Moving On in Cell Culture

Record-breaking titer outputs in mammalian cell culture systems in the past few years have pushed the industry to a new crisis of sorts: resolving the downstream bottleneck. However, the cell culture and fermentation groups at biopharmaceutical companies aren’t yet ready to sit back and rest on their laurels. Instead, they are moving forward, tackling the downstream issue with upstream modifications and continuing their drive for more cost-efficient processing. The Cell Culture and Upstream Processing track will focus on cell culture media development, clone selection, cell line engineering, alternative expression systems, scale-up strategies, and streamlining process development and optimization.

Nicole Borth, professor in the department of biology at the Institute of Applied Microbiology in Vienna, Austria, will speak about the benefits of flow cytometry and cell sorting in improving understanding of cellular performance.

“Flow cytometry is based on measuring the fluorescent properties of single cells as they pass an optical system,” explained Borth. “As the cells pass by, you get a measurement of each single cell, and the important thing is that you really get a distribution of properties within the population.” Additionally, you can measure several parameters simultaneously from each single cell, which shows correlations between different properties in each. Using this information, you can select cells that have properties favorable to production.

“In the past when you talked to cell line development people, most of them were concerned in getting good transcription,” explained Borth. “But in recent years, by looking at cells and their behavior it’s become more and more clear that the translation part and the processing part are also very important in the overall output of the product.”

Therefore, it may no longer be enough to have a high level of messenger RNA. Borth observed that now it is clear that much of the translation of regulation is not at the level of transcription, but also includes which and how much, messenger RNA is being translated. That and the relationship to assembly and formation of disulfide bonds and glycosylation are all important parts of the whole production process, so they are the key areas of interest for cell culture and upstream process developers.

Majid Mehtali, chief scientific officer and vice president of research and development at Vivalis SA, commented that the new expression technologies are focused on improved glycosylation profiles. Additionally, new expression technologies beyond the “big three” (CHO, NS0, and Per.C6), include several microbial technologies.

“Less advanced, but highly exciting is the possibility to combine the advantages of microorganisms, such as cost-effective culture and production, with higher eukaryotic cells’ ability to produce complex proteins, by engineering yeast,” said Mehtali. “This is in particular what companies such as Glycofi are attempting to achieve, with promising data already published confirming the ability to engineer Pichia pastoris to produce proteins such as Epoetin alfa with complex sugars, displaying terminally sialylated glycans.”

Mehtali’s talk is focused on an alternative expression system in avian EBx cells for production of vaccines and therapeutic proteins. He described the advantages of the platform in an interview with BioProcess International. “First, this is a cell line of avian origin growing in suspension in serum-free media to high cell densities. As such, it can be used to produce most, if not all human and animal vaccines currently produced in chicken eggs.” Mehtali further explained that because of the proprietary process involved in the derivation of the stem cells, the cells are immortal and genetically stable and diploid. This unique circumstance has advantages to other immortal cell lines that are usually genetically unstable and tumorogenic. The other advantage is that the glycosylation of avian cells is similar to human cells, with the difference being the low content of fucose in the avian cells.

“Today we use our EBx cell lines beyond the vaccine field for the production of human antibodies, and we have confirmed that we can produce such antibodies with lower fucose and enhanced ADCC (antibody-dependent, cell-mediated cytoxicity) activity with no engineering of the cell line,” explained Mehtali. He explained that the limitations of the technology include viral vaccines that do not grow on avian cells, such as gene therapy products based on human adenoviruses or lentiviruses.

Business strategy appears to favor licensed cell lines, and according to Mehtali, licensing of cell lines supports innovation in the area. “Companies with proprietary technologies invest more efforts and financial resources to develop their technologies according to the need of the market, and also with the regulatory guidelines and expectations in mind,” said Mehtali. “If the technology is promising, it allows for licensing deals with larger companies, which provides more means and leads to more players developing such technologies.”