• Titer and Purity - What you should know


    Viral vectors used in research as well as gene therapy applications provide a multitude of advantages. The area of gene and cell therapy based medicines is experiencing a significant resurgence, notably due to the introduction of transfer viral vectors including adenovirus, adeno-associated virus (AAV) and lentivirus. Vector quality as well as purity and potency are extremely important. Titer determination, as part of the testing package for viral vectors, is a critical measurement.

    High Titer, High Purity Production Options

    AAV packaging and purification

    AAV custom cloning

    Lentivirus packaging and purification

    Lentivirus custom cloning

    Adenovirus production, amplification and purification

    Trans-splicing system for large gene delivery

    GMP grade viral vectors

    Troubleshooting Tips: Achieving Titer and Purity

    Viruses are complicated biological units. They perform intricate yet complicated bioligcal tasks. What do we do when we are presented with technical hurdles, namely, no expression. Below we share some of the common obstacles you may come across, and actions you can take to remedy the problem. For specific troubleshooting of your AAV, adenovirus and/or lentivirus please refer to their corresponding webpage or explore our Product Manuals, Handbooks & Vector Maps. Additionally, refer to our FAQs.

    Preventing loss of titer by minimizing freeze-thaw cycles

    Viral stocks may have a sensitivity to freeze thaw cycles, thereby reducing the end titer. To avoid the possibility of reduced titer due to freeze-thaw cycles, storage should be at -80C upon receipt and for long term storage. Storage for short periods of time at -20 or +4°C. Whenever possible, vectors should be aliquoted into single use portions to avoid repeated freeze/thaw cycles. Please aliquot in at least 10ul per tube and use low protein binding tubes to avoid loss of virus.

    Viral titer has an affect on transduction efficiency

    Unfortunately, some vectors will give a low titer. Additionally, the titering method\ chosen can produce differing titer results between the physical versus the functional titer. To overcome a low titer, Increasing low titer can be done by concentrating viral stock, accomplished by ultracentrifugation, followed by resuspension in a smaller volume. Alternative methods of concentrating viral stock can be accomplished by using smaller volumes of culture.

    Increasing transduction efficiency

    Variety of reagents are available that can increase transduction efficiency. This is accomplished by adsorption of a virus particle to a target cell.

    Unsuccessful transductions

    First step is to check if your virus is being generated by the transfected packaging cells. Perform antibiotic selection 72 hours post transfection. This can be easily done by taking advantage of the selectable antibiotic resistance gene, which is encoded in the viral vector. This method should give rise to a surviving population, approximately 20–50%, of packaging cells after 1–3 days of selection.

    Focus on Discovery, Not Viral Gene Delivery

    Vigene Biosciences provides high purity, high titer viral vectors for both life science research, gene therapy and cell therapy purposes.

    Viral Services: Turnaround time, Titer and Price

    Viral Service

    Expected Minimum Titer

    Price

    Turnaround time

    Viral Vector
    Construction

    Please inquire

    7-30 days

    AAV
    Packaging

    Small scale: 1x10E12GC/ml, 500µl
    Large scale: 1x10E13GC/ml, 500µl

    $545
    $1398

    7-14 days

    Lentivirus
    Packaging

    Small scale: 1x10E8 IU/ml, 100µl
    Large scale: 1x10E9 IU/ml, 200µl

    $595
    $1495

    7-21 days

    Adenovirus
    Packaging

    Small scale: 1x10E10vp/ml, 500µl
    Large scale: 10E12-13vp/ml, 500µl

    $545 see note
    $1398 see note

    28-35 days

    Turnaround time: Fluctuations in turn around time may occur and are dependent on individuals project needs. Factors that may affect turnaround time, may include but are not limited to, size of the packaged sequence, vector contains sequences of very high GC content, custom vector design and construction.

    Viral Production

    Viruses are generated using standard methods optimized for each specific virus; adenovirus, adeno-associated virus (AAV) and lentivirus. Following production, all virus preparations are subjected to quality control including titer determination before being distributed to Vigene customers. Details about Vigene's production protocols, titering methods, and additional quality control measures are breifly described below.

    AAV

    AAV packaging involves transfections using the transfer plasmid, a plasmid encoding rep and serotype-specific cap, and a plasmid encoding adenoviral helper sequences. For more detailed information see our section on AAV production.

    Lentivirus

    Lentiviral particles are produced using the 3rd generation packaging system. For more detailed information see our section on Lentivirus production.

    Adenovirus

    The Production of adenoviral vectors is both a tedious and time-consuming process involving the construction of the adenovirus shuttle plasmid, primary stock generation and amplification. For more detailed information see our section on Adenovirus production.

    Viral Titer

    AAV

    AAV particles are titered by real-time quantitative PCR using primers targeting the ITR. The amplicons are detected using SYBR green technology. Titer values are then determined by comparison to a standard curve of a plasmid sample of known concentration. The qPCR assay and corresponding titer determinations are also validated using AAV Reference Material (ATCC).

    Lentivirus

    Titering of lentiviral preparations is performed on product that has been stord at -80C and thawed. This method helps to account for any loss of titer associated with the recipient's initial thaw.

    Viral Purity

    AAV

    AAV Purity is determined by comparing the relative stoichiometric ratios of the viral capsid proteins VP1, VP2 and VP3. This is performed by taking small aliquots of the viral preparation and running them through polyacrylamide gel electrophoresis (PAGE), subsequently followed by silver staining. The molecular weight and relative intensity of the viral capsid proteins are analyzed.

    Further Technical Support

    Please also refer to our frequently asked questions section, or one of our product manuals as the answers to your question(s) may be there.

    Contact Information

    ● Via email: custsupport@vigenebio.com

    ● Call us on (800)485-5808. Office hours are 9:00AM-5:30PM ET.

    ● You may place an order or request a quote here