Physical stress testing of insulin suspensions and solutions.

Insulin pen-cartridge devices have evolved in order to increase patient compliance and convenience of use in a portable, multiple dosage device. With the advent of a portable insulin containing device, stability considerations have evolved from standard chemical indicators to include the effects of temperature and agitation on physical characteristics. To address these issues, two automated physical stress tests were developed based on market research data and input from regulatory authorities to understand the effect of thermomechanical stress on the product. First, the temperature cycling and resuspension test (TCRT) includes temperature cycling (25-37 degreesC) combined with agitation. The high temperature and extreme agitation test (HTEAT) includes continuous high temperature (37 degreesC) exposure combined with 4 h daily agitation. The total stress exposure is a function of the temperature, agitation, and time. The tests range from moderate stress (TCRT) to considerable stress (HTEAT) determined from the number of cartridge inversions and average daily temperature. Physical stress testing of both insulin suspensions and solution formulations in cartridges were performed and interpreted with respect to multiple endpoints. For suspensions, prolonged exposure to extreme stress caused the protein to form agglomerates, either in the suspension or adhered to the cartridge walls. In contrast, protein solutions subjected to the same extreme stress conditions did not exhibit any visually detectable change. Visual changes in the product under physical stress conditions can increase dose-potency result variability as well as exhibit acid-insoluble aggregates.

Fluorescence signaling of ligand binding and assembly in metal-chelating lipid membranes.

BACKGROUND: Chemical information is sometimes transmitted across cell membranes by ligand-induced assembly of receptors. We have previously designed a series of lipids with metal-chelating headgroups that can serve as general receptors for proteins containing accessible histidines. Such lipids can also be derivatized with pyrene, a fluorescent probe that has a different emission maximum when it is aggregated (excimer fluorescence) from that seen for the monomer. We set out to examine whether lipids of this kind would produce a signal in response to ligand binding. RESULTS: A model ligand, poly-L-histidine (poly(His)), bound specifically to pyrene-labeled Cu(II)-iminodiacetate lipid (Cu-PSIDA) within a membrane matrix. Binding of poly(His) induces the redistribution of Cu-PSIDA, so that it forms pyrene-rich domains that are detectable by the increased ratio of excimer to monomer fluorescence. Using rhodamine-labeled poly(His), we have shown that the receptor lipid domains correspond to poly(His)-rich domains below the lipid interface. CONCLUSIONS: The Cu-PSIDA receptor signals binding of the macromolecular ligand through its excimer fluorescence and allows the resulting domains formed by ligand assembly to be imaged. Fluorescent Cu-PSIDA can thus serve as an optical reporter of ligand-induced lipid reorganization.

Chiral copper-chelate complexes alter selectivities in metal affinity protein partitioning.

Proteins can be distinguished by exploiting complementarity between a histidine's microenvironment and a metal-chelate ligand in metal-affinity separations. The partitioning behavior of three myoglobins was investigated in aqueous two-phase polyethylene glycol-dextran systems containing polyethylene glycol derivatized with Cu(II) complexes of the L- and D-isomers of methionine and aspartate. TSK chromatographic supports derivatized with the methionine complexes were used to study retention of these proteins in metal-affinity chromatography. In the partitioning studies, the amino acid metal chelates exhibit selectivities for the myoglobins that are different from that of Cu(II)-iminodiacetate. Significant differences in selectivity based on the chiral nature of the amino acid complexes were also observed. The chromatographic selectivities of the chelating ligands exhibit little variation, however, suggesting that interactions occurring in solution but not on a surface play an important role in protein binding to the Cu(II)-amino acid-PEG complexes. In solution, the Cu(II)-amino acid complexes are sensitive probes of the microenvironments of surface histidines. The choice of the metal chelate affinity ligand offers a powerful means by which the selectivity of metal-affinity separations can be altered.