Drug Formulation Impact on Prefilled Syringe Functionality and Autoinjector Performance.

Given the surging interest in developing prefilled syringe and autoinjector combination products, investment in an early compatibility assessment is critical to prevent unwarranted drug/container closure interactions and avoid potential reformulation during late stages of drug development. In addition to the standard evaluation of drug stability, it is important to consider container closure functionality and overall device performance changes over time because of drug-container closure component interaction. This study elucidated the mechanisms that cause changes in syringe glide force over time and the impact on the injection duration. It was an expansion of the previous work, which indicated that drug formulation variables such as formulation excipients and pH affect syringe functionality over time. The current study described an investigative process for troubleshooting prolonged and variable autoinjector injection time caused by an increased syringe glide force variability over time. This increase in glide force variability stems from two root causes, namely plunger dimensional variation and syringe silicone oil change over time. The results demonstrated (a) the underlying factors of silicone oil change in the presence of drug formulation matrices, (b) accelerated stability of syringe glide force as a good indicator of long-term, real-time stability, and (c) that buffer matrix-filled syringes can be used to predict the syringe functionality and stability of drug product-filled syringes. Based on the experimental findings of a variety of orthogonal characterization techniques including contact angle, interfacial tension, and calculation of Hansen solubility parameters, it is proposed that silicone oil change is caused by formulation excipients and a complex set of phenomena summarized as "wet, wash, and delube" processes.

Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective.

Biologic products encounter various types of interfacial stress during development, manufacturing, and clinical administration. When proteins come in contact with vapor-liquid, solid-liquid, and liquid-liquid surfaces, these interfaces can significantly impact the protein drug product quality attributes, including formation of visible particles, subvisible particles, or soluble aggregates, or changes in target protein concentration due to adsorption of the molecule to various interfaces. Protein aggregation at interfaces is often accompanied by changes in conformation, as proteins modify their higher order structure in response to interfacial stresses such as hydrophobicity, charge, and mechanical stress. Formation of aggregates may elicit immunogenicity concerns; therefore, it is important to minimize opportunities for aggregation by performing a systematic evaluation of interfacial stress throughout the product development cycle and to develop appropriate mitigation strategies. The purpose of this white paper is to provide an understanding of protein interfacial stability, explore methods to understand interfacial behavior of proteins, then describe current industry approaches to address interfacial stability concerns. Specifically, we will discuss interfacial stresses to which proteins are exposed from drug substance manufacture through clinical administration, as well as the analytical techniques used to evaluate the resulting impact on the stability of the protein. A high-level mechanistic understanding of the relationship between interfacial stress and aggregation will be introduced, as well as some novel techniques for measuring and better understanding the interfacial behavior of proteins. Finally, some best practices in the evaluation and minimization of interfacial stress will be recommended.

Novel Ricin Subunit Antigens With Enhanced Capacity to Elicit Toxin-Neutralizing Antibody Responses in Mice.

RiVax is a candidate ricin toxin subunit vaccine antigen that has proven to be safe in human phase I clinical trials. In this study, we introduced double and triple cavity-filling point mutations into the RiVax antigen with the expectation that stability-enhancing modifications would have a beneficial effect on overall immunogenicity of the recombinant proteins. We demonstrate that 2 RiVax triple mutant derivatives, RB (V81L/C171L/V204I) and RC (V81I/C171L/V204I), when adsorbed to aluminum salts adjuvant and tested in a mouse prime-boost-boost regimen were 5- to 10-fold more effective than RiVax at eliciting toxin-neutralizing serum IgG antibody titers. Increased toxin neutralizing antibody values and seroconversion rates were evident at different antigen dosages and within 7 days after the first booster. Quantitative stability/flexibility relationships analysis revealed that the RB and RC mutations affect rigidification of regions spanning residues 98-103, which constitutes a known immunodominant neutralizing B-cell epitope. A more detailed understanding of the immunogenic nature of RB and RC may provide insight into the fundamental relationship between local protein stability and antibody reactivity.

Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen.

There is great interest in the design and development of highly thermostable and immunogenic protein subunit vaccines for biodefense. In this study, we used two orthogonal and complementary computational protein design approaches to generate a series of single-point mutants of RiVax, an attenuated recombinant ricin A chain (RTA) protein subunit vaccine antigen. As assessed by differential scanning calorimetry, the conformational stabilities of the designed mutants ranged from 4°C less stable to 4.5°C more stable than RiVax, depending on solution pH. Two more thermostable (V18P, C171L) and two less thermostable (T13V, S89T) mutants that displayed native-like secondary and tertiary structures (as determined by circular dichroism and fluorescence spectral analysis, respectively) were tested for their capacity to elicit RTA-specific antibodies and toxin-neutralizing activity. Following a prime-boost regimen, we found qualitative differences with respect to specific antibody titers and toxin neutralizing antibody levels induced by the different mutants. Upon a second boost with the more thermostable mutant C171L, a statistically significant increase in RTA-specific antibody titers was observed when compared with RiVax-immunized mice. Notably, the results indicate that single residue changes can be made to the RiVax antigen that increase its thermal stability without adversely impacting the efficacy of the vaccine.

Surfactant-laden soft contact lenses for extended delivery of ophthalmic drugs.

Eye drops are inefficient means of delivering ophthalmic drugs because of limited bioavailability and these can cause significant side effects due to systemic uptake of the drug. The bioavailability for ophthalmic drugs can be increased significantly by using contact lenses. This study focuses on the development of surfactant-laden poly-hydroxy ethyl methacrylate (p-HEMA) contact lenses that can release Cyclosporine A (CyA) at a controlled rate for extended periods of time. We focus on various Brij surfactants to investigate the effects of chain length and the presence of an unsaturated group on the drug release dynamics and partitioning inside the surfactant domains inside the gel. The gels were imaged by cryogenic scanning electron microscopy (cryo-SEM) to obtain direct evidence of the presence of surfactant aggregates in the gel, and to investigate the detailed microstructure for different surfactants. The images show a distribution of nano pores inside the surfactant-laden hydrogels which we speculate are regions of surfactant aggregates, possibly vesicles that have a high affinity for the hydrophobic drug molecule. The gels are further characterized by studying their mechanical and physical properties such as transparency, surface contact angle and equilibrium water content to determine their suitability as extended wear contact lenses. Results show that Brij surfactant-laden p-HEMA gels provide extended release of CyA, and possess suitable mechanical and optical properties for contact lens applications. The gels are not as effective for extended release of two other hydrophobic ophthalmic drugs, dexamethasone (DMS) and dexamethasone 21 acetate (DMSA) because of insufficient partitioning inside the surfactant aggregates.