APPLICATION NOTE
Novel approach for quantitative
real-time particle analysis of lentiviral vectors

Authors

Abstractaratinga.bio

Lentiviral vectors are efficient vehicles for stable genetransfer in dividing and non-dividing cells. They tend to
be increasingly used as a powerful tool to introduce
genes into cells ex vivo, for instance in CAR-T celltherapies.

Victoire Barailles, Alice Pailleret, Aurélie
Perier, Nicolas Delacroix, Cécile Bauche,
Renaud Vaillant

During manufacturing and production of lentiviralvectors, relevant quality control is necessary to allowbatch release (1). Among standard quality control
methods that can be used, quantification of lentiviral
vector particles – or physical titer – is one of the mostimportant. Up to now, this characterization can beachieved either indirectly with p24 protein quantification
or with physical methods like Tunable Resistive Pulse
Sensing (TRPS) for example, both methods implying priorpreparation of samples (lysis, dilution or filtration). These
two methods thus show important limitations as theycannot accurately reflect the true nature of the product,in addition to being relatively time-consuming (2).
Myriade, a French company created in 2017, isdeveloping Videodrop, a new optical techniqueperforming real-time, user-friendly, and label-freemeasurement of lentiviral vector physical titer. Thismethod, based on full-field interferometry (3), wastested on various lentiviral vector samples: in a context
of Drug Product (DP) release as well as in-processcontrols.

MyriadeMatthieu Greffet, David Poizat

We compared three lentiviral physical titration methods
on aratinga.bio productions: p24 ELISA, qNano andVideodrop – Myriade instrument. The correlation
between Videodrop analysis and the other two methods
appeared to be robust, with high R² values. These results
suggest that Myriade technology is relevant for DP
release as well as in-process controls, offering the ability
to be a tool for continuous improvement. It is an easy-touse and fast alternative to the standard more complex
and time-consuming physical titration methods.

1Introduction

filtration even in the presence of large cellular debris.
The instrument is essentially a microscope, therefore
objects in the micrometer range will be seen the sameway as with a conventional microscope, allowing tocontrol the purity of the sample. The samples werediluted in DPBS (Gibco), and measurements were
performed on 7 µL droplets, in triplicate.

aratinga.bio is a preclinical stage biotechnology
company developing an off-the-shelf CAR-T therapy
allowing the in vivo targeting and transduction of T-cells,
relying on the use of lentiviral vectors.
In the context of the production of pilot batches and
GMP batches of lentiviral vectors, aratinga.bio is looking
for innovative and relevant solutions to bettercharacterize its viral particles. To quantify the physicalviral titer, two techniques are used as internalreferences: p24 ELISA and Tunable Resistive Pulse
Sensing (TRPS) with qNano. Myriade is a French
company developing Videodrop: a new type of
microscope that measures the concentration of
nanoparticles from a single drop of sample (4,5). Theinterest of this method lies in its simplicity: themeasurement is real-time, label-free, filtration-free,
non-destructive and only requires 5 to 10 µL sample
volume. This technology can therefore be particularlyadapted to evaluate a lentiviral vector titer.

Figure 1. Lentiviral vector sample image obtained with
Myriade technology.

p24 protein quantification
Cell Biolabs’ QuickTiter™ HIV Lentiviral Quantitation Kit(HIV p24 ELISA) was used for virus quantification. It is anenzyme immunoassay developed for detection andquantitation of the HIV-1 p24 core protein. A mousemonoclonal antibody to HIV-1 p24 is coated onto strip
wells of microtiter plate. The quantity of HIV p24 antigen
is determined by comparing its absorbance with that ofknown recombinant p24 antigen standard curve1.

Here we compare back to back Myriade technology withp24 protein quantification and TRPS on two identified
needs: characterization of the final lentiviral vector
product – after purification and concentration – and inprocess controls during Upstream Process (USP) andDownstream Process (DSP) steps.

TRPSMethods

qNano is an equipment relying on tunable resistive pulse
sensing (TRPS). It is based on the Coulter principle,
which states that particles pulled through a pore, while
an electric current is applied, produce a change inimpedance that is proportional to the volume of theparticle traveling through the pore (6). The samples
were analyzed using NP150 nanopores (Izon ScienceLtd., New Zealand), after prior dilution in DPBS (Gibco)
and 0,22 µm filtration. Measurements were performedon 35 µL of sample, in triplicate.

Three techniques were used to evaluate the lentiviralvector physical titer.

Myriade technology
Videodrop consists of a custom microscope relying on
an interferometer phenomenon to produce images
where nanoparticles in solution can be detected (3). The
instrument records videos where nanoparticles andmicroparticles can be seen. Figure 1 shows diffraction
limited spots of lentiviral vectors – approximately 110
nm diameter. The recorded films can be used to countthe nanoparticles and estimate the sample
concentration as well as tracking their movement toestimate their hydrodynamic radius. The microscopemagnification and camera speed allow for small sample
volumes (down to 5 µL) and fast measurement, under
one minute. The samples do not need either labeling or

All three techniques were performed on the samples ofinterest, stored at -80°C after production and thawed atroom temperature.

1The calculation is based on the fact that there areapproximately 2000 molecules of p24 per Lentiviral Particle(LP), therefore, 1 LP contains: 2000 x 24 x 103 / (6 x 1023) g of
p24 = 8 x 10-5 pg of p24

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Results and discussion
Characterization of the final lentiviralvector product(APIs) and intermediates (7). Researchers should
characterize the key production steps in the processcontrol and define the limits of critical parameters inorder to avoid process deviation (1). This approach also
allows process improvement in real-time and ensures
reproducibility over time.

Prior to the release of raw materials used for CAR-T cell
products production, appropriate tests must be carriedout to ensure that the products fulfil regulatory criteria(1). When a lentiviral vector batch is released, the titerof the fraction of interest – thereafter called Drug
Product (DP) – must be precisely determined. It can also
be compared with that of previous batches, in order todemonstrate reproducibility.

During USP/DSP steps – involving production,clarification, purification, concentration, diafiltrationand filtration – the lentiviral vector physical titer, as wellas the infectious titer, can be strongly impacted.
Therefore, in order to monitor in real time theproduction quality and to calculate yields, the titerevolution has to be evaluated during the process
through in-process control.

The physical titer of 8 Drug Products was measured withp24 ELISA, qNano and Videodrop. The results of p24
ELISA and Myriade instrument show a strong correlation
together (correlation coefficient R² = 0.95), and slightly
less with the qNano (correlation coefficient of R² = 0.85).

The physical titer of 6 fractions of interest in 4 different
batches was measured with p24 ELISA, qNano andVideodrop. The evaluated fractions were: harvested
bulk (HB), clarified harvested bulk (HCB), clarified
harvested bulk after benzonase treatment (HCBB), AEX
eluate (E2), drug substance (DS) – after tangential flow
filtration – and drug product (DP) – after 0,22 µm
filtration.

A

p24 ELISA and qNano titers correlate particularly wellwith Myriade instrument results (correlation coefficient
R2 = 0.96 and R2 = 0.94 respectively).

AB

BFigure 2. Comparison of methods measuring physical titer onDrug Products. (A) Correlation between p24 ELISA andVideodrop. (B) Correlation between qNano and Videodrop.

In-process controls
In-process control tests are now the most widely used
tests/criteria to monitor the progress during
manufacturing of active pharmaceutical ingredients

Figure 3. Comparison of methods measuring physical titer on
Drug Products. (A) Correlation between p24 ELISA andVideodrop. (B) Correlation between qNano and Videodrop.

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Conclusion

References

In this study, a comparison of three methods to measurelentiviral vector physical titer for DP release andUSP/DSP in-process control has been performed.
Measurements achieved with Videodrop correlate wellwith qNano, with correlation coefficients R2 = 0.95 andR2 = 0.85 for in-process controls and DP releaserespectively. The correlation was even higher with p24
ELISA results, with R2 = 0.96 for DP and R2 = 0.93 for inprocess control on various USP/DSP fractions of interest.

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method, consuming low volumes of product. Hence it
could be used for Drug Product release as well as offline
in-process controls.

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© 2019 aratinga.bio and Myriade. The informationin this application note is current of October 2019,
unless otherwise noted.

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