Presented by Adam Elhofy (chief science officer, Bio-Ess Laboratories) 2:00–2:20 pm

“What we call biomanufacturing,” said Elhofy, “is all about cells. When cells get cranked up, they start to have deficiencies — and those deficiencies lead to different protein quality, to aggregates, and to a loss of consistency in protein quality. So how do you improve productivity and protein quality?” Bio-Ess Laboratories (formerly Essential Pharmaceuticals) has a technology to address the role that lipids play in cell productivity.

The company has existed for over a decade, making media products and medical devices. Cell-Ess medium is made to good manufacturing practice (GMP) standards and is free of animal components. It is chemically defined and can be used to increase titers in serum-free systems or to replace serum in other systems.

Proteins are formed on the lipid membranes of each cell’s endoplasmic reticulum (ER), then transported to Golgi apparati for posttranslational modification on another lipid membrane. They are secreted through a membrane vesicle. Protein quality attributes happen at each point of that process.

To increase expression titers, the objective is usually for more cell division and higher cell densities. But Golgi apparati can stop working properly, leading to improper glycosylation, improper protein folding, and formation of aggregates. A recent discovery showed that the more sylated a product is, the longer it will stay in circulation, and the longer the monoclonal antibodies (MAbs) will remain active. Here is where Cell-Ess medium comes in.

Scientists knew that they should add lipids in the right ratios, but doing so was difficult because lipids are not soluble in media. Precipitation leads to clogged filters. Oxidized lipids can destroy cells in culture. Some bioprocessors have tried adding free fatty acids or cholesterol, but after three days those will oxidize and start killing off cells. Free fatty acids can degrade and adhere to vessel walls.

Merck studied adherence in 2,000-L single-use bags, wondering whether lipids could be added. The team found that after 72 hours added cholesterol was 98% gone, and free fatty acids (cholesterol and linoleic acid) were bound up by polymer films in six hours. Thus, lipid addition hasn’t been successful in single-use bags. With steel and glass vessels, again after 72 hours the cholesterol was mostly gone (75%), and after 14 days the lipids were gone. In continuous bioprocessing up to 200 days, lipids either degrade or are oxidized and causing complete cellular destruction.

Bio-Ess Laboratories has a patented lipid-delivery technology designed for use in combination with other supplements and feeds. It is already used in preclinical work in process development. But it could be brought in at a later stage because it is manufactured under CGMP guidelines and will not change process parameters.

Open-ended, independent experiments have determined the effects of Cell-Ess media with different feed strategies on titer and glycosylation in a scalable model. Both titer and productivity increased with the addition of Cell-Ess media, with no change in process parameters. Viable cell density remained the same, as did glucose, glutamine, ammonia, and lactate values. Using Cell-Ess media increases titer by following a different pathway from that of traditional glycolytic methods. And it increases yield by 20% or more across different media backbones. The method is scalable and works at small and medium volumes thus far. Yields were increased for different clones and media backbones — and in single-use bags — by increasing productivity of individual cells. Better cell function improves culture consistency. So you don’t have to choose between increased titer or increased galactosylation.

Questions and Answers
Is the number of cell divisions important when you add Cell-Ess media? “Yes and no.” It doesn’t matter how many cell divisions there have been when you add Cell-Ess medium as long as it remains long enough to be incorporated into the cells. Most other serum-free media have no cholesterol. If cells have cholesterol available, they won’t waste time and energy making their own rather than synthesizing proteins. So they need Cell-Ess medium to be present when they start to divide. Thus, it is best to add it on the first day of production.