Using PLS to understand potential sources of process variation & assessing medium components to alter afucosylation

Understanding potential sources of process performance and product quality variation in a manufacturing process may benefit from the use of multivariate data analysis techniques. PLS analysis identified some potential sources of process performance variation. For products where antibody-dependent cell-mediated cytotoxicity (ADCC) is part of the mechanism of action, total afucoslyated glycans (Afuc) variation may be important. This work was also directed at understanding current process variability for Afuc and the effect of process parameters that may vary during routine production. In addition, a series of small-scale experiments were conducted to screen several medium components for their ability to shift Afuc. Components studied included substrates and co-factors of enzymes involved in the relevant pathways (de novo and salvage production of GDP-fucose and fucosylation). Several medium components were shown be effective for altering levels of Afuc, however the majority of these also resulted in a loss in productivity. Supplementing zinc (Zn) and cobalt (Co) to production culture medium reduced Afuc without negatively impacting process performance

Mutually-Reactive, Fluorogenic Hydrocyanine/Quinone Reporter Pairs for In-Solution Biosensing via Nanodroplet Association

Mutually reactive, fluorogenic molecules are presented as a simple and novel technique for in-solution biosensing. The hypothesis behind this work was that aggregating droplets into close proximity would cause rapid mixing of their contents. To take advantage of this effect, a novel pair of fluorogenic redox molecules were designed to remain in lipid-stabilized oil droplets but mix once aggregated. First, the hydrophobic cyanine dye 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI) was reduced with sodium borohydride to form a nonfluorescent analog (HDiI). Hydrophobic quinone derivatives were then screened as oxidizing agents, and it was found that <i>p</i>-fluoranil oxidized nonfluorescent HDiI back to fluorescent DiI. Next, HDiI and <i>p</i>-fluoranil were loaded into NEOBEE oil nanodroplets of average diameter 600 nm that were stabilized by a monolayer of 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol (PEG), and DSPE-PEG-biotin. Addition of streptavidin caused aggregation of droplets and the appearance of red fluorescent aggregates within 30 min. Next, Nanoparticle Tracking Analysis was used to record the fluorescence of the droplets and their aggregates. By integrating the fluorescence emission of the tracked droplets, streptavidin could be detected down to 100 fM. Finally, the droplets were reformulated to sense for vascular endothelial growth factor (VEGF), a biomarker for tumor metastasis. Using anti-VEGF aptamers attached to DSPE-PEG incorporated into the nanodroplet monolayer, VEGF could also be detected down to 100 fM