Integrated Method for Purification and Single-Particle Characterization of Lentiviral Vector Systems by Size Exclusion Chromatography and Tunable Resistive Pulse Sensing
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Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes. In addition to viral marker protein and total protein analysis, a well-established single-particle characterization technology, termed tunable resistive pulse sensing, was employed to analyze fractions of highest particle load and purity and characterize the preparations by size and surface charge/electrophoretic mobility. With this study, we propose an integrated platform combining size exclusion chromatography and tunable resistive pulse sensing for monitoring production and purification of viral particles.
KeywordsViral vectors Virus-like particles Lentivirus Extracellular vesicles Gene therapy Vaccine development Size exclusion chromatography Tunable resistive pulse sensing Single particle analysis
Nanoparticle tracking analysis
Product-enhanced reverse transcriptase assay
Resistive pulse sensing
Size exclusion chromatography
Tunable resistive pulse sensing
Several virus families can be employed for biotechnological or biomedical applications, among them adenoviruses [1, 2], adeno-associated viruses , and retro- or lentiviruses . Lentivirus (LV) vectors, derived mostly from human immunodeficiency virus (HIV), are widely used for research (e.g., to express recombinant proteins or generate transgenic animals)  and biomedical applications (e.g., in gene therapy or for vaccine development) [6, 7]. Preparations need to be appropriately pure for any envisaged use. Purification strategies for LV particles include density gradient ultracentrifugation, ultrafiltration, precipitation, and different chromatography approaches including affinity-based systems [8, 9, 10, 11]. Choice of the method is usually dictated by the stability of the virus, scalability, available infrastructure, and economic considerations.
As a model system LV producer cell lines based on the STAR system generating HIV-like particles optionally modified with a retroviral surface glycoprotein from murine leukemia virus (amphotropic 4070A MLV Env) [38, 39] were used, to see whether differences can be determined as a result of the presence of the viral surface protein. To our knowledge, LV particles (with and without Env) were subjected to the size exclusion chromatography (SEC) purification system developed for extracellular vesicles, termed qEV, for the first time. Using TRPS we have shown that the size, concentration, and electrophoretic mobility of the viral preparations can be measured in a quick and straightforward fashion. We have successfully extended the range of analytes to LV and herpes virus particles. In this report, we thus demonstrate the potential of a single workflow, combining SEC by qEV and TRPS in monitoring the production of LV (e.g., for size, concentration).
Materials and Methods
Viral Vector Preparation
STAR and STAR-A-HV (ECACC Nos. 04072119 and 04072115, respectively) are lentiviral producer cell lines based on HEK293T cells. Herpesvirus preparations were produced by de novo infection of CrFK cells (ATCC accession CCL-94™) with feline herpesvirus type 1 (FHV-1), as described previously . Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. Concentration of viruses previous to purification was either carried out by ultracentrifugation or by ultrafiltration as described previously .
Size exclusion Purification of Vector Preparations
For purification of virus samples, commercially available size exclusion columns (qEV, Izon Science, New Zealand) were used. The procedure was carried out as described by the manufacturer. In brief: 0.5–1 ml of clarified cell culture medium (centrifuged at 2200×g and filtered through 0.45-µm syringe filters) was overlaid on qEV size exclusion columns (Izon Science Ltd, New Zealand) followed by elution with PBS. 0.5 ml fractions were collected (and used immediately or stored at −80 °C before further analysis). The columns contain 10-ml resin material (with a pore size of approximately 75 nm) and has a void volume of 3 ml and a nominal separation size of 70 nm.
Total Protein Concentration Measurement
Levels of protein were determined using the BioRad ProteinDC quantification kit according to the manufacturer’s instructions. Samples were analyzed in microtiter plates using a Tecan Genios plate reader.
Samples were subjected to SDS-PAGE using 10% gels and a Laemmli buffer system. Silver staining was carried out as described previously .
Samples were subjected to SDS-PAGE using 10% gels and a Laemmli buffer system. Proteins were electro-transferred onto PVDF membranes (GE Healthcare) and incubated o/n in blocking buffer (4% milk powder w/v; 1% bovine serum albumin w/v in tris-buffered saline containing 0.1% Tween 20). Primary antibody purchased from Polymun (Vienna, Austria) was used in a 1:1000 dilution. HRP-labeled anti-murine secondary antibody (from DakoCytomation) was used in dilutions of 1:10,000. ECL detection kits (GE Healthcare) were used for generating signals, which were developed and recorded using an AGFA Curix 60 developer and film (GE Healthcare).
Product-Enhanced Reverse Transcriptase (PERT) Assay
PERT assays infer viral particle numbers from the levels of reverse transcriptase enzyme activity. Assays were carried out as described previously .
Tunable Resistive Pulse Sensing (TRPS) Analysis
All measurements were conducted using the qNANO (Izon Science Ltd., New Zealand). The lower fluid cell always contained the electrolyte buffer (75 μl). The upper fluid cell always contained 40 μl of sample (that was suspended in the buffer) when a measurement was being completed with pressure. Prior to TRPS analysis, all samples were vortexed for 30 s. Between each sample run, the system was washed by placing Izon Science’s Solution Q or PBS containing 0.1% Tween 20 (40 μl) into the upper fluid cell several times with various pressures applied to ensure there were no residual particles remaining and therefore no cross contamination between samples. To ensure the most efficient measurements, all solutions are filtered using 0.45-µm filters before use. Also, degassing of all solutions is recommended. Increased purity will facilitate measurements. A detailed description of such a tunable resistive pulse sensing device can be found in Willmott et al. and Vogel et al. [21, 25, 29, 42]. The concentration, size distribution, and electrophoretic mobility of particles were analyzed using a NP100 nanopore (Izon Science Ltd., New Zealand) at a 45-mm stretch. The choice of the right nanopore size is critical for efficient measurements. The concentration of particles was standardized using multi-pressure calibration with 70-nm carboxylated polystyrene beads at a concentration of 1.5 × 1011 particles/ml (Izon Science Ltd., New Zealand).
Calculations and Software
Relative data were calculated by dividing absolute values by the levels in the starting material and multiplying by one hundred to obtain percentages. Recovery rates were determined by combining the values from fractions containing the most viral markers and dividing by levels in the starting material, followed by multiplication with one hundred to obtain percentages. Purity was calculated by dividing a measure of concentration of viral marker with a measure of total protein content, providing a measure for viral marker per µg total protein. The higher the value, the more pure is the preparation. Enrichment is calculated by dividing the purity value of a given fraction by the purity value of the starting material. All TRPS measurements were processed with proprietary data capture and analysis software (Izon Control Suite v.3.3). Densitometry analysis on p24 immunoblots and silver-staining images were carried out using ImageJ .
Biochemical Analysis of Purified Virus
Concentration, Size Distribution, and Charge Analysis of Virus by TRPS
178 ± 23
145 ± 13
178 ± 7
137 ± 7
223 ± 1
222 ± 1
Recovery and Purity of qEV-Purified Lentiviral Samples
Purity of selected fractions
By p24/total proteina
By PERT/total proteinc
Our studies using qEV columns indicated the potential for this method in the fractionation (and subsequent characterization) of lentiviral preparations by size exclusion. A clear separation effect between viral proteins and bulk total proteins was observed, probably most vividly recognizable in images from silver-stained PAGE gels (see Fig. 2b, upper panel). Signals for virus-specific markers from PERT and immunoblots are also observed in the higher fraction, indicating lower molecular weight components. These represent viral debris or non-aggregated proteins, not associated with or organized in complete virion particles . Quantitative differences between these two peaks (particulate and debris) can be indicative of the quality of a viral preparation. The more particulate and the less debris viral markers are observed, the higher the quality of the preparation is.
TRPS data for viral concentration matches well with the biochemical analysis, i.e., fractions 8 and 9 contain the greatest number of particles (see Fig. 3a). The higher numbers observed for the STAR-A-HV derived particles compared to STAR particles are also reflected in the starting material (see Fig. 3a, columns before qEV). This may either be a consequence of the production capabilities of the respective producing cell lines, or reflect stability of the particles. The lack of the MLV 4070A Env protein may weaken the integrity of the viral envelope. In addition to this, while the size of particles derived from STAR and STAR-A-HV cells is quite similar, the observed standard deviation is increased for STAR compared to STAR-A-HV derived particles (see Table 1). Again, this may suggest greater heterogeneity and/or decreased stability of the Env-less viral variant. When compared to a different method for single particle analysis, NTA, both results and observed challenges were comparable. Representative outcomes for two viral preparations (STAR and FHV-1) are depicted in Supplementary Fig. 2. Size measurements agree exceptionally well: for STAR a mode diameter of 145 nm by TRPS is matched by 131 nm by NTA; for FHV-1 the respective values are 222 nm (TRPS) and 216 nm (NTA) (see also Table 1 and Supplementary Fig. 2). For further comparison of SPA techniques, additional recent information is available [13, 15].
An interesting side aspect emerged from the analysis of electrophoretic motility. The charge, or zeta potential (as measured by DLS and RPS) of enveloped viruses, such as the lenti- or herpesviruses used in the study is considered low (roughly at ±5 mV) . Alternatively, isoelectric points can be determined for intact virus particles . Not surprisingly, there was no significant difference observed between STAR and STAR-A-HV derived particles because they only differ in one determinant present in low copy number (the MLV 4070A env; see Fig. 3c, compare yellow and black dots). However, a clear difference was visible between STAR-derived and DTT-treated herpesvirus particles. The blockade duration is shifted toward longer intervals, most likely indicating a reduced electrophoretic mobility as a consequence of reduced zeta potential or charge (see Fig. 3c; compare full and broken ellipsoids, indicating virus populations). This strongly suggests that biochemical differences between viral species and/or chemical changes to viral surfaces can be detected by TRPS. This fact can be further developed as an analytical tool to differentiate between different virus types or states.
Measurement differences especially between TRPS, PERT, and p24 analysis are best seen in recovery rates (see Table 2) and probably indicate the stringency levels of the techniques . While PERT requires a functional RT molecule, presence of the p24 capsid protein epitope is sufficient for detection. In TRPS measurements, all particles of a certain size are counted, irrespective of their origin. The good agreement of data between TRPS and p24 recovery rates (57–48%) indicates that the vast majority of counted particles are indeed viral particles. Enrichment values for the fraction eight containing most viruses were in good agreement (60–57%) for the two measurement methods (see Table 3, in bold). However, other fractions do not agree (i.e., fractions 7 or 9). When measured values get smaller, the quantitative content of the measurement is reduced. This is especially true for the p24 immunoblot density data. This introduces a stronger variation in the weaker bands, most likely leading to the irregularities in enrichment data.
In this study we have demonstrated the application of commercially available SEC columns (Izon science qEV) to the purification of lentiviral preparations. Protein contaminants can be efficiently removed from the samples in a fast and cost-friendly manner. Additionally, we employed TRPS (Izon qNANO) to characterize viral preparations for titer, size and electrophoretic mobility simultaneously, demonstrating important potential for virus production processes.
Open access funding provided by Austrian Science Fund (FWF). The authors would like to thank Dimitri Aubert and John A. Dangerfield for critically reading the manuscript. This study was supported by a grant from the Austrian Science Fund (FWF Grant No. P25297). NTA measurements were conducted with Haider Sami at the University of Vienna Division of Clinical Pharmacy and Diagnostics.
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