We recently finished our Ask the Expert discussion, “Maximizing Transient Protein Production”. During this Ask the Expert session, we discussed factors that influence transfection, troubleshooting, and optimizing productivity. Specific topics included host cell choice, target types, factors that impact transfection efficiency, increasing titers, and improving quality.
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 transfection 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.
This Ask the Expert session, was hosted 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.
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Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – Maximizing Transient Protein Production.
What factors influence selection of a host cell for transient 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.
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.
How is MaxCyte’s electroporation technology different from other electroporation instruments?
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.