Why is the titer of my virus so low?

Based on our experience, if a customer buys viral vectors from VectorBuilder but chooses to package virus on their own rather than using our virus packaging service, there is a greater than 50% chance that the resulting titer is significantly below the optimal level. This could lead to a great deal of unhappiness. In many cases, customers found that once they utilized our packaging services, they received the high-titer virus they needed. VectorBuilder has extensive experience, and we've shared some of our tips and tricks to achieving high viral titer in the VectorAcademy post Slipping Under Titer.

VectorBuilder’s virus packaging service employs extensively optimized protocols and many proprietary reagents and techniques to ensure high titer, high purity and consistence even in the case of difficult vectors. When customers do their own packaging, they typically use standard protocols that have not been optimized. This, coupled with a lack of experience, can lead to low titer. We often see customers make low-level mistakes such as using the wrong packaging cells or helper plasmids. For example, some customers mistakenly used the 2nd generation lentiviral packaging system to package our vectors (all VectorBuilder lentiviral vectors are 3rd generation).

Technical competence of personnel aside, some viral vectors are inherently hard to package due to various reasons. One of the most common causes is that the viral vector carries a gene that is toxic to packaging cells such that the packaging cells are killed or very unhealthy. For some of these cases, VectorBuilder has developed proprietary technologies to overcome the toxic gene effect so the virus can still be made. Another common cause is that the size of the insert fragment is too big for the viral packaging machinery to handle. We recommend that you limit the insert size to 6.4 kb for lentiviral vectors, 8.3 kb for adenoviral vectors, 4.7 kb for AAV vectors, and 5.5 kb for MMLV vectors. An insert fragment exceeding the above listed cargo limit may result in compromised viral production. Also, based on our experience, if the viral vector contains very high GC sequence (> 70% across a few hundred bases), it can reduce packaging efficiency.

Properly packaged high-titer virus could also lose titer over time. This is especially true for lentivirus, which is unstable and can quickly lose infectivity if not frozen at -80°C or if subjected to repeated freeze-thaw cycles. Adenovirus and AAV are much more stable but may also lose infectivity if left unfrozen for a very long period of time or otherwise mishandled.

Sometimes, the experimental circumstance in which VectorBuilder’s virus preparation is used may give a customer the false impression that the viral titer is much lower than what is indicated. This can happen if the customer is using the virus on a cell type that is much harder to transduce than what we used to measure titer. It also happens frequently that the customer estimates titer from the number of transduced cells that express either a fluorescent or drug-selection marker carried on the viral vector. This approach could severely under-estimate titer because a fluorescent or drug-selection marker may fail to be expressed at detectable levels in some host cells due to silencing or some other effect, and also because one cell may be infected by multiple viral particles. For these reasons, it is important to first perform small-scale tests of a virus preparation on the cells of interest at several multiplicity of infection (MOI) to find an optimal MOI before conducting large-scale transduction experiments.

We offer virus packaging services for lentivirus, adenovirus, AAV and MMLV. We strongly suggest that you choose our packaging service because our proprietary packaging reagents, packaging cells and protocols are optimized for our viral vectors. As a result, we can generally produce higher titer, better purity, and more robust live virus than our customers can on their own. You will also find that our prices are highly competitive and likely lower than the cost of making virus on your own.