Pulmonary Application of Novel Antigen-Loaded Chitosan Nano-Particles Co-Administered with the Mucosal Adjuvant C-Di-AMP Resulted in Enhanced Immune Stimulation and Dose Sparing Capacity.

The most successful medical intervention for preventing infectious diseases is still vaccination. This effective strategy has resulted in decreased mortality and extended life expectancy. However, there is still a critical need for novel vaccination strategies and vaccines. Antigen cargo delivery by nanoparticle-based carriers could promote superior protection against constantly emerging viruses and subsequent diseases. This should be sustained by the induction of vigorous cellular and humoral immunity, capable of acting both at the systemic and mucosal levels. Induction of antigen-specific responses at the portal of entry of pathogens is considered an important scientific challenge. Chitosan, which is widely regarded as a biodegradable, biocompatible and non-toxic material for functionalized nanocarriers, as well as having adjuvant activity, enables antigen administration via less-invasive mucosal routes such as sublingual or pulmonic application route. In this proof of principle study, we evaluate the efficacy of chitosan nanocarriers loaded with the model antigen Ovalbumin (OVA) co-administrated with the STING agonist bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) given by pulmonary route. Here, BALB/c mice were immunized with four doses of the formulation that stimulates enhanced antigen-specific IgG titers in sera. In addition, this vaccine formulation also promotes a strong Th1/Th17 response characterized by high secretion of IFN-γ, IL-2 and IL-17, as well as induction of CD8+ T cells. Furthermore, the novel formulation exhibited strong dose-sparing capacity, enabling a 90% reduction of the antigen concentration. Altogether, our results suggest that chitosan nanocarriers, in combination with the mucosal adjuvant c-di-AMP, are a promising technology platform for the development of innovative mucosal vaccines against respiratory pathogens (e.g., Influenza or RSV) or for therapeutic vaccines.

Towards a Continuous Manufacturing Process of Protein-Loaded Polymeric Nanoparticle Powders.

To develop a scalable and efficient process suitable for the continuous manufacturing of poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing ovalbumin as the model protein. PLGA nanoparticles were prepared using a double emulsification spray-drying method. Emulsions were prepared using a focused ultrasound transducer equipped with a flow cell. Either poly(vinyl alcohol) (PVA) or poloxamer 407 (P-407) was used as a stabilizer. Aliquots of the emulsions were blended with different matrix excipients and spray dried, and the yield and size of the resuspended nanoparticles was determined and compared against solvent displacement. Nanoparticle sizes of spray-dried PLGA/PVA emulsions were independent of the matrix excipient and comparable with sizes from the solvent displacement method. The yield of the resuspended nanoparticles was highest for emulsions containing trehalose and leucine (79%). Spray drying of PLGA/P-407 emulsions led to agglomerated nanoparticles independent of the matrix excipient. PLGA/P-407 nanoparticles pre-formed by solvent displacement could be spray dried with limited agglomeration when PVA was added as an additional stabilizer. A comparably high and economically interesting nanoparticle yield could be achieved with a process suitable for continuous manufacturing. Further studies are needed to understand the robustness of a continuous process at commercial scale.

Combining R-DOTAP and a particulate antigen delivery platform to trigger dendritic cell activation: Formulation development and in-vitro interaction studies.

The utilization of the cationic lipid R-DOTAP as immune cell stimulant (e.g. its stimulating effects on immature dendritic cells) and correspondingly as possible adjuvant for vaccination is well known. Likewise, it is described in literature that solid polymer particles loaded with antigens can be size-tailored in a manner to be suitable for phagocytosis by antigen presenting cells. The effects of DOTAP-microparticle combinations, however, are not well understood. This study aimed therefore to explore the potential of R-DOTAP stabilized microparticles (MP) to act as a carrier platform for antigens e.g. for cancer vaccination. It was investigated whether or not a combination of R-DOTAP and PLGA leads to a boosted adjuvant effect in dendritic cell maturation. For proper comparison, neutral and negatively charged MPs of comparable sizes were developed. Toxicity, uptake, routing and maturation of the MP platform was assessed in-vitro on human immature dendritic cells (iDCs). Interestingly, none of the tested placebo formulations (without antigen) was capable to induce DC maturation when compared to LPS as positive control. This is in contrast to experiments previously reported in literature, where R-DOTAP (e.g. in liposomal form) triggered iDC maturation even without antigen. Possible reasons and further approaches are discussed in the paper.

Antigen delivery via hydrophilic PEG-b-PAGE-b-PLGA nanoparticles boosts vaccination induced T cell immunity.

Here, we evaluate the use of hydrophilic PEG-b-PAGE-b-PLGA (PPP) for the preparation of antigen loaded nanoparticles (NPs) as a platform for prophylactic vaccination. To investigate the suitability of PPP-NPs for antigen delivery, we used the double emulsion evaporation technique to prepare NPs of different sizes, antigen-loading efficiencies and -release kinetics for the model antigen Ovalbumin (OVA). Prior to applying the PPP-NPs in biological in vitro or in vivo models, all materials were tested for absence of cytotoxicity and endotoxins. While the uptake of NPs in antigen presenting cells was size but not polymer dependent, the efficiency of cross presentation of NP-associated antigen on MHC I molecules for CD8 T cell activation depended on the polymer type. T cell activation by antigen-presenting cells was significantly increased in vitro if antigen was delivered via PPP NPs compared to PLGA NPs or soluble OVA, although antigen content was the same in all tested formulations. Subcutaneous application of PPP-OVA-NPs even without adjuvants led to generation of potent CD8 T cell-mediated OVA-specific cytotoxicity in vivo that was more pronounced than after application of OVA alone or PLGA-OVA-NPs. Our data suggest that PPP-NPs can serve as platform for antigen-delivery in future vaccination formulations. Although PPP-NPs already bear intrinsic adjuvant-function, the complementation with TLR ligands loaded inside NPs may further strengthen the immune response to a point, where it might be possible to use it as a therapeutic vaccine to break immune tolerance in chronic disease states.

Investigation of the stabilizer elimination during the washing step of charged PLGA microparticles utilizing a novel HPLC-UV-ELSD method.

Quantification of stabilizer content in microparticles and other products is of great importance for formulation development, drug product quality control as well as for reproducible manufacturing. A fast and sensitive HPLC method with evaporative light scattering detection (ELSD) capable of detecting docusate sodium (DOSS), poly (lactic-co-glycolic acid) (PLGA; Resomer RG 503 H) and R-1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP) in a single run was successfully developed. In contrast to previously described methods, hydrolysis of PLGA as pretreatment is not necessary, thereby enabling accurate quantification of stabilizer next to the intact matrix polymer. This method was used to investigate the impact of washing procedures of polymeric microparticles manufactured either with anionic stabilizer DOSS or with cationic stabilizer DOTAP. High amounts of DOSS were detected in the washing water. This finding was consistent with the result that no DOSS could be detected in the washed and dried microparticles (

Focused Ultrasound as a Scalable and Contact-Free Method to Manufacture Protein-Loaded PLGA Nanoparticles.

PURPOSE: Although nanomaterials are under investigation for a very broad range of medical applications, only a small fraction of these are already commercialized or in clinical development. A major challenge for the translation of nanomedicines into the clinic is the missing scalability of the available lab scale preparation methods and, ultimately, non-identical samples during early and late research. METHODS: Protein-loaded PLGA nanoparticles using focused ultrasound in an emulsion solvent diffusion method were prepared in different batch sizes to evaluate achievable mean size, protein loading, and yield. RESULTS: Using the same equipment, nanoparticles could be prepared in batch sizes from 1 mg to 2.5 g. Size and yield were directly controllable by the amount of incident energy with good reproducibility. The nanoparticles displayed similar mean size, protein loading, and nanoparticle yield in batch sizes over three orders of magnitude. A scalable purification method based on diafiltration was established. CONCLUSIONS: The proposed method enables for feasibility studies during early research using just a small amount of polymer and protein, while at the same time it allows for larger scale production at later stages. As the proposed method further relies on contact-free energy transmission, it is especially suited for the preparation of clinical research samples.

Spatial separation of the processing and MHC class I loading compartments for cross-presentation of the tumor-associated antigen HER2/neu by human dendritic cells.

Cross-presentation is the process by which professional antigen presenting cells (APCs) (B cells, dendritic cells (DCs) and macrophages) present endocytosed antigens (Ags) via MHC-I to CD8+ T cells. This process is crucial for induction of adaptive immune responses against tumors and infected cells. The pathways and cellular compartments involved in cross-presentation are unresolved and controversial. Among the cells with cross-presenting capacity, DCs are the most efficient, which was proposed to depend on prevention of endosomal acidification to block degradation of the epitopes. Contrary to this view, we show in this report that some cargoes induce strong endosomal acidification following uptake by human DCs, while others not. Moreover, processing of the tumor-associated antigen HER2/neu delivered in nanoparticles (NP) for cross-presentation of the epitope HER2/neu369-377 on HLA-A2 depended on endosomal acidification and cathepsin activity as well as proteasomes, and newly synthesized HLA class I. However, the HLA-A*0201/HER2/neu369-377 complexes were not found in the endoplasmic reticulum (ER) nor in endolysosomes but in hitherto not described vesicles. The data thus indicate spatial separation of antigen processing and loading of MHC-I for cross-presentation: antigen processing occurs in the uptake compartment and the cytosol whereas MHC-I loading with peptide takes place in a distinct subcellular compartment. The findings further elucidate the cellular pathways involved in the cross-presentation of a full-length, clinically relevant tumor-associated antigen by human DCs, and the impact of the vaccine formulation on antigen processing and CD8+ T cell induction.

Comparison of different protein concentration techniques within preformulation development.

Highly concentrated antibody solutions are of increasing importance in the pharmaceutical industry. During production highly concentrated solutions are usually prepared by tangential flow filtration (TFF). Since this technique is often not applicable in the early phase of formulation development, where the available amounts of protein are commonly very small, small scale techniques like dialysis or ultrafiltration with stirred cells or centrifugal filters have to be employed. In this study the small scale techniques were compared to tangential flow filtration, with regard to the quality and stability of the concentrated products. The achievable concentration of a protein, when starting from a model antibody solution with 10mg/ml, was also assessed. Concentrations above 100mg/ml could be obtained with all techniques, however with different product qualities. The stability of the highly concentrated solutions (100 mg/ml) was analyzed by turbidity measurements, size exclusion chromatography (SEC), SDS-PAGE and isoelectric focusing (IEF) after storage at 25 and 40°C for 8 weeks. Solutions prepared by dialysis exhibited the smallest degree of instability, whereas those manufactured by centrifugal filtration revealed the best comparability to products obtained by tangential flow filtration with regard to the results of isoelectric focusing, turbidity measurements (UV-vis) and size exclusion chromatography. Stability differences were observed within all analytical methods, primarily after storage and not directly after the concentration process.

Investigating the effect of moisture protection on solid-state stability and dissolution of fenofibrate and ketoconazole solid dispersions using PXRD, HSDSC and Raman microscopy.

Enhanced dissolution of poorly soluble active pharmaceutical ingredients (APIs) in amorphous solid dispersions often diminishes during storage due to moisture-induced re-crystallization. This study aims to investigate the influence of moisture protection on solid-state stability and dissolution profiles of melt-extruded fenofibrate (FF) and ketoconazole (KC) solid dispersions. Samples were kept in open, closed and Activ-vials(®) to control the moisture uptake under accelerated conditions. During 13-week storage, changes in API crystallinity were quantified using powder X-ray diffraction (PXRD) (Rietveld analysis) and high sensitivity differential scanning calorimetry (HSDSC) and compared with any change in dissolution profiles. Trace crystallinity was observed by Raman microscopy, which otherwise was undetected by PXRD and HSDSC. Results showed that while moisture protection was ineffective in preventing the re-crystallization of amorphous FF, KC remained X-ray amorphous despite 5% moisture uptake. Regardless of the degree of crystallinity increase in FF, the enhanced dissolution properties were similarly diminished. Moisture uptake above 10% in KC samples also led to re-crystallization and significant decrease in dissolution rates. In conclusion, eliminating moisture sorption may not be sufficient in ensuring the stability of solid dispersions. Analytical quantification of API crystallinity is crucial in detecting subtle increase in crystallinity that can diminish the enhanced dissolution properties of solid dispersions.

Nanoparticle formation and growth during in vitro dissolution of ketoconazole solid dispersion.

The aim of this study is to examine the physical mechanisms during the dissolution of a solid dispersion, so as to provide further understanding behind the enhanced dissolution properties. X-ray amorphous solid dispersions of ketoconazole (KC), a poorly aqueous soluble drug, were prepared by melt extrusion with polyvinlypyrrolidone 17 (PVP 17) and PVP-vinyl acetate (PVP-VA64) copolymer. Prior to dissolution, Raman mapping showed a fully homogeneous spatial distribution of KC in polymer and possible drug dispersion at molecular level, whereas Fourier transform infrared spectroscopy revealed no drug-polymer chemical interaction. During in vitro dissolution test, a burst release followed by a gradual decline in dissolution could be explained by the release of KC in molecular form followed by formation of drug nanoparticles and their subsequent growth to micron size range as shown by dynamic light scattering analysis. Observations using transmission electron microscopy and cryogenic scanning electron microscopy provided support to the suggested mechanisms. The results suggested that the release of KC from the solid dispersions was carrier controlled initially, and PVP 17 PF is more efficient in inhibiting particle growth as compared with PVP-VA64. The particle growth inhibition during dissolution may be an important consideration to achieve the full benefits of dissolution enhancement of solid dispersions.

Detection of trace crystallinity in an amorphous system using Raman microscopy and chemometric analysis.

A novel analytical method to detect and characterize active pharmaceutical ingredient (API) trace crystallinity in an amorphous system using Raman microscopy and chemometric methods, namely band-target entropy minimization (BTEM) and target transformation factor analysis (TTFA) is developed. The method starts with Raman mapping measurements performed on some random areas of the amorphous system. This is followed by chemometric data analysis. In the case of a system without any a priori information, the BTEM algorithm is used to recover a set of pure component Raman spectral estimates followed by component and/or crystal structure identification. In the case of a system with some a priori information, TTFA is used to predict the presence or existence of a suspected component and/or crystal structure in the observed system. Four different amorphous systems were used as models. It is demonstrated that combined Raman microscopy and chemometric methods (BTEM or TTFA) outperformed powder X-ray diffraction (PXRD) in detecting trace crystallinity in amorphous systems. The spatial distributions of drug and polymer can also be directly obtained in order to study the homogeneity of the APIs in the solid dispersions. The present methodology appears very general and applicable to many other types of systems.

Relevant shaking stress conditions for antibody preformulation development.

In protein formulation development, shaking stress is often employed to assess the physical stability of antibody formulations against aggregation. Since there are currently no guidelines describing suitable test conditions, very different shaking stress designs are used. These different designs may influence the resulting stability data. The aim of this study was to establish a shaking stress design within the protein range of 2-5mg/ml which can rapidly distinguish between antibody formulations of poor stability and those with potential for further development. Small scale shaking stress experiments were performed with different monoclonal IgG antibodies (as buffered solutions or marketed formulations). Variables were the filling degree of the sample containers, the container type and size and the shaking intensity. The stability of the samples was assessed by visual inspection, UV-VIS spectrophotometric turbidity measurements and size exclusion chromatography. All tested parameters had a strong influence on the stability results. The most discriminating conditions were obtained when shaking of the formulations was performed at 200rpm in a 2ml injection vial filled with 1ml protein solution. This experimental setup led to clearly different stability results for buffered solutions and marketed products. Moreover, this setup required only relatively small amounts of protein solution which is advantageous in prefomulation studies.