Cell culture media are complex products that are inherently difficult to analyze. Yet, media analysis is critical for assuring product quality. A group at National University of Ireland-Galway led by Alan Ryder, Ph.D., is investigating rapid, “holistic” fluorescence analysis for screening commercially used cell culture media.
“Holistic means taking on overall measurement of the media, rather than going down the separations route and identifying or quantifying every component,” Dr. Ryder explains.
His group has been working with Bristol-Myers Squibb (BMS) and other leading biomanufacturers on the technique, which is designed for Chinese hamster ovary (CHO) cell cultures. Eventually, it will become available to all biotech companies.
The technique uses readily available fluorescence instrumentation, costing about $20,000, and MATLAB, a fourth-generation engineering software package from MathWorks (www.mathworks.com). It further relies on chemometric analysis such as multiway robust principal component analysis (MROBPCA), n-way partial least-squares discriminant analysis, and regression (NPLS-DA).
MROBPCA assesses data variance, determines its statistical significance, and seeks to understand the source of the spectral variation. “If the variation is significant we use NPLS-DA to build identification models to discriminate different media based on their spectra, or NPLS to quantify the relationship between spectral data variation and production data, for example yield.”
This approach was developed empirically, by correlating samples of cell culture media from BMS with productivity measurements of manufacturing batches employing those specific media.
These sophisticated statistical tools are required since, while it identifies medium components through their natural fluorescence, this approach does not quantify ingredients individually. Rather, it provides a fluorescence map or signature that quantifies media quality and predicts its success. “We look at overall changes, not specific components.” Dr. Ryder claims the technique predicts volumetric productivity to within 0.13 g/L.
Chromatographic analysis of media components is possible but extremely time-consuming and expensive. Dr. Ryder’s rapid, low-cost technique is suitable for screening media rapidly and then, if needed, analyzing only select formulations later by HPLC for quality or trouble-shooting purposes.
The ability to observe compositional changes in media and predict product yield before production has enormous potential, Dr. Ryder says. “It should effectively eliminate one of the major process variables, thus leading to more consistent product quality and yield.”
When to Optimize?
CHO lines have become the workhorse cells for the production of monoclonal antibodies—arguably the most successful class of biologic drug. The stakes for efficient CHO media development are, therefore, high but the trend toward chemically defined media burdens companies with multiple mAb products. A one-size-fits-all approach to media formulation ignores significant phenotypic differences between cell lines, even those arising from a common parental cell, while creating unique media for specific clones is time-consuming and costly.
Genentech has solved this dilemma through creation of a platform medium that serves all its CHO-based products from early development through Phase II testing. If necessary, cell-line-specific media optimization occurs once a molecule enters late-stage development. This approach saves time and effort during the riskiest stages of drug development, while applying extensive quality-by-design science to late-stage compounds.
Feng Li, Ph.D., pilot plant manager at Genentech, explains that extensive differences among clones warrants this approach. “Clones can be remarkably different, even when they arise from the same parental cell lines, and even if you transfect with the same gene.” Some cells grow rapidly, others slowly, and some show markedly different metabolism, for example lactate production. “Gene integration during transfection is a random process.”
To develop a platform media that can accommodate different clones’ needs, Dr. Li’s group tested multiple model cell lines with various growth and metabolic properties to balance nutrient supplement.
Genentech often divides projects into early and late phases based on clinical stage. “Only during late-stage development when high titer is required, do we then conduct intensive media optimization,” Dr. Li says. “Due to the high attrition rate during biopharmaceutical development, it is important to have a good platform medium to fill the gap between early- and late-stage development.”
Having a platform medium, in which cells are expected to grow and express proteins well, if not robustly, greatly aids in clone selection and early-stage process development. And while it is true that optimization before clone selection might uncover some pleasant productivity surprises, “no company can afford individual medium optimization, at this stage, for so many clones.”
Dr. Li conducts optimization the old fashioned way, by applying a design-of-experiment approach to key media ingredients but mainly though trial and error. Two or three iterations are usually sufficient to achieve optimization. “If you have a decent platform medium and well-behaved cell lines the process can last from three to six months,” Dr. Li says, depending on the target expression level, basal performance, and project timelines. “But each cell line is different.”