FabRICATOR^® in capillary zone electrophoresis-native mass spectrometry

The use of capillary electrophoresis for the analysis of therapeutic antibodies and other biopharmaceuticals is growing in popularity. A new article by researchers from CNRS in France showed how capillary zone electrophoresis-native mass spectrometry could be used for the quality control of intact therapeutic monoclonal antibodies. The authors show for the first time the use of a triple layer coated (PB-DS-PB) capillary with mAbs, which helps prevent mAbs adsorption. The intact therapeutic mAb, Infliximab, was analyzed under non-denaturing conditions to retain conformational heterogeneity and avoid denaturation. A middle up approach using FabRICATOR digestion was used to characterize dimer association detected in the stressed mAbs preparation. Digestion below the hinge region of the mAb produced F(ab’)2 and Fc/2 fragments which was subsequently analysed by capillary zone electrophoresis-native mass spectrometry. Using this approach the authors were able to see nature of the dimer association while maintaining the non-covalent interactions of the Fc/2 fragments.

For more information on FabRICATOR please visit the following pages:

• FabRICATOR Product Page
• Generation of Antibody Fragments

FabRICATOR^® driven middle-down glycan analysis using NMR

Analysis of the glycosylation of therapeutic antibodies and other biopharmaceuticals is typically done by LC or LC/MS-based methods. However, each analytical technique has its strengths and weaknesses which makes the development of orthogonal methodologies crucial for in-depth characterization. In this study, researchers from the FDA present a middle-down NMR approach for studying glycosylation of therapeutic antibodies. Analogous to middle-down MS methods, the antibodies were digested using FabRICATOR to yield Fc/2 fragments. After chemical denaturation, these exhibited high enough solubility and sufficiently fast molecular dynamics to allow for glycan analysis by 2D-NMR without the need for isotope labeling or glycan release. Using this method, the authors were able to determine the chemical structure, glycosidic linkage position and anomeric configuration of each monosaccharide unit of the major Fc N-glycan structures and were able to quantify important quality attributes such as galactosylation and fucosylation.

For more information on FabRICATOR please visit the following pages:

• FabRICATOR Product Page
• Generation of Antibody Fragments

SmartEnzymes™ assist MALDI in-source decay FT-ICR Mass Spectrometry analysis

The Consortium for Top-down Proteomics is currently conducting a large inter lab study. They are developing methods for the analysis of intact mAbs and antibody subunits generated by digestion with either FabRICATOR or GingisKHAN. In this paper, van der Burgt et al. present a novel method for the analysis of mAbs based on MALDI in-source decay fragmentation coupled with high resolution FT-ICR mass spectrometry. The standard method for antibody sequence confirmation by MS is based on fragmentation using electron transfer dissociation (ETD). The MALDI-ISD based method yielded complementary fragments to those observed in ETD experiments, translating to increased sequence coverage. Using digestion with either FabRICATOR or GingisKHAN, a higher total sequence coverage could be achieved than for the intact mAbs. FabRICATOR digestion also allowed for direct analysis of Fc glycosylation by MALDI-FT-ICR without the need for LC separation.

Meet the Scientist

We got the opportunity to interview the last author of the paper, Dr Simone Nicolardi at Leiden University Medical Center.

Tell us about yourself?

I am a senior researcher at the Center for Proteomics and Metabolomics at Leiden University Medical Center (https://www.lumc.nl/org/proteomics-metabolomics/). My work is focused on the development of ultrahigh resolution mass spectrometry-based methods for the analysis of biomolecules such as glycans, peptides, and proteins. This includes the structural characterization of monoclonal antibodies for determination of primary amino acid sequence and post-translational modifications.

What is new with the MALDI in-source decay fragmentation you have published?

Our newly developed method combines the advantage of ultrahigh resolution mass measurements, obtained using high-end instrumentation and novel spectra processing software, with the efficient fragmentation provided by MALDI-in-source decay using 1,5-diaminonaphthalene as a reducing MALDI matrix. The main advantages are complementary sequence information compared to other mass spectrometric fragmentation techniques and the use of minimal sample preparation procedure that does not require separation techniques such as liquid chromatography.

What are the advantages of using FabRICATOR or GingisKHAN?

In MALDI-ISD experiments singly charged ions are generated from the protein backbone. Thus, a wide m/z-range is needed for the detection of all fragments generated from large compounds, such as monoclonal antibodies. Even in high-end MS instrumentation, this m/z-range is limited and sequence information is obtained from N- and C-terminal protein portions only. The use of FabRICATOR or GingisKHAN allows extending sequence information of heavy chains to more internal protein regions. In addition, after digestion mAb Fc portion can be analyzed directly by MALDI-MS allowing the detection of most abundant Fc glycoforms.

How do you view sample preparation prior to MS analysis of mAbs?

The structure characterization of mAbs is generally performed using a multi-level approach based on different analytical methods. Many of these methods are based on liquid chromatography (LC) electrospray ionization (ESI) MS. LC is used to separate mAb subunits and to remove ESI-non-compatible compounds such as salts. Our method is based on MALDI which is known to be more tolerant to salts in the sample. Thus, MALDI-ISD MS avoids laborious sample preparation steps that can lead to artificial modification of mAbs. Also, it comes with short analysis times in contrast to other chromatographic techniques.

What are you working on now?

Our attention is now on bispecific mAbs. We apply our MALDI-based method for the simultaneous analysis of all six different polypeptide chains generated after treatment with FabRICATOR and chemical reduction of disulfide bonds. Our aim is to develop a fast method for the monitoring of chemically induced mAb modifications.

For more information on FabRICATOR or GingisKHAN please visit the following pages:

• FabRICATOR and GingisKHAN Product Page
• Generation of Antibody Fragments