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Hydrolysis of Flexible Linkers

Digestion of Fusion Proteins with Flexible Linkers

Fusion proteins are created by joining the functionalities of two or more proteins or peptides that are originally coded by separate genes by gene fusion. This allows for the generation of tailor-made molecules to specifically address a therapeutic challenge. The most common way of linking such protein or peptide domains is by inclusion of a flexible linker. These linkers are commonly glycine-rich, for example solely glycine residues (G) or glycine residues interspersed with serine residues (GS) to form repeating sequences. This gives the linker enough flexibility as not to interfere with the function of the connected protein domains. Furthermore, G and GS linkers have been shown to be resistant to degradation by common proteases.

It has previously been shown with mAbs that middle-level analysis strategies offer a rapid way to improve the quality of mass spectra. This allows for the monitoring of many different product quality attributes simultaneously as well as providing domain-specific information. However, new modalities pose new analytical challenges and require novel tools to facilitate characterization and product quality monitoring. GlySERIAS is an enzyme that digests flexible glycine-rich linkers. This allows for separation of the different functional domains and enables middle-level approaches for detailed characterization of fusion proteins, multi-specific antibodies and various mAb fragments containing glycine-rich linkers.

GlySERIAS Digestion

Digestion of a GS-linked Fusion Protein

The fusion protein dulaglutide consists of two glucagon-like peptide-1 (GLP-1) molecules linked to an Fc region of human IgG4 via flexible GS linkers. To study the peptides and Fc region separately and thereby be able to identify specific PTMs, dulaglutide was digested with GlySERIAS at 37°C for 1 hour. To reduce the sample complexity, the Fc glycans were removed using the endoglycosidase GlycINATOR and the interchain disulfide bridges were reduced with DTT (Fig. 1a). Analysis of the sample by reversed-phase LC-MS showed that the peptides were completely removed from the Fc region upon linker digestion using GlySERIAS. The multitude of glycine residues in the linker offers many different potential cleavage sites for GlySERIAS which is why both the Fc/2 and the GLP-1 peptide were detected as several variants with different numbers of glycine and serine residues attached (Fig. 1b, c). GLP-1 was present as two variants, with four and five glycine residues respectively. In addition, an oxidation modification on the GLP-1 peptide was identified. The Fc/2 subunit was present as three main variants: Fc/2 with one remaining serine residue, Fc/2 with a SG3 linker tail and Fc/2 with a SG4S linker tail. Triplicate digests showed repeatable results in the relative amount of the different Fc/2 variants obtained (Fig. 1d), despite GlySERIAS digesting at several sites simultaneously (Fig. 1e).

Figure 1. Digestion of dulaglutide using GlySERIAS. The flexible GS linker of dulaglutide was digested with GlySERIAS for 1 hour at 37°C under native conditions, and the Fc glycans were concurrently hydrolyzed using GlycINATOR to reduce sample complexity. To stop the GlySERIAS reaction, 1 mM ZnCl2 was added. The interchain disulfide bonds were reduced with 20 mM DTT for 30 minutes at 37°C. The digest was performed in triplicate. a) Illustration of the sample preparation workflow. The samples were analyzed by reversed-phase LC-MS. b) Deconvoluted mass spectrum of the Fc/2 subunit. c) Deconvoluted mass spectrum of the GLP-1 peptide. d) Relative amount of the identified Fc/2 variants, displaying the mean value between the triplicate digests and error bars representing the standard deviation. The digestion products were separated by reversed-phase chromatography (BioResolve™ RP mAb Polyphenyl, 450 Å, 2.7 µm 2.1 x 100 mm, Waters™) and analyzed with ESI-QTOF MS (Bruker Impact II). e) Schematic image of the flexible linker, connecting the GLP-1 peptide to the Fc/2 subunit, and the identified digestion sites.

GlySERIAS Digestion

Digestion of a Polyglycine-linked Fusion Protein

Romiplostim consists of four identical thrombopoietin (TPO) receptor binding peptides and one human IgG1 Fc region, linked together by flexible polyglycine sequences. This protein was digested with GlySERIAS at 37°C, both for 1 hour and overnight, and the interchain disulfide bridges were reduced with DTT (Fig. 2a). After 1 hour of digestion, small amounts of Fc/2 linked to one TPO peptide remained (Fig 2b). This enabled the study of the linker region between the Fc/2 and the first peptide without interference from the second peptide. However, in the majority of the material, both TPO peptides were cleaved off from the Fc/2, leaving Fc/2 with four glycine resides and the TPO peptide with one to seven glycine residues as the main digestion products (Fig. 2c). Overnight digestion resulted in a more homogenous sample, in which both the TPO peptides were completely cleaved off from the Fc/2 subunit. The resulting peptides contained one to five glycine residues each. This workflow shows that GlySERIAS can be used for analysis of flexible polyglycine linkers of fusion proteins as well as to digest fusion proteins with several linked domains, to allow for a more detailed analysis of the individual domains (Fig. 2d).


 

Figure 2. Digestion of romiplostim using GlySERIAS. The polyglycine linkers of romiplostim were digested with GlySERIAS for 1 hour and overnight at 37°C under native conditions. The interchain disulfide bonds were reduced with 20 mM DTT for 30 minutes at 37°C. a) Illustration of the sample preparation workflow. The samples were analyzed by reversed-phase LC-MS. b) Deconvoluted mass spectrum of the Fc/2 subunit. c) Deconvoluted mass spectrum of the TPO receptor binding peptide. The glycine residues can be present at both ends of the peptide. The digestion products were separated by reversed-phase chromatography (BioResolve™ RP mAb Polyphenyl, 450 Å, 2.7 µm 2.1 x 100 mm, Waters™) and analyzed with ESI-QTOF MS (Bruker Impact II). d) Schematic image of the two flexible linkers, connecting first the Fc/2 subunit to one TPO receptor binding peptide and then to the second TPO receptor binding peptide, and the identified digestion sites.

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Hydrolysis of Flexible Linkers

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