Effect of the spraying conditions and nozzle design on the shape and size distribution of particles obtained with supercritical fluid drying.

In the perspective of production of dry therapeutic protein formulations, spray drying of lysozyme (as a model protein) into supercritical carbon dioxide was studied. The effects of the nozzle (i.e.

Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation.

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP-SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488nm was recorded on excitation at 385nm. By combining HP-SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions.

Efficacy of pulmonary insulin delivery in diabetic rats: use of a model-based approach in the evaluation of insulin powder formulations.

The potential of N-trimethyl chitosan (TMC) with two degrees of quaternization (DQ), TMC20 (DQ 20%, as a mucoadhesive) and TMC60 (DQ 60%, as a mucoadhesive and a permeation enhancer), and dextran (as a non-mucoadhesive and non-permeation enhancer) microparticles as carriers for pulmonary delivery of insulin was studied in diabetic rats. The impact of the powder formulation on insulin bioavailability and its pharmacological effect was evaluated using a population pharmacokinetic-pharmacodynamic (PKPD) model. Insulin-loaded microparticles were prepared by a supercritical fluid (SCF) drying technique.

Rational design of nasal vaccines.

Nasal vaccination is a promising alternative to classical parental vaccination, as it is non-invasive and, in principle, capable of eliciting strong systemic and local immune responses. However, the protective efficacy of nasally administered antigens is often impaired because of delivery problems: free antigens are readily cleared from the nasal cavity, poorly absorbed by nasal epithelial cells and generally have low intrinsic immunogenicity. In this review paper, we describe the main physiological hurdles to nasal vaccine delivery, survey the progress made in technological approaches to overcome these hurdles and discuss emerging opportunities for improving nasal vaccines.

Extrinsic fluorescent dyes as tools for protein characterization.

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation.

Quality control of routine, experimental and real-time aged diphtheria toxoids by in vitro analytical techniques.

Physicochemical and immunochemical techniques can be used to assess the quality of diphtheria toxoid vaccines. In a previous paper [Metz B, Jiskoot W, Hennink WE, Crommelin DJA, Kersten GFA. Physicochemical and immunochemical techniques predict the quality of diphtheria toxoid vaccines.

Supercritical fluid drying of carbohydrates: selection of suitable excipients and process conditions.

The processibility of 15 carbohydrates, more or less commonly used, was investigated as excipients in supercritical fluid drying. The focus was on the ability to produce amorphous powder, the stability of the powders towards crystallisation, and the residual water and ethanol content. The aqueous solutions were sprayed into a pressurised carbon dioxide-ethanol mixture flowing cocurrently through a coaxial two-fluid nozzle.

Self-assembly of recombinant amphiphilic oligopeptides into vesicles.

The aim of the present study was to design amphiphilic oligopeptides that can self-assemble into vesicular structures. The ratio of hydrophilic to hydrophobic block length was varied, and peptides were designed to have a hydrophobic tail in which the bulkiness of the amino acid side groups increases toward the hydrophilic domain (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu(2/7)-COOH). These peptides were recombinantly produced in bacteria as an alternative to solid-phase synthesis.

Diphtheria toxoid-containing microparticulate powder formulations for pulmonary vaccination: preparation, characterization and evaluation in guinea pigs.

In this study, the potential of N-Trimethyl chitosan (TMC, degree of quaternization 50%) and dextran microparticles for pulmonary delivery of diphtheria toxoid (DT) was investigated. The antigen-containing microparticles were prepared by drying of an aqueous solution of polymer and DT through a supercritical fluid (SCF) spraying process. The median volume diameter of the dry particles, as determined by laser diffraction analysis, was between 2 and 3 microm and the fine particle mass fractions smaller than 5 microm, as determined by cascade impactor analysis, were 35 and 56% for the dextran and TMC formulations, respectively.

Stable sugar-based protein formulations by supercritical fluid drying.

The aim of this work was to produce stable, sugar-containing protein formulations by supercritical fluid (SCF) drying. Lysozyme solutions with and without added sucrose or trehalose were dried by spraying them in an SCF composed of CO(2) and ethanol or CO(2) only. The protein-to-sugar ratio was varied between 1:0 and 1:10 (w/w).

Characterization of a cyclosporine solid dispersion for inhalation.

For lung transplant patients, a respirable, inulin-based solid dispersion containing cyclosporine A (CsA) has been developed. The solid dispersions were prepared by spray freeze-drying. The solid dispersion was characterized by water vapor uptake, specific surface area analysis, and particle size analysis.

Preparation and physicochemical characterization of supercritically dried insulin-loaded microparticles for pulmonary delivery.

In the search for non-invasive delivery options for the increasing number of therapeutic proteins, pulmonary administration is an attractive route. Supercritical fluid (SCF) drying processes offer the possibility to produce dry protein formulations suitable for inhalation. In this study, insulin-loaded microparticles suitable for pulmonary administration were prepared and characterized.

Stabilization of IgG by supercritical fluid drying: optimization of formulation and process parameters.

The aim of this study was to stabilize human serum immunoglobulin G (IgG) by a supercritical fluid (SCF) drying process. Solutions containing IgG (20mg/ml) and trehalose or hydroxypropyl-beta-cyclodextrin in a 1:4 (w/w) ratio were sprayed into a SCF phase consisting of CO(2) and ethanol at 100bar and 37 degrees C. Initially, a set of drying conditions previously developed to successfully stabilize lysozyme and myogobin formulations was used [N.

ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity.

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol.

Cytosolic delivery of liposomally targeted proteins induced by photochemical internalization.

Purpose: The application of therapeutic proteins is often hampered by limited cell entrance and lysosomal degradation, as intracellular targets are not reached. By encapsulation of proteins into targeted liposomes, cellular uptake via endocytosis can be enhanced. To prevent subsequent lysosomal degradation and promote endosomal escape, photochemical internalization (PCI) was studied here as a tool to enhance endosomal escape.

Effect of excipients on the encapsulation efficiency and release of human growth hormone from dextran microspheres.

The possibility was investigated to modulate the encapsulation efficiency and release of human growth hormone (hGH) from hydroxyl ethyl methacrylated dextran (dex-HEMA) hydrogel microspheres by using excipients. Microspheres were prepared by polymerization of dex-HEMA in an aqueous two-phase system of this polymer and PEG with or without excipients (Tween 80, pluronic F68, sucrose, NaCl, urea or methionine). High hGH encapsulation efficiencies (50-70%) were obtained for microspheres prepared without excipients and with Tween 80, NaCl or methionine.

Recombinant gelatin hydrogels for the sustained release of proteins.

A recombinant gelatin (HU4) containing part of the amino acid sequence of the alpha1-chain of human type I collagen was used for preparing hydrogels for the sustained release of proteins. HU4 gelatin was modified with methacrylate residues for chemical crosslinking and gel formation. Methacrylated gelatins with degrees of substitution (DS; defined as fraction of methacrylate residues with respect to the total number of primary amines) of 0.

Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile red.

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein beta-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of beta-galactosidase below and above the protein's unfolding temperature of 57.

A role for protein misfolding in immunogenicity of biopharmaceuticals.

For largely unknown reasons, biopharmaceuticals evoke potentially harmful antibody formation. Such antibodies can inhibit drug efficacy and, when directed against endogenous proteins, cause life-threatening complications. Insight into the mechanisms by which biopharmaceuticals break tolerance and induce an immune response will contribute to finding solutions to prevent this adverse effect.

In vitro degradation behavior of microspheres based on cross-linked dextran.

The aim of this study was to investigate the in vitro degradation of hydroxyl ethyl methacrylated dextran (dex-HEMA) microspheres. Dextran microspheres were incubated in phosphate buffer pH 7.4 at 37 degrees C, and the dry mass, mechanical strength, and chemical composition of the microspheres were monitored in time.

N-trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model.

In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of a monovalent influenza subunit vaccine was investigated. The antigen-loaded nanoparticles were prepared by mixing a solution containing TMC and monovalent influenza A subunit H3N2 with a tripolyphosphate (TPP) solution, at ambient temperature and pH 7.4 while stirring.

Near-infrared imaging for studying homogeneity of protein-sugar mixtures.

Purpose: To investigate the applicability of near-infrared (NIR) imaging for assessing the homogeneity of dried protein-sugar formulations.

Methods: Physical mixtures of lysozyme and trehalose in different ratios were prepared and analyzed by near-infrared (NIR) imaging with a spatial resolution of 10 or 40 microm. To define and select the best imaging strategy, besides visual inspection of the images, several approaches for data processing were tested: single wavelength intensity, peak/height ratio of two specific wavelengths, correlation coefficient with a reference spectrum and principal component analysis (PCA).

Static light scattering and small-angle neutron scattering study on aggregated recombinant gelatin in aqueous solution.

Recombinant gelatins are currently evaluated as new excipients for pharmaceutical formulations. They can differ from nonrecombinant gelatins because of intentional alteration of the amino acid sequence and specific properties of the expression systems used. This may affect their solution behavior.