Viral vector producer cell lines: Learning from mAbs

Where we are now with viral vector manufacturing for gene therapies

The field of gene therapy has blossomed since 2017, and numerous treatments for rare diseases have been approved. With the proven success for treating uncommon inherited conditions, potential therapeutic indications are expanding into more prevalent conditions, such as age-related macular degeneration and diabetic vascular complications (1). With one exception, the approved treatments use viral vector-based gene delivery. Adenovirus and adeno-associated virus (AAV) vectors collectively accounted for approximately 80% of gene therapy clinical trials in 2021 (2). AAV vectors have emerged as a frontrunner because they offer the advantages of low immunogenicity, a broad range of infectivity, and a low biosafety rating compared with adenovirus.

Groundbreaking gene therapies have reached the market using technology and processes geared for speed but not necessarily scalability and efficiency. Current AAV production methods are not sufficient to meet the demand for new and existing therapies and broaden patient access across the globe. Fortunately, the journey of monoclonal antibody (mAb) production over the last decades provides many lessons that we can draw from in the next generation of viral vector processing.

 

How mAb production has evolved — and what it means for viral vectors

Monoclonal antibodies have dramatically changed the landscape of biologics available today. In fact, the 100th mAb was approved by the US FDA in 2021 (3). But blockbuster status didn’t happen overnight. Transient transfection has been explored for expressing recombinant mAbs; however, it’s not used for clinical supply (4). Over the years, technological advances have led to leaps in process improvements for mAb production, with Chinese hamster ovary (CHO) cells emerging as a common host. In this article we’ll focus on cell line development and cell culture process development where vector design, clone selection, media and process optimization, as well as critical quality attribute control are all key enablers to this success.

Although other cell types are in use, a large percentage of AAV processes are based on human embryonic kidney (HEK) cells grown in adherent mode with genes delivered by triple-plasmid transient transfection. Beyond 2D flatware, these processes can be adapted to single-use fixed-bed bioreactors, and there’s been much improvement recently in scaling the transient transfection reproducibly at large scale. HEK293 cell line development is taking a similar journey for viral vector production as CHO cell line development took for mAbs.

The touchstone to the ‘revolution’ of mAb therapy production starts before the upstream process, with CHO host cells adapted to suspension and producer cell lines. The transition to suspension and stirred-tank bioreactors is well underway for viral vectors. However, achieving a true producer cell line — which stably integrates rep, cap, helper, and the gene of interest (GOI) into the host genome — is a technically complex endeavor. Complicated, but not impossible.

 

Learnings from decades of therapeutic mAb production

Start with the right cell line

We’ve learned from mAbs that the choice of cell line can have a huge impact on product yield, quality, and ultimate ability to scale up in good manufacturing practices (GMP) environments. According to Susan Riley, General Manager of bioprocessing cell culture at Cytiva, mAb manufacturers vary in what they need from a cell line. For some applications a lower-producing cell line may be sufficient; expression levels can then be enhanced by pairing it with an optimized cell culture medium. But if cost is an issue, as it is with biosimilars, levels of protein expression must be high. Riley explains, “It all starts with the cell line...All the media optimization in the world isn't going to completely compensate for a lousy cell line.”

David Mainwaring, Principal Scientist, Fast Trak™ Process Development Services at Cytiva concurs, adding that it’s well worth the time to choose a viral vector cell line that’s fit for purpose, whether that’s for transient or stable production. Otherwise, “you will spend an awful lot of time hitting your head against a brick wall.” He draws this conclusion from his years of experience working alongside customers to refine their viral vector processes. David explains that not only will choosing wisely streamline process development, but it will also avoid the need to go back to square one and rework the process with a different cell line. So, starting with the right cell line can save time and labor and avoid costly delays.

Keep the final scale in mind

To ensure that the developed process can be transferred to manufacturing, it’s important to think beyond what works at the bench. This learning applies to the entire end-to-end process, but here we highlight an example around the timing of pumping fluids. In the mAb world, slow feed addition can be readily achieved at small scale with highly accurate pumps. However, mirroring those conditions in a good manufacturing practices (GMP) environment at scale isn’t easy and could lead to deviations in the process. For viral vectors, a small volume of transfection reagent and plasmids can be added to a one liter shake flask in seconds. But at 200 liter scale it takes much longer for transient transfection, where the timing of mixing the DNA and reagents together is absolutely critical. Careful consideration of the ultimate scale early in development can avoid the surprise of having to redevelop a process.

Use platform processes where possible

“Not reinventing the wheel every time is a really good learning from mAbs,” says Mainwaring. Beginning with the cell line, a platform process facilitates speed to obtain the initial clinical material. And it can help with technology (tech) transfer, whether internal or external, because staff gain confidence from the familiarity. Not introducing new aspects helps to get processes transferred quickly and robustly. A platform process also accelerates understanding of what the critical process parameters may be, and of how any deviations affect product quality. Insights from one process can be applied to other processes to save time in process development for each new asset.

Understand the operating space

Another parallel with the mAb industry is that having a good understanding of where to start a process helps to ensure successful process development and manufacturing. Within the total design space, staying within the operating space allows ”reasonably good results without being close to an edge of failure”, states Mainwaring. Knowing the parameters to stay within this design space reduces the need to repeat experiments. Mainwaring adds that he’s seen a shift in the past few years from adherent to suspension mode and a growing interest in defining that ’safe space’ for cell culture process development and the rest of the workflow.

 

The path forward for viral vector cell line development

Applying what we’ve learned from mAbs, stable production in suspension cells is expected to enable robust and reproducible results that can be scaled to thousands of liters. Host cell line engineering can pave the way by integrating rep/cap into the host cell genome (5). Using the engineered packaging cell line, AAV production can be simplified into a single-plasmid transfection process designed to improve reproducibility and increase efficiency compared with triple-plasmid transient transfection.

Further engineering to integrate all four genes into the genome will provide additional advantages over a packaging cell line. Such a true stable producer cell line could then be used as a cell bank for future GMP batches, which would complete the path to success that mAb development took. As was true with mAbs, cell line development timelines can be shortened by using advances in screening technologies and automation to speed up clone selection.

Both packaging and producer cell lines enable a scalable, platform approach. Packaging cell lines simplify screening of different GOIs with the same capsid, which saves time as various assets are evaluated in the race to the clinic and market. Transient transfection will likely continue to have its place in early research and development and for low dose and low prevalence diseases. For those use cases, it’s still worthwhile to continue developing cell lines to enhance viral vector titer and quality. All in all, choosing the cell line that’s appropriate for a given application will save time and effort later.

The next generation of viral vector manufacturing will support development of new gene therapies for common and ultimately prevalent diseases. Among the many lessons from mAbs, stably integrating genes into host cells should help to enhance reproducibility and robustness. But transient transfection is more complicated for viral vectors than for mAbs, as multiple genes are required. Eliminating plasmids and transfection reagents — even reducing three plasmids to one — drives down not only costs but also labor. Emmanuelle Cameau, Genomic Medicine Strategic Technology Partnership Leader at Cytiva, echoes Riley’s and Mainwaring’s insights on starting with the right cell line. Cameau says, “If you don't have a good cell that produces a good product, then you have to fix it with the downstream, and that's where your cost increases. The answer to decrease the cost is all in the upstream. That's the key.” And technology providers must do their part to help manufacturers reduce cost of goods. Because everyone deserves access to the latest gene therapies.

Learn more about a true stable producer cell line for viral-vector based gene therapies.