Evaluation of Different Quality-Relevant Aspects of Closed System Transfer Devices (CSTDs).

PURPOSE: Health care professionals can be exposed to hazardous drugs such as cytostatics during preparation of drugs for administration. Closed sytem transfer devices (CSTDs) were introduced to provide protection for healthcare professional against unintended exposure to hazardous drugs. The interest in CSTDs has significantly increased after USP <800> monograph was issued. The majority of the studies published so far on CSTDs have focused on their "containment" function. However, other important attributes for CSTDs with potential importance for product quality impact are not yet fully evaluated. METHODS: In the current study, we evaluated four sytems from different suppliers, in combination with different container closure systems (CCS), using solutions of different viscosity and surface tension. The different CSTD / CCS combinations were tested for (a) containment (integrity) using a highly sensitive helium leak test, (b) the force required for mounting the vial adaptor, (c) contribution to visible and subvisible particles as well as (d) the hold-up volume. RESULTS: Results show that the majority of CSTDs may have leaks varying in size, and that some of them generated visible particles due to stopper coring and subvisible particles, both due to silicon oil and particulate contaminations of the Devices. Finally, the holdup volume was up to 1 mL depending on the CSTD type, vial size and solution viscosity. CONCLUSION: These results show that there is a need to evaluate the compatibility of CSTD systems to select the best system for the intended use and that CSTDs may adversely impact product quality and delivered dose.

A Survey on Handling and Administration of Therapeutic Protein Products in German and Swiss Hospitals.

Protein products in hospitals often have to be compounded before administration to the patient. This may comprise reconstitution of lyophilizates, dilution, storage, and transport. However, the operations for compounding and administration in the hospital may lead to changes in product quality and possibly even impact patient safety. We surveyed healthcare practitioners from three clinical units using a questionnaire and open dialogue to document common procedures and their justification and to document differences in handling procedures. The survey covered dose compounding, transportation, storage and administration. One key observation was that drug vial optimization procedures were used for some products, e.g., use of one single-use vial for several patients. This included the use of spikes and needles or closed system transfer devices (CSTDs). Filters or light protection aids were used only when specified by the manufacturer. A further observation was a different handling of the overfill in pre-filled infusion containers, possibly impacting total dose. Lastly, we documented the complexity of infusion administration setups for administration of multiple drugs. In this case, flushing procedures or the placement and use of filters in the setup vary. Our study has revealed important differences in handling and administration practice. We propose that drug developers and hospitals should collaborate to establish unified handling procedures.

Impact of the Design of Different Infusion Containers on the Dosing Accuracy of a Therapeutic Drug Product.

Residual volumes of infusion solutions vary greatly due to container and dimensional variances. Manufacturers use overfill to compensate, but the exact amounts vary significantly. This variability in overfill - when carrier solutions are used to dilute other parenteral preparations - may lead to variable concentrations and dosing, hence, potential risk for patients. We analyzed the overfill and residual volume of 22 pre-filled infusion containers and evaluated the impact on the (simulated) dosing accuracy of a therapeutic drug product for different handling scenarios. In addition, compendial properties of the diluents (i.e. sub-visible particles, pH, color and opalescence) were assessed. The overfill and residual volume between different containers for the same diluent varied. As container size increased, the relative volume of overfill decreased while the residual volume remained constant. The design and material of the containers (e.g. port systems) defined the residual volume. Different handling scenarios led to differences in dosing accuracy. As a result, no universal approach applicable for all containers can be defined. To ensure the right dose, it is recommended to pre-select the preferred diluent, evaluate fill volumes of carrier solutions, and assess in-use compatibility of the product solution with its diluent in terms of concentration and volume.

Characterization of Silicone from Closed System Transfer Devices and its Migration into Pharmaceutical Drug Products.

Closed System Transfer Devices (CSTDs) are increasingly used in healthcare settings to facilitate compounding of hazardous drugs but increasingly also therapeutic proteins. However, their use may significantly impact the quality of the sterile product. For example, contamination of the product solution may occur by leaching of silicone or particulates from the CSTDs. It was therefore the aim of the present study to identify and quantify the types of silicone oil in a panel of typically used CSTDs. Particles found after simulated CSTD compounding processes were evaluated using Light Obscuration and Micro-Flow Imaging and were confirmed to be silicone oil particles. The number of particulates shed from CTSDs was in single cases exceeding pharmacopeial limits for a final parenteral product. Using X-ray microtomography, lubrication was shown to be primarily applied at connecting parts of the CSTD. Quantitative and qualitative analysis by Fourier transform infrared spectroscopy (FTIR) revealed a total released amount between 0.8 and 16 mg per CSTD of polydimethylsiloxane or polymethyltrifluoropropylsiloxane per CSTD. While pronounced differences in total silicone content between CSTDs were observed, it did not fully correlate with particle contamination in the test solutions, potentially due to variations in CSTD design. The impact of typical surfactants in biological formulations on silicone migration into product was additionally evaluated. We conclude that CSTDs may compromise final product quality, as (different types of) silicone oil may be released from these devices and contaminate the administered product.

The role of polysorbate 80 and HPßCD at the air-water interface of IgG solutions.

PURPOSE: To test the hypothesis of surface displacement as the underlying mechanism for IgG stabilization by polysorbates and HPßCD against surface-induced aggregation. METHODS: Adsorption/desorption-kinetics of IgG-polysorbate 80/-HPßCD were monitored. Maximum bubble pressure method was used for processes within seconds from surface formation. Profile analysis tensiometry was applied over long periods and to assess surface rheologic properties. Additionally, the kinetics of adsorption, desorption and surface displacement was followed by a double-capillary setup of the profile analysis tensiometer, allowing drop bulk exchange. RESULTS: Weak surface activity for HPßCD vs. much higher surface activity for polysorbate 80 was shown. Protein-displacement when exceeding a polysorbate 80 concentration close to the CMC and a lack of protein displacement for HPßCD was observed. The drop bulk exchange experiments show IgG displacement by polysorbate 80 independent of the adsorption order. In contrast, HPßCD coexists with IgG at the air-water interface when the surface layer is built from a mixed IgG-HPßCD-solution. Incorporation of HPßCD in a preformed IgG-surface-layer does not occur. CONCLUSIONS: The results confirm surface displacement as the stabilization mechanism of polysorbate 80, but refute the frequently held opinion, that HPßCD stabilizes proteins against aggregation at the air-water interface in a manner comparable to non-ionic surfactants.

N-nitrosamine Mitigation with Nitrite Scavengers in Oral Pharmaceutical Drug Products.

N-nitrosamines are likely human carcinogens. After N-nitrosamine contaminants were detected in pharmaceutical products in 2018, regulatory authorities set a framework for the risk assessment, testing and mitigation of N-nitrosamines in drug products. One strategy to inhibit the formation of N-nitrosamines during the manufacture and storage of drug products involves the incorporation of nitrite scavengers in the formulation. Diverse molecules have been tested in screening studies including the antioxidant vitamins ascorbic acid and α-tocopherol, amino acids, and other antioxidants used in foods or drugs, for inclusion into drug products to mitigate N-nitrosamine formation. This review article outlines key considerations for the inclusion of nitrite scavengers in oral drug product formulations.

Comparative investigations on in vitro serum stability of polymeric micelle formulations.

PURPOSE: Stability of polymeric micelles upon injection is essential for a drug delivery system but is not fully understood. We optimized an analytical test allowing quantification of micellar stability in biofluids and applied it to a variety of block copolymer micelles with different hydrophobic block architechtures. METHODS: Polymeric micelles were prepared from four different polymers and investigated via encapsulation of two fluorescent dyes. Samples were incubated in human serum; changes in Foerster Resonance Energy Transfer (FRET) were recorded as a function of time. This fluorescence-based approach was supported semi-quantitatively by results from Asymmetrical Flow Field-Flow-Fractionation (AF4). RESULTS: After incubation experiments, micellar stability was determined by calculation of two stability-indicating parameters: residual micellar fractions (RMFs) and in vitro serum half-lives. Both parameters showed that PEG-PVPy micelles rapidly destabilized after 3 h (RMF < 45%), whereas PEG-PLA, PEG-PLGA and PEG-PCL micelles were far more stable (RMFs 65 to 98%). CONCLUSION: This FRET-based assay is a valuable tool in evaluating and screening serum stability of polymeric micelles and revealed low serum stability of PEG-PVPy micelles compared to polyester-based micelles.

An Industry Perspective on Compatibility Assessment of Closed System Drug-Transfer Devices for Biologics.

The Formulation Workstream of the BioPhorum Development Group (BPDG), an industry-wide consortium, has identified the increased use of closed system drug-transfer devices (CSTDs) with biologics, without an associated compatibility assessment, to be of significant concern. The use of CSTDs has increased significantly in recent years due to the recommendations by NIOSH and USP that they be used during preparation and administration of hazardous drugs. While CSTDs are valuable in the healthcare setting to reduce occupational exposure to hazardous compounds, these devices may present particular risks that must be adequately assessed prior to use to ensure their compatibility with specific types of drug products, such as biologic drugs, which may be sensitive. The responsibility of ensuring quality of biologic products through preparation and administration to the patient lies with the drug product sponsor. Due to the significant number of marketed CSTD systems, and the large variety of components offered for each system, a strategic, risk-based approach to assessing compatibility is recommended herein. In addition to traditional material compatibility, assessment of CSTD compatibility with biologics should consider additional parameters to address specific CSTD-related risks. The BPDG Formulation Workstream has proposed a systematic risk-based evaluation approach as well as a mitigation strategy for establishing suitability of CSTDs for use.

Tracking the urinary excretion of high molar mass poly(vinyl alcohol).

The fate of poly(vinyl alcohol) (PVA) of weight average molar mass of 125,000 g/mol after administration into the peritoneum of rabbits has bean studied by various methods. PVA was spin-labeled with a nitroxide radical and then detected in urine using electron spin resonance (ESR) spectroscopy. Furthermore, unlabeled polymer was also administered to rabbits, then the urine was collected, dialyzed, precipitated, and the excretion of PVA was confirmed by size exclusion chromatography (SEC), FTIR spectroscopy, and (1)H NMR spectroscopy. ESR and SEC results show that, despite its relatively high molar mass, PVA is excreted through the kidneys without significant molar mass changes. Nevertheless, NMR and FTIR spectra show slight differences between the excreted and neat PVA. Possible causes of these discrepancies are discussed.

Protein Adsorption to In-Line Filters of Intravenous Administration Sets.

Ensuring compatibility of administered therapeutic proteins with intravenous administration sets is an important regulatory requirement. A low-dose recovery during administration of low protein concentrations is among the commonly observed incompatibilities, and it is mainly due to adsorption to in-line filters. To better understand this phenomenon, we studied the adsorption of 4 different therapeutic proteins (2 IgG1s, 1 IgG4, and 1 Fc fusion protein) diluted to 0.01 mg/mL in 5% glucose (B. Braun EcoFlac; B. Braun Melsungen AG, Melsungen, Germany) or 0.9% sodium chloride (NaCl; Freeflex; Fresenius Kabi, Friedberg, Germany) solutions to 8 in-line filters (5 positively charged and 3 neutral filters made of different polymers and by different suppliers). The results show certain patterns of protein adsorption, which depend to a large extent on the dilution solution and filter material, and to a much lower extent on the proteins' biophysical properties. Investigation of the filter membranes' zeta potential showed a correlation between the observed adsorption pattern in 5% glucose solution and the filter's surface charge, with higher protein adsorption for the strongly negatively charged membranes. In 0.9% NaCl solution, the surface charges are masked, leading to different adsorption patterns. These results contribute to the general understanding of the protein adsorption to IV infusion filters and allow the design of more efficient compatibility studies.

Loading and mobility of spin-labeled insulin in physiologically responsive complexation hydrogels intended for oral administration.

Poly(methacrylic acid-g-ethylene glycol) copolymers are pH-responsive complexation hydrogels that have shown promise in in vitro and in vivo results as oral insulin delivery carriers. With the aim of gaining more detailed insight into their performance to further improve the carriers, we spin-labeled insulin and used electron spin resonance (ESR) spectroscopy to follow the loading of the spin-labeled insulin into the copolymer microparticles. A flow through system was developed to monitor continuously and non-invasively the dynamics of the spin-labeled insulin and its surrounding microviscosity during release. Using these methods, the loading efficiency of insulin was determined and was found to match previous HPLC measurements. Additionally, the protein-friendly nature of the hydrogels was demonstrated. The monitoring of the dynamics during flow through provided a rationalization for the unwanted initial burst release in an acidic environment. These studies will aid in the optimization of the system, and will be a basis for subsequent in vivo ESR investigations.

Physico-chemical characterization of polysaccharide-coated nanoparticles.

A series of amphiphilic copolymers (PCL-DEX) made of poly(epsilon-caprolactone) (PCL) side chains grafted onto a dextran (DEX) backbone, was used to modify the surface of PCL nanoparticles. PCL-DEX nanoparticles were prepared by a technique derived from emulsion-solvent evaporation. The purpose of the present study was to investigate the DEX coating (quantification, conformation, mobility) in order to better understand particle surface-protein interactions. The DEX coating was deeply examined using different complementary methods: zeta potential measurement, specific degradation of the DEX shell by dextranase, energy-filtering transmission electron microscopy coupled to image-spectrum electron energy-loss spectroscopy, electronic paramagnetic resonance, high performance size exclusion chromatography as well as nonspecific bovine serum albumin adsorption. All our data together supported a core-shell structure of the nanoparticles, DEX moieties constituting the external coating. The amount of DEX located on the nanoparticle surface was estimated to 70%. The organisation of the shell including chains density and mobility was found to be dramatically influenced by DEX molar mass. The steric repulsion conferred by the presence of DEX at the surface of the nanoparticles decreased the adsorption of albumin. The nanoparticle-protein interaction was, however, greatly influenced by the polysaccharide conformation onto the surface.

Calculation of injection forces for highly concentrated protein solutions.

Protein solutions often manifest a high viscosity at high solution concentrations, thus impairing injectability. Accordingly, accurate prediction of the injection force based on solution viscosity can greatly support protein formulation and device development. In this study, the shear-dependent viscosity of three concentrated protein solutions is reported, and calculated injection forces obtained by two different mathematical models are compared against measured values. The results show that accurate determination of the needle dimensions and the shear-thinning behavior of the protein solutions is vital for injection force prediction. Additionally, one model delivered more accurate results, particularly for solutions with prominent shear-thinning behavior.

Non-spherical micro- and nanoparticles: fabrication, characterization and drug delivery applications.

INTRODUCTION: Micro- and nanoparticles in drug and vaccine delivery have opened up new possibilities in pharmaceutics. In the past, researchers focused mainly on particle size, surface chemistry and the use of various materials to control particle characteristics and functions. Lately, shape has been acknowledged as an important design parameter having an impact on the interaction with biological systems. AREAS COVERED: In this review, we report on the latest developments in fabrication methods to tailor particle geometry, summarize analytical techniques for non-spherical particles and highlight the most important findings regarding their interaction with biological systems and their potential applications in drug delivery. EXPERT OPINION: The impact of shape on particle internalization into different cell types and particle biodistribution has been extensively studied in the past. Current research focuses on shape-dependent uptake mechanisms and applications for tumour therapy and vaccination. Different fabrication methods can be used to produce a variety of different particle types and shapes. Key challenges will be the transfer of new non-spherical particle fabrication methods from lab-scale to industrial large-scale production. Not all techniques may be scalable for the production of high quantities of particles. It will also be challenging to transfer the promising in vitro findings to suitable in vivo models.

Influence of particle size, an elongated particle geometry, and adjuvants on dendritic cell activation.

Modern subunit vaccines have many benefits compared to live vaccines such as convenient and competitive large scale production, better reproducibility and safety. However, the poor immunogenicity of subunit vaccines usually requires the addition of potent adjuvants or drug delivery vehicles. Accordingly, researchers are investigating different adjuvants and particulate vaccine delivery vehicles to boost the immunogenicity of subunit vaccines. Despite the rapidly growing knowledge in this field, a comparison of different adjuvants is sparsely found. Until today, little is known about efficient combinations of the different adjuvants and particulate vaccine delivery vehicles. In this study we compared three adjuvants with respect to their immune stimulatory potential and combined them with different particulate vaccine delivery vehicles. For this reason, we investigated two types of polyI:C and a CL264 base analogue and combined these adjuvants with differently sized and shaped particulate vaccine delivery vehicles. A high molecular weight polyI:C combined with a spherical nano-sized particulate vaccine delivery vehicle promoted the strongest dendritic cells activation.

Head to head comparison of the formulation and stability of concentrated solutions of HESylated versus PEGylated anakinra.

Although PEGylation of biologics is currently the gold standard for half-life extension, the technology has a number of limitations, most importantly the non-biodegradability of PEG and the extremely high viscosity at high concentrations. HESylation is a promising alternative based on coupling to the biodegradable polymer hydroxyethyl starch (HES). In this study, we are comparing HESylation with PEGylation regarding the effect on the protein's physicochemical properties, as well as on formulation at high concentrations, where protein stability and viscosity can be compromised. For this purpose, the model protein anakinra is coupled to HES or PEG by reductive amination. Results show that coupling of HES or PEG had practically no effect on the protein's secondary structure, and that it reduced protein affinity by one order of magnitude, with HESylated anakinra more affine than the PEGylated protein. The viscosity of HESylated anakinra at protein concentrations up to 75 mg/mL was approximately 40% lower than that of PEG-anakinra. Both conjugates increased the apparent melting temperature of anakinra in concentrated solutions. Finally, HESylated anakinra was superior to PEG-anakinra regarding monomer recovery after 8 weeks of storage at 40°C. These results show that HESylating anakinra offers formulation advantages compared with PEGylation, especially for concentrated protein solutions.

Freeze-drying of HESylated IFNα-2b: Effect of HESylation on storage stability in comparison to PEGylation.

A comparison of lyophilized PEGylated and HESylated IFNα was carried out to investigate the influence of protein conjugation, lyoprotectants as well as storage temperature on protein stability. Results show that PEG tends to crystallize during freeze-drying, reducing protein stability upon storage. In contrast, HESylation(®) drastically improved the stability over PEGylation by remaining totally amorphous during lyophilization, with and without lyoprotectants while providing a high glass transition temperature of the freeze-dried cakes.

Influence of particle geometry and PEGylation on phagocytosis of particulate carriers.

Particle geometry of micro- and nanoparticles has been identified as an important design parameter to influence the interaction with cells such as macrophages. A head to head comparison of elongated, non-spherical and spherical micro- and nanoparticles with and without PEGylation was carried out to benchmark two phagocytosis inhibiting techniques. J774.A1 macrophages were incubated with fluorescently labeled PLGA micro- and nanoparticles and analyzed by confocal laser scanning microscope (CLSM) and flow cytometry (FACS). Particle uptake into macrophages was significantly reduced upon PEGylation or elongated particle geometry. A combination of both, an elongated shape and PEGylation, had the strongest phagocytosis inhibiting effect for nanoparticles.

Characterization and compatibility of hydroxyethyl starch-polyethylenimine copolymers for DNA delivery.

Hydroxyethyl starch (HES) has been proposed as a biodegradable polymer for shielding of DNA polyplexes, where the feasibility of this approach was shown both in vitro and in vivo. In this study, we report on the physicochemical characterization, the in vitro cytocompatibility and hemotoxicity of HES-decorated polyplexes. For this purpose, various HES molecules were coupled to a 22 kDa linear polyethylenimine (LPEI22) to produce a library of nine different HES-PEI conjugates. Particle analysis using dynamic light scattering showed that, neither the molar mass of HES nor the amount of HES in the polyplexes affected the particle diameter, as it was consistently around 70-80 nm. Imaging using atomic force microscopy and transmission electron microscopy showed that, both naked and HESylated polyplexes were in the same size range and had a spherical morphology. Meanwhile, the HES-mediated particle-shielding effect, manifested as reduction in the surface charge, strongly correlated with the molar mass of HES, where the charge decreased linearly with the increase in molar mass. Ethidium bromide binding assay showed that HES-PEI did not negatively affect DNA condensation at N/P ratios higher than 4. HES conjugation also showed a stabilizing effect against salt-induced particle disassembly, and particle aggregation in protein-containing media. Compatibility tests included cellular viability, as well as erythrocyte aggregation and hemolysis assays. HES-PEI conjugates showed lower cytotoxicity, no aggregation, and much lower hemolysis compared to unmodified PEI. In conclusion, these results show that the HES-PEI conjugates are promising gene delivery polymers with favorable physicochemical properties and compatibility profile.