The utility of microcalorimetry as a rapid screening tool for assessing the solution stability of high molecular weight pharmaceutical proteins was evaluated using model recombinant antibodies, Protein I and Protein II. Changes in the transition midpoint, Tm, were monitored as a function of pH and/or in the presence of excipients, and results were compared with traditional accelerated stability data from samples that were analyzed by size exclusion chromatography (SEC). The data from microcalorimetry were well correlated with those from SEC for predicting both optimal solution pH as well as excipient effects on solution stability. These results indicate that microcalorimetry can be an efficient screening tool useful in identifying optimal pH conditions and excipients to stabilize pharmaceutical proteins in solution formulations.
During the past two decades, there has been a rapidly increasing interest in development and commercialization of protein-based therapeutics. One of the greatest challenges during development for such products is the stabilization of proteins in solution. To address this issue, many proteins are formulated as a lyophile that must be reconstituted with a suitable vehicle just prior to use. However, the preference for simpler administration procedures and reduced production costs make development of ready-to-use (RTU) solutions a particularly attractive approach for clinical and commercial drug product formulations, provided sufficient solution stability and adequate shelf life can be achieved. In addition, a large number of protein-based bulk drug substances are provided to development in solubilized form following purification, making identification of an appropriate buffer composition for storage and handling of the bulk protein an important step in the early development process. The studies required to support storage buffer recommendations and RTU solution development for protein pharmaceuticals can be timeconsuming and tedious and often require a significant amount of drug substance to conduct. The design of traditional solution stability studies involves storage of protein solutions of different concentrations in various buffer systems (with or without added excipients) under several stress temperature and/or lighting conditions.
Samples of the solutions are then withdrawn periodically for analysis by one or more methods such as size exclusion chromatography (SEC), gel electrophoresis, and enzyme-linked immunosorbent assay (ELISA). This process can require several weeks or months and grams of bulk drug substance to complete. In an effort to improve efficiency of solution stability investigations for biologics, analytical techniques such as microcalorimetry have been explored as potentially useful screening tools, especially for early evaluation and resolution of possible physical stability issues. Microcalorimetric studies require relatively small amounts of material,1 which can be a particular advantage in early stages of development when drug substance availability is usually limited. Since unfolding of the native protein to a denatured state followed by aggregation and/or chemical degradation in solution is, by far, the most common pathway of protein inactivation, a thorough investigation of variables affecting this process is critical to successful formulation development. Differential scanning calorimetry (DSC)/ microcalorimetry has been used as a method of characterizing heat induced changes in protein conformation and mechanisms of protein unfolding and stabilization in solution.2-10 In this technique, the energy required to maintain a sample cell containing a protein solution at the same temperature as a reference cell is measured as the system undergoes heating at a constant rate. Energy changes in the sample cell are associated with various thermal transitions including protein denaturation, aggregation, and precipitation and can be monitored as a function of solution variables such as pH, ionic strength, buffer type, etc. Protein unfolding is typically observed as a sharp endotherm positioned at a given temperature known as the transition midpoint, or Tm. The thermal stability of the protein in solution can be estimated by shifts in the position of Tm.11-13 Shifts of Tm to higher temperatures indicate greater protein conformational stability, while shifts to lower temperature indicate increased susceptibility of the protein to thermal denaturation. DSC/microcalorimetry has been useful in the study of protein-protein interactions and protein-ligand binding.3, 11, 14-17 It has also been employed to determine the effect of pH and excipients on the solution stability of polypeptides and proteins.11, 18-29 The current studies were conducted to evaluate the use of microcalorimetry for rapid preliminary screening of pH stability and/or potential RTU solution formulations for two recombinant antibodies, Protein I and Protein II, each with molecular weights of approximately 150 kDa.
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Malvern Instruments provides the materials and biophysical characterization technology and expertise that enable scientists and engineers to 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 materials characterization 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 and our commitment to maximizing the potential of established techniques. 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. Headquartered in Malvern, UK, Malvern Instruments has subsidiary organizations in all major European markets, North America, Mexico, China, Japan and Korea, a joint venture in India, a global distributor network and applications laboratories around the world. www.malvern.com severine.michel@malvern.com
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