Articles by Felicia Mejàre

Antibodies Conjugated with GlyCLICK for Super Resolution Imaging

Super resolution microscopy techniques such as stimulated emission depletion microscopy (STED) improves imaging resolution compared to conventional light microscopy. In STED microscopy, super resolution is achieved using photoactivatable dyes that are excited and de-excited selectively with a laser to restrict fluorescense to a specific focal point. While such super resolution methods in combination with immunostaining advances the quality of imaging, limitations related to dyes and conjugation strategies remain.

 

In this paper, Weber and colleagues at University Göttingen synthesized a new fluorophore suitable for super resolution microscopy techniques. The ONB-2SiR dye is photoactivatable while being stable against two-photon activation, routinely used in STED microscopy. The scientists also present an efficient conjugation and purification protocol for labeling antibodies with hydrophobic and aggregating dyes using the GlyCLICK technology and phase separation.

 

To obtain a defined degree of labeling (DOL) and compensate the hydrophobic nature of the fluorophore, a conjugation and purification protocol suitable for aggregating and poorly soluable dyes was developed. Using the GlyCLICK technology, antibodies were labeled specifically at the Fc glycan sites, generating conjugates with preserved affinity carrying two labels per antibody. Compared to other conjugation methods, GlyCLICK resulted in conjugates with a definite DOL of 2 while also reducing aggregation. The conjugates were then purified using a tree-phase separation technique, allowing the scientists to purify the hydrophobic dye from the labeled antibodies.

 

In summary, the scientists present a new fluorophore suitable not only for STED, but for other single-molecule localization microscopy techniques such as PALM and MINIFLUX. This work also highlights the importance of high-quality conjugation, which is obtained using GlyCLICK.

 

Weber et al., 2020. Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels. Chemistry – A European Journal. doi: 10.1002/chem202004645

 

GlyCLICK
Site-specific labeling of native IgG using a single kit.

 

 

 

Charge Heterogeneity Analysis of Antibody Subunits Generated by FragIT

Charge heterogeneity of monoclonal antibodies is an important critical quality attribute that requires close monitoring due to its potential impact on antibody efficacy and immunogenicity. Since the heterogeneity is mostly caused by post translational modifications such as C-terminal lysine clipping, deamidation, glycation, sialic acid or adduct formation, these modifications can pose significant challenges to the analytical scientists.

 

In a recently published article by Jaag et al., scientists from the Institute of Pharmaceutical Sciences at University at Tübingen, Germany, present a new approach to charge variant analysis at the intact and sub-unit level by 2D-LC separation. In this workflow, the first-dimension separation is based on strong-cation chromatography (SCX) and the second-dimension separation is based on desalting reversed phase liquid chromatography (RP-HPLC) which enables combination with the mass analysis by mass spectrometric (MS) detection.

 

The analysis at the subunit level was achieved by digesting humanized IgG1K with FragIT MicroSpin columns from Genovis. These columns contain immobilized FABRICATOR enzyme which digests the antibody below the hinge and generates two fragments, Fc/2 and F(ab´)2. By further reduction with TCEP, three subunits were generated: Fc/2, Fd and the light chain.

 

The three subunits allowed for a fast separation by RP-HPLC. All three fragments were separated within 30 seconds. Further on, the Fd fragment resulted in two peaks which suggests different intrachain disulphide forms or D (P) clipping. As the aim of the analysis was to achieve a separation of as many charge variants as possible, the subunit level analysis enabled the extraction of detailed information about the molecule. Moreover, the main glycoforms were successfully identified using MS and middle-up approach by 2D-LC-ESI-MS analysis. In comparison to the intact mAb analysis, the 2D -LC method also contributed to orthogonal selectivity for the separation of fragments.

 

Jaag, S. Shirokikh, M. and Lämmerhofer, M., 2020. Charge variant analysis of protein-based biopharmaceuticals using two-dimensional liquid chromatography hyphenated to mass spectrometry. Journal of Chromatography A, 1636, p.461786.

 

 

 

FragIT
Generation of F(ab’)2 and Fc/2 fragments.

FragIT kit
Generation and purification of F(ab’)2 and Fc/2 fragments.

Generating Antibody Mimetics with GingisKHAN

Antibodies formulated as solid-state depots can potentially be used for local treatments and minimize the need for large systemic doses. Bevacizumab may for instance be administered locally to control post-operative scarring following glaucoma filtration surgery. A solid-state form would however be required in order to obtain a proper slow release of the antibody.

 

In order to study the possibility of such a solid-state formulation, scientists at the School of Health in London developed antibody mimetics and compared the stability after lyophilization to that of bevacizumab. Consisting of two Fabs linked together by a flexible polyethylene glycol (PEG) scaffold, the mimetics functionality is similar to that of IgG, but without the Fc-mediated effector functions. In addition, the mimetic format enhances binding affinity while reducing the propensity for aggregation.

 

To prepare the antibody mimetics, Fab fragments from bevacizumab were obtained by digestion above the hinge using either papain or GingisKHAN®. While both enzymes were able to produce Fabs without impacting the binding site, LC-MS analysis showed that the unspecific papain digestion resulted in three different isomers, and a free light chain due to disulfide reduction. In contrast, the GingisKHAN enzyme generated a homogenous pool of Fab fragments as was indicated by one single mass peak.

 

Antibody mimetics produced with the different Fab fragments were then subjected to freeze-drying for a head-to-head comparison with lyophilized bevacizumab. The results showed that only the mimetic produced with GingisKHAN generated Fabs was unsusceptible to aggregation and remained intact without heavy or light chain dissociation. In short, the scientists concluded that the antibody mimetic was more stable than IgG when subjected to lyophilization, and that pure homogenous Fabs could play an important part for the stability of the final product.

 

Khalili. 2020. Using different proteolytic enzymes to digest antibody and its impact on stability of antibody mimetics. Journal of Immunological Methods. doi: 10.1016/j.jim.2020.112933

 

 

 

 

GingisKHAN
Generation of human IgG1 Fab fragments.

 

GingisKHAN Fab kit
Generation and purification of human IgG1 Fabs.

 

 

SmartEnzymes in de novo Sequencing of Antibodies

Two complementary determining regions (CDR3) are considered major determinants of antigen-binding specificity that give rise to the human immunoglobulin repertoire with billions of unique antibodies. For de novo sequencing of the human repertoire, circulating antibodies can be analyzed by mass spectrometry after proteolytic cleavage. Complex mixtures such as plasma derived samples are however challenging to analyze due to the increased complexity that may prevent accurate assignments.

 

In this paper, scientists at Utrecht University explored de novo sequencing by analysis at the antibody subunit level using electron capture dissociation (ECD) and mass spectrometry. To decrease complexity and target the variable CDR3s regions, antibodies were digested to obtain homogenous F(ab’)2 or Fab subunits using the FabRICATOR® or FabALACTICA® enzymes respectively. The unique capability of FabRICATOR to digest human IgG1-4 allowed the scietists to process multiple antibodies for this analytical approach.

 

Sample complexity was decreased by removing the Fc part containing PTMs including glycosylation and lysine clipping, while the F(ab’)2 fragments were subjected to fragmentation and mass spectrometry analysis. The use of EDC allowed for amino acid sequence ladders spanning the CDR3 region of both the light and heavy chains, with gaps only observed for proline containing sequences. By studying different antibodies and subclasses, the authors were able to observe correlations between subclass-specific features and fragmentation patterns.

 

This work by den Boer et al. is a first step in predicting the fragmentation patterns observed in ECD fragmentation of IgGs, which may greatly contribute to future tools for de novo sequencing of antibodies.

 

den Boer et al., 2020. Selectivity over coverage in de novo sequencing of IgGs. Chem. Sci. doi: 10.1039/d0sc03438j

 

 

FabRICATOR

Generate F(ab’)2 and Fc/2 from IgG.

 

FabALACTICA

Generate Fab and Fc from human IgG1.

 

FabRICATOR in Efficient Structural Characterization of mAbs

In contrast to small generic molecules, therapuetic monoclonal antibodies (mAbs) exhibit inherent heterogeneity that may arise during production and formulation or due to the storage conditions. Therefore, it is essential to characterize the structural heterogeneity of mAbs with respect to properties including conformational changes, aggregation and post-translational modifications. In this work, Zhu et al. at the Chinese Academy of Medical Sciences & Peking Union Medical College present an integration strategy for structural characterization of mAbs by combining intact mass and middle-down analysis using only a high-resolution Q-TOF mass spectrometer.

 

The mAbs trastuzumab and adalimumab were analyzed at intact level using native SEC-MS and denatured RPLC-MS to measure the molecular mass, detect heterogenous modified protein species and to obtain a relative quantification of all the major Fc glycoproteoforms. In order to obtain a more detailed structural confirmation of the protein sequence and glycosylation profile, antibodies were digested using the FabRICATOR® (IdeS) enzyme and reduced to generate homogenous LC, Fd’ and Fc/2 fragments for middle-down analysis.

 

The optimized native and denatured methods were suitable for rapidly assessing the structural heterogeneity while the combined CID and ETD middle-down analysis enhanced the sequence coverage of the fragments from both mAbs. The integrated workflow resulted in quantitative and qualitative glycosylation profiling while better resolving the overall heterogeneity caused both N-glycosylation and other modifications such as C-terminal processing. This integrated strategy can easily be implemented for in-depth structural characterization of mAbs during pharmaceutical development and quality control.

 

Zhu et al., 2020. Integrating Intact Mass Analysis and Middle-Down Mass Spectrometry Approaches to Effectively Characterize Trastuzumab and Adalimumab Structural Heterogeneity. J. Proteome Res. doi: 10.1021/acs.jproteome.0c00373

 

 

 

FabRICATOR (Ides) 
Cysteine protease for below hinge digestion of IgG

 

 

 

FabULOUS Middle-Level Analysis of Murine Polyclonal Antibodies

Important advances in top-down and middle-level analytical LC-MS strategies have arisen in recent years, focused on the characterization of therapeutic monoclonal antibodies. Similar strategies to analyze polyclonal IgG in regard to subclass abundance and glycosylation patterns may provide new insight into immune regulatory processes. However, challenges associated with molecular heterogeneity due to inherent sequence variability of such samples persist.

 

Analysis of the Fc glycosylation pattern can be exploited as determinant of the IgG subclass and may be performed on human IgG by specific proteolytic cleavage using the FabRICATOR® (IdeS) enzyme. In this work, the scientists investigate the feasibility of middle-level analysis by FabULOUSTM (SpeB) digestion and LC-MS of polyclonal mouse IgG with extensive sequence variability. The FabULOUS enzyme was exploited for the generation of Fc/2 subunits from all murine IgG subclasses (1/1i, 2a/c, 2b/2bi, and 3), which all were proven amenable for proteolytic cleavage in 3 hours under mild reducing conditions. The obtained subunits enabled the dissociation of Fc and Fab domains needed to tackle the sequence heterogeneity.

 

Middle-down analysis by HPLC-MS of the Fc/2 subunits allowed the assignment of both murine IgG subclasses and isotypes, while simultaneous middle-up analysis provided quantitative information on the subclasses as well as their respective glycosylation variants. The workflow thereby permitted a global analysis of polyclonal murine IgGs with respect to subclass abundances including closely related isotypes as well as glycosylation profiles and other PTMs such as oxidation and lysine variants. Finally, the authors demonstrated the capabilities of this workflow in a pilot study where murine polyclonal IgG from mouse serum after immunization with pollen allergen.

 

In summary, the described middle-level workflow provides comprehensive information obtained in a single analysis involving swift sample preparation, standard LC-MS analysis, and straightforward data evaluation as an attractive extension to the toolbox of analytical strategies for antibodies.

 

Blöchl et al., 2020. Towards middle‑up analysis of polyclonal antibodies: subclass‑specific N‑glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC‑MS. Scientific Reports. doi:10.1038/s41598-020-75045-1

 

 

 

FabULOUS Enzyme
Generation of IgG Fab fragments from several species.

 

FabULOUS Fab kit
Generation and purification of mouse IgG Fab fragments.

 

The Crystal Structure of OpeRATOR Reveals O-glycan Substrate Specificity 

October 8, 2020 | Products, References |

The O-glycoprotease OpeRATOR® from Genovis has spurred great interest from the community and has become a valuable tool for characterizing O-glycosylated biopharmaceuticals. Through a collaboration with the lab of Marcelo Guerin, the structure of OpeRATOR has now been solved and published in Nature Communications.  

 

OpeRATOR originates from the commensal gut bacterium Akkermansia muciniphila, and as the name suggests this bacterium is capable of digesting mucins. The bacterium is associated with beneficial effects on health, likely through the regulation of the thickness of the mucus layer, a role in which the OpeRATOR enzyme may play a role.  

 

In the paper, the high-resolution X-ray crystal structure of OpeRATOR is solved together with the ligated form to a glycopeptide substrate and the resulting product. The combined data set gives indications to the catalytic cycle and is complemented with glycopeptide chemistry and enzyme kinetics measurements to characterize the molecular mechanism and the substrate specificity. The structure and the kinetics data clearly shows that OpeRATOR digestion is occurring at sites modified with core 1 O-glycans and to a limited degree core 3 and α2,3 sialylated core 1 structures.  

 

This new paper answers many questions regarding the specificities of the OpeRATOR O-glycoprotease and functional studies will provide further insights into the role of this enzyme in the host interactions of A. muciniphila. Meanwhile, the new findings in this paper make an important contribution to the application of this enzyme in analytical workflows for characterization of complex O-glycosylated biopharmaceuticals.  

 

Trastoy, B. et al., 2020. Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila. Nature communications, pp.1–14. 

 

FabULOUS and IgGZERO in Study of Antibody Pathogenesis in Blood Transfusions

September 17, 2020 | Applications, References |

Scientists at the University of Amsterdam use SmartEnzymes in a new study of the biological and structural properties of antibody pathogenesis in blood transfusions.

 

Blood transfusions are a vital part of healthcare but can in some cases lead to severe conditions such as anti-leukocyte antibodies in the transfusion product that cause transfusion-mediated acute lung injury (TRALI). Even though TRALI is the leading cause of transfusion-associated fatalities, only supportive measures can be provided and no treatment is currently available. To date, the antibody characteristics responsible for causing TRALI remain unknown and the pathogenesis is hard to decipher.

 

Only some antibodies have been observed to induce TRALI, while others are incapable of doing so and are thereby deemed resistant. To study the structural and biological characteristics between TRALI resistant or inducing antibodies, the scientists analyzed different anti-MHC antibodies for affinity to antigens and IgG-Fc receptors, as well as the ability to activate the classical complement pathway. To determine if binding was Fc-mediated, the antibdoies were digested with the FabULOUSTM enzyme to generate intact Fab and Fc fragments. The role of Fc glycosylation in TRALI was further analyzed by deglycosylation using deGlycIT spin columns, containing the immobilized IgGZERO enzyme that specifically trims the antibody N-glycans.

 

The authors found no substantial differences in binding affinity for antibodies to FcϒRs that could explain the TRALI inducing or resistant properties. However, when studying complement activation, the scientists observed significant differences in binding to the C1q complex. The SmartEnzymes-assisted antibody fragmentation and deglycosylation allowed the authors to determine that complement activation was fully Fc-mediated and independent of Fc glycosylation. With these results, the scientists were able to conclude that TRALI induction correlated to increased antibody Fc-mediated complement activation.

 

Link to Paper: Zeeuw van der Laan et al., 2020. Biological and structural characterization of murine TRALI antibody reveals increased Fc-mediated complement activationBlood advances. doi: 10.1182/bloodadvances.2020002291

 

 

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

 

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