Polymorphic distribution of proteins in solution by mass spectrometry: The analysis of insulin analogues.

The characterization of conformational and oligomeric distribution of proteins is of paramount importance for the understanding of the correlation between structure and function. Among the bioanalytical approaches currently available, the electrospray ionization-mass spectrometry (ESI-MS) coupled to ion mobility spectrometry (IMS) is the best suited for high resolution identification with high sensitivity, allowing the in situ separation of oligomeric and conformational species. We tested the performance of the ESI-MS technique along with the IMS separation approach on a broad variety of insulin and insulin analogues with distinct oligomeric distribution pattern. The measurement of commercial insulin allowed the identification of species ranging from monomers to hexamers and their complexes with zinc ions. Dissimilar distribution profile for regular insulin as a function of formulation component and among the insulin analogues were observed by ESI-IMS-MS but not by ESI-MS along, crystallographic assays or size-exclusion chromatography. These data suggest the additional suitability of ESI-IMS-MS in conformational and oligomeric profiling of biomacromolecules and biopharmaceuticals. The easiness of the technique provides further motivation for its application in the characterization of both raw and formulated protein biopharmaceuticals in routine and comparability exercises.

Assignment of polymorphic species of insulin analogues in ion mobility mass spectroscopy.

Electrospray ionization - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) allows the identification of protein polymorphic distribution of protein conformers and oligomers. We report the detailed identification of the species observed with commercially available pharmaceutical preparation of wild-type, regular human insulin.

Macromolecular confinement of therapeutic protein in polymeric particles for controlled release: insulin as a case study

ABSTRACT Sustained release systems for therapeutic proteins have been widely studied targeting to improve the action of these drugs. Molecular entrapping of proteins is particularly challenging due to their conformational instability. We have developed a micro-structured poly-epsilon-caprolactone (PCL) particle system loaded with human insulin using a simple double-emulsion w/o/w method followed by solvent evaporation method. This formulation is comprised by spheric-shaped microparticles with average size of 10 micrometers. In vitro release showed a biphasic behavior such as a rapid release with about 50% of drug delivered within 2 hours and a sustained phase for up to 48 h. The subcutaneous administration of microencapsulated insulin showed a biphasic effect on glycemia in streptozotocin-induced diabetic mice, compatible with short and intermediate-acting behaviors, with first transition peak at about 2 h and the second phase exerting effect for up to 48h after s.c. administration. This study reveals that a simplified double-emulsion system results in biocompatible human-insulin-loaded PCL microparticles that might be used for further development of optimized sustained release formulations of insulin to be used in the restoration of hormonal levels.

Molecular confinement of human amylin in lipidic nanoparticles.

Amylin is a pancreatic hormone involved in the regulation of glucose metabolism and homeostasis. Restoration of the post-prandial and basal levels of human amylin in diabetic individuals is a key in controlling glycemia, controlling glucagon, reducing the insulin dose and increasing satiety, among other physiologic functions. Human amylin has a high propensity to aggregate. We have addressed this issue by designing a liposomal human amylin formulation. Nanoparticles of multilamellar liposomes comprising human amylin were obtained with 53% encapsulation efficiency. The in vitro kinetic release assay shows a biphasic profile. The stabilization of the lipidic nanoparticle against freeze-drying was achieved by using mannitol as a cryoprotectant, as evidenced by morphological characterization. The effectiveness of the human amylin entrapped in lipidic nanoparticles was tested by the measurement of its pharmacological effect in vivo after subcutaneous administration in mice. Collectively these results demonstrate the compatibility of human amylin with the lipidic interface as an effective pharmaceutical delivery system.

Monoconjugation of Human Amylin with Methylpolyethyleneglycol.

Amylin is a pancreatic hormone cosecreted with insulin that exerts unique roles in metabolism and glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable in diabetes mellitus. Protein conjugation with the biocompatible polymer polyethylene glycol (PEG) has been shown to extend the biological effects of biopharmaceuticals. We have designed a PEGylated human amylin by using the aminoreactive compound methoxylpolyethylene glycol succinimidyl carbonate (mPEGsc). The synthesis in organic solvent resulted in high yields of monoPEGylated human amylin, which showed large stability against aggregation, an 8 times increase in half-life in vivo compared to the non-conjugated amylin, and pharmacological activity as shown by modulation of cAMP production in MCF-7 cell line, decrease in glucagon and modulation of glycemia following subcutaneous administration in mice. Altogether these data reveal the potential use of PEGylated human amylin for the restoration of fasting amylin levels.

Preparation and characterization of PEGylated amylin.

Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono- and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys(1). The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono- and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.

Amylin conjugation with methoxyl polyethyleneglycol.

We modified amylin chemically by conjugating methoxyl polyethyleneglycol succinimidyl carbonate (mPEGSC) of varying molecular weights (1 kDa, 2 kDa and 5 kDa). The reaction occurred within a few minutes, resulting in at least four distinct PEGylated products. The reaction products were separated by reversed-phase chromatography and identified by mass-spectrometry. The monoPEGylated and diPEGylated amylin products were generated rapidly through conjugation to the two amino groups of the N-terminal lysine residue. Both PEGylated amylin products bound to the receptor activity-modifying protein 1 (RAMP1). Pharmacological evaluation by subcutaneous administration in mice of monoPEGylated and diPEGylated amylin obtained with mPEG-SC 5 kDa revealed that both compounds modulated glycemia for longer times than unmodified amylin. Collectively, these data demonstrate the potential of bioconjugation with mPEG for the design of amylin therapeutics with sustained action.

Polymeric particles for the controlled release of human amylin.

Since its discovery the therapeutic use of the pancreatic hormone amylin has been limited due to its poor water solubility and propensity for amyloid aggregation. We have entrapped the human amylin protein in polymeric nanoparticles, using a single emulsion-solvent evaporation method and investigated its effectiveness in the controlled release of the peptide. Typical preparations composed of poly-ε-caprolactone had a mean particle size of approximately 200 nm, low polydispersity index, high protein entrapment efficiency (80%) and process yield (90%), and spherical and smooth surfaces. These nanoparticles presented a controlled release in vitro for approximately 240 h. Pharmacological evaluation in vivo by subcutaneous administration in fasting mice demonstrated the bioactivity and effectiveness of the released human amylin, resulting in reduced glycemia lasting for at least 36 h. These features indicate the potential for the use of a confined particulate system in the therapeutic controlled and sustained release of human amylin.