ISO/FDIS 6631

ISO/DISFDIS 6631:2025(en)
ISO/TC 150/SC 7
Secretariat: JISC
Date: 2025-07-28
Tissue-engineered medical products — Quantification of bovine type
I collagen marker peptide with liquid chromatography — Tandem
mass spectrometry
Date: 2024-05-28
FDIS stage
ISO/DISFDIS 6631:2025(en)
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iii
Contents
Foreword . v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principles and significances . 2
4.1 Principles . 2
4.2 Significances . 2
5 Reagent and apparatus . 3
5.1 Reagents and preparation . 3
5.2 Apparatuses . 4
6 Experimental procedures . 5
6.1 Sample preparation . 5
6.2 Denaturation treatment . 5
6.3 Trypsin digestion . 5
6.4 LC-MS/MS detection . 6
7 Data collection and calculation . 8
7.1 Standard curve . 8
7.2 Result calculation . 8
8 Acceptable criteria of experimental result . 10
9 Test report . 10
Bibliography . 11

© ISO #### 2025 – All rights reserved
iv
ISO/DISFDIS 6631:2025(en)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has been
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The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types of
ISO documentsdocument should be noted. This document was drafted in accordance with the editorial rules
of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
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represent the latest information, which may be obtained from the patent database available at
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For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 150, Implants for surgery, Subcommittee SC 7,
Tissue-engineered medical products.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
Introduction
Collagen is a primary biopolymer in the extracellular matrix of mammals, and provides a scaffold for cell
attachment and migration, as well as specific mechanical properties. It is prevalent in tendons, skin, blood
vessel, cornea and coordinates with minerals to construct bones, teeth and cartilage. The collagen molecule is
formed by three chains building a triple helix. According to their amino sequences and structures, collagen
can be categorized into different types. So far, at least 28 different types of collagen have been identified in
vertebrates. The different collagen types are characterized by considerable complexity and diversity in their
structure, their splice variants, and the presence of additional non-helical domains, their assembly and their
function. The most abundant and widespread family of collagens with about 90 % of the total collagen is
[ , ]
represented by the fibril-forming collagens, among which type I is the most common fibrillar collagen. 0 0. .
(4,5)
The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and
weak antigenicity, made collagen as one of the most useful biomaterials. It was widely used as raw materials
for constructing tissue-engineered medical products (TEMPs) for tissue regeneration, reconstruction or
replacement, such as skin graft, bone substitutes, and artificial blood vessels and valves, etc. Collagen based
wound dressing include hydrogel, haemostatic sponges/powder, membranes and film. The main applications
of collagen as drug delivery systems are collagen shields in ophthalmology, sponges for burns/wounds, mini-
pellets and tablets for protein delivery, gel formulation in combination with liposomes for sustained drug
delivery, as controlling material for transdermal delivery, and nanoparticles for gene delivery and basic
matrices for cell culture systems. To improve collagen physical and chemical properties, chemical
[ ]
modification, crosslinking, or hybridizing with synthetic polymer are also used. 0 . Collagen-based
(6)
biomaterials are expected to become a widely used matrix substance for various biomedical applications in
the future. Therefore, quality control of collagens is of great importance from safety, regulatory, and process
control perspectives. The specific peptide content of collagen products, is important parameter that related to
collagen purity, identification (whether is bovine specific or/and type I collagen-specific),) and is useful for
quality control of collagen.
Among all the collagen types, type I collagen is the most common used in the fields of TEMPs and regenerative
medicine. Type I collagen is normally a heterotrimeric molecule, composed of two α1 (I) chains and one α2 (I)
chain. Most collagens with native states are insoluble in water. Collagen extracted from different animal
sources shows similarity in structure and properties. Thus, it is difficult to qualify a particular type or animal
source (species) of collagen. To date, several ways for quantifying collagen and/or collagen types or even
particular collagen chains have been reported, such as the quantification of hydroxyproline, which accounts
for approximately 10 % of the collagen molecule, or a method based on a spectrophotometric assay of collagen
stained by a strong anionic dye in picric acid solution. However, since hydroxyproline is a common amino acid
in all types of collagen, the hydroxyproline quantification method shows low specificity. Other methods
include the radioactive labelling of proline and enzyme immunoassays using antibodies specific to each
collagen type, which are very expensive and require many complicated steps, or not fit for some formulations
of collagen-containing biomaterials. Any type of collagen has its particular amino acid sequences in their
chains, which are different from other types from the same animal. Same type collagen from different species
of animals also shows different sequences in their chains. Thus, the sequential differences can be used for
characterization of its type or animal source. Enzymatic digestion of collagen coupled with various separation
methods such as liquid chromatography (LC), will be able to achieve some marker reference peptides, which
is particular for a specific-type and/or specific-animal sources collagen. Marker peptides can be used for
[ ]
qualitative and quantitative analysis of the original collagen with mass spectrometry. 0 .
(7)
Qualitative identification and quantification of type I collagen using mass spectrometry has been applied in
many fields, including quantification of collagen in some tissues, identification of animal source of gelatins, or
[ - ]
metabolic kinetic analysis in vivo of collagen-containing medicine. 0 0 . The method for determination of
(8-10)
donkey collagen type I for product quality control can be found in the Pharmacopoeia of People's Republic of
China, identification method of Ajiao, donkey skin-based gelatin, traditional Chinese medicine (Volume one,
[ ]
2020 edition). 0) . The mass spectrometry method also has been widely used in detection of ageing-related
(1)
changes of collagen in tissues, investigation of collagen disorder or degradation for some diseases.
© ISO #### 2025 – All rights reserved
vi
ISO/DISFDIS 6631:2025(en)
Currently, there is no international standard that focus on the marker peptide quantification of type I collagen
from bovine. The mass spectrometry method was validated for both native collagen and degraded collagen in
[ - , - ]
several year application. 0 0 0 0 . A standardized method for marker peptide quantification of bovine
(7-10, 11-14)
type I collagen has been developed and some methodology validation study of this method has been
[ ] [ ]
published, 0 , and this method has been issued in Chinese industry standard. 0 . To develop an
(15) (2)
international standard for quantification of marker peptide of bovine type I collagen, the principle,
requirements and standard operating procedures for determination is provided in this document.
vii
DRAFT International Standard ISO/DIS 6631:2025(en)

Tissue-engineered medical products - — Quantification of bovine type
I collagen marker peptide with liquid chromatography - tandem—
Tandem mass spectrometry
1 Scope
This document provides a method for quantification of marker peptide of type I collagen which was purified
products extracted from bovine tissues with liquid chromatography - tandem mass spectrometry (LC-MS/MS).
The method described in this document is intended to be used for marker peptide detection of purified bovine
type I collagen which will be used for constructing tissue-engineered medical products (TEMPs) or other
collagen-based biomaterials, for product quality control.
This method also can be used for qualitative analysis and quantitative detection of bovine-specific and/or type
I-specific collagen in the samples mixed with other animal sources and or other type collagen.
This document does not exclude other possible methods for quantifying type I collagen, such as
hydroxyproline quantification, that can evaluate the total amount of collagen regardless of type.
NOTE 1 The collagen has been known there are greater than 28 types and with the dif
...