Mapping the O-glycoproteome Using Site-Specific Extraction of O-linked Glycopeptides

August 20, 2020 | Applications, References |

The Tn-antigen is characterized by the presence of a single N-Acetylgalactosamine (GalNAc) residue linked to serine or threonine residues on proteins. It is an immature glycoform without extension to form any of the common O-glycan core structures. The Tn antigen is a hallmark of many forms of cancer and rarely found in healthy tissue. Mapping and comparing Tn-antigens on proteins is therefore of great interest but current methods are laborious and inefficient. 

 

 

Scientists at Johns Hopkins School of Medicine have developed an ingenious new approach for mapping Tn-antigens from a number of complex samples (Yang et al 2020a). It is based on a technique called ExoO (Yang et al 2018) where tryptic peptides are covalently immobilized on a solid support and digested with OpeRATOR®. As this enzyme can only digest when an O-glycan is present, this results in the specific release of O-glycopeptides which can be analyzed in-depth using LC-MS.

 

However, OpeRATOR does not digest at sites modified with Tn antigens. To circumvent this limitation, Yang et al. first treated their samples with recombinant C1GalT1, an enzyme able to elongate the Tn antigen into a core 1 structure (Gal-β1,3- GalNAc). Sites modified with core 1 O-glycans are efficiently digested by OpeRATOR and can therefore be mapped in depth using EXoO and LC-MS. To distinguish the Tn-antigen sites from naturally occurring core 1 glycosylation sites, the C1GalT1 reaction was performed in the presence of a heavy isotope labelled donor substrate (13C-UDP-Gal). This introduces a unique mass signature that specifically marks the Tn-antigen sites and is easily detectable by mass spectrometry. Using this new approach, termed EXoO-Tn, Yang et al. were able to identify an approximately 10-fold higher number of Tn-glycosylation sites compared to previous studies. 

 

Link to paper, Yang, W. et al., 2018. Mapping the O-glycoproteome using site-specific extraction of O-linked glycopeptides (EXoO). Molecular systems biology, 14(11), p.e8486. 
For a detailed description on how to use
OpeRATOR for in-depth mapping of O-glycosylation sites using the EXoO workflow, check also this recent publication in Nature Protocols (Yang et al 2020b) 

Easy IgG Isotype Fingerprinting with FabRICATOR® and Ion-Mobility MS

June 17, 2020 | References |

Researchers at Strasbourg University and Pierre-Fabre highlight the benefits of a middle-level approach to IgG isotype fingerprinting using native ion-mobility mass spectrometry.

 

FabRICATOR and ion mobility mass spectrometry

FabRICATOR and ion mobility mass spectrometry (Botzanowski et al. 2020)

 

In this new article by Botzanowski et al., a middle-level approach was compared to more classical intact methods for distinguishing IgG isotypes using native ion mobility mass spectrometry. Ion mobility mass spectrometry and collision induced unfolding (CIU) at the intact level is hampered by minimal variations that can be observed. The authors used FabRICATOR to digest adalimumab, panitumumab, and natalizumab down to Fc/2 and F(ab’)2 domains. The Fc domain provided only limited isotype information due to sequence similarity. However, the stability profile of F(ab’)2 and its’ unfolding pattern measured by CIU uncovered very clear differences between the isotypes that could not be achieved with full length, intact mAb.

 

Eculizumab, a humanized IgG2/4 hybrid, which gives conflicting isotype patterns using classical approaches, was also tested. With the middle-level CIU approach, eculizumab could be easily distinguished from reference isotypes. In summary, the authors clearly show how easy, reliable and clear-cut classification of mAb isotypes can be achieved using a middle-level approach with FabRICATOR digestion.

 

The full article is available here:

Botzanowski, T. et al., 2020. Middle Level IM-MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting. Analytical Chemistry. 10.1021/acs.analchem.0c00293

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.


“Most ADCs are produced with non-selective bioconjugation of drug payloads to lysine or cysteine residues creating a wide variety of drug-antibody ratios (DAR). In the frame of new ADC product development, we believe that having control over the DAR and drug load distribution (DLD) is of crucial importance, as is the ability to accurately monitor these two CQAs. Therefore, the combination of the GlyCLICK technology to create homogeneous site-specific ADCs with the middle-up LC/HRMS approach to rapidly determine both the DLD and DAR has a great potential for ADC development.”

 

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. https://doi.org/10.1016/j.jpba.2020.113107

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. https://doi.org/10.1038/s41598-020-60438-z

 

Genovis Business Continuity

March 31, 2020 | Uncategorized |

 

Genovis continues operation and remain committed to serve our customers during the ongoing pandemic crisis. Manufacturing and distribution of the Genovis SmartEnzymes™ are running according to our business continuity plan to meet our customers’ needs and to secure supply.

 

We are monitoring the situation carefully and will notify you immediately if we experience disruptions in fulfilling customer orders. Genovis is fully committed to providing uninterrupted supply of our products to safely keep your research on track.

 

With these uncertain times “scientists enabling scientists” takes on new meaning.  Toward this goal, we continue to provide convenient online access to our customer service, technical support and sales teams. Should you have any questions or concerns, do not hesitate to contact us at info@genovis.com.

 

Most importantly, we hope your staff as well as ours stay healthy and safe during this time.

Fully automated 3D LC-MS workflow for the characterization of antibody glycosylation

March 19, 2020 | References |

Genentech used FabRICATOR®-HPLC to develop a fast and automated 3D LC-MS workflow for the characterization of glycosylation on therapeutic antibodies.

 

The glycosylation profile is a critical quality attribute of biopharmaceuticals including antibodies. This heterogenous quality attribute can be tackled using a range of different analytical technologies including separation of released glycans , intact glycoprotein mass analysis, subunit mass analysis and many more. An antibody subunit approach using FabRICATOR (IdeS) combined with separation on hydrophilic interaction chromatography (HILIC) offers a promising strategy for characterization of IgG glycosylation. Typically, the FabRICATOR digestion is done manually, offline, prior to LC-MS. In a growing number of cases, like online monitoring of glycosylation, it is desirable to automate such a workflow.

 

Researchers at Genentech in collaboration with University of Geneva recently published an automated workflow using three dimensional separation coupled to mass spectrometry. To automate the workflow, they employed the FabRICATOR-HPLC column to deliver online antibody subunit generation. In their setup, this was followed by on column reduction and separation of the subunits using a reversed phase column, coupled to HILIC column to separate the Fc/2 glycoforms. In summary, the system incorporated three columns controlled by two valves: FabRICATOR-HPLC x Reduction/RPLC x HILIC/MS (Fig. 1 below).

 

Camperi et al. compared their automated workflow to the standard manual digestion by testing two of their candidate mAbs. The authors concluded that the 3D automated workflow, which took 95 minutes per sample, could deliver comparable data for all N-glycan variants for the Fc/2 subunit and therefore could be applied to the analysis of not only mAbs but also antibody drug conjugated (ADCs) and bispecific antibodies. Finally, the scientists emphasize that the key strength of the 3D approach was not just the automation aspect but also the fact that no tedious sample preparation was required, and that it is applicable to routine analysis tasks.
 


Fig 1. 3D-LC Workflow for automated middle-up analysis of mAbs (Camperi et al., 2020.)
 

Camperi et al., 2020. Development of a 3D–LC/MS workflow for fast, automated and effective characterization of glycosylation patterns of biotherapeutic products. Anal. Chem. 2020, 92, 6, 4357-4363. doi:10.1021/acs.analchem.9b05193

FabRICATOR® presents F(ab’)2 PET-tracers for Prognostic Imaging

March 18, 2020 | References |

Scientists at Minerva Imaging demonstrate the use of FabRICATOR® to generate a 64Cu-labeled F(ab’)2 PET-tracer for the detection of changes in T Cell response to combined radiation and immunoblockade therapy.
 

Current immunotherapy response-evaluation criteria are limited in discriminating responsive patients eligible for immunotherapy from non-responders. Accurate evaluations of progression are limited by pseudo-progression, a phenomenon where therapy-induced inflammatory events temporarily increase tumor volume, thereby delaying evidence of response. Routine and standardized clinical evaluations are needed that require predictive biomarkers combined with reproducible methods for monitoring biomarker expression and dynamics.
 

Targeting T cells as essential players in the anti-tumor immune response, Kristensen et al. developed a 64Cu-labeled F(ab’)2 against murine CD8a+ T cells. The scientists further demonstrate its potential as a prognostic PET-imaging biomarker

for immunotherapy response in mouse models of colorectal cancer. Rat anti-mouse CD8a antibodies were digested using the FabRICATOR enzyme to generate homogenous F(ab’)2 and Fc/2 subunits. Purification by preparative HPLC yielded isolated F(ab’)2 subunits for random chelation with p-SCN-Bn-NOTA and radiolabeling using the 64Cu isotope. Treatment response assessment was conducted using in vivo PET-imaging of tumor-bearing mice subjected to combined radiation and anti-CTLA-4 therapy.
 

The scientists found that the tumor-to-heart ratio of the PET-tracer increased with combined therapy, this was also confirmed by flow cytometry and IHC analysis showing increased tumor infiltration by CD8+ T cells. The prognostic value of the PET-tracer was further demonstrated using in vivo imaging as the scientists were able to distinguish responsive mice from non-responders prior to treatment-induced variations in tumor volume.
 

Kristensen et al., 2020. Monitoring CD8+ T Cell Responses to Radiotherapy and CTLA-4 Blockade Using [64Cu]NOTA-CD8a PET imaging. Mol Imaging Biol (2020).