevant FDA guidance for allowable levels and information gen that has been denatured during purification steps such as
regardingvalidationofendotoxinassaysshouldbeconsultedif acid and base treatment, solvent treatment, and so forth.
human trials are contemplated (Interim Guidance for Human Trypsin will digest that portion of the collagen and can be
andVeterinaryDrugProductsandBiologicals).Theuserisalso measured by assaying the hydroxyproline content of the
directed to CFR610.13(b) and Food and DrugAdministration supernatant. Triple helical collagen is resistant to digestion by
F2212 − 20
TABLE 1 Characterization Methods for Type I Collagen
most proteases. Susceptibility to trypsin or other appropriate
proteasesisdeterminedbyexposingthecollagentotheenzyme Characterization Method Applicable to
and assaying the digest for degradation. There are several Chemical
Appearance Soluble or Insoluble
methods for this test.
Concentration Soluble or Insoluble
Purity Soluble or Insoluble
5.14 Differential Scanning Calorimetry (DSC) determines
Amino acid analysis Soluble or Insoluble
dissociation temperature of collagens in fibrils, as well as
Peptide mapping Soluble or Insoluble
detecting microfibrils and denatured collagen at lower melting Impurities profile, including Soluble or Insoluble
Heavy Metal Analysis
temperatures. (See also 5.8, crosslinking reactions with colla-
Carbohydrate analysis Soluble or Insoluble
gen).
Trypsin resistance Soluble or Insoluble,
Mainly Insoluble
5.15 Viscosity is more applicable to gels or suspensions but
Collagenase resistance Soluble or Insoluble,
may be useful with collagen configured in forms such as, but Mainly Insoluble
pH of implantable Soluble or Insoluble
notlimitedto,pastesorfilms (18).Viscosityofcollagen-based
Additives (cross-linkers, Soluble or Insoluble
materials depends on a number of factors which may include,
lubricants, drugs, sterilents)
but are not limited to, the following: solution or dispersion/
Physical
suspension, concentration, temperature, operating condition,
Shrink Temperature (DSC) Insoluble
and so forth. It is not feasible to determine the viscosity of
Viscosity Mainly soluble
TEM Insoluble
films.This is a routine test performed with a viscometer (not a
SDS-PAGE Soluble or Insoluble
u-tube). The user must clearly state the conditions of the test.
Moisture Content, dependent Insoluble
on storage environment
5.16 Transmission electron microscopy may be used to
Electron Micrograph (native Insoluble
show the quality of collagen fibers. Unraveling or changes in
banded 640 Å
structure for fibrils)
banding will be obvious.
5.17 DNA sequence data on recombinant or transgenic Biochemical
Endotoxin level Soluble or Insoluble
source cells: Verify sequence data for expression gene, that is,
Bioburden Soluble or Insoluble
COL1A1 or COL1A2.
% Type I collagen/Total Protein Soluble or Insoluble
% Other Types Collagen and Soluble or Insoluble
5.18 Characterization Methods for Type I Collagen (Table
List of
1)—The collagen material shall have specifications for an which Types present
Total DNA (ppm or %) Soluble or Insoluble
extensive set of chemical and physical properties such as, but
Total Lipid Soluble or Insoluble
not limited to, those listed in Table 1. The table represents
% native collagen (by trypsin Soluble or Insoluble
resistance,
methods which may or may not be appropriate for character-
circular Dichroism)
izing a particular collagen sample. Not all the methods listed
may be required to characterize the sample, and the specificity
Abbreviations in Table:
andsensitivityvaryamongthemethodslisted.Theusershould DSC = Differential Scanning Calorimetry
TEM = Transmission Electron Microscopy
befamiliarwiththelimitationsoftheappropriatetestmethods.
SDS-PAGE = Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
5.19 Analysis for Type II, IV, and Other Collagens—Tissues
commonly used to isolate Type I collagen typically contain
likely, an assessment should be completed for collagens that
someTypeIIIcollagenwhichcoexistsinmanytissues.TypeII
have the potential to generate adverse reactions. The extent of
collagenisfoundprimarilyincartilage,whileTypeIVcollagen
analysis required will depend upon the risk of other collagen
is found in basement membranes and has been associated with
types being present as impurities in a particular collagen
Goodpasture’s Syndrome (Wieslander, J., et al, (19)). The
product.
purity of collagen is important in determining the potential for
safety problems and providing criteria for the consistency of
6. Product Development Considerations
the manufacturing process. For example, skin collagen is
6.1 StorageConditions/ShelfLifeStabilityofCollagen—For
composedofapproximately90%TypeI,8–10%TypeIII,and
collagen, the most relevant stability-indicating parameters are
theremainderismadeupoftraceamountsofthelessabundant
those related to the functionality of the polymer. Depending
collagens, primarily Type IV, V, and VI (Holbrook and Smith,
upon what function the collagen will have in the final
(20)). As all collagens contain triple helical domains, the
formulation, parameters such as viscosity (apparent and intrin-
properties of different collagen types can be very similar.
sic) and biological activity, along with other parameters
Detection byWestern Blot analysis, therefore, requires the use
deemed relevant, may also be considered. Storage conditions
of antibodies that recognize epitopes in the more diverse
are important, especially for collagen solutions. International
non-helical regions.Antibodies are available for Types I to VI
Conference on Harmonization (ICH) guidance documents
collagens, and possibly others, for use in Western Blot or
should be consulted for information on stability testing of
ELISAanalyses following SDS gel electrophoresis.Validation
pharmaceuticals (that is, ICH Q1A).
of antibody specificity, as well as the test procedure, using
suitablestandards,shouldbeconductedpriortoanalysis.Arisk 6.2 Sterilization Method, if Applicable, and Effects of Ster-
assessment should be performed on the potential for other ilization on the Product—The user should verify that the
collagens in the product. If the presence of other collagens is sterilization method does not adversely affect the collagen end
F2212 − 20
product. Collagen can be sterilized by gamma irradiation, (ICH) documents: Guidance for Industry and for FDA
electron beam, or by ethylene oxide, or prepared using aseptic Reviewers, Medical Devices Containing Materials Derived
processing steps. Potential degradation of the collagen or
fromAnimalSources(Exceptfor In VitroDiagnosticDevices);
sterilization residuals should be evaluated to determine the U.S. Food and DrugAdministration (FDA) and Committee for
impact on the product. Solutions of collagen may be (1) filter
Proprietary Medicinal Products (CPMP), 1998 International
sterilizediftheviscosityofthecollagensolutionpermits;or(2) Conference on Harmonization (ICH), Quality of Biotechno-
gamma-irradiated. Any changes in viscosity may reflect an
logical Products: Viral Safety Evaluation of Biotechnology
alteration of the molecular mass and should be evaluated. The ProductsDerivedfromCellLinesofHumanorAnimalOrigin,
methodofsterilizationisprimarilydictatedbytheeffectonthe
ConsensusGuidelineICHViralSafetyDocument:Step5;U.S.
producteffectiveness.Themethodchosenmustbevalidatedto
Food and Drug Administration (FDA) Center for Biologics
determine the effectiveness of sterilization. The reader should
Evaluation and Research (CBER), 1993 Points to Consider in
refer to the most current version of the relevant standards
the Characterization of Cell Lines Used to Produce Biologi-
regarding the sterilization of healthcare products by radiation,
cals;andU.S.FoodandDrugAdministration(FDA)Centerfor
steam, and ethylene oxide gas, such as AAMI TIR 19:1998;
Biologics Evaluation and Research (CBER), 1997 Points to
ANSI/AAMI/ISO 14160:2011; ANSI/AAMI ST67:2011; and
Consider in the Manufacture and Testing of Monoclonal
The European Agency for the Evaluation of Medicinal
Antibody Products for Human Use, 94D-0259. The European
Products,(EMEA),CommitteeforProprietaryMedicinalProd-
Pharmacopoeia 5.0 has a monograph describing methods to
ucts (CPMP) Guidance Document for Decision Trees for the
minimizeTSErisks(5.2.8MinimisingtheRiskofTransmitting
Selection of Sterilisation Methods (CPMP/QWP/054/98 corr
Animal Spongiform Encephalopathy Agents Via Human and
2000) and Annex to Note for Guidance on Development
Veterinary Medicinal Products).
Pharmaceutics (CPMP/QWP/155/96).
6.4.1 Viral Clearance—The sources of raw materials from
humans or animals should be screened for known viral
6.3 Sourcing—The criteria to consider for safe sourcing
include appropriate human or animal donor selection and the pathogens to reduce or eliminate the potential infectivity. For
tissue collection procedures to ensure that the source material material of animal origin to be considered, see also ISO
is unlikely to contain TSE infectivity. Recombinant sources of 22442–1. Viral clearance methods can include, but not be
collagen, which provide a good option from purity and limited to, methods such as detergent treatment, high or low
integrity standpoints, should also be considered. Additional pH, urea treatment, other chemical treatments, and filtration or
information can be obtained from the following documents: other purification methods. However, even these harsh treat-
ISO 22442–1; ISO 22442–2; ISO 22442–3; 21 CFR Parts 207, ments may not ensure complete viral inactivation. Viral clear-
807, and 1271; 21 CFR Part 820; 21 CFR Part 1271, Part C;
ance should be demonstrated by an appropriately validated
Federal Register Vol. 43, No. 141; Federal Register Vol. 62,
viral clearance study protocol (see USP30/NF19<1050>).The
No.145;FederalRegisterVol.66,No.5;FederalRegister,Vol.
user should verify that the viral clearance procedure is com-
66, No. 13; ISO 13408–1; Council Directive 93/42/EEC;
patiblewiththestartingmaterialortheconfiguredendproduct.
Commission Directive 2003/32/EC; and EMEA/410/01-rev.2.
For human tissue sources for manufacturing into collagen, the
Additional documents may be available. The user should use
observance of Current Good Tissue Practices should be con-
the most current version of all documents.
sidered.
6.3.1 For further information, the user is referred to Appen-
6.4.2 TSE Sourcing Issues and TSE Clearance—Careful
dixX2,SourcingIssues:X2.1TissueforCollagenorCollagen-
attention should be given to the sourcing of raw materials,
ContainingMedicalDevices;X2.2RequirementsforaClosed-
process design to remove potential TSE agents and treatments
Herd; and X2.3 Documentation.
to inactivate TSE agents for those products which can with-
6.3.2 The collagen can be isolated from tissues or cell
stand the harsh treatments required to inactivate TSE agents.
cultures by any method, including, but not limited to, extrac-
Th
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F2212 − 19 F2212 − 20
Standard Guide for
Characterization of Type I Collagen as Starting Material for
Surgical Implants and Substrates for Tissue Engineered
Medical Products (TEMPs)
This standard is issued under the fixed designation F2212; 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 (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Collagen-based medical products are becoming more prevalent, especially in the area of soft tissue
augmentation. The use of collagen in surgery dates back to the late 1800s, with the use of catgut
sutures, human cadaveric skin, and fascia. More recently, collagen has been used in hemostatic
sponges, dermal equivalents, injectables for soft tissue augmentation, as a matrix for cell-based
products, and as a vehicle for drug delivery. It is because of the versatility of collagen in medical
applications that specific characterizations should be performed as a way to compare materials.
1. Scope
1.1 This guide for characterizing collagen-containing biomaterials is intended to provide characteristics, properties, and test
methods for use by producers, manufacturers, and researchers to more clearly identify the specific collagen materials used. With
greater than 20 types of collagen and the different properties of each, a single document would be cumbersome. This guide will
focus on the characterization of Type I collagen, which is the most abundant collagen in mammals, especially in skin and bone.
Collagen isolated from these sources may contain other types of collagen, for example, Type III and Type V. This guide does not
provide specific parameters for any collagen product or mix of products or the acceptability of those products for the intended use.
The collagen may be from any source including, but not limited to, animal or cadaveric sources, human cell culture, or recombinant
sources. The biological, immunological, or toxicological properties of the collagen may vary, depending on the source material.
The properties of the collagen prepared from each of the above sources must be thoroughly investigated, as the changes in the
collagen properties as a function of source materials is not thoroughly understood. This guide is intended to focus on purified Type
I collagen as a starting material for surgical implants and substrates for tissue engineered medical products (TEMPs); some
methods may not be applicable for gelatin or tissue implants. This guide may serve as a template for characterization of other types
of collagen.
1.2 The biological response to collagen in soft tissue has been well documented by a history of clinical use (1, 2) and laboratory
studies (33-6, 4, 5, 6). Biocompatibility and appropriateness of use for a specific application(s) is the responsibility of the product
manufacturer.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
This guide is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee F04.42
on Biomaterials and Biomolecules for TEMPs.
Current edition approved Sept. 1, 2019Dec. 1, 2020. Published December 2019January 2021. Originally approved in 2002. Last previous edition approved in 20112019
as F2212 – 11F2212 – 19. . DOI: 10.1520/F2212-19.10.1520/F2212-20.
The boldface numbers in parentheses refer to the list of references at the end of this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2212 − 20
1.4 Warning—Mercury has been designated by EPA and many state agencies as a hazardous material that can cause central
nervous system, kidney, and liver damage. Mercury, or its vapor, may be hazardous to health and corrosive to materials. Caution
should be taken when handling mercury and mercury-containing products. See the applicable product Material Safety Data Sheet
(MSDS) for details and EPA’s website (http://www.epa.gov/mercury/faq.htm) for additional information. Users should be aware
that selling mercury or mercury-containing products, or both, in your state may be prohibited by state law.
1.5 The following precautionary caveat pertains only to the test method portion, Section 5, of this guide. 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, health, and environmental practices and determine the applicability of regulatory limitations prior
to use.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
F619 Practice for Extraction of Materials Used in Medical Devices
F720 Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F749 Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
F756 Practice for Assessment of Hemolytic Properties of Materials
F763 Practice for Short-Term Screening of Implant Materials
F813 Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
F895 Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
F981 Practice for Assessment of Compatibility of Biomaterials for Surgical Implants with Respect to Effect of Materials on
Muscle and Insertion into Bone
F1439 Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
F1903 Practice for Testing for Cellular Responses to Particles in vitro
F1904 Practice for Testing the Biological Responses to Particles in vivo
F2148 Practice for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay (LLNA)
2.2 ISO Standards:
ISO 10993–1 Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing within a Risk Management Process
ISO 10993–9 Framework for Identification and Quantification of Potential Degradation Products
ISO 10993–10 Biological Evaluation of Medical Devices—Part 10: Tests for Irritation and Skin Sensitization
ISO 10993–17 Establishment of Allowable Limits for Leachable Substances Using Health-Based Risk Assessment
ISO 13408–1 Aseptic Processing of Health Care Products—Part 1: General Requirements
ISO 14971 Medical Devices—Application of Risk Management to Medical Devices
ISO 22442–1 Animal Tissues and their Derivatives Utilized in the Manufacture of Medical Devices—Part 1: Application of Risk
Management
ISO 22442–2 Animal Tissues and their Derivatives Utilized in the Manufacture of Medical Devices—Part 2: Controls on
Sourcing, Collection, and Handling
ISO 22442–3 Animal Tissues and their Derivatives Utilized in the Manufacture of Medical Devices—Part 3: Validation of the
Elimination and/or Inactivation of Viruses and Transmissible Spongiform Encephalopathy (TSE) Agents
2.3 U. S. and European Pharmacopeia Documents:
European Pharmacopeia 5.0
United States Pharmacopeia (USP), Edition XXX (30)
USP 30/NF 19 Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin
2.4 Code of Federal Regulations:
9 CFR 113 Standard Requirements
21 CFR 312 Investigational New Drug Application
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva,
Switzerland, http://www.iso.org.
Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
F2212 − 20
21 CFR Part 820 Quality System Regulation
21 CFR Parts 207, 807, and 1271 Human Cells, Tissues and Cellular and Tissue-Based Products, Establishment Registration and
Listing
21 CFR Part 1271, Part C Suitability Determination for Donors of Human Cell and Tissue-based Products, Proposed Rule
CFR 610.13(b) Rabbit Pyrogen Assay
Current Good Tissue Practice for Manufacturers of Human Cellular and Tissue-Based Products, Inspection and Enforcement.
Proposed Rule. Federal Register/Vol. 66, No. 5/January 8, 2001/Proposed Rules, pp. 1552-1559
Guidance for Screening and Testing of Donors of Human Tissue Intended for Transplantation, Availability. Federal
Register/Vol. 62, No. 145/July 29, 1997/Notices
Guidance for Industry and for FDA Reviewers, Medical Devices Containing Materials Derived from Animal Sources (Except
for In Vitro Diagnostic Devices), November 6, 1998, U.S. Department of Health and Human Services, Food and Drug
Administration, Center for Devices and Radiological Health
Federal Register Vol. 43, No. 141, Friday, July 21, 1978
Federal Register, Vol. 66, No. 13, Jan 19, 2001/Rules and Regulations, p. 5447
Federal Register, Vol. 72, No. 8, Jan. 12, 2007, pp. 1581–1619, Proposed Rule: Use of Materials Derived from Cattle in Medical
Products Intended for Use in Humans and Drugs Intended for Use in Ruminants
2.5 ICH Documents:
ICH M3(R2) Guidance for Industry M3 Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing
Authorizations for Pharmaceuticals 62 FR 62922 (2009)
ICH S1A Guideline for Industry S1A The Need for Long-term Rodent Carcinogenicity Studies of Pharmaceuticals. 61 FR 8153
(1996)
ICH S1B Guidance for Industry S1B Testing for Carcinogenicity of Pharmaceuticals. 63 FR 8983 (1998)
ICH S1C Guideline for Industry S1C Dose Selection for Carcinogenicity Studies of Pharmaceuticals. 60 FR 11278 (1995)
ICH S1C(R) Guidance for Industry Addendum to Dose Selection for Carcinogenicity Studies of Pharmaceuticals: Addition of
a Limit Dose and Related Notes. 62 FR 64259 (1997)
ICH S2A Guideline for Industry S2A Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. 61 FR 18199
(1996)
ICH S2B Guidance for Industry S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals 62 FR
62472 (1997)
ICH S5A Guideline for Industry S5A Detection of Toxicity to Reproduction for Medicinal Products. 59 FR 48746 (1994)
ICH S5B Guidance for Industry S5B Detection of Toxicity to Reproduction for Medicinal Products: Addendum on Toxicity to
Male Fertility. 61 FR 15360 (1996)
ICH Q1A(R2) Harmonized Tripartite Guideline for Stability Testing of New Drug Substances and Products (February 6, 2003)
2.6 FDA Documents:
FDA Guidance for Industry, Pyrogen and Endotoxins Testing: Questions and Answers, June 2015, U.S. Department of Health
and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics
Evaluation and Research, Center for Veterinary Medicine, Center for Devices and Radiological Health, Office of Regulatory
Affairs
FDA Interim Guidance for Human and Veterinary Drug Products and Biologicals, Kinetic LAL Techniques, DHHS, July 15,
U.S. Food and Drug Administration (FDA) and Committee for Proprietary Medicinal Products (CPMP), 1998 International
Conference on Harmonization (ICH), Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology
Products Derived from Cell Lines of Human or Animal Origin, Consensus Guideline ICH Viral Safety Document: Step 5
U.S. Food and Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER), 1993 Points to Consider in
the Characterization of Cell Lines Used to Produce Biologicals
U.S. Food and Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER), 1997 Points to Consider in
the Manufacture and Testing of Monoclonal Antibody Products for Human Use, 94D-0259
2.7 AAMI Documents:
AAMI TIR 19:1998 Guidance for ANSI/AAMI/ISO 10993-7:1995, Biological Evaluation of Medical Devices—Part 7: Ethylene
Oxide Sterilization Residuals
ANSI/AAMI/ISO 11737-1:2018 Sterilization of Healthcare Products—Microbiological Methods—Part 1: Determination of a
Population of Microorganisms on Products
ANSI/AAMI/ISO 11737-2:2009 Sterilization of Medical Devices—Microbiological Methods—Part 2: Tests of Sterility
Performed in the Definition, Validation and Maintenance of a Sterilization Process
Available from International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH Secretariat, 9,
chemin des Mines, P.O. Box 195, 1211 Geneva 20, Switzerland, http://www.ich.org.
Available from U.S. Food and Drug Administration (FDA), 10903 New Hampshire Ave., Silver Spring, MD 20993, http://www.fda.gov.
Available from Association for the Advancement of Medical Instrumentation (AAMI), 4301 N. Fairfax Dr., Suite 301, Arlington, VA 22203-1633, http://www.aami.org.
F2212 − 20
ANSI/AAMI/ISO 14160:2011/(R) 2016 Sterilization of Health Care Products—Liquid Chemical Sterilizing Agents for
Single-Use Medical Devices Utilizing Animal Tissues and Their Derivatives—Requirements for Characterization,
Development, Validation and Routine Control of a Sterilization Process for Medical Devices
ANSI/AAMI ST67:2011/(R) 2017 Sterilization of Health Care Products—Requirements and Guidance for Selecting a Sterility
Assurance Level (SAL) for Products Labeled “Sterile”
2.8 Other References:
Draft Guidance for Preclinical and Clinical Investigations of Urethral Bulking Agents Used in the Treatment of Urinary
Incontinence, November 29, 1995. (ODE/DRARD/ULDB), Document No. 850
Council Directive 93/42/EEC, with Respect to Medical Devices Using Tissues of Animal Origin
Commission Directive 2003/32/EC, with Respect to Medical Devices Manufactured Using Tissues of Animal Origin
EMEA/410/01-rev.2, Committee for Proprietary Medical Products, Note for Guidance on Minimizing the Risk of Transmitting
Animal Spongiform Encephalopathy Agents via Human and Veterinary Medical Products
The European Agency for the Evaluation of Medicinal Products, (EMEA), Committee for Proprietary Medicinal Products
(CPMP) Guidance Document for Decision Trees for the Selection of Sterilisation Methods (CPMP/QWP/054/98 corr 2000)
and Annex to Note for Guidance on Development Pharmaceutics (CPMP/QWP/155/96)
3. Terminology
3.1 Definitions:
3.1.1 adventitious agent, n—an unintentionally introduced microbiological or other infectious contaminant.
3.1.1.1 Discussion—
In the production of TEMPs, these agents may be unintentionally introduced into the process stream or the final product, or both.
3.1.2 biocompatibility, n—a material may be considered biocompatible if the material performs with an appropriate host response
in a specific application (7).
3.1.3 collagen, n—a family of at least 20 genetically different secreted proteins that serve a predominantly structural function and
possess a unique triple helical structure configuration of three polypeptide units known as alpha chains.
3.1.4 degradation, n—change in chemical, physical, or molecular structure or appearance (that is, gross morphology) of a material.
3.1.5 endotoxin, n—pyrogenic high molar mass lipopolysaccharide (LPS) complex associated with the cell wall of gram-negative
bacteria.
3.1.5.1 Discussion—
Though endotoxins are pyrogens, not all pyrogens are endotoxins. Endotoxins are specifically detected through a Limulus
amebocyte lysate (LAL) test.
3.1.6 medical product, n—any diagnostic or therapeutic treatment that may be regulated as a device, biologic, drug, or combination
product.
3.1.7 microorganism, n—bacteria, fungi, yeast, mold, viruses, and other infectious agents.
3.1.7.1 Discussion—
However, it should be noted that not all microorganisms are infectious or pathogenic.
3.1.8 solubility, n—a measure of the extent to which the material can be dissolved.
3.1.8.1 Discussion—
Any colloidal system without obvious phase separation can be considered soluble. In the context of collagen, solubility refers to
the dissociation of the fibrillar aggregates of collagen molecules into a solution. Native Type I collagen, which is soluble in dilute
Available from U.S. Food and Drug Administration (FDA), 10903 New Hampshire Ave., Silver Spring, MD 20993, http://www.fda.gov.
Available from Office for Official Publications of the European Communities—European Law, 2, rue Mercier, L-2985, Luxembourg, http://eur-lex.europa.eu/en/
’index.htm.
Available from European Medicines Agency (EMEA), 7 Westferry Circus, Canary Wharf, London E14 4HB, U.K., http://www.eudora.org/emea.html, and
http://www.emea.europa.eu/pdfs/human/bwp/TSE%20NFG%20410-rev2.pdf.
Available from European Medicines Agency (EMEA), 7 Westferry Circus, Canary Wharf, London E14 4HB, U.K., http://www.eudora.org/emea.html, and
http://www.emea.europa.eu/pdfs/human/qwp/005498en.pdf.
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acids, but not soluble at physiological conditions, is termed “insoluble” or “acid soluble,” while simple aggregates of non-fibrillar
collagen soluble in neutral salt solutions are termed “neutral salt soluble.” Post translational surface charge modifications may alter
the solubility of collagen in neutral pH condition.
3.1.9 sterilization, n—the destruction or removal of all microorganisms in or about an object (for example, by chemical agents,
electron beam, gamma irradiation, or filtration).
3.1.9.1 Discussion—
If the medical product collagen permits, terminal sterilization is preferential to aseptic processing.
3.1.10 suspension, n—the dispersion of a solid through a liquid with a particle size large enough to be detected by purely optical
means.
4. Significance and Use
4.1 The objective of this guide is to provide guidance in the characterization of Type I collagen as a starting material for surgical
implants and substrates for tissue engineered medical products (TEMPs). This guide contains a listing of physical and chemical
parameters that are directly related to the function of collagen. This guide can be used as an aid in the selection and characterization
of the appropriate collagen starting material for the specific use. Not all tests or parameters are applicable to all uses of collagen.
4.2 The collagen covered by this guide may be used in a broad range of applications, forms, or medical products, for example (but
not limited to) medical devices, tissue engineered medical products (TEMPs) or cell, drug, or DNA delivery devices for
implantation. The use of collagen in a practical application should be based, among other factors, on biocompatibility and physical
test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success in any tissue engineered
medical product or drug delivery application.
4.3 The following general areas should be considered when determining if the collagen supplied satisfies requirements for use in
TEMPs. These are source of collagen, chemical and physical characterization and testing, and impurities profile.
4.4 The following documents or other appropriate guidances from appropriate regulatory bodies relating to the production,
regulation, and regulatory approval of TEMPs products should be considered when determining if the collagen supplied satisfies
requirements for use in TEMPs:
FDA CFR:
21 CFR 3: Product Jurisdiction:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=3
21 CFR 58: Good Laboratory Practice for Nonclinical Laboratory Studies:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=58
FDA/CDRH CFR and Guidances:
21 CFR Part 803: Medical Device Reporting:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=803
21 CFR 812: Investigational Device Exemptions:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=812
21 CFR 814: Premarket Approval of Medical Devices :
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=814
21 CFR 820: Quality System Regulation:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=820
Design Control Guidance for Medical Device Manufacturers:
http://www.fda.gov/cdrh/comp/designgd.pdf
Preproduction Quality Assurance Planning Recommendations for
Medical Device Manufacturers (FDA 90-4236):
http://www.fda.gov/cdrh/manual/appende.html
The Review and Inspection of Premarket Approval Applications under the
Bioresearch Monitoring Program—Draft Guidance for Industry and FDA Staff:
http://www.fda.gov/cdrh/comp/guidance/1602.pdf
FDA/CDRH Search Engines:
CDRH Guidance Search Engine:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfggp/search.cfm
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CDRH Premarket Approval (PMA) Search Engine:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMA/pma.cfm
CDRH 510(k) Search Engine:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.cfm
CDRH Recognized STANDARDS Search Engine :
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/search.cfm
FDA/CBER CFR and Guidances:
21 CFR 312: Investigational New Drug Application :
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=312
21 CFR 314: Applications for FDA Approval to Market a New Drug:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=31
21 CFR 610: General Biological Products Standards:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=610
21 CFR 1271: Human Cells, Tissues and Cellular and Tissue-Based
Products:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=1271
Cellular & Gene Therapy Guidances and Other Publications:
http://www.fda.gov/cber/genetherapy/gtpubs.htm
Human Tissue Guidances and Other Publications:
http://www.fda.gov/cber/tissue/docs.htm
CBER Product Approval Information:
http://www.fda.gov/cber/efoi/approve.htm
21 CFR 600, 601 BLA Regulations:
http://www.access.gpo.gov/nara/cfr/waisidx_07/21cfrv7_
07.html
21 CFR 210, 211 GMP Regulations:
http://www.access.gpo.gov/nara/cfr/waisidx_07/21cfr210_07.html
5. Chemical and Physical Characterizations
5.1 General Comments on Chemical and Physical Characterization of Collagen—These methods are suggested assays; however,
other validated assay methods may be used. Selection of assay systems will vary, depending on the configuration of the collagen
(that is, soluble or insoluble). The user should ensure that the method selected is reliable and commonly accepted in protein
chemistry. A review of collagen materials may be found in Li, 2000 (8), while a review of the collagen family of proteins may
be found in Refs (9-14). When selecting an appropriate test method, the user should note that impurities in highly purified collagen
are low or lower than 1 to 2 %, so sensitive test methods need to be utilized. For soluble collagen, the following represents a
non-inclusive list of assay systems available: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE); peptide
mapping; and physico-chemical analysis. A similar list for insoluble collagen may include, but not be limited to, assay methods
for hexosamine (that is, detection of glycoproteins), lipid, total sugar, desmosine (that is, elastin), and amino acid composition (that
is, collagen composition profile; non-collagenous amino acids). Additionally, methods such as transmission electron microscopy
may be helpful in characterizing the collagen fibers or collagen superstructure.
5.2 The concentration of collagen should be expressed in mass/volume or mass/mass. Colorometric assays or amino acid analysis
for hydroxyproline are commonly used methods to measure collagen content.
5.3 Amino acid analysis will provide information on the composition of the amino acids of collagen (that is, the amino acids must
be within the range of published data for highly purified collagen preparations, generally in the acid soluble form). Amino acid
analysis is routinely performed on hydrolyzed collagens by reverse phase High Performance Liquid Chromatography (HPLC). This
method can be used to quantify hydroxyproline, tyrosine, tryptophan, and cysteine. There are other methods available for amino
acid analysis.
5.4 Purity of soluble collagen can be analyzed by SDS-PAGE, either on the collagen directly or after digestion of the collagen
with purified bacterial collagenase to detect any remaining proteins.
5.5 Elastin Assay—Elastin can be a component of the impurities in an insoluble collagen preparation. One method to assay for
elastin, although other methods are available, involves the detection of desmosine (15). These impurities can be detected by
Western blots, enzyme-linked immunosorbent assays (ELISAs), and other types of assays.
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5.6 Peptide mapping is one possible method to identify Type I collagen. The most commonly used peptide mapping method
utilizes Cyanogen Bromide (CNBr) digestion. The digest can be analyzed by SDS-PAGE or HPLC.
5.7 Impurities Profile—The term impurity relates to the presence of extraneous substances and materials in the collagen. These
impurities can be detected by Western blots, ELISAs, gas chromatography/mass spectrometry (GC-MS), and other types of assays.
If there is a concern for the presence of processing aids or other impurities associated with the collagen, they should be addressed
with the supplier. The major impurities of concern include, but are not limited to the following: endotoxins, glycosaminoglycans,
elastin, lipids, improperly aligned collagen molecules, host cell contaminants, cell culture contaminants, heavy metals, bioburden,
viruses, transmissible spongiform encephalopathy (TSE) agents, cross-linking and enzymatic agents, and components used in
extraction or solubilization (for example, acids, surfactants, solvents, and so forth). Type III collagen may also be associated with
Type I collagen. While its presence may have no adverse effect on product quality, levels should be evaluated and controlled for
lot-to-lot consistency. The inclusion of urea can be used to resolve Type I and Type II collagen alpha 1 chains (16). Assessment
of collagens other than Type I and III is discussed in 5.19. At a minimum, any protein impurity of greater than 1 % in the final
collagen preparation should be identified and quantified.
5.8 Crosslinking Reactions with Collagen—Collagen is a very stable protein due to its triple-helical structure, imparting resistance
to most proteolytic enzymes. It is still sensitive to collagenase, however. The stability can be enhanced by crosslinking the
molecule by physical or chemical means. Both inter- and intrachain crosslinking can occur due to the propensity of collagen fibers
to naturally crosslink. Crosslinking agents and methods include aldehydes, carbodiimides, epoxides, diisocyanates, non-enzymatic
glycosylation, dehydrothermal treatment (DHT), radiation (for example, gamma, electron beam), and ultraviolet light. For
chemical crosslinking, excess crosslinker should be removed and quantitated before or at the final product stage. A crosslinker may
be cytotoxic or mutagenic, or both, and any component in the final product needs to be quantitated. There are several methods
available, including liquid chromatography/mass spectrophometry (LC/MS), GC/MS, or other assays. Any crosslinker used that
has the potential of reacting with DNA should be considered a mutagen. A mutagenic assay should at least be performed on the
final product in that case. A cytotoxicity assessment will also provide a measure of acceptable crosslinker levels. Physical
crosslinking may result in unwanted changes to the structure of the collagen molecule and should be assessed with qualification
assays appropriate to the clinical indication under consideration. Direct measurement of collagen crosslinking can be performed
by looking at the altered amino acid composition and using methods appropriate for the crosslinker. One method, for example
(other methods exist), to measure degree of crosslinking when lysine residues are involved is to detect free lysines and
hydroxylysines by labeling the ε-amino acid groups with 2.4.6 trinitrobenzenesulfonic acid (TNBS), where the TNBS-labeled
amino acids absorb at 345 nm with a molar absorptivity of 1.46 × 10 L/mole × cm. Amino acid composition can also be examined
by analysis of sodium borohydride-treated collagen. The thermal denaturation characteristics can also be measured by Differential
Scanning Calorimetry (DSC) (17). The thermal denaturation characteristics can sometimes be correlated with the crosslink density.
The % water uptake (% swell), using the equation (W –W )/W , where W = dry weight and W = wet weight, is also an
W D W D W
indirect measure of collagen crosslinking. The tensile strength can be altered by crosslinking. Measurements using a universal
testing machine (UTM) or a rheometer will note a change in properties after crosslinking. Collagen crosslinking imparts a
resistance to the proteolytic enzyme collagenase. Collagenase is the one enzyme that will digest triple-stranded collagen. When
collagen is crosslinked, it is more resistant to breakdown and extensive crosslinking will afford the greatest resistance to
collagenase.
5.9 Endotoxin Content—Endotoxin contamination is difficult to prevent because it is ubiquitous in nature, stable, and small enough
to pass through sterilizing filters (0.22 μm). Endotoxin tests for collagen include the gel clot, endpoint assay, and the kinetic assay
(Food and Drug Administration Guidance for Industry, Pyrogen and Endotoxins Testing: Questions and Answers). The gel clot test
is the simplest and easiest of the Limulus amebocyte lysate (LAL) test methods, although much less sensitive than the kinetic assay.
The quantitative kinetic assay, which measures the amount of time required to reach a predetermined optical density, is the most
sensitive. Each new lot of reagents should meet acceptance criteria established by appropriate qualification or validation studies
(for investigational or licensed/cleared products, respectively). The endotoxin level in collagen will ultimately be critical to its use
in biomedical applications where there are regulatory limits to the amount of endotoxin that can be implanted into humans.
Relevant FDA guidance for allowable levels and information regarding validation of endotoxin assays should be consulted if
human trials are contemplated (Interim Guidance for Human and Veterinary Drug Products and Biologicals). The user is also
directed to CFR 610.13(b) and Food and Drug Administration Guidance for Industry, Pyrogen and Endotoxins Testing: Questions
and Answers for information pertaining to the rabbit pyrogen assay.
5.10 Heavy Metal Content by the USP Method—This test is provided to demonstrate that the content of heavy metal impurities
does not exceed a limit in the individual product specification. This method is based on <231> Heavy Metals, 1st and 6th
Supplement USP-NF. Substances that typically respond to this test are lead, mercury, bismuth, arsenic, antimony, tin, cadmium,
F2212 − 20
silver, copper, and molybdenum. Under the specified test conditions, the limit is determined by a concomitant visual comparison
of metals that are colored by sulfide ion with a control prepared from a Standard Lead Solution. Additional heavy metal
contaminants may be present due to processing. If necessary, the user may detect these contaminants by various methods that may
include, but are not limited to, spectrographic, chromatographic, and flame atomic absorption techniques.
5.11 Microbiological Safety—Bacteria, viruses, and fungi are also contaminants that can arise in a biological sample. The user will
validate sterilization and characterize its effect on the product. The presence of bacteria may also contribute to the presence of
endotoxins. The following Microbiological Tests in USP 30 are of particular relevance: Microbial Limit Tests <61>, Sterility Tests
<71>, Sterilization and sterility assurance of compendial articles <12211>, the Biological Tests and Assays: Bacterial Endotoxins
Tests <85>, and viral validation studies <1050>. The user should also consider other relevant standards, such as, but not limited
to, Association for the Advancement of Medical Instrumentation (AAMI) standards and international standards, of which the
following are examples: ANSI/AAMI/ISO 11737-1:2018; ANSI/AAMI/ISO 11737-2:2009; and ISO 13408–1. The collagen is first
dissolved in a sterile, aqueous solution, then filtered using sterile techniques through a 0.45 μm membrane filter. The filters are
subsequently incubated on Tryptic Soy Agar to determine the presence of bacteria, and on Sabouraud Dextrose Agar to determine
the presence of yeast and mold. If collagen products are intended to serve as a barrier to microorganisms, this function will need
to be validated with specific experiments.
5.12 Carbohydrate analysis of collagens can be carried out by classical gas-liquid chromatographic methods or spectrophoto-
metric methods. Novel sources of collagen may result in a different glycosylation pattern or sugars that differ from human collagen,
or both. A potential risk of autoimmune disease may be present. If a novel source of collagen is used and the carbohydrate pattern
is unknown, a risk assessment should be performed. This should include an analysis of the sugars present on the collagen. If
necessary, the gylcosylation properties of the collagen should be examined and an assessment of the autoimmunity potential
performed.
5.13 Trypsin susceptibility will detect that portion of collagen that has been denatured during purification steps such as acid and
base treatment, solvent treatment, and so forth. Trypsin will digest that portion of the collagen and can be measured by assaying
the hydroxyproline content of the supernatant. Triple helical collagen is resistant to digestion by most proteases. Susceptibility to
trypsin or other appropriate proteases is determined by exposing the collagen to the enzyme and assaying the digest for
degradation. There are several methods for this test.
5.14 Differential Scanning Calorimetry (DSC) determines dissociation temperature of collagens in fibrils, as well as detecting
microfibrils and denatured collagen at lower melting temperatures. (See also 5.8, crosslinking reactions with collagen).
5.15 Viscosity is more applicable to gels or suspensions but may be useful with collagen configured in forms such as, but not
limited to, pastes or films (18). Viscosity of collagen-based materials depends on a number of factors which may include, but are
not limited to, the following: solution or dispersion/suspension, concentration, temperature, operating condition, and so forth. It
is not feasible to determine the viscosity of films. This is a routine test performed with a viscometer (not a u-tube). The user must
clearly state the conditions of the test.
5.16 Transmission electron microscopy may be used to show the quality of collagen fibers. Unraveling or changes in banding will
be obvious.
5.17 DNA sequence data on recombinant or transgenic source cells: Verify sequence data for expression gene, that is, COL1A1
or COL1A2.
5.18 Characterization Methods for Type I Collagen (Table 1)—The collagen material shall have specifications for an extensive set
of chemical and physical properties such as, but not limited to, those listed in Table 1. The table represents methods which may
or may not be appropriate for characterizing a particular collagen sample. Not all the methods listed may be required to characterize
the sample, and the specificity and sensitivity vary among the methods listed. The user should be familiar with the limitations of
the appropriate test methods.
5.19 Analysis for Type II, IV, and Other Collagens—Tissues commonly used to isolate Type I collagen typically contain some Type
III collagen which coexists in many tissues. Type II collagen is found primarily in cartilage, while Type IV collagen is found in
basement membranes and has been associated with Goodpasture’s Syndrome (Wieslander, J., et al, (19)). The purity of collagen
is important in determining the potential for safety problems and providing criteria for the consistency of the manufacturing
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TABLE 1 Characterization Methods for Type I Collagen
Characterization Method Applicable to
Chemical
Appearance Soluble or Insoluble
Concentration Soluble or Insoluble
Purity Soluble or Insoluble
Amino acid analysis Soluble or Insoluble
Peptide mapping Soluble or Insoluble
Impurities profile, including Soluble or Insoluble
Heavy Metal Analysis
Carbohydrate analysis Soluble or Insoluble
Trypsin resistance Soluble or Insoluble,
Mainly Insoluble
Collagenase resistance Soluble or Insoluble,
Mainly Insoluble
pH of implantable Soluble or Insoluble
Additives (cross-linkers, Soluble or Insoluble
lubricants, drugs, sterilents)
Physical
Shrink Temperature (DSC) Insoluble
Viscosity Mainly soluble
TEM Insoluble
SDS-PAGE Soluble or Insoluble
Moisture Content, dependent Insoluble
on storage environment
Electron Micrograph (native Insoluble
banded 640 Å
structure for fibrils)
Biochemical
Endotoxin level Soluble or Insoluble
Bioburden Soluble or Insoluble
% Type I collagen/Total Protein Soluble or Insoluble
% Other Types Collagen and Soluble or Insoluble
List of
which Types present
Total DNA (ppm or %) Soluble or Insoluble
Total Lipid Soluble or Insoluble
% native collagen (by trypsin Soluble or Insoluble
resistance,
circular Dichroism)
Abbreviations in Table:
DSC = Differential Scanning Calorimetry
TEM = Transmission Electron Microscopy
SDS-PAGE = Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
process. For example, skin collagen is composed of approximately 90 % Type I, 8–10 % Type III, and the remainder is made up
of trace amounts of the less abundant collagens, primarily Type IV, V, and VI (Holbrook and Smith, (20)). As all collagens contain
triple helical domains, the properties of different collagen types can be very similar. Detection by Western Blot analysis, therefore,
requires the use of antibodies that recognize epitopes in the more diverse non-helical regions. Antibodies are available for Types
I to VI collagens, and possibly others, for use in Western Blot or ELISA analyses following SDS gel electrophoresis. Validation
of antibody specificity, as well as the test procedure, using suitable standards, should be conducted prior to analysis. A risk
assessment should be performed on the potential for other collagens in the product. If the presence of other collagens is likely, an
assessment should be completed for collagens that have the potential to generate adverse reactions. The extent of analysis required
will depend upon the risk of other collagen types being present as impurities in a particular collagen product.
6. Product Development Considerations
6.1 Storage Conditions/Shelf Life Stability of Collagen—For collagen, the most relevant stability-indicating parameters are those
related to the functionality of the polymer. Depending upon what function the collagen will have in the final formulation,
parameters such as viscosity (apparent and intrinsic) and biological activity, along with other parameters deemed relevant, may also
be considered. Storage conditions are important, especially for collagen solutions. International Conference on Harmonization
(ICH) guidance documents should be consulted for information on stability testing of pharmaceuticals (that is, ICH Q1A).
6.2 Sterilization Method, if Applicable, and Effects of Sterilization on the Product—The user should verify that the sterilization
method does not adversely affect the collagen end product. Collagen can be sterilized by gamma irradiation, electron beam, or by
ethylene oxide, or prepared using aseptic processing steps. Potential degradation of the collagen or sterilization residuals should
F2212 − 20
be evaluated to determine the impact on the product. Solutions of collagen may be ((1)1) filter sterilized if the viscosity of the
collagen solution permits; or ((2)2) gamma-irradiated. Any changes in viscosity may reflect an alteration of the molecular mass
and should be evaluated. The method of sterilization is primarily dictated by the effect on the product effectiveness. The method
chosen must be validated to determine the effectiveness of sterilization. The reader should refer to the most current version of the
relevant standards regarding the sterilization of healthcare products by radiation, steam, and ethylene oxide gas, such as AAMI TIR
19:1998; ANSI/AAMI/ISO 14160:2011; ANSI/AAMI ST67:2011; and The European Agency for the Evaluation of Medicinal
Products, (EMEA), Committee for Proprietary Medicinal Products (CPMP) Guidance Document for Decision Trees for the
Selection of Sterilisation Methods (CPMP/QWP/054/98 corr 2000) and Annex to Note for Guidance on Development
Pharmaceutics (CPMP/QWP/155/96).
6.3 Sourcing—The criteria to consider for safe sourcing include appropriate human or animal donor selection and the tissue
collection procedures to assureensure that the source material is unlikely to contain TSE infectivity. Recombinant sources of
collagen, which provide a good option from purity and integrity standpoints, should also be considered. Additional information can
be obtained from the following documents: ISO 22442–1; ISO 22442–2; ISO 22442–3; 21 CFR Parts 207, 807, and 1271; 21 CFR
Part 820; 21 CFR Part 1271, Part C; Federal Register Vol. 43, No. 141; Federal Register Vol. 62, No. 145; Federal Register Vol.
66, No. 5; Federal Register, Vol. 66, No. 13; ISO 13408–1; Council Directive 93/42/EEC; Commission Directive 2003/32/EC; and
EMEA/410/01-rev.2. Additional documents may be available. The user should use the most current version of all documents.
6.3.1 For further information, the user is referred to Appendix X2, Sourcing Issues: X2.1 Tissue for Collagen or Collagen-
Containing Medical Devices; X2.2 Requirements for a Closed-Herd; and X2.3 Documentation.
6.3.2 The collagen can be isolated from tissues or cell cultures by any method, including, but not limited to, extraction by dilute
acids or dilute salt solutions or by enzymatic digestion of the tissue (21-24). The user should be aware that even though Type III
collagen is less abundant, it is often associated with Type I, except in bones and tendons. Type V collagen is also associated with
Type I.
6.4 Viral and Transmissible Spongiform Encephalopathy (TSE) Agent Inactivation—Viruses and TSE agents can be introduced
into a product as a result of raw materials sourcing or through adventitious means. Appropriate measures should be taken so that
the resultant product is free from viruses and TSE agents. For further guidance on viral or TSE clearance, or both, the user is
directed to the references throughout this guide as well as USP 30/NF 19 <1050>, and other pertinent references, as appropriate.
Additional information may be found in the following FDA Guidance Document, FDA points to consider and International
Conference on Harmonization (ICH) documents: Guidance for Industry and for FDA Reviewers, Medical Devices Containing
Materials Derived from Animal Sources (Except for In Vitro Diagnostic Devices); U.S. Food and Drug Administration (FDA) and
Committee for Proprietary Medicinal Products (CPMP), 1998 International Conference on Harmonization (ICH), Quality of
Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or An
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