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7.9 Sample Stability Considerations 183
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500 copies/reaction. This spike‐in approach is particularly useful for liquid specimens, such as urine, saliva, blood, plasma, and semen, which are monitored in
clinical vector shedding studies. While feasible, this approach faces obvious difficulties with solid samples. These difficulties may be overcome by preparing multiple specimen portions to accommodate single extractions at each stability time
point, and by adapting testing procedures to lyse stability specimens directly in
their original containers.
To assess stability concurrently to or after a study, biological specimens from
GTx‐treated individuals can be extracted and tested using a primary PCR test that
serves as baseline assessment, followed by multiple secondary extractions and
PCR retests of the same specimen (or aliquots/portions thereof) at defined stability intervals. This would not require spiking any vector material and resembles
incurred sample reanalysis (ISR), which is sometimes performed for validation of
other pharmacokinetic methods. This retest approach is useful for tissue specimen in biodistribution studies, where spiked surrogate vector DNA or intact GTx
vector capsids may not fully recapitulate the biological conditions that affect episomal vector DNA within the nuclei of transduced cells. In particular, linearized
surrogate vector DNA spiked onto tissue specimens may represent more of a
worst‐case scenario for analyte stability, since circularly closed vector episomes
within cells would be expected to be more stable than open‐ended surrogates that
could be exposed to extracellular DNases in tissue samples. The obvious disadvantage of using secondary PCR tests of incurred specimens for stability studies is
that the results cannot inform study design, and thus sample storage conditions
and duration are decided at risk. Another consideration is limited sample quantity
in small animal studies and the need to plan for collecting multiple specimen
aliquots/portions to facilitate evaluation after various storage intervals without
incurring freeze–thaws.
As for extracted DNA stability, most PCR workflows aim to test shortly after
DNA samples have been extracted from biological specimens. In addition, the stability of purified DNA frozen at low temperature is relatively well established, and
thus long‐term stability studies may not be needed. Considering the possibility of
PCR retests following a failed initial run, freeze–thaw assessments combined with
short‐term stability periods (e.g. 1month) appear to be more useful. This evaluation can be performed pre‐study using spiked surrogate vector DNA, or post‐study
by repeated PCR testing of extracted DNA samples after the primary test was completed on study.
In‐assay short‐term stability assessments differ between qPCR and ddPCR. In
qPCR, one would typically proceed from sample preparation to amplification and
generate results in real time. Using Bio‐Rad’s QX200 ddPCR system, it may be possible to “pause” the run for a period of time following thermal‐cycling of the generated droplets (e.g. overnight). This allows for greater analyst flexibility and
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184
increased throughput, since test plates can be prepared and thermal‐cycled on
Day 1 and read out on Day 2, with runs staggered by one day. Furthermore, holding droplets at 12
more droplets will meet the instrument’s droplet quality metrics [24]. A higher
number of acceptable droplets translates into a higher probability that low abundance targets will be detected.
Stability acceptance criteria for PCR‐based methods vary. Measured GTx vector
quantities in stability samples can be compared back to baseline samples to reveal
any trends of degrading sample integrity. Alternatively, a comparison to nominal
spike levels can be made. The latter approach lends itself more easily to extracted
DNA samples spiked with known amounts of vector, while the former approach
is more applicable for biological specimens, where extraction variability and
unknown vector quantities preclude the use of nominal concentrations.
When comparing results from stability time points to those from baseline samples, a threshold needs to be established for the biological specimen to be considered stable. In other words, what relative difference from baseline results indicates
sample instability? No consensus exists but reasonable acceptance ranges may fall
between 0.5 and 0.2 times baseline value. To justify a threshold, the variability of
both DNA extraction efficiency and PCR measurement needs to be considered.
For example, if extraction efficiency varies by a factor of 2, then halving of PCR‐
measured vector quantities would not be conclusive for specimen instability.
Assuming a potential negative bias of −50% for both extraction efficiency and
PCR measurements, one would obtain a theoretical lower limit for overall analytical variability of 0.5 × 0.5 = 0.25. Thus, after adding a small margin of tolerance, a
value of 0.2 times baseline could be a reasonable threshold, below which biological specimens are considered instable.
°C for a minimum of 4 hours can increase assay sensitivity, since
7.10 Data Reporting Formats, Acceptance Criteria,
and Trending
Depending on study context, PCR data may be reported in various formats: In
nonclinical biodistribution studies, VG copies per microgram of gDNA are commonly used, and VG copies per mg tissue may also be reported. The former unit
can be converted to VG copies per cell by multiplying with the approximate mass
of a diploid genome (6 pg in humans/mice, 5 pg in NHP). Potentially higher
degrees of ploidy, for example in liver and heart cells[25–27], may need to be
considered as appropriate. In shedding studies, reporting as VG copies per mL
biological fluid or mg solid specimen is oftentimes more clinically relevant, since
volumes and masses are more easily visualized. For clinical specimens with sufficient DNA content, such as whole blood and seminal fluid, reporting as VG copies per microgram gDNA could additionally be provided.
7.10 Data Reporting Formats, Acceptance Criteria, and Trending 185
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Exemplary acceptance criteria for regulated PCR sample testing are provided in
Tables7.3 and7.4. The standard curve and low‐level quality control (QC) within
1log of the LLOQ are routinely included, together with various negative controls
that monitor for potential cross‐contamination. Medium and high QCs can be
added to increase confidence in accurate and precise sample quantification.
The range of acceptable accuracy for qPCR‐based methods is typically broader
(−50% to +100% relative error, RE) than that of other pharmacokinetic methods, as
discussed above. When performing qPCR, it can be useful to verify that no sample‐
mediated inhibition occurred, in particular for samples without detectable increase
in fluorescence that would be reported as negative. Sample‐mediated inhibition
can be assessed by spiking a low quantity of vector DNA (50–500
copies/reaction)
into a designated test sample replicate and comparing with a spike volume control
(SVC) sample. The SVC sample contains the same number of vector DNA
Table7.3 Exemplary standard curve, QC, and run acceptance criteria forPCR.
Acceptance criteria qPCR sample
Parameter
Standard
Curve
Regression
Precision The CV of replicate Cq or Ct values
Accuracy The RE for each standard
Negative
controls
analysis
The standard curve should have an
2
R
≥ 0.98 and a slope between −3.1
and −3.6 (corresponding to
90–110% amplification efficiency).
for each calibration point and
quality control (QC) sample should
be ≤3%. OR: The CV of interpolated
VG copies for each standard curve
point and QC samples should be
≤30%, except for LLOQ‐QC where
CV should be ≤50%. Outlier
exclusion: 1 out of 3 replicates may
be omitted, if the Cq or Ct
differences between the omitted
replicate and each of the other two
replicates are greater than 1.
calibration point and QC sample
should be within −50% and +100%
of the nominal value.
Negative extraction controls,
no‐template controls, and sentinel
controls should not show any
increase in fluorescence OR:
remain below the LOD.
Acceptance criteria dPCR sample
analysis
Not applicable, absolute
quantification mode alleviates the
need for a standard curve
The CV of measured VG copies for
QC samples should be ≤30%, except
for LLOQ‐QC where CV should be
≤50%. Outlier exclusion: 1 out of 3
replicates may be omitted, using an
appropriate statistical method (e.g.
Median Absolute Deviation, MAD).
The RE for QC samples should be
within ±30% of the nominal value,
except for LLOQ‐QC where RE
should be within ±50% of the
nominal value.
Negative extraction controls,
no‐template controls, and sentinel
controls should not have VG copies
greater than LOD.
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copies/reactions in the same spike volume added to a non‐interfering matrix or
plain buffer. If the Cq or Ct value of the spiked test sample replicate is significantly
higher than that of the SVC, sample‐mediated inhibition has occurred and adequate remediation procedures may be needed, as detailed in Table7.4.
Table7.4 Exemplary test sample acceptance criteria forPCR.
Acceptance criteria qPCR sample
Parameter
Sample‐
mediated
inhibition
Precision Samples should be tested in
Sample result
determination
analysis
Samples should be non‐inhibitory.
Samples are considered inhibitory
if the spiked replicate exhibits a Cq
or Ct value that is greater than
1.73
+ the mean Cq or Ct value of
the spike volume control (SVC).
Inhibitory samples shall be
retested. If a sample remains
inhibitory upon retest, it should be
reextracted from biological
specimens. If re‐extraction is not
feasible, the sample should be
reported as not determinable (ND).
triplicate or duplicate. The CV of
replicate Cq or Ct values for a
sample above the LLOQ should
be ≤3%. OR: the CV of
interpolated VG copies for a
sample above the LLOQ should
be ≤30%. Outlier exclusion: 1 out
of 3 replicates may be omitted, if
the Cq or Ct differences between
the omitted replicate and each
ofthe other two replicates are
greater than 1.
Samples are considered positive if
the level of fluorescence after 40
cycles rises above the run‐specific
threshold and is indicative of true
amplification. Otherwise, samples
are considered negative. OR:
Samples are considered positive if
interpolated mean VG copies fall
at or above the LOD. Otherwise,
samples are considered negative.
Acceptance criteria dPCR sample
analysis
Samples should be non‐inhibitory.
Samples are considered inhibitory
if the spiked replicate exhibits
measured VG copies that are more
than 30% lower than those in the
SVC. Inhibitory samples shall be
retested. If a sample remains
inhibitory upon retest, it should
be reextracted from biological
specimens. If re‐extraction is not
feasible, the sample should be
reported as ND.
Samples should be tested in
triplicate or duplicate. Samples
with mean VG copies above 2
times the LLOQ must have CV
≤30%. Samples with mean VG
copies between 2 times the LLOQ
and the LLOQ must have CV
≤50%. Outlier exclusion: 1 out of
3 replicates may be omitted, using
appropriate statistical methods
(e.g., Median Absolute Deviation,
MAD).
Samples are considered positive if
mean VG copies fall at or above
the LOD. Otherwise, samples are
considered negative.
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Parameter
Reporting
ranges
Acceptance criteria qPCR sample
analysis
Positive samples should be
quantified and reported with a
numerical result if they fall
within the limits of quantification
(LLOQ, ULOQ) established in
assay validation. Samples above
the ULOQ should be diluted
adequately and retested. Positive
samples below the LLOQ should
be reported as falling below the
lower limit of quantification
(BLQ). Negative samples should
be reported as 0 VG copies or
below the limit of detection
(BLD).
Acceptance criteria dPCR sample
analysis
Positive samples should be
quantified and reported with a
numerical result if they fall
within the limits of quantification
(LLOQ, ULOQ) established in
assay validation. Samples above
the ULOQ should be diluted
adequately and retested. Positive
samples below the LLOQ should
be reported as falling BLQ.
Negative samples should be
reported as 0 VG copies or BLD.
Routine clinical vector shedding assays should be monitored for consistent performance. For qPCR methods, the Cq or Ct values of the standard curve could be
trended and should typically fall within two standard deviations from the mean.
VG copies for QCs can also be trended, which may be the most appropriate way to
monitor ddPCR performance over time. The proportion of droplets that pass
internal instrument quality checks may also be monitored in ddPCR since a significant and prolonged change in accepted droplets used for quantification could
impact assay performance.
7.11 Immunocapture qPCR: AnUltra-Sensitive
Method toDetect Intact AAV Capsids
The main objectives of clinical vector shedding studies are to evaluate the potential risk of horizontal transmission and release into the environment. AAV‐based
GTx are generally designed to have minimal risk, given that they are replication‐
incompetent, non‐pathogenic, encode nontoxic gene products, have weak or
moderate promoters/enhancers, do not carry antibiotic resistance genes, and
rarely integrate into the host genome. Nonetheless, there is a desire to limit any
unintended exposure or release. Monitoring vector shedding can inform the duration of potential pre‐cautionary measures and is therefore mandated by regulatory agencies during clinical studies.
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If standard PCR‐based methods are used to monitor shedding, encapsidated
and non‐encapsidated vector DNA cannot be distinguished. This distinction,
however, could be informative, since the potential risk associated with each of
these two forms of vector DNA is not equal. On the one hand, encapsidated vector
DNA is contained in structurally intact capsid particles that may have the capability to enter cells, i.e. this form of vector DNA is potentially transduction competent. On the other hand, non‐encapsidated vector DNA could stem from low
amounts of degraded capsid particles or emerge during uncoating within transduced cells and be released following cell death. No meaningful transduction
competence would therefore be expected for non‐encapsidated vector DNA,
which decreases any potential risk.
To measure intact GTx vector capsids, cell‐based transduction assays or ELISA‐
based immunoassays may be used[3, 28]. In clinical practice, however, the limited
sensitivity of these methods poses a significant challenge to detecting low residual
levels of GTx capsids in shedding matrices or blood[29, 30]. Recently, a more sensitive ELISA‐based method named VIRELISA was described, which employs a
recombinant high‐affinity AAV receptor (AAVR) to capture and detect AAV2 capsids [31]. With an estimated sensitivity of detecting as low as 5E+05
of AAV2 capsids in buffered aqueous solution, this method may warrant further
investigation, even though the sensitivity for matrixed clinical samples and other
AAV serotypes remains unclear.
To overcome the hurdles imposed by limited sensitivity of existing methods, an
ultra‐sensitive assay for detecting structurally intact, and thus potentially transduction‐
competent capsids has recently been developed for an AAV5‐based GTx [3]. This
two‐step methodology, named immunocapture‐qPCR (iqPCR), features a monoclonal antibody (ADK5a/b) that specifically recognizes a conformational epitope on
AAV5 capsids and facilitates magnetic bead‐mediated pull‐down, followed by capsid
denaturation and PCR amplification of released vector DNA. The major advantage
of iqPCR is the quantum leap in analytical sensitivity for detecting AAV capsids
across various clinical matrices. IqPCR exceeds the sensitivity of both cell‐based
methods and advanced immunoassay platforms (including single‐molecule counting, or SMC‐Erenna), and approximates detection limits of standard PCR‐based
methods. Compared to VIRELISA, iqPCR is at least 40 times more sensitive.
Another advantage of iqPCR over ELISA‐based methods is its dually assured
specificity for intact GTx capsids, (1) by using a conformation‐specific antibody
for a particular AAV serotype and (2) by ensuring that captured capsids contain
specific vector DNA. ELISA‐based methods, such as VIRELISA, detect AAV capsids regardless of whether they are full (“heavy”) or empty (“light”), or regardless
of whether they contain vector DNA or a wild‐type AAV genomes. Hence, one
may expect potential interference from natural AAV infections for ELISA‐based
capsid detection methods if used in clinical studies.
vg/mL
References 189
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A notable limitation of iqPCR (as well as ELISA‐based capsid detection methods) is interference from AAV antibodies. These antibodies may compete for
epitopes with the monoclonal antibody reagent used to capture capsids, and consequently iqPCR has a low tolerance to AAV antibodies present in plasma or
semen[3]. This interference is of lesser concern within the first few days after GTx
administration, since patients have usually been pre‐screened to have low or no
pre‐existing humoral immunity to AAV. While treatment‐emergent AAV antibodies can impact capsid quantification in iqPCR, they are generally also highly neutralizing[32–34]. Hence, AAV capsid/antibody complexes that remain undetectable
by iqPCR would also be less likely to mediate horizontal transmission or environmental impact, as compared to “free” transduction‐competent AAV capsids.
IqPCR has been successfully implemented in clinical GTx studies and used to
describe clearance kinetics of transduction‐competent “free” GTx vector in
human biological fluids[3]. This helped inform the duration of precautionary
measures deemed necessary to limit unintended exposure or release. In summary,
PCR‐based methods to detect vector DNA in tissues and biological fluids are
essential components of bioanalytical portfolios that support the development of
novel AAV GTx.
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