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. Read more »
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. Read more »
Launching NEW GlyCLICK® ADC kits!
Genovis launches new GlyCLICK kits for site-specific generation of custom ADCs carrying unique 2-step cleavable linker-payloads from Glykos Finland.
Antibody-drug conjugates (ADCs) comprise a new generation of antibody-based biologics that carry drug payloads directly into target cells, allowing for a broadened therapeutic window. The drawbacks of conventional antibody conjugation strategies are rapidly being surpassed by site-specific methods, where conjugation at the Fc-glycan sites using GlyCLICK has proven to be an attractive option for labeling of native antibodies without genetic engineering.
The GlyCLICK conjugation technology results in site-specific incorporation of 2.0 drugs per antibody, for this reason the GlyCLICK ADC kits offer conjugation with highly potent payloads functionalized with DBCO to enable click-chemistry to azide activated antibodies. GlyCLICK ADC kits can be used to combine native IgG with a two-step cleavable linker carrying either MMAE or PNU for the desired cytotoxic effect on targeted cells (Fig. 1).
Learn more about the GlyCLICK technology for ADC development.
GlyCLICK ADC kit MMAE – Site-specific ADC generation with cleavable linker-payloads carrying MMAE.
GlyCLICK ADC kit PNU – Site-specific ADC generation with cleavable linker-payloads carrying PNU.
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. Read more »
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. Read more »
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. Read more »
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. Read more »
The Crystal Structure of OpeRATOR Reveals O-glycan Substrate Specificity
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. Read more »
FabULOUS and IgGZERO in Study of Antibody Pathogenesis in Blood Transfusions
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.
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.”
