This application note presents an example of how calorimetry aided the formulation development for ProX by providing insight into excipient-protein interactions. Polysorbate-80 and phenol are examined here as potential additives to the formulation buffer for ProX. Polysorbate-80 is a commonly used surfactant to prevent non-specific adsorption and aggregation of proteins and has been shown previously to interact with proteins (2, 3). It protects the protein against surface-induced aggregation by binding to exposed hydrophobic regions on the protein molecule surface (3). The most commonly used levels of polysorbate-80 are 0.002 to 0.1% (w/v). Phenol is used as an antimicrobial agent for formulations intended to be dosed more than once from the same container. Phenol is toxic and therefore the concentration used in the formulation buffer must be minimized. The most commonly used levels are 0.3 to 0.5% (v/v).
Introduction
Development of protein-based therapeutics is a challenging task due to inherent protein instabilities, which can manifest as physical instability (unfolding, aggregation, adsorption), and chemical degradation (oxidation, deamidation, cleavage). Such instabilities can lead to reduced activity of the protein or even generation of potentially immunogenic species. One approach to protein stabilization is changing the properties of the solvent in contact with it, which can be accomplished by careful selection of the buffer system, adjustment of pH, and addition of excipients/additives (i.e., creating an optimal formulation). One important factor in solvent-based stabilization of protein drugs is the choice of appropriate excipients; equally important is the optimization of excipient concentrations that provide extended shelf life while also ensuring highest safety to patient. Thus, a critical part of the protein drug formulation development is the selection of excipients that are suitably soluble and nontoxic, maintain the structural integrity of the protein, enable an acceptable product shelf-life, and preserve the biological activity of the product. These excipients can include amino acids, salts, metals, surfactants, sugars and polyols, and polymers. They can serve as stabilizers, surface-active agents, antimicrobial agents, or antioxidants. Their stabilizing effects are usually protein-specific and concentration-dependent. Together with the excipient selection, information needs to be gathered not only on optimum excipient concentration, but also on the interactions between various formulation components. Optimizing the selection and concentration of each excipient can be a labor-intensive task that involves extensive formulation screening and stability studies. While general principles of stabilization have emerged from the literature over the past decade (1), the mechanisms by which excipients can improve the stability of a protein during storage are still not completely understood. Knowledge of the mechanisms of excipient interactions with proteins would eliminate a purely empirical screening approach and allow a rational design and optimization of protein formulation, thus decreasing the time and material requirements for protein product development. Moreover, information on the strength and type of protein-excipient interactions would help predict the protein drug behavior in vivo. Biophysical analysis techniques have proven to be extremely useful in excipient screening for formulation development. In particular, calorimetry is one of the most efficient methods for assessing protein stability and interactions, as it allows a complete thermodynamic characterization of the system, provided all events are reversible. Calorimetric investigations to explore protein-excipient interactions have been increasingly applied toward the design and optimization of biopharmaceutical formulations. Calorimetry is based on the principle of determining the energetics and stoichiometry of macromolecular interactions by measuring the heat changes resulting from association, dissociation, and/or unfolding processes. ITC is primarily used to determine thermodynamic binding parameters such as binding affinity and dissociation constants, as well as stoichiometry, enthalpy, entropy, and Gibbs free energy of binding under isothermal conditions. It can also be used to determine heat capacity changes upon binding by performing experiments over a range of temperatures. DSC data supplies the thermodynamic parameters of protein unfolding, including the midpoint unfolding temperature, enthalpy, entropy, Gibbs free energy, and heat capacity changes upon unfolding. These thermodynamic parameters are commonly used to compare protein stability in different formulations, or to determine the relative stabilizing or destabilizing effects of certain excipients. >> Download the full White Paper as a PDFMalvern provides the materials and biophysical characterization technology and expertise that enables scientists and engineers to investigate, understand and control the properties of dispersed systems. These systems range from proteins and polymers in solution, particle and nanoparticle suspensions and emulsions, through to sprays and aerosols, industrial bulk powders and high concentration slurries. Used at all stages of research, development and manufacturing, Malvern’s instruments provide critical information that helps accelerate research and product development, enhance and maintain product quality and optimize process efficiency. Our products reflect Malvern’s drive to exploit the latest technological innovations. They are used by both industry and academia, in sectors ranging from pharmaceuticals and biopharmaceuticals to bulk chemicals, cement, plastics and polymers, energy and the environment. Malvern systems are used to measure particle size, particle shape, zeta potential, protein charge, molecular weight, mass, size and conformation, rheological properties and for chemical identification, advancing the understanding of dispersed systems across many different industries and applications. www.malvern.com Material relationships http://www.malvern.com/en/
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