Multidimensional methods for the formulation of biopharmaceuticals and vaccines

Abstract

Determining and preserving the higher order structural integrity and conformational stability of proteins, plasmid DNA and macromolecular complexes such as viruses, virus-like particles and adjuvanted antigens is often a significant barrier to the successful stabilization and formulation of biopharmaceutical drugs and vaccines. These properties typically must be investigated with multiple lower resolution experimental methods, since each technique monitors only a narrow aspect of the overall conformational state of a macromolecular system. This review describes the use of empirical phase diagrams (EPDs) to combine large amounts of data from multiple high-throughput instruments and construct a map of a target macromolecule's physical state as a function of temperature, solvent conditions, and other stress variables. We present a tutorial on the mathematical methodology, an overview of some of the experimental methods typically used, and examples of some of the previous major formulation applications. We also explore novel applications of EPDs including potential new mathematical approaches as well as possible new biopharmaceutical applications such as analytical comparability, chemical stability, and protein dynamics.

Improved data visualization techniques for analyzing macromolecule structural changes

“…no reversibility is implied), but rather simple, empirical representations of the physical behavior of the macromolecule. [1][2][3] As shown in Fig-ure 5(B), a small pink-colored region between the native and unfolded state is also observed in the three-index EPD. The calculated structural indices used for the construction of the three-index EPD for the protein antigen SP1732 are shown in Figure 6(G-I; see Methods section).…”

“…This includes the lack of meaningful relationship between color itself and actual molecular features (in EPDs, structural changes are represented by color transitions 1,2,9 ) as well as limitations resulting from color deficiencies in vision and blindness, which are possessed by a substantial portion of the human population. Red-green color vision defects are found in about 8% of males and 0.5% of females among people of Northern European origin, 5% among Chinese and Japanese populations, and 4% or less among individuals of African origin.…”

“…Therefore, stability data should first be converted to the corresponding structural indices using Eqs. (1) and (2). In each environmental condition (e.g., solution pH and temperature), the values in the associated data indices are mapped to points in the polar coordinates of the radar chart.…”

“…Using singular value decomposition (SVD), the three largest contributions to a vector are obtained and reduced to color based on an RGB scheme. 1,2 Although the actual colors are somewhat arbitrary, changes in color are useful, because they represent structural changes. In addition, differences between EPDs can be directly compared (e.g., between mutants of the same protein) by processing all the data together.…”

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

“…The long term loss of monomer is generally predicted through quantitative monitoring of aggregation under accelerated and stress conditions over weeks to months. Recent progress has been made in: (i) the development of detailed kinetic models [2,4,5,34,42,49,60,96]; (ii) correlating aggregation kinetics with protein structure and folding [11,15,16,32,35,52,53,54,67,88]; (iii) and with native-state protein-protein interaction measurements [36,46,57,74,75,79,82].…”