Navigating the Challenges – and Opportunities – of Lentiviral Vectors
Lentiviruses are growing in popularity within the gene therapy and gene-modified cell therapy landscape, but their production comes with unique challenges, including scalability, performance optimization, and regulatory considerations. Here’s what experts at Sartorius have to say on the matter.
| 7 min read | Interview
sponsored by Sartorius
Featuring Marcel Fueger, Product Manager for Separation Technologies - Filtration, and Geraldine Guérin-Peyrou, Head of Product Management for Advanced Therapy Solutions, both at Sartorius
What are the biggest trends and conversation points in cell and gene therapies (CGT)?
Marcel Fueger (MF): Although CGT is a relatively new field, there have been many breakthroughs, but to meet increasing patient demand there are several key challenges to address, including the need to reduce costs and improve scale. Viral vectors are essential for CGT, which is why their manufacture using efficient and scalable production methods is a key topic in the industry. The most commonly used viral vectors are adeno-associated viruses and lentiviruses, but each vector has its own characteristics and advantages/disadvantages. It’s our job at Sartorius to help our customers overcome all types of bioprocessing challenges, including those related to viral vector production.
Geraldine Guérin-Peyrou (GG-P): Viral vectors are definitely a big talking point in CGT. The manufacture of viral vectors – particularly on a commercial scale – is a complex process with many steps that require a high level of expertise. Safety is also paramount. You have to ensure that your vectors are not carrying unwanted gene insertions or other risks that could lead to cancer or life-threatening illnesses.
Why are lentiviruses receiving increasing attention?
MF: Lentiviruses are highly effective at delivering therapeutic genes to cells and can permanently integrate genetic material into the host cells’ genome to provide longterm and stable gene expression, which is highly beneficial for treating chronic diseases. Unlike many other types of viral vectors, lentiviruses can transduce both non-dividing and dividing cells, expanding their applicability to a wider range of cell types, including T-cells. They also have a large packaging capacity to deliver genes of interest. In short, they have many unique capabilities compared with other viral vectors – and will only improve with further innovations in vector design.
What are the manufacturing considerations for lentiviral vectors?
GG-P: You first need to transfect HEK 293 cells, typically using three or four plasmids to express the different components of the vectors, including your gene of interest. After the cells have been cultured, you can proceed with collection and purification – both of which can be tricky because of the sensitivity of lentiviruses.
Scalability during the transfection step is also a significant challenge. You must mix the plasmids with the transfection reagent before adding them to your cells – and that means the stability of transfection complexes is critical to ensure you have enough time for mixing, especially when working at industrial scales.
Even at the earliest stages, you need to be considering how you will achieve the process quality and robustness required for the commercial scale. In reality, it is not at all straightforward to go from a flask to a bioreactor; key aspects, such as mixing and oxygenation, are very different. However, there are plug and play solutions available that can be used early on to facilitate later scale up.
MF: From my perspective as a clarification and filtration expert, it is interesting to see how approaches used in the production of mAbs are being employed for viral vectors. But the challenges are not the same. Lentiviruses are large particles, around 100 nm in size, and are very sensitive to shear stress, temperature, and time. Additionally, the feed stream often consists of highdensity cell cultures, and therefore shows demanding turbidities and is processed in volumes of around 200 liters. In other words, scalability is essential – especially during the clarification steps. One common issue with filtration, particularly in prefiltration, is the potentially low capacity of filters when it comes to challenging feed streams. Low capacity can result in high cost of goods. Not choosing the optimal filter chemistry leads to product loss caused by adsorptive effects, which is a frequent challenge when filtering lentiviral feeds.
What about regulatory considerations?
GG-P: There is a lack of clear regulatory guidelines and standards for producing lentiviral vectors. For example, there is no guidance on the quality grade of transfection reagents or plasmids; however, many people in the industry expect regulatory scrutiny to increase in the coming years as the CGT field matures, and the focus will almost certainly be on higher-quality materials. Even in the absence of guidelines, using the highest quality plasmids and transfection reagents possible is a wise choice. I recommend GMP-compliant – and ideally ICH Q7 compliant – components to improve consistency and to reduce the risk of introducing impurities or contaminants that may affect the safety or efficacy of the final product. And by using high quality components and working to the highest standards, you don’t have to worry about regulations changing in the future.
How can Sartorius help customers with lentiviral vector production?
GG-P: Sartorius has acquired several specialist businesses to build out a full portfolio in CGT, including transfection reagents, plasmids, filters, bioreactors, and beyond. We have solutions that span from the bench all the way to commercialization. We also have a strong regulatory affairs team that can support customers, as well as technical support specialists that can help set up design of experiments (DOE) and optimize processes. Scaling a process can be tricky – but you don’t have to do it alone!
Sartorius has successfully tackled all types of projects over the years. Sometimes, customers come to us after they’ve started working with products that aren’t GMPcompliant at larger scales. In those cases, we help them revamp their entire process to ensure GMP compliance. We can also support customers right from the start of their projects, which is recommended. After all, when we are involved early, it’s much easier to streamline the scalingup process because we can use the same products at larger volumes.
MF: Lentiviral vectors will remain a key cornerstone of CGT for the foreseeable future. However, as the field is relatively new, many biopharma companies may find that their internal expertise is lacking or still developing. Our team has the knowledge to support in finding the best fitting solutions and overcoming challenges. Since a lentivirus is handled as Biosafety level 2 material, many customers come to us for closed, single-use solutions. This approach protects the user and the environment from exposure to harmful agents, while also minimizing the possibility of cross-contamination.
For this procedure our filter trains are a popular solution. For example, a first step may use Sartopure® PP3, a highly porous, polypropylene-based filter that minimizes product loss caused by adsorption, while accommodating large particle sizes. This could be followed by Sartopore® 2 XLG, a sterilizing-grade filter to ensure the removal of smaller contaminants. Together, these filtration steps protect subsequent processes, especially the expensive chromatography stage, by preventing clogging and maintaining product integrity.
What are your top tips for success?
GP-P: As I noted earlier, optimizing the transfection step is crucial but often overlooked. Many people tend to use a single plasmid ratio, a fixed amount of DNA, and a standard volume of transfection reagents without considering alternatives. I strongly recommend adopting a DOE approach to optimize conditions. The Sartorius Ambr® system is a powerful tool for this purpose because it allows simultaneous analysis at different points. And when combined with Sartorius’ MODDE® software, it becomes an even more effective solution. It’s important to remember that all parameters should be optimized together; if you are changing the media, you need to re-optimize. And if you are changing the gene of interest, then you need to re-optimize.
As a side note, I came to Sartorius through the acquisition of Polyplus. And when I was at Polyplus, I would not shy away from recommending certain Sartorius products, such as MODDE, because I knew they were so good!
MF: The goal for clarification is low turbidity, but high yields are needed for the complete manufacturing process, which should be robust with the right production and purification techniques. As Geraldine explained, optimization is really important – and you need a process that can be scaled. I recommend testing and trialling alongside partners to find what works best for your application. Manufacturing and filtration processes used to produce one viral vector may not work for another because viruses behave so differently.
As the CGT field continues to mature, there will be more innovation – and more regulatory changes – aimed at increasing safety and manufacturing efficiency. Right now, we don’t know what the gold standard will be. But what I do know is that Sartorius will be at the forefront – continuing to investigate and invest in new solutions so that we are ready to support our customers.
