October 15, 2009 (Vol. 29, No. 18)

Emerging Insights on Metabolomics, Media, and 3-D Structures Could Lead to Improvements

The CHI conference “Optimizing Cell Culture Technology,” which was held in Boston recently, featured several speakers whose work directly affects the biopharmaceutical industry.

Yubing Xie, Ph.D., assistant professor at the College of Nanoscale Science and Engineering at SUNY Albany, discussed nanoparticle methods for delivering genes to cells and a hydrogel technique for creating 3-D tissue cultures whose microenvironments replicate complex tissue physiology. Applications include tissue regeneration, artificial organs, and drug screening.

“Cells in our bodies are in a 3-D environment,” noted Dr. Xie. “The idea here is to use nanotechnology and nanoengineering to mimic that environment, including extracellular factors such as the tissue matrix.”

Dr. Xie’s mouse embryonic stem cell  model can be programmed to take on the characteristics of tissue-specific cells for organ reconstruction. Similarly, they can be differentiated into hormone-secreting cells to replace malfunctioning organs, for example, in diabetes or into cytokine-secreting cells for treating cancer and other diseases.

The key is the microenvironment, or niche, that stem cell experts say is required (along with the right chemical stimulus) to coax cells into differentiating into their final forms. Dr. Xie hopes to replicate these niches through nanoengineering.

3-D microstructure also plays a role in how well cell cultures perform during drug screens. Drug discovery scientists spend billions of dollars per year on animal models because plated cells, which exist in flat or 2-D configurations, lack the form and function of living tissue. Where test drugs bathe cells cultured in monolayers in Petri dishes or microtiter plates, they must penetrate into living tissues. 3-D structures replicate the difficulties of penetration more closely and “can greatly reduce usage of test animals, which is always welcome,” Dr. Xie added.

Zhaohui Geng, Ph.D., principal scientist in culture process development at Pfizer, described her work on analysis of intracellular and extracellular metabolites that could predict the quality of proteins expressed in cell culture.

Dr. Geng first noticed that, in 1 L bioreactors, elevated pH caused lactate concentration to rise in CHO cell cultures. In an effort to understand the relationships between pH, lactate, and energy-production pathways, she submitted samples from cultures with elevated pH and control cultures, for metabolomic analysis to Metabolon.

The study uncovered significant differences in concentrations of metabolites related to energy production, particularly lipids. For example, glycerophosphorocholine, which is released from cells to combat osmotic stress, was particularly elevated in high pH cultures. This raised an interesting question: was pH or high osmolality to blame for lactate accumulation and concomitant changes in glucose and amino acid metabolism?

The metabolomic study quantified hundreds of metabolites, globally, unbiased, and without any pathway focus. Some, like pH, are measured daily in cell cultures, but many others are not. “We wondered if there were other trends, in measurements we did not take daily, that we should know about,” said Dr. Geng.

After plotting all the variables, she found that pH rose during the entire culture time, but the metabolic changes did not occur until the osmolality rose. “The metabolic change curve matched the osmolality change rather than the pH change.” She confirmed this finding by raising osmolality without pH changes by addition of sodium chloride, and, sure enough, found a salt concentration-related rise in lactate. 

“I think the osmolality-related metabolic changes are universal across cell lines and cultures,” Dr. Geng explained. “But the more interesting question is whether metabolic changes are related to changes in product quality. This question is currently unanswerable by looking only at daily reaction monitoring data.”

Mouse embryonic stem cells grown in a 3-D nanostructured bioreactor at SUNY Albany. (Yubing Xie, Ph.D.)

Perfusion Process

Kathryn Golden, associate scientist III at Percivia, presented data on a high-titer perfusion process for PER.C6 cells. The process is based on the high-titer, high-density XD™ perfusion process, which was developed by DSM and is being commercialized by Percivia, a joint venture between DSM and Crucell.

XD reportedly achieves cell densities of more than 200 million cells/mL—about ten times the density of conventional cultures—and antibody production titers of 40 g/L of cell-free supernatant over a 17-day culture. Because the cell volumes are substantial, the volumetric productivity are only 27 g/L of harvest when the correction is made, reported John Chon, Ph.D., director of process development.

“Moreover, the cells are healthy throughout the culture time,” he noted. “Cells in standard fed-batch cultures tend to be quite healthy in the beginning, but die off as a result of not removing metabolites.” In the XD process, metabolites are constantly removed and the cells are recirculated. Unlike standard perfusion cultures, product is retained within the medium, which provides substantial benefits during purification by keeping volumes low, Dr. Chon added.

XD has been demonstrated at the 50 L scale which, due to the high cell density and volumetric productivity, performs about as well as a reactor ten times as large, Dr. Chon said. Percivia is working to bring the culture volume up to 250 L.

XD is based on the ATF™ cell culture system from Refine Technology. ATF consists of a pump and hollow fiber membrane that removes media from the culture but returns cells and products. The ATF system connects to any bioreactor (stainless steel or disposable) and can handle cultures of up to 2,000 L working volume. “The great benefit is that you don’t need to retool your manufacturing plant to use XD technology,” noted Dr. Chon.

Percivia’s XD™ process takes advantage of the PER.C6® cell’s ability to withstand high shear and grow to high concentrations.

Minimal Media

Ferruccio Messi, Ph.D., CEO of Cell Culture Technologies, another speaker at the Cambridge event, is a huge advocate of what he calls minimal cell culture media. Minimal media contain only water and small molecule chemicals with Chemical Abstract Services or European Inventory of Existing Commercial Chemical Substances numbers—no animal-derived sera, extracts, peptones, hydrolysates, or other undefined substances. “We only use compounds with a defined chemical identity,” said Dr. Messi.

According to Dr. Messi, 15 companies have performed “remarkable work” on the cultivation of CHO cells in minimal media. He described one line, the CHO-easyC (derived from CHO-K1), as “one of the most promising host cell lines for development of next-generation bioprocesses.

“Many people talk about chemically defined media, but when you try to go beyond that definition you realize that chemically defined means everything and therefore nothing. People are using chemically defined lipid mixtures, and the user doesn’t know what’s in there. In my presentation, I showed the complete composition of a minimal medium, and everybody knew what I was talking about.”

Even more than media that take half-measures to achieve chemical definition, minimal media replace all vestiges of black boxes and “fairy dust” from cell culture with hard science—only what cells actually need to thrive and produce.” The most obvious benefit is batch-to-batch consistency and the opportunity for science-based optimization—goals that can only be achieved when processors know precisely what media consist of. Minimal media are also less likely to require esoteric purification techniques and more readily satisfy regulators.

Processors add extracts, hydrolysates, and other ingredients, explained Dr. Messi, because their basal media are not optimized. “They didn’t fulfill the cell’s basic nutritional requirements, so they compensated by adding boosters. But you can easily grow any kind of cell in minimal media if you knew the identity of every molecule required by cells, and their proper balance.”

Cell Culture Technologies provides its expertise through long-standing collaborations with client companies. The average such relationship, which involves scientists from Dr. Messi’s company working at clients’ facilities, lasts 2.7 years. “It’s a lot more complex than filling a bottle with media and selling it,” Dr. Messi explained. 

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