Pepfinder software for peptide mapping of monoclonal antibodies

Zhang Xiaoxi 1, Sutton, Jennifer N.2

1 Thermo Fisher Scientific (China) Co., Ltd.

2 Thermo Fisher Scientific, West Palm Beach, FL, USA

1 Introduction

Peptide mapping is an important part of the quality control requirements for protein drug products. Peptide mapping analysis includes the identification and quantification of protein amino acid sequences and related modifications. Thermo Fisher and Amgen have jointly developed PepFinderTM software based on the characteristics of protein drug mass spectrometry and the specific needs of biopharmaceutical applications.

PepFinder not only has the most basic peptide sequence confirmation, coverage analysis, modification site identification and quantification, disulfide bond analysis, map comparison and peptide CID/HCD/ETD spectrum analysis, but also has its unique features, such as Unknown modified search function and amino acid mutation search function [1-5]. These two features help analysts quickly identify possible sequence mutations, greatly reducing the risk of quality risks. At the same time, PepFinder has more practical functional design, such as built-in CHO cell line glycoform, with peptide identification credibility evaluation function, and can also predict the theoretical fragmentation spectrum of peptide based on fragmentation and energy intelligence, and use Visually contrast with the experimental map to aid analysis. These features not only reduce the intensity of the work, but also reduce the analyst's understanding of the depth of the mass spectrometry data. For amino acid immobilization settings, unlike other similar software (fixed modifications can only be accurate to a certain class of amino acids), in PepFinder, specific amino acid sites can be specified. This feature provides great convenience for certain specific analytical needs of protein drugs.

2 PepFinder function introduction

2.1 PepFinder basic functions

Figure 1 shows the sequence coverage results from the PepFinder search library, where the different colors represent signal strength. As can be seen from the figure, the light and heavy chain coverage of the monoclonal antibody standard is 100%; PepFinder can also quantify the deamidation, which is very useful for biomedical analysts. Figure 2 shows a summary of the modification generated by PepFinder; Figure 3 shows a qualitative and quantitative case of the deamidated modified peptide SNWEAGNTFTCSVLHEGLHNHHTEK .

2.2 New Features in PepFinder2.0

In version 2.0 of the PepFinder software, De Novo was added (snap-sequenced, referring to the use of bioinformatics calculations for species without protein sequences, techniques for resplicing and assembling sequences to obtain protein sequences based on mass spectrometry information), De Novo Sequencing can be performed on all peptides (Fig. 4), or De Novo Sequencing (Fig. 5) for peptides corresponding to a single MS/MS spectrum.

In PepFinder2.0 software, the mgf format file output function has been added for HCP analysis by MASCOT users. The Pinpoint txt file output function has also been added for Pinpoint users to perform absolute protein quantification; it is convenient for biopharmaceutical users to use different software to data. In-depth analysis greatly reduces the time and effort that staff spend on data processing.

As can be seen from Figure 3, the use of PepFinder software can obtain continuous peptide fragment ions, which improves the reliability of the identification results; respectively, the N416 and N429 sites that may undergo deamidation modification are quantified, and N416 can be obtained. The deamidation ratio was 2.28%, and the deamidation ratio of N429 was 2.90%. For biomedical industry analysts, the change in the amount of deamidation modification is an important parameter for drug stability testing. PepFinder software provides accurate de-amidation information to the site, providing a powerful reference for analysts.

3 PepFinder unique features introduced

In the PepFinder software, as long as the sequence of the monoclonal antibody is introduced, the molecular weight of the heavy chain end K of the antibody is lost, the reduced (unreduced) light/heavy chain molecular weight, the sugar-free/de-sugared heavy chain molecular weight, and Various common glycotype heavy chain molecular weights (Figure 6); currently, this feature is unique to PepFinder software, and other similar software does not yet have this feature.

PepFinder software also predicts secondary spectra. In similar software, PepFinder software predicts that the secondary spectrum is closest to the true fragmentation spectrum (Figure 7).

Figure 1 PepFinder sequence coverage results

Figure 2 Summary of the modification generated by PepFinder

Figure 3 Deamidation identification and quantification of peptide SNWEAGNTFTCSVLHEGLHNHHTEK

Figure 4 De Novo Sequencing for all peptides in PepFinder 2.0

Figure 5 De Novo Sequencing for an MS/MS spectrum

Figure 6 Calculating the various commonly used molecular weights of monoclonal antibodies in PepFinder software

Figure 7 Comparison of the secondary spectrum predicted by the PepFinder software with the measured secondary spectrum. On, predict the secondary spectrum; below, the measured secondary spectrum.

4 Conclusion

In this article, we show various applications of PepFinder software for routine peptide mapping of monoclonal antibodies. The results show that the results of sequence coverage analysis can be obtained quickly and easily using PepFinder software, and important post-translational modifications in biopharmaceutical quality control such as deamidation can be identified and quantified. PepFinder's unique monoclonal antibody molecular weight calculation function and secondary spectrum intelligent prediction function greatly reduce the time spent by analysts on data processing; in PepFinder version 2.0, the new De Novo Sequencing function and mgf format file, The pinpoint txt file output feature helps analysts dig deeper into the data on different platforms. By generating fast, comprehensive peptide characterization and automated grooming site summary reports, PepFinder software dramatically reduces the time and effort required for detailed characterization of biological products.

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