ISO/FDIS 16921-2

ISO/DISFDIS 16921-2
ISO/TC 276/SC 1
Secretariat: ANSI
Date: 2025-09-25
Biotechnology — Gene delivery systems —
Part 2:
Quantification methods for viral vectors
DISFDIS stage
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without notice and may not be referred to as an International Standard.
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ISO/DISFDIS 16921-2:20242025(en)
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ISO/DISFDIS 16921-2:20242025(en)
Contents
Foreword . vi
Introduction .vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Concepts for viral vector titer . 1
4.2 Physical titer measurements . 6
4.3 Activity assays . 10
5 Considerations for viral vector quantification . 13
5.1 Selection of fit for purpose attribute . 13
5.2 Consideration for selection of a fit for purpose assay . 14
5.3 Sampling of viral vectors for titer . 14
5.4 Preparation of samples for titer determination . 15
5.5 Considerations for cell-based assays. 16
5.6 Performing a measurement . 17
6 Qualification, validation, and verification . 17
6.1 Instrument qualification . 17
6.2 Method qualification . 17
6.3 Method validation and verification . 18
6.4 Reference materials . 21
7 Data processing, analysis, and reporting . 22
7.1 Data processing and analysis . 22
7.2 Reporting . 22
Annex A (informative) Quantification methods of viral vector titer . 24
Annex B (informative) General methods for the quantification of viral vector titer . 26
Annex C (informative) Example workflow of lentivirus (LV) transducing titer determination
method . 32
Bibliography . 35
Foreword . vi
Introduction .vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Concepts for viral vector titer . 1
5 Considerations for viral vector quantification . 13
6 Qualification, validation, and verification . 17
7 Data processing, analysis, and reporting . 22
Annex A (informative) Quantification methods of viral vector titer . 24
Annex B (informative) General methods for the quantification of viral vector titer . 26
Annex C (informative) Example workflow of lentivirus (LV) transducing titer determination
method . 32
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ISO/DISFDIS 16921-2:20242025(en)
Bibliography . 35

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ISO/DISFDIS 16921-2:20242025(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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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 document 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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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, Subcommittee SC 1,
Analytical methods.
A list of all parts in the ISO 16921 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.
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ISO/DISFDIS 16921-2:20242025(en)
Introduction
Modern and emerging biotechnology is underpinned byrelies on the ability to manipulate the genes and
genomes ofin living systems. Gene delivery systems are foundational to genome engineering. Gene delivery
technology is evolving rapidly with numerous types of gene delivery systems providing a comprehensive set
of tools and capabilities for in vitro or in vivo targeted delivery.
The ISO 16921 series consists of multiple parts to provide common understanding, guides, analytical methods
and, data reporting for characterizing these emerging biotechnology tools. ISO 16921-1 of the series specifies
Vocabulary related to gene delivery systems. This document (ISO 16921-2) focuses on quantification of one
type of gene delivery system, viral vectors.
Viral vectors are engineered viruses for delivering the desired genetic payload into target cells. Viral vectors
are powerful molecular biology tools and have been increasingly used in broad biotechnology applications
and products. Various types of viral vectors are used as advanced gene therapies, as vaccines, and as critical
reagents for cellular therapies. They have also been increasingly used in genome editing applications. Viral
vector titer (titre) is central to all applications. Robust measurements for the quantification and reporting of
viral vector titer are important for the industry. This document provides general guidance for viral vector titer
as well as aspects of functional analysis including method selection, sample preparation, measurement,
qualification and validation, data analysis and reporting.
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ISO/DISFDIS 16921-2:20242025(en)
Biotechnology — Gene delivery systems —
Part 2:
Quantification methods for viral vectors
1 Scope
This document specifies minimum requirements for quantifying viral vectors in terms of physical titer and
their associated activity. This document specifies key considerations for quantification methods for viral
vector titer as well as activity, including method selection, measurement process, data analysis, and reporting.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 16921-1, Biotechnology — Gene Delivery Systems — Part 1: Vocabulary
ISO 20395, Biotechnology — Requirements for evaluating the performance of quantification methods for nucleic
acid target sequences — qPCR and dPCR
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16921-1 and the following 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/obphttps://www.iso.org/obp
— — IEC Electropedia: available at https://www.electropedia.org/https://www.electropedia.org/
3.1
direct particle counting
counting method in which one signal is (or several signals are) detected for each single event
Note 1 to entry: Each single event represents a single viral particle in an idealized measurement
3.2
indirect particle counting
counting method during which a signal (or a set of signals) is measured from a population of viral particles
and that signal is then related to viral titer based on a measurement-specific mathematical model (e.g.
calibration curve)
4 Concepts for viral vector titer
4.1 General viral vector concepts

Viral vectors are genetically engineered viruses designed to deliver nucleic acid into a cell. As such, viral
vectors are almost always replication incompetent, so that no new viral vectors can be produced within the
target cell.
ISO/DISFDIS 16921-2:20242025(en)
NOTE 1 Viral vectors can be made to be replication competent in the presence of a helper virus or complementary
replicative genes in a cell.
NOTE 2 Replication competent viral vectors are generally not used in therapeuticaltherapeutic applications due to
safety concerns.
A functional viral vector generally consists of three components:
— the protein capsid with or without the envelope that encapsulates the payload, and defines the vector’s
tissue or cell tropism and antigen recognition;
— the gene of interest, which when expressed in cells, serves to confer a desired functional outcome; and
— the regulatory components including the combined enhancer, promoter, and auxiliary elements that
control stable or transient somatic expression of the transgene as an episome or as a chromosomal
integrant.
NOTE 3 Some viral vectors designs do not precisely follow the transgene and regulatory cassette descriptions stated
above. Examples include those utilizing micro RNAs, guide RNAs, multiple transgenes/promoters, and dual vectors
(which utilize in vivo splicing).
The process to engineer and manufacture viral vectors is complex and evolving. Current technologies
generally produce a mixture of functional viral vectors that can deliver the appropriate payload into the target
cells as well as non-functional viral vectors and free capsid proteins (Figure 1).0). Other potential impurities
in the viral vector mixture include free DNA from the host cell or residual plasmid components, all of which
may be encapsulated as well.
Key
1 functional particles
2 nonfunctional particles (empty and immature particles)
3 free capsid proteins
ISO/DISFDIS 16921-2:20242025(en)
Figure 1 — Products from viral vector production that include functional particles, nonfunctional
particles, and free capsid protein. Adapted from “Understanding viral titration- behind the
[1] ]
science” .0 .
Non-functional viral vector particles do not have all the necessary components to complete the transduction
process, express the intended protein, and result in the functional outcome. Non-functional viral vector
particles include various types of defects, including for example:
— vectors with defective capsids that maycan affect its tissue or cell tropism and antigen recognition,
— vectors that contain missing or defective payloads,
— vectors that contain missing or defective regulatory components.
Viral vectors are characterized according to critical quality attributes, such as quantity, potency, safety, and
identity. This document focuses on quantity. General methods for a) quantification of physical titer of viral
vectors and b) functional characterization of viral vectors using cell-based measurements are shown in figure
2.0.
ISO/DISFDIS 16921-2:20242025(en)

ISO/DISFDIS 16921-2:20242025(en)
a
Additional potency assays for titer quantification are often specific to the product and mechanism of action and is beyond the
scope of this document.
Key
a
Additional potency assays for titer quantification are often specific to the product and mechanism of action
and is beyond the scope of this document
Figure 2 — Analytical methods for quantification of viral vectors based on physical methods or their
interactions with cells. This figure of quantification methods is not exhaustive and can be further
developed.
Physical titer of viral vectors refers to the particle count, which is determined by direct particle counting or
indirect particle counting methods.
Direct particle counting involves the recording of a signal or a set of signals from individual viral particles
(3.1Error! Reference source not found.). Direct particle counting requires well-dispersed particles for
optimal performance. The presence of debris and aggregated or agglomerated particle maycan lead to over-
or underestimated virus titer. Whenever possible, a process should be established to prepare well- dispersed
samples with minimized debris, aggregate, and agglomerate content.
Indirect particle counting (3.2)Error! Reference source not found.) methods use a surrogate measure to
evaluate the count. The accuracy of these methods depends on the accuracy of the measurement and the
accuracy of a calibration curve, if a calibration curve is needed. Indirect particle counting methods include:
— the use of a calibration to estimate a count (e.g. fluorescence, qPCR, ELISA, or another method to back
calculate count using a calibration curve);
— the measuring a component of the particle to infer the count (e.g. quantifying the total capsid protein
concentration to estimate particle count).
Activity methods are used to measure the interaction between viral vector particles and target cells. Activity
measures determine the concentration of functional particles in a viral vector preparation. Activity
measurements can be further divided into transduction and other potency measures to gain insight into
general transduction activity and product specific potency.
Transduction assays measure viral vector transgene entry into target cells for non-replication competent viral
systems. Vector copy number per cell or transduction efficiency can be measured in the target cells. A dilution
series of virus to cells is generally used to specify at which dilutions the multiplicity of infection to transduction
efficiency is linear. The transduction units in the original virus preparation can be determined from the
specified range of the dilution series.
Infectivity assays may include a viral vector, a helper virus, and a complementary cell line that enables the
viral vector to replicate. Viral replication can result in target cells undergoing cytopathic effect. Replication
and cytopathic effect can be visualized and quantified as plaques or foci in the cells. Viral vector replication in
target cells enables quantification of virus activity and infectivity assay titer.
Potency assays measure transgene expression and transgene activity. Transduction assays can be considered
one part of potency measures which can measure transgene insertion, integration, and expression. Additional
potency measures beyond transduction and transgene expression are often production specific measures of
transgene mechanism of action. Due to the product specific nature of such assays, use of potency assays for
viral vector quantification are beyond the scope of this document.
See Table A.10 for attributes, biological properties, analytical methods, measurement principles, and units for
the quantification of viral vector titer.
ISO/DISFDIS 16921-2:20242025(en)
4.2 Physical titer measurements
4.2.1 General physical titer concepts
Physical titer refers to the total concentration of viral vectors or the concentration of a predefined, subset of
viral vectors.
Physical titer of viral vectors shall be expressed as amount per volume (1/mL).
Physical titer can be determined via several analytical methods, including direct particle counting methods
and indirect particle counting methods.
An appropriate measurement shall be selected to determine the total quantity or the quantity of a predefined,
subset of viral vectors.
Assessment of the quality of the viral vectors may involve the use of two or more titers, such as filled to empty
ratio.
See Annex B.1.1See Annex B.2.1 for additional physical titer methods.
4.2.2 Direct viral vector titer measurement
Direct viral vector titer methods identify and enumerate single particles. Direct viral vector titer can be used
for total viral vector enumeration or enumeration of particles of a specific predefined attribute.
Direct viral vector titer measurements can include electron microscopy-based counting, flow virometry,
resistive pulse sensing, nanoparticle tracking analysis, and other methods capable of capturing single entities
(i.e., viral vector particles).
Results of direct particle counting methods shall be reported in terms of particles/mL or specified units of
volume.
Due to sample heterogeneity, direct viral vector counting methods maycan inadvertently count impurities,
such as extracellular vesicles, as viral vectors. Counting methods targeting only the viral vectors within a
sample may therefore be more appropriate. Combining methods specific for impurities, such as flow
cytometry that targets extracellular vesicles, can also be used to correct direct particle count measurements
to be more accurate for viral particles.
4.2.3 Indirect viral vector titer measurement
4.2.3.1 General indirect viral vector titer concepts
Indirect viral vector titer measurements include all methods to extrapolate the viral vector quantity not
associated with enumeration.
Indirect methods generally involve the use of a calibration curve to determine quantity. These include
methods that quantify a defined attribute of the viral vector, such as capsid protein quantification and genome
quantification methods.
4.2.3.2 Capsid titer quantification
Capsid titer or capsid concentration is a measurement of protein quantification used to characterize viral
vectors. An example of a method used to determine capsid titer is the enzyme-linked immunosorbent assay
(ELISA). Other example methods for capsid titer can be found in Table A.1.0. ELISA capsid protein
measurements reported as virus particles/mL is ambiguous to what the actual measurand is. Capsid titer shall
be reported as capsids/mL. The specific capsid protein (e.g. p24 for lentivirus) being targeted shall be included
ISO/DISFDIS 16921-2:20242025(en)
in the measurement reporting. The method of determining the capsids/mL and associated calculations shall
be documented. A standard curve shall be included for capsid titer determination using a well characterized
and known concentration of the target protein.
NOTE 1 Capsid protein measurements measure free soluble capsid protein and encapsulated capsid protein as a
quality measure of a viral vector preparation.
NOTE 2 ELISA measurements report absorbance values for the samples being measured. Depending on the viral
vector, the absorbance values can be converted to mass and then to capsids/mL, or directly from absorbance values to
capsids/mL.
The calculations to perform these conversions are based on literature assumptions and should be considered
when evaluating the accuracy for capsid quantification. For example, p24 capsid quantification by ELISA relies
[2][ ]
on the assumption that there are approximately 2000 2 000 molecules of p24 per lentivirus particle. 0 . The
accuracy of this estimate affects the calculated capsid titer.
4.2.3.3 Genome titer quantification
4.2.3.3.1 General genome titer quantification
Indirect physical titer methods for genome titer include absorbance, fluorescence, and qPCR/dPCR.
4.2.3.3.2 Absorbance
Absorbance can be used to measure total nucleic acid content of the viral vector. Absorbance at OD260 nm
represents all nucleic acids species present in a sample (DNA, RNA, free nucleotides). Absorbance
measurements are calculated as nucleic acid concentration and shall be reported as ng/µL or µg/mL. The
vector genome concentration can be determined from absorbance, the molecular mass of the viral vector, and
the extinction coefficient as vector genome (vg)/mL.
Nucleic acid quantification using absorbance is impacted by impurities in the sample, such as proteins, and
shall be considered when interpreting results (ISO 20395:2019, 5.2). In some cases, viral particles are lysed
using a detergent, such as SDS, to denature proteins and release viral nucleic acid to allow for nucleic acid
quantification. The absorbance of viral vectors at OD260 nm depends on the molecular mass of the vector
[3][ ]
DNA and the amount of capsid protein. 0 .
Acceptable purity for applying absorbance measurement can be assessed by inspecting the ratios of
absorbance at different wavelengths that are altered by common contaminants. The purity of a particular viral
vector preparation, such as AAV and AdV, can be determined based on the OD 260/280 nm ratio which
accounts for the nucleic acid and the capsid protein content. These ratios can provide insight into the quality
of the viral vector preparation.
NOTE It is possible that the OD 260/280 nm ratio is not appropriate for certain viral vectors, such as LVV. In
addition, OD measurements can vary due to other factors such as pH and ionic strength.
4.2.3.3.3 Fluorescence
Similar to absorbance, fluorescence can be used to measure nucleic acid content of viral vectors. For the
nucleic acid content to be quantified, fluorescent stains may be used.
[4,5] [ , ]
NOTE 1 Common nucleic acid fluorescent stains include PicoGreen or GelGreen. . 0 0
A standard curve shall be used for fluorescent assays using a calibrant nucleic acid material. Examples of
calibrants include plasmid DNA, genomic nucleic acid, and fragmented nucleic acid. The type of calibrant used
(e.g. single-stranded DNA or double-stranded DNA) shall be taken into consideration based on the viral vector
being analyzed.
ISO/DISFDIS 16921-2:20242025(en)
Using a standard curve, fluorescent units are converted to nucleic acid concentration and shall be reported as
ng/µL or µg/mL. Nucleic acid concentration can then be converted to vector genome concentration as vg/mL
based on the molecular mass of the viral vector.
NOTE 2 The accuracy of the conversion from nucleic acid concentration to vector genome concentration depends on
the sample purity.
4.2.3.3.4 Quantitative PCR (qPCR) and Digital PCR (dPCR) Methods
For viral vector titer determination, genome counting methods (i.e., dPCR) do not directly enumerate virus
particles but rather targeted nucleic acid content and therefore is considered an indirect physical titer method.
ISO 20395 shall be referred to for information on qPCR and dPCR quality control, methodologies, and minimal
information needed for reporting.
Specific primers that target a known sequence in the viral vector shall be optimized and tested for optimal
parameters, including concentration, annealing temperature, and cycling parameters. A probe can be used to
complement the primers to enhance the specificity of the assay. Reverse transcription-PCR (RT-PCR) is
needed to convert RNA to cDNA for quantitation of RNA samples. The efficiency of the reverse transcriptase
reaction shall be determined.
For qPCR, a calibration or standard curve of known concentration or copies of DNA shall be used to obtain
DNA quantitation for an unknown sample. The efficiency of the PCR assay shall be determined based on the
data from the standard curve and unknown samples. The DNA standard curve should match the unknown
sample in terms of conformation and structure to not bias PCR efficiency between standard and sample. PCR
efficiency is impacted by supercoiled or linear states and proximity of the amplicon to hairpins and other
secondary structure in the DNA. Plasmids shall be linearized prior to performing qPCR.
When using purified DNA, e.g. plasmid as a standard, the number of target copies in the plasmid shall be taken
into account when quantifying the target copy number in the genomic DNA (e.g. ratio of the number of ITR
sequences per linearized plasmid versus number of ITR sequences in the genomic DNA).
NOTE 1 Indirect titer measurements assume the sample to be free of any impurities or obstacles that interfere with
the intended measurement purpose. PCR efficiency is affected by inhibitors in the sample and nucleic acid configuration.
Samples shall be assessed for PCR inhibitors by performing a dilution series and looking for quantitation
across the dilutions.
Digital PCR partitions the PCR reaction into nanolitre sized reactions (generally in droplets or chambers) and
uses Poisson statistics to determine concentration. Absolute quantitation of amplifiable nucleic acid in a
sample can be performed using dPCR without a standard curve.
For dPCR assays involving intact viral vectors without the need for nucleic acid extraction, aggregation, buffer
selection, capsid lysis, and enzyme treatment shall be considered when designing dPCR methods for accurate
[6,7][ , ]
absolute quantitation. 0 0 .
For dPCR assays where nucleic acid extraction is needed, extraction efficiency shall be determined with
[6,8][ , ]
appropriate controls. 0 0 .
Results from qPCR and dPCR genome titer methods shall be reported as copies/mL and shall include the name
of the target and the associated sequence. The results of copies/mL can be converted to vector genomes/mL
and the method used shall be reported. The sample to be measured shall fall within the concentration range
of the standard curve for qPCR.
NOTE 2 PCR-based genomic titer measurements detect a specific target sequence determined by primers and probes
selected. The sequence is generally ~100 to 150 base pairs long. PCR-based genomic titer measures target copies and
ISO/DISFDIS 16921-2:20242025(en)
does not measure the entire genome. Multiplexed PCR targeting of multiple target sequences can enable more
quantitative measures of vector genome copies and genomic integrity.
Other units that can be used for genome titer include vector genomes (vg)/mL, genome copies (gc)/mL, and
DNAse-resistant particles (DRP)/mL. For accuracy and to enable comparability, the conversion from
copies/mL to these other units shall be reported.
Proper controls shall be included in each assay to assess the quality of the assay. Positive controls, such as a
reference material with a known concentration if available, shall be included to generate a standard curve in
qPCR and be used in dPCR to ensure the primers, probes, and other reagents are working properly. Negative
controls shall be included to ensure PCR reagents are not contaminated with the test material or other
unwanted reagents. Additional negative controls shall be included for each nucleic acid target for all qPCR and
dPCR assays. Examples of negative controls include PCR grade water or appropriate buffer in place of the test
material. Negative controls shall remain negative for the presence of the target nucleic acid.
[9] ] [10,11] , ]
Minimum information requirements documented in the MIQE guidelines for qPCR 0 and dPCR 0 0 shall
[12] [ ]
be reported according to data formatting guidelines. 0 .
NOTE 3 There can be commercial kits and reagents that contain proprietary information which can result in some
information not being able to be included in the reporting guidelines listed in the MIQE.
4.2.4 Physical titer assay matrix
A matrix of assays should be considered when determining viral vector titer. A matrix of assays can be used
to infer purity attributes in the population.
The ratio of genomic titer to capsid titer can be used to infer the presence of empty and full particle
populations. This can be determined from the ratio of genomic PCR to capsid ELISA measurements, but also
the ratio of absorbance at 260 nm to 280 nm.
Additionally, the ratio of the capsid titer and total particle count can be used to infer presence of free capsid
particles in solution.
Measurement of free nucleic acid in solution and encapsulated nucleic acid contained within viral particles as
measured by fluorescence can be used to measure the encapsulation ratio of a preparation.
Counts for full and empty particles using electron microscopy and mass photometry can determine the capsid
content ratio.
Methods such as analytical ultracentrifugation and charge detection mass spectrometry can characterize the
percentage of empty, partial, and full viral vectors in a preparation but do not provide a titer and may require
an additional method to determine total particles and quantify the titer of the subpopulations.
Such measurements of purity attributes can be used to measure changes in viral vector quality through
production and downstream processing to quantify product and process related impurities. Large
discrepancies between assays can suggest the presence of impurities and biases in results.
Viral vector heterogeneity including full, partial, and empty particles (Figure 10) can impact purity and
agreement of physical titer measurements. Partially filled particles with partial genomes might not be
detected by genomic titer tests. Full particles contaminated by host cell DNA (detected using a separate assay)
are not detected by genomic titer tests but would be included in the capsid titer. Capsid titer measurement
would differ from total particle count if the capsid assay cannot distinguish between assembled and
unassembled capsid proteins.
The physical titer measurements are biased for measuring different attributes. Genome titer methods measure
viral vectors containing the full target sequence whereas the capsid titer measurements measure protein
ISO/DISFDIS 16921-2:20242025(en)
content. In a sample of 100 % full particles without any impurities, genome titer and capsid titer
measurements maycan have high agreement. However, in samples with impurities, such as soluble capsid
protein or partial filled particles, there maycan be low correlation and agreement. The measurement bias of
these different methods should be considered when characterizing the viral vector. Selection of assay
measurands and implications of measurands in the context of a process pipeline should be considered.
4.3 Activity assays
4.3.1 General activity assay concepts
Assays to determine the activity of viral vectors provide a measure of the concentration of viral vector
particles that can transduce cells and/or induce the desired function or phenotypic change.
Assays for activity of viral vectors involve the use of cells. This can include in vitro and in vivo assays such as
PCR, in vivo fluorescence, and immunohistology staining. Results for viral activity assays are cell line and
protocol dependent. The measured activity can be different for the sample tested on the same cells if a
different protocol is used. Additionally, protocol considerations such as inoculum volume and resulting
[13] ]
diffusion distance between viruses and cells can affect the reproducibility of viral activity assay results .0 .
The timing of an activity assay is application specific and shall be recorded along with other pertinent assay
protocol details. Experimental metadata shall be documented. Metadata should include information such as
cell passage number, media components, reagent lot numbers, and expiration dates.
Reagents shall be qualified for use in activity assays (including cells) for identity, the presence of adventitious
agents, expiration dates, etc. as these maycan affect many aspects of the assay including reproducibility (see
5.55.5).
Reported viral vector activity titer shall be derived in the assay’s specified range. The specified range can be
determined using a dilution series of virus to cells where identified dilutions demonstrate a linear response.
Refer to 5.55.5 for activity assay cell qualification and controls. See Annex B.1.3See Annex B.3 for activity assay
readout methods and Figure B.10 for example usage in a lentivirus activity assay.
4.3.2 Assay for transduction
4.3.2.1 General concepts for transduction assays
Transduction assays measure the concentration of viral vectors capable of delivering a transgene in a viral
vector preparation through a cell-based assay. Virus transduction assays are used to assess activity for non-
replication competent viral vectors, such as recombinant lentivirus and adeno-associated virus. Target cells
can be assessed for transgene expression using flow cytometry, qPCR or dPCR, and microscopy methods.
The assays involve taking viral vectors containing a transgene and exposing target cells to the viral vector. A
dilution series of virus to cells is generally used to specify at which dilutions the multiplicity of infection to the
measure of transgene expression is linear. The transduction units in the original virus preparation can be
determined from the specified range of the dilution series. Assuming a single virus particle delivers a
transgene to a cell, the number of cells expressing the transgene is used to determine the number of virus
particles in a virus preparation.
4.3.2.2 PCR methods
Quantitative PCR and dPCR can be used to measure transgene expression in the target cell. In the case of
lentivirus or other genome integrating viruses, measurement of the transgene copies per cell is known as
vector copy number (VCN). Using a dilution series experiment of known volumes of virus to vector copy
number per cell, one can determine the transduction units in the original viral vector preparation.
ISO/DISFDIS 16921-2:20242025(en)
ISO 20395 shall be referenced for extensive information on qPCR and dPCR quality control and methodologies.
The resulting measure of transduction shall be reported as transducing units (TU)/mL as determined by
copies/mL for a particular target. The target shall be specific to a sequence on the product transgene or be
specific to a regulatory element of the viral vector. The method, target, primers, and probes used shall be
[9] ]
reported. Minimum information requirements documented in the MIQE guidelines for qPCR 0 and
[10,11] , ] [12][ ]
dPCR 0 0 shall be reported according to data formatting guidelines. 0 .
For additional details regarding controls and calibration, see 4.2.3.4.3.4.2.3.4.3.
4.3.2.3 Flow cytometry methods
Flow cytometry is a measurement procedure which involves using a flow cytometer instrument to quantify
viral vector transduction in cells. Expression of a transgene protein can be quantified using a fluorescently
tagged antibody against the transgene protein. Additionally, a reporter protein (e.g. fluorescence reporter)
can be expressed simultaneously with the transgene to report expression of the transgene.
Measurement of transduction at the population level shall be determined as percent transduction efficiency.
In an experiment using a dilution series of virus to target cells, the percent transduction efficiency can be used
to determine the number of transducing units in the viral vector preparation.
Transduction assay results using flow cytometry shall be reported as transducing units (TU)/mL. The method,
target and antibody used to measure transduction by flow cytometry shall be reported. Minimum information
requirements for flow cytometry measurements should follow MIFlowCyt and data formatting
[12,14][ , ]
guidelines. 0 0 . Experimental metadata shall be documented. The metadata should include the cell line,
media components, any drug selection information, and cell passage number at the time of the assay.
4.3.2.4 Microscopy methods
Similar to flow cytometry, microscopy approaches can be used to perform automated imaging and image
analysis of virus transduced cells. This includes identification of the transgene expression by a fluorescence
reporter or antibody staining for the transgene protein. Individual cells or cell area can be enumerated to
determine the total number of cells. The ratio of the fluorescently stained cells to the total number of cells can
determine the percentage of transduced cells in the total population.
Measurement of transduction at the population level shall be determined as percent transduction efficiency.
The percent transduction efficiency can be used to determine the number of transducing units in the viral
vector preparation. These experiments often involve the use of a dilution series of virus to target cells to give
higher confidence in the determined transducing titer.
Transduction assay results using microscopy shall be reported as transducing units (TU)/mL. If using
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automated microscopy, reporting should follow data formatting guidelines . 0 The method, target and
antibody used to measure transduction by microscopy shall be reported. Experimental metadata shall be
documented. Metadata should include the cell line, media components, any drug selection information, and
cell passage number at the time of the assay.
4.3.2.5 Colony forming assay
A specific type of transduction assay utilizing microscopy is the colony forming assay. In cases where the viral
vector transgene contains an antibiotic resistance marker, a cell-based colony formation assay can be used to
quantify transduction.
A monolayer of target cells is prepared and then exposed to viral vector to allow for viral vector transduction.
Following transduction, the target cells are challenged with exposure to antibiotics. Cells not transduced by
the viral vector will die from antibiotic exposure. Cells successfully transduced by the viral vector with the
ISO/DISFDIS 16921-2:20242025(en)
antibiotic selection marker will survive and continue to proliferate as a cell colony. For virus transduction
performed at a sufficiently low dilution where each cell colony is formed by a single antibiotic resistant cell,
microscopic counting of colonies can be used to determine colony forming units.
The colony forming units can be used to determine the transducing units in the viral vector preparation. The
dilutions used in the assay shall enable quantification of individual colonies. Multiple dilutions with coun
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