Viral Vector Industry Boosts Product Quality and Quantity

The viral vector and plasmid DNA manufacturing market is one of the fastest growing global biotechnology segments. Pegged at $321.3 million in 2017, the market is expected to reach $1.1 billion by 2023, according to Allied Market Research. This growth, which is being driven by an increase in funding for R&D related to gene therapy, could be slowed if gene therapy costs remain high or if gene therapy applications bring unacceptable risks of mutagenesis. Nonetheless, Allied Market Research expects that market players will be presented with lucrative opportunities.

Later this month, viral vectors will be a cornerstone of the 10th Spring Meeting of the International Society for BioProcess Technology select ISBioTech) in Norfolk, VA. One of the speakers, Maurizio Cattaneo, PhD, founder and CEO of Artemis Biosystems, plans to deliver a talk describing how continuous perfusion can achieve an order-of-magnitude increase in lentiviral vector select LV) production. During a preconference interview, he noted that “current adherent cell systems for producing viral vectors are not scalable.”

“Suspension cell systems are scalable,” he added, “but they suffer from the cell-density effect where the LV vector production stays the same independent of how high the cell density is. One million cells/mL gives the same cell productivity as 80 million cells/mL on a volume basis.”

At higher cell densities, vector production faces some challenges. “Due to an unknown limitation or inhibition, the specific productivity of infected batch cultures is found to decrease significantly with increasing cell densities,” Cattaneo stressed. This challenge can be addressed in part by growing cells in a perfusion bioreactor, where continuous supplementation of fresh media and removal of metabolic waste products allow productivity to increase with the cell density.

In the world of producing viral vectors, manufacturers want to make as much as possible. “The main challenge in lentiviral production is the low yield of LV vector,” Cattaneo pointed out. To address that challenge, Cattaneo and his colleagues developed Artemis Biosystems’ VHU Perfusion System, which can make clinical or commercial levels of viral vectors from mammalian cells, including HEK 293 or HeLa cells. By design, the VHU Perfusion System can be easily scaled up or customized, Cattaneo asserted. “Using our VHU Perfusion technology,” he continued, “we can increase the total LV yield 30-fold compared to a batch operation.”

Purification platform

In addition to LV vectors, biomanufacturers and biopharmaceutical companies produce adeno-associated viral select AAV) vectors. This side of vector production will also be covered at the ISBioTech meeting. For example, an AAV-related presentation is expected from Pete Gagnon, chief scientific officer at BIA Separations.

In a preconference interview, Gagnon noted that the most challenging part of purifying AAV comes from an invisible contaminant. “It is host DNA, hiding in the form of chromatin,” Gagnon pointed out. Making purification even more complicated is the high host DNA to AAV ratio—50–100 times higher in comparison to proteins like immunoglobulin G. “A bigger DNA burden going into a process reduces purification performance at every step and often leads to excess DNA in the final product,” Gagnon explained. “[Our] methods remove most of the host-cell DNA in advance.”

The next challenge is that empty capsids—ones without vector DNA—must be removed from the full ones. “The amount of chemical differentiation between full and empty capsids is modest at best,” Gagnon noted. “Developing tools to exploit those differences is genuinely difficult, but improvements are possible if you have enough determination and the right resources.” The goal, Gagnon continued, “is to make density gradient ultracentrifugation unnecessary.”

His presentation, Gagnon said, will describe “two new approaches to sample preparation that enable clinical-quality AAV purification with two chromatography steps.” According to Gagnon, Bia Separations has demonstrated “universal select meaning all serotypes) capture on a cation-exchange monolith that supports repeated sanitization with one molar sodium hydroxide.”

Gagnon plans to unveil “preliminary data on a new method for separating empty and full capsids that offers better performance than the known method of salt-gradient elution on strong anion exchangers.” He also intends to give examples that of a new generation of multidetector HPLC assays that “accelerate process development and validation, and also streamline in-process testing during commercial manufacture.”

In some cases, these new assays can provide results in minutes or hours, Gagnon asserted, rather than the days or weeks that would ordinarily be required. “The main outcome,” he emphasized, “is a fast, universal platform for purification of all AAVs—all serotypes, all cultures, all lysis methods—plus the fast assays to document it.”

Off-the-shelf advances

Many methods of gene therapy involve plasmid DNA. This point will be covered at the ISBioTech meeting by James Brown, PhD, vice president of corporate development at Aldevron. His presentation is expected to emphasize the most pressing plasmid DNA challenges in gene therapy, which are, in Brown’s view, speed of delivery, capacity, and throughput.

According to Brown, “The number of programs in gene therapy is growing, the quantities of plasmid they require are increasing, and the programs are rapidly progressing into and through the clinic.” In reference to the issues with plasmid DNA supply, Aldevron and other companies keep ramping up their capabilities.

“We’ve been expanding our capacity and throughput, resulting in immediate availability” of our GMP-Source plasmid DNA,” he said. GMP-Source plasmid DNA can be used as an ancillary or critical raw material for producing protein and viral candidates, typically for use in early-phase studies. “Generation of a master cell bank—MCB—takes weeks, and scaled-up plasmid is turned around in two to three months,” Brown noted. “As for manufacture of GMP MCB’s and plasmid, lead times—though variable—have also been greatly reduced.”

To add a crucial improvement to the entire process, Brown and his colleagues optimize the production for the intended use of the material. As he explained, “The specifications for a viral vector and the plasmid should be tailored to how these components are being used.”

For example, a plasmid that will be directly injected into a person must meet extremely stringent specifications. On the other hand, if a plasmid is a raw material—being used to make something that will be delivered as a treatment—that plasmid could meet less stringent specifications, but still not impact the safety or efficacy of the final product.

In gene therapies based on AAV or LV vectors, some of the same components—various plasmids—can be used to make a variety of viral vectors. So, Aldevron makes some of these critical materials, including pALD-Lenti packaging plasmids and pALD-X80, which is an adenoviral helper plasmid.

“We have these ready to ship,” Brown declared. “If you want clinical-grade helper and packaging plasmids to make LV or AAV vectors, we can ship that tomorrow.” By manufacturing these more universal plasmids at large scale, Aldevron can “drive down the price and reduce the turnaround time to basically zero,” Brown explained. Additionally, Aldevron offers clients research-grade samples of the off-the-shelf plasmids.

As the experts interviewed here reveal, there are many new tools and techniques for viral vectors that may help biomanufacturers scale-up production, improve purification, or employ off-the-shelf parts. These techniques promise to advance several fields, especially gene-based therapies.

With these new techniques, biopharmas working on new gene therapies can more easily obtain the supplies of viral vectors that are needed. In fact, biopharmas can get more vectors in more forms—all on a faster timetable.