Secondary structure characterization of proteins using amide I band Raman spectroscopy: Methods development and applications to bioprocessing

The stability of protein-based therapeutic agents during processing and storage is of prime concern to the bioprocess industry. Due to their labile nature, proteins are susceptible to structural changes during various stages of bioprocessing. These structural changes often lead to irreversible losses of active protein. Improvements in the yield of active protein product could translate into significant economic and strategic gains for a biotech company. Accurate characterization of protein structure in bioprocessing environments could help in understanding why proteins lose their activity in such systems and to determining how to minimize physical and chemical degradation of proteins during bioprocessing.; Raman spectroscopy, due to its capability to analyze samples in solid, solution, suspension, adsorbed and immobilized forms, is particularly suited for the characterization of protein structure. In the current investigation, an algorithm was developed to estimate the secondary structure of proteins from their amide I band Raman spectra in a reliable and accurate manner. First, using Raman spectra of several well-characterized crystalline proteins, amide I spectra for pure classes of secondary structures, viz. ordered-helix, unordered-helix and sheet, turn and unordered structures, were obtained. The calculation of these reference spectra was achieved through simultaneous subtraction of all aromatic peaks, buffer and fluorescence peaks as well as baseline contributions and normalization and deconvolution of the amide I regions. The procedure doesn't limit the amide I region to any specific range of frequencies, nor does it assume equal scattering efficiencies for different secondary structures. The possibility that bound-water spectra are different from bulk water spectra was recognized; this was accounted for in the reference set through the inclusion of two broad Gaussian-Lorentzian peaks. The suitability of the calculated reference spectra for the estimation of secondary structures of proteins in solution, suspension and dry crystalline forms was demonstrated. The agreement between the structure estimates and corresponding estimates from x-ray structure characterization is good.; The structure estimation protocol was used to characterize protein behavior in two different model environments relevant to bioprocessing. (1) Proteins adsorbed on chromatographic surfaces: Information was used in concert with retention studies to identify driving forces for denaturation in chromatographic columns. Potential protein-specific mechanisms for adsorption induced denaturation were also identified. (2) Proteins in Frozen Solutions: Results were used to understand freezing-induced secondary structural changes in proteins and the role of cryoprotectants in preventing them.