ASTM F3089-23

Standard Guide for Characterization and Standardization of Polymerizable Collagen-Based Products and Associated Collagen-Cell Interactions

SIGNIFICANCE AND USE
4.1 The objective of this document is to provide guidance in the production, characterization, testing, and standardization of: (1) polymerizable collagen starting materials; and (2) collagen polymeric materials produced with polymerizable collagen formulations, used for surgical implants, substrates for TEMPs, vehicles for therapeutic cells and molecules, and 3D in-vitro tissue systems for basic research, drug development, and toxicity testing. This guide can be used as an aid in the selection, characterization, and standardization of the appropriate polymerizable collagen starting formulations as well as collagen polymeric materials prepared from polymerizable collagens for a specific use. Not all tests or parameters are applicable to all uses of collagen and users are expected to select and justify a subset of the tests for characterization purposes.  
4.2 This guide can be used by the following types of users:  
4.2.1 Manufacturers of polymerizable collagens and collagen polymeric materials who wish to set specifications for their products or provide characterization data for customers or users. They may also use the terminology and characterization sections to specify and differentiate the properties of polymerizable collagens and collagen polymeric materials.  
4.2.2 Producers of collagen polymeric materials that use polymerizable collagen as starting materials. Producers may use this guide to evaluate and characterize multiple sources of polymerizable collagen. They may also use this guide to assist with evaluation and comparison of single or multiple sources of polymerizable collagen and collagen polymeric materials.  
4.2.3 Researchers may use this guide as a reference for properties and test methods that can be used to reproducibly evaluate polymerizable collagens and collagen polymeric materials.  
4.3 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) wound an...
SCOPE
1.1 This guide is intended to provide characteristics, properties, test methods, and standardization approaches for evaluation and identification of specific polymerizable collagen formulations and collagen polymeric materials produced with these formulations.  
1.2 This guide focuses on characterization of purified polymerizable forms of type I collagen, which is the most abundant collagen in mammalian connective tissues and organs, including skin, bone, tendon, and blood vessels. Polymerizable type I collagen may be derived from a variety of sources including, but not limited to, animal or cadaveric tissues, cell culture, recombinant cell culture, and chemical synthesis.  
1.2.1 This guide covers evaluation of polymerizable collagens and collagen polymeric materials prepared from polymerizable collagens for use as a starting material for wound and hemostatic dressings, surgical implants, substrates for tissue-engineered medical products (TEMPs), delivery vehicles for therapeutic cells or molecules, and 3D in-vitro tissue systems for basic research, diagnostics, drug development, and toxicity testing. Most collagen products on the market today are regulated as devices since their primary intended purpose is not achieved through chemical action within or on the body. However, a medical product comprising polymerizable collagens or collagen polymeric materials may be regulated as a device, biologic, drug, or combination product depending on its intended use and primary mode of action.  
1.2.2 Polymerizable collagen or collagen self-assembly implies that the collagen composition exhibits spontaneous macromolecular assembly from its components without the addition of exogenous factors such as cross-linking agents. Polymerizable collagens may include but are not limited to: (1) tissue-derived monomeric collagens, including tropocollagen or atelocollagen, and oligomeric collagens; (2) collagen proteins and peptides produce...

General Information

• Status: Published
• Publication Date: 31-Dec-2022
• 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 F2914-12(2024) - Standard Guide for Identification of Shelf-Life Test Attributes for Endovascular Devices
• Effective Date: 15-Jan-2024
• Refers: ASTM F749-20 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Feb-2020
• Refers: ASTM F2914-12(2018) - Standard Guide for Identification of Shelf-life Test Attributes for Endovascular Devices
• Effective Date: 01-Nov-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 F895-11(2016) - Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
• Effective Date: 01-Apr-2016
• Refers: ASTM F748-16 - Standard Practice for Selecting Generic Biological Test Methods for Materials and Devices
• Effective Date: 01-Apr-2016
• Refers: ASTM E4-14 - Standard Practices for Force Verification of Testing Machines
• Effective Date: 01-Jun-2014
• 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 F749-13 - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Mar-2013
• Refers: ASTM F749-98(2012) - Standard Practice for Evaluating Material Extracts by Intracutaneous Injection in the Rabbit
• Effective Date: 01-Oct-2012

Overview

ASTM F3089-23 is a comprehensive guide developed by ASTM International for the characterization and standardization of polymerizable collagen-based products and the assessment of collagen-cell interactions. The primary focus of this standard is on purified, polymerizable forms of type I collagen- the most prevalent collagen in mammalian connective tissues-used in a wide array of biomedical and research applications. This document provides critical guidance for manufacturers, producers, and researchers in selecting, characterizing, and standardizing collagen starting materials and derived polymeric products. The standard is highly relevant for those involved in tissue engineering, medical devices, regenerative medicine, drug development, and in-vitro tissue systems.

Key Topics

• Characterization of Polymerizable Collagen:
  The guide outlines methods for evaluating the unique properties of type I collagen from various sources, including animal tissues, recombinant technologies, and chemical synthesis.

• Standardization Protocols:
  ASTM F3089-23 describes protocols and test methods that enable consistent identification and evaluation of polymerizable collagen formulations, ensuring comparability between products.

• Collagen-Cell Interactions:
  The standard highlights the importance of properties such as microstructure, mechanical integrity, degradation behavior, immunogenicity, and biological compatibility, which are crucial for cell adhesion, differentiation, and tissue integration.

• Product Format Flexibility:
  Guidance is provided for a diverse range of product forms-such as injectable polymerizable solutions, preformed scaffolds, gels, sheets, coatings, sponges, and films-supporting both medical and research use cases.

• Test Selection and Justification:
  Users are encouraged to determine and justify the subset of tests most relevant to their particular application, as not all characterization methods are universal.

• Terminology and Definitions:
  To support clear communication and differentiation, the guide includes standardized terminology for polymerizable collagen and related materials.

Applications

Polymerizable collagen-based products evaluated under ASTM F3089-23 are essential in many healthcare and research fields. Typical applications include:

• Surgical Implants:
  Collagen scaffolds and matrices are widely used as surgical implants for tissue repair and reconstruction due to their biocompatibility and capacity for promoting cell-guided tissue regeneration.

• Wound and Hemostatic Dressings:
  Collagen-based dressings leverage the material’s inherent compatibility to support hemostasis and promote natural healing in acute and chronic wounds.

• Tissue-Engineered Medical Products (TEMPs):
  The standard supports the development of substrates and scaffolds for TEMPs that facilitate cellular attachment, proliferation, and differentiation.

• Drug Delivery and Therapeutic Vehicles:
  Collagen polymers are employed as carriers or vehicles for the controlled release of therapeutic cells and molecules.

• 3D In-Vitro Tissue Systems:
  Collagen-based 3D tissue models are indispensable in basic research, drug discovery, and toxicity testing, enabling more physiologically relevant cell and tissue studies.

• Device and Combination Products:
  Depending on intended use and primary mode of action, products may be regulated as medical devices, biologics, drugs, or combination products, making standardization critical for regulatory compliance.

Related Standards

ASTM F3089-23 references a wide array of international and regulatory standards to promote harmonization and best practices, including:

• ASTM Standards

  • ASTM F619, F720, F748, F756, F813, F895, F981, F1439, F2148, F2914, and others for evaluation of material compatibility, biological safety, and mechanical properties.

• ISO Standards

  • ISO 10993 series for biological evaluation of medical devices
  • ISO 22442 series for risk management of animal-derived materials
  • ISO 14971 for risk management

• Pharmacopeia and Regulatory Documents

  • United States Pharmacopeia (USP) chapters
  • European Pharmacopeia
  • Code of Federal Regulations (21 CFR Parts 312, 820, 1271, and others)

These referenced documents collectively ensure a robust and internationally accepted framework for the characterization and standardization of polymerizable collagen-based products for medical and research use.

ASTM F3089-23 is central to advancing safe, effective, and innovative collagen-based biomaterial products worldwide.

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: F3089 − 23
Standard Guide for
Characterization and Standardization of Polymerizable
Collagen-Based Products and Associated Collagen-Cell
Interactions
This standard is issued under the fixed designation F3089; 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
The collagen family of proteins represents the major structural and mechanical component of the
in-vivo extracellular matrix of human tissues and organs. Type I collagen is the most abundant and as
such, it is an ideal candidate for medical materials, tissue-engineered medical products, delivery of
therapeuticcells/molecules,and in-vitrocell/tissuecultureapplications.Furthermore,itisnowevident
that specific collagen material properties, including microstructure, mechanical integrity (stiffness,
strength), cell adhesion, immunogenicity, and resorption (degradation) are major determinants of the
interfacial properties between cells and collagen-based materials, including guidance of fundamental
cell behaviors that contribute to recapitulation and/or restoration of tissue structure and function.
Advanced understanding of collagen self-assembly, as occurs in vivo and in vitro, is contributing to
a rapid expansion of commercial and laboratory-produced purified collagen formulations that
polymerize (self-assemble) or exhibit transitions from solution to semi-solid material (for example,
gel, scaffold). Most recent developments have focused on polymerizable collagen formulations that
supporttherationaldesignandcustomfabricationofcollagenpolymericmaterialsforimprovedtissue
integration, guidance of cell fate, and tissue response outcomes. Unfortunately, the term “collagen” is
applied generally to describe various collagen types and formulations (soluble, insoluble, monomeric,
gelatin/peptides, oligomeric, tropocollagen, atelocollagen) that vary significantly in their molecular
compositions, polymerization capacity and properties, and ability to interact with cells. As such, the
need exists for an expanded set of characterization and standardization strategies to facilitate
comparison, safety and efficiency testing, and translation of the next generation polymerizable
collagen formulations and associated collagen polymeric materials produced with these formulations.
1. Scope PolymerizabletypeIcollagenmaybederivedfromavarietyof
sources including, but not limited to, animal or cadaveric
1.1 This guide is intended to provide characteristics,
tissues, cell culture, recombinant cell culture, and chemical
properties, test methods, and standardization approaches for
synthesis.
evaluationandidentificationofspecificpolymerizablecollagen
formulations and collagen polymeric materials produced with 1.2.1 This guide covers evaluation of polymerizable colla-
these formulations.
gens and collagen polymeric materials prepared from polym-
erizable collagens for use as a starting material for wound and
1.2 This guide focuses on characterization of purified po-
hemostatic dressings, surgical implants, substrates for tissue-
lymerizable forms of type I collagen, which is the most
engineered medical products (TEMPs), delivery vehicles for
abundant collagen in mammalian connective tissues and
therapeutic cells or molecules, and 3D in-vitro tissue systems
organs, including skin, bone, tendon, and blood vessels.
for basic research, diagnostics, drug development, and toxicity
testing. Most collagen products on the market today are
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
regulatedasdevicessincetheirprimaryintendedpurposeisnot
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs.
achieved through chemical action within or on the body.
Current edition approved Jan. 1, 2023. Published January 2023. Originally
However, a medical product comprising polymerizable colla-
approved in 2014. Last previous edition approved in 2014 as F3089 – 14. DOI:
gens or collagen polymeric materials may be regulated as a
10.1520/F3089-23.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F3089 − 23
device, biologic, drug, or combination product depending on E4 Practices for Force Calibration and Verification of Test-
its intended use and primary mode of action. ing Machines
1.2.2 Polymerizable collagen or collagen self-assembly im- F619 Practice for Extraction of Materials Used in Medical
plies that the collagen composition exhibits spontaneous mac- Devices
romolecular assembly from its components without the addi- F720 PracticeforTestingGuineaPigsforContactAllergens:
tion of exogenous factors such as cross-linking agents. Guinea Pig Maximization Test
Polymerizablecollagensmayincludebutarenotlimitedto:(1) F748 PracticeforSelectingGenericBiologicalTestMethods
tissue-derived monomeric collagens, including tropocollagen for Materials and Devices
or atelocollagen, and oligomeric collagens; (2) collagen pro- F749 Practice for Evaluating Material Extracts by Intracuta-
teins and peptides produced through in vitro cell culture, with neous Injection in the Rabbit
or without using recombinant technology; and (3) chemically F756 Practice for Assessment of Hemolytic Properties of
synthesized collagen mimetic peptides. It should be noted that Materials
the format of collagen polymeric material products also will F763 Practice for Short-Term Intramuscular Screening of
vary and may include injectable solutions that polymerize in Implantable Medical Device Materials
situ as well as preformed sheets, particles, spheres, fibers, F813 Practice for Direct Contact Cell Culture Evaluation of
sponges, matrices/gels, coatings, films, and other forms. Materials for Medical Devices
1.2.3 This guide may serve as a template for characteriza- F895 TestMethodforAgarDiffusionCellCultureScreening
tion and standardization of type I fibrillar collagen or other for Cytotoxicity
collagen types that demonstrate polymerization or self- F981 Practice for Assessment of Compatibility of Biomate-
assembly. rials for Surgical Implants with Respect to Effect of
Materials on Muscle and Insertion into Bone
1.3 This guide does not provide a significant basis for
F1439 Guide for Performance of Lifetime Bioassay for the
assessing the biological safety (biocompatibility) of polymer-
Tumorigenic Potential of Implant Materials
izable collagens and collagen polymeric materials. While the
F1903 Practice for Testing for Cellular Responses to Par-
ability of collagen polymeric materials to guide cellular re-
ticles in vitro
sponses through provision of cellular adhesion and proteolytic
F1904 Practice for Testing the Biological Responses to
domains as well as physical constraints (for example,
Particles in vivo
structural, cell-matrix traction force) has been well docu-
F1983 Practice forAssessment of Selected Tissue Effects of
mented through extensive clinical and basic research studies
Absorbable Biomaterials for Implant Applications
(1-5), usersaredirectedtotheISO10993seriesforevaluating
F2914 Guide for Identification of Shelf-life Test Attributes
biological risks of medical devices. The biocompatibility and
for Endovascular Devices
appropriateness of use for a specific application is the respon-
2.2 ISO Standards:
sibility of the product manufacturer.
ISO 5725-1 Accuracy (Trueness and Precision) of Measure-
1.4 The values stated in SI units are to be regarded as
ment Methods and Results—Part 1: General Principles
standard. No other units of measurement are included in this
and Definitions
standard.
ISO 5725-2 Accuracy (Trueness and Precision) of Measure-
1.5 The following precautionary caveat pertains only to the ment Methods and Results—Part 2: Basic Method for the
test method portion, Sections 6 and 7, of this guide: This
Determination of Repeatability and Reproducibility of a
standard does not purport to address all of the safety concerns, Standard Measurement Method
if any, associated with its use. It is the responsibility of the user
ISO 5725-3 Accuracy (Trueness and Precision) of Measure-
of this standard to establish appropriate safety, health, and ment Methods and Results—Part 3: Intermediate Mea-
environmental practices and determine the applicability of
sures of the Precision of a Standard Measurement Method
regulatory limitations prior to use. ISO 5725-4 Accuracy (Trueness and Precision) of Measure-
1.6 This international standard was developed in accor-
mentMethodsandResults—Part4:BasicMethodsforthe
dance with internationally recognized principles on standard- DeterminationoftheTruenessofaStandardMeasurement
ization established in the Decision on Principles for the
Method
Development of International Standards, Guides and Recom- ISO 5725-5 Accuracy (Trueness and Precision) of Measure-
mendations issued by the World Trade Organization Technical
ment Methods and Results—Part 5: Alternative Methods
Barriers to Trade (TBT) Committee. for the Determination of the Precision of a Standard
Measurement Method
2. Referenced Documents
ISO 5725-6 Accuracy (Trueness and Precision) of Measure-
ment Methods and Results—Part 6: Use in Practice of
2.1 ASTM Standards:
Accuracy Values
ISO 10993-1 Biological Evaluation of Medical Devices—
The boldface numbers in parentheses refer to a list of references at the end of
Part 1: Evaluation and Testing with a Risk Management
this standard.
Process
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 International Organization for Standardization (ISO), 1, ch. de
the ASTM website. la Voie-Creuse, CP 56, CH-1211 Geneva 20, Switzerland, http://www.iso.org.
F3089 − 23
ISO 10993-3 Biological Evaluation of Medical Devices— <71> Sterility Tests
Part 3:Tests for Genotoxicity, Carcinogenicity and Repro- <85> Bacterial Endotoxins Test
ductive Toxicity <161> Transfusion and Infusion Assemblies and Similar
ISO 10993-4 Biological Evaluation of Medical Devices— Medical Devices
Part 4: Selection of Tests for Interactions with Blood <232> Elemental Impurities—Limits
ISO 10993-5 Biological Evaluation of Medical Devices— <233> Elemental Impurities—Procedures
Part 5: Tests for In Vitro Cytotoxicity <791> pH
ISO 10993-6 Biological Evaluation of Medical Devices— <1050> Viral Safety Evaluation of Biotechnology Products
Part 6: Tests for Local Effects after Implantation Derived from Cell Lines of Human or Animal Origin
ISO 10993-7 Biological Evaluation of Medical Devices— <1058> Analytical Instrument Qualification
Part 7: Ethylene Oxide Sterilization Residuals <1225> Validation of Compendial Procedures
ISO 10993-9 Biological Evaluation of Medical Devices— <1211> Sterilization and Sterility Assurance of Compendial
Part 9: Framework for Identification and Quantification of Articles
Potential Degradation Products European Pharmacopeia 11.0
ISO 10993-10 Biological Evaluation of Medical Devices— 2.4 Code of Federal Regulations:
Part 10: Tests for Skin Sensitization 9 CFR Part 113 Standard Requirements
ISO 10993-11 Biological Evaluation of Medical Devices— 21 CFR Part 312 Investigational New Drug Application
Part 11: Tests for Systemic Toxicity 21 CFR 610.13(b) Rabbit Pyrogen Assay
ISO 10993-12 Biological Evaluation of Medical Devices— 21 CFR Part 812 Investigational Device Exemption
Part 12: Sample Preparation and Reference Materials 21 CFR Part 820 Quality System Regulation
ISO 10993-13 Biological Evaluation of Medical Devices— 21 CFR Parts 207, 807, and 1271 Human Cells, Tissues and
Part 13: Identification and Quantification of Degradation Cellular and Tissue-Based Products, Establishment Reg-
Products from Polymeric Medical Devices istration and Listing
ISO 10993-17 Biological Evaluation of Medical Devices— 21 CFR Part 1271, Subpart C Donor Eligibility
Part 17: Establishment ofAllowable Limits for Leachable Federal Register Vol. 43 No. 141, Friday, July 21, 1978
Substances Federal Register, Vol. 66 No. 13, Jan. 19, 2001/Rules and
ISO 10993-18 Biological Evaluation of Medical Devices— Regulations, p. 5447
Part 18: Chemical Characterization of Materials Federal Register, Vol. 72 No. 8, Jan. 12, 2007, pp.
ISO 10993-20 Biological Evaluation of Medical Devices— 1581–1619, Proposed Rule: Use of Materials Derived
Part 20: Principles and Methods for Immunotoxicology from Cattle in Medical Products Intended for Use in
Testing of Medical Devices Humans and Drugs Intended for Use in Ruminants
ISO 13408-1 Aseptic Processing of Health Care Products— Current Good Tissue Practice for Manufacturers of Human
Part 1: General Requirements Cellular and Tissue-Based Products Inspection and En-
ISO 14971 Medical Devices—Application of Risk Manage- forcement. Proposed Rule. Federal Register/Vol. 66, No.
ment to Medical Devices 5/January 8, 2001/Proposed Rules, pp. 1552–1559
ISO 22442-1 Medical Devices UtilizingAnimal Tissues and 2.5 ICH Documents:
Their Derivatives—Part 1: Application of Risk Manage- ICH M3(R2) Nonclinical Safety Studies for the Conduct of
ment Human Clinical Trials and Marketing Authorizations for
ISO 22442-2 Medical Devices UtilizingAnimal Tissues and Pharmaceuticals 62 FR 62922 (2009)
Their Derivatives—Part 2: Controls on Sourcing, ICH Q1A Stability Testing of New Drug Substances and
Collection, and Handling Products
ISO 22442-3 Medical Devices UtilizingAnimal Tissues and ICHQ2 ValidationofAnalyticalProcedures:TextandMeth-
Their Derivatives—Part 3: Validation of the Elimination odology
and/or Inactivation of Viruses and Transmissible Spongi- ICH Q3A Impurities in New Drug Substances
form Encephalopathy (TSE) Agents ICH Q3B Impurities in New Drug Products
ISO/TR 22442-4 Medical Devices Utilizing Animal Tissues ICH Q3C Guideline for Residual Solvents
and Their Derivatives—Part 4: Principles for Elimination ICH Q3D Guideline for Elemental Impurities
and/or Inactivation of Transmissible Spongiform En- ICHQ5A QualityofBiotechnologicalProducts:ViralSafety
cephalopathy (TSE) Agents and Validation Assays for Evaluation of Biotechnology Products Derived from Cell
Those Processes Lines of Human or Animal Origin
2.3 U.S. and European Pharmacopeia Documents: ICH S1A The Need for Carcinogenicity Studies of Pharma-
U.S. Pharmacopeia (USP) General Chapters ceuticals
<61> Microbiological Examination of Nonsterile Products:
Microbial Enumeration Tests
AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
U.S Pharmacopeia available from U.S. Pharmacopeia (USP), 12601Twinbrook Available from International Conference on Harmonisation of Technical
Pkwy., Rockville, MD 20852-1790, http://www.usp.org. European Pharmacopeia Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH
available from EDQM Council of Europe, 7 allée Kastner, CS 30026, F-67081 Secretariat, 9, chemin des Mines, P.O. Box 195, 1211 Geneva 20, Switzerland,
Strasbourg, France, Tel. [phone], http://pheur.edqm.eu. http://www.ich.org.
F3089 − 23
ICH S1B Testing for Carcinogenicity of Pharmaceuticals AAMI TIR 19:1998 Guidance for ANSI/AAMI/ISO
ICH S1C Dose Selection for Carcinogenicity Studies of 10993-7: 1995, Biological Evaluation of Medical
Pharmaceuticals Devices—Part 7: Ethylene Oxide Sterilization Residuals
ICH S2 Guidance on Genotoxicity Testing and Data Inter- AAMI/ISO14160:2011(R2016) SterilizationofHealthCare
pretation for Pharmaceuticals Intended for Human Use Products—Liquid Chemical SterilizingAgents for Single-
ICH S5 Detection of Reproductive and Developmental Tox- Use Medical Devices Utilizing Animal Tissues and Their
icity for Human Pharmaceuticals Derivatives—Requirements for Characterization,
Development, Validation and Routine Control of a Steril-
2.6 FDA Documents:
ization Process for Medical Devices
U.S. Food and Drug Administration (FDA) Center for
AAMI ST67:2019 Sterilization of Health Care Products—
Devices and Radiological Health (CDRH) and Center for
Requirements and Guidance for Selecting a Sterility
Biologics Evaluation and Research (CBER), 2020 Use of
Assurance Level (SAL) for Products Labeled “Sterile”
International Standard ISO 10993-1, “Biological evalua-
AAMI ST72:2019 Bacterial Endotoxins—Test Methods,
tion of medical devices – Part 1: Evaluation and testing
Routine Monitoring, and Alternatives to Batch Testing
withinariskmanagementprocess.”GuidanceforIndustry
and Food and Drug Administration Staff
2.8 Other References:
U.S. Food and Drug Administration (FDA) Center for Drug Council Directive 93/42/EEC, with Respect to Medical De-
Evaluations and Research (CDER), Center for Biologics
vices Using Tissues of Animal Origin
Evaluation and Research (CBER), Center for Veterinary
CommissionDirective2003/32/EC, withRespecttoMedical
Medicine (CVM), Center for Devices and Radiological
Devices Manufactured Using Tissues of Animal Origin
Health (CDRH), Office of Regulatory Affairs (ORA),
EMA/410/01-rev.3 Committee for Proprietary Medical
2012 Guidance for Industry. Pyrogen and Endotoxins
Products, Note for Guidance on Minimizing the Risk of
Testing: Questions and Answers
Transmitting Animal Spongiform Encephalopathy Agents
U.S. Food and Drug Administration (FDA) Center for
via Human and Veterinary Medical Products
Devices and Radiological Health, 2019 Medical Devices
European Medicines Agency, (EMA/CHMP/CVMP/QWP/
Containing Materials Derived from Animal Sources (Ex-
850374/2015) Guideline on the Sterilisation of the Me-
cept for In Vitro Diagnostic Devices). Guidance for
dicinal Product, Active Substance, Excipient and Primary
Industry and for Food and Drug Administration Staff
Container
U.S. Food and Drug Administration (FDA) Center for
Automotive Industry Action Group (AIAG) Measurement
Biologics Evaluation and Research (CBER), 1993 Points
Systems Analysis Reference Manual, 4th Edition
to Consider in the Characterization of Cell Lines Used to
National Institute of Standards and Technology
Produce Biologicals
(NIST) NIST/SEMATECH e-Handbook of Statistical
U.S. Food and Drug Administration (FDA) Center for
Methods, http://www.itl.nist.gov/div898/handbook/,
Biologics Evaluation and Research (CBER), 1997 Points
Chapter 2: Measurement Process Characterization
toConsiderintheManufactureandTestingofMonoclonal
Antibody Products for Human Use
3. Terminology
U.S. Food and Drug Administration (FDA) Center for Drug
3.1 Definitions:
Evaluation and Research (CDER) and Center for Biolog-
3.1.1 adventitious agent, n—an unintentionally introduced
ics Evaluation and Research (CBER), 2015 Analytical
microbiological or other infectious contaminant.
Procedures and MethodsValidation for Drugs and Biolog-
ics. Guidance for Industry 3.1.1.1 Discussion—In the production of TEMPs, these
U.S. Food and Drug Administration (FDA) Division of agents may be unintentionally introduced into the process
Small Manufacturers Assistance Office of Training and stream, the final product, or both.
Assistance Center for Devices and Radiological Health,
3.1.2 atelocollagen, n—triple helical molecule in which the
1991 Shelf Life of Medical Devices
telopeptide regions have been partially or completely removed
2.7 AAMI Documents:
fromtropocollagen(seeFig.1).Suchpreparationsaretypically
ANSI/AAMI/ISO 11737-1:2018 Sterilization of Healthcare
the outcome of enzyme-based (for example, pepsin) collagen
Products—Microbiological Methods—Part 1: Determina-
extraction procedures from tissues.
tion of a Population of Microorganisms on Products
ANSI/AAMI/ISO 11737-2:2009 Sterilization of Medical
Devices—Microbiological Methods—Part 2:Tests of Ste-
Available from Office for Official Publications of the European
rility Performed in the Definition, Validation, and Main-
Communities—European Law, 2, rue Mercier, L-2985, Luxembourg, http://eur-
tenance of a Sterilization Process lex.europa.eu/en/’index.htm.
Available from European Medicines Agency (EMA), Domenico Scarlattilaan
6, 1083 HS Amsterdam, The Netherlands, and https://www.ema.europa.eu/en/
documents/scientific-guideline/guideline-sterilisation-medicinal-product-active-
Available from Food and Drug Administration (FDA), 10903 New Hampshire substance-excipient-primary-container_en.pdf.
Ave., Silver Spring, MD 20993-0002, http://www.fda.gov. Available from European Medicines Agency (EMA), Domenico Scarlattilaan
Available from Association for the Advancement of Medical Instrumentation 6, 1083 HS Amsterdam, The Netherlands, and https://www.ema.europa.eu/en/
(AAMI), 4301 N. Fairfax Dr., Suite 301, Arlington, VA 22203-1633, http:// documents/scientific-guideline/minimising-risk-transmitting-animal-spongiform-
www.aami.org. encephalopathy-agents-human-veterinary-medicinal_en.pdf.
F3089 − 23
3.1.14.1 Discussion—However, it should be noted that not
all microorganisms are infectious or pathogenic.
3.1.15 permeability, n—a measure of the ability of porous
materials to transmit fluids; the rate of flow of a liquid through
a porous material.
3.1.16 polymerization, n—a chemical reaction in which two
FIG. 1 Schematic of Procollagen Molecule and Associated
or more molecules combine to form larger molecules that
Propeptide, Telopeptide, and Triple Helical Regions. Enzymatic
contain repeating structural units.
Removal of Amino- and Carboxy-terminal Propeptide Ends of
Procollagen Molecule by Procollagenases Yields Tropocollagen.
3.1.17 procollagen, n—collagen molecule comprising three
hydroxylated prototcollagen (alpha) chains; amino- and
carboxy-terminal propeptide ends are intact (Fig. 1).
3.1.3 biocompatibility, n—a material may be considered
3.1.18 propeptides, n—amino- and carboxy-terminal
biocompatibleifthematerialperformswithanappropriatehost
nontriple-helical domains of individual collagen protocollagen
response in a specific application (6).
(alpha) chains that direct triple-helix folding and formation of
procollagen molecules (Fig. 1); propeptide removal is required
3.1.4 biomaterial, n—a synthetic or natural substance or
for collagen fibrillogenesis and self-assembly.
composite used for a biological or biomedical application.
3.1.19 protocollagen, n—single collagen alpha polypeptide
3.1.5 collagen, n—a family of at least 20 genetically differ-
chain as produced by ribosomes.
ent secreted proteins that serve a predominantly structural
function and possess a unique triple helical structure configu-
3.1.20 scaffold, n—a two- or three-dimensional structural
ration of three polypeptide units known as alpha chains.
matrix that provides a conductive surface that enables the
3.1.6 diffusion, n—the random thermal motion of atoms, attachment, survival, proliferation, migration, and/or differen-
tiation of local or transplanted cells, and thereby facilitates the
molecules, clusters of atoms, etc., in gases, liquids, and some
solids. distributionofatissueformationresponsethroughoutadesired
surface or tissue volume. Medically, scaffolds may be used to
3.1.7 endotoxin,n—pyrogenichighmolarmasslipopolysac-
replace, repair, augment, or regenerate tissues.
charide (LPS) complex associated with the cell wall of
gram-negative bacteria.
3.1.21 self-assembly, n—the process by which a complex
3.1.7.1 Discussion—Although endotoxins are pyrogens, not macromolecule (as collagen) or a supramolecular system (as a
all pyrogens are endotoxins. Endotoxins are specifically de- virus) spontaneously assembles itself from its components.
tected through a test using the Limulus Amebocyte Lysate
3.1.22 solution, n—a type of homogenous mixture in which
(LAL) or recombinant Factor C (rFC) reagents.
atoms, ions, or molecules (the solute) are distributed uniformly
3.1.8 extracellular matrix (ECM), n—a composite medium, throughout another substance (the solvent) and which does not
where cells reside, remodel, and interact. ECM promotes cell
separate upon standing.
adhesion, spreading, survival, proliferation, migration,
3.1.23 sterilization, n—the destruction or removal of all
differentiation, and/or other functions over a range of dimen-
microorganisms in or about an object (for example, by chemi-
sional scales to maintain cell/tissue homeostasis, growth, and
cal agents, electron beam, gamma irradiation, or filtration).
remodeling.
3.1.23.1 Discussion—If the medical product collagen
3.1.8.1 Discussion—The ECM component of mammalian
permits, terminal sterilization is preferential to reliance on
tissues is produced and assembled by cells and often has
aseptic processing.
collagen as a predominant component.
3.1.24 stiffness, n—a general term describing the extent to
3.1.9 fibrillogenesis, n—the process of tropocollagen mono-
which a material resists deformation in response to an applied
mers assembling into mature fibrils and associated fibril-
force; specific measures of stiffness depend upon the material
network structures.
loading format (for example, tension, compression, shear,
3.1.10 fibrosis, n—an in situ process of tissue repair result-
bending).
ing in a relatively avascular and collagen rich tissue.
3.1.25 suspension, n—the dispersion of a solid through a
3.1.11 gel, n—the three-dimensional network structure aris-
liquidwithaparticlesizelargeenoughtobedetectedbypurely
ing from intermolecular polymer chain interactions.
optical means.
3.1.11.1 Discussion—Such chain interactions may be
3.1.26 telopeptide, n—amino- and carboxy-terminal
covalent, ionic, hydrogen bond, or hydrophobic in nature.
nontriple-helicaldomainsoftropocollagenstrandsknowntobe
3.1.12 mechanotransduction, n—process by which cells
important to fibrillogenesis and intermolecular cross-link for-
convert mechanical stimuli into a chemical response.
mation (Fig. 1).
3.1.13 medical product, n—any diagnostic or therapeutic
3.1.27 tissue engineered medical product (TEMP), n—a
treatment that may be regulated as a device, biologic, drug, or
manufactured or manipulated article that consists of cells, with
combination product.
or without a synthetic and/or naturally derived scaffold, used
3.1.14 microorganism, n—bacteria, fungi, yeast, mold, for repair, replacement, restoration, or regeneration of the
viruses, and other infectious agents. structure or function of a recipient’s cells, tissues, or organs.
F3089 − 23
3.1.28 tissue regeneration, n—an in-situ process of tissue 3.2.10.1 Discussion—In situations where there is an inflam-
repairwherethereisapartialorcompleterestorationofnormal matory response to implanted materials, immune cells, includ-
tissue structure and function. ing neutrophils, macrophages, lymphocytes, and giant cells,
can actively participate in resorption through material phago-
3.1.29 tissue repair, n—a process of partial or complete
cytosis and/or proteolysis processes.
restoration of tissue structure and/or function.
3.2.11 solubility, n—a measure of the extent to which a
3.1.30 tropocollagen, n—collagen molecule comprising
material can be dissolved.
three alpha chains with amino- and carboxy-terminal propep-
3.2.11.1 Discussion—In the context of collagen polymers,
tide ends removed (Fig. 1); carboxy- and amino-terminal
solubility refers to collagen molecules (partial, full, or mul-
non-helical telopeptide ends are intact; able to undergo self-
tiples) or peptides in a solution; further qualification of
assembly into fibrillar matrix.
solubility may include “acid-soluble” and “neutral salt-
3.2 Definitions of Terms Specific to This Standard:
soluble” which describes compositions that are soluble in
3.2.1 adhesion, n—steady or firm attachment; in the context
dilute acids and neutral salt solutions, respectively.
of collagen, adhesion refers to the ability of cells to physically
attach or bind to collagen molecules and macromolecular 4. Significance and Use
assemblies of collagen via cell surface proteins like integrins.
4.1 The objective of this document is to provide guidance in
3.2.2 collagen mimetic peptides, n—specific amino acid
the production, characterization, testing, and standardization
sequences representing the triple helical portion of collagen,
of: (1) polymerizable collagen starting materials; and (2)
often –(Pro–Hyp–Gly)x–, forms a triple helix conformation
collagen polymeric materials produced with polymerizable
that resembles that found in natural collagens.
collagen formulations, used for surgical implants, substrates
for TEMPs, vehicles for therapeutic cells and molecules, and
3.2.3 collagen polymeric material, n—a composition
3D in-vitro tissue systems for basic research, drug
formed by polymerization or self-assembly and consisting
development, and toxicity testing.This guide can be used as an
essentially of repeating collagen structural units.
aid in the selection, characterization, and standardization of the
3.2.4 degradation, n—change in chemical, physical, or mo-
appropriate polymerizable collagen starting formulations as
lecular structure or appearance (that is, gross morphology) of
well as collagen polymeric materials prepared from polymer-
material.
izable collagens for a specific use. Not all tests or parameters
3.2.4.1 Discussion—Degradation of collagen under physi-
are applicable to all uses of collagen and users are expected to
ologic conditions involves site-specific cleavage within the
select and justify a subset of the tests for characterization
central triple helical region by proteolytic enzymes known as
purposes.
collagenases.Collagenasesaremembersofthelargerfamilyof
proteases known as matrix metalloproteases. 4.2 This guide can be used by the following types of users:
4.2.1 Manufacturers of polymerizable collagens and colla-
3.2.5 matrix, n—loose meshwork within which cells are
genpolymericmaterialswhowishtosetspecificationsfortheir
embedded or arrangement of connected things. In the context
products or provide characterization data for customers or
of collagen, matrix refers to a composite material comprised of
users. They may also use the terminology and characterization
an insoluble collagen-fibril network or amorphous nanostruc-
sections to specify and differentiate the properties of polymer-
ture surrounded by an interstitial fluid phase.
izable collagens and collagen polymeric materials.
3.2.6 monomer, n—individual tropocollagen molecule (Fig.
4.2.2 Producers of collagen polymeric materials that use
1).
polymerizable collagen as starting materials. Producers may
3.2.7 oligomer, n—two or more tropocollagen molecules
use this guide to evaluate and characterize multiple sources of
covalently attached by a naturally occurring intermolecular
polymerizable collagen. They may also use this guide to assist
cross-link.
with evaluation and comparison of single or multiple sources
3.2.8 polymerizable collagen, n—purified type I collagen
of polymerizable collagen and collagen polymeric materials.
formulation that demonstrates the capacity to self-assemble or
4.2.3 Researchers may use this guide as a reference for
polymerize into higher order structures (macromolecular as-
properties and test methods that can be used to reproducibly
semblies) in absence of exogenous agents such as cross-
evaluate polymerizable collagens and collagen polymeric ma-
linkers.
terials.
3.2.9 recombinant collagen protein/peptide, n—collagen or
4.3 The collagen covered by this guide may be used in a
collagen-like polypeptide produced by recombinant methods,
broad range of applications, forms, or medical products, for
such as by expression of a nucleotide sequence encoding the
example (but not limited to) wound and hemostatic dressings,
protein or peptide in a microorganism, insect, plant, or animal
surgical implants or injectables (including in-situ forming),
host. Such compositions often comprise Gly-X-Y triplets
hybrid medical devices, TEMPs, injectable (including in-situ
where Gly is the amino acid glycine and X and Y can be the
forming) or implantable delivery vehicles for therapeutic cells,
same or different, are often proline or hydroxyproline, but can
molecules, and drugs, and 3D in-vitro tissue systems or models
be any known amino acid.
for basic research, drug development, and toxicity testing. The
3.2.10 resorption, n—removal by gradual breakdown into practical application of polymerizable collagens and collagen
component materials; a loss of substance by lysis, or by polymeric materials should be based, among other factors, on
physiologic or pathologic means. biocompatibility, application-specific performance measures,
F3089 − 23
as well as chemical, physical, and biological test data. Recom-
21 CFR 610: General Biological Products Standards:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
mendations in this guide should not be interpreted as a
CFRSearch.cfm?CFRPart=610
guarantee of success for any specific research or medical
21 CFR 1271: Human Cells, Tissues and Cellular and Tissue-Based
application. Products:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
4.4 The following general areas should be considered when
CFRSearch.cfm?CFRPart=1271
Cellular & Gene Therapy Guidances and Other Publications:
determining if the collagen supplied satisfies requirements for
http://www.fda.gov/cber/genetherapy/gtpubs.htm
use in the above mentioned medical and research applications:
Human Tissue Guidances and Other Publications:
source of polymerizable collagen, impurities profile, and com-
http://www.fda.gov/cber/tissue/docs.htm
CBER Product Approval Information:
prehensive chemical, physical, and biological characterization
http://www.fda.gov/cber/efoi/approve.htm
and testing.
21 CFR 600, 601 BLA Regulations:
http://www.access.gpo.gov/nara/cfr/waisidx_07/21cfrv7_07.html
4.5 The following documents or other relevant guidance
21 CFR 210, 211 GMP Regulations:
documents from appropriate regulatory bodies relating to the
http://www.access.gpo.gov/nara/cfr/waisidx_07/21cfr210_07.html
production, regulation, and regulatory approval of devices,
biologics, drugs, and combination products should be consid-
5. Standardization of Polymerizable Collagens and
ered when determining if the collagen supplied satisfies re-
Collagen Polymeric Material Products
quirements for use in medical and research products, including
5.1 Master File and Product Specifications—For the pur-
TEMPs, therapeutic delivery vehicles, and 3D in-vitro tissue
poses of standardizing and characterizing polymerizable colla-
systems:
gens and collagen polymeric materials prepared from polym-
FDA CFR:
erizable collagens, manufacturers should compile
21 CFR 3: Product Jurisdiction:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
specifications and certificates of analysis with information on
CFRSearch.cfm?CFRPart=3
theimportantpropertiesandperformanceparametersdescribed
21 CFR 58: Good Laboratory Practice for Nonclinical Laboratory
inthefollowingsections.Usersofpolymerizablecollagenmay
Studies:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
choose to characterize properties when data is not available
CFRSearch.cfm?CFRPart=58
from the manufacturer. Some properties are important to
understand the end performance of the materials, while others
FDA/CDRH CFR and Guidances:
21 CFR Part 803: Medical Device Reporting:
are important for the use of the materials. Manufacturers are
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
recommended to notify regular users when revising product
CFRSearch.cfm?CFRPart=803
21 CFR 812: Investigational Device Exemptions: specifications. Collagen for use in biomedical and pharmaceu-
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
tical applications including TEMPs should ideally be docu-
CFRSearch.cfm?CFRPart=812
mented in a master file to which end users may obtain a letter
21 CFR 814: Premarket Approval of Medical Devices:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
of cross reference from suppliers of collagen. Such a master
CFRSearch.cfm?CFRPart=814
file should be submitted to the relevant national and interna-
21 CFR 820: Quality System Regulation:
tional regulatory authorities. ISO 14971 should also be refer-
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=820
enced when appropriate.
Design Control Guidance for Medical Device Manufacturers:
http://www.fda.gov/cdrh/comp/designgd.pdf
5.2 The characterization methods outlined below and in
Preproduction Quality Assurance Planning Recommendations for
Tables 1 and 2 represent suggested chemical, physical, and
Medical Device Manufacturers (FDA 90-4236):
biological assays or analyses; however, other validated assays
http://www.fda.gov/cdrh/manual/appende.html
The Review and Inspection of Premarket Approval Applications
and analyses may be used (7). Method selection will vary
under the Bioresearch Monitoring Program—Draft Guidance
depending on the formulation and source of the collagen (for
for Industry and FDA Staff:
http://www.fda.gov/cdrh/comp/guidance/1602.pdf example, tissue-derived molecular collagen or collagen pep-
tides produced synthetically). The user should ensure that the
FDA/CDRH Search Engines:
methodselectedisreliableandcommonlyacceptedforprotein,
CDRH Guidance Search Engine:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfggp/
polymer, biological, and biomaterial analyses. In addition, the
search.cfm
test should have appropriate dynamic range, detection limits,
CDRH Premarket Approval (PMA) Search Engine:
specificity, and sensitivity.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMA/
pma.cfm
5.3 Test Method Development and Validation—Testing that
CDRH 510(k) Search Engine:
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/
is performed to demonstrate conformance to specifications
pmn.cfm
should be done, when possible, using well-characterized and
CDRH Recognized STANDARDS Search Engine:
reliablemethods.Soundandvalidatedscientificmethodologies
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/
search.cfm
should be applied to ensure production of consistent, accurate,
and meaningful results that are insensitive (robust) to changes
FDA/CBER CFR and Guidances:
21 CFR 312: Investigational New Drug Application: in environment, equipment and analytical instrumentation,
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
personnel, sampling procedure, and test specimen format. It is
CFRSearch.cfm?CFRPart=312
beyond the scope of this document to provide references
21 CFR 314: Applications for FDA Approval to Market a New Drug:
applicable to all the methods herein, but users should be aware
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=31
of the most common practices.
F3089 − 23
TABLE 1 Characterization Methods for Type I Polymerizable Collagens
Example Methods
Parameter Qualitative Quantitative
(not comprehensive or exclusive)
Physical/Chemical/Biochemical
Form and Appearance Visual inspection X
Collagen Concentration or Content Spectrophotometric (A ; Sirius Red Assay; Hydroxyproline Assay) X
Gel Electrophoresis X X
Solids Content (Loss on Drying Methods) X
pH pH meter X
pH indicator X
pH test papers X
Viscosity Viscometry X
Rheology X
Purity, Including Collagen Type Composition MS X
FTIR X
Amino Acid Analysis X
ELISA X
Circular Dichroism X
Gel Electrophoresis; Western Blot; Peptide Mapping X X
Impurities Profile, Including Heavy Metals GC X
Analysis HPLC X
ICP X
Gel Electrophoresis X X
Specific Chemical Assays X X
TGA (Thermogravimetric Analysis)
Degree of Cross-linking (Natural Intermolecular MS X
Cross-links or Exogenous Cross-links) HPLC X
DSC X
Gel Electrophoresis X X
Colorimetric X X
TGA (Thermogravimetric Analysis) X
Molecular Mass; Molecular Mass Distribution; Viscometry X
Average Polymer Molecular Weight Rheology X
LC X
DLS X
Analytical Ultracentrifugation X
Enzyme Susceptibility (e.g., trypsin, collagenase) Digestion Assay X X
Additives (e.g., light/heat stabilizers, viscosity HPLC XX
modifiers, antimicrobial agents, cross-linking GC XX
agents, other biomolecules, drugs) MS XX
Polymerization Kinetics Spectrophotometric X
Rheometric X
5.3.1 Equipment—Common practices pertaining to test FDAGuidance forAnalytical Procedures and Methods Valida-
equipment include routine calibration, more frequent measure- tion for Drugs and Biologics, and USP <1225>. For analytical
ment verifications, periodic preventative maintenance, and methods, these documents generally provide recommendations
formalequipmentqualifications.Anexampleofthesepractices
to document a method’s performance in terms of accuracy,
is provided in USP <1058>. More specific practices also exist specificity, sensitivity, range, linearity, and precision/
forsomeapplications,suchasPracticeE4forforceverification
reproducibility. Method validation includes ongoing activities
of testing machines.
such as routine system suitability tests.
5.3.2 Personnel and Procedures—The performance of per-
5.3.4.2 Physical Method Guidance—Method validations for
sonnel and the procedures assigned to them is ensured through
physical tests generally focus on characterizing the accuracy
training and tests to demonstrate proficiency. Computational or
and precision of a method. The most prevalent approach is a
analyticaltasksshouldbeincludedalongsideoperationaltasks.
Gauge R&R study. Recommendations applicable to these
5.3.3 Control Materials and Calibration Curve—For meth-
methods are found in ISO 5725 (Parts 1 through 6), theAIAG
ods that are sensitive to interference or background noise,
Measurement Systems Analysis Reference Manual, and the
usually analytical or thermal tests, well-characterized control
NIST/SEMATECH Engineering Statistics Handbook, Chapter
samples are included alongside each batch of test samples to
2: Measurement Process Characterization. As with analytical
demonstrate ongoing accuracy and precision as far as possible.
methods, validated physical methods should include ongoing
Similarly, a set of known reference samples may be used to
activities such as routine system suitability tests.
create a calibration curve for each sample batch that can relate
direct sensor measurements (such as fluorescence intensity) to
6. Characterization of Polymerizable Collagens
an estimate of sample concentration.
5.3.4 Method Validation—A test method validation is a 6.1 Form and Appearance—Lyophilized polymerizable col-
lagen is often a white friable or flocculent solid or powder.
formalprocessofdemonstratingthecapabilityofatestmethod
to meet the needs of its intended use. Polymerizable collagen in aqueous solution is often colorless.
5.3.4.1 Analytical Method Guidance—Guidance for test Dependingonitscomposition,thesolutionmaybeviscousand
method validation of analytical methods include ICH Q2, U.S. appear transparent (clear), translucent, or opaque.
F3089 − 23
TABLE 2 Characterization Methods for Collagen Polymeric Materials Prepared from Type I Polymerizable Collagens
Example Methods
Parameter Qualitative Quantitative
(not comprehensive or exclusive)
Chemical/Biochemical
Form and Appearance Visual inspection X
Collagen Concentration or Content Spectrophotometric (Hydroxyproline Assay) X
Gel Electrophoresis X X
Solids Analysis (Loss on Drying Methods) X
Purity, Including Collagen Type Composition MS X
FTIR X
Amino Acid Analysis X
ELISA X
Circular Dichroism X
Gel Electrophoresis; Western Blot; Peptide Mapping X X
Impurities Profile, including Heavy Metals Analysis GC X
HPLC X
ICP X
Gel Electrophoresis X X
Specific Chemical Assays X X
Degree of Cross-linking (Natural Intermolecular Cross- MS X
links or Exogenous Cross-links) HPLC X
DSC X
Gel Electrophoresis X X
Colorimetric X X
TGA X
Enzyme Susceptibility (e.g., trypsin, collagenase) Digestion Assay X X
Additives (e.g., light/heat stabilizers, viscosity modifiers, HPLC X X
antimicrobial and cross-linking agents, other GC XX
biomolecules, drugs) MS XX
Physical
Viscosity Viscometry X
Rheology X
Nano-/micro-structure Microscopy, including TEM, SEM, Confocal, and AFM X X
Transport Properties 
...

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: F3089 − 14 F3089 − 23
Standard Guide for
Characterization and Standardization of Polymerizable
Collagen-Based Products and Associated Collagen-Cell
Interactions
This standard is issued under the fixed designation F3089; 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
The collagen family of proteins represents the major structural and mechanical component of the
in-vivo extracellular matrix of human tissues and organs. Type I collagen is the most abundant and as
such, it is an ideal candidate for medical materials, tissue-engineered medical products, delivery of
therapeutic cells/molecules, and in-vitro cell/tissue culture applications. Furthermore, it is now evident
that specific collagen material properties, including microstructure, mechanical integrity (stiffness),
(stiffness, strength), cell adhesion, immunogenicity, and biodegradation resorption (degradation) are
major determinants of the interfacial properties between cells and collagen-based materials, including
guidance of fundamental cell behaviors that contribute to recapitulation and/or restoration of tissue
structure and function. Advanced understanding of collagen self-assembly, as occurs in vivo and in
vitro, is contributing to a rapid expansion of commercial and laboratory-produced purified collagen
formulations that polymerize (self-assemble) or exhibit solution to gel (matrix) transition. transitions
from solution to semi-solid material (for example, gel, scaffold). Most recent developments have
focused on polymerizable collagen polymer formulations with tunable features to formulations that
support the rational design and custom fabrication of collagen polymeric materials for improved tissue
integration and integration, guidance of cell fate. fate, and tissue response outcomes. Unfortunately,
the term “collagen” is applied generally to describe various collagen types and formulations (soluble,
insoluble, monomeric, gelatin/peptides, oligomeric, tropocollagen, atelocollagen) that vary signifi-
cantly in their molecular compositions, self-assemblypolymerization capacity and properties, and
ability to interact with cells. As such, the need exists for an expanded set of characterization and
standardization strategies to facilitate comparison, safety and efficiency testing, and translation of the
next generation polymerizable collagen polymer formulations and associated self-assembled collagen-
basedcollagen polymeric materials produced with these formulations.
1. Scope
1.1 This guide is intended to provide characteristics, properties, test methods, and standardization approaches for evaluation and
identification of specific polymerizable collagen formulations and collagen polymeric materials produced with these formulations.
1.2 This guide for characterizing polymerizable collagens is intended to provide characteristics, properties, test methods, and
standardization approaches for use by producers, manufacturers, and researchers to identify specific collagen polymer formulations
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 May 1, 2014Jan. 1, 2023. Published July 2014January 2023. Originally approved in 2014. Last previous edition approved in 2014 as F3089 – 14.
DOI: 10.1520/F3089-14.10.1520/F3089-23.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F3089 − 23
and associated self-assembled collagen-based products produced with these formulations. This guide will focus on the
characterization of polymer forms of Typefocuses on characterization of purified polymerizable forms of type I collagen, which
is the most abundant collagen in mammalian connective tissues and organs, including skin, bone, tendon, and blood vessels. Type
Polymerizable type I collagen may be derived from a variety of sources including, but not limited to, animal or cadaveric tissues,
cell culture, recombinant, recombinant cell culture, and chemical synthesis. This guide is intended to focus on purified Type I
collagen polymers as a starting material for wound and hemostatic dressings, surgical implants, substrates for tissue-engineered
medical products (TEMPs), delivery vehicles for therapeutic cells or molecules, and 3D in-vitro tissue systems for basic research,
drug development, and toxicity testing. Polymerizable or self-assembly implies that the collagen composition exhibits spontaneous
macromolecular assembly from its components in the absence of the addition of exogenous factors including cross-linking agents.
Self-assembling collagen polymers may include, but are not limited to: (1) tissue-derived atelocollagens, monomers, and
oligomers; (2) collagen proteins and peptides produced using recombinant technology; and (3) chemically synthesized collagen
mimetic peptides. It should be noted that the format of associated self-assembled collagen-based products also will vary and may
include injectable solutions that polymerize in situ as well as preformed sheets, particles, spheres, fibers, sponges, matrices/gels,
coatings, films, and other forms. This guide may serve as a template for characterization and standardization of other fibrillar
collagen types that demonstrate polymerization or self-assembly.
1.2.1 This guide covers evaluation of polymerizable collagens and collagen polymeric materials prepared from polymerizable
collagens for use as a starting material for wound and hemostatic dressings, surgical implants, substrates for tissue-engineered
medical products (TEMPs), delivery vehicles for therapeutic cells or molecules, and 3D in-vitro tissue systems for basic research,
diagnostics, drug development, and toxicity testing. Most collagen products on the market today are regulated as devices since their
primary intended purpose is not achieved through chemical action within or on the body. However, a medical product comprising
polymerizable collagens or collagen polymeric materials may be regulated as a device, biologic, drug, or combination product
depending on its intended use and primary mode of action.
1.2.2 Polymerizable collagen or collagen self-assembly implies that the collagen composition exhibits spontaneous macromo-
lecular assembly from its components without the addition of exogenous factors such as cross-linking agents. Polymerizable
collagens may include but are not limited to: (1) tissue-derived monomeric collagens, including tropocollagen or atelocollagen, and
oligomeric collagens; (2) collagen proteins and peptides produced through in vitro cell culture, with or without using recombinant
technology; and (3) chemically synthesized collagen mimetic peptides. It should be noted that the format of collagen polymeric
material products also will vary and may include injectable solutions that polymerize in situ as well as preformed sheets, particles,
spheres, fibers, sponges, matrices/gels, coatings, films, and other forms.
1.2.3 This guide may serve as a template for characterization and standardization of type I fibrillar collagen or other collagen types
that demonstrate polymerization or self-assembly.
1.3 The ability of self-assembled collagen This guide does not provide a significant basis for assessing the biological safety
(biocompatibility) of polymerizable collagens and collagen polymeric materials. While the ability of collagen polymeric materials
to guide cellular responses through provision of cellular adhesion and proteolytic domains as well as physical constraints (for
example, structural, cell-matrix traction force) has been well documented through extensive clinical (1, 2)and basic research studies
(31-5,), 4).users are directed to the ISO 10993 series for evaluating biological risks of medical devices. The biocompatibility and
appropriateness of use for a specific application(s)application is the responsibility of the product manufacturer.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 Warning—Mercury has been designated by the Environmental Protection Agency (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 the EPA 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, SectionSections 56 and 7, of this guide.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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
The boldface numbers in parentheses refer to thea list of references at the end of this standard.
F3089 − 23
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:
E1298E4 Guide for Determination of Purity, Impurities, and Contaminants in Biological Drug ProductsPractices for Force
Calibration and Verification of Testing Machines (Withdrawn 2014)
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 Intramuscular Screening of Implantable Medical Device 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
F1251 Terminology Relating to Polymeric Biomaterials in Medical and Surgical Devices (Withdrawn 2012)
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
F1905 Practice For Selecting Tests for Determining the Propensity of Materials to Cause Immunotoxicity (Withdrawn 2011)
F1906 Practice for Evaluation of Immune Responses In Biocompatibility Testing Using ELISA Tests, Lymphocyte Proliferation,
and Cell Migration (Withdrawn 2011)
F1983 Practice for Assessment of Selected Tissue Effects of Absorbable Biomaterials for Implant Applications
F2148F2914 PracticeGuide for Evaluation of Delayed Contact Hypersensitivity Using the Murine Local Lymph Node Assay
(LLNA)Identification of Shelf-life Test Attributes for Endovascular Devices
2.2 ISO Standards:
ISO 5725-1 Accuracy (Trueness and Precision) of Measurement Methods and Results—Part 1: General Principles and
Definitions
ISO 5725-2 Accuracy (Trueness and Precision) of Measurement Methods and Results—Part 2: Basic Method for the
Determination of Repeatability and Reproducibility of a Standard Measurement Method
ISO 5725-3 Accuracy (Trueness and Precision) of Measurement Methods and Results—Part 3: Intermediate Measures of the
Precision of a Standard Measurement Method
ISO 5725-4 Accuracy (Trueness and Precision) of Measurement Methods and Results—Part 4: Basic Methods for the
Determination of the Trueness of a Standard Measurement Method
ISO 5725-5 Accuracy (Trueness and Precision) of Measurement Methods and Results—Part 5: Alternative Methods for the
Determination of the Precision of a Standard Measurement Method
ISO 5725-6 Accuracy (Trueness and Precision) of Measurement Methods and Results—Part 6: Use in Practice of Accuracy
Values
ISO 10993–110993-1 Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing with a Risk Management
Process
ISO 10993–310993-3 Biological Evaluation of Medical Devices—Part 3: Tests for Genotoxicity, Carcinogenicity and
Reproductive Toxicity
ISO 10993-4 Biological Evaluation of Medical Devices—Part 4: Selection of Tests for Interactions with Blood
ISO 10993-5 Biological Evaluation of Medical Devices—Part 5: Tests for In Vitro Cytotoxicity
ISO 10993-6 Biological Evaluation of Medical Devices—Part 6: Tests for Local Effects after Implantation
ISO 10993-7 Biological Evaluation of Medical Devices—Part 7: Ethylene Oxide Sterilization Residuals
ISO 10993–910993-9 Biological Evaluation of Medical Devices—Part 9: Framework for Identification and Quantification of
Potential Degradation Products
ISO 10993–1010993-10 Biological Evaluation of Medical Devices—Part 10: Tests for Irritation and Delayed-Type Hypersen-
sitivity Skin Sensitization
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), 1, ch. de la Voie-Creuse, CP 56, CH-1211 Geneva 20, Switzerland, http://www.iso.org.
F3089 − 23
ISO 10993-11 Biological Evaluation of Medical Devices—Part 11: Tests for Systemic Toxicity
ISO 10993-12 Biological Evaluation of Medical Devices—Part 12: Sample Preparation and Reference Materials
ISO 10993-13 Biological Evaluation of Medical Devices—Part 13: Identification and Quantification of Degradation Products
from Polymeric Medical Devices
ISO 10993–1710993-17 Methods for Biological Evaluation of Medical Devices—Part 17: Establishment of Allowable Limits
for Leachable Substances Using Health-Based Risk Assessment
ISO 10993-18 Biological Evaluation of Medical Devices—Part 18: Chemical Characterization of Materials
ISO 10993-20 Biological Evaluation of Medical Devices—Part 20: Principles and Methods for Immunotoxicology Testing of
Medical Devices
ISO 13408–113408-1 Aseptic Processing of Health Care Products—Part 1: General Requirements
ISO 14971 Medical Devices—Application of Risk Management to Medical Devices
ISO 22442–122442-1 Medical Devices Utilizing Animal Tissues and their Derivatives Utilized in the Manufacture of Medical
Devices—Part 1: Analysis and Management of RiskTheir Derivatives—Part 1: Application of Risk Management
ISO 22442–222442-2 Medical Devices Utilizing Animal Tissues and their Derivatives Utilized in the Manufacture of Medical
Devices—Part Their Derivatives—Part 2: Controls on Sourcing, Collection, and Handling
ISO 22442–322442-3 Medical Devices Utilizing Animal Tissues and their Derivatives Utilized in the Manufacture of Medical
Devices—Part 3: Validation andTheir Derivatives—Part 3: Validation of the Elimination and/or Inactivation of Virus and
Transmissable Viruses and Transmissible Spongiform Encephalopathy (TSE) Agents
ISO/TR 22442-4 Medical Devices Utilizing Animal Tissues and Their Derivatives—Part 4: Principles for Elimination and/or
Inactivation of Transmissible Spongiform Encephalopathy (TSE) Agents and Validation Assays for Those Processes
2.3 U.S. and European Pharmacopeia Documents:
United States Pharmacopeia (USP), Edition XXX (30)U.S. Pharmacopeia (USP) General Chapters
<61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
<71> Sterility Tests
<85> Bacterial Endotoxins Test
<161> Transfusion and Infusion Assemblies and Similar Medical Devices
<232> Elemental Impurities—Limits
<233> Elemental Impurities—Procedures
<791> pH
USP 30/NF 19<1050> Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin
<1058> Analytical Instrument Qualification
<1225> Validation of Compendial Procedures
<1211> Sterilization and Sterility Assurance of Compendial Articles
European Pharmacopeia 5.0 11.0
2.4 Code of Federal Regulations:
9 CFR Part 113 Standard Requirements
21 CFR Part 312 Investigational New Drug Application
21 CFR 610.13(b) Rabbit Pyrogen Assay
21 CFR Part 812 Investigational Device Exemption
21 CFR Part 820 Quality System Regulation
Federal Register Vol. 43 No. 141, Friday, July 21, 1978
21 CFR Parts 207, 807, and 1271 Human Cells, Tissues and Cellular and Tissue-Based Products, Establishment Registration and
Listing
21 CFR Part 1271, Subpart C Donor Eligibility
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
21 CFR Part 1271, Part C Suitability Determination for Donors of Human Cell and Tissue-based Products, Proposed Rule
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. 1552-15591552–1559
Guidance for Screening and Testing of Donors of Human Tissue Intended for Transplantation Availability. Federal Register/Vol.
62, No. 145/July 29, 1997/Notices 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
Available U.S Pharmacopeia available from U.S. Pharmacopeia (USP), 12601 Twinbrook Pkwy., Rockville, MD 20852-1790, http://www.usp.org. European
Pharmacopeia available from EDQM Council of Europe, 7 allée Kastner, CS 30026, F-67081 Strasbourg, France, Tel. [phone], http://pheur.edqm.eu.
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.
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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
CFR 610.13(b) Rabbit Pyrogen Assay
2.5 ICH Documents:
ICH M3M3(R2) Guidance for Industry M3 Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing
Authorizations for Pharmaceuticals 62 FR 62922 (1997)(2009)
ICH S2AQ1A Guideline for Industry S2A Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals 61 FR 18199
(1996)Stability Testing of New Drug Substances and Products
ICH S2BQ2 Guidance for Industry S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals 62 FR
62472 (1997)Validation of Analytical Procedures: Text and Methodology
ICH S5AQ3A Guideline for Industry S5A Detection of Toxicity to Reproduction for Medicinal Products 59 FR 48746
(1994)Impurities in New Drug Substances
ICH S5BQ3B Guidance for Industry S5B Detection of Toxicity to Reproduction for Medicinal Products: Addendum on Toxicity
to Male Fertility 61 FR 15360 (1996)Impurities in New Drug Products
ICH Q3C Guideline for Residual Solvents
ICH Q3D Guideline for Elemental Impurities
ICH Q5A Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of
Human or Animal Origin
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)S2 Guidance for Industry Addendum to Dose Selection for Carcinogenicity Studies of Pharmaceuticals: Addition
of a Limit Dose and Related Notes 62 FR 64259 (1997)on Genotoxicity Testing and Data Interpretation for Pharmaceuticals
Intended for Human Use
ICH Q1A ICHS5 Harmonized Tripartite Guidance for Stability Testing of New Drug Substances and Products (September 23,
1994)Detection of Reproductive and Developmental Toxicity for Human Pharmaceuticals
2.6 FDA Documents:
U.S. Food and Drug Administration (FDA and Committee for Proprietary Medicinal Products (CPMP), 1998(FDA) Center for
Devices and Radiological Health (CDRH) and Center for Biologics Evaluation and Research (CBER), 2020 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 5Use
of International Standard ISO 10993-1, “Biological evaluation of medical devices – Part 1: Evaluation and testing within a
risk management process.” Guidance for Industry and Food and Drug Administration Staff
FDAU.S. Food and Drug Administration (FDA) Center for Drug Evaluations and Research (CDER), Center for Biologics
Evaluation and Research (CBER), Center for Veterinary Medicine (CVM), Center for Devices and Radiological Health
(CDRH), Office of Regulatory Affairs (ORA), 2012 Guidance for IndustryIndustry. Pyrogen and Endotoxins Testing:
Questions and Answers, DHHS, June 2012Answers
U.S. Food and Drug Administration (FDA) Center for Devices and Radiological Health, 2019 Medical Devices Containing
Materials Derived from Animal Sources (Except for In Vitro Diagnostic Devices). Guidance for Industry and for Food and
Drug Administration Staff
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-0259Use
U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) and Center for Biologics
Evaluation and Research (CBER), 2015 Analytical Procedures and Methods Validation for Drugs and Biologics. Guidance for
Industry
FDAU.S. Food and Drug Administration (FDA) Division of Small Manufacturers Assistance Office of Training and Assistance
Center for Devices and Radiological Health, 1991 Interim Guidance for Human and Veterinary Drug Products and
Biologicals, Kinetic LAL techniques, DHHS, July 15, 1991Shelf Life of Medical Devices
Available from International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH Secretariat, c/o
IFPMA, 15 ch. Louis-Dunant, 9, chemin des Mines, P.O. Box 195, 1211 Geneva 20, Switzerland, http://www.ich.org.
Available from Food and Drug Administration (FDA), 10903 New Hampshire Ave., Silver Spring, MD 20993-0002, http://www.fda.gov.
F3089 − 23
2.7 AAMI Documents:
ANSI/AAMI/ISO 11737-1: 200611737-1:2018 Sterilization of Medical Devices—MicrobiologicalHealthcare Products—
Microbiological Methods—Part 1: Estimation of Bioburden on ProductDetermination of a Population of Microorganisms on
Products
ANSI/AAMI/ISO 11737-2: 199811737-2:2009 Sterilization of Medical Devices—Microbiological Methods—Part 2: Tests of
Sterility Performed in the Validation Definition, Validation, and Maintenance of a Sterilization Process
AAMI TIR No. 19-199819:1998 Guidance for ANSI/AAMI/ISO 10993-7: 1995, Biological Evaluation of Medical Devices—
Part 7: Ethylene Oxide Sterilization Residuals
AAMI/ISO 14160-199814160:2011 (R2016) Sterilization of Health Care Products—Liquid Chemical Sterilizing Agents for
Single-Use Medical Devices Incorporating Materials of Animal Origin—Validation and Utilizing Animal Tissues and Their
Derivatives—Requirements for Characterization, Development, Validation and Routine Control of Sterilization by Liquid
Chemical Sterilantsa Sterilization Process for Medical Devices
AAMI ST67/CDV-2: 1999ST67:2019 Sterilization of Medical Devices—Requirements for Health Care Products—
Requirements and Guidance for Selecting a Sterility Assurance Level (SAL) for Products Labeled “Sterile”
AAMI ST72:2019 Bacterial Endotoxins—Test Methods, Routine Monitoring, and Alternatives to Batch Testing
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,EMA/410/01-rev.3 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, (EMA),European Medicines Agency, (EMA/CHMP/CVMP/
QWP/850374/2015) Committee for Proprietary Medicinal Products (CPMP) Guidance Document for Decision Trees for the
Selection of Sterilization Methods (CPMP/QWP/054/98 corr 2000) and Annex to Note for Guidance on Development
Pharmaceutics (CPMP/QWP/155/96)Guideline on the Sterilisation of the Medicinal Product, Active Substance, Excipient and
Primary Container
Automotive Industry Action Group (AIAG) Measurement Systems Analysis Reference Manual, 4th Edition
National Institute of Standards and Technology (NIST) NIST/SEMATECH e-Handbook of Statistical Methods, http://
www.itl.nist.gov/div898/handbook/, Chapter 2: Measurement Process Characterization
3. Terminology
3.1 Definitions:
3.1.1 adventitious agents,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, the final product, or both.
3.1.2 atelocollagen, n—triple helical molecule in which the telopeptide regions have been partially or completely removed from
tropocollagen (see Fig. 1). Such preparations are typically the outcome of enzyme-based (for example, pepsin) collagen extraction
procedures from tissues.
3.1.3 biocompatibility, n—a material may be considered biocompatible if the material performs with an appropriate host response
in a specific application (6).
3.1.4 biomaterial, n—a synthetic or natural substance or composite used for a biological or biomedical application.
3.1.5 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.
Available from Association for the Advancement of Medical Instrumentation (AAMI), 4301 N. Fairfax Dr., Suite 301, Arlington, VA 22203-1633, http://www.aami.org.
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.(EMA), Domenico Scarlattilaan 6, 1083 HS Amsterdam, The Netherlands, and https://
www.ema.europa.eu/en/documents/scientific-guideline/guideline-sterilisation-medicinal-product-active-substance-excipient-primary-container_en.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.(EMA), Domenico Scarlattilaan 6, 1083 HS Amsterdam, The Netherlands, and https://www.ema.europa.eu/en/
documents/scientific-guideline/minimising-risk-transmitting-animal-spongiform-encephalopathy-agents-human-veterinary-medicinal_en.pdf.
F3089 − 23
FIG. 1 Schematic of Procollagen Molecule and Associated Propeptide, Telopeptide, and Triple Helical Regions. Enzymatic Removal of
Amino- and Carboxy-terminal Propeptide Ends of Procollagen Molecule by Procollagenases Yields Tropocollagen.
3.1.4 collagen mimetic peptides, n—specific amino acid sequences representing the triple helical portion of collagen, often
–(Pro–Hyp–Gly) –, forms a triple helix conformation that resembles that found in natural collagens.
x
3.1.5 collagen polymer, n—purified Type I collagen formulation that demonstrates the capacity to self-assemble or polymerize into
higher order structures (macromolecular assemblies) in absence of exogenous agents such as cross-linkers.
3.1.6 diffusion, n—the random thermal motion of atoms, molecules, clusters of atoms, etc., in gases, liquids, and some solids.
3.1.7 endotoxin, n—pyrogenic high molar mass lipopolysaccharide (LPS) complex associated with the cell wall of gram-negative
bacteria.
3.1.7.1 Discussion—
Although endotoxins are pyrogens, not all pyrogens are endotoxins. Endotoxins are specifically detected through a test using the
Limulus Amebocyte Lysate (LAL) test (USP<85> Bacterial Endotoxin Tests).or recombinant Factor C (rFC) reagents.
3.1.8 extracellular matrix (ECM), n—a composite medium, where cells reside, remodel, and interact. ECM promotes cell
adhesion, spreading, survival, proliferation, migration, differentiation, and/or other functions over a range of dimensional scales
to maintain cell/tissue homeostasis, growth, and remodeling.
3.1.8.1 Discussion—
The ECM component of mammalian tissues is produced and assembled by cells and often has collagen as a predominant
component.
3.1.9 fibrillogenesis, n—the process of tropocollagen monomers assembling into mature fibrils and associated fibril-network
structures.
3.1.10 fibrosis, n—an in situ process of tissue repair resulting in a relatively avascular and collagen rich tissue.
3.1.11 gel, n—the three-dimensional network structure arising from intermolecular polymer chain interactions.
3.1.11.1 Discussion—
Such chain interactions may be covalent, ionic, hydrogen bond, or hydrophobic in nature.
3.1.12 mechanotransduction, n—process by which cells convert mechanical stimuli into a chemical response.
3.1.13 medical product, n—any diagnostic or therapeutic treatment that may be regulated as a device, biologic, drug, or
combination product.
3.1.14 microorganism, n—bacteria, fungi, yeast, mold, viruses, and other infectious agents. However, it should be noted that not
all microorganisms are infectious or pathogenic.
3.1.14.1 Discussion—
However, it should be noted that not all microorganisms are infectious or pathogenic.
3.1.15 permeability, n—a measure of the ability of porous materials to transmit fluids; the rate of flow of a liquid through a porous
material.
3.1.16 polymerization, n—a chemical reaction in which two or more molecules combine to form larger molecules that contain
repeating structural units.
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3.1.17 procollagen, n—collagen molecule comprising three hydroxylated prototcollagen (alpha) chains; amino- and carboxy-
terminal propeptide ends are intact (Fig. 1).
3.1.18 propeptides, n—amino- and carboxy-terminal nontriple-helical domains of individual collagen protocollagen (alpha) chains
that direct triple-helix folding and formation of procollagen molecules (Fig. 1); propeptide removal is required for collagen
fibrillogenesis and self-assembly.
3.1.19 protocollagen, n—single collagen alpha polypeptide chain as produced by ribosomes.
3.1.20 recombinant collagen protein/peptide, scaffold, n—collagen or collagen-like polypeptide produced by recombinant
methods, such as by expression of a nucleotide sequence encoding the protein or peptide in a microorganism, insect, plant, or
animal host. Such compositions often comprise Gly-X-Y triplets where Gly is the amino acid glycine and X and Y can be the same
or different, are often proline or hydroxyproline, but can be any known amino acid.a two- or three-dimensional structural matrix
that provides a conductive surface that enables the attachment, survival, proliferation, migration, and/or differentiation of local or
transplanted cells, and thereby facilitates the distribution of a tissue formation response throughout a desired surface or tissue
volume. Medically, scaffolds may be used to replace, repair, augment, or regenerate tissues.
3.1.21 self-assembly, n—the process by which a complex macromolecule (as collagen) or a supramolecular system (as a virus)
spontaneously assembles itself from its components.
3.1.22 solution, n—a type of homogenous mixture in which atoms, ions, or molecules (the solute) are distributed uniformly
throughout another substance (the solvent) and which does not separate upon standing.
3.1.23 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).
3.1.23.1 Discussion—
If the medical product collagen permits, terminal sterilization is preferential to reliance on aseptic processing.
3.1.24 stiffness, n—a general term describing the extent to which a material resists deformation in response to an applied force;
specific measures of stiffness depend upon the material loading format (for example, tension, compression, shear, bending).
3.1.25 suspension, n—the dispersion of a solid through a liquid with a particle size large enough to be detected by purely optical
means and which separates or settles upon standing.means.
3.1.26 telopeptide, n—amino- and carboxy-terminal nontriple-helical domains of tropocollagen strands known to be important to
fibrillogenesis and intermolecular cross-link formation (Fig. 1).
3.1.27 tissue engineered medical product (TEMP), n—a manufactured or manipulated article that consists of cells, with or without
a synthetic and/or naturally derived scaffold, used for repair, replacement, restoration, or regeneration of the structure or function
of a recipient’s cells, tissues, or organs.
3.1.28 tissue regeneration, n—an in-situ process of tissue repair where there is a partial or complete restoration of normal tissue
structure and function.
3.1.29 tissue repair, n—a process of partial or complete restoration of tissue structure and/or function.
3.1.30 tropocollagen, n—collagen molecule comprising three alpha chains with amino- and carboxy-terminal propeptide ends
removed (Fig. 1); carboxy- and amino-terminal non-helical telopeptide ends are intact; able to undergo self-assembly into fibrillar
matrix.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 adhesion, n—steady or firm attachment; in the context of collagen, adhesion refers to the ability of cells to physically attach
or bind to collagen molecules and macromolecular assemblies of collagen via cell surface proteins like integrins.
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3.2.2 collagen mimetic peptides, n—specific amino acid sequences representing the triple helical portion of collagen, often
–(Pro–Hyp–Gly)x–, forms a triple helix conformation that resembles that found in natural collagens.
3.2.3 collagen polymeric material, n—a composition formed by polymerization or self-assembly and consisting essentially of
repeating collagen structural units.
3.2.4 degradation, n—change in chemical, physical, or molecular structure or appearance (that is, gross morphology) of material;
degradation of collagen under physiologic conditions involves site-specific cleavage within the central triple helical region by
proteolytic enzymes known as collagenases. Collagenases are members of the larger family of proteases known as matrix
metalloproteases.material.
3.2.4.1 Discussion—
Degradation of collagen under physiologic conditions involves site-specific cleavage within the central triple helical region by
proteolytic enzymes known as collagenases. Collagenases are members of the larger family of proteases known as matrix
metalloproteases.
3.2.5 matrix, n—loose meshwork within which cells are embedded or arrangement of connected things. In the context of collagen,
matrix refers to a composite material comprised of an insoluble collagen-fibril network or amorphous nanostructure surrounded
by an interstitial fluid phase.
3.2.6 monomer, n—individual tropocollagen molecule (Fig. 1).
3.2.7 oligomer, n—two or more tropocollagen molecules covalently attached by a naturally occurring intermolecular cross-link.
3.2.8 polymerizable collagen, n—purified type I collagen formulation that demonstrates the capacity to self-assemble or
polymerize into higher order structures (macromolecular assemblies) in absence of exogenous agents such as cross-linkers.
3.2.9 recombinant collagen protein/peptide, n—collagen or collagen-like polypeptide produced by recombinant methods, such as
by expression of a nucleotide sequence encoding the protein or peptide in a microorganism, insect, plant, or animal host. Such
compositions often comprise Gly-X-Y triplets where Gly is the amino acid glycine and X and Y can be the same or different, are
often proline or hydroxyproline, but can be any known amino acid.
3.2.10 resorption, n—removal by gradual breakdown into component materials; a loss of substance by lysis, or by physiologic or
pathologic means.
3.2.10.1 Discussion—
In situations where there is an inflammatory response to implanted materials, immune cells, including neutrophils, macrophages,
lymphocytes, and giant cells, can actively participate in resorption through material phagocytosis and/or proteolysis processes.
3.2.11 solubility, n—a measure of the extent to which a material can be dissolved; in the context of collagen polymers, solubility
refers to collagen molecules (partial, full, or multiples) or peptides in a solution; further qualification of solubility may include
“acid-soluble” and “neutral salt-soluble” which describes compositions that are soluble in dilute acids and neutral salt solutions,
respectively.dissolved.
3.2.11.1 Discussion—
In the context of collagen polymers, solubility refers to collagen molecules (partial, full, or multiples) or peptides in a solution;
further qualification of solubility may include “acid-soluble” and “neutral salt-soluble” which describes compositions that are
soluble in dilute acids and neutral salt solutions, respectively.
4. Significance and Use
4.1 The objective of this document is to provide guidance in the production, characterization, testing, and standardization of: ((a)1
collagen polymers as a starting) polymerizable collagen starting materials; and (2 material ) collagen polymeric materials produced
with polymerizable collagen formulations, used for surgical implants, substrates for tissue-engineered medical products (TEMPs),
TEMPs, vehicles for therapeutic cells and molecules, and 3D in-vitro tissue systems for basic research, drug development, and
toxicity testing; and testing. (b) self-assembled collagen-based materials produced with collagen polymer formulations. This guide
can be used as an aid in the selection, characterization, and standardization of the appropriate polymerizable collagen polymer
starting materialformulations as well as associated self-assembled collagen-based products collagen polymeric materials prepared
F3089 − 23
from polymerizable collagens for a specific use. Not all tests or parameters are applicable to all uses of collagen.collagen and users
are expected to select and justify a subset of the tests for characterization purposes.
4.2 This guide can be used by the following types of users:
4.2.1 Manufacturers of polymerizable collagens and collagen polymeric materials who wish to set specifications for their products
or provide characterization data for customers or users. They may also use the terminology and characterization sections to specify
and differentiate the properties of polymerizable collagens and collagen polymeric materials.
4.2.2 Producers of collagen polymeric materials that use polymerizable collagen as starting materials. Producers may use this
guide to evaluate and characterize multiple sources of polymerizable collagen. They may also use this guide to assist with
evaluation and comparison of single or multiple sources of polymerizable collagen and collagen polymeric materials.
4.2.3 Researchers may use this guide as a reference for properties and test methods that can be used to reproducibly evaluate
polymerizable collagens and collagen polymeric materials.
4.3 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) wound and hemostatic dressings, surgical implants or injectables, injectables (including in-situ forming), hybrid
medical devices, tissue-engineered medical productsTEMPs, injectable (including (TEMPs),in-situ injectableforming) or implant-
able delivery vehicles for therapeutic cells, molecules, and drugs, and 3D in-vitro tissue systems or models for basic research, drug
development, and toxicity testing. The practical application of the collagen polymers and associated self-assembled collagen-based
polymerizable collagens and collagen polymeric materials should be based, among other factors, on biocompatibility,
application-specific performance measures, as well as chemical, physical, and biological test data. Recommendations in this guide
should not be interpreted as a guarantee of success for any specific research or medical application.
4.4 The following general areas should be considered when determining if the collagen supplied satisfies requirements for use in
the above mentioned medical and research applications: source of collagen polymer,polymerizable collagen, impurities profile, and
comprehensive chemical, physical, and biological characterization and testing.
4.5 The following documents or other relevant guidances guidance documents from appropriate regulatory bodies relating to the
production, regulation, and regulatory approval of devices, biologics, drugs, and combination products should be considered when
determining if the collagen supplied satisfies requirements for use in medical and research products, including TEMPs, therapeutic
delivery vehicles, and 3D in-vitro tissue systems:
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 Plannin
...