Articles by Felicia Mejàre

GlyCLICK Ref Blog

Improved antibody-PET tracers for in vivo imaging with GlyCLICK®

 

Radioactively labelled antibodies are excellent immuno-PET tracers for evaluating in vivo distribution and performance of therapuetic agents. Site-specific conjugation at the antibody Fc glycan site by enzymatic remodeling allows for a uniform label distribution of such PET-tracers, compared to conjugates generated with conventional random labelling strategies.

 

In an article by Kristensen et al. (2019), the authors evaluated the stability, immunoreactivity and in vivo biodistribution of the radioactively labelled mAb Trastuzumab (Herceptin). Using GlyCLICK, the antibody was enzymatically modified with GlycINATOR (EndoS2) and conjugated with a SCN-Bn-DFO chelator prior to 89Zr radioactive labelling. Comparing the GlyCLICK technology with ß-galactosidase remodelled conjugates and two random labelling techniques, the authors obtained valuable data on the overall performance of the various PET-tracers.

 

Antibodies subjected to site-specific labelling showed significantly increased in vitro stability and immunoreactivity compared to randomly labeled Trastzumab. Furthermore, using in vivo immuno-PET imaging, these conjugates also displayed superior tumor-targeting properties based on the successful detection of HER2-positive tumors in mouse models. These results highlight the advantages of site-specific antibody conjugation.
 

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Reference:
Kristensen, L. et al., 2019. Site-specifically labeled 89Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake for immuno-PET in a subcutaneous HER2-positive xenograft mouse model. Theranostics, 9(15). pp.4409-4420.

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SmartEnzymes™ in Multiplexed Middle-Down MS for targeted structure analysis

 

 

 In a recent article by Srzentic et al. (2018) the authors present a multiplexed middle-down MS workflow with improved performance for targeted protein structure analysis. Using GingisKHAN for antibody digestion, the authors analysed the F(ab) subunits of a therapeutic mAb. By implementing spectral and transient averaging of mass spectra across several LC-MS experiments, the authors revealed valuable information on chain pairing in the mAb.

 

To make the analysis, the therapeutic mAb trastuzumab was digested above the hinge using the GingisKHAN enzyme to generate intact F(ab) subunits. Intact myoglobin was subjected to analysis in a top-down MS approach to benchmark the workflow. The GingisKHAN-generated F(ab) subunits were then analysed using the middle-down MS workflow to compare the performance of data averaging approaches.

 

The results show the performances of spectral and transient averaging for tandem mass spectra as separate software tools for structural protein analysis. The transient averaging provided the most extensive sequence coverage for the F(ab) subunits, followed by spectral averaging. Furthermore, utilizing the multiplexed middle-down MS workflow for subunit analysis, the authors detected low-abundance branched product ions revealing valuable information about the light and heavy chain connectivity.

 

GingisKHAN® (Kgp enzyme) is a cysteine protease that digests human IgG1 at a specific site above the hinge region. The enzyme generates intact Fc and Fab subunits in 60 minutes.

 
Learn more about GingisKHAN

 
Srzentic et al., 2018. Multiplexed Middle-Down Mass Spectrometry Reveals Light and Heavy Chain Connectivity in a Monoclonal Antibody

Sequence Analysis

Antibody Sequence Analysis using GingisKHAN® and FabRICATOR®

September 28, 2018 | Applications, References |

  In an article by Luca Fornelli & Kristina Srzentic et al. recently published in Analytical Chemistry the authors present a workflow for antibody sequence determination by combining top-down and middle-down LC/MS. The authors analyzed the therapeutic antibody rituximab in its intact and fragmented form, using FabRICATOR and GingisKHAN to generate antibody subunits. By combining the performance of multiple ion activation techniques and a new software tool with top-level and middle-level strategies, the authors achieved extensive sequence coverage and obtained valuable information on key quality attributes.

  Rituximab was fragmented using members of the SmartEnzymes™ family for the generation of various antibody subunits. GingisKHAN was used for generating intact Fc and Fab subunits by site-specific cleavage of IgG1 above the hinge region. In order to obtain antibody subunits Fc/2, Fd and LC the authors used FabRICATOR-digestion followed by reduction. The intact antibody and the antibody subunits were analyzed using reversed phase LC/MS coupled with three separate ion activation techniques, and analyzed using a new software tool for fragment ion deconvolution.

  The complementing features of the ion activation techniques provided high quality information for a low number of LC/MS experiments. The authors achieved sequence coverage equivalent to what is obtainable with bottom-up strategies. In addition, the authors were able to analyze quality attributes such as PTMs, chain pairing and intact antibody mass determination – properties otherwise lost after extended proteolysis. These results highlight the benefits of combining top-level and middle-level strategies for applications currently performed by bottom-level strategies.

GingisKHAN® (Kgp enzyme) is a cysteine protease that digests human IgG1 at a specific site above the hinge region. The enzyme generates intact Fc and Fab subunits in 60 minutes.

Learn more about GingisKHAN

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.

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FabRICATOR® in service for in-depth 2D-LC MS profiling of therapeutic mAbs

 

In an article by Stroll et al. (2018), the authors demonstrate a striking in-depth characterization of three therapeutic mAbs, achieved by combining FabRICATOR® (IdeS) digestion with an online two-dimensional LC-MS approach. The authors generate a highly resolved separation and detection of FabRICATOR-digested N-glycosylated mAb subunits by implementing Active Solvent Modulation (ASM), a method for valve-based effluent dilution between the first and second dimension separations.

Multidimensional Liquid Chromatography constitutes a powerful technology for in-depth profiling of therapeutic proteins, capable of generating rapid and highly resolved separations. The authors demonstrate the advantages of implementing ASM in an online 2D-LC system for deep profiling of antibody glycosylations, subjecting mAbs to FabRICATOR digestion followed by HILIC x RP separation and ESI Mass Spectrometry (ESI-MS) detection.

Three therapeutic antibodies displaying diverse N-glycosylation patterns were submitted to digestion using FabRICATOR for a single site-specific proteolytic cleavage below the hinge, generating Fc/2 and F(ab’)2 fragments. Further reduction of the interchain disulphide bonds of the F(ab’)2 subunit was carried out on the FabRICATOR-digested samples for the additional generation of LC and Fd fragments.

Implementing the ASM method on antibody subunits, the authors achieved a significant increase in detection sensitivity for Fc/2 and Fd fragments, without detectable breakthrough, otherwise associated with larger loading volumes in the second-dimension separation. Furthermore, the authors demonstrated the resolving power of HILIC x RP for analyzing the extent of glycosylations present in heavily glycosylated mAbs, the method showing increased separation and detection for both high and low abundant glycan species, compared to 2D-LC combining CEX and RP separations.

FabRICATOR is a protease with a single digestion site below the hinge of IgG. The enzyme is widely used in middle-level analytical workflows for characterization of antibody based biopharmaceuticals.

 
Learn more about FabRICATOR

 
Stoll, D.R. et al., 2018. Development of Comprehensive Online Two-Dimensional Liquid Chromatography-Mass Spectrometry using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies. Analytical Chemistry, pp.acs.analchem.8b00776–9.