Articles in the Category ”References”

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

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.

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.

OpeRATOR™ Decodes O-glycans; Publication by FDA and Genovis

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Scientist at the Center for Biologics Evaluation and Research, Food and Drug Administration, have, in collaboration with Genovis, developed a method for analyzing O-glycosylated proteins based on a solid phase chemical modification and followed by OpeRATOR digestion. Using this method, up to 8-fold more O-glycosites were discovered as compared to previously reported data.


The method uses an on-bead system to capture tryptic peptides deglycosylated using PNGaseF from a glycoprotein mixture. First, the tryptic peptides are bound via the N-terminus to the beads, and subsequent modifications to the sugars can be carried out. Secondly, the OpeRATOR enzyme is applied to digest the peptide bond, N-terminal of the O-glycosylated serine or threonine. In this way, only O-glycosylated peptides will be cleaved off and enriched. The OpeRATOR digested peptides were then analyzed using LC-MS/MS.


OpeRATOR was launched at the American Society for Mass Spectrometry 2017 and the FDA team quickly became interested in this novel tool. The enzyme originates from Akkermansia muciniphila and has been engineered by Genovis for biotech applications and analytical workflows and denoted OpeRATOR. The enzyme binds to musin type O-glycans and cuts the protein backbone, N-terminally of the O-glycosylated site. OpeRATOR can be used to study site occupancy and composition of O-glycans on biopharmaceuticals and for O-glycomic workflows.


We establish the method on standard glycoproteins, confirming known O- glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity” (Shuang et al 2018)


The paper has been selected Editor’s choice in Analytical Chemistry and is available using the link below:


Shuang Yang et al., “Deciphering Protein O‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment,” Analytical Chemistry, June 3, 2018, 1–9, doi:10.1021/acs.analchem.8b01834.


More information on OpeRATOR and its applications:



Interview with Valegh Faid at LFB Biotechnologies in France


Unique enzymatic digestions in study of antibody disulphides


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). 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, Fab and Fc/2 fragments. These fragments were resolved using RP-HPLC and mass spectrometry and enabled analysis of antibody disulphide bridges and other quality attributes.



Interview with Valegh Faid, Scientist at LFB and first author of the paper:


Why are antibody disulphide bonds important?


Disulphide bonds are highly important because of their critical role in the stabilization of protein conformations. Breaking and/or scrambling of disulphide bond occur during manufacturing and storage of biotherapeutics which is a concern in terms of safety and efficacy. The monitoring of these product-derived impurities is mandatory during development operations in order to minimize these forms.


How did you come up with the idea to combine FabRICATOR (IdeS) and FabALACTICA (IgdE)?


We have been using IdeS for many years in order to cleave IgG’s below the hinge; following DTT reduction, more amenable fragments for RP-HPLC/MS analysis are generated as previously published by our laboratory (Chevreux et al., 2011). This middle-up analysis is fast and very informative regarding the protein sequence integrity and post-translational modifications. However, investigating the oxidative state of disulfide bridges is tricky and often involved a time-consuming peptide mapping in non-reducing conditions.

In this context, IgdE is an interesting enzyme that cleaves specifically IgGs above the hinge and without requiring reducing conditions as papain do. The combination of IdeS and IgdE in non-reducing conditions presents the advantage to generate specifically three fragments i.e. hinge, Fc/2 and Fab that are both easily separated by RP-HPLC and analysed by MS.


How does the new enzymatic assay compare to previous methods to study antibody disulphide bonds?


Peptide mapping in non-reducing condition is the gold standard to investigate disulphide bonding of biotherapeutics. However, data interpretation is time consuming even if dedicated software to improve the treatment of data has largely improved. Although being slightly less informative than peptide mapping, this combined IdeS/IgdE middle-up approach increases the throughput for the investigation of free thiols and disulphide scrambling. Considering that other CQAs can also be monitored in the same experiment, it should be more applicable to routine use in process optimization, formulation screening and stability studies.


Would the assay be used in a QC setting relying solely on liquid chromatography separation?


The analytical workflow is robust and requires mere handlings of the antibody samples. Once the identification of each peak of the chromatogram is confirmed by MS, quantitation based on the UV detection is a current practice. Such analytical configuration involving an HPLC and a UV detection is actually common in most of QC labs and thus easily and robustly implementable.


How are you implementing this assay at LFB Biotechnologies?


This assay is integrated in our portfolio of analytical approaches for the analysis of mAbs currently in development, for process optimisation, batch characterization and stability studies.



Read more about FabALACTICA and FabRICATOR.




Chevreux, G. et al., 2011. Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry. Anal Biochem. 15;415(2): pp. 212-4.


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.



Study on Glycoform Heterogeneity using Enzymatic Digestion and Native Mass Spectrometry

In a study by Wohlschlager et al. (2018), FragIT™ kit was used to digest the Fc-fusion protein etanercept, and the resulting fragments were analyzed using high-resolution native mass spectrometry (MS). Native MS offers a higher spatial resolution at a lower charge state, enabling studies of glycan heterogeneity, and FragIT digestion reduces sample complexity, enabling a detailed annotation of glycoforms on complex compounds.


A detailed knowledge about structure and post-translational modifications (PTMs) is required for biopharmaceuticals to be approved for clinical use, and an important quality attribute that may affect both the efficacy and safety of biopharmaceuticals is glycosylation.


Etanercept is a highly glycosylated Fc-fusion protein that is used to treat autoimmune diseases such as rheumatoid arthritis, and it consists of the TNF-? receptor domain fused to the Fc domain of human IgG1. FragIT – an immobilized version of the FabRICATOR® (IdeS) enzyme – digests IgG from several species and subclasses at a specific site below the hinge region. The resulting fragments are easily purified using the Fc-specific affinity resin that, together with FragIT, comprises the FragIT kit.


In this study, the researchers analyzed the glycosylation of etanercept on both the intact level, and on the middle-down level after FragIT digestion. By combining native MS analysis with enzymatic remodelling of etanercept, a detailed annotation of glycoforms could be achieved and transferred from subunit to whole protein level.


The authors end by concluding:

Comprehensive information on glycoform heterogeneity, fast analysis with minimal sample preparation and product-characteristic fingerprints render our method highly attractive for the quality control of biologics as well as for comparability studies following changes in the manufacturing process.”(Wohlschalger et al. 2018).


Read more about FragIT and FabRICATOR




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.


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.

Subunit Comparability Analysis of Etanercept and Biosimilar

February 16, 2018 | References |

FabRICATOR+Enbrel-rakResearchers at the Free University of Berlin have performed a comparability study of the Fc-fusion protein etanercept and a biosimilar using FabRICATOR® and subunit analysis. The etanercept molecule consists of an IgG1 Fc domain fused to a tumor necrosis factor alpha receptor (TNFaR) and is used for autoimmune diseases such as rheumatoid arthritis. The originator etanercept (Enbrel®) was compared to its biosimilar Altebrel™ (AryoGen Pharmed), that has been launched in Iran.

The scientists used FabRICATOR to digest the Fc-fusion protein and studied the subunits, TNFaR and Fc/2 separately using middle-up mass spectrometry. Interestingly, differences in the glycosylation pattern,  the level of C-terminal lysine clipping and oxidation status of the two biopharmaceuticals were observed. The c-terminal lysine clipping was only observed in the originator molecule whereas the biosimilar showed no lysine clipping. Looking at the Fc/2 glycosylation profile using middle-up is a rapid way of determining the glycan content and the relative abundance of the species. In this case, the pattern was similar although the peak intensities differed, indicating a variation between the originator and the biosimilar.

Taken together, this paper highlights the use of FabRICATOR for comparability assessment of Fc-fusion proteins and shows that the middle-level approach can be used for fingerprinting of originator and biosimilar biopharmaceuticals.

Find the article using this link:

Montacir, O. et al., 2018. Physicochemical Characterization, Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept. The protein journal, 8(6), pp.1136–16.