ISO/TS 20853:2026

Biotechnology — Bioprocessing — General requirements for bacteriophage preparation for therapeutic use

This document specifies the minimum requirements for bacteriophage preparation processing including assessment on the titre and quality control. The document applies to data processing of bacteriophage isolation, culture, characterization and storage. This document applies to the quality evaluation/assessment of bacteriophage used for therapy.

Biotechnologie — Bioprocédés — Exigences générales pour la préparation de bactériophages à usage thérapeutique

General Information

• Status: Published
• Publication Date: 04-Feb-2026
• ICS: 07.100.10 - Medical microbiology
• Technical Committee: ISO/TC 276 - Biotechnology
• Drafting Committee: ISO/TC 276 - Biotechnology
• Current Stage: 6060 - International Standard published
• Start Date: 05-Feb-2026
• Due Date: 05-Oct-2026
• Completion Date: 05-Feb-2026

Overview

ISO/TS 20853:2026 - Biotechnology - Bioprocessing - General requirements for bacteriophage preparation for therapeutic use establishes a standardized framework for the preparation of bacteriophage products intended for human therapeutic applications. Developed by the International Organization for Standardization (ISO), this technical specification defines the minimum requirements for the processes of phage isolation, culture, characterization, quality control, and storage. With the growing interest in phage therapy as an alternative or complement to antibiotics-especially in the fight against multidrug-resistant (MDR) bacteria-this standard supports safe, effective, and high-quality phage product development for clinical practice.

Key Topics

• Bacteriophage Isolation and Enrichment
  Outlines procedures for extracting phages from diverse environmental and clinical sources, focusing on environments rich in the target host bacteria, such as hospital samples, soils, or sewage.

• Phage Characterization and Selection
  Details steps for identification, genome sequencing, and bioinformatic analysis to ensure absence of undesirable genes (e.g., virulence, drug resistance, lysogeny) in final therapeutic phage preparations.

• Phage Amplification and Storage
  Defines requirements for phage amplification methods and titre determination, as well as best practices for short- and long-term storage, maintaining high viability and traceability of phage stocks.

• Quality Control Procedures
  Specifies critical quality markers, including:

  • Removal of harmful bacterial toxins (endotoxins, exotoxins)
  • PFU (Plaque-Forming Unit) titration and concentration standards
  • Endotoxin quantification using validated methods
  • Sterility, absence of bacterial proteins and DNA contaminants
  • pH verification for clinical safety and product stability

• Documentation and Traceability
  Emphasizes comprehensive record keeping at every stage, from sampling to storage, to ensure reproducibility, transparency, and regulatory compliance.

Applications

ISO/TS 20853:2026 is vital for developers, manufacturers, and researchers working in biopharmaceuticals, biotechnology, and clinical microbiology focused on phage-based therapies. Applications include:

• Therapeutic Phage Product Manufacturing
  Ensures robust quality management systems for large-scale production of clinical-grade phages, supporting regulatory approvals and consistent therapeutic efficacy.

• Clinical Trials and Hospital Use
  Standardizes the preparation and quality assessment of phage preparations deployed in clinical studies or individualized therapies targeting antibiotic-resistant infections.

• Research and Development
  Provides a baseline for academic and industrial research dedicated to phage discovery, characterization, and optimization for medical use.

• Phage Banks and Biorepositories
  Guides the establishment and maintenance of reliable phage collections by defining stringent preservation and documentation practices.

By following this standard, organizations can improve the reproducibility and global acceptance of their bacteriophage products, supporting advances in precision bio-therapeutics and the growing field of phage therapy.

Related Standards

For comprehensive bacteriophage preparation and quality assessment, ISO/TS 20853:2026 refers to or aligns with several other key international standards, including:

• ISO 29701:2010 – Nanotechnologies - Endotoxin test on nanomaterial samples for in vitro systems
• ISO 10705-3:2003 – Water quality - Detection and enumeration of bacteriophages
• ISO 21702:2019 – Measurement of antiviral activity on plastics and other non-porous surfaces
• ISO 11737-3:2023 – Sterilization of medical devices - Tests of sterility
• ISO 10993-11:2017 – Biological evaluation of medical devices - Tests for systemic toxicity
• ISO 20397-2:2021 – Biotechnology - Data processing and bioinformatics for genome sequencing
• USP <71> / EP 2.6.1 – Sterility tests for pharmaceuticals

Adherence to ISO/TS 20853:2026 and these related standards ensures a globally harmonized approach to the safe and effective development of bacteriophage therapies, supporting quality, safety, and innovation in biotechnology and bioprocessing.

Standards Content (Sample)

Technical
Specification
ISO/TS 20853
First edition
Biotechnology — Bioprocessing
2026-02
— General requirements for
bacteriophage preparation for
therapeutic use
Biotechnologie — Bioprocédés — Exigences générales pour la
préparation de bactériophages à usage thérapeutique
Reference number
© ISO 2026
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or ISO’s member body in the country of the requester.
ISO copyright office
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Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Processing workflow . 2
5 Phage isolation and purification from diverse environments . 4
5.1 Preliminary preparations .4
5.2 Phage enrichment .4
5.3 Phage isolation .4
5.4 Phage amplification .4
5.5 Phage suspension titration .4
5.6 Phage storage . .4
5.7 Genome sequencing .5
5.8 Bioinformatic analysis .5
5.9 Phage selection .5
6 Quality control for phage or phage cocktail . 5
6.1 Removal of harmful bacterial toxins .5
6.1.1 Endotoxin removal .5
6.1.2 Exotoxin removal .5
6.2 PFU titration .5
6.3 Endotoxin quantification.6
6.4 Sterility testing .6
6.5 Bacterial proteins checking .6
6.6 Residual bacterial DNA detection .6
6.7 pH determination .6
Annex A (informative) Examples for sewage pre-treatment methods . 8
Annex B (informative) Example of a method for obtaining a single bacterial colony . 9
Annex C (informative) Examples for amplification methods .10
Annex D (informative) Example of a method for titre determination .11
Annex E (informative) Example of a method for phage concentration .12
Annex F (informative) Example of a method for phage screening .13
Annex G (informative) Example of a method for endotoxin removal .16
Bibliography . 17

iii
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
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The procedures used to develop this document and those intended for its further maintenance are described
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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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
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This document was prepared by Technical Committee ISO/TC 276, Biotechnology.
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
Introduction
Phage therapy has been attempted for over a century. It has essentially been re-discovered by the increasing
occurrence of multidrug-resistant (MDR) bacteria and the lack of new antibiotics. Currently, phages are not
only researched as an additional tool for combatting antibiotic resistant infections, but also used directly to
kill MDR bacteria. Due to the universal decline in the effectiveness of antibiotics and the widespread of MDR
bacteria species, phage therapy is considered as one of the most promising alternative strategies against
[7]
MDR bacterial infections .
Phages are generally applied in liquid form and stored preferably refrigerated. Oral, topical, intravenous,
nebulized, and several individualized phage therapies have already been utilized successfully to treat multi-
drug-resistant bacteria clinically. Phage cocktails (phage mixtures) are generally prepared to improve
the chances of successful treatment due to the host specific of phage and complicated infections of multi-
drug-resistant bacteria. Phage or phage cocktails with high therapeutic applicability depend on the type
of bacterial infections, for example, patients with respiratory infections would give priority to considering
nebulized phage therapy.
With the continuous and expanded utilization of phage therapy during clinical trials of life-threatening
[8-11]
conditions, approaches have been developed for phage preparations for clinical practice. Besides, they
have shown several pitfalls, including ineffective phage, inadequate endotoxin removal, high bacterial gross
protein impurities, and the addition of toxic chemicals, etc. Therefore, the demand for high-quality and
clinically safe phage preparations is increasingly required.
Standardization and large-scale good manufacturing practices (GMP) production of phage preparations will
likely meet the growing demands for phage therapy. In addition, this will further promote drug approval
of phage preparations in medicine by authorities. Therefore, this document has been developed to help
standardize the safe preparation of phages.

v
Technical Specification ISO/TS 20853:2026(en)
Biotechnology — Bioprocessing — General requirements for
bacteriophage preparation for therapeutic use
1 Scope
This document specifies the minimum requirements for bacteriophage preparation processing including
assessment on the titre and quality control.
The document applies to data processing of bacteriophage isolation, culture, characterization and storage.
This document applies to the quality evaluation/assessment of bacteriophage used for therapy.
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
endotoxin unit
EU
standard unit of endotoxin activity
Note 1 to entry: The endotoxin unit was defined by the World Health Organization (WHO) Expert Committee on
Biological Standardization (ECBS) in 1996, relative to the activity of 0,1 ng of WHO reference standard endotoxin
(RSE) from Escherichia coli 0113:HK10:K(-) or 10 EU/ng.
Note 2 to entry: EU is equal to international unit (IU) of endotoxin.
[SOURCE: ISO 29701:2010, 2.4]
3.2
phages
bacteriophages
bacterial viruses that are capable of infecting bacterial strains
Note 1 to entry: Bacteriophages produce visible plaques (clearance zones) in a confluent lawn of the host strain grown
under appropriate culture conditions.
[SOURCE: ISO 10705-3:2003, 3.1, modified – ‘phages’ added as a preferred term.]
3.3
phage cocktail
therapy method mixture of at least two phages to a bacterial infection

3.4
plaque-forming unit
PFU
unit expressed as the concentration of the infectious viral particles per unit volume (ml)
[SOURCE: ISO 21702:2019, 3.7]
3.5
titre
infectivity titre
number of infectious phage particles present per unit volume in a suspension
3.6
triple streaking method
microbiological technique used to isolate individual bacterial colonies on agar plates
3.7
spot test
method to determine whether a phage can infect a bacterium by placing a small drop or ‘spot’ of phage
suspension onto a plate inoculated with the bacterium
4 Processing workflow
Phages can be isolated from multiple environments. If phages are isolated successfully, phage purification,
suspension titration, storage, and phage concentration, if necessary, shall be conducted afterwards. Whole
genome sequencing and bioinformatic analysis shall be performed. Then, quality control of phages or the
phage cocktail should be carried out as shown in Figure 1.

Figure 1 — Working flow of phage preparation

5 Phage isolation and purification from diverse environments
5.1 Preliminary preparations
Phage particles can be isolated from diverse environments, including but not limited to bacterial samples
of patients from hospitals and a variety of natural environments where bacteria exist, such as oceans, soil,
and sewage (e.g. living sewage, hospital sewage, farm sewage). The selected isolation source should be an
environment where the target host bacteria is commonly abundant and free from substances that can cause
bacterial death or inhibit growth, such as antibiotics or disinfectants. The location and date of sampling
shall be documented.
A method for sewage pre-treatment should be selected according to where the particle originates from, see
Annex A.
The bacterium selected as the host for isolating phages shall be streaked on a solid medium, and a single
colony (see Annex B) shall be used to inoculate a liquid broth for overnight culture. The logarithmic phase
bacteria should be prepared afterwards.
5.2 Phage enrichment
Phage enrichment involves co-incubating an exponentially growing bacterial host with the pre-treated
environmental sample. For volume-volume co-incubation, a 2X culture medium, i.e., a culture medium
containing twice the usual concentration of nutrients, needs to be prepared. The selection of culture medium
should align with the specific bacterial host requirements and can include specific supplements (e.g., CaCl
and MgSO ). Co-incubation usually takes place overnight, but the duration (shorter or longer) should be
adjusted according to the bacterial host.
The co-incubation mixture shall be centrifuged, and the supernatant shall be filtered and stored at 4 °C.
The enrichment should include a negative control group with sterile water instead of the environmental
sample.
5.3 Phage isolation
The isolation of phages relies on their capacity to form plaques on a solid medium. This can be achieved
[12]
using either the direct overlay method or the double-layer agar method .
The isolation step shall be repeated 4 times to 5 times until the morphology of all plaques (plaque size,
transparency, halo) is consistent.
5.4 Phage amplification
The amplification shall use a liquid method or solid method, see Annex C.
5.5 Phage suspension titration
The amplification of phages should ideally yield a suspension with a concentration of ≥ 10 PFU/ml. When
this titre cannot be reached, large volume of lysates should be concentrated.
NOTE Annex E includes an example of a suitable phage concentration method.
Phage titre determination should be performed using the propagating host strain to ensure consistency. For
the phage titre determination, the method in Annex D should be used.
5.6 Phage storage
The phage titre shall be determined before storage. For storage, the titre should be ≥ 10 PFU/ml. For the
titre determination, the method in Annex D should be used.

Phage preservation:
a) Each phage should be stored as a triplicate in a 4 °C refrigerator as a working solution. The viability of
refrigerated phages should be checked periodically (e.g., weekly, monthly, quarterly, or annually).
b) Each phage should be stored as a triplicate mixed with glycerol (the final concentration of glycerol is
[13,14]
15 % ~ 30 %) or dimethyl sulfoxide (DMSO) in a -80 °C freezer as a preservation solution. Both
working and preservation solution tubes shall be marked with the name, preservation date, the name
of the depositor, and other essential and valuable information. The working solution, also known as a
working phage stock (WPS), shall be used first for experiments, e.g., a spot test to check whether a
phage can infect a bacterium offered, see Annex F. The preservation solution, usually referred to as a
master phage stock (MPS), should be stored as part of the phage bank for long-term preservation if
necessary.
5.7 Genome sequencing
Genomes of phage lysates should be extracted following standard protocols or utilizing commercial kits. It
is advised to pre-treat phage lysates with DNAse and RNAse (e.g., DNase I and RNase A) in order to digest
nucleic acids from the bacterial host.
Phage genome sequencing, ideally through next-generation sequencing (NGS), shall be performed.
5.8 Bioinformatic analysis
[4]
Quality control of raw sequencing data should follow the guidelines outlined in ISO 20397-2:2021, 4.3,
using tools such as SOAPnuke and Fastp for filtering low-quality sequences. Genome assembly should utilize
SPAdes to generate a single, complete contig. Annotation should employ phage-specific tools and curated
databases, such as UniProt and Swiss-Prot. The use of Prodigal for open reading frame (ORF) prediction
is recommended, followed by BLASTp and hmmscan searches against curated phage protein libraries. Key
annotation focus areas include identifying potential virulence factors, antibiotic resistance genes, and genes
associated with lysogeny, which should be flagged for further therapeutic consideration.
5.9 Phage selection
[15]
Phages used for therapeutics shall be free of virulence genes (e.g., toxins and effector proteins ), drug
[16] [17]
resistance genes (e.g., β-lactamases ), and lysogenic genes (e.g., integrase ) in the genome.
6 Quality control for phage or phage cocktail
6.1 Removal of harmful bacterial toxins
6.1.1 Endotoxin removal
Endotoxin removal is for Gram-negative bacteria. Endotoxin levels shall be reduced to a level that corresponds
at least to applicable regulatory requirements on the maximum allowed concentration of endotoxin for the
applicable mode of administration. An example method of endotoxin removal is given in Annex G.
6.1.2 Exotoxin removal
Exotoxins from Gram-positive bacteria should be removed from phage preparations. Methods such as
ultrafiltration, dialysis, and affinity chromatography should be considered for this purpose.
6.2 PFU titration
The therapeutic dose of phage or phage cocktail should be ≥10 PFU/ml.

6.3 Endotoxin quantification
Trace amounts of endotoxin can impede the use of phages for therapeutic purposes. To detect the
endotoxin concentration, appropriate methods shall be applied according to their application range.
Guidance on methods and their application range can be found in ISO 29701:2010, ISO 11737-3:2023, and
[1,5,6]
ISO 10993-11:2017. These methods include:
a) Limulus Amebocyte Lysate (LAL) Assay: typical respective limits are 0,5 EU/ml reagent;
b) Recombinant Factor C (rFC) Assay: An alternative to the LAL assay, using recombinant Factor C to detect
endotoxins, reducing reliance on animal-derived materials and providing a more sustainable option for
endotoxin quantification, range of 0,005 EU/ml – 5,0 EU/ml;
c) Gel Clot Assay: normally between 0,01 EU/ml and 0,1 EU/ml;
d) Chromogenic Method: usually between 0,01 EU/ml and 0,1 EU/ml;
e) Turbidimetric Method: usually between 0,01 EU/ml and 0,1 EU/ml;
f) Fluorometric Method: typically ≤ 0,001 EU/ml;
g) Bioassay: inflammation tests in rabbits or mice: typical respective limits are 5 EU/kg rabbit body mass
[18,19]
administered in one hour .
NOTE Regulatory requirements regarding the maximum allowed concentration of endotoxins can apply and can
vary depending on the type of application (e.g., intravenous injection or ingestion).
6.4 Sterility testing
All phage preparations intended for therapeutic use should undergo sterility testing to confirm the absence
of bacterial and fungal contaminants. Testing should
...