Abstract
In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.
Highlights
TO COMPARABILITY ASSESSMENTS Protein based drugs are structurally complex, highly specific macromolecules used therapeutically to compensate for body deficiencies, as medical treatments as well as to prevent diseases (Aggarwal, 2012)
In 2003, a new data visualization method was introduced as a tool for analysis of protein physical stability data obtained from high throughput biophysical measurements (Kueltzo et al, 2003). This method known as the Empirical Phase Diagram (EPD), is a data visualization tool that is based on using data sets from multiple low-resolution biophysical techniques to construct a color coded diagram reflecting the different structural states that a protein would experience during at least two applied stress conditions
There are a number of higher resolution analytical tools potentially available to examine and compare the higher-order structure of protein molecules, many of these techniques have practical limitations in their routine use including technical complexities, low-throughput, high costs and difficult interpretability
Summary
TO COMPARABILITY ASSESSMENTS Protein based drugs are structurally complex, highly specific macromolecules used therapeutically to compensate for body deficiencies (e.g., hormones and clotting factors), as medical treatments (e.g., cytokines and monoclonal antibodies) as well as to prevent diseases (e.g., polyclonal antiserum and certain vaccines) (Aggarwal, 2012). The assessment of higher order structural integrity and conformational stability of proteins in comparability studies is best performed using multiple biophysical and calorimetric techniques.
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