NEW! We’re Launching GlySERIAS Immobilized

GlySERIAS is a unique enzyme that digests flexible glycine-rich fusion protein linkers such as Gly₄Ser and Gly_xSer_y (GS), and polyglycine (G) linkers. Today, we’re happy and proud to be launching this unique SmartEnzyme in a new format – GlySERIAS Immobilized – for digestion of flexible linkers in easy-to-use spin columns!
 
The enzyme enables separation of the individual domains of multi-functional fusion proteins to facilitate characterization and increase the understanding of these molecules. Middle-level analysis of fusion proteins serves to both reduce the overall sample complexity and allow for domain-specific identification and monitoring of post-translational modifications.
 
For improved linker removal, we recommend GlySERIAS Immobilized.
For linker characterization, we recommend GlySERIAS Lyophilized.
 

• Unique enzyme digesting fusion proteins with flexible linkers
• A middle-level approach reduces complexity of fusion protein characterization
• Reliable and reproducible digestion of complex fusion proteins

A research grade biosimilar of blinatumomab, a BiTE molecule with specificity towards CD19 and the T-cell marker CD3, was digested with GlySERIAS Immobilized overnight at room temperature (Fig. 4a). Direct analysis of the digested sample by LC-MS showed that all three linker regions were digested, with the α-CD3 V_L and V_H chains eluting as one chromatographic peak, and the α-CD19 V_H and V_L chains eluting as separate peaks (Fig. 4b). A small peak from the linked α-CD3 V_H and α-CD19 V_H domains revealed that this short linker was not completely digested, indicating that GlySERIAS has a lower activity on short linkers. The separation of the four domains provided high quality spectra with monoisotopic resolution, as compared to the intact protein analysis (Fig. 4c, d). A few different variants of each domain could be observed, with different numbers of linker residues remaining. The α-CD3 V_H domain could not be fully baseline resolved from the α-CD19 V_H domain during chromatographic separation, resulting in some co-elution which could be observed in the associated mass spectra. The separation of the four domains yielded information about the location of protein modifications identified on the intact protein level. For example, a protein modification of 37 Da, which was also identified on the intact level, was assigned to the α-CD3 V_H chain. Furthermore, two modifications of 240 Da and 320 Da, respectively, were assigned to the α-CD3 V_L domain. The latter also contained a His-tag with five or six histidine residues. These data demonstrate the power of middle-level workflows using GlySERIAS Immobilized for quality control of fusion proteins containing several flexible linkers.
 

 
Figure 4. Middle-level analysis of BiTE using GlySERIAS Immobilized. The GS linkers of a biosimilar of blinatumomab, a BiTE molecule with specificity towards CD19 and CD3, were digested with GlySERIAS Immobilized overnight at room temperature under non-denaturing conditions. a) Illustration of the sample preparation workflow. The samples were analyzed by reversed-phase LC-MS. b) Chromatographic separation of the digest, total ion chromatogram. c) Deconvoluted mass spectrum of the intact BiTE. d) Deconvoluted mass spectra of the four protein domains separated after GlySERIAS digestion. The glycine and serine residues can be present at both ends of the heavy chains. The processed samples 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).

GlySERIAS Immobilized – Process 5 x 0.2 mg or 10 x 0.2 mg fusion protein.