Simple and fast antibody drug disulfide bond analysis based on Q-Exactive platform and BioPharma Finder software

Nie Aiying

Thermo Fisher Scientific (China) Co., Ltd.

Key words

Q-Exactive; BioPharma Finder; monoclonal antibody; disulfide bond analysis

1 Introduction

At present, monoclonal antibody drugs have become the most dazzling pearl in the field of biomedicine. These antibody drugs have the characteristics of specificity, high specificity and low toxic and side effects, which represent the latest development direction in the field of drug therapy, and have been strongly promoted in the field of anti-tumor and autoimmune system defects treatment. China's antibody drug industry is also in full swing. In the early development and production quality control of antibody drugs, the structural molecular characteristics, such as the complete molecular mass, light and heavy chain molecular mass, peptide map, sugar chain, disulfide bond, etc. Strict and rapid monitoring and quality control are required to ensure the effectiveness and safety of antibody drugs.

Among them, the disulfide bond acts as a covalent bond to crosslink two intracysteines or two cysteines between the chains, which plays an important role in stabilizing the spatial structure of the protein, maintaining the correct spatial folding conformation, and maintaining and regulating its biological activity. The role, especially for biopharmaceuticals, the location and attachment of disulfide bonds plays a key role in the spatial structure of the biopharmaceutical, drug activity, and the like. X-ray diffraction crystal structure analysis method, multi-dimensional nuclear magnetic resonance method, disulfide bond isomerization and mutation analysis method, enzymatic hydrolysis method and chemical cleavage method can be used for disulfide bond localization analysis, but these methods are generally slow in detection. The sample requires high purity and complicated manual operation. However, with the continuous improvement of mass detection range, resolution, sensitivity, accuracy and analysis speed, mass spectrometry can be applied simply and quickly. In the localization analysis of disulfide bonds, not only the confirmation of known disulfide bonds, but also the discovery and identification of unknown disulfide bonds can be achieved, which is far superior to other detection methods.

Currently, the Q-Exactive HF instrument platform based on advanced quadrupole technology and ultra-high field Orbitrap mass analyzer provides faster, sensitive and accurate disulfide bonds, molecular weights, peptide maps, sugar chains, etc. for biopharmaceutical analysis. Important information. At the same time, BioPharma Finder biopharmaceutical software, developed by biopharmaceutical companies, jointly developed by Amgen and Thermo Fisher, the world's leading biopharmaceutical companies, can provide biopharmaceutical customers with disulfide bonds, molecular weights, peptide maps and sugars related to antibody drugs. Qualitative and quantitative information on chains, amino acid mutation sites, and various post-modification sites. Here we mainly introduce the simple and rapid analysis of antibody drug disulfide bonds based on Q-Exactive HF instrument platform and BioPharma Finder biopharmaceutical software.

2. Experimental part

2.1 Instruments and reagents

Mass Spectrometry Instruments: Q-Exactive HF (Thermo Fisher Scientific, USA);

Chromatography instrument: Easy Nano1000 LC system (Thermo Fisher Scientific, USA);

Column: Homemade reversed phase Nano column (C18, 2 μm, 75 × 150 μm, 100 Å);

Reagents: chromatographic grade formic acid, secondary deionized water, chromatographic grade acetonitrile, guanidine hydrochloride (Sigma-Aldrich, USA), ammonium bicarbonate (Sigma-Aldrich, USA), Trypsin (Promega, USA).

2.2 sample preparation

The IgG1 type of monoclonal antibody was placed in a 6 M guanidine hydrochloride system and denatured at room temperature for 1 hour. The solution system was replaced with an ultrafiltration tube. The final solution was 50 mM ammonium bicarbonate, and then the mass ratio of Trypsin, enzyme and protein was 1. :40,37 °C overnight digestion, add 1% formic acid to stop digestion, spin freeze-dried, and set aside. (The denaturation time and enzymatic hydrolysis time can be adjusted according to the inconsistency of the sample)

2.3 Instrument method

Chromatographic conditions: see Table 1 for details;

Mass spectrometry conditions: see Table 2 for details;

Table 1. Chromatographic conditions for disulfide bond analysis

Table 2. Mass spectrometry conditions for disulfide bond analysis

2.4 Data Analysis Method

The original spectrum was analyzed by disulfide bond using BioPharma Finder 1.0 software. The database is the amino acid sequence corresponding to the antibody drug. The search parameters are basically set by the default setting of the software. Check the disulfide bond search without specifying the possible link position of the disulfide bond. ,As shown in Figure 1.

Figure 1. Disulfide bond setup module in BioPharma Finder 1.0 software

3. Results and discussion

The most commercially available monoclonal antibody is the IgG1 type of monoclonal antibody. In this type of antibody, there are mainly 16 pairs of disulfide bonds, including 8 pairs inside the heavy chain and 4 pairs inside the light chain. Light and heavy chains There are 2 pairs between the chains, and there are 2 pairs between the heavy chains. Considering the symmetry of the mAb structure, finally, there are 9 pairs of non-redundant disulfide bonds. Taking the monoclonal antibody being tested as an example, the theoretical disulfide bond connection is shown in Figure 1.

Figure 1. Disulfide-bonded version of the IgG1 type of monoclonal antibody and specific linkage information for the theoretical disulfide bond of the monoclonal antibody being tested.

Thanks to the ultra-high sensitivity, ultra-high precision and ultra-fast acquisition speed of Q-Exactive HF, the 9-pair disulfide linkage of the test antibody drug is 100% only in the peptide product of Trypsin digestion. The identification coverage covers the powerful qualitative capabilities of Q-Exactive HF.

First, the disulfide linkage of HC22-HC96 in the heavy chain is taken as an example. As shown in Fig. 2, the secondary fragment information of the peptides AEDDAVYYCAK and LSCAASGFTFDDYAMHWVR is very rich, and the mass deviation of the primary parent ion is 1.58 ppm, so it can be accurate. Quickly determine the connection of disulfide bonds.

Figure 2. Disulfide-linked secondary fragmentation of the peptides AEDDAVYYCAK and LSCAASGFTFDDYAMHWVR (HC22-HC96)

Secondly, taking the disulfide bond of LC23-LC88 in the light chain as an example, as shown in Figure 3, the secondary fragment information of the peptides VTITCR and FSGSGSGTDFTLTISSLQPEDVATYYCQR is also very rich, and the mass deviation of the primary parent ion is 1.73 ppm. The connection of disulfide bonds can be determined accurately and quickly.

Figure 3. Disulfide-linked secondary fragmentation of the peptides VTITCR and FSGSGSGTDFTLTISSLQPEDVATYYCQR (LC23-LC88)

Finally, taking two pairs of similar disulfide bonds HC230-HC230 and HC233-HC233 in the heavy chain hinge region as an example, as shown in Figure 4, the secondary fragment information of the peptides THTCPPCPAPELLGGPSVFLFPPKPK and THTCPPCPAPELLGGPSVFLFPPKPK is also very rich, and the primary parent ion The mass deviation is 1.70 ppm, so the connection of the two pairs of disulfide bonds can be determined accurately and quickly.

Figure 4. Peptide segments THTCPPCPAPELLGGPSVFLFPPKPK and THTCPPCPAPELL

Disulfide-linked secondary fragmentation spectra of GGPSVFLFPPKPK (HC230-HC230 and HC233-HC233)

4 Conclusion

Based on Q-Exactive instrument platform and BioPharma Finder data analysis software, this paper realizes simple and rapid disulfide bond analysis of monoclonal antibody, and provides an efficient and reliable analytical test method for initial development and production quality control of monoclonal antibody. In addition, the Q-Exactive series of mass spectrometers, with their ultra-high resolution, ultra-fast scanning speed and high mass accuracy, will certainly help deep mass spectrometry in the biopharmaceutical industry.