Demonstrating comparability of secondary structure composition as part of higher order structure (HOS) in therapeutic proteins is a significant challenge. Previously, we showed that the variability of second derivative amide I Fourier transform infrared (FTIR) spectra were small enough that significant differences in secondary structures could be seen for a variety of model proteins. Those comparisons used spectral overlap and spectral correlation coefficients to quantify spectral differences. However, many of the excipients used in downstream purification process, drug substance, and drug product formulation, such as free amino acids and sugars, can interfere with the absorbance in the amide I region. In this study, analysis of amide II FTIR spectra is shown as an alternative to using spectral data from the amide I region to analyze protein secondary structure to assess their HOS. This research provided spectral overlap and spectral correlation coefficient mathematical approaches for analysis of amide II FTIR spectra to demonstrate comparability of protein secondary structure. Spectral overlap and spectral correlation coefficients results show strong correlations between changes in the second derivative of amide II and amide I FTIR spectra for various model proteins under different conditions, which demonstrate the applicability of using amide II FTIR spectra for the comparability of protein secondary structure. These results indicate that the analysis of the second derivative of amide II FTIR spectra may be used to monitor and demonstrate comparability of protein secondary structure during downstream process and formulation development of protein therapeutics.

Short peptides are important biopharmaceuticals as agonistic or antagonistic ligands, aggregation inhibitors, and vaccines, as well as in many other applications. They behave differently from globular proteins in solution. Many short peptides are unstructured and tend to aggregate and undergo structural transition in response to changes in solvent environment, including pH, temperature, ionic strength, presence of organic solvents or surfactants, and exposure to lipid membranes. Such structural transitions are often associated with fibril or β-amyloid formation. These structural characteristics of short peptides have drastic impact on their function, immunogenicity, and storage stability.

Microparticles prepared by the precipitation with a compressed antisolvent (PCA) process were evaluated for their potential in targeting an ionizable prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), for sustained delivery of INH to alveolar macrophages (AMs). The charged prodrug was ion-paired with two different hydrophobic cations (tetrapentylammonium (TPA)— and tetraheptylammonium (THA)—bromide), and loaded separately into the poly(l-lactide) (PLA) microparticles. The drug/polymer particles were spherical in shape and between 1 and 3 μm in diameter. The choice of hydrophobic cations did not affect drug incorporation efficiencies or the release kinetics of INH from the microparticles. Using a sensitive liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay developed for INH, high level of INH was detected in NR8383, a rat AM cell line, following exposure of these cells to drug-loaded microparticles. To confirm the microparticles can target AMs in vivo, we compared the INH levels in lavaged bronchoalveolar macrophages by LC-MS/MS after the Sprague–Dawley rats were administered either INHMS in PLA microparticles by intra-tracheal instillation or INH solution by gavage or intra-tracheal instillation. As expected, only microparticles provided sustained and targeted delivery of INH to AMs. Most importantly, this method of delivery led to substantial reduction in the blood levels of acetylisoniazid (AcINH), a major and potential toxic metabolite of INH.

Cisplatin is one of the most widely used and effective chemotherapeutic drugs ever discovered against certain forms of cancer. However, its use is limited by toxicity. A more potent form might allow lower doses to be used and would diminish the toxicity. A new analog of cisplatin has been synthesized by stoichiometric replacement of the chloride ligands with the anionic surfactant, Aerosol OT (AOT). The new compound has a very low aqueous solubility (about 2 mg/l) and a log P value of 2.17, which is more than 4 log units higher than cisplatin itself, indicating a dramatic increase in hydrophobicity. While hydrophobic cisplatin analogs have been synthesized previously, this is the first one with readily dissociable ligands replacing the chlorides. The resultant AOT complex is able to penetrate cellular membranes more efficiently, resulting in a threefold to fivefold increase in intracellular platinum levels. These increased intracellular concentrations correlate with lower IC50 values in a number of cancer and normal cell lines. These findings suggest that further development of the AOT complex as a chemotherapeutic agent is warranted, given its marked increase in potency over the parent compound.

Plasmid DNA encoding the green lantern protein was ion-paired with 1,2-dioleoyl, 3-trimethylammonium propane (DOTAP) at a (+/−) charge ratio of (1:1) to form a hydrophobic ion-pair (HIP) complex using the Bligh and Dyer method, and transferred into methylene chloride. Precipitation with a compressed antisolvent (PCA) was then employed to encapsulate plasmid DNA into poly(L-lactide) (PLLA) microspheres. The hydrophobicity of DOTAP:DNA complexes allowed consistently high encapsulation efficiencies (>70%) to be achieved. Release of the DOTAP:DNA complex from PLLA microspheres exhibitedminimal burst and a short (ca. 1 week) lag phase, followed by sustained release over a 20 week period. Release kinetics were consistent with a simple Fickian diffusion model. No correlation was identified between release rate of soluble poly(L-lactide) species (≤10 lactate units) from PLLA and the DNA release kinetics. Only ~12% of the polymer was degraded into soluble poly(L-lactide) over the time frame where ~90% of the plasmid load had been released.

Benzyl alcohol, an antimicrobial preservative, accelerates aggregation and precipitation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in aqueous solution. The loss of native monomer during incubation at 37 degrees C was determined by analysis of sample aliquots with size exclusion high performance liquid chromatography (SE-HPLC). Benzyl alcohol caused minor perturbation of the tertiary structure of the protein without changing its secondary structure, documenting that the preservative caused a minor shift in the protein molecular population toward partially unfolded species. Consistent with this conclusion, in the presence of benzyl alcohol the rate of H-D exchange was accelerated and the fluorescence of 1-anilinonaphthalene-8-sulfonic acid in the presence of rhIL1ra was increased. Benzyl alcohol did not alter the free energy of unfolding based on unfolding experiments in urea or guanidine HCl. With differential scanning calorimetry it was determined that benzyl alcohol reduced the apparent Tm of rhIL-1ra, but this effect occurred because the preservative lowered the temperature at which the protein aggregated during heating. Isothermal calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra is relatively weak and hydrophobically driven. Thus, benzyl alcohol accelerates protein aggregation by binding to the protein and favoring an increase in the level of partially unfolded, aggregation-competent species. Sucrose partially inhibited benzyl alcohol-induced aggregation and tertiary structural change. Sucrose is preferentially excluded from the surface of the protein, favoring most compact native state species over expanded aggregation-prone forms.

La presente invention a trait a des formulations pour administration pulmonaire comprenant une proteine et un tensioactif non physiologique au niveau ou au-dessus du niveau de la concentration critique pour la formation de micelles, et des procedes de preparation et d'utilisation de telles formulations.

Recombinant proteins and polypeptides continue to be the most important class of biotechnology-derived agents in today's pharmaceutical industry. Over the past few years, our fundamental understanding

This unit provides a summary of some of the issues that researchers face when attempting to store purified proteins. It briefly explains the stresses that induce protein aggregation the major causes for chemical degradation. It also discusses how to use various storage strategies to increase the long-term stability of proteins. When appropriate it points out critical mistakes to avoid. This unit provides a summary of some of the issues that researchers face when attempting to store purified proteins.