Monoclonal antibody (mAb) is one of the fastest-growing treatment methods in the pharmaceutical industry. The high-order structure (HOS) of mAb affects the binding specificity of drugs and targets, thus affecting the therapeutic effect and side effects. If storage leads to changes in HOS, such as protein misfolding and aggregation, it will lead to reduced stability, loss of efficacy or possible immunogenicity. Therefore, monitoring HOS is very important to ensure the effectiveness and safety of mAb therapy. X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy can provide atomic resolution, but they have the disadvantage of time-consuming and sample consuming; Biophysical techniques, such as differential scanning calorimetry (DSC), dynamic light scattering (DLS), fluorescence spectroscopy, infrared (IR) spectroscopy and circular dichroism (CD) spectroscopy, can only provide low-resolution overall conformation.
As an electrophilic reagent, diethyl pyrocarbonate (DEPC) can modify the solvent accessible nucleophilic side chains (Cys, his, Lys, THR, Tyr, Ser) and the N-terminus of proteins. The carboxylated products produced by these residues have a mass transfer of +72.021da. After protein hydrolysis digestion, liquid chromatography and tandem mass spectrometry, specific protein modification sites can be identified and semi quantitatively analyzed. When one condition (such as natural) is compared with another condition (such as heating), the change of covalent labeling degree at a specific residue can be used to detect the change of HOS of proteins (Fig. 1). In this article, the author uses DEPC covalent labeling coupled with mass spectrometry, and takes rituximab as the model of monoclonal antibody drugs, in order to specifically detect subtle changes in HOS at a temperature far below the melting point of mAb therapeutic drugs, which is verified by activity determination.
Figure 1 The process of analyzing the structure of McAbs by DEPC labeling and mass spectrometry
Before studying heat stressed rituximab by covalent labeling, the authors used CD spectra, fluorescence spectra and dynamic light scattering (DLS) to identify the interference of heating on protein structure. It was found that when rituximab was heated at a temperature below its melting point for 4 hours, these three techniques could not detect significant structural changes at 45 ° C or 55 ° C, but only showed slight changes at 65 ° C.
The author team then used DEPC cl-ms to detect subtle structural changes in rituximab. In the rituximab samples at 45 ° C pressure, it was found that the changes of DEPC labeling level were less, and most of the changes were due to the increase of labeling caused by protein thermal unfolding (Fig. 2), and the changes in the variable region were much less than those in the constant region. More than 70%of the marker changes occurred at Tyr, Ser and thr residues, while the marker changes at his and Lys residues were always less than 20%. Labeling changes showed that the structural changes at 45 ° C were mainly changes in the local microenvironment, rather than large structural changes with significant differences in solvent accessibility, that is, the modification sites were dispersed in the whole protein structure, rather than concentrated in some regions of the protein.
Figure 2 The change of DEPC modification degree after 45 ° C thermal stress for 4 h. The pie chart shows the proportion of modified residues with significant label changes in each domain of rituximab. Red represents an increase in markings, while blue represents a decrease. The bar graph shows the number of residues with low (L), medium (m) and high (H) covalent marker changes.
Activity determination can reflect the effect of structural changes on the activity of rituximab to a certain extent, so as to verify the results of DEPC labeling. The results of bridging ELISA showed that after preheating to 45 ° C, the Fc binding activity of rituximab did not change significantly (Fig. 3a), the CDC activity in the Fc region was estimated to remain unchanged after 45 ° C heat stress (Fig. 3b), and the Fab binding activity of rituximab was estimated to be no different from that of the control sample (Fig. 3C). The results of activity determination showed that there was no significant structural change of protein at 45 ° C. In fab and Fc regions, the number of residues with marker changes is relatively small, and the main markers are Tyr, Ser and thr residues that are more sensitive to local microenvironment changes. The modified sites were dispersed in the whole protein, and had little effect on the conformation of Fab and Fc regions, which was consistent with the results of covalent labeling mass spectrometry.
Figure 3 The structural changes revealed by cl-ms experiments were verified by monoclonal antibody activity assay. The structural integrity of the Fc region was assessed by (a) rituximab bridging ELISA to measure Fc binding to capture antibodies and (b) alamarblue assay to measure complement dependent cytotoxicity. The structural integrity of Fab region was evaluated by (c) Raji cell pull-down test, measuring the binding of fab to B cell CD20 antigen.
After heating at 55 ° C for 4h, the residue modification degree of all domains of rituximab changed significantly, especially the VH and VL domains in fab region. (Fig. 4) when heated to 55 ° C, the labeling changes at his and Lys residues were almost twice that at 45 ° C, indicating that the protein expanded in these regions; The marker level of Fab region changed significantly, especially in VH, VL and Cl domains. This indicates that there are local structural changes in the Fab region of rituximab, which is also reported to be the most sensitive region of IgG1 molecule to heat stress. No similar residue aggregation with marker changes was observed in the Fc region, and most of the marker changes at Tyr, Ser and thr were moderate or high. These results indicate that the protein topology may change.
Figure 4 The change of DEPC modification degree after 4 hours of thermal stress at 55 ° C. The pie chart shows the proportion of modified residues with significant label changes in each domain of rituximab. Red represents an increase in markings, while blue represents a decrease. The bar graph shows the number of residues with low (L), medium (m) and high (H) covalent marker changes.
Size exclusion chromatography (SEC) measurements showed the presence of high molecular weight substances at 65 ° C. After applying DEPC cl-ms method to rituximab under 65 ° C thermal stress, it was found that the labeling of all rituximab domains changed significantly (Fig. 5), mainly reflected in the reduction of labeling, which may be due to protein aggregation. Residue clusters with reduced labeling were found in the Fab and Fc regions of rituximab. The activity determination results showed the decrease of Fc binding and CDC activity (Fig. 3), indicating the labeling changes in the Fc region, especially the CH3 domain, which was consistent with the results of DEPC labeling.
Figure 5 The change of DEPC modification degree after thermal stress at 65 ° C for 4 h. The pie chart shows the proportion of modified residues with significant label changes in each domain of rituximab. Red represents an increase in markings, while blue represents a decrease. The bar graph shows the number of residues with low (L), medium (m) and high (H) covalent marker changes.
The structural resolution and sensitivity of DEPC labeling technology are sufficient to detect subtle protein conformational changes. This technology combined with mass spectrometry can reveal subtle HOS changes in rituximab at temperatures below TM, which is complementary to classical biophysical technology. In general, in view of the simplicity and sensitivity of cl-ms, this method will be applicable to the structural study of other antibody drugs.