Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling

The neurotrophins are a small group of dimeric proteins that profoundly affect the development of the nervous system of vertebrates. Recent studies have established clear correlations between the survival requirements for different neurotrophins of functionally distinct subsets of sensory neurons. The biological role of the neurotrophins is not limited to the prevention of programmed cell death of specific groups of neurons during development. Neurotrophin-3 in particular seems to act on neurons well before the period of target innervation and of normally occuning cell death. In animals lacking functional neurotrophin or receptor genes, neuronal numbers do not seem to be massively reduced in the CNS, unlike in the PNS. Finally, rapid actions of neurotrophins on synaptic efficacy, as well as the regulation of their mRNAs by electrical activity, suggest that neurotrophins might play important roles in regulating neuronal connectivity in the developing and in the adult central nervous system.

Physiology of the neurotrophins

Purpose
To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates.

/pdf/critical-evaluation-of-nanoparticle-tracking-analysis-nta-by-ly650nvx5j.pdf

Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates

Life on earth is almost entirely solar-powered. We can get some idea of the enormous quantity of energy received from the sun by noting that during daylight hours, the sun provides several thousand times more power to the surface of the U.S.A. than is produced by all of the nation’s electrical power stations. 1,2 Around 50% of the radiation that reaches the earth’s surface, roughly the visible region, is of a frequency useful to photosynthetic organisms. Oxygenic photosynthetic organisms convert this radiation into chemical energy, in the form of carbohydrate and dioxygen, at an optimal efficiency of something like 25%. 3 These products together sustain the rest of aerobic life, with carbohydrate acting as a source of high-energy electrons and dioxygen providing a lower-energy destination for these electrons. The overall equation of oxygenic photosynthesis is given in eq 1, where (CH2O) represents carbohydrate:

Water-splitting chemistry of photosystem II.

Fibroblast growth factors (FGFs) are small polypeptide growth factors, all of whom share in common certain structural characteristics, and most of whom bind heparin avidly. Many FGFs contain signal peptides for secretion and are secreted into the extracellular environment, where theycan bind to the heparan-like glycosaminoglycans (HLGAGs) of the extracellular matrix (ECM). From this reservoir, FGFs mayact directlyon target cells, or theycan be released through digestion of the ECM or the activityof a carrier protein, a secreted FGF binding protein. FGFs bind specific receptor tyrosine kinases in the context of HLGAGs and this binding induces receptor dimerization and activation, ultimatelyresulting in the activation of various signal transduction cascades. Some FGFs are potent angiogenic factors and most playimportant roles in embry onic development and wound healing. FGF signaling also appears to playa role in tumor growth and angiogenesis, and autocrine FGF signaling maybe particularlyimportant in the progression of steroid hormone-dependent cancers to a hormone-independent state.

/pdf/fibroblast-growth-factors-their-receptors-and-signaling-4rbv35g0yr.pdf

Fibroblast growth factors, their receptors and signaling.

Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions

Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.

Role of Arginine in Protein Refolding, Solubilization, and Purification

Aggregation or reversible self-association of protein therapeutics can arise through a number of different mechanisms. Five common aggregation mechanisms are described and their relations to manufacturing processes to suppress and remove aggregates are discussed.

/pdf/mechanisms-of-protein-aggregation-1l469vyqig.pdf

Mechanisms of protein aggregation.

A Method for Directly Fitting the Time Derivative of Sedimentation Velocity Data and an Alternative Algorithm for Calculating Sedimentation Coefficient Distribution Functions

Protein-based pharmaceuticals exhibit a wide range of aggregation phenomena, making it virtually impossible to find any one analytical method that works well in all cases. Aggregate sizes cover a range from small oligomers to visible “snow” and precipitates, and generally only the smaller species are reversible. It is less widely recognized that aggregates also exhibit a broad spectrum of lifetimes, and the lifetime has important consequences for detection methods. The fact that the measurement itself may destroy or create aggregates poses a major analytical challenge and is a key determinant for method selection. Several examples of some interesting aggregation phenomena and the analytical approaches we have used are presented. In one case, an “aggregate” seen by SEC in stressed samples was shown to actually be a partially denatured monomer using both size-exclusion chromatography with online multiangle laser light scattering (SEC-MALLS) and sedimentation velocity. In a second case, freeze/thaw stress generates transient, metastable oligomers that are extremely sticky and difficult to measure by SEC. By using sedimentation velocity as the “gold standard” a much improved SEC method was developed and used to investigate the temperature-dependent dissociation of these oligomers. For problems with visible particulates, dynamic light scattering has been effective, in our hands, at detecting the precursors to the large, visible particles and tracking the source of stress or damage to particular manufacturing steps.

/pdf/is-any-measurement-method-optimal-for-all-aggregate-sizes-376fn7tlzg.pdf

John S. Philo

Papers

Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions

Role of Arginine in Protein Refolding, Solubilization, and Purification

Mechanisms of protein aggregation.

A Method for Directly Fitting the Time Derivative of Sedimentation Velocity Data and an Alternative Algorithm for Calculating Sedimentation Coefficient Distribution Functions

Is any measurement method optimal for all aggregate sizes and types