ISO 20688-1:2020

INTERNATIONAL ISO
STANDARD 20688-1
First edition
2020-02
Biotechnology — Nucleic acid
synthesis —
Part 1:
Requirements for the production
and quality control of synthesized
oligonucleotides
Biotechnologie – Synthèse des acides nucléiques —
Partie 1: Exigences relatives à la production et au contrôle qualité des
oligonucléotides synthétisés
Reference number
©
ISO 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Design and selection of suitable oligonucleotides that are fit for purpose .2
5 General quality management requirements . 2
5.1 General requirements . 2
5.2 Oligonucleotide grading . 3
5.3 Control of documents . 3
5.4 Quality management system . 3
5.5 Personnel and training . 3
5.6 Safety control . 3
6 Resource management . 3
7 Requirements for production process . 4
7.1 General . 4
7.2 Oligonucleotide synthesis . 4
7.3 Purification . 4
7.4 Quality control inspection . 4
7.5 Drying . 5
7.6 Formulation . 5
8 Requirements for quality control process . 5
8.1 General . 5
8.2 Validation and verification of analytical methods . 5
8.3 Identity and purity . 5
8.3.1 Identity of the base sequence . 5
8.3.2 Purity . 5
8.3.3 Impurity . . . 6
8.4 Quantity . 6
8.4.1 General. 6
8.4.2 Concentration . 6
8.4.3 Mass . 7
8.5 Molar mass and/or base length . 7
8.6 Melting temperature (T ) . 8
m
8.7 Report . 8
8.8 V erification of corrective action and improvements . 9
9 Additional requirements for synthetic oligoribonucleotides (RNA) .9
Annex A (informative) Process checklist .10
Annex B (informative) List of equipment and devices and their control standards .12
Annex C (informative) Calculation of molar mass and molar numbers .14
Annex D (informative) Verification of oligonucleotide sequences using mass analysis .17
Annex E (informative) Accuracy control of measurement instrument with certified
reference materials — Examples.23
Annex F (informative) Calculation method of T values .26
m
Bibliography .28
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
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 documents 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).
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iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 276, Biotechnology.
A list of all parts in the ISO 20688 series can be found on the ISO website.
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.
iv © ISO 2020 – All rights reserved

Introduction
Single stranded, linear bio-polymers made up of nucleotides which are called “synthetic oligonucleotides”
or ‘oligos’ are indispensable components for biotechnology. For example, they are used as polymerase
chain reaction (PCR) amplification primers, microarray, real time PCR or next generation sequencing
(NGS) capture probes, and as input starting materials for the creation of entire target genes.
Control of quality in production is important in the synthesis of oligonucleotides. The quantification
of the size range, concentration and contaminants is necessary to ensure that quality requirements
are met for end-use applications. Considering that oligonucleotides are used in biologically active
applications, their quality, particularly sequence and conformation, will affect fitness or function, for
example molecular recognition of cognate binding site, chemical behaviour. The specific requirements
for each end-use application can differ.
This document defines common quality attributes of synthetic oligonucleotides and addresses their
quantification and assessment for end-use.
It is intended to help improve quality management and demonstrate product quality.
International, national or regional regulations or requirements can also apply to specific topics covered
in this document. For example, when synthesized oligonucleotides are used as investigational drugs or
pharmaceutical agents, regional regulations and/or good manufacturing practices (GMP) may need to
be considered.
INTERNATIONAL STANDARD ISO 20688-1:2020(E)
Biotechnology — Nucleic acid synthesis —
Part 1:
Requirements for the production and quality control of
synthesized oligonucleotides
1 Scope
This document specifies minimum requirements for the production and quality control of synthesized
oligonucleotides (nominally up to 250 bases).
This document also describes general quality attributes for synthesized oligonucleotides as well as
common methods for evaluating quality attributes.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
certified reference material
CRM
reference material (3.4) characterized by a metrologically valid procedure for one or more specified
properties, accompanied by an RM certificate that provides the value of the specified property, its
associated uncertainty, and a statement
...