Turning Tox Screening toward Biologics

Technology Advances

As pharmaceuticals turn more toward biologics, companies face challenges in finding ways to screen these new products for toxicokinetics. In preclinical safety studies of small molecules, pharmaceutical companies typically used liquid chromatography (LC) followed by mass spectrometry (MS) to measure a compound’s toxicokinetic profile and check for reactive metabolites. Biologics and their metabolites, however, pose a different challenge because the current analytical techniques are primarily based on ligand-binding assays. “This requires a different approach to toxicokinetic profiling. For example, serum-binding profiles are often carrier-protein specific for biologics compared to small molecules, which tend to ubiquitously bind to serum albumin,” says Rohan Thakur, PhD, director of small molecule solutions at Thermo Fisher Scientific in Waltham, Mass. “Proteins are different because these are rarely chemically-synthesized, but rather biologically-produced.” He adds, “Heterogeneity and hence purity is always in question.”

Part of the problem in assessing the potential toxicokinetics of a biologic is that it often depends on its heterogeneity. “Glycosylation and phosphorylation types of heterogeneity may change target-receptor kinetics, affecting toxicokinetics,” says Thakur. “For example, dephosphorylation of the native protein due to a change in the partition coefficient can make it active again, thereby influencing the toxicokinetic profile.” Such complexities of biologics are pushing toxicity testing from ligand-binding assays to LC/MS. But even here, technology faces some new challenges. For instance, proteins usually require a higher mass-to-charge range than small molecules, because proteins tend to have multiple charges, whereas small molecules usually have a mass-to-charge ratio of one. In addition, the endogenous nature of biologics often means that chemical background becomes an issue. Consequently, LC-MS for testing biological must be more specific. (See “Seeing More in HPLC.”)

“We recently launched our TSQ Vantage,” says Thakur, “and the whole idea behind the TSQ Vantage was S-Lens technology coupled to the HyperQuads, which leads to an increase in signal but reduced the transmission of chemical noise, thereby enhancing the signal-to-noise ratio, a vital factor in quantitative analysis.” Thakur adds that the TSQ series of triple-stage quadrupoles provides highly-selective, reaction monitoring (H-SRM). “This gives far better specificity compared to basic SRM methodology for the quantitative analysis of peptides,” says Thakur.

Seeing more in HPLC Testing a new compound’s properties, including toxicity, often requires high-performance liquid chromatography (HPLC). Phenomenex in Torrance, Calif., recently released several products that turn up the potential of HPLC for stability and toxicology studies. As Terrell Mathews, product manager at Phenomenex, says, “In degradation studies, for instance, you need to detect peaks that are 0.1 percent of the parent peaks. That requires sensitivity and a way to compress the main peak, which takes high surface area materials.” That’s why Phenomenex introduced a 2.5 micrometer particle in their Synergi HPLC line that provides 475 square meters of surface area per gram. In addition, during very early in vitro and in vivo toxicology studies, pharmaceutical companies require fast and accurate testing of low-concentration compounds in cellular and plasma matrices. “The goal at this early stage is to not do any sample preparation, but just put it right on an LC/MS system,” says Mathews. Such raw samples, though, can clog some columns. Phenomenex’s new two millimeter internal diameter Onyx, however, is a high throughput monolithic column for HPLC. “It’s very highly permeable,” says Mathews, “so it does not clog with dirty samples and produces no carry-over.” In short, says Mathews, “The Onyx provides consistent results with little clean up ahead of time.”

About the Author
Mike May, PhD, is a publishing consultant for science and technology based in Minnesota.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 11, November, 2008, pp. 14.

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Filed Under: Drug Discovery

 

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