ISO/DTS 20853
(Main)Biotechnology — Bioprocessing — General requirements for bacteriophage preparation for therapeutic use
Biotechnology — Bioprocessing — General requirements for bacteriophage preparation for therapeutic use
This document describes the minimum requirements for bacteriophage preparation processing including the assessment of the titer and quality control. This document applies to data processing of bacteriophage isolation, culture, purification 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
Standards Content (Sample)
FINAL DRAFT
Technical
Specification
ISO/TC 276
Biotechnology — Bioprocessing
Secretariat: DIN
— General requirements for
Voting begins on:
bacteriophage preparation for
2025-10-21
therapeutic use
Voting terminates on:
2025-12-16
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
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TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
Technical
Specification
ISO/TC 276
Biotechnology — Bioprocessing
Secretariat: DIN
— General requirements for
Voting begins on:
bacteriophage preparation for
therapeutic use
Voting terminates on:
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland Reference number
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 detection .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) Examples 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
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. 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).
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) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
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 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 multi-drug resistant bacteria and the lack new antibiotics. Currently, phages are not only
researched as an additional tool for combatting antibiotic resistant infections, but also used directly to
kill multi-drug resistant bacteria. Due to the universal decline in the effectiveness of antibiotics and the
widespread of multidrug-resistant bacteria species, phage therapy is considered as one of the most
[7]
promising alternative strategies against multidrug-resistant (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
FINAL DRAFT Technical Specification ISO/DTS 20853:2025(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 (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 exemple 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
...
ISO/TC 276/ WG 4
Secretariat: DIN
Date: 2025-07-18
Biotechnology — Bioprocessing — General requirements for the
bacteriophage preparation for therapeutic use
DTS stage
Warning for WDs and CDs
This document is not an ISO International Standard. It is distributed for review and comment. It is subject to change
without notice and may not be referred to as an International Standard.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which
they are aware and to provide supporting documentation.
ISO #####-#:####(X/DTS 20853:(en)
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication
may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying,
or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
at the address below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
EmailE-mail: copyright@iso.org
Website: www.iso.orgwww.iso.org
Published in Switzerland
ii © ISO #### 2025 – All rights reserved
ii
Contents
Foreword . vi
Introduction .vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Processing workflow . 2
5 Phage isolation and purification from diverse environments . 5
6 Quality control for phage or phage cocktail . 6
Annex A (informative) Examples for sewage pre-treatment methods . 9
Annex B (informative) Example of a method for obtaining a single bacterial colony . 10
Annex C (informative) Examples for amplification methods . 11
Annex D (informative) Examples of a method for titre determination . 12
Annex E (informative) Example of a method for phage concentration . 13
Annex F (informative) Example of a method for phage screening . 14
Annex G (informative) Example of a method for endotoxin removal . 18
Bibliography . 20
Foreword .vii
Introduction . viii
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 . 5
6.4 Sterility testing . 6
6.5 Bacterial proteins checking . 6
© ISO 2025 – All rights reserved
iii
ISO #####-#:####(X/DTS 20853:(en)
6.6 Residual bacterial DNA detection . 6
6.7 pH detection . 6
Annex A (informative) Exemplary methods for sewage pretreatment . 7
A.1 Sewage pretreatment . 7
A.2 Coprecipitation . 7
A.3 Resuspension . 7
Annex B (informative) Exemplary method for obtaining a single bacterial colony . 8
B.1 Single bacterial colony obtaining . 8
B.2 Bacteria storage in a bacteria bank . 8
B.3 Bacteria sequencing . 8
B.4 Bioinformatic analysis . 8
Annex C (informative) Exemplary amplification methods . 9
C.1 Liquid method . 9
C.2 Solid method . 9
Annex D (informative) Exemplary titer determination method . 10
Annex E (informative) Exemplary method of phage concentration . 11
Annex F (informative) Exemplary method of phage screening . 12
F.1 Phage screening . 12
F.1.1 General . 12
F.1.2 Preparation for logarithmic growth bacteria . 12
F.1.3 Preparation for double-layer agar/agarose plates . 12
F.1.4 Phage screening by spot tests . 12
F.2 High-throughput phage screening . 12
F.2.1 General . 12
F.2.2 Phage arrangement . 13
F.2.3 Preparation for logarithmic growth bacteria . 13
F.2.4 Preparation for double-layer agar/agarose plates . 13
F.2.5 Phage screening from phage collections (or phage biobank) . 13
F.2.5.1 Phage titration . 13
F.3 High-throughput automation phage screening . 13
F.3.1.1 General . 13
F.3.2 Phage arrangement . 14
F.3.3 Preparation for logarithmic growth bacteria . 14
F.3.4 Preparation for agar/agarose plates . 14
F.3.5 Phage automation titration . 14
Annex G (informative) Exemplary Endotoxin removal method . 16
Bibliography . 18
iv © ISO #### 2025 – All rights reserved
iv
© ISO 2025 – All rights reserved
v
ISO #####-#:####(X/DTS 20853:(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
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. 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).
Field Code Changed
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)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents.www.iso.org/patents. ISO shall not be held responsible for identifying any or all such
patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
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.
Field Code Changed
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.htmlwww.iso.org/members.html.
vi © ISO #### 2025 – All rights reserved
vi
Introduction
Phage therapy has been attempted for over a century. It has essentially been re-discovered by the increasing
occurrence of multi-drug resistant bacteria and lackingthe lack new antibiotics. Nowadays, phage isCurrently,
phages are not only mediatized and researched as an additional tool for combatting antibiotic resistant
infections, but also used directly to kill multi-drug resistant bacteria. Due to the universal decline in the
effectiveness of antibiotics and the widespread of multidrug-resistant bacteria species, phage therapy is
considered as one of the most promising alternative strategies against multidrug-resistant (MDR) bacterial
[ ]
infections [7]. [1] .
Phage isPhages 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
[ - ]
conditions, approaches have been developed for phage preparations for clinical practice [8-11]. 0 0 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 Practicesgood 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 authority agency.authorities.
Therefore, we develop this document has been developed to help standardize the normalized and safe phage
preparationspreparation of phages.
© ISO 2025 – All rights reserved
vii
Biotechnology — Bioprocessing — General requirements for the
bacteriophage preparation for therapeutic use
1 Scope
This document describesspecifies the minimum requirements for bacteriophage preparation processing
including assessment on the titertitre 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
Field Code Changed
— IEC Electropedia: available at https://www.electropedia.org/
Field Code Changed
3.1
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 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 3.3
phage cocktail
therapy method mixture of at least two phages to a bacterial infection
© ISO 2025 – All rights reserved
3.4 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
titer
3.5
titre
infectivity titertitre
number of infectious phage particles present per unit volume in a suspension
3.53.6 3.6
triple streaking method
microbiological technique used to isolate individual bacterial colonies on agar plates
3.63.7 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.
2 © ISO #### 2025 – All rights reserved
© ISO 2025 – All rights reserved
Figure 1 — Working flow of phage preparation
4 © ISO #### 2025 – All rights reserved
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 (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. 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)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 using
[ ]
either the direct overlay method or the double-layer agar method [12]. 0 .
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.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 titertitre cannot be reached, large volume of lysates should be concentrated.
NOTE Annex E includes an example of a suitable phage concentration method.
Phage titertitre determination should be performed using the propagating host strain to ensure consistency.
For the phage titertitre determination, the method in Annex DAnnex D should be used.
© ISO 2025 – All rights reserved
5.6 Phage storage
The phage titertitre shall be determined before storage. For storage, the titertitre should be ≥ 10 PFU/ml. For
the titertitre determination, the method in Annex DAnnex 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 15 %
[ , ]
~ 30 %) [13, 14]%) 0 0 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.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
[ ]
Quality control of raw sequencing data should follow the guidelines outlined in ISO 20397-2:2021, 4.3[4],, 0
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
[ ]
Phages used for therapeutics shall be free of virulence genes (e.g., toxins and effector proteins [15]), 0 ), drug
[ ] [ ]
resistance genes (e.g., β-lactamases [16]), 0 ), and lysogenic genes (e.g., integrase [17]) 0 ) 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 theapplicable regulatory requirements abouton the maximum allowed concentration of endotoxin
for the applicable mode of administration. An exemplaryexemple method of endotoxin removal is given in
Annex G.Annex G.
6 © ISO #### 2025 – All rights reserved
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 ISO 10993-
[ , , ]
11:2017[1, 5, 6]. 0 0 0 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
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