Articles in the Category ”Applications”

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.




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





Generation of human IgG1 Fab fragments.


GingisKHAN Fab kit
Generation and purification of human IgG1 Fabs.



Antibody Mixtures Digested using GingisREX

The formulation of antibodies in mixtures has revealed significant clinical advantages but causes increased analytical challenges. Long-term studies of formulated antibody mixtures over time are both difficult and time consuming. An example of a post-translational modification that could occur during storage is the oxidation of tyrosine that may induce conformational changes of an antibody.


In a recent article by scientist at Merck in New Jersey, USA, new analytical workflows were explored to study a formulated mixture of antibodies of subclass IgG1, IgG2 and IgG4. To study conformational changes in single antibodies and mixtures, the scientists utilized fluorescent labeling of oxidized tyrosine to 3,4-dihydroxyphenylalanine for analysis using LC-MS.


In a complex sample containing antibody mixtures with similar primary sequences, the authors found that by using the arginine specific protease GingisREX®, longer peptides could be generated which resulted in the formation of unique peptides. The GingisREX protease is highly specific for c-terminal digestion after arginine residues and has showed improved performance over Arg-C in a direct comparison (GingisREX Applications).


Using the GingisREX based LC-MS workflow, the scientists could study a particular oxidation hotspot in peptide 20 of the IgG4 antibody that only occurred when the antibodies were studied in a mixture. The authors conclude that orthogonal methods for study of antibody mixtures are needed and that the combination of fluorescent labeling of oxidized residues in combination with mass spectrometry is a promising approach.



Mozziconacci, O. et al., 2020. Probing Protein Conformation Destabilization in Sterile Liquid Formulations through the Formation of 3,4-Dihydroxyphenylalanine. Molecular Pharmaceutics, 17(10), pp.3783–3793.


GingisREX Box




Learn more about GingisREX

Protease that digests C-terminally of Arginines






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




Generate F(ab’)2 and Fc/2 from IgG.



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.


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



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)