A major concern for process scaleup, according to Oren Beske, Ph.D., vp of laboratory services at Aragen Bioscience is having a robust scaled-down model.
“Whether it’s a transient transfection in HEK cells or stable expression in CHO cell lines, a scaledown model helps predict what is likely to occur at larger scale,” says Dr. Beske. “The presumption is, of course, that you will obtain similar cell growth characteristics, productivity, and product quality at large scale as was observed at small scale.”
Aragen is a CRO specializing in molecular biology, cell-line development, process development, and preclinical studies in rodents. Aragen’s scaledown models typically begin at about 20 mL, with the goal of predicting results up to 100 liters, but they occasionally perform work in microtiter formats as well. The factors that are typically controlled and evaluated at the smaller scale are surface area to volume ratio, mixing/agitation rates, feeds and feed schedules, and temperature.
Several vendors offer scaled-down systems, or microbioreactors, that control even more variables. Manufacturers for such systems include Dasgip, Sartorius Stedim Biotech, HexaScreen, New Brunswick Scientific, TAP Biosystems, and Applikon.
“These products are wonderful tools for process development on late-stage clones and, in some cases, earlier clone screening,” Dr. Beske says.
Yet as a CRO, Aragen does not enjoy the luxury of multimonth or even multiweek experimentation.
“Those higher-end technologies are useful when you’re investigating and defining nuances of cell culture processes, to tweak and obtain the highest expression levels. However, many of our customers need two grams in two weeks. We can’t very well ask them to give us six months to come up with an optimized process,” notes Dr. Beske.
But he admits that Aragen is moving toward these types of scaledown technologies. “We’ve entertained acquiring microbioreactor technologies as growth of our cell culture process development business justifies it.”
Stem Cells and Biosimilars
Scaling up stem cell cultures presents a maze of challenges quite different from those facing the expansion of mammalian or insect production cells, bacteria, or yeast. The obvious distinction is that therapeutic stem cells are the product itself, not merely production vehicles.
Moreover the concept of “batch” is turned on its head. “With proposed applications involving one batch per patient in many cases, we’re talking much smaller-volume production runs,” points out Ruedi Stoffel, who heads Roche’s cellular analysis business.
Stoffel compares the step up in complexity from traditional biomanufacturing to stem cell culture to the differences between small molecule drugs and biotherapeutics. Nonbiotech drugs are quality-checked using conventional chemical analysis. Biomolecules are much tougher since their characterization involves much more complex chemistry and biological activity.
“With therapeutic cells, production variability can mean treatment failures. In biomanufacturing you always retain the option of analyzing the product before release, and potentially remediating any production variability inefficiencies,” according to Stoffel.
The most critical steps in biomanufacturing occur during cell-line development and afterward in designing a feed strategy. This is true to some degree for stem cells as well, as investigators must select precursors capable of differentiating into the desired cells, and devise a differentiation strategy based on “feeding” the proper growth factors and cytokines.
But at the comparable point where biomanufacturers stop caring about cells, therapeutic cell producers’ interests peak.
The cell systems and scaleup strategies also differ in terms of economics. Traditional biotech is characterized by high R&D outlays, low manufacturing costs, and high market value. By contrast stem and therapeutic cell production is expensive at all levels and stages, and that is unlikely to change despite advances in manufacturing automation and the use of disposable equipment.
Finally, producers of protein drugs have developed both process- and unit operation-level platform technologies for production and purification. For therapeutic cells, aside from harvest and washing, production is always a custom, labor-intensive activity. “The steps are similar from product to product, but the timing and exact formulation of growth factors will always be different from one type of cell to the next,” says Stoffel.
“Four years ago Roche launched the Cellavista system, a microtiter plate reader that characterizes cells using bright field and fluorescence imaging. This automated technique provides extremely high-quality images of cells. By reading the contents of 96 wells in less than four minutes, Cellavista provides significant time savings compared with conventional microscopy.”
A number of researchers and producers of therapeutic (and protein-producing) cells have adopted this analytical platform to monitor the effects of growth conditions. Most recently, Dutch researchers used it to characterize mouse embryonic stem cell renewal.
Scaleup can be trickier for biosimilars than for originator molecules. “Developers must fit through a very narrow window of similarity,” notes Patrick Daoust, a consultant with Viropro which specializes in biosimilars. The company is working on follow-on versions of rituxumab and several other products.
U.S. regulations on biosimilars are notoriously fuzzy. Various working groups grapple with what constitutes chemical similarity, similarity in biological activity, and the relative importance of the two. The boundary developers of biosimilars do not wish to cross is the point of dissimilarity—again, poorly defined—that will trigger new clinical studies. Here Hatch-Waxman rules (or whatever emerges as the biosimilars guidance documents) will be of little comfort.
With small molecule drugs you either have the chemical structure (and to a lesser extent secondary characteristics and formulation) or you don’t. With biologics, as the process changes so does the product. And that includes secondary characteristics, like post-translational modifications, which may or may not affect the product’s effectiveness or safety.
Since nobody knows for sure, most experts agree that U.S. regulators will cite noticeable changes in such secondary characteristics.
Daoust notes an example of a process that provided high yield but slight differences in glycosylation relative to the comparator molecule. The company decided to forego huge cost-of-goods advantages and stick with the process that was less efficient but more faithful to the original molecule’s chemical composition.
“It’s a tradeoff that all developers of biosimilars will have to face,” he says. Developers have to begin thinking of products from an analytic perspective, and work with process development on analysis from very early on, to avoid surprises during scaleup.” That includes, he says, when the biosimilar has therapeutic efficacy that is superior to the original molecule.