With thanks to: Andreas Nägeli and Magdalena Widgren-Sandberg (Genovis, Kävlinge, Sweden)

Summary

This application note describes the use of the GlySERIAS™ protease in a middle-level analysis of the fusion protein Blinatumomab, processed by the intact workflow in Byos®.

Protein Digestion: The GlySERIAS™ protease was applied to digest the glycine-rich linkers in Blinatumomab, resulting in smaller protein fragments for analysis.
Mass Spectrometry (LC-MS/MS): The generated fragments were analyzed by LC-MS/MS, with the Byos intact workflow used to deconvolute and identify fragment masses automatically.
Middle-Level Sequencing: Sequencing was used to confirm cleavage sites. This middle-level approach allowed for more effective analysis of protein fragments via MS2 when compared to the more challenging top-down approach using the intact protein.

Introduction

Middle-level LC-MS analysis has become a standard method for the characterization of monoclonal antibody therapeutics. This approach involves digesting the protein into well-defined subunits that are small enough to generate high-quality mass spectra but large enough to provide domain-specific insights into product quality. As the development of new protein formats continues, the demand for novel proteases grows to create fragments of appropriate size for effective analysis.

Fusion proteins, such as Blinatumomab, combine multiple protein domains to tackle specific therapeutic challenges. These domains are often joined by linkers rich in glycine (G) or glycine-serine (GS) sequences, which are designed to resist degradation by traditional proteases, which makes them challenging molecules to analyze using standard LC-MS methods.

The protease GlySERIAS™ was developed to specifically hydrolyze these flexible glycine-rich linkers in fusion protein such as Blinatumomab.

Another key challenge in middle-level LC-MS analysis is the interpretation of complex, multiply charged spectra. To address this, an integrated workflow is required that can handle both deconvolution and primary sequence confirmation.

This article describes a middle-level strategy to streamline sample preparation and data analysis for the characterization of the fusion protein Blinatumomab.

In brief, an immobilized form of GlySERIAS was used to ensure complete digestion of the linkers. This digestion process made the protein fragments more amenable to analysis. Following LC-MS/MS analysis, the intact workflow in Byos was used to perform advanced deconvolution, MS identification (including detection of clipped species), and MS2 sequence confirmation to precisely locate the site of cleavage. This integrated workflow significantly enhances the efficiency and depth of fusion protein characterization, providing a robust tool for analyzing complex protein formats like Blinatumomab.

Figure 1. Summary of the middle-level approach used to characterize Blinatumomab

Experimental

Sample Collection and Preparation:

A research grade biosimilar of Blinatumomab, exhibiting specificity for CD19 and the T-cell marker CD3, was reduced and subjected to digestion using GlySERIAS Immobilized (Genovis). This enzyme selectively targeted all three GS linkers of the molecule, resulting in only a minimal amount of the linked α-CD3-α-CD19 fragment.

Mass Spectrometry:

The digestion products were separated by reversed-phase chromatography (ACQUITY Premier
Protein BEH C4, 300 Å, 1.7 µm 2.1 x 100 mm, Waters™) and analyzed with
ESI-QTOF MS (Bruker Impact II).

Data processing:

-Deconvoluting isotopically resolved data

High-resolution LC-MS analysis of the subunits produced isotopically resolved spectra, providing greater accuracy than what is typically observed in intact protein analysis. To preserve this isotopic resolution in the uncharged spectrum, the default advanced deconvolution settings in the Byos software were modified. Specifically, the parameters for m/z, mass smoothing, and spacing were adjusted to lower values (Figure 2), ensuring that the isotopic detail remained intact during the deconvolution process. This fine-tuning allowed for more precise and accurate identification of the protein subunits, enhancing the overall analysis.

Figure 2. Advanced deconvolution parameters required to preserve the isotopic resolution in the uncharged spectrum

In order to direct the software to calculate the monoisotopic mass, the following advanced
parameters were entered in the processing node section of the workflow:

[Intact]

IsotopeEnvelopeFilter=true

MaxMonoisotope=30000

AdvancedMonoCalculation=true

ShowObservedMono=true

SteppingMethod=Linear

-Identifying Clipped Sequences

The sequences of the individual subunits (including linker regions) were entered into the sequence combinations box. As this was a reduced digest, the disulfide count was set to 0 (Figure 3).

The presence of multiple cleavage sites within the glycine-rich linkers leads to a heterogeneous mixture of hydrolysis products, making their identification particularly challenging. To address this, the clip function in Byos was utilized. This function is specifically designed to detect cleavage products originating from either the N- or C-termini of the protein. For this analysis, the clip function was configured to search for cleavages at specific motifs, such as GG, GS, or SG sequences. This targeted approach enables more efficient identification of the diverse products generated from the hydrolysis, improving the overall clarity and accuracy of the analysis of these cleavage products.

Figure 3. Workflow set-up: Sequences and masses tab

Results

The resulting isotopically resolved MS data was processed using the intact protein workflow in Byos. This workflow facilitates the deconvolution of isotopically resolved low molecular weight subunits and implements a novel clip function to identify cleavage at any position within the protein chain. Furthermore, by choosing monoisotopic mass matching (as opposed to average), the peaks were characterized with increased confidence. The workflow has the capability to simultaneously deconvolute and identify subunits at the intact level, while also verifying the primary sequence through the annotation of MS/MS spectra, reducing the requirement to perform an additional peptide mapping experiment and to process data in a separate workflow.

The Byos Intact workflow (inspection view) is shown in Figure 4. This workflow enabled the efficient identification and validation of the major proteolytic fragments separated by LC-MS. Deconvoluted masses were validated by direct comparison with the raw data (E and F).

Figure 4. Inspection view layout:

A. Trace peak table – Details of the four integrated TIC peaks.

B. Trace plot – TIC showing manually editable peak boundaries.

C. Masses table – Deconvoluted masses found in peak 1. Masses identified based on theoretical monoisotopic masses and clipped species.

D. MS2 Plot – Annotated MS2 spectrum of subunit CD3_VL, confirming sequence and location of cleavage.

E. MS1 Spectrum – Colored dots in raw spectrum link multiply charged masses to the deconvoluted masses in F.

F. Deconvoluted spectrum – Isotopic resolution preserved on deconvolution. Monoisotopic masses labeled with yellow diamonds.

Clipped versions of VL and VH subunits were identified using the built-in clip function. Cleavage sites were confirmed through annotation of MS2 spectra (Figure 5).

Figure 5. Identification summary

Conclusion

Heterogeneous mixtures of subunits resulting from GlySERIAS digestion were primarily identified through monoisotopic mass assignments of deconvoluted MS1 data. This process was facilitated by utilizing the clip function in the Byos intact workflow, which automatically detected cleavages between GG, GS and SG residues.

The sequencing capability in this workflow, operating at middle-level, enabled the confirmation of sequences from all major subunits by annotating MS2 data. This also provided a more confident assignment of the cleavage sites.

By employing this middle-level strategy with GlySERIAS, protein fragments became more amenable to analysis by MS2 compared to full top-down approach using the intact protein.

In summary, the combination of GlySERIAS with a middle-level workflow enabled more detailed and manageable MS analysis of fusion proteins like Blinatumomab.