Flash BioSolutions has optimized a purification and concentration platform specifically for viral vector production. Our production processes are customized based on the target cell type, ensuring high titer and exceptionally pure viral particles which allow to fully maintain target cell viability, phenotype, and proliferation capacities.

During the production of viral vector, both efficient and non-efficient particles are produced. Efficient and complete particles are quantified by qPCR as integrated genome (IG) and by cytometry as transduction unit (TU). Physical Particles (PP) are quantified by p24 ELISA and represent all particles, i.e., not only efficient particles but also damaged/empty particles and free p24 protein (see figure below). Consequently, the PP/TU or PP/IG ratio is a quality control criterion for the vector product, as it is better to have a minimum of damaged/empty PP in the viral supernatants to retain only the efficient particles. Reliable and discriminating titration methods are therefore needed for the different types of particles.

A titer in TU/ml or IG/ml is required for an accurate calculation of effective vectors applied to the cells. The IG/mL or TU/mL titer provided by Flash BioSolutions is a precise titer of effective viral particles. It represents the titer determination of integrative particles only. Calculating the volume of vectors to be used using such titration is then more accurate, and experimental results can be extrapolated and compared from one experiment to another.

The titer information provided by Flash BioSolutions is a functional titer determined by qPCR measurement of only the viral particles that are able to transduce cells (Integrated Genome/mL), giving a measure of the vector’s potency. Specifically, IG corresponds to the number of integrated genomes generated by transducing HCT116 cells with several serial dilutions of viral supernatant. Data are reviewed and validated as part of quality control to ensure that the viral vectors meet the required specifications.

The titer information provided by Flash BioSolutions can be a functional titer determined by flow cytometry measurement of only the viral particles that are able to transduce HCT116 titration cells (TU transducing Unit/mL). Specifically, the titer deduced from FACS experiments corresponds to the level of expression of the protein of interest and, as such, can be considered as a functional titer. Data are reviewed and validated as part of quality control to ensure that the viral vectors meet the required specifications.

Our viral vector platforms are tailored to meet the specific needs of different applications, with each platform offering distinct levels of purity and quality control measures to ensure optimal performance. We offer a comprehensive range of options designed to meet different vector production needs. The entry-level VectaStart offers reliable performance for a wide range of applications. It is optimized for larger, high-throughput needs where extreme purity is not as critical. It is suitable for initial research, high-volume screening? and applications where cost-effectiveness is a priority over the highest possible purity levels. VectaPremium is the advanced offering in the Vecta series, engineered to deliver superior purity and quality. It undergoes enhanced purification processes to guarantee higher levels of vector purity, including advanced filtration techniques that minimize contaminants and residues, ideal for applications requiring the highest purity standards, where even minimal contaminants could impact outcomes or safety.

In addition, the production report of the transfer plasmid batch used for viral vector production is provided, as well as the plasmid cloning report, when the transfer plasmid has been constructed on demand.

Because the titer of each batch of viral particles varies, the volume required for transduction will also vary. The titer can be found on the Certificate of Analysis (CoA) provided with each viral vector batch. Use the formula detailed in the Customer Recommendations document to calculate the volume of integrative viral vector for transduction. Please refer to the other customer recommendations manual for additional information. 

Viral vectors volume required (µl) = Number of cells to transduce / Viral vectors Titer (IG/ml) x Multiplicity of Infection (M.O.I) x 1000

These products are designated as biosafety level 2 biological agents. Follow the biosafety procedures for use of retroviral-derived vectors in accordance with all applicable regulations. All Flash BioSolutions manufactured viral vectors are produced from separate transfer, envelope, and packaging components onto different plasmids, which results in the generation of replication-incompetent vectors. Furthermore, all transfer plasmids include two additional safety features, with the use of a chimeric tat-independent 5’LTR and the self-inactivating (SIN) modification of the 3’ LTR. As a result, while the viral vectors maintain the ability to transduce a wide range of cell types, they are not capable of producing replicative particles from transduced cells, as assessed by RCL/RCR testing. Please refer to the MSDS documents for more information.

The viral vectors must be stored at -80°C immediately upon receipt.

Vectors must be taken out of the -80°C freezer at the last moment. On removal from the -80°C freezer, viral vectors must be placed on ice to begin the thawing protocol. Once thawed, the vectors must be rapidly used for transduction. It is also essential to avoid thermal shock when handling viral vectors. If vectors are to be diluted in cell culture medium, use medium at room temperature once the vector has thawed, to avoid thermal shock and ensure particle efficiency.

Thaw the particles according to the volume of your vial, as described below.
For a volume of viral vectors higher than 1mL: Just before transduction, take the tubes of viral vectors out of the -80°C freezer and thaw them at room temperature or in a 37°C water bath. Before the last ice-cube disappears, place the tube at room temperature to complete thawing and start transduction.

For a volume of viral vectors between 1mL and 100µL:  Just before transduction, take the tubes of viral vectors out of the -80°C freezer and thaw them at room temperature or in a 37°C water bath. Before the last ice-cube disappears, place the tube at room temperature to complete thawing and start transduction.

For a volume of viral vectors lower than 100µL: Just before transduction, take the tubes of viral vectors out of the -80°C freezer and thaw them on ice at 4°C. 5 minutes before transduction take the tubes out of the ice and allow them to warm to room temperature.

Viral vectors are sensitive to freeze-thaw cycles. Flash BioSolutions therefore recommends avoiding freeze-thaw cycles for your viral vectors, which is why they are packaged in several aliquots. If absolutely necessary, we recommend aliquoting viral vectors on ice in working volumes for your experiments, when you perform your first transduction experiment. An increasing reduction in titer for every freeze-thaw cycle should be expected.

To avoid this aliquoting step, you can request a specific aliquoting for your project when you place your order.

We do not recommend using the same aliquot of viral vectors more than once. In addition, the remaining volume of any thawing viral vectors should be discarded after the transduction experiment.

The optimal media will vary based on the cell type. The best medium to use is usually the medium used for your cells’ culture. No need for any specific medium related to transduction using viral vectors.

The CRISPR-Cas9 system is widely used for functional genomic screening, with single-guide RNA (sgRNA) libraries, targeting thousands of genes and allowing screening for gene activation or inhibition. These libraries are commonly packaged into viral vectors. When multiple transductions are required, how long should I wait before the second transduction? If necessary, we recommend waiting a minimum of 48 hours before performing a second transduction.

Is it possible to perform specific cell targeting with your vectors? We developed a range of viral vectors allowing specific gene expression in particular tissues, cell types or cell states. With reporter expression under the control of specific promoters, these vectors enable to label specific tissues (liver, pancreas,…), subcell types in a tissue (neurons, astrocytes, germline cells,…) or cells in a specific state (proliferative, senescent, …).

We can produce pooled viral sgRNA libraries, starting from a provided transfer plasmid library, expressing the different sgRNAs (as well as Cas9 coding sequence, if required). The vector concentration grade and final volume will have to be adapted to the library complexity, to ensure a sufficient number of integrations per sgRNA in your screening experiment.

We achieve excellent transduction rates for all cell types, including those difficult to transduce, such as primary cells and stem cells. For difficult-to-transduce cells or sensitive cells, we recommend using the Premium grade to achieve the highest transduction efficiency.