PROCESS OPTIMIZATION AND VALIDATION OF AN ASSAY FOR HIGH-THROUGHPUT SCREENING

A biological assay is designed to set up a rapid and robust drug-screening system on a small scale. An assay is considered as a single unit of a platform to screen various compounds for aiding in drug discovery. Each assay is carried out in a 96-well plate, each of whose wells consists of the biological component called the Spheroids. The value of each assay lies in it facilitating for versatile screening applications. The spheroid is considered as a micro-structural product. And the addition of various compounds for testing is performed in each well (consisting of the spheroids). The focus has been to put forth the production principles and validation strategies to run the biological assay and test its efficacy to be used for screening in high volumes. The assay development illustrates processing and validation techniques. The goal is to develop optimized standards to process the assay, addressing various quality control issues, from the raw material to the end-product stage. Such an approach also brings interesting analogies of biological process in a manufacturing scenario. The developed system incorporates a value stream approach, by pulling the product from the customer end. The process involves simply encapsulating HUVECs (Human Umbelical Vein Endothelial cells) from the raw material stage, culturing to form the spheroid and transferring the component to assemblage in a 96-well format undergoing stages of heat treatments. The small scale screening system allows the use of small amounts of drug, which is especially essential for new drug synthesis or in rapid decision making to find out any unknown potent compounds. The design of optimal processes in product development of the spheroid assay is illustrated. Thus in light of the value of this assay, developing the production system has been pivotal so as to produce quality spheroids in the 96-well plate formats. The quantification of the stimulatory and inhibitory effects of the different agents is required to help understand the complex biological behavior involved. The goal is to validate the data using image analysis software. The image analysis helps determine the quantification to be accurate, objective, and consistent. The quality of the product is tested by the reproducibility and robustness of the assay

Small molecule antiangiogenic probes of the ubiquitin proteasome pathway: potential application to choroidal neovascularization. Invest Ophthalmol Vis Sci 47

PURPOSE. To characterize the angiogenic and inflammatory responses of human choroidal endothelial cells (HCECs) to stimulators and inhibitors of the ubiquitin proteasome pathway (UPP). METHODS. The regulation of the UPP by the inhibitor withaferin A and its congener, withanolide D, two natural products derived from the medicinal plant Withania somnifera was assessed in the three-dimensional endothelial cell sprouting assay (3D-ECSA), by using HCEC-and human umbilical vein endothelial cell (HUVEC)-derived spheroids embedded in a collagen I matrix. Western blot analysis was used to investigate the effect of withanolides on IB-␣, polyubiquitination, and heme oxygenase (HO)-1 regulation in HCEC and HUVEC cultures. RESULTS. HCECs, like HUVECs, responded to fibroblast growth factor-2, vascular endothelial growth factor, and tumor necrosis factor (TNF)-␣ stimulation and sprouted vessel-like structures in collagen I matrix. However, HCECs were slower to generate these sprouting vessels, when compared with HUVECs. The extent of inhibition of endothelial cell sprouting in 3D matrix, the blockade of TNF-␣-induced IB-␣ degradation, levels of global polyubiquitinated proteins, and induced production of HO-1 in response to treatment by the withanolides in cultured endothelial cells was similarly regulated between HCECs and HUVECs. CONCLUSIONS. HCECs share with HUVECs a similar response to UPP inhibitors, suggesting that this well-conserved pathway that regulates angioinflammatory mechanisms could be exploited for drug-targeting in the development of novel agents for CNV treatment. (Invest Ophthalmol Vis Sci. 2006;47: 4138 -4145