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Quadrupole Dalton-Based Controlled Proteolysis Method for Characterization of Higher Order Protein Structure

Cao, Xiang, Flagg, Shannon C., Li, Xue, Chennamsetty, Naresh, Balakrishnan, Gurusamy, Das, Tapan K.
Analytical chemistry 2019 v.91 no.8 pp. 5339-5345
antibodies, bioactive properties, circular dichroism spectroscopy, denaturation, differential scanning calorimetry, drugs, fluorescence, instrumentation, liquid chromatography, mass spectrometry, monitoring, peptides, protein structure, proteins, proteolysis
The higher order structure (HOS) of proteins plays a critical role in the efficacy and stability of biological drugs. Perturbation of the regional structure of proteins can affect biological activity and cause instability. Characterization of HOS has become an integral part of biological drug development and is expected from regulatory agencies. The commonly used techniques for HOS characterization, such as circular dichroism, Fourier-transform infrared, differential scanning calorimetry, intrinsic fluorescence, and hydrogen–deuterium exchange mass spectrometry, have their limitations ranging from lack of sensitivity and specificity to the need of high-level expertise and poor access to instrumentation due to high cost. In this study, we demonstrated a novel controlled proteolysis-based LC-QDa method for the detection of HOS change. By digesting proteins directly without denaturation and reduction, the HOS information can be revealed through the digested peptides. After optimizing the digestion conditions and the detection procedures, we identified 13 signature peptides that can monitor various antibody domains for any HOS changes caused by external stress. By comparing the peptide peak areas between unknown samples and a native control sample, any regional structural changes in unknown samples can be detected. The method was subsequently applied to a wide range of forced degradation samples to demonstrate higher sensitivity compared to the near-UV CD method that is frequently used for monitoring tertiary structural changes. By further reducing the number of signature peptides to five and optimizing liquid chromatography gradient duration, a streamlined, high-throughput, and controlled proteolysis method was successfully established. This method can be used to support process and formulation development as well as potentially for stability testing.