Impact of injection time and protein modality on particle formation when using closed system transfer devices.

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. 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 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 1mL depending on the CSTD type, vial size and solution viscosity. 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.

Overcoming Challenges of Implementing Closed System Transfer Device Clinical In-Use Compatibility Testing for Drug Development of Antibody Drug Conjugates.

A Systematic Study of CSTD-Generated Stress on Different Biomolecular Modalities

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

Determination of Holdup Volume and Transient Contact Compatibility of Closed System Transfer Devices for a Reconstituted Lyophilized Drug Product

Challenges of Using Closed System Transfer Devices With Biological Drug Products: An Industry Perspective.

Background and ImportanceInvestigational drug products (IDP) should be treated as hazardous drugs (HD) as it is not frequent to have hazard studies available or the information about safety is usually...

The time-course and extent of visible particle (VP) and sub-visible particle (SVP) formation was monitored as a function of interfacial area (IA) for a model bioconjugate. To facilitate particle formation, the bioconjugate was agitated in a glass vial and exposed to IAs up to 478mm2. Since vials had equal fill and headspace volumes, the area of the air-water interface was varied by placing vials on angled blocks at 0°, 30°, 60°, or 90° from the horizontal. A significant increase in visible and sub-visible particle formation was observed with increasing air-water IA. Exposure to IAs below ∼305mm2 resulted in the formation of very few particles, while IAs > ∼305mm2 resulted in substantial particle formation. Visible and sub-visible particle morphology varied with interfacial area and time. The sub-visible particles initially increased with time but did not reach steady state; instead the initial increase was followed by complete depletion. These phenomena indicate that visible particle formation likely increased at the expense of the sub-visible particle population and demonstrate a potential link between the two particle populations for this model bioconjugate. Initiation of particle formation did not result in corresponding decreases in protein concentration or increases in soluble aggregates. However, extended agitation time resulted in a significant decrease in protein concentration.

Vaccine formulations may contain visible and/or subvisible particles, which can vary in both size and morphology. Extrinsic particles, which are particles not part of the product such as foreign contaminants, are generally considered undesirable and should be eliminated or controlled in injectable products. However, biological products, in particular vaccines, may also contain particles that are inherent to the product. Here we focus on the characterization of visible and subvisible particles in a live, replication-deficient viral vaccine candidate against HSV genital herpes in an early developmental stage. HSV-2 viral vaccine was characterized using a panel of analytical methods, including Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, liquid chromatography-mass spectrometry (LC-MS), light microscopy, transmission electron microscopy (TEM), micro-flow imaging (MFI), dynamic light scattering (DLS), right angle light scattering (RALS), and intrinsic fluorescence. Particles in HSV-2 vaccine typically ranged from hundreds of nanometers to hundreds of micrometers in size and were determined to be inherent to the product. The infectious titer did not correlate with any trend in subvisible particle concentration and size distribution as shown by DLS, MFI, and TEM under stressed conditions. This suggested that particle changes in the submicron range were related to HSV-2 virion structure and had direct impact on biological activity. It was also observed that subvisible and visible particles could induce aggregation in the viral product. The temperature induced aggregation was observed by RALS, intrinsic fluorescence, and DLS. The increase of subvisible particle size with temperature could be fitted to a two-step thermokinetic model. Visible and subvisible particles were found to be inherent to the HSV-2 viral vaccine product. The mechanism of protein aggregation was discussed and a two-step thermokinetic aggregation profile was proposed. The approaches reported in this study may be applied to a variety of vaccines and other biological products, as a way to assess the consistency of the manufacturing process and identify key product quality attributes.

Evaluation of the Effect of Syringe Surfaces on Protein Formulations

The subvisible and visible particles present in a solution are often classified based on size, and are quantified by the actual number of particles present rather than by weight or molar amounts. The analysis of these particles in protein therapeutics are governed by compendial methods and the regulatory agencies, and the methods available to measure them originally evolved focusing on potential safety issues, including capillary occlusion and immunogenicity, that might arise from their presence. Ultracentrifugation, size exclusion chromatography, etc., discussed in previous articles, can be used to analyze aggregates of less than 0.10 microns. This article will focus on methods for analyzing and quantitating sub visible particles (SbVP) of 2 microns or larger. At the present time there is no routine method for quantitating sub visible particles (SbVP) between 0.1 microns and 2 microns. The most common technique for quantitating the amount of subvisible particles between 2 and 100 microns is the light obscuration method. This technique can determine size and amount of particles, but cannot differentiate between the types of particles, such as protein particles, foreign material, micro bubbles or silicone oil droplets, that can be present in protein solutions. The difficulties in adapting this method, originally developed for small molecule drugs for IV administration, to protein therapeutics delivered subcutaneously is discussed. The flow imaging techniques can determine morphology and optical characteristics of the particles, but still not identify the chemical composition. Other methods that can also be used, but are applicable for characterization purposes only, are discussed. The primary method for quantitating visible particles is visual inspection, a method that can be subjective and relies on adequate training of the human inspectors. Automated methods for visible particle determination are being developed. Identification of the chemical composition of isolated particles greater than about 50 microns is possible using several micro-spectroscopic methods, and these will also be discussed.

Visible particles must be monitored as part of the control strategy for parenteral biopharmaceutical drug products. In these products, formation of protein particles is a natural occurrence. All protein drugs contain particles that vary greatly in visibility and size from invisible (sub-micron) to visible (millimeter), and pharmaceutical companies are required to monitor and minimize the presence of visible and sub-visible particles in their products. There is an industry-wide unmet need for particle standards for visual inspection of protein drugs. A new, semi-quantitative method using particle standards for assessing the levels of small, naturally occurring visible particles is presented. This method can be used during drug development to identify a formulation that minimizes particle formation and also during testing of final clinical or commercial drug product to monitor and control naturally occurring proteinaceous visible particles.

The effect of injection time on rates of epileptogenic zone localization using SISCOM and STATISCOM

There is an increased interest from industry, academia and regulators for protein aggregates, subvisible and visible particles due to possible biological consequences, such as immunogenicity, altered bioactivity and modified pharmacokinetic profiles. Aggregates, subvisible and visible particles are important product instabilities, which might be present in every formulation of biotherapeutic products like monoclonal antibody solutions. Especially, the presence of subvisible particles in biotherapeutic products is currently a hot topic and it constantly gains more importance. The ultimate goal of this thesis is to develop tools and techniques in order to be able to characterize well-defined size fractions of proteinaceous subvisible particles with various desired oxidation profiles using in vivo transgenic mouse model. Up to now only a few articles were published and the available data from in vitro and in vivo experiments on aggregates and subvisible particles is often conflicting and fragmented, which impedes the development of sound conclusions. Moreover, complex mixtures of monomers, aggregates, particles and other degradants were used to draw conclusions. Only estimated values of protein particle density were used up to now in published studies although it is required to know the density of the measured particles in order to accurately calculate their dimensions and mass. The first aim of this thesis was therefore to develop a method to measure experimentally the protein particle density without extrapolation (Chapter 1). The density for commercially available standard beads (polystyrene, polymethacrylate and melamine) and a large bench of stressed proteinaceous samples was determined with the use of the resonant mass measurement instrument (RMM, Archimedes) and its ability to measure the buoyant mass of individual particles. Various fluids with increasing densities were implemented in order to determine the neutral buoyant mass where the particle density equals the fluid density. Chapter 2 reports the development of a process to isolate well-defined subvisible fractions using differential centrifugation for a model IgG1 antibody. The process to separate four fractions in the submicron and micrometer size range was developed and successfully optimized through the use of a design of experiments. The centrifugation technique was compared to an already published fractionation method using a preparative fluorescence-activated cell sorter. Efficiency, advantages and drawbacks for both methods were compared and discussed (Chapter 2). Oxidation profile of aggregates and subvisible particles seem to be an important attribute regarding induced biological consequences. That is why the next goal was to develop a method for selective oxidation of methionine and tryptophan residues in a model mAb in order to be able to delineate the effects and the contribution of individual protein modifications in the primary structure. This included a large set of experiments where different reaction conditions such as temperature of incubation, reaction time, type and concentration of oxidant (t-BHP, H2O2, AAPH) were evaluated in presence (or not) of a large excess of anti-oxidant (free amino acids) in order to protect the corresponding amino acid in a model antibody of the IgG1 subtype (Chapter 3). To complete the work, unfractionated materials and well-defined size fractions (with well-established oxidation profile) were prepared using the established tools (Chapter 1-3). Those samples were deeply characterized and injected subcutaneously into wild type and transgenic mice for immunization. Anti-drug antibody levels were measured following ELISA in order to assess the immunogenic potential of those preparations (Chapter 4).

As the packaging of choice for many therapeutic proteins, prefilled syringes have been widely used in biopharmaceutical industry as primary containers, where silicone oil is applied to ensure their proper functionality. Adequate lubrication from sufficient amount of silicone oil and its appropriate distribution across syringe barrels is crucial for successful administration of drug product (DP) from the prefilled syringes; however, silicone oil is also susceptible to leaching from the syringe surface into the formulation with the potential to interact with therapeutic proteins, which could lead to the formation of visible and sub-visible aggregates and/or particles that are potentially immunogenic. Accurate determination and careful control of silicone oil levels in both empty syringes and protein drug products are therefore critical in process development to ensure syringe functionality, drug product quality, and patient safety. On the other hand, analysis of silicone oil can be challenging especially when the analysis is performed on formulated protein drug products, where matrix effects could be significant. It is demonstrated in this study that silicone oil in empty syringes or formulated drug products can be extracted effectively using organic solvents and quantitatively determined using high-performance liquid chromatography (HPLC) coupled with a universal detector. It was also shown that direct extraction of silicone oil from formulated protein drug products can be very challenging, but pretreatment of the protein drug products with pepsin enzymatic digestion facilitated the extraction process, which enabled the analysis of silicone oil in the drug product at low ppm levels.