Articles in the Category ”Applications”

GlyCLICK® and Middle-up LC-MS Enables Robust ADC Development

Scientists at the University of Geneva and CNRS present site-specific ADCs generated using the GlyCLICK technology and an analytical middle-up LC-HRMS workflow as a potential core module for ADC development.


Antibody-drug conjugates (ADCs) are efficient therapeutic agents that possess the cell-targeting properties of monoclonal antibodies combined with the potency of cytotoxic drugs. Early generation ADCs were predominantly obtained through non-selective conjugation methods by incorporation of a drug payload at randomly distributed sites. Such methods result in highly heterogenous subpopulations of varying antibody-drug ratio (DAR) leading to potential loss of efficacy and impaired pharmacokinetics. While alternative strategies exploring genetic engineering have emerged for conjugation at non-natural amino acids, challenges related to both production and analytical characterization persist.


Glycan-mediated bioconjugation using the GlyCLICK technology is an attractive option to overcome the challenges of conventional bioconjugation without the need for genetic engineering to produce custom ADCs. By utilizing a unique combination of enzymes, the conserved Fc-glycans are remodeled and site-specifically conjugated using click chemistry for ADCs carrying two payloads per antibody (DAR=2.0) having controlled drug stoichiometry and preserved immunoreactivity. In this paper, Duivelshof et al. developed a site-specific ADC by coupling trastuzumab to DM1 using the GlyCLICK technology and evaluated the quality of the conjugation process using complementary reversed phase (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS).


The trastuzumab antibody was site-specifically conjugated to DBCO-functionalized DM1 (DBCO-PEG4-Ahx-DM1) using the GlyCLICK technology. To reduce sample complexity, the antibodies were digested with FabRICATOR® (Ides) or FabALACTICA® (IgdE) and reduced for comparison of native and GlyCLICK conjugated trastuzumab at the subunit level. The complementary HILIC and RPLC workflow allowed the authors to observe the significant shift in retention between the lipophilic drug payloads on the ADC and the hydrophilic N-glycans on native trastuzumab. These results enabled the scientists to confirm site-specific conjugation at the Fc-glycans sites, while hyphenation to HRMS detection allowed accurate determination of a DAR of 2.0 for GlyCLICK conjugated trastuzumab, which was not possible at the intact ADC level.


Duivelshof et al., 2020. Glycan-mediated technology for obtaining homogenous site-specific conjugated antibody-drug conjugates: synthesis and analytical characterization by using complementary middle-up LC/HRMS analysis. Analytical Chemistry. doi: 10.1021/acs.analchem.0c00282


FabRICATOR® in complete characterization of seven therapeutic monoclonal antibodies

Monoclonal antibodies (mAb) are used in various treatments and new potential targets continue to arise. To ensure a high specificity of the antibody to the intended antigen each mAb needs to be well characterized since they are often heterogeneous. In this paper, Giorgetti et al. combined bottom-up, middle-level and intact level analysis to get the complete structure of seven mAbs with worldwide approval. For the middle-level analysis, FabRICATOR digestion followed by a reduction step was the preferred approach.


On-line capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) compatible with all three levels of analysis was used for the characterization. The overall heterogeneity and high mass post-translational modifications (PTMs) were determined at the intact level. Confirming the results, the middle-level approach expanded on this with more advanced PTM data and information about the backbone structure. Finally, bottom-up analysis provided the precise location of the PTMs and the relative quantity of micro-heterogeneities in the proteoforms. However, while bottom-up data provided the most detailed information it required the most sample preparation which might lead to artifacts. The intact and middle-level analysis avoided this problem. Therefore, the research group suggested that a combination of the three levels efficiently analyzed with CE-ESI-MS was ideal for a representative, complete characterization of the structure of the mAbs.


For middle-level analysis the mAbs were digested with FabRICATOR yielding F(ab)’2 and Fc/2 fragments of about 100 kDa and 25 kDa. This allowed PTMs such as methionine oxidation and lysine clipping to be observed. The N-glycan profile was also determined. Reduction of the F(ab)’2 fragment to light chain (LC) and Fd fragments further reduced complexity to make small PTMs such as asparagine deamidation detectable. In addition, the speed and efficiency along with the high accuracy was appreciated by the researchers.



Giorgetti et al., 2020. Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry.  Journal of Pharmaceutical and Biomedical Analysis. 2020, 182, 113107.

IgGZERO® used to determine the core fucosylation of antibodies in bioprocess

Scientists at NIBRT in Dublin together with scientists at the University of Manitoba, Winnipeg, have investigated the effect of two different methods to control the level of fucosylation of a model antibody during expression in CHO cells.


Production of biopharmaceuticals in mammalian cells requires that critical quality attributes are controlled for safety and therapeutic efficacy. The efficacy of an IgG antibody for cancer immunotherapy is dependent on its ability to elicit effector functions such as antibody-dependent cell cytotoxcicity (ADCC). The absence of fucose on the core GlcNAc of the Fc glycan in the antibody increases the ADCC and hence there is a desire to control the level of fucosylation during the manufacturing process.


The model antibody investigated in this work was a camelid heavy-chain human Fc fusion of about 80 kDa in size. During expression of the antibody in CHO cells, two factors were were evaluated: the effect of adding a fucosyltransferase inhibitor and the impact of overexpression of a gene that deflects the fucose de novo pathway to a dead-end. The antibodies were harvested from the cell culture supernatant by a protein A column. The N-glycans of the antibodies were then released using PNGaseF, labelled with 2-AB and analyzed by HILIC-HPLC. The fucosylation pattern of the antibodies was identified by electrospray ionization mass spectroscopy (ESI-MS) of the intact control antibody after treatment with IgGZERO (EndoS). Hydrolysis with IgGZERO results in three possible antibody variants with two, one or no fucose per antibody. Since IgGZERO specifically removes the Fc glycans leaving the core GlcNAc (+/- fucose), the observed shift in mass of -146 Da and -292 Da revealed antibody species where one or two fucoses were missing. Using this approach, it was shown that the inhibitor for fucosyltransferase decreased the addition of fucose on the inner GlcNAc during the expression of the antibody in a concentration dependent manner.


By combining data from the released glycan analysis with the mass data of intact antibody after IgGZERO treatment, crucial information about the glycan profile and fucosylation pattern was revealed and evaluated to support the bioprocess design.



IgGZERO® Turns a Toxic Antibody into a Novel Treatment for Sepsis

April 16, 2020 | Applications, References |

Genovis SmartEnzyme IgGZERO was used in this recent study by researches from the University of Pennsylvania and the Philadelphia Children’s Hospital.


Sepsis is a dysregulated immune response to an infection that leads to very high levels of inflammation resulting in tissue damage and potential for multiorgan failure. It therefore leads to a high rate of mortality and morbidity. Neutrophil extracellular traps are part of the innate immune systems defence against infections where neutrophils rupture and release DNA, histones and many antimicrobial proteins. This however comes at a cost as NETs are degraded by circulating DNase and toxic degradation products are formed.


In a recent study published in Blood, researchers from the University of Pennsylvania and the Philadelphia Childrens Hosptial used a monoclonal antibody that binds to NETs to stabilize the traps, therefore significantly reducing the collateral tissue damage induced by NET degradation products. However, the mAb also activated the complement system and platelets, therefore negating the positive effect of NET stabilization. Using IgGZERO, the authors were able to remove the Fc glycosylation from their mAb, thereby impairing its ability to elicit an immune response. The deglycosylated mAb was still able to stabilize NETs and its administration lead to a significantly improved outcome in a murine sepsis model.


Fc glycosylation is a major determinant for which effector functions a monoclonal antibody is able to elicit and therefore its mode of action. Genovis IgG-specific endoglycosidases (GlycINATOR and IgGZERO) provide an easy way to remove the Fc glycans from mAbs.


Gollomp et al, 2020. Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis. Blood, 135(10), 743–754. doi:10.1182/blood.2019002329 


Investigating IgG Delivery Across the Blood-Brain Barrier with GlycINATOR®

Scientists from the University of Delaware demonstrate the use of GlycINATOR for studying transcytosis of IgG in an in vitro model of the blood-brain barrier.

Brain endothelial cells (BECs) are important structural components of the blood-brain barrier with a unique physiology that restricts permeability of blood-borne molecules such as therapeutic antibodies to the brain. The neonatal fragment crystalline receptor (FcRn) is known to mediate IgG recycling and transcytosis in peripheral epithelium, but the role of FcRn in transcytosis of antibodies in BECs remains uncertain.

In this paper, Ruano-Salguero and Lee study the role of FcRn in transcytosis of IgG across the blood-brain barrier in BEC-like cells (iBECs) derived from induced human pluripotent stem cells. Using microscopy-based methods, different antibody species and subunits were compared to investigate the role of FcRn on transcytosis of IgG. To specifically determine the impact of Fc-glycosylation on permeability, all glycoforms on human IgG1 was removed using the GlycINATOR enzyme and the deglycosylated antibodies analyzed in iBECs using live-cell microscopy. Finally, the authors also investigated the impact of biophysical properties such as charge and size on transcytosis mechanisms.

Using the in vitro blood-brain barrier model, the scientists found that FcRn mediates both recycling and reduced lysosomal accumulation of IgG in iBECs. Transcytosis of antibodies across the in vitro blood-brain barrier exhibited non receptor-medicated mechanisms that were unaffected by human FcRn-binding motifs and Fc-glycoforms as demonstrated by the different species and deglycosylated human IgG1. Investigations of intracellular trafficking by FcRn binding or other IgG-specific mechanisms were further observed to be non-saturable, indicating fluid-phase permeability. Interestingly, the authors found that biophysical changes enhanced permeability of molecules with positively charged isoelectric points. These results highlight the potential for use of in vitro models as well as characterization and modification of biophysical properties to improve therapeutic delivery to the brain.

Deglycosylation of IgG using the GlycINATOR enzyme decreases binding to Fc-receptors (FcRs) enabling bifunctional assays to study glycan-mediated interactions such as ADCC activity. The binding to FcRn is however preserved with GlycINATOR, allowing recycling and increased circulation in vivo of deglycosylated IgG and GlyCLICK conjugated ADCs.


















Ruano-Salguero and Lee, 2020. Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn. Sci Rep 10, 3685.


SmartEnzymes™ in Quality Control of Commercial Antibodies

In a recent paper, Sokolowska and colleagues at Janssen Research and Development qualified and covalidated a subunit LC-MS method for quality control and stability testing of the oxidation status of commercial antibodies.


LC-MS is commonly used for therapeutic antibody development and characterization within the biopharmaceutical industry due to the inherent strengths to provide site-specific identification and quantitation of post-translational modifications. However, the implementation of LC-MS methods to commercial QC labs is challenging, since there are not many options for fully GMP compliant systems. In addition, the methods often require extensive MS expertise and suffer from time-consuming sample preparation and lack of robustness. To counter these obstacles, Sokolowska et al. have developed an LC-MS method that requires minimum analyst training. It uses validated GMP compliant software and is based on subunit analysis, which is proved to be faster and more robust compared to peptide mapping.


The assay uses FabRICATOR® (IdeS) and  IgGZERO® (EndoS) enzymes to generate deglycosylated IgG subunits suitable for MS analysis. FabRICATOR digests the antibody below the hinge and IgGZERO hydrolyzes the Fc N-glycans. The subunits are analyzed using reversed phase-ultraperformance liquid chromatography coupled to a quadrupole time-of-flight (RP-UPLC-QTOF) MS to monitor antibody oxidation for stability testing and commercial product release.


The developed subunit LC-MS assay was covalidated in three laboratories and showed comparable performance. The robustness was tested by varying both the LC-MS settings and the sample preparation. The enzymatic conditions included variations in protein concentration, enzyme lots, enzyme-to-protein ratio, digestion time and temperature, reduction time and temperature, and reagent concentrations. Minor variations in sample preparation all led to measured Fc oxidation within the method variation +/- 0.9%.


Figure 1. mAb subunit oxidation assay using FabRICATOR and IgGZERO (Sokolowska et al., 2020.)

The approval of this method opens the door for implementing other subunit LC-MS and multiattribute methods in QC laboratories to modernize commercial QC and stability testing.


Learn more by reading the full paper, follow the link below.



















SmartEnzymes™ in a new approach to characterize ADCs

October 25, 2019 | Applications, References |

Antibody drug conjugates (ADCs) consist of monoclonal antibodies chemically linked to a cytotoxic agent. The target specificity of the monoclonal antibody in combination with the potency of the cytotoxic drug make ADCs promising therapeutic agents. However, the molecules are often complex, making evaluation of the quality attributes for the ADC challenging.


In order to characterize the ADCs, the predominant analysis of choice is peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry. However, the sample preparation steps in a bottom-up approach are often time-consuming and a comprehensive view of ADCs with different sequence variants and post-translational modifications is lacking.


In this recently published article by Chen et al., a middle-down RPLC-MS strategy with electron transfer disscociation (ETD) was developed to analyze lysine and cysteine conjugated ADCs at the subunit level. FabRICATOR® (IdeS) and GingisKHAN® (KGB) were used to generate the subunits. FabRICATOR digests below the hinge, generating F(ab’)2 and Fc/2 fragments, and GingisKHAN digests above the hinge, generating intact Fab and Fc fragments. For the deglycosylation, the IgG-specific endoglycosidase GlycINATOR® (EndoS2) was used.


This middle-down approach enabled high-resolution evaluation of several ADC quality attributes at the subunit level, including drug to antibody ratio (DAR), conjugation sites and micro-variants. The approach shows great potential for investigating quality attributes during the development and characterization of novel ADCs.


Read more about FabRICATOR, GingisKHAN and GlycINATOR.


Chen, B et al., 2019. Middle-Down Multi-Attribute Analysis of Antibody-Drug Conjugates with Electron Transfer Dissociation. Anal. Chem. 91(18). 11661-11669.

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.

Free Thiols using FabRICATOR® and FabALACTICA®

In biopharmaceutical product development and manufacturing, free thiol content is one of the product quality attributes of interest as its presence could impact structure, stability and function of the product.

At Biogen, Yi Pu et al have optimized a label-free LC (UV) / MS method for free thiol quantification at a subunit level of IgG1 and IgG4. The new method, which is based on a method developed by Faid et al*, was compared to two conventional approaches, Ellman’s assay and peptide mapping.

It is very challenging to identify free thiol forms by mass spectrometry at the intact antibody level. By combining the highly specific proteolytic enzymes FabALACTICA (IgdE) and FabRICATOR (IdeS) the authors generated the subunits Fab, hinge and Fc/2, suited for confident mass determination. The subunits were subsequently separated on a polyphenyl reversed phase column in order to separate free thiol forms from their corresponding disulphide bond-linked form. A baseline or near baseline separation was obtained making it possible to calculate the free thiol content on each subunit.

The result of the quantification of free thiols from all three methods were comparable and showed similar trends even though the peptide mapping approach generally gave a higher free thiol content.

The authors conclude that compared to Ellman’s assay, the subunit approach is more sensitive, requires less sample and provides domain-specific information of the free thiol content. Compared to peptide mapping, the subunit method is faster, less labour intensive and lacks dependence on labelling efficiency. Finally, it demonstrated promise in the quantification of free thiols in a high throughput manner with domain specific information available.

The developed method has successfully been applied to several in-house IgG1 mAbs with different hydrophobicity and isoelectric points.


*V. Faid Y. Leblanc N. Bihoreau G. Chevreux Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls, J. Pharm. Biomed. Anal. 149 (2018) 541-546,


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