A New Way of Gauging Baculovirus Residue in Gene Therapy Products 

In baculovirus expression systems, microbes can unintentionally get introduced into the manufacturing process. This new assay helps weed them out more efficiently 

It might seem strange harnessing a pathogen that preys on insects to churn out gobs of another virus valuable in gene therapy. But that’s what baculovirus does.

A baculovirus is an enveloped double-stranded DNA virus that belongs to the Baculoviridae family, which affects approximately 600 different insect species. (1) These viruses have been exploited for their recombinant protein expression since 1983—about 10 years after the first recombinant proteins were made—enabling the development of a diverse range of therapeutics. (2) The most widely used baculovirus species for basic virology and biotechnology is Autographa californica multiple nucleopolyhedrovirus or AcMNPV, which encodes approximately 156 genes and is highly pathogenic to a variety of larval moths, butterflies and kippers. (1)

Baculovirus expression system enables fast, flexible, and scalable protein production. It generates recombinant proteins in insect cells at high production levels. These systems enable high expression of very complex, glycosylated proteins that cannot be produced in other protein expression systems like the E. coli virus or in yeast cells. (3) Importantly, the first gene therapy to reach the clinic — Glybera, from Amsterdam-based uniQure — was produced from baculovirus-infected insect cells for both clinical testing and commercial release.

The big baculovirus challenge: Host cell proteins

Yet every tool has its challenges and baculovirus is no exception. Arguably the biggest drawback with baculovirus is the high risk of adventitious and endogenous virus contamination— industry-speak for microorganisms (viruses as well as bacteria and other pathogens) that unintentionally get introduced into the manufacturing process.  During production, a high level of residual baculovirus from the host cell sometimes remains in the intermediate and final product. Although not fatal to humans, if left undetected the residual baculovirus has the potential to cause inflammation and other immunogenic responses in patients.

Since a sizeable number of our clients’ gene therapy products are reliant on baculovirus SF9 protein expression system, our lab moved last year to develop and validate an assay to clear both adventitious and endogenous proteins robustly.

The conventional method we had been using during the viral clearance process—the TCID50 assay—involves spiking a model virus into the load material and performing the downstream procedures at small scale under conditions equivalent to the commercial scale. (4) While effective, significant amounts of background baculovirus still remain in the material, and the amount of residual virus can overwhelm the amount of model virus that has been spiked.

A baculovirus assay that is more efficient 

The new assay developed by our lab works differently. Material with and without Baculovirus was used to detect these residuals through a process evaluation cell system and titration assays. What we ultimately found was that the assay was sufficient to replace the legacy assay as the primary test method.

Although the broader macroeconomic impacts that this assay will bring to the biopharmaceutical industry are currently being assessed internally, the impact that it will have on the development process is already evident.

By inactivating and removing baculovirus with detergent treatment and affinity chromatography in early down-stream processing, the newly developed baculovirus assay will achieve better log reduction values of residual virus. Moreover, from a regulatory perspective, we anticipate that the inclusion of this assay will result in a smoother, more efficient approval process when our clients file their product with the FDA.

Once implemented into the manufacturing process, we hope to reduce the overall number of purification steps needed to achieve the desired result which will help decrease the time and costs associated with the development of next generation biotherapeutics.

So, what is the takeaway? Certainly, that a new assay is removing some wrinkles from the use of this viral expression on system. But the larger point is the importance of working the science. Whether you are designing a cell therapy or manufacturing it, there will always be roadblocks blocking progress and savvy labs willing to find alternative routes.

Nick Bartosik is a Sr. Marketing Specialist for Biologics Solutions at Charles River Laboratories. His main focus is on the design, development, and implementation of global marketing strategies. Nick grew up in St. Louis, Missouri where he graduated from Southern Illinois University in Edwardsville and currently resides in Grand Rapids, Michigan. Nick has held various marketing roles in the pharmaceutical manufacturing and biotech industry and is a passionate advocate for the user experience.