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Lentiviral vectors (LVVs) are used as a starting material to generate chimeric antigen receptor (CAR) T cells. Therefore, LVVs need to be carefully analyzed to ensure safety, quality, ...and potency of the final product. We evaluated orthogonal and complementary analytical techniques for their suitability to characterize particulate matter (impurities and LVVs) in pharmaceutical LVV materials at development stage derived from suspension and adherent manufacturing processes. Microfluidic resistive pulse sensing (MRPS) with additional manual data fitting enabled the assessment of mode diameters for particles in the expected LVV size range in material from adherent production. LVV material from a suspension process, however, contained substantial amounts of particulate impurities which blocked MRPS cartridges. Sedimentation-velocity analytical ultracentrifugation (SV-AUC) resolved the LVV peak in material from adherent production well, whereas in more polydisperse samples from suspension production, presence of particulate impurities masked a potential signal assignable to LVVs. In interferometric light microscopy (ILM) and nanoparticle tracking analysis (NTA), lower size detection limits close to ∼ 70 nm resulted in an apparent peak in particle size distributions at the expected size for LVVs emphasizing the need to interpret these data with care. Interpretation of data from dynamic light scattering (DLS) was limited by insufficient size resolution and sample polydispersity. In conclusion, the analysis of LVV products manufactured at pharmaceutical scale with current state-of-the-art physical (nano)particle characterization techniques was challenging due to the presence of particulate impurities of heterogeneous size. Among the evaluated techniques, MRPS and SV-AUC were most promising yielding acceptable results at least for material from adherent production.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a ...challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (R
) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae
(T), conditioned culture media of microalgae
(P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In R
, ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from
flagella (T) did not have a globular shape, so the interpretation by R
of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from
conditioned culture media (P), which provides an explanation for the considerably larger R
obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and R
of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Virus particle concentration is a critical piece of information for virology, viral vaccines and gene therapy research. We tested a novel nanoparticle counting device, “Videodrop”, for its efficacy ...in titering and characterization of virus particles. The Videodrop nanoparticle counter is based on interferometric light microscopy (ILM). The method allows the detection of particles under the diffraction limit capabilities of conventional light microscopy. We analyzed lenti-, adeno-, and baculovirus samples in different concentrations and compared the readings against traditional titering and characterization methods. The tested Videodrop particle counter is especially useful when measuring high-concentration purified virus preparations. Certain non-purified sample types or small viruses may be impossible to characterize or may require the use of standard curve or background subtraction methods, which increases the duration of the analysis. Together, our testing shows that Videodrop is a reasonable option for virus particle counting in situations where a moderate number of samples need to be analyzed quickly.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Research on extracellular vesicles (EVs) and bacteriophages (phages) has been steadily expanding over the past decades as many of their roles in medicine, biology, and ecosystems have been unveiled. ...Such interest has brought about the need for new tools to quantify and determine the sizes of these biological nanoparticles. A new device based on interferometric light microscopy (ILM), the Videodrop, was recently developed for this purpose. Here, we compared this new device to two nanoparticle tracking analysis (NTA) devices, the NanoSight and the ZetaView, for the analysis of EVs and phages. We used EVs isolated from bacteria, fecal samples, bovine milk and human cells, and phages of various sizes and shape, ranging from 30 to 120 nm of diameter. While NTA instruments correctly enumerated most phages, the Videodrop detected only the largest one, indicating a lower sensitivity threshold compared to the NTA devices. Nevertheless, the performance of the Videodrop compared favourably to that of the NTA devices for the determination of the concentration of eukaryotic EV samples. The NanoSight instrument provided the most precise size distributions but the Videodrop was by far the most time‐saving device, making it worthy of consideration for studies conducted on a large number of samples composed of nanoparticles larger than 90 nm.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by ...differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000×
contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 μm) and meso-sized particles (MSPs) (cca 400 nm-2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300×
. The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
There is a constant need for direct counting of biotic nanoparticles such as viruses to unravel river functioning. We used, for the first time in freshwater, a new method based on interferometry ...differentiating viruses from other particles such as membrane vesicles. In the French Marne River, viruses represented between 42 and 72% of the particles. A spring monitoring in 2014 revealed their increase (2.1 × 107 to 2.1 × 108 mL−1) linked to an increase in algal biomass and diversity of bacterial plankton. Predicted virus size distributions were in agreement with transmission electron microscopy analysis suggesting a dominance of large viruses (≥60 nm).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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