Micro-Flow Imaging: Flow Microscopy Applied to Sub-visible Particulate Analysis in Protein Formulations

The need to monitor, measure, and control sub-visible proteinaceous particulates in biopharmaceutical formulations has been emphasized in recent publications and commentaries. Some of these particulates can be highly transparent, fragile, and unstable. In addition, for much of the size range of concern, no practical measurement method with adequate sensitivity and repeatability has been available. A complication in measuring protein particulates in many formulations is the simultaneous presence of other particle types such as silicone micro-droplets, air bubbles, and extrinsic contaminants. The need has therefore been identified for new analytical methods which can accurately measure and characterize sub-visible particulates in formulations. Micro-flow imaging has been shown to provide high sensitivity in detecting and imaging transparent protein particles and a unique capability to independently analyze such populations even when other particle types are present

The Mechanism of Enhanced Insulin Amyloid Fibril Formation by NaCl Is Better Explained by a Conformational Change Model

The high propensity of insulin to fibrillate causes severe biomedical and biotechnological complications. Insulin fibrillation studies attain significant importance considering the prevalence of diabetes and the requirement of functional insulin in each dose. Although studied since the early years of the 20th century, elucidation of the mechanism of insulin fibrillation has not been understood completely. We have previously, through several studies, shown that insulin hexamer dissociates into monomer that undergoes partial unfolding before converting into mature fibrils. In this study we have established that NaCl enhances insulin fibrillation mainly due to subtle structural changes and is not a mere salt effect. We have carried out studies both in the presence and absence of urea and Gdn.HCl and compared the relationship between conformation of insulin induced by urea and Gdn.HCl with respect to NaCl at both pH 7.4 (hexamer) and pH 2 (monomer). Fibril formation was followed with a Thioflavin T assay and structural changes were monitored by circular dichroism and size-exclusion chromatography. The results show salt-insulin interactions are difficult to classify as commonly accepted Debye-Hückel or Hofmeister series interactions but instead a strong correlation between the association states and conformational states of insulin and their propensity to fibrillate is evident

Observance of polymorphic behaviour during dissolution of insulin and lysozyme

Although protein crystallization is a unit operation with potentially high separation factors, it has not been widely used in industry. Protein crystallization studies and practices have hitherto been largely limited to crystallography protocols. Knowledge of the behaviour of protein in solution would help to overcome empiric limitations in protein crystallisation. Thus, dissolution of porcine insulin and hen egg white lysozyme was studied and an unusual variation in solute concentration, with a concentration peak for short dissolution times, was verified. Polymorphic behaviour of protein in solution was observed, which altered physical properties such as solubility