The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

As the number of therapeutic proteins produced by mammalian cell cultures in the pharmaceutical industry continues to increase, the need to improve productivity and ensure consistent product quality during process development activities becomes more significant. Rational medium design is known to improve cell culture performance, but an understanding of nutrient consumption and metabolite accumulation within the medium is required. To this end, we have developed a technique for using 1D (1)H NMR to quantitate nonprotein feed components and metabolites in mammalian cell cultures. We refer to the methodology as "Fermentanomics" to differentiate it from standard metabolomics. The method was found to generate spectra with excellent water suppression, signal-to-noise, and resolution. More importantly, nutrient consumption and metabolite accumulation was readily observed. In total, 50 media components have been identified and quantitated. The application of Fermentanomics to the optimization of a proprietary CHO basal medium yielded valuable insight regarding the nutrient levels needed to maintain productivity. While the focus here is on the extracellular milieu of CHO cell cultures, this methodology is generally applicable to quantitating intracellular concentrations and can be extended to other mammalian cell lines, as well as platforms such as yeasts, fungi, and Escherichia coli.

Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools.

ABSTRACT:The pharmacokinetics and metabolism of anacetrapib (MK-0859), a novel cholesteryl ester transfer protein inhibitor, were examined in rats and rhesus monkeys. Anacetrapib exhibited a low clearance in both species and a moderate oral bioavailability of ϳ38% in rats and ϳ13% in monkeys. The area under the plasma concentrationtime curve in both species increased in a less than dose-proportional manner over an oral dose range of 1 to 500 mg/kg. After oral administration of [ 14 C]anacetrapib at 10 mg/kg, ϳ80 and 90% of the radioactive dose was recovered over 48 h postdose from rats and monkeys, respectively. The majority of the administered radioactive dose was excreted unchanged in feces in both species. Biliary excretion of radioactivity accounted for ϳ15% and urinary excretion for less than 2% of the dose. Thirteen metabolites, resulting from oxidative and secondary glucuronic acid conjugation, were identified in rat and monkey bile. The main metabolic pathways consisted of O-demethylation (M1) and hydroxylation on the biphenyl moiety (M2) and hydroxylation on the isopropyl side chain (M3); these hydroxylations were followed by O-glucuronidation of these metabolites. A glutathione adduct (M9), an olefin metabolite (M10), and a propionic acid metabolite (M11) also were identified. In addition to parent anacetrapib, M1, M2, and M3 metabolites were detected in rat but not in monkey plasma. Overall, it appears that anacetrapib exhibits a low-to-moderate degree of absorption after oral dosing and majority of the absorbed dose is eliminated via oxidation to a series of hydroxylated metabolites that undergo conjugation with glucuronic acid before excretion into bile.A significant amount of effort is being put into developing therapeutic agents that are capable of lowering low-density lipoprotein (LDL) levels because increased circulating levels of LDL have been demonstrated to increase risk for cardiovascular disease and associated clinical sequelae (Kannel et al., 1979;Castelli et al., 1983;Stamler et al., 1988). Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) remain the cornerstone of LDL-lowering therapy and have been demonstrated to reduce cardiovascular risk in humans (Shepherd et al., 1995). However, there remains a high incidence of residual cardiovascular events even after aggressive treatment with LDL-lowering drugs (Bays and Stein, 2003). It has recently been shown that, in addition to LDL, high-density lipoprotein (HDL) cholesterol is an independent factor that may modulate the risk of cardiovascular disease. Epidemiological evidence suggests that plasma HDL cholesterol levels are inversely correlated with atherosclerosis and cardiovascular risk (Asztalos et al., 2004;Cobain et al., 2007). However, there are no clinical outcome data to support this hypothesis at the present time. One class of dyslipidemia agents that affect HDL cholesterol levels are the inhibitors of cholesteryl ester transfer protein (CETP) (Hesler et al., 1987;Linsel-Nitschke and Tall, 2005). CETP is a plasma glycop...

MK-0524 ( Fig. 1) is a selective antagonist of prostaglandin D2 receptor 1 (DP1) (Sturino et al., 2007) that is undergoing preclinical and clinical development for the treatment of niacin-induced flushing. Niacin (nicotinic acid), a member of the vitamin B complex, has been used as a dietary supplement in milligram quantities and as a successful plasma lipid-modifying agent when administered in gram quantities. Niacin decreases plasma concentrations of cholesterol, free fatty acids, and triglycerides in humans, increases plasma high-density lipoprotein cholesterol (Shepherd et al., 1979), and decreases plasma very low-density lipoprotein and low-density lipoprotein cholesterol (Knopp, 1999). Furthermore, studies have shown that niacin can be effectively combined with statins to treat patients with low highdensity lipoprotein cholesterol (Rubenfire, 2004;Zhao et al., 2004). The major side effect with niacin therapy is the mild to severe cutaneous flushing experienced by patients that is mediated by vasodilation (Vogt et al., 2006). MK-0524, a potent DP1 antagonist, has an IC 50 value of 1.1 nM in a mouse DP1 functional assay (C. Sturino, G. The objective of the present study is to investigate the absorption, metabolism, and excretion of [ 14 C]MK-0524 in six male human volunteers. In preclinical species (rats and dogs), MK-0524 was eliminated primarily via glucuronidation, followed by excretion of the acyl glucuronic acid conjugate of the parent compound (M2) in bile . The in vitro metabolism of MK-0524 in nonclinical species and humans has been described elsewhere . The major metabolite in hepatocytes from all the species was M2, whereas phase I metabolites (hydroxylated and keto derivatives) collectively comprised a minor component of the metabolic profiles . Materials and Methods Chemicals