Companies are turning to transient production of proteins to accelerate timelines, gain quick access to protein for early go-no go decisions, delay stable cell line generation, and reduce costs. Expanding the role of transient protein production within the biotherapeutic discovery and development workflow is highly dependent on the ability to supply the required quantities of quality proteins — generally ranging from milligram to multiple grams of protein — in the required timeframes. Thus, increasing productivity and process scalability are aspects of production that must be addressed.
There are a wide variety of factors that influence productivity – – from pre-transfection factors such as cell type, vector design, and cell health to post-transfection factors such as seed densities, media additives, and feed schedules. In addition, researchers must consider the method of transfection which affects both productivity (cell health & transfection efficiency) and process scalability.
Recently, Cell Culture Dish has published several poster and webinar articles discussing the use of MaxCyte’s electroporation-based delivery platform for cell engineering, specifically in the areas of transient and stable protein production. There was a high level of interest from readers regarding CHO-based, gram-level protein production, and so during this week’s Ask the Expert session, we will be joined by Dr. Weili Wang, the Director of Cell Culture at MaxCyte, to answer questions regarding cell engineering and culturing that can maximize transient protein production. Dr. Wang has over 20 years of biopharma industry experience focusing on process development, stable cell line generation, tech transfer and scale up/scale down modeling to support cGMP manufacturing. Prior to joining MaxCyte, Dr. Wang was the upstream manager at Human Genome Sciences, MacroGenics and Amplimmune. Dr. Wang received his Ph. D degree from Texas A&M University, a Master degree from Florida International University and Bachelor degree from East China University of Science & Technology.
Are differences in cell viability levels from one transfection method to another large enough to actually affect protein titers?
Yes, high cell viability is critical for achieving high titers. Of course, it is essential to use cells that exhibit high viability pre-transfection (>95%), but it should also be emphasized that if cell viability is impacted by the transfection process, then cells will also exhibit reduced productivity. We consistently observed that post transfection viability >90% leads to titers that are significantly higher compared to cells that show viability <90% post transfection.
The ideal host cells for protein production aimed at supporting biotherapeutic development should exhibit robust cell growth to allow scalability, and they should produce proteins that meet customers' standards for quality and posttranslational modification. CHO cell lines, which are most commonly used for commercial protein manufacturing, typically meet these criteria. Thus, starting the biotherapeutic development process in CHO cells, rather than in HEK cells, will harmonize early stage discovery efforts with later stage development activities, which greatly reduces risks that might arise from misleading data due to host cell variability. It should be noted that even some commercially available CHO cell lines may present issues with protein quality (e.g., degradation or increased levels of host cell proteins) that are not observed in the CHO cells that will be used for manufacturing. Therefore, it is very important to perform testing of host cell lines to ensure there are no issues with protein quality before moving into later stage of the drug development process. In other words, analyzing and developing your biotherapeutics by using the same host cell line from the beginning to the end of the development spectrum can really help you to avoid/reduce risk and accelerate timelines.
It has been shown that lipids, including cholesterol, in the medium can significantly impact transfection efficiency. That's why screening and selection of the basal growth medium is important to achieve good transfection efficiency. Besides chemical components, healthy, low passage cells are also critical for maximizing transfection results.
Just as process development is critical for achieving maximum productivity from a stable cell line, optimization of culture conditions is very important to achieve high protein titers following transient transfection, regardless of the transfection method. Those conditions include the balance of nutrients with a good feed strategy and strict control of CO2, dissolved oxygen, pH and metabolites. Some chemical based transfection methods are impacted by media components, which may prevent use of optimal production conditions following transfection. It is also important to note that culture conditions are only one part of the equation. High titers can be achieved only if transfection efficiency and viability are both greater than 90%. Therefore, it is important to choose a transfection method that is amenable to many different culture conditions and that achieves high transfection efficiency and viability with a range of host cell lines. We find that the high efficiency and viability of MaxCyte Flow Electroporation™ Technology as well as its cell type and media flexibility are critical for enabling our users to maximize productivity.
Using your system have you been able to express a variety of antibody types, including ones with kappa or lambda light chains?
Yes. Both MaxCyte scientists and our clients have expressed a range of antibodies, antibody-like molecules and Fc fusion proteins from a variety of species including human, rabbit and mouse. In comparison studies with other transfection methods, flow electroporation generally leads to higher titers independent of the type of antibody molecule that is being expressed. GFP control transfections indicate that our technology results in higher efficiencies and cell viability, which very likely is a key contributing factor to the increased titers.
There are several key differences between MaxCyte technology and other electroporation instruments that have important roles during transient protein production. 1) MaxCyte systems generally have higher transfection efficiencies and cell viabilities compared to other electroporation-based systems. Even small changes to either of these parameters can have significant impacts on transient yields. 2) MaxCyte's delivery platform has a much larger capacity to scale. The MaxCyte STX system can transfect up to 2e10 cells in a single run, while the MaxCyte VLX can further scale up to 2e11 cells in a single 30-minute run. 3) MaxCyte transfection systems can generate cGMP-grade materials and are the only electroporation-based instruments supported by an FDA Master File which can therefore support toxicology and clinical studies.
Does the quality of the expressed protein decrease if you push transient expression to gram/L titers?
The quality of expressed proteins is not necessarily related to high titer. Many factors contribute to protein quality including the molecule itself, transfection conditions, production conditions, and the host cell line. Recently, we observed issues specifically with protein degradation when using the ExpiCHO cell line for several proteins even when using MaxCyte flow electroporation. Interestingly, this issue was not observed for the same protein when using the same transfection method and production process but a different CHO cell line. Thus the selection of a host cell line should not be overlooked as an important factor for minimizing risk for your biotherapeutic candidates.
Have you produced viral vectors using CHO? Have you seen any differences or benefits over using HEK?
In general, we find our clients are using HEK, Vero, or insect cells for producing viral vectors or VLPs. Thus, our research scientists do not have direct experience transfecting CHO cells specifically for viral vector production. We certainly have produced a variety of vaccines and viral vectors in other commonly used cell lines.
We find similar transfection efficiencies and cell viabilities for suspension and adherent CHO cells. When possible, however, I would recommend the use of suspension CHO cells. It is far easier to conduct the transfection using a chemically-defined production process (animal component free process) for suspension cells as well as allows significantly easier scalability.
No. Our system delivers DNA exceptionally well with no need for helper plasmids. If your helper plasmid can further activate the transcriptional activity for your system, then co-transfection is also acceptable.
I read your post on host cell choice, but I was wondering is there ever a time when HEK is best or are there certain applications where there is a clear reason to use one over another. Would you ever use a cell line other than CHO or HEK, for instance if the manufacturing cell line was going to be NS0 or SP2/0
Hybridoma cells like NS0 and SP2/0 were used as the cell line of choice very early on for the development of therapeutic recombinant protein drugs. CHO cell lines have now been well characterized, have post translational modification profiles closer to human cells than hybridoma cells, and have a regulatory track record of use in manufacturing biotherapeutics. Thus, many companies continue to rely on stable CHO cell lines for manufacturing. We do, however, have clients that have chosen to use other cell lines for bioproduction including BHK21, Vero, CAP-T and insect cells. We generally recommend that early-stage research be conducted in the cell type identified for manufacturing. Ideally, a company’s transfection platform has the performance and flexibility to allow them to use whatever cell type is most scientifically relevant, rather than their transfection platform dictating what cell type they must use.
Cell aggregation is influenced by a variety of factors, including basal culture medium, media additives, culture vessels and agitation rates. It is also important to note that certain cell strains are more prone to clumping than others. Screening and selection of the right culture medium is a good way to reduce cell aggregation. Adding surfactants, such as pluronic F-68 to the culture can also help to alleviate aggregation. Continuous culturing of cells with non-aggregated cells is another way to minimize cell aggregation issues. PEI should not be a major factor for cell aggregation. However, not all culture media and additives are suitable for PEI transfection. Thus, for cells that are prone to aggregation, it may be better to use a transfection method, such as electroporation, that is compatible with many different types of culture media and conditions.
Would you recommend a media change post transfection or could you tell me how to avoid having to change the media. How long to culture post transfection?
After transfection, we typically do not change media, unless we are performing selection for stable pool or clone generation. The high level of cell viability following MaxCyte electroporation allows not only for rapid protein production, but also for longer-term culturing to maximize total protein yield. Thus, the length of post transfection culture depends on the amount of protein that is needed. Some customers prefer to harvest after only 5-7 days of culture. However, using fed-batch cultures, we have seen transient titers steadily increase for up to 3 weeks post transfection.
Yes, codon optimization can certainly improve titers. Other elements of vector design, including transcriptional enhancers and matrix attachment regions, can also contribute to enhanced transient titers.
Cells transfected via MaxCyte electroporation do not require the use of a specialized media or media additive. During electroporation, calls are suspended in MaxCyte electroporation buffer (a single buffer for all cell types) that is a balanced salt solution containing no animal-derived components or other macromolecules.
There are a variety of factors that should be considered when determining total protein production costs for different transfection methods including - the required scale, the level of cell productivity post transfection, the requirement for use of specialized media or additives, as well as the ability to use higher cell densities which impacts both upstream and downstream process costs. Secondary costs include reliability of the method and flexibility to use a single system for multiple proteins and cell types.
I liked your article, is the 2.5 g/l average you mentioned pretty reproducible for most CHO lines or do you see lots of variation in the titers?
Antibody yields following MaxCyte electroporation using a given expression system and downstream culture conditions is highly reproducible. In terms of the ability to achieve g/L productivity, it is dependent on a variety of factors including the vector, the protein being expressed, and as you inquired, is also dependent on the cell line being used (including different CHO lines). MaxCyte electroporation has yielded titers exceeding 1 g/L without process optimization (i.e., using commercial base media/feed with no adjustments to the metabolites or pH) in several CHO cell lines, including CHO-S, CHO-K1 and CHOK1SV. We can further increase productivity to multi-gram per liter titers following basic process optimization of feed strategy and media additives. Some CHO cell lines are more suited to transient production than others. For example, DHFR deficient CHO cells, such as DG44, can be transfected with high efficiency using the MaxCyte system, but sustained viability is more difficult, which results in lower transient titers compared to other CHO lines.
What kind of target types using electroporation, i.e. non-antibody proteins, antibody fragments, fusion proteins?
Electroporation can be used to engineer cells to express a range of proteins from secreted proteins, membrane proteins or cytosolic proteins. In terms of protein biotherapeutics, MaxCyte clients have expressed recombinant antigens, full IgGs from multiple species, bi- and tri-specific antibody, FcSv, Fab and Fc, or other fusion proteins. One benefit of electroporation over other transfection technologies is the ability to easily control the ratio of plasmids during cotransfection, a property particularly useful for multi-subunit proteins. MaxCyte systems are the only FDA approved instruments and thus can produce cGMP grade materials which can be used for toxicology and clinical studies.