APPLICATION NOTE

Double down with lentivirus titer and RNA content
on Leprechaun
Introduction
Getting an early read on the number of intact,
RNA-containing lentivirus (LV) particles in a batch
makes a real difference when optimizing cell therapy
development and production. Knowing post-harvest
which batches have the highest titer of structurally
complete viral particles, and which have the lowest,
ensures you don’t waste time on preps which are poor
quality to begin with. Additionally, increasing focus
from the FDA and other regulators on the need foranalytical development and product characterization
for LV based therapies makes understanding exactly
what’s in your LV prep more important than ever for
those wishing to avoid costly delays.

Figure 1: Leprechaun is the only platform to provide completebiophysical characterization of individual LV particles.

Leprechaun is the only platform to provide completebiophysical characterization of individual LV particles,
regardless of pseudotype, at every step of theproduction process (Figure 1).

Compared to other viral vectors, the analyticaltechniques available for LV characterization are still
underdeveloped. Widely used assays, such as thep24 ELISA and qRT-PCR, provide limited information
as they are destructive, detect only one part of theviral structure, and analyze the total amount of capsid or gene of interest without checking how much is
inside viral particles. On the other hand, technologies
which rely on non-specific particle analysis can detectand count individual particles but make assumptions
on their content without directly checking what’s
inside or on the surface.

Lentivirus w/ capsid& RNA

Lentivirus w/ capsid
w/o RNA

The Lentivirus VSVG RNA Luni consumable ispre-coated with a highly specific antibody against
VSVG to capture viral particles straight from crude
samples, without prior purification (Figure 2). For
analysis of non-VSVG pseudotyped virus theLentivirus Flex RNA Luni allows the capture step to becustomized with your own antibody for the envelope

Lentivirus w/o capsid
w/ RNA

Lentivirus w/o capsid
w/o RNA

Aggregates

Figure 2: LV captured on the RNA Luni is sized, then stained with anti-p24-CF647 and SYTO14 to detect viral capsid and RNA. Get n≥3
technical replicates for each output from a single Luni.

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protein of your choice. Once bound, Leprechaun uses
single-particle interferometry to measure the sizeof each viral particle to check whether it is intact,aggregated or a fragment. Antibodies confirm thepresence of the viral envelope and p24 capsid, while
fluorescent RNA dye checks for the all-importantnucleic acid. Regardless of how dirty your sample is,Leprechaun can measure the titer of RNA-containing
lentivirus down to 1x107 particles/mL.

CF647 (1:250). Lunis were washed, dried and run on
Leprechaun using the Lentivirus RNA application.

Benzonase Treatment
25 U/µL benzonase stock (Sigma-Aldrich) was
diluted 1:8 in benzonase incubation buffer (20 mM
Tris-HCl, 4 mM MgCl2, 20 mM NaCl), 1 µL LV sample added per 2 µL, and the mixture incubated at
37 °C for 1 hour. For Leprechaun analysis, SYTO14(or an equal volume of PBS for non-dye samples)
was added to benzonase treated samples and Luni
preparation performed as per above method.

This app note describes how Leprechaun’s Lentivirus
RNA application quantifies RNA and capsid-containing LV in crude and pure samples, and demonstrates
the value of LV structural analysis for both process
and analytical development.

Benzonase functionality was verified by adding
1 mg/mL calf liver RNA (Sigma-Aldrich) to 25 U/µL benzonase (in benzonase incubation buffer)and 10 µM SYTO14. For the non-benzonase treatedcontrol, benzonase was replaced with an equivalent
volume of buffer (20 mM Tris-HCl, 2 mM MgCl2, 20mM NaCl and 50% glycerol). Samples were incubatedat 37 °C for 1 hour using the Isothermal application onUncle (Unchained Labs) and fluorescence emission
monitored between 500 – 630 nm.

Methods
Leprechaun Analysis
LV samples were acquired from five vendors andLeprechaun analysis performed using the Lentivirus
VSVG RNA Kit (Unchained Labs). LV was diluted
between 1:12 and 1:2000 in manufacturer supplied
Incubation Solution with added 1% FBS. Kit supplied
SYTO14 was diluted to 10 µM in Incubation Solution
and added to the diluted LV sample at a dye to sample ratio of 1:10. The dye-sample mixture was incubated for 16 hours at 37 °C. Post dye incubation, 50µL SYTO14 labelled sample was incubated for 1 hourat RT on Lentivirus VSVG RNA Lunis. After fixationand permeabilization by Solution C and D, Lunis wereincubated for 1 hour at RT with kit supplied anti-p24-Particle type

Capture antibody

LV w/ capsid & RNA

qRT-PCR Analysis
Analysis was performed by SydLabs using theLenti-X qRT-PCR Titration Kit (Takara Bio), according
to the kit protocol. Three dilutions of each sample
were run in duplicate and results averaged to obtaingenomic titer.

Particle definition
Size (nm)Component detectedanti-VSVG/custom

35 – 130

VSVG+ p24+ RNA+

LV w/ capsid w/o RNA

anti-VSVG/custom

35 – 130

VSVG+ p24+ RNA-

LV w/o capsid w/ RNAanti-VSVG/custom

35 – 130

VSVG+ p24- RNA+

LV w/o capsid w/o RNA

anti-VSVG/custom

35 – 130

VSVG+ p24- RNA-

Aggregatesanti-VSVG/custom>130 – 200

VSVG+ p24± RNA±

Viral p24

anti-VSVG/custom

N/A

VSVG+ p24+ RNA±

Soluble p24

anti-p24

N/A

p24+ VSVG-

Table 1: Definitions for LV particle types analyzed on the VSVG and Flex RNA Lunis. Default size ranges can be customized during analysis. In
addition to the viral capture spots, p24 is also present on the Luni surface providing quantification of soluble p24 alongside viral titer.

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Lentivirus
w/ capsid w/o RNA

Lentivirus
w/ capsid & RNA

Figure 3: VSVG capture spot on the VSVG RNA Luni. Sample was incubated with SYTO14 (blue), then fixed and permeabilized to allow
probing for encapsulated p24 (red). LV containing both a p24 capsid and RNA is purple, LV with capsid but without RNA is red and virus
with RNA but no capsid is blue.

Counts are only detectable in the blue channel
when both a LV sample and the SYTO14 dye arepresent. When dye is removed, and the viral sample
analyzed alone there is no detectable colocalizationof blue signal with VSVG positive particles. This
results in all viral particles being classified as‘without RNA’ and confirms that signal is specificto SYTO14 binding to RNA, not a result of autofluorescence from the sample or bleed through fromother fluorescence channels. Furthermore, no blue
signal is detectable when Lunis are incubated withdye alone, verifying that SYTO14 does not stick
non-specifically to the Luni surface.

Results
How it all comes together
Viral particles are immuno-captured on the surface of the RNA Luni via their envelope protein, then
their structure is analyzed using a combination ofinterferometry and fluorescence microscopy. Single
particle interferometry uses the signal enhancing
silicon dioxide surface of the Luni to maximize
interference of light reflected from the Luni surface
and scattered by the captured virus. This resultsin high resolution sizing of single particles, down
to 35 nm in diameter. Establishing viral size allows
Leprechaun to separate viral fragments andaggregates from single viruses.

SYTO14 was selected for LV RNA detection since itis cell permeable and able to cross the viral envelope without compromising the viral structure.
To confirm SYTO14 is binding to RNA inside theSize alone is not enough to identify a particle as an intact LV. Fluorescent staining for p24 and RNA checks
for the key structural components (Table 1). Samples
are incubated with the membrane permeable
RNA dye SYTO14 prior to immobilization on theLuni, labelling viral RNA. Once bound and after amild fixation and permeabilization, CF647-labelled
anti-p24 detects virally encased capsid (Figure 3).

Validated RNA Detection
SYTO14 emission peaks at 530 nm upon binding
to RNA, which is detected by Leprechaun’s bluechannel. To confirm that blue channel fluorescenceis specific to the binding of SYTO14 to LV particles,
Lunis were incubated with four conditions: with/without virus and with/without dye (Figure 4).

Figure 4: Negative controls confirm that blue signal is specificto binding of SYTO14 with LV. Error bars are standard deviation,n = 3 for each condition.

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A

B

Figure 5: (A) Confirmation of nuclease activity. SYTO14 and RNA were incubated in the presence and absence of benzonase and theresulting fluorescence measured on Uncle. (B) Impact of nuclease treatment on the titer of LV which contains RNA as analyzed onLeprechaun. For both graphs error bars are standard deviation, n = 3 for each condition.

virus, rather than nucleic acid stuck to the surface
or free in solution, LV samples were treated withthe nuclease benzonase prior to incubation onthe Luni. Benzonase activity was first tested byincubating RNA and SYTO14 in the presence andabsence of the enzyme, and the resulting fluorescence signal measured on Uncle (Figure 5A).
Addition of benzonase led to a 95% reductionin fluorescence, verifying nuclease functionality.
Benzonase treatment had no impact on thenumber of LV particles identified as containing
RNA by Leprechaun (Figure 5B), confirming thatSYTO14 is binding to RNA inside viral particles.

Uptake of small molecule dyes, such as SYTO14,
across viral protein capsids is known to be time
and temperature dependent1,2. To ensure a highefficiency of RNA staining SYTO14 was incubatedwith LV for increasing lengths of time, and atdifferent temperatures. The percentage of virus
found to contain RNA increases with time whenincubated at 37 °C, before levelling off after
16 hours (Figure 6A). Lowering the incubation
temperature to 25 °C or 4 °C significantly reduces
SYTO14 staining of LV particles (Figure 6B). Taken
together this data confirms that a high temperature and overnight incubation period aids efficient
uptake of SYTO14 by the virus and access to theencapsulated RNA.

AB

Figure 6: (A) Time dependence of SYTO14 staining of LV RNA. Sample was incubated with 10 µM dye for between 1.5 and 22 hours at37 °C. (B) Temperature dependence of SYTO14 staining. Sample was incubated with 10 µM dye for 16 hours at 4 °C, 25 °C and 37 °C. For
both graphs error bars are standard deviation, n = 3 for each condition.

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Figure 7: Linearity of LV titer as measured by Leprechaun. Error bars are SD.

Particle typew/ RNA as measured on Leprechaun revealed
a positive correlation (R2 = 0.95) for purified
samples (Figure 8A).

Dynamic range

LV w/ capsid & RNA
LV w/ capsid w/o RNA
LV w/o capsid w/ RNAInterestingly, the same correlation was not observedin crude samples (Figure 8B). This strongly suggests
there are a significant number of particles released
from the producer cell which are the same size as
LV, express VSVG, and contain RNA other than theintended viral genome. Most likely these particles
are vesicles released alongside LV particles, andwhich contain cytoplasmic RNA. Leprechaun handles this population by probing for capsid protein
at the same time as RNA to help ensure you’re
only counting LV which have undergone thecorrect assembly process. It is therefore vital thatany technique relying on generic RNA detection foranalysis of early-stage LV samples also checks forthe presence of p24 capsid.

1x107 – 5x108
particles/mL

LV w/o capsid w/o RNA
Aggregates
Soluble p24

5 – 10,000 pg/mL

Viral p24

1,000 - 50,000 pg/mL

Table 2: Dynamic range for all outputs provided by theLeprechaun Lentivirus VSVG/Flex RNA Kits.

Linear and Precise
Envelope specific capture of LV particles on theLuni surface stops non-viral particles from binding, preventing contaminants from interfering withviral titer. It is this step that means Leprechaun
can maintain a linear range for lentiviral titer down
to 1x107 particles/mL in both crude and purified
samples (Figure 7, Table 2), with a typical % CV of 8%.

Track Purification
Leprechaun allows you to track the quality ofyour LV batch throughout the production process,revealing if you’re enriching for that crucial capsid& RNA-containing LV and removing incorrectly
assembled viral-like particles. Samples from thesame LV batch, but from different steps of thepurification process – crude harvest, post PEG
precipitation and final product (PEG, UC and UFpurified) – were analyzed on Leprechaun (Figure 9A).

Correlation with Genomic Titer
Crude and purified LV samples from a range ofsuppliers, with different viral genomes, were analyzed by both Leprechaun and qRT-PCR. Comparison of genomic titer with the overall titer of LV
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A

B

Figure 8: Relationship between the titer of LV w/ RNA as measured by Leprechaun and genomic titer from qRT-PCR in pure (A) andcrude (B) samples.

Checking the structure of individual particles on
Leprechaun reveals that although total LV titerincreases with each purification step, the proportion of structurally complete viral particles isno higher in the final product than in the crude
harvest (Figure 9A - blue line). This indicates thatwhile standard LV purification techniques successfully concentrate particles which have the size anddensity of LV, they are not necessarily effective atenriching for structurally complete LV.
The total p24 content of the three samples wasanalyzed on Leprechaun and titer calculatedusing the conversion of 1x104 physical LV particles

per pg of p24 to produce a comparable output
to a p24 ELISA3. p24 concentration increases
with each step, implying an increase in viral titer(Figure 9B). However, this output is misleading
without the context of how many p24 negativeviral particles are still present (Figure 9A – gray,yellow bars) – information not provided by a traditional ELISA. These particles express the sameenvelope protein, may contain RNA and have thepotential to compete with intact LV for transduction of target cells. When p24 negative particles
are taken into consideration the final product hasa similar proportion of capsid-containing LV to thecrude harvest.

A

B

Figure 9: (A) Leprechaun analysis of a LV batch at different stages of purification. (B) p24 based titer as measured on Leprechaun. Titeris calculated from the sum of the concentration of viral and soluble p24. Error bars = SD, n = 3.

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A

B

Figure 10: (A) Leprechaun analysis of the structural composition of three crude LV samples. (B) p24 based LV titer as determined byLeprechaun. Error bars = SD, n = 3.

sample, it cannot differentiate between the qualityof batch 1 and 2 due to the lack of information on
both capsid negative LV and the RNA content of
the capsid positive particles.

ID a Good Batch
Leprechaun also provides the capability to screen
LV samples at harvest to check if a batch has ahigh titer of structurally complete LV, and is worth
continuing with, or whether you should cut yourlosses as there is a low titer of intact LV indicatingthat the batch is mostly junk. Crude samples fromthree LV batches were analyzed on Leprechaun
and their compositions compared (Figure 10A).

Conclusion
Traditional purification methods are effective atconcentrating LV but can struggle to remove LV
particles which are the correct size and density butlack the key structural components – specifically a
p24 capsid & RNA. These particles have the sameenvelope protein as intact LV and can compete fortransduction of target cells but are not capableof delivering the desired payload and may indeed
carry RNA other than the viral genome. Delivery of
unknown RNA sequences to cells intended for treatment of patients needs to be avoided to ensure safeand effective cell therapies.

At first glance batch 2 has the highest overall
titer of LV particles, however a closer look at thestructural composition of these particles showsthat a significant proportion of batch 2 are empty
viral particles containing neither capsid nor RNA.
Batch 1 and 2 have comparable titers of LV w/
capsid & RNA, but batch 1 contains far fewer
structurally incomplete LV contaminants, meaning
it is significantly more enriched for LV w/ capsid& RNA (39% v 14% of total LV particles). As viralparticles missing capsid and/or RNA are difficult
to remove during processing, selecting a starting
material with a low proportion of these particles iscritical, in this case batch 1.

Current LV analytical techniques only look for either
capsid or RNA, and therefore fail to provide any
information on how many structurally incomplete
viral particles are present. Even cell based functional
assays focus exclusively on particles with the correct
viral genome, and do not measure the impact ofparticles carrying other RNA or cargo proteins. Take
your process development to the next level with thehelp of Leprechaun, the only platform to show you
the complete picture of your LV sample, including

As with the previous set of samples, the p24 content
was measured by Leprechaun and converted toa physical titer comparable to that provided by
ELISA (Figure 10B). While p24 content alone issuccessful in identifying batch 3 as the lowest titer
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how many viruses are missing p24 capsid, RNA orboth. For the first time you can truly know what’s
in your sample and get a detailed insight into theeffectiveness of each step of your LV production
process to make fully informed decisions about
your process development.

References
1 Binding of fluorescent dye to genomic RNA inside
intact human rhinovirus after viral capsid penetration investigated by capillary electrophoresis.
Kremser, L. et al. Anal Chem, 2004. 76: 882-887
2 Quantitative evaluation of protein heterogeneity
within herpes simplex virus 1 particles. El Bilali, N. etal. J Virol, 2017. 91:e00320-17

Leprechaun puts you in the best position to produce
a high-quality final product by showing you which
upstream material is highest in LV w/ capsid & RNA,and low in structurally incomplete LV which may
alter your target cells in unknown ways. Identifying
the top samples early on is critical to overcoming
the limitations in LV purification techniques that arepresent in even the best production processes. As
regulatory demand for LV characterization grows,
revolutionize your LV analytics with Leprechaun andensure your LV preps always meet the gold standard.

3 Production of high-titer lentiviral vectors. Zufferey,
R. and D. Trono. Curr Protocols in Neuroscience,
2001. 12: 4.21.1-4.21.12.

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