High-Throughput Quantification and Glycosylation Analysis of Antibodies Using Bead-Based Assays.

A novel version of bead -based assays with fluorescence detection enables the high-throughput analysis of antibodies and proteins. The protocols are carried out in special 384-well plates, require very few manual interventions, and are easy to automate. Here we describe how the technology can be used to determine antibody titers and screen for product glycosylation, a critical quality attribute, early in cell line and bioprocess development.

Permeation through phospholipid bilayers, skin-cell penetration, plasma stability, and CD spectra of α- and ß-oligoproline derivatives.

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-ß(3)-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-ß(2)h- and oligo-ß(3)h-prolines without and with fluorescence labels (1-8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2-4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6-8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9-11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.