Automated, High-Throughput Infrared Spectroscopy for Secondary Structure Analysis of Protein Biopharmaceuticals.

Protein higher order structure (HOS) is an important product quality attribute that governs the structure-function characteristics, safety, and efficacy of therapeutic proteins. Infrared (IR) spectroscopy has long been recognized as a powerful biophysical tool in determining protein secondary structure and monitoring the dynamic structural changes. Such biophysics analyses help establish process and product knowledge, understand the impact of upstream (cell culture) and downstream (purification) process conditions, create stable formulations, monitor product stability, and assess product comparability when process improvements are implemented (or establish biosimilarity to originator products). This paper provides an overview of a novel automated mid-IR spectroscopic technique called microfluidic modulation spectroscopy (MMS) for the characterization of protein secondary structure. The study demonstrates that MMS secondary structure analysis of therapeutic monoclonal antibodies (mAb) is comparable with a conventional Fourier transform infrared (FTIR) method. More importantly the study shows MMS exhibits higher sensitivity and repeatability for low concentration samples over FTIR, as well as provides automated operation and superior robustness with simplified data analysis, increasing the utility of the instrument in determination of mAb secondary structure. Therefore, we propose that the MMS method can be widely applied in characterization and comparability/biosimilarity studies for biopharmaceutical process and product development.

α-Sheet secondary structure in amyloid ß-peptide drives aggregation and toxicity in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the deposition of ß-sheet-rich, insoluble amyloid ß-peptide (Aß) plaques; however, plaque burden is not correlated with cognitive impairment in AD patients; instead, it is correlated with the presence of toxic soluble oligomers. Here, we show, by a variety of different techniques, that these Aß oligomers adopt a nonstandard secondary structure, termed "α-sheet." These oligomers form in the lag phase of aggregation, when Aß-associated cytotoxicity peaks, en route to forming nontoxic ß-sheet fibrils. De novo-designed α-sheet peptides specifically and tightly bind the toxic oligomers over monomeric and fibrillar forms of Aß, leading to inhibition of aggregation in vitro and neurotoxicity in neuroblastoma cells. Based on this specific binding, a soluble oligomer-binding assay (SOBA) was developed as an indirect probe of α-sheet content. Combined SOBA and toxicity experiments demonstrate a strong correlation between α-sheet content and toxicity. The designed α-sheet peptides are also active in vivo where they inhibit Aß-induced paralysis in a transgenic Aß Caenorhabditis elegans model and specifically target and clear soluble, toxic oligomers in a transgenic APPsw mouse model. The α-sheet hypothesis has profound implications for further understanding the mechanism behind AD pathogenesis.