Articles in the Category ”Products”

Characterizing ADCs using FabRICATOR and middle-down MS

September 24, 2019 | Applications, Products |

The process of characterizing an antibody drug conjugate (ADC) requires the evaluation of critical quality attributes including primary sequence analysis and drug conjugation assessment. Addressing glycoprofile determination as well as drug load distribution and drug-to-antibody ratio (DAR) is however challenging using peptide mapping. In addition, further challenges arise from the increased hydrophobicity of the ADC and the risk of drug-linker dissociation in an MS/MS experiment.


In an article by Hernandez-Alba et al. (2019) the authors characterized a site-specific ADC using middle-down MS. They combined three different fragmentation strategies for improved sequence coverage and drug conjugation assessment. The site-specific ADC (DAR=4) was digested using FabRICATOR and reduced to generate homogenous Fc/2, Fd’ and LC fragments for analysis by multiple ion activation techniques. By combining MS/MS data obtained with HDC (hydrodynamic chromatography), ETD (electron-transfer dissociation) and UVPD (ultraviolet photodissociation) fragmentation modes, the scientists obtained valuable information with the advantages of minimal sample preparation and analysis time using middle-down MS. 


The UVPD mode showed better performance compared to ETD and HDC. This indicated that the performance of this activation technique was unaffected by the hydrophobicity of the ADC. The complementarity between UVPD and ETD was further highlighted for drug conjugation assessment by allowing primary sequence validation and accurate identification of drug conjugation and glycosylation sites. These results highlight the potential of middle-down MS as a complement in next-generation strategies for the characterization of  mAb-based compounds including ADCs. 


Read more about FabRICATOR and Applications of FabRICATOR.


Hernandez-Alba, O. et al., 2019. A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate using Middle-Down Mass Spectrometry. American Society for Mass Spectrometry, 30(8). pp. 1-11. 

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 DIBO-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.
For more information on GlyCLICK please visit

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.

Genovis Launches GlycOCATCH™

April 26, 2018 | Applications, Products |

Please welcome GlycOCATCH™ to the Genovis SmartEnzymes™ family!


GlycOCATCH is an enrichment resin for affinity purification of O-glycosylated proteins and peptides. The resin is designed to bind proteins and peptides carrying O-glycans with high affinity, and it is provided in convenient spin columns to allow easy-to-use O-glycoprotein enrichment.


The applications of GlycOCATCH involve glycomics, specific enrichment or removal of O-glycoproteins and peptides, studies of O-glycosylation in complex samples and characterization of biopharmaceuticals.


The GlycOCATCH product is available for purchasing today.


Read more about GlycOCATCH here

A New Assay to Study IgG Galactosylation in Serum

March 21, 2018 | Applications, Products |


In a study by Vanderschaeghe et al. (2018), a new assay to measure IgG galactosylation in serum has been developed. The setup includes hydrolysis of IgG Fc glycans using the IgG-specific endoglycosidase IgGZERO® (EndoS).


A reduced level of IgG galactosylation in serum is a promising biomarker to evaluate the severity, determine the treatment and assess the efficacy of the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Crohn’s disease.


Traditionally, it has been difficult to study IgG galactosylation in serum because of the requirement to purify the antibodies, a procedure that is both complex and time-consuming. However, Vanderschaeghe et al. demonstrate a new assay where IgGZERO is used to efficiently hydrolyze serum IgG Fc glycans before analyzing galactosylation on high-throughput DNA sequencers. IgGZERO works on natively folded IgG, meaning that the assay can be performed on complex serum without first needing to purify the IgG, a feature that renders the assay both fast and simple.


The authors conclude by describing their new IgGZERO-based assay:

“… an important breakthrough towards the clinical implementation of the proposed biomarker” (Vanderschaeghe et al. 2018).


Read more about IgGZERO


Find the full text of the paper here:

Vanderschaeghe et al., 2018. Clinical assay for direct assessment of IgG galactosylation in serum using endoglycosidase S. BioRxiv.


Unique Enzymatic Digestions Allow Study of Antibody Disulphides

December 12, 2017 | Products, References |


Valegh Faid and colleagues at LFB Biotechnologies in France have developed and published an assay to study antibody disulphide bonds using middle-up LC-MS (Faid et al. 2017). In the paper, the combination of FabRICATOR® for digestion below the hinge and FabALACTICA™ for digestion above the hinge, generated three fragments from a human IgG1 antibody; the hinge peptide, the Fab and the Fc/2 fragments. These fragments were separated using RP-HPLC on a diphenyl column and analyzed using mass spectrometry. This setup enabled analysis of both inter- and intrachain disulphide bonds as well as other quality attributes.


The disulphide bonds of a therapeutic antibody can serve as indicators for misfolded antibodies during antibody manufacturing and has also been directly linked to mAb stability and are considered a quality parameter during antibody manufacturing. Traditionally, the methods for investigating the disulphide bonds of antibodies include labelling free cysteine residues using Ellman’s reagent or fluorescent dyes, resulting in a measure of the overall free sulfhydryls. The new assay using FabALACTICA and FabRICATOR is based on the unique digestion sites of the enzymes and utilizes the fact that both enzymes works under non-reducing conditions. The authors conclude:


“This method is easy to use, generic for mAbs and presents the advantage to deal with the whole complexity of a single mAb molecule by generating three well separated smaller and less heterogeneous fragments. Besides a direct application to the detection of intra- and inter-chain free sulfhydryls, this combined digestion could be promising to investigate other mAbs quality attributes“ (Faid et al. 2017).

Try the assay! Order 2000 units of FabRICATOR and 2000 units of FabALACTICA and get 15% off using the following product number: A0-FSS-040. This offer is valid until January 31, 2018.
Order online here.


Read more about FabALACTICA and FabRICATOR.


Faid, V. et al., 2017. Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls. Journal of Pharmaceutical and Biomedical Analysis, 149, pp.541–546.


GingisKHAN Used in Antibody Lead Identification – Publication by Roche

September 11, 2017 | Products, References |


“GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies.”


In this paper, the scientists at Roche Innovation Center Munich, Moelleken et al, discuss the use of GingisKHAN in antibody lead selection screening and affinity measurements. The binding strength of an antibody is called affinity and when developing an antibody based drug, the selection of the best lead candidate involves measuring the affinity to its target antigen. A traditional antibody has two binding regions and the characteristics of the binding involves either one or two binding sites, depending on the target molecule. For this reason, the binding of a single Fab fragment is important to study during lead selection. Traditional methods of generating Fab fragments includes unspecific digestion with enzymes such as papain or LysC, mild reduction, or recombinant expression of the Fab fragment. Methods involving unspecific proteases suffer from low reproducibility, unspecific and incomplete digestion, and requires optimization for each IgG molecule to obtain a homogenous pool of Fab fragments. Another drawback is that none of the traditional methods allow high-throughput generation of Fabs.


In this setting Moelleken et al turned to GingisKHAN and studied the digestion efficiency, specificity, and ease of use for antibodies, bispecific antibodies, and new molecules with more than two antigen binding domains. The conclusions from the paper indicate that GingisKHAN has a high degree of specificity above the hinge of human IgG1 with no overdigestion or incomplete digestion. The protocol using GingisKHAN can be used as a platform method since there is very limited optimization needed. Due to the specificity of GingisKHAN, Moelleken et al were able to study the binding of Fab fragments directly from the digestion mixture without the need for purification. This feature shortens the analysis time and allows higher throughput.


The authors conclude the following:

“In summary, we have shown that the GingisKHAN protease is highly suited to differentiate between affinity- and avidity-driven binding of human IgG1s monoclonal antibodies and bispecific antibody formats in a lean and efficient manner”. (Moelleken et al, 2017)


GingisKHAN is an enzyme from Genovis for specific digestion above the hinge of human IgG1 and generation of homogenous Fab and Fc fragments.


Read more about GingisKHAN

  • Specific digestion of human IgG1
  • 1 h incubation at 37°C
  • Available in 2000 units or as a Fab preparation kit



Moelleken, J. et al., 2017. GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies. mAbs, pp.1–12.

Generate and purify Fab fragments from human and mouse IgG using Genovis SmartEnzymes

Antibodies are important tools in several scientific research areas, and sometimes it is necessary to cleave antibodies to generate antigen binding (Fab) fragments. The Fab fragments can be used within e.g. imaging, removal of effector functions, infection biology, binding studies, and mass spectrometry.

In a recently published book (“Bacterial Pathogenesis, Methods and Protocols”, Humana Press, 2017), the Genovis Team has written a chapter of how to generate and purify Fab fragments from human and mouse IgG by using the bacterial proteases IdeS (FabRICATOR), SpeB (FabULOUS) and Kgp (GingisKHAN).


  • The FabRICATOR enzyme is derived from Streptococcus pyogenes, and generates F(ab’)2 fragments in all human IgG subclasses. A homogenous pool of Fab’ fragments can be generated under mild reducing conditions. Read more about FabRICATOR here.
  • The FabULOUS enzyme is also derived from Streptococcus pyogenes, and can be used to digest mouse IgG. Using light chain affinity resins, Fab fragments can be purified. Read more about FabULOUS here.
  • The GingisKHAN enzyme is derived from Porphyromonas gingivalis, digests the upper hinge and generates intact Fab fragments from human IgG1 in one step. Using a CH1-specific affinity resin, the Fab fragments can be purified. Read more about GingisKHAN here.

Pathogenesis Book
Find the full text paper here:
Sjögren, J. et al., 2017. Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp. Methods in Molecular Biology, 1535, pp.319–329.

FabULOUS Fab Kit – Intact Fab fragments from mouse IgG

June 7, 2016 | Products |


The FabULOUS (SpeB) enzyme digests IgG in the hinge region. The FabULOUS Fab kit is designed for digestion and purification of Fab fragments from mouse IgG antibodies. Incubation at optimized reducing conditions generates intact Fab, easily purified with spin columns.

  • Digestion in the hinge with FabULOUS
  • Affinity purification of Fab fragments using the light chain
  • Preparation of Fab fragments from mouse IgG in less than 2 h

Read more about FabULOUS Fab kit and order now

GingisREX – New Enzyme from Genovis


Genovis expands the protease portfolio with GingisREX – a new enzyme with unique specificity for arginine residues. The GingisREX protease can be used for peptide mapping, protein sequencing and post-translational modification analysis using mass spectrometry.

Press release

Read more about GingisREX

Now Available – FragIT™Z & FragIT™Z Kit

May 28, 2015 | Products |

We like to introduce FragIT Z – the immobilized version of FabRICATOR Z. Digest your mouse IgG2a more rapidly without having to remove the enzyme from the final preparation.

For separation of the generated antibody domains we also offer the convenient FragIT Z Kit which enables rapid isolation of F(ab’)2 and Fc.

Read more about these products here