Rapid multi-attribute characterization of intact bispecific antibodies by a microfluidic chip-based integrated icIEF-MS technology.

Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip-based integrated imaged capillary isoelectric focusing-mass spectrometry (icIEF-MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob-into-hole bispecific antibody.

Evaluation of an icIEF-MS system for comparable charge variant analysis of biotherapeutics with rapid peak identification by mass spectrometry.

Protein therapeutics are usually produced in heterogeneous forms during bioproduction and bioprocessing. Heterogeneity results from post-translational modifications that can yield charge variants and require characterization throughout product development and manufacturing. Isoelectric focusing (IEF) with UV detection is one of the most common methods to evaluate protein charge heterogeneity in the biopharmaceutical industry.

Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear Stress.

The role of nanotopographical extracellular matrix (ECM) cues in vascular endothelial cell (EC) organization and function is not well-understood, despite the composition of nano- to microscale fibrillar ECMs within blood vessels. Instead, the predominant modulator of EC organization and function is traditionally thought to be hemodynamic shear stress, in which uniform shear stress induces parallel-alignment of ECs with anti-inflammatory function, whereas disturbed flow induces a disorganized configuration with pro-inflammatory function. Since shear stress acts on ECs by applying a mechanical force concomitant with inducing spatial patterning of the cells, we sought to decouple the effects of shear stress using parallel-aligned nanofibrillar collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients.

Multiplexed Fluid Flow Device to Study Cellular Response to Tunable Shear Stress Gradients.

Endothelial cells (ECs) line the interior of blood and lymphatic vessels and experience spatially varying wall shear stress (WSS) as an intrinsic part of their physiological function. How ECs, and mammalian cells generally, sense spatially varying WSS remains poorly understood, due in part to a lack of convenient tools for exposing cells to spatially varying flow patterns. We built a multiplexed device, termed a 6-well impinging flow chamber, that imparts controlled WSS gradients to a six-well tissue culture plate.

Nek2 phosphorylates and stabilizes β-catenin at mitotic centrosomes downstream of Plk1.

β-Catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt signaling, and the centrosome cycle. Whereas the regulation of β-catenin in cell-cell adhesion and Wnt signaling are well understood, how β-catenin is regulated at the centrosome is not. NIMA-related protein kinase 2 (Nek2), which regulates centrosome disjunction/splitting, binds to and phosphorylates β-catenin.

Microvascular endothelial cells migrate upstream and align against the shear stress field created by impinging flow.

At present, little is known about how endothelial cells respond to spatial variations in fluid shear stress such as those that occur locally during embryonic development, at heart valve leaflets, and at sites of aneurysm formation. We built an impinging flow device that exposes endothelial cells to gradients of shear stress. Using this device, we investigated the response of microvascular endothelial cells to shear-stress gradients that ranged from 0 to a peak shear stress of 9-210 dyn/cm(2).