Working within the Design Space: Do Our Static Process Characterization Methods Suffice?

The Process Analytical Technology initiative and Quality by Design paradigm have led to changes in the guidelines and views of how to develop drug manufacturing processes. On this occasion the concept of the design space, which describes the impact of process parameters and material attributes on the attributes of the product, was introduced in the ICH Q8 guideline. The way the design space is defined and can be presented for regulatory approval seems to be left to the applicants, among who at least a consensus on how to characterize the design space seems to have evolved.

Preclinical evaluation of a chemically detoxified pneumolysin as pneumococcal vaccine antigen.

The use of protein antigens able to protect against the majority of Streptococcus pneumoniae serotypes is envisaged as stand-alone and/or complement to the current capsular polysaccharide-based pneumococcal vaccines. Pneumolysin (Ply) is a key virulence factor that is highly conserved in amino acid sequence across pneumococcal serotypes, and therefore may be considered as a vaccine target. However, native Ply cannot be used in vaccines due to its intrinsic cytolytic activity.

Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein.

Species and tissue distribution of the regulatory protein of glucokinase.

Rat liver is known to contain a regulatory protein that inhibits glucokinase (hexokinase IV or D) competitively versus glucose. This inhibition is greatly reinforced by the presence of fructose 6-phosphate and antagonized by fructose 1-phosphate and by KCl. This protein was now measured in various rat tissues and in the livers of various species by the inhibition it exerts on rat liver glucokinase.

Heterogeneity in glucose sensitivity among pancreatic beta-cells is correlated to differences in glucose phosphorylation rather than glucose transport.

Rat beta-cells differ in their individual rates of glucose-induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta-cell subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.

Binding of sorbitol 6-phosphate and of fructose 1-phosphate to the regulatory protein of liver glucokinase.

Using a binding assay in which the ligand-protein complex is separated from free ligand by precipitation with poly(ethylene glycol) 6000, we found that the regulatory protein of rat liver glucokinase bound close to 1 mol of radiolabelled sorbitol 6-phosphate, a negative effector, or of fructose 1-phosphate, a positive effector, per mol of regulatory protein. Scatchard plots were linear, the dissociation constant being 0.3 microM for both phosphate esters.

Mechanism of the stimulatory effect of a potassium-rich medium on the phosphorylation of glucose in isolated rat hepatocytes.

We have investigated the mechanism by which the replacement of a Na(+)-rich medium by a K(+)-rich medium causes an increase in the apparent affinity of glucokinase (hexokinase IV or D) for glucose in isolated hepatocytes [Bontemps, F., Hue, L. & Hers, H.

The regulatory protein of liver glucokinase.

Fructose, sorbitol and D-glyceraldehyde stimulate the rate of glucose phosphorylation in isolated hepatocytes. This effect is mediated by fructose 1-phosphate, which releases the inhibition exerted by a regulatory protein on liver glucokinase. In the presence of fructose 6-phosphate, the regulatory protein binds to, and inhibits, liver glucokinase.

Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation.

In the absence of fructose 6-phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15-fold) lower potency than in the presence of a saturating concentration of fructose 6-phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6-phosphate. In the presence of regulatory protein, sorbitol 6-phosphate, a potent analog of fructose 6-phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein.

Competitive inhibition of liver glucokinase by its regulatory protein.

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half-saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate).

The mechanism by which rat liver glucokinase is inhibited by the regulatory protein.

The fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J.

Regulation of glucokinase by a fructose-1-phosphate-sensitive protein in pancreatic islets.

In the post-microsomal supernatant of pancreatic islets, prepared from fasted or fed rats, D-fructose 1-phosphate increased the activity of glucokinase by 20-30% as measured in the presence of D-glucose 6-phosphate and D-fructose 6-phosphate. Such an activation was less marked than that found in liver extracts. The islet cytosol was also found to inhibit purified liver glucokinase, and this effect was antagonized by D-fructose 1-phosphate.

Fructose 2,6-bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii.

Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose.

Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae.

The properties of yeast trehalose-6-phosphate synthase were reinvestigated in relation with the recent claim made by Panek et al. [Panek, A. C.

Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes.

The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system.