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

Abstract

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.

Native peptide mapping – A simple method to routinely monitor higher order structure changes and relation to functional activity

“…Limited proteolysis sample treatment, and especially the assumption that the protease accessibility in native-like conditions gives information about the HOS of the molecule, is increasingly accepted by the scientific community, [33][34][35][38][39][40][41][42][43][44] particularly to monitor the HOS of a mAb. 32,36,45 1 and Figure 1 results suggest that the order of appearance of cleaved peptides is linked to: 1) the primary structure because of the specific cleavage sites of the trypsin, and 2) the HOS of the molecule due to the accessibility of the enzyme to cleavable sites present on the molecule. These assumptions are in line with previous publications 32,36 with an IgG4 mAb, but have never been demonstrated for an IgG1 subtype mAb with such a method (see Table 1 in supplementary material).…”

“…32,36,45 1 and Figure 1 results suggest that the order of appearance of cleaved peptides is linked to: 1) the primary structure because of the specific cleavage sites of the trypsin, and 2) the HOS of the molecule due to the accessibility of the enzyme to cleavable sites present on the molecule. These assumptions are in line with previous publications 32,36 with an IgG4 mAb, but have never been demonstrated for an IgG1 subtype mAb with such a method (see Table 1 in supplementary material).…”

“…This routine UPLC-MS system has already been used by Xu et al and Cao et al for the development of routine-driven methods for product characterization, product development and quality control of biologics. 36,37 The peptide list, which was made during the development stage during MALDI-HRMS analysis, was used to aid this change of instrument.…”

“…This and other limited proteolysis methods have been used to discriminate stressed and unstressed material. 32,36 Here, we have further developed the method so that, in addition to its routine use to support process, formulation development and stability studies, the method yields predictive information on the biological activity of the drug. The initial work was conducted to identify all the peptides that are cleaved by the trypsin in native-like conditions by matrixassisted laser desorption ionization mass spectrometry (MALDI) coupled with high-resolution mass spectrometry.…”

Response to “Molecular ion: A more contemporary definition”

“…Many practitioners of mass spectrometry do not have access to instrumentation capable of accurate mass measurement, and numerous applications of nominal m/z measurement, where the m/z of the ion and mass of the molecule are the same numerical value, still exist. [3][4][5][6][7][8][9][10][11][12][13] Therefore, the advancements in instrument capability are not universally available and do not justify amending the definition of "molecular ion".…”