Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS.

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS-lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.

Alternative routes of administration for systemic delivery of protein pharmaceuticals.

Non-invasive and patient-friendly delivery of proteins are important targets for protein formulation development. Traditionally, a lyophilized cake for reconstitution followed by s.c. or i.v. administration has been used. At present, several protein pharmaceuticals delivered by alternative routes of administration are under investigation. The long-term effects of protein delivery by these alternative routes are not known. Nevertheless, these alternative administration routes can in the near future lead to new, successful protein pharmaceuticals on the market.:

Preliminary studies of the physical stability of a glucagon-like peptide-1 derivate in the presence of metal ions.

The physical stability and the secondary structure of a glucagon-like peptide-1 derivative were investigated in the presence of the metal ions Al(3+), Zn(2+), Mg(2+), and K(+), known as possible leachables from container-closure systems. Metal ions were investigated in concentrations of 0-50 ppm. Test solutions of the peptide were exposed to elevated temperature (25 degrees C) and rotation (37 degrees C) for up to 4 weeks. The samples were examined by nephelometry, thioflavine T fluorescence, and Fourier-transform infrared spectroscopy. Readily prepared test solutions were examined by tryptophan fluorescence. The stability profiles were unchanged after addition of Mg(2+) and K(+) in 0-50 ppm concentrations. However, a concentration-dependent increase in thioflavine intensities was observed after addition of Al(3+) and Zn(2+). The destabilising effect of Al(3+) and Zn(2+) was furthermore confirmed by FTIR as the secondary structure of the peptide changed from predominantly alpha-helix to a higher beta-sheet content. Additionally Al(3+) changed the secondary structure of the peptide using Trp fluorescence.

Stability, liposome interaction, and in vivo pharmacology of ghrelin in liposomal suspensions.

Ghrelin is an appetite-stimulating peptide hormone. It is a pharmacologically interesting peptide because of its involvement in e.g. appetite and metabolism, but it has a very short half-life in the body. Ghrelin carries a Ser-3-octanoyl group, and it has previously been suggested that acylated peptides can bind to or be incorporated into liposomes. Therefore, neutral dipalmitoylphosphatidylcholine (DPPC) liposomes and phosphatidylcholine:cholesterol (PC:Chol) (70:30) liposomes as well as negatively charged dipalmitoylphosphatidylcholine:dipalmitoylphosphatidylserine (DPPC:DPPS) liposomes (70:30) were prepared, and ghrelin was added. The chemical and physical stability of ghrelin was examined. Affinity capillary electrophoresis (ACE) revealed an interaction between ghrelin and the negatively charged (DPPC:DPPS) liposomes, whereas only very small affinities were discerned in the other liposomal formulations of ghrelin. Differential scanning calorimetry showed no changes in phase transitions (T(m)). In vivo pharmacokinetics following subcutaneous administration of ghrelin in buffer and in the liposomal formulations was examined in rats. The PC:Chol formulation had a longer-lasting effect as compared to the ghrelin buffer solution and the other two liposomal formulations. The prolonged effect of the PC:Chol formulation is suggested not to be caused by association between ghrelin and the liposome.

Recent trends in stabilising peptides and proteins in pharmaceutical formulation - considerations in the choice of excipients.

In the area of peptide and protein pharmaceuticals, both the physical and chemical stability of biopharmaceuticals are critical and need to be optimised when formulating a drug product, in order to optimise the outcome after processing and storage. This review focuses on the effects on the stability from various types of excipient and the choices that have to be made during formulation of drug products containing peptides or proteins. It is illustrated, through examples, how the choice of one excipient over another can affect the stability of a protein drug formulation, along with other problems linked to this choice. The excipients used in pharmaceutical preparations are limited and from an academic point of view there is a clear requirement for new excipients.

Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis.

In this manuscript we report the binding affinity between two model proteins, human serum albumin (HSA) and ribonuclease A (RNase A), and negatively charged polyelectrolytes, two different heparin fractions and dextran sulfate, by means of partial filling and affinity capillary electrophoresis. The apparent dissociation constants, K(d), obtained by use of the partial-filling method, between HSA and heparin (17kDa), heparin (3kDa) and dextran sulfate (8kDa) were 33 and 307microM, respectively. A new method was developed to determine affinities that take in account different migration directions between the protein and the polyelectrolyte, which was required to study RNase A. By use of this affinity capillary electrophoresis two K(d) values were observed for the interaction between RNase A and heparin 17kDa, yielding a high affinity binding with K(d1) 0.0075microM, and a lower affinity binding with K(d2) 8.7microM. For dextran sulfate 8kDa these K(d) values were 0.027 and 10.4microM, respectively. Heparin 3kDa only showed a single K(d) value of 0.52microM. The results show that the magnitude of the binding affinity depends on the type of polyelectrolyte and its molecular weight.