Nutritional and Process Strategies
Once a cell line has been engineered and/or set by cloning, nature’s input ends; the nurture part begins in earnest immediately and continues at some level for the remainder of the product’s life cycle.
On the process side of the continuum, Avid achieves yield improvements by optimizing physical setpoints such as pH, osmolality, and dissolved oxygen in bioreactors while media and feed strategy experiments are carried out at small scale.
Avid is familiar with most commercial media, but sometimes clients ask the company to explore specific media and feeds that worked well in early development. Zaidi calls such projects “informed scouting.” Her group performs small-scale experiments and applies software statistical packages to assure that the project proceeds “in the right direction.”
Numerous companies now offer media and feed development services. One of the more comprehensive is BD Biosciences’ BD AutoNutrientSM Media Design Service (AMDS).
AMDS optimizes chemically defined media and supplementation ingredients for cells that have already been optimized for innate protein production. An AMDS project takes between 3 and 12 months.
Although BD does not perform cell-line engineering, it recognizes the importance of nature in achieving optimal productivity. “We receive all types of cell lines, both low- and high-producers,” says Jon Wannlund, Ph.D., R&D director. Two daughter lines, selected from different clones, can have radically different productivity profiles. With some, no amount of nutritional supplementation or feeding strategies can improve productivity. But many, particularly those that express above approximately 1 µg/mL, can, through media strategies, become high producers.
In one AMDS project two years ago, nutritional optimization provided a 1.7-fold increase in IgG production; peptone supplementation improved this by another 2.1-fold, while feed strategies improved productivity by an additional 30%, for an overall 4.5-fold increase in productivity. Another project saw a fourfold improvement from media and supplementation alone (no feed work was done on these cells), to about 3.5 g/L. The client upped productivity to 6 g/L through process optimization.
But as the advertisement says, “mileage may vary.” The highest-producing cells tend to benefit most from nurturing strategies, but some lines do not respond at all, and those showing the greatest improvement or the highest productivity in the end may not be those with the highest output before media/feed strategies are applied.
A standard approach to the in-process nurture strategies for cell-line optimization is to monitor fermentation for a relatively narrow panel of analytes such as pH or dissolved oxygen. This technique is related to process analytics.
Similarly, metabolomics—the emerging omics discipline that quantifies metabolites and other small molecules—may serve as an indirect measure (as opposed to measuring protein concentration) of cell health and productivity. “Metabolomics tells you which nutrients are being used, which metabolites are getting into the cell, and what types of waste products are being produced,” notes Denise Sonntag, Ph.D., senior scientist at Biocrates Life Sciences.
The metabolome of a mammalian cell is typically large—upward of 3,000 compounds. Specialists like to mention that this is far fewer than the number of relevant genes or proteins, which is true, but concentration-dynamic ranges of small molecules of interest can be huge, a fact that complicates quantification. Nevertheless, the analysis can focus on the disposition of specific nutrients and cofactors, or take in a subset of biogenic amines, amino acids (and their metabolites), lipids, fatty acids, or energy-related metabolites. Profiles of a predefined, select subset of the metabolome give a precise snapshot of what is occurring in the cell at a specific time, for example during growth or expression phases.
Considering that cell-based biomanufacturing seeks to produce proteins that are foreign to the expression systems, in bioreactors instead of intact organisms, it is no wonder so much effort is expended on optimization. One may argue the relative merits of hard-wiring cells for productivity, or coaxing them through media/feed strategies later on, but it is clear that success depends on both strategies.
“Nature and nurture need to come together,” opines Dr. Seewoester. But there are limits to what may be achieved with poorly producing cells. “Without natural expression capabilities, nurturing through media and process conditions will be limited.” On the other hand, every cell can be improved through selection and engineering. “We have not seen one example of a wild-type host cell in the mammalian or microbial world that can’t be effectively and significantly improved through targeted optimization on the genetic or physiological level,” he says.