Articles in the Category ”Applications”

mAb Deamidation Study using FabRICATOR® Digestion and HIC Separation


Hydrophobic interaction chromatography (HIC) is often used in characterization of therapeutic antibody products due to its ability to separate direct or indirect structural changes in the studied protein. Scientists at Alexion have published a study where FabRICATOR (IdeS) was used to generate Fc and F(ab’)2 fragments of an antibody to study conformational changes of a monoclonal antibody (King et al. 2018).


Separation of the intact antibody on HIC reveled two major peaks that were collected and subjected to FabRICATOR digestion. After digestion, the Fc and F(ab’)2 fragments separated well, and the heterogeneity was localized to the F(ab’)2 domain. Variations in the Fc were observed and attributed to oxidation modifications. Peptide mapping of the domains were carried out and a 1 Da difference was localized , indicating deamidation of Asn to either Asp or isoAsp in the complementarity-determining region (CDR) of the light chain. The observed difference in HIC separation pattern was also linked to changes in antigen binding, since the deamidation of the Asn residues reduced the binding of the antibody to its target antigen.


Taken together, this paper indicates that a single deamidation in the light chain changed the hydrophobicity profile of the antibody and impacted the antigen binding. The use of FabRICATOR (IdeS) digestion and HIC separation could serve as a quick screening assay to study deamidation changes in the F(ab’)2 domain.



King, C. et al., 2018. Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis. Journal of Chromatography B, 1085, pp.96–103.


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.


Antibody Glycation Study using Intact LC-MS


A new study from Janssen by Mo et al, demonstrates the use of intact mass spectrometry to determine the levels of glycation on therapeutic antibodies. To perform the assay, the authors used IgGZERO for rapid removal of the Fc-glycans.


Glycation occurs when reducing sugars such as glucose, galactose or fructose, reacts with protein amino acids through the Maillard reaction, and results in attachment of sugars to the protein. For therapeutic antibodies, glycation not only increases the heterogeneity of the drug but may also affect safety and efficacy.


To study the level of glycation on antibodies, the authors used both intact mass of the reduced antibody and peptide mapping to find the +162 Da mass shift indicating an addition of a hexose sugar. The Fc-glycan of an antibody contain 0, 1 or 2 galactose sugars that also gives a mass shift of 162 Da. To specifically remove the Fc-glycans, the scientist used IgGZERO (EndoS) from Genovis. Using this enzymatic pretreatment, the authors could determine glycation levels using intact mass spectrometry.


The authors found the peptide mapping and the intact LC-MS to give correlating results but conclude: “intact LC- MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing”(Mo et al. 2018).



Find the full text of the paper here:

Mo, J. et al., 2018. Quantitative analysis of glycation and its impact on antigen binding. mAbs, 154, pp.1–10.

ADC Subunit Characterization of Drug Load and Glycosylation using HILIC-MS


In a collaboration headed by Davy Guillarme at University of Geneva, scientists have explored the characterization of subunits derived from antibody drug conjugates (ADCs) using hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (D’Atri et al. 2018).
The scientists used brentuximab vedotin (BV, Adcetris®), an approved ADC for treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). The BV consists of an antibody directed towards CD30, coupled to the vedotin toxin using cysteine conjugation chemistry. The random cysteine conjugation method results in a heterogeneous attachment of the drug, with differences in efficacy depending on the drug load. For this reason, the amount of conjugated toxins requires careful characterization. A key quality attribute of both antibodies and ADCs is the glycosylation profile, that may affect the stability, efficacy and safety. In this paper, a method to study ADC drug load and glycan profiling in a single experiment was demonstrated.


The intact ADC is around 150 kDa, which makes it very complicated to study details with high resolution. For this reason, D’Atri and colleagues used FabRICATOR digestion and reduction to generate specific antibody subunits of around 25 kDa, with increased resolution in both separation and mass determination. New wide-pore HILIC phase has enabled separation of larger molecules such as antibody subunits, and the team has already published a glycoprofiling strategy using HILIC on naked antibodies (Periat et al. 2016).


The coupling of HILIC separation to MS of subunits resulted in more detailed characterization of the subunits as compared to reverse phase separation (RP-HPLC). The relative percentage of each subunit aligned well with both methods of separation. However, additional positional isomers of the Fd’ fragment were observed using HILIC separation. Also, the glycoforms of the Fc/2 fragments were chromatographically separated, making mass deconvolution and determination easier. The authors conclude the middle-up HILIC-MS method to be orthogonal to RP-MS with the benefit that the methodology allows simultaneous characterization of drug load and glycosylation of the antibody drug conjugate.


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.



D’Atri, V. et al., 2018. Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry. Journal of Chromatography B, 1080, pp.37–41.

Periat, A. et al., 2016. Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals. Journal of chromatography. A, 1448, pp.81–92.

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.


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.




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



[New Reference] Using HILIC to study antibody subunits

Researchers at the University of Geneva led by Davy Guillarme has used a novel form of HPLC column, based on wide-pore hybrid silica bonded with amide ligand, and developed an HILIC analytical workflow for characterization of biopharmaceuticals. The scientists used FabRICATOR to fragment trastuzumab to subunits and studied the fragments using the new column with HILIC separation. The results showed that the method effectively separated important glycoforms of the Fc/2 fragment. The developed method is compatible with mass spectrometry, does not need high mobile phase temperatures, and allows serial coupling of columns.

Read more on this approach in Journal of Chromatography

More on the FabRICATOR enzyme

Real-time Monitoring of Antibody Quality Control using FabRICATOR

September 23, 2015 | Applications, References |

The production of antibody-based therapeutics requires careful monitoring of quality attributes. Researchers at Amgen developed a technology to model and monitor the high-mannose content of the antibody in real-time using FabRICATOR digestion (Zupke et al. 2015). Read more »

Study of Antibody Quality Attributes on Cell Culture Supernatant

September 23, 2015 | Applications, References |

The production of antibody-based therapeutics requires careful monitoring of quality attributes. Researchers at Institute Pasteur in collaboration with LFB Biotechnologies have used FabRICATOR to monitor important quality attributes of a therapeutic antibody, directly on the cell culture supernatant (Henninot et al. 2015).
Read more »