Articles in the Category ”Applications”

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 FabULOUS (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.

 

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 FabULOUS 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

 

 

Inter-laboratory Study of mAb Characterisation using Top-Down and Middle-Down Mass Spectrometry

September 15, 2020 | Applications, References |

Srzentic et al. present an interlaboratory study investigating the current state of top-down and middle-down mass spectrometry methods for the characterization of therapeutic monoclonal antibodies and explore the potential added value of these methods to the more commonly used bottom-up mass spectrometry.

 

In the growing market of therapeutic monoclonal antibodies (mAbs), there is an increasing demand to precisely and reproducibly characterize the structural heterogeneity of mAbs. At present, bottom-up mass spectrometry (MS) approaches, using peptide mapping of tryptic peptides, is widely employed for this. In this study, initiated by the Consortium of Top-Down Proteomics, the use of top-down and middle-down MS methods were explored as potential complements to bottom-up MS approaches in regard to primary structure confirmation, variable domain sequencing and post-translational modification (PTM) identification on mAbs.

 

The three commercial mAbs: SiLuLite, NIST mAb standard and Herceptin were centrally prepared in three different forms before being distributed to 20 participating laboratories worldwide; intact for top-down analysis, and digested using either of the IgG proteases GingisKHAN (above the hinge) or FabRICATOR (below the hinge) for middle-level analysis. The participating laboratories were asked to analyze these using their top-down/middle-down MS workflow of choice.

 

The study revealed the wide variety of techniques and expertise available among the participants. Nevertheless, top-down/middle-down approaches proved to be a fast and valuable compliment to bottom-up approaches in achieving a high amino acid sequence coverage, avoiding artifacts and confirming bottom-up derived PTM profiling and stoichiometry. They could also deduce if protein modifications observed in bottom-up assays were naturally occurring or sample preparation mediated. Middle-level analysis, using FabRICATOR or GingisKHAN generated subunits, proved especially beneficial for assigning correct monoisotopic masses and identifying glycosylation and glycation sites. The use of IgGZERO to remove the Fc N-glycans and eliminate glycosylation related heterogeneity prior to top-down/middle-down MS analysis was also suggested. Overall, the study shows the added value of top-down/middle-down approaches for mAb analysis and the great potential for further advances in top-down and middle-down MS techniques.

 

Link to Paper: Srzentić et al., 2020. Inter-laboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry. Anal. Chem. doi: 10.1021/jasms.0c00036

 

Enhanced CE-MS analysis of bispecific and LALA antibodies using SmartEnzymes

September 3, 2020 | Applications, References |

 

Gstöttner et al. used SmartEnzymes for their CE-MS analysis of highly homologous bispecifics, one of them containing LALA mutations. 

 

Bispecific antibodies (BsAb) offer an attractive alternative to traditional mAbs since they can bind to two different epitopes simultaneously. Because they are composed of four different peptide chains, rather than two, they are subject to additional macro- and microheterogeneity which requires proper analysis and monitoring. Bottom-up approaches together with mass spectrometry (MS) have been employed to address such heterogeneity but such methods are unable to handle minor mass shifts and does not provide domain localization information.

 

In a new article by Gstöttner and colleagues at Leiden University Medical Center together with Roche Penzberg, sheathless capillary electrophoresis (CE) coupled to MS was used to probe two highly homologous BsAb. They looked at both intact BsAbs and their subunits generated using SmartEnzymes. FabRICATOR was used for below hinge digestion in one of the BsAb while the second BsAb proved to be more challenging due to a LALA mutations and required above hinge digestion using FabULOUS, highlighting the versatility of the SmartEnzymes family.

 

Gstöttner et al., CE-MS analysis of highly homologous bispecifics.

 

The nature of the sheathless CE approach meant that the authors could collect and compare both the intact and subunit data using the same instrument setup. From that, marco- and microheterogeneity could be assessed. Differences between the two BsAb could be detected such as incomplete assemblies and free chains, along with PTMs such as glycation. Combining the observations, the authors conclude that the different engineering processes for each BsAbs results in varying heterogeneity. They also underscore the wide applicability of sheathless CE. Finally, they reveal the importance of SmartEnzymes and subunit analysis.

 

Link to Paper: Gstöttner et al. Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS. Analytica Chimica Acta 1134 (2020) p. 18-27

 

 

 

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) 

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