Obtaining high sequence coverage and confident post-translational modification (PTM) analysis of biotherapeutics using an electron activated dissociation (EAD)-based middle-down workflow

Featuring the EAD-based middle-down workflow using the ZenoTOF 7600 system and Biologics Explorer software from SCIEX

Haichuan Liu and Zoe Zhang
SCIEX, USA

Abstract

This technical note highlights the power of a single-injection, EAD-based middle-down workflow to achieve consistently high sequence coverages (70%-85%) between injections or across different monoclonal antibodies (mAbs). This streamlined workflow leverages information-rich EAD fragmentation with automated data analysis using Biologics Explorer software to offer confident sequence confirmation and accurate localization of PTMs, such as glycosylation and oxidation. 

Introduction

Sequence confirmation and PTM analysis are essential for the comprehensive characterization of therapeutics to ensure drug safety and efficacy. ¹ Middle-down mass spectrometry (MS) combines the advantages of bottom-up and top-down approaches and offers high sequence coverages of protein therapeutics following a simple sample preparation. ^2-4 Traditionally, a middle-down workflow requires extensive method development and often involves multiple fragmentation techniques and/or injections to obtain high sequence coverage. This challenge can be addressed using a single-injection, EADbased middle-down workflow. ⁵ This streamlined workflow provided consistently high sequence coverage of NISTmAb subunits, enabling sequence and PTM confirmations. ⁵

In this technical note, the EAD-based middle-down workflow (Figure 1) was used to characterize multiple available biotherapeutics. This powerful workflow achieved high sequence coverages (70%-85%) and enabled the localization of important PTMs, including glycosylation and oxidation.

Key features of the EAD-based middle-down workflow
 

• Single-injection platform method: The EAD-based middledown workflow provides high sequence coverages in a single injection with 1 fragmentation technique
  
  
• Reproducible high sequence coverage: Sequence coverages ranging from 70% to 85% are obtained between runs for the subunits of various biotherapeutics
  
  
• Localization of PTMs: High sequence coverage and preservation of labile modifications by EAD allows for comprehensive PTM analysis
  
  
• Streamlined: The workflow consists of simple sample preparation, efficient data acquisition and automatic data analysis, with limited method development required

Methods

Sample preparation: The 10-25 µg/µL stock solutions of mAbs, including NISTmAb, adalimumab, bevacizumab, cetuximab and trastuzumab, were diluted in water to concentrations ranging from 0.5 to 1 µg/µL. The IdeS protease (Promega) with a concentration of 50 units/µL was added to the diluted solutions and the mixture was incubated at 37°C for 2 hours. After IdeS treatment, a solution of 7.6M guanidine hydrochloride (HCl) and 50mM Tris-HCl (pH=7.4) was added, followed by reduction using dithiothreitol. The mixture was incubated at 60°C for 30 minutes. The reaction was terminated by adding 10% formic acid (FA). The final solution contained 0.2-0.5 µg/µL of the Fc/2, LC and Fd subunits. 2-10 µL aliquots of the final solutions (1-2 µg of each subunit) were injected for LC-MS analysis. Oxidation of mAbs occurred during the storage of the digested samples in the autosampler for an extended period of time.

Chromatography: The IdeS subunits of mAbs were separated using an ACQUITY UPLC Protein BEH C4 column (2.1 × 50 mm, 1.7 µm, 300 Å, Waters). The subunits of NISTmAb, adalimumab and trastuzumab were separated using the Gradient 1 parameters shown in Table 1, whereas the cetuximab subunits were separated using the Gradient 2 parameters. Bevacizumab subunits were separated using a modified version of the Gradient 1 parameters (not shown), in which %B was 25% at 2 min and 35% at 9 min. A flow rate of 0.3 mL/min was used for all LC runs. The column was kept at 60°C in the column oven of an ExionLC system (SCIEX). Mobile phase A was 0.1% FA in water and mobile phase B was 0.1% FA in acetonitrile. 

Mass spectrometry: MRM^HR EAD experiments were performed in SCIEX OS software using the ZenoTOF 7600 system. Two or 3 charge states were targeted per subunit for EAD fragmentation. The key TOF MS and MRM^HR settings used are listed in Tables 2 and 3, respectively.

Data processing: MRM^HR data were analyzed using a new topdown workflow template in the Biologics Explorer software, as previously described. ⁵

EAD-based middle-down workflow

The middle-down MS approach combines the advantages of the bottom-up and top-down methods by providing high sequence coverage of mAb subunits with minimal interference from sample preparation-related modification artifacts. Traditionally, the success of middle-down MS relied on extensive method optimization and/or multiple fragmentation techniques,^2-4 limiting its implementation for routine analysis. A single-injection, EADbased middle-down workflow was developed to achieve consistently high sequence coverage of NISTmAb with minimal effort needed for method development and optimization. As a result, this workflow can be quickly adopted for sequence confirmation and PTM analysis by users with varying levels of LC-MS experience.

The EAD-based middle-down workflow leverages reproducible and information-rich fragmentation by EAD and automated data analysis by Biologics Explorer software. Figure 2 shows a snapshot of Biologics Explorer software that displays multiple tabs in 1 window, facilitating data inspection and results review. The software provides an easy-to-use pre-built workflow template that is specifically designed for biotherapeutic sequence confirmation and PTM analysis using the middle-down approach. This workflow template includes user instructions and optimized parameters to process middle-down EAD data (Figure 2A).The coverage map (Figure 2B) offers a quick assessment of the sequence coverage, whereas the annotated mass spectrum (Figure 2C) and cluster table (Figure 2D) allow users to perform manual inspection and verification of fragment assignments.

Reproducible middle-down characterization of protein therapeutics

It was demonstrated that the EAD-based middle-down workflow provided reproducible high sequence coverages (70%-80%) of Fc/2, LC and Fd subunits of NISTmAb, enabling confirmation of sequences and the site of N-linked glycosylation on the Fc/2 subunit. 5 In this work, the EAD-based middle-down workflow was extended to characterize multiple protein therapeutics, including adalimumab, bevacizumab, cetuximab and trastuzumab, using the same EAD parameters. Similar to the NISTmAb results, 5 consistently high sequence coverages (70%-85%) were obtained for these biotherapeutics, demonstrating the reproducibility and broad applicability of the EAD-based middle-down workflow for biotherapeutic characterization.

The high sequence coverages of the EAD-based middle-down workflow can be attributed to the information-rich spectra generated by EAD. A representative EAD spectrum of the cetuximab LC subunit with fragment annotation is shown in Figure 3. EAD produced many sequence fragments across the full mass range (Figure 3), leading to high sequence coverage (84%) of the cetuximab LC subunit (Figure 4). High sequence coverages (79% and 73%) were also achieved for cetuximab Fc/2 and Fd subunits containing glycans G0F and G2FGal2, respectively (Figure 4).

The EAD-based middle-down workflow provided reproducible high sequence coverages between runs or across different protein therapeutics. Figure 5 shows the high sequence coverages (70-85%) of Fc/2, LC and Fd subunits that were obtained consistently between 6 runs or across 4 different mAbs.

This result demonstrates that the EAD-based middle-down workflow can be employed as a single-injection method for confident biotherapeutic characterization.

EAD-based middle-down workflow for comprehensive PTM analysis

PTMs are important for the function of protein therapeutics and are often considered critical quality attributes of protein therapeutics. ¹ PTMs occurring in unexpected locations or at undesirable levels might have a significant impact on the safety and efficacy of therapeutics. Although the bottom-up MS approach offers a complete characterization of PTMs, modification artifacts might be introduced during complicated sample preparations. Top-down MS requires little or no sample preparation, however, its low sequence coverage is often insufficient to analyze modifications that are not near the termini. Middle-down MS allows superior PTM analysis compared to topdown MS while reducing the artificial modifications from sample preparation compared to bottom-up MS.

The EAD-based middle-down workflow was used to confirm the location of N-linked glycosylation in the Fc/2 subunits of all mAbs and the Fd subunit of cetuximab. Figure 6 shows the signature fragments (c₆₀ and c₆₁) generated for the Fc/2 subunits of bevacizumab, trastuzumab and cetuximab for the localization of G0F. The detection of non-glycosylated c₆₀ and glycosylated c₆₁ ions confirmed the glycosylation of Asn⁶¹ (Figure 6). The detailed characterization of N-linked glycosylation in Fc/2 and Fd subunits of cetuximab will be described in a separate technical note.

Oxidation might occur at different stages of biotherapeutic manufacturing, storage or characterization. The localization of oxidation can facilitate understanding the hot spots in a protein therapeutic. Figure 7 shows 2 examples in which the EAD-based middle-down workflow localized 2 main oxidation sites in NISTmAb LC and Fc/2 subunits. The detection of an oxidized c₄ (Figure 7B) confirmed the oxidation of Met⁴ in the LC subunit. The detection of non-oxidized c₁₅ and oxidized c₁₆ fragments (Figures 7C and 7D) indicated that the Met¹⁶ residue was the main oxidation site in the Fc/2 subunit. In-depth characterization of oxidation in different protein therapeutics by the EAD-based middle-down workflow will be demonstrated in a separate technical note.

In summary, these results demonstrate that the streamlined, EAD-based middle-down workflow can achieve consistent high sequence coverages of mAb subunits and confident PTM analysis in a single injection.

Conclusion
 

• A streamlined EAD-based middle-down workflow enabled confident sequence confirmation and PTM analysis in a single injection
  
  
• High sequence coverages ranging from 70% to 85% were obtained for the Fc/2, LC and Fd subunits consistently between injections or across multiple protein therapeutics
  
  
• Middle-down MS allows for more accurate characterization of a protein therapeutic with a lower risk of introducing artificial modifications compared to a complete protein digestion
  
  
• The detection of signature fragments enabled the confirmation of N-linked glycosylation on the Fc/2 and Fd subunits
  
  
• The EAD-based middle-down workflow confirmed the 2 main oxidation sites in the LC and Fc/2 subunits of NISTmAb
  
  
• Biologics Explorer software offers an optimized middle-down workflow and provides automated data analysis with high accuracy and efficient results review for improved user experience

References
 

1. Anna Robotham and John Kelly. (2020) LC-MS characterization of antibody-based therapeutics: recent highlights and future prospects. Approaches to the Purification, Analysis and Characterization of AntibodyBased Therapeutics. Chapter 1: 1-33.
   
   
2. Milos Cejkov et al. (2021) Electron transfer dissociation parameter optimization using design of experiments increases sequence coverage of monoclonal. J. Am. Soc. Mass Spectrom. 32(3): 762-771.
   
   
3. Luca Fornelli et al. (2018) Accurate sequence analysis of a monoclonal antibody by top-down and middle-down Orbitrap mass spectrometry applying multiple ion activation techniques. Anal. Chem. 90(14): 8421-8429.
   
   
4. Kristina Srzentic et al. (2020) Interlaboratory study for characterizing monoclonal antibodies by top-down and middle-down mass spectrometry. J. Am. Soc. Mass Spectrom. 31(9): 1783-1802.
   
   
5. A streamlined single-injection middle-down workflow using electron activated dissociation (EAD) for biotherapeutics characterization. SCIEX technical note, MKT-26997-A.