Scalability of Sartobind Rapid A Membrane for High Productivity Monoclonal Antibody Capture.

Improved upstream titres in therapeutic monoclonal antibody (mAb) production have shifted capacity constraints to the downstream process. The consideration of membrane-based chromatographic devices as a debottlenecking option is gaining increasing attention with the recent introduction of high-capacity bind and elute membranes. We have evaluated the performance and scalability of the Sartobind Rapid A affinity membrane (1 mL) for high-productivity mAb capture.

Effect of natural biopolymers on amyloid fibril formation and morphology.

Amyloid fibrils are associated with the pathogenesis of protein misfolding diseases such as Alzheimer's disease. These fibrils typically exhibit different morphologies when grown in vitro, and this has been known to affect their biological properties and cytotoxicity. The formation kinetics and resultant morphology of fibrils formed from the model proteins Bovine Insulin and Hen Egg White Lysozyme have been measured.

A generic class of amyloid fibril inhibitors.

Amyloid fibrils are large ordered fibrillar aggregates formed from mis-folded proteins. A number of human diseases are linked to the presence of amyloid deposits, including Alzheimer's disease, Parkinson's disease and type II diabetes. One therapeutic strategy for treating amyloid related diseases involves inhibiting fibril formation.

Electric field induced changes in protein conformation.

The effect of a low strength oscillating electric field on the conformation of Bovine Serum Albumin (BSA) and Lysozyme in solution has been measured. A purpose built cell has been used to measure the real time autofluorescence and Circular Dichroism of the protein solutions exposed to electric fields of differing strength and frequency. Exposure to the electric fields results in protein unfolding for both Lysozyme and BSA.

The effects of shear flow on protein structure and function.

Protein molecules are subjected to potentially denaturing fluid shear forces during processing and in circulation in the body. These complex molecules, involved in numerous biological functions and reactions, can be significantly impaired by molecular damage. There have been many studies on the effects of hydrodynamic shear forces on protein structure and function.

Shear flow induced changes in apolipoprotein C-II conformation and amyloid fibril formation.

The misfolding and self-assembly of proteins into amyloid fibrils that occur in several debilitating diseases are affected by a variety of environmental factors, including mechanical factors associated with shear flow. We examined the effects of shear flow on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Shear fields (150, 300, and 500 s(-1)) accelerated the rate of apoC-II fibril formation (1 mg/mL) approximately 5-10-fold.

Tyrosine autofluorescence as a measure of bovine insulin fibrillation.

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers --> monomers --> partially unfolded monomers --> oligomeric aggregates --> fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence.

Shear-induced deformation of bovine insulin in Couette flow.

We have applied a uniform, shear-driven flow field (Couette flow) to study the effect of shear on the structure and conformation of aqueous bovine insulin, in situ and in real time, using intrinsic Tyr fluorescence and circular dichroism (CD) spectroscopy. The morphology of post-shear insulin samples was analyzed using atomic force microscopy (AFM). Both fluorescence and CD data show a shear-dependent deformation of bovine insulin in Couette flow.

How does shear affect Abeta fibrillogenesis?

The fibrillogenesis of Abeta1-40 proceeds via three main stages: (i) formation of aggregates from monomers, (ii) linear association of these aggregates to form "beaded" protofibrils, and (iii) fusion and structural reorganization of protofibrils into mature fibrils. We have studied the effect of shear on the rate of each of these steps through a combination of fluorescence, atomic force microscopy, and circular dichroism experiments. We find that shear increases the rate of the first two stages (aggregation and protofibril formation) and inhibits the third.