Consortium for Top Down Proteomics uses FabRICATOR and GingisKHAN

October 13, 2017 | Applications |


The Monoclonal Antibody Project initiated by The Consortium for Top Down Proteomics (CTDP) aims to assess top-down mass spectrometry approaches for antibody characterization. It is a multicentre study, where the participants use several workflows (for example top-down sequencing, middle-down sequencing and intact mass analysis) to investigate the detailed structure and modifications of three monoclonal antibodies. Both intact antibodies and antibody subunits are thoroughly investigated in the study, and Genovis FabRICATOR and GingisKHAN enzymes are used to produce the subunits. FabRICATOR digests antibodies into F(ab’)2 and Fc fragments, and GingisKHAN into intact Fab and Fc fragments, and detailed protocols on how to use these enzymes are available from our website (Genovis IgG Proteases) and also from CTDP’s newly launched website (Top Down Proteomics Methods). The project is coordinated by Yury Tsybin from Spectroswiss Sarl, Joe Loo from UCLA and Kristina Srzentic from Northwestern University, and preliminary results are planned to be presented this fall! For updates about the study results, visit Top Down Proteomics.


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.

Middle level analysis of site-specific ADC

May 29, 2017 | References |

In a collaboration between Universite de Strasbourg, Pierre-Fabre, and Catalent Biologics a multilevel mass spectrometry analysis of a site-specific conjugated ADC was recently published in the journal mAbs. As a first step in the analysis, FabRICATOR (IdeS) was used to perform middle-up analysis and confirm the drug to antibody ratio, DAR. The scientists write on p.7: “Middle level analysis, which is now the first line strategy used in most laboratories for mAb and ADC analytical characterization, revealed a highly homogeneous sample in terms of drug load, with mainly DAR4 detected and low amounts of DAR3” (Botzanowski et al. 2017).

For intact mass analysis using top-down mass spectrometry, the ADC was deglycosylated using IgGZERO for rapid removal of the glycan heterogeniety on the intact ADC. For this approach the newer and improved GlycINATOR enzyme could have been used. Studies have shown that GlycINATOR (EndoS2) removes all glycans from IgG whereas IgGZERO (EndoS) leaves the high-mannose glycans (Sjögren et al. 2015).



Botzanowski, T. et al., 2017. Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates. mAbs, ePublished ahead of print.

Sjögren, J. et al., 2015. EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans. Glycobiology, 25(10), pp.1053–1063.

Glycan analysis by LC/MS in regulated environments

April 28, 2017 | References |

A team of scientist from Quality Assistance in collaboration with Alain Beck from Pierre Fabre, have published a detailed article highlighting the analytical strategies for both N- and O-linked glycan analysis of biotherapeutics using LC/MS. The workflows for glycan analysis on antibodies includes FabRICATOR digestion and study of the Fc/2 fragment for identification of the Fc glycoforms. The researchers used widepore HILIC-MS to separate the individual glycoforms of the Fc/2 subunit of adalimumab and obtained the glycoprofile with increased chromatography and MS resolution as compared to intact analysis. When applying the same workflow to cetuximab, site specific glycan profiles of both the Fc and Fd glycosylation sites were obtained.


In the same paper, a workflow combining GlycINATOR (EndoS2) and FabRICATOR (IdeS) was applied to study the level of core afucosylation. This digestions, that can be carried out simultaneously in 30 min, results in a dramatic reduction of the complexity of the Fc/2 subunit. After this simple sample processing the level of GlcNAc, GlcNAc + Fucose, or aglycosylated Fc/2 fragments can be quantified using LC/MS.


FabRICATOR_Logo_Gubbe GlycINATOR_Logo_Gubbe

Find the paper available in open access here:

Largy, E. et al., 2017. Orthogonal liquid chromatography-mass spectrometry methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level. Journal of chromatography. A, 1498, pp.128–146.

Biosimilar Comparability Assesment using FabRICATOR

March 31, 2017 | References |

Biosimilars are gaining in popularity as patents of innovator drugs are expiring. The analytical strategies to characterize and assess the similarity between the innovator product and the biosimilar often involve liquid chromatography and mass spectrometry (LC/MS). In a recent paper from the Freie Universität Berlin, Montacir et al. studied Rituximab and follow-on molecules using FabRICATOR digestion, reduction, and middle-down LC/MS. Using this approach the researchers found differences in the level of c-terminal lysine clipping of the Fc/2 fragment, a sequence error in the Fd fragment (as previously reported by Beck et al 2014), and a pyro-glutamic acid formation in the light chain.

The comparability assessment of biosimilars and innovator drugs using middle-down LC/MS with FabRICATOR digestion have also been published by the FDA a couple of years ago (Wang et al. 2013). Wang et al. argues that the FabRICATOR middle-down approach is very suitable for rapid fingerprinting of complex molecules, due to the high robustness and specificity of the enzyme.



Montacir, O. et al., 2017. Comparability study of Rituximab originator and follow-on biopharmaceutical. Journal of Pharmaceutical and Biomedical Analysis. E-published ahead of print.

Wang, B. et al., 2013. Structural comparison of two anti-CD20 monoclonal antibody drug products using middle-down mass spectrometry. The Analyst, 138(10), pp.3058–3065.


Assessment of Oxidation using FabRICATOR and LC/MS

Oxidation of methionine residues in the Fc region of a therapeutic antibody may affect the binding of the antibody to Protein A and FcRn leading to difficulties in purification or increased clearence in vivo. For the variable regions of the antibody, oxidation may affect antigen binding or lead to increased immunogenicity. For these reasons, the propensity of an IgG molecule to become oxidized is a critical quality attribute to consider early in the selection of therapeutic antibody candidates. The team at Adimab have developed an high-throughput assay based on FabRICATOR digestion and LC/MS analysis to evaluate the oxidation levels of 121 clinical stage antibodies. The antibodies were digested with FabRICATOR for 30 min at 37°C and reduced with DTT to obtain Fc, Fd and LC, prior to analysis on LC/MS, an approach called middle-down. The scientists correlated the observed oxidations with a model of predicted solvent-exposed methionine residues. They authors discovered oxidation at antibodies experimentally that were not predicted in the model,  probably due to inaccurate crystal structures or differences in expression host.


Taken together, the paper by Yang et al demonstrates the robustness of the oxidation assay based on FabRICATOR digestion and subunit analysis. The 121 antibodies analyzed in the paper indicated this method applicable to early clone selection for evaluation of antibody oxidation at the subunit level.




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.

First GingisKHAN Publication

February 27, 2017 | References |

Researchers at LFB Biotechnologies have published the first paper using the Genovis SmartEnzyme GingisKHAN. GingisKHAN digests human IgG1 antibodies in the upper hinge, resulting in intact Fab and Fc fragments. The paper published in Journal of Chromatography describes how charge variants of therapeutic antibodies are characterized in a long term stability study using ion exchange chromatography coupled to native mass spectrometry. FabRICATOR was applied to digest antibodies below the hinge region and generated good separation of the Fc fragments. To specifically investigate any variations in the Fab domain, GingisKHAN was applied to digest the antibodies in the upper hinge and generate Fab fragments of about 50 kDa. The specific digestion site in combination with native MS allowed both oxidations (+16 Da) and deamidations (+1 Da) to be characterized.


Leblanc, Y. et al., 2017. Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: Case study after a long-term storage at +5°C. Journal of Chromatography B, 1048, pp.130–139.


Monitoring mAb afucosylation using GlycINATOR


The level of core fucosylation on therapeutic antibodies have major impact on antibody dependent cell cytotoxicity. To study the core fucosylation, Suli Liu and Li Zang at Biogen have published a method using deglycosylation by GlycINATOR, reduction of the antibody and LC-MS analysis. Using this methodology the scientist could quantify the fucosylation within 1 h, including sample preparation and analysis. Liu and Zang argue that this methodology is particularly well suited for high-troughput analysis cell line development and process development of therapeutic mAbs.


Liu, S. & Zang, L., 2016. Rapid quantitation of monoclonal antibody N-glyco-occupancy and afucosylation using mass spectrometry. Analytical Biochemistry, pp.1–10.



Read more about GlycINATOR



Genovis and Promega sign global license agreement and settle litigation

Genovis has reached a settlement in its patent infringement lawsuit with Promega Corp. and agreed to a royalty bearing, worldwide license to the patents-in-suit.

The settlement ends all ongoing patent-infringement claims between the parties and Promega has agreed to a royalty bearing, worldwide license to the patents-in-suit, thereby ending the lawsuit between the parties. The specific terms of the contract are confidential.

“I am pleased we were able to reach a settlement with a favorable business solution that accommodates Genovis’ long term growth strategy,” says Fredrik Olsson, CEO of Genovis.