Overcoming Limitations with Transient Protein Expression to Accelerate Development

Overcoming Limitations with Transient Protein Expression to Accelerate Development

October 11, 2022PAO-10-022-CL-02

In this Q&A, Cenk Sumen, Ph.D., Chief Scientific Officer at non-viral cell engineering platform provider MaxCyte, discusses the importance of failing fast and confidently in early development, how delaying the transition from transient to stable expression can help, and how MaxCyte’s technology and approach allow the company to work as a manufacturing partner to pharma companies, biotech startups, and CDMOs, enabling production of any protein at scale, in conversation with Pharma’s Almanac Editor in Chief David Alvaro, Ph.D.

David Alvaro (DA): Can you discuss some of the current inefficiencies or bottlenecks that you see in development that underlie the importance of ensuring that candidates that fail do so as fast as possible?

Cenk Sumen (CS): The drug development process is becoming more elaborate, with more assays and QC and toxicology work that needs to be done, including on complex models. It’s always important to ensure that proteins are manufactured in the right amounts and using the right tools so that developers can consider a broader repertoire of protein candidates moving quickly through the selection process. In these early stages, the objective is to produce as much material as fast and cost-effectively as possible.

You might have several candidates, and you need to narrow the range down quickly while still considering a broad repertoire of proteins for manufacturability and paying attention to a host of factors, like glycosylation, binding assays, safety, and titer.

This is a big challenge, especially now when things are moving toward automation and rapid, high-throughput screening. There’s a growing need for very stable, consistent transfection methods that can be applied to any cell type. Just with CHO cells, many different variants are available now, each offering features that enable the production of different types of protein. The ideal would be to develop a transfection system that works across various cell lines and various types of proteins and vectors.

It’s always best to fail fast and narrow your range of options quickly with confidence. Before specific candidates can be eliminated from consideration, developers must feel confident they can test them using multiple methods and assays — to fail not only fast but to fail confidently. To do that, you need to produce enough material, typically on the scale of grams of protein. Of course, when you move downstream to purification, we know there will always be some yield loss, so we have to factor in the need to make enough protein upstream that there will be enough left over downstream of sufficient purity to use for these assays.

To be able to fail fast and confidently, you need to produce enough reliable material to run these series of tests, while also considering as large a repertoire of candidates as possible. For a single antibody candidate, we often have to consider hundreds of different variants of that protein. Your transfection technique should create a blank slate into which you can plug your diverse candidates and let the best ones win.

DA: What are the fundamental mechanistic challenges in establishing transient expression, and how are they best overcome?

CS: The first task is getting molecules across the plasma membrane. For example, let’s take the case of plasmid DNA. It has to be shuttled across the membrane, but membranes have evolved to prevent or at least resist molecules passing through them, particularly polar molecules. There are a few ways to make the membrane more amenable to the shuttling of these molecules. There are chemical methods that will coat the cargo and help it interact with the membrane and allow these coated complexes to enter. We can use liposomes in a similar manner. There are also more esoteric methods, including microinjection, analogous to how fertility clinics inject sperm into oocytes. Still, you simply can’t do that individually for the billions of cells you need to put into a bioreactor. In order to make the cells more permeable, we can also bombard them with particles, waves, lasers, beads, and so on.

Many of these biochemical methods are interesting but experimental — they will work in controlled environments using controlled cargo and cells, but once you start to apply them to different cells and different cargo, translational scientists often realize that these methods are not robust enough for biomanufacturing or lack the ability to scale up production consistently within the context of transient transfection. Typically, chemical methods are used.

A suitable method must be robust and scalable, but there are also some novel, post-pandemic considerations, primarily supply chain shortages. We’ve encountered supply chain issues with common components, such as human platelet lysate and other key ingredients, as well as basic chemicals like lipids and salts, many of which are overwhelmingly manufactured in certain geographic locations. It is challenging to confidently advance candidates without securing the supply chains. Supply chains have to be simplified to the point where you can de-risk, so you want to avoid single-source chemicals or ingredients for which you anticipate supply chain shortages. I think that’s another critical point. Any opportunity to use a “one buffer, one pot, one method” approach is desirable, because you can apply it across multiple situations without worrying about maintaining a sufficient stock of a specific chemical to last throughout the development cycle, which can take years.

DA: In a more typical approach, you would use transient expression early before transitioning to stable expression. What was the rationale for where that shift was traditionally positioned, and why do you think it should be reconsidered?

CS: The industry benefits from cell line engineering, design, and development: the engineering of specific cell lines dedicated to producing a single protein. However, companies cannot dedicate enough bandwidth to produce dedicated cell lines for every candidate, which could be on the order of tens or even hundreds for a given project.

Cell line development unquestionably has its place, but it may take over a year and millions of dollars or more for each cell line. This is not a problem once you are confident about what you want to make, but most developers begin with a large number of candidates and need to determine which to pursue. You don’t want to proceed with a therapeutic candidate and only realize years later, in late-stage clinical trials, that it would have been better to use a variant of that protein with a slight modification.

The idea is to generate enough material to make those decisions confidently. If we had unlimited budgets, we could make hundreds of different cell lines simultaneously. However, being nimble is one of the keys to success in this industry. Transient transfection allows developers to stay agile and produce enough of each candidate to thoroughly evaluate whether to move forward.

DA: How and when would you suggest that developers transition from transient to stable expression?

CS: Much of this depends on budget and time. A large pharma company that enjoys the benefits of scale can have its own cell line development teams in place, potentially moving this technology into stable lines faster. However, drug development today mainly occurs in small to midscale biotechs with limited budgets. For them, there is real value in trying to get the most out of transient transfection. Suppose you can produce sufficient material using transient transfection to assay and hopefully reduce a list of candidates by tenfold or more. In that case, that will be far faster and cheaper than pursuing cell line development to achieve stable expression.

I view transient transfection as a natural complement to stable cell line development. The key consideration then is what method to use for transient transfection and when to transition to stable expression. As we discussed, making that switch early costs considerably more resources and time.

I do not want to imply that stable expression is necessarily a must from a scientific, regulatory, or biomanufacturing perspective. It is possible to use transient transfection for the manufacture of commercial therapeutics — we have seen this for biologics and vaccines — it is just that, in the current paradigm, it still makes sense in most situations to switch to stable expression once the supply requirements are finalized for clinical trials and for the commercial product life cycle.

DA: Are there cases for which there would be very clear advantages to sticking with transient expression all the way to commercial production?

CS: It comes down to the proteins that you are making. For some proteins, it takes more work to develop cell lines that perform. However, it may be a worthwhile investment: if this protein can treat a lot of patients and make a significant impact in the market, it may make sense to make that investment. Now, with AlphaFold and other bioinformatics methods, we have access to areas within the protein space that have not been well explored by pharma, which is very exciting. But to address that whole ecosystem of therapeutic proteins, therapeutic developers need to leverage methods and systems that work consistently, even with difficult or unusual proteins and different cell lines.

Not everyone uses CHO and HEK293 cells. There is a push to use cell lines that are less demanding in terms of cost and bioreactor design. We have seen people use Sf9 insect cell lines, even fungal cell lines. However, these systems have stayed niche for the most part because CHO cells work; they have earned their status as workhorse cell lines. They enable glycosylation, which is very important, and we’ve increased yields considerably over the past 10 years. But there are many different options just over the horizon.

We need to develop methods that work across a broad range of cell lines, where you don’t have to keep tinkering with the method. You determine your settings once for that cell line, and you can use the same parameters at different scales. I think that can provide a lot of confidence for developers who are willing to move further with transient transfection.

DA: Are there clear-cut reasons why it might make sense to transition to stable expression sooner: certain types of proteins or projects where you would just need to invest that time and cost?

CS: In certain instances, if a very large amount of product is required, and the developer knows that they want to use a stable cell line to lock in this protein and have mapped out the requirements for clinical trials, as well as the commercial life cycle, it may make sense. This could be the case for very complex proteins or even whole viruses. For example, we’ve seen this with lentivirus. A developer creates a modification — a specific chimeric antigen receptor (CAR) for a cancer marker — and may then decide to use this specific CAR throughout clinical and commercial phases. They may want to make a really big batch of lentivirus, which will cover all those needs.

If you’re in the position where you’re willing to make enough material for the entire journey ahead or at least most of it, it may make sense to make the investment and schedule the time and the resources to be able to achieve a robust, stable cell line. Economies of scale at more prominent companies can also play into making commitments earlier in a developmental timeline — they can afford to make these investments into these cell lines, because they know that they will make enough material over the next 10–15 years. 

DA: Is this idea of extending transient expression further gaining traction? Are people beginning to explore it in a serious way, or are we still looking at the seed of this idea?

CS: In a sense, stable cell line development is the newer piece of this methodology. Transient expression has historically been the leading technology for engineering cells to make specific proteins or viruses or any kind of therapeutics. But typically, transient expression was done at lower scales, and stable cell lines enabled the seeding of larger and larger bioreactors: hundreds of liters, maybe thousands. These large bioreactors are typically seeded with stable cell lines that are already clonal and engineered to efficiently make that identical protein, batch after batch. But our ability to engineer cell lines is still developing, while transient transfection has been around for decades, probably over half a century.

What we’re envisioning is the ability for transient expression to be able to carry more of the workload for that development so that developers can invest accordingly. If you can consistently get to your desired candidates quickly and with fewer resources, you can be more confident making investments into your stable cell lines. This can get the therapeutic material to patients faster and more inclusively. I really see these two approaches to biomanufacturing as complementary. It’s more of a question of considering all the attributes of the protein and the program as a whole and determining the most effective moment for that inflection point from transient to stable.

DA: In that recalibration, who do you think should or likely will be the driver? The pharma companies themselves or CDMOs?

CS: I come from the CDMO world, and I think it’s important for the CDMO to take the lead in process development and scale-up and to work closely with their partners to determine the best path forward. We increasingly see CDMOs acting as process consultants creating strategic manufacturing workflows for their customers. It’s often more costly and resource-hungry to carry out these projects inside a typical large pharma company than to let the CDMO determine the most efficient way forward. This puts a lot of responsibility on CDMOs, which must balance a diverse group of customers and projects, facing very different risk ratios than the pharma companies.

In an inclusive sense, I see a role for CDMOs in leading the investigation of different methods and processes and non-competitively granting access to different players. If a CDMO cuts its teeth on difficult projects, the smaller companies get a chance to work with them and can follow a path that has already been established. I think it helps the entire ecosystem by enabling smaller companies to access this know-how through the CDMO versus trying to hire the right experts and build it in-house.

DA: You mentioned some limitations to the ability to use transient expression at very large scales. Do you see a straightforward path to overcoming those limitations?

CS: Classically, the cells had to be prepared for transient transfection — analogous to the process of making competent E. coli with which you and I and most biologists are familiar. We want to find ways to avoid that: to be able to just take CHO cells from a seed bioreactor, transiently transfect them, and then put them back into a bioreactor and scale them up. Whatever change needs to be made to the cell membrane, it can’t be cumbersome, and it should be efficient and work on different cell types.

I think electroporation will likely play an important role in this and that it should be considered for all types of transient transfection because the process is simple; it just requires optimizing the electrical parameters for a given cell line and a reliable technology platform. At MaxCyte, we’ve already optimized our system to work in a very effective way for a very large number of cell types, including CHO and HEK293, and we have a universal buffer that works with all the cell types that we use. That reduces complexity, as well as potential supply chain issues.

In the past, that could be done reliably only at smaller scales. Today, instruments are being developed — and we’re launching one this year, the VLX — where you can take the same parameters that you used with a smaller number of cells at smaller volumes and scale them right up without making any changes — the same electroporated conditions, same buffer, same cell type, up into the billions of cells. But in reality, these technologies have been around, and the question has been how to achieve scale consistently.

DA: Does the fact that electroporation has been less explored for mammalian cells mostly stem from a scale issue, or are there other fundamental challenges to using electroporation for mammalian cells that needed to be resolved?

CS: It’s important to consider how far electroporation has come and how well we’ve understood the impact of different electrical parameters and intensities on what happens to the cells. There are ways to introduce electrical currents into cells that preserve their function, so it’s really about establishing the parameters to maximize the amount of vector you can get into the cell gently.

Over the last 20 years at MaxCyte, we have become experts at controlling electricity and designing our technology accordingly, and flow electroporation is an important approach to overcome some of the limitations of cuvette design that affect the lab-scale methods.

Combining pumps and electrodes allowed us to achieve a consistent flow of electroporation; the next challenge was figuring out how to do that across hundreds of cycles consistently and be able to collect all the viable cells at the end. Ideally, the process is so consistent that you can consider all cells generated in this way as one single batch, because each cycle is nearly identical to the next cycle.

DA: Can you expand a bit on MaxCyte and how your technology has evolved?

CS: I officially joined MaxCyte in March of this year, but I’ve followed MaxCyte for well over a decade. Their foundational technology dates back to the 1990s, although there were many advances in the 2000s. In the 2010s, MaxCyte started to form partnerships with companies moving through the clinic into commercial — not just as a provider of instrumentation but as a manufacturing partner that will advance with the company through clinical scale-up and commercial development. One of the major differences of MaxCyte is that the technology is a foundational platform that enables the stable engineering of cell types to produce therapeutics.

We work with manufacturers that use engineered cells to produce their therapeutics, but we also work with cell therapy manufacturers for whom the cell itself is the therapy. We have a lot of experience working with almost every cell type out there that is being considered for therapeutic use. Our technology platform has enabled us to reach scales of hundreds of billions of cells, which is truly astounding, considering that electroporation started with tiny populations of cells in small cuvettes. Now, we’re bringing it into the biomanufacturing and bioproduction market and enabling CDMOs, biotechs, and pharma companies to access this technology.

Today, MaxCyte is growing rapidly. Last year, it was very helpful to have access to the public markets, which enabled us to achieve scale and develop our technologies. We are now at a phase of our journey where we think that we can take on the entire bioproduction field and market rather than focusing on individual niches. We can work with anyone making any protein and ensure that we can reliably and consistently produce these proteins at scale. I think that’s really powerful. We know it’s been a very tough couple of years for biotech startups, and we hope that partners like us are encouraging these startups and providing them with a good path forward. I came from a small company myself, so I know how important it is to have companies like MaxCyte that are not just out there selling instruments but providing partnerships in biomanufacturing and working side by side with clients to gain their trust, all the way through clinical and commercial phases of biomanufacturing novel therapies for patients.  

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