Single-molecule resolution of protein dynamics on polymeric membrane surfaces: the roles of spatial and population heterogeneity.

Although polymeric membranes are widely used in the purification of protein pharmaceuticals, interactions between biomolecules and membrane surfaces can lead to reduced membrane performance and damage to the product. In this study, single-molecule fluorescence microscopy provided direct observation of bovine serum albumin (BSA) and human monoclonal antibody (IgG) dynamics at the interface between aqueous buffer and polymeric membrane materials including regenerated cellulose and unmodified poly(ether sulfone) (PES) blended with either polyvinylpyrrolidone (PVP), polyvinyl acetate-co-polyvinylpyrrolidone (PVAc-PVP), or polyethylene glycol methacrylate (PEGM) before casting. These polymer surfaces were compared with model surfaces composed of hydrophilic bare fused silica and hydrophobic trimethylsilane-coated fused silica.

Multipronged approach to managing beta-glucan contaminants in the downstream process: control of raw materials and filtration with charge-modified nylon 6,6 membrane filters.

(1→3)-β-D-Glucans (beta-glucans) have been found in raw materials used in the manufacture of recombinant therapeutics. Because of their biological activity, beta-glucans are considered process contaminants and consequently their level in the product needs to be controlled. Although beta-glucans introduced into the cell culture process can readily be removed by bind-and-elute chromatography process steps, beta-glucans can also be introduced into the purification process through raw materials containing beta-glucans as well as leachables from filters made from cellulose.

Impact of antibody aggregation on a flowthrough anion-exchange membrane process.

The impact of typical anion-exchange flowthrough conditions on the IgG mass loading of an anion-exchange membrane scale-down unit (Mustang Q coin) was investigated. High performance size-exclusion chromatography and multiangle laser light scattering results suggested the presence of a small fraction of IgG aggregates with average radius >100 nm under anion-exchange flowthrough conditions. The small filtration area presented by the 0.

Sequence-specific recognition of DNA by hydrophobic, alanine-rich mutants of the basic region/leucine zipper motif investigated by fluorescence anisotropy.

We generated minimalist proteins capable of sequence-specific, high-affinity binding of DNA to probe how proteins are used and can be used to recognize DNA. In order to quantify binding affinities and specificities in our protein-DNA system, we used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) domain to DNA duplexes containing target sites AP-1 (5'-TGACTCA-3') or ATF/CREB (5'-TGACGTCA-3'). We simplified the alpha-helical bZIP molecular recognition scaffold by alanine substitution: 4A, 11A, and 18A contain four, eleven, and eighteen alanine mutations in their DNA-binding basic regions, respectively.

Manipulation of temperature to improve solubility of hydrophobic proteins and cocrystallization with matrix for analysis by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) requires cocrystallization of analyte with a large excess of matrix, which must be mutually soluble in a solvent that encourages crystal growth upon evaporation. MALDI-MS of hydrophobic proteins can be difficult, because they tend to aggregate in polar solutions. High concentrations of denaturants and salts are often employed to combat protein aggregation, but this can result in signal suppression.