Articles tagged ”FabRICATOR”

Easy IgG Isotype Fingerprinting with FabRICATOR® and Ion-Mobility MS

June 17, 2020 | References |

Researchers at Strasbourg University and Pierre-Fabre highlight the benefits of a middle-level approach to IgG isotype fingerprinting using native ion-mobility mass spectrometry.

 

FabRICATOR and ion mobility mass spectrometry

FabRICATOR and ion mobility mass spectrometry (Botzanowski et al. 2020)

 

In this new article by Botzanowski et al., a middle-level approach was compared to more classical intact methods for distinguishing IgG isotypes using native ion mobility mass spectrometry. Ion mobility mass spectrometry and collision induced unfolding (CIU) at the intact level is hampered by minimal variations that can be observed. The authors used FabRICATOR to digest adalimumab, panitumumab, and natalizumab down to Fc/2 and F(ab’)2 domains. The Fc domain provided only limited isotype information due to sequence similarity. However, the stability profile of F(ab’)2 and its’ unfolding pattern measured by CIU uncovered very clear differences between the isotypes that could not be achieved with full length, intact mAb.

 

Eculizumab, a humanized IgG2/4 hybrid, which gives conflicting isotype patterns using classical approaches, was also tested. With the middle-level CIU approach, eculizumab could be easily distinguished from reference isotypes. In summary, the authors clearly show how easy, reliable and clear-cut classification of mAb isotypes can be achieved using a middle-level approach with FabRICATOR digestion.

 

The full article is available here:

Botzanowski, T. et al., 2020. Middle Level IM-MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting. Analytical Chemistry. 10.1021/acs.analchem.0c00293

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

 

ADC Biotransformation Analysis using FabRICATOR and LC-MS

March 11, 2020 | References |

Current strategies for analyzing in vivo biotransformation of antibody-drug conjugates (ADCs) are limited by the site of conjugation, extensive sample preparation and insufficient sensitivity. In this paper by Kotapati et al., the authors developed a universal affinity capture method for assessing the effects of biotransformation on any site-specific ADC using generic reagents and LC-HRMS analysis.

 

Antibody-Drug Conjugates (ADCs) can undergo in vivo biotransformation where the payload can be metabolized to an inactive species or be subjected to deconjugation releasing the payload into systemic circulation. Strategically selected conjugation sites can minimize proteolytic cleavage or steric hindrance of the surrounding mAb domains, ultimately improving the potency and stability in vivo. The process of screening for optimal conjugation sites is therefore an important part of ADC discovery and development.

 

ADCs prepared from various antibodies and payloads with site-specific conjugation sites at the LC, HC-Fab and HC-Fc were prepared and analyzed using a mono- or dual affinity capture method. Streptavidin magnetic beads coated with anti-human F(ab’)2 captured ADCs from mouse serum and were processed on a KingFisher Flex automated magnetic extraction instrument. The captured ADCs were then, according to conjugation site, either subjected to reduction, on-bead digestion with only the FabRICATOR enzyme or in combination with PNGaseF for complete Fc-deglycosylation. The samples were then either reduced or eluted directly for analysis using high resolution LC-TOF mass spectrometer.

 

With this method, the authors were able to successfully study biotransformation of site-specific ADCs independent of antibody type, conjugation type or linker-payload chemistry. Using the site-specific FabRICATOR enzyme, HC-Fab and HC-Fc ADCs were digested below the hinge into homogenous F(ab’)2 and Fc subunits for the generation of antibody fragments. Compared to intact ADC analysis, this middle-level approach increased the resolution and sensitivity for identification of the conjugated payload and its metabolites at exceptional sensitivity and resolution.

 

Kotapati et al., 2020. Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies. Analytical Chemistry (92). pp. 2065-2073. doi: 10.1021/acs.analchem.9b04572

 

SmartEnzymes™ in Quality Control of Commercial Antibodies

In a recent paper, Sokolowska and colleagues at Janssen Research and Development qualified and covalidated a subunit LC-MS method for quality control and stability testing of the oxidation status of commercial antibodies.

 

LC-MS is commonly used for therapeutic antibody development and characterization within the biopharmaceutical industry due to the inherent strengths to provide site-specific identification and quantitation of post-translational modifications. However, the implementation of LC-MS methods to commercial QC labs is challenging, since there are not many options for fully GMP compliant systems. In addition, the methods often require extensive MS expertise and suffer from time-consuming sample preparation and lack of robustness. To counter these obstacles, Sokolowska et al. have developed an LC-MS method that requires minimum analyst training. It uses validated GMP compliant software and is based on subunit analysis, which is proved to be faster and more robust compared to peptide mapping.

 

The assay uses FabRICATOR® (IdeS) and  IgGZERO® (EndoS) enzymes to generate deglycosylated IgG subunits suitable for MS analysis. FabRICATOR digests the antibody below the hinge and IgGZERO hydrolyzes the Fc N-glycans. The subunits are analyzed using reversed phase-ultraperformance liquid chromatography coupled to a quadrupole time-of-flight (RP-UPLC-QTOF) MS to monitor antibody oxidation for stability testing and commercial product release.

 

The developed subunit LC-MS assay was covalidated in three laboratories and showed comparable performance. The robustness was tested by varying both the LC-MS settings and the sample preparation. The enzymatic conditions included variations in protein concentration, enzyme lots, enzyme-to-protein ratio, digestion time and temperature, reduction time and temperature, and reagent concentrations. Minor variations in sample preparation all led to measured Fc oxidation within the method variation +/- 0.9%.

 

Figure 1. mAb subunit oxidation assay using FabRICATOR and IgGZERO (Sokolowska et al., 2020.)

The approval of this method opens the door for implementing other subunit LC-MS and multiattribute methods in QC laboratories to modernize commercial QC and stability testing.

 

Learn more by reading the full paper, follow the link below.

https://pubs.acs.org/doi/full/10.1021/acs.analchem.9b05036

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Monitoring Glycation Levels on Bispecific Biologics using FabRICATOR®

November 18, 2019 | References |

Bispecific monoclonal antibodies (BsAbs) are multi-functioning and complex biologics with the ability to recognize two different epitopes for improved therapeutic properties. Characterizing protein modifications such as glycation on biologics is vital to ensure consistency in stability and function. The structural complexity of BsAbs requires robust analytical methods, where conventional top-down and bottom-up strategies may lack in sensitivity or even introduce further modifications. Middle-level analysis using site-specific proteases such as GingisKHAN®(Kgp) and FabRICATOR®(IdeS) is an intermediate strategy that enables complementary analysis of intact or reduced Fab and Fc fragments.

 

In a recent article by Gstöttner et al. (2019) from Leiden University together with Roche Pharma Technical Development, the authors analyzed a BsAb for protein modification levels, N- and C-terminal sequencing and modification localization using top-down, middle-level and bottom-up strategies. The BsAb was analyzed for changes in glycation levels over time using middle-up FT-ICR MS on Fc/2, LC and Fd’ fragments obtained by FabRICATOR digestion. The scientists also localized glycation hot spots on the heavy chain backbone of the FabRICATOR-digested BsAb using sequential in source decay (ISD) MALDI fragmentation.

 

Using FabRICATOR in a novel middle-up MS strategy, the scientists were able to analyze all antibody subunits in a single high-resolution mass spectrum. By implementing the method in a forced-glycation experiment, changes in glycation levels were successfully monitored over time. The authors were also able to localize several glycation hot spots by intact top-down and FabRICATOR-assisted middle-down analyses. The use of middle-level strategies in combination with conventional MS-based methods successfully provided complementary data for monitoring the level of glycation.

 

Learn more about FabRICATOR and our other proteases.

 

Gstöttner, C. et al., 2019. Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh resolution MALDI FT-ICR mass spectrometry. mAbs. doi: 10.1080/19420862.2019.1682403.

SmartEnzymes™ in a new approach to characterize ADCs

October 25, 2019 | Applications, References |

Antibody drug conjugates (ADCs) consist of monoclonal antibodies chemically linked to a cytotoxic agent. The target specificity of the monoclonal antibody in combination with the potency of the cytotoxic drug make ADCs promising therapeutic agents. However, the molecules are often complex, making evaluation of the quality attributes for the ADC challenging.

 

In order to characterize the ADCs, the predominant analysis of choice is peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry. However, the sample preparation steps in a bottom-up approach are often time-consuming and a comprehensive view of ADCs with different sequence variants and post-translational modifications is lacking.

 

In this recently published article by Chen et al., a middle-down RPLC-MS strategy with electron transfer disscociation (ETD) was developed to analyze lysine and cysteine conjugated ADCs at the subunit level. FabRICATOR® (IdeS) and GingisKHAN® (KGB) were used to generate the subunits. FabRICATOR digests below the hinge, generating F(ab’)2 and Fc/2 fragments, and GingisKHAN digests above the hinge, generating intact Fab and Fc fragments. For the deglycosylation, the IgG-specific endoglycosidase GlycINATOR® (EndoS2) was used.

 

This middle-down approach enabled high-resolution evaluation of several ADC quality attributes at the subunit level, including drug to antibody ratio (DAR), conjugation sites and micro-variants. The approach shows great potential for investigating quality attributes during the development and characterization of novel ADCs.

 

Read more about FabRICATOR, GingisKHAN and GlycINATOR.

 

Chen, B et al., 2019. Middle-Down Multi-Attribute Analysis of Antibody-Drug Conjugates with Electron Transfer Dissociation. Anal. Chem. 91(18). 11661-11669.

Free Thiols using FabRICATOR® and FabALACTICA®

In biopharmaceutical product development and manufacturing, free thiol content is one of the product quality attributes of interest as its presence could impact structure, stability and function of the product.

At Biogen, Yi Pu et al have optimized a label-free LC (UV) / MS method for free thiol quantification at a subunit level of IgG1 and IgG4. The new method, which is based on a method developed by Faid et al*, was compared to two conventional approaches, Ellman’s assay and peptide mapping.

It is very challenging to identify free thiol forms by mass spectrometry at the intact antibody level. By combining the highly specific proteolytic enzymes FabALACTICA (IgdE) and FabRICATOR (IdeS) the authors generated the subunits Fab, hinge and Fc/2, suited for confident mass determination. The subunits were subsequently separated on a polyphenyl reversed phase column in order to separate free thiol forms from their corresponding disulphide bond-linked form. A baseline or near baseline separation was obtained making it possible to calculate the free thiol content on each subunit.

The result of the quantification of free thiols from all three methods were comparable and showed similar trends even though the peptide mapping approach generally gave a higher free thiol content.

The authors conclude that compared to Ellman’s assay, the subunit approach is more sensitive, requires less sample and provides domain-specific information of the free thiol content. Compared to peptide mapping, the subunit method is faster, less labour intensive and lacks dependence on labelling efficiency. Finally, it demonstrated promise in the quantification of free thiols in a high throughput manner with domain specific information available.

The developed method has successfully been applied to several in-house IgG1 mAbs with different hydrophobicity and isoelectric points.

 

*V. Faid Y. Leblanc N. Bihoreau G. Chevreux Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls, J. Pharm. Biomed. Anal. 149 (2018) 541-546, https://doi.org/10.1016/j.jpba.2017.11.046

 

For more information on FabRICATOR and FabALACTICA please visit the following pages:

The full text paper is available online:

FabRICATOR, SialEXO and OglyZOR in Middle-up HILIC/HRMS Approach

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In an article by Valentina D’Atri et al. recently published in Analytical Chemistry (2019), the scientists developed a middle-up HILIC/HRMS workflow for detailed characterisation of the Fc fusion protein etanercept.  The etanercept molecule consists of an IgG1 Fc domain fused to a tumour necrosis factor receptor (TNFR) and is used in the treatment of autoimmune diseases such as rheumatoid arthritis. The protein is highly glycosylated and contains numerous O- and N-glycosylation sites that require extensive characterization.

 

To develop a strategy that would work with a mass spec instrument of limited resolution, the authors used FabRICATOR enzyme to specifically digest the etanercept molecule and generate TNFR and Fc/2 subunits. Combinations of the O- and N- glycosidases SialEXO, OglyZOR and PNGaseF were applied to allow evaluation of the O- and N-glycosylation patterns of TNFR and Fc/2 respectively. In addition, complete deglycosylation allowed for primary structure analysis. By using a wide-pore HILIC stationary phase, appropriate separation of the subunits with different degrees of remaining glycans was achieved, and this significantly facilitated spectra deconvolution.

 

Applying this workflow, D’Atri and colleagues were able to assess the main PTMs, the subunit distribution of glycans, the overall N/O glycan composition and the sialylation profiles of each subunit.

 

Read more about the SmartEnzymes in this publication

 

Reference:

D’Atri, V. et al., 2018. Orthogonal Middle-up Approaches for Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High-Resolution Mass Spectrometry. Analytical Chemistry, 91(1), pp.873–880.

Antibody Sequence Analysis using GingisKHAN® and FabRICATOR®

September 28, 2018 | Applications, References |

  In an article by Luca Fornelli & Kristina Srzentic et al. recently published in Analytical Chemistry the authors present a workflow for antibody sequence determination by combining top-down and middle-down LC/MS. The authors analyzed the therapeutic antibody rituximab in its intact and fragmented form, using FabRICATOR and GingisKHAN to generate antibody subunits. By combining the performance of multiple ion activation techniques and a new software tool with top-level and middle-level strategies, the authors achieved extensive sequence coverage and obtained valuable information on key quality attributes.

  Rituximab was fragmented using members of the SmartEnzymes™ family for the generation of various antibody subunits. GingisKHAN was used for generating intact Fc and Fab subunits by site-specific cleavage of IgG1 above the hinge region. In order to obtain antibody subunits Fc/2, Fd and LC the authors used FabRICATOR-digestion followed by reduction. The intact antibody and the antibody subunits were analyzed using reversed phase LC/MS coupled with three separate ion activation techniques, and analyzed using a new software tool for fragment ion deconvolution.

  The complementing features of the ion activation techniques provided high quality information for a low number of LC/MS experiments. The authors achieved sequence coverage equivalent to what is obtainable with bottom-up strategies. In addition, the authors were able to analyze quality attributes such as PTMs, chain pairing and intact antibody mass determination – properties otherwise lost after extended proteolysis. These results highlight the benefits of combining top-level and middle-level strategies for applications currently performed by bottom-level strategies.

GingisKHAN® (Kgp enzyme) is a cysteine protease that digests human IgG1 at a specific site above the hinge region. The enzyme generates intact Fc and Fab subunits in 60 minutes.

Learn more about GingisKHAN

Fornelli et. al., 2018. Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques.

Interview with Valegh Faid at LFB Biotechnologies in France

 

Unique enzymatic digestions in study of antibody disulphides

 

Valegh Faid and colleagues at LFB Biotechnologies in France have developed and published an assay to study antibody disulphide bonds using middle-up LC-MS (Faid et al., 2017). The combination of FabRICATOR® for digestion below the hinge and FabALACTICA™ for digestion above the hinge, generated three fragments from a human IgG1 antibody; the hinge peptide, Fab and Fc/2 fragments. These fragments were resolved using RP-HPLC and mass spectrometry and enabled analysis of antibody disulphide bridges and other quality attributes.

 

 

Interview with Valegh Faid, Scientist at LFB and first author of the paper:

 

Why are antibody disulphide bonds important?

 

Disulphide bonds are highly important because of their critical role in the stabilization of protein conformations. Breaking and/or scrambling of disulphide bond occur during manufacturing and storage of biotherapeutics which is a concern in terms of safety and efficacy. The monitoring of these product-derived impurities is mandatory during development operations in order to minimize these forms.

 

How did you come up with the idea to combine FabRICATOR (IdeS) and FabALACTICA (IgdE)?

 

We have been using IdeS for many years in order to cleave IgG’s below the hinge; following DTT reduction, more amenable fragments for RP-HPLC/MS analysis are generated as previously published by our laboratory (Chevreux et al., 2011). This middle-up analysis is fast and very informative regarding the protein sequence integrity and post-translational modifications. However, investigating the oxidative state of disulfide bridges is tricky and often involved a time-consuming peptide mapping in non-reducing conditions.

In this context, IgdE is an interesting enzyme that cleaves specifically IgGs above the hinge and without requiring reducing conditions as papain do. The combination of IdeS and IgdE in non-reducing conditions presents the advantage to generate specifically three fragments i.e. hinge, Fc/2 and Fab that are both easily separated by RP-HPLC and analysed by MS.

 

How does the new enzymatic assay compare to previous methods to study antibody disulphide bonds?

 

Peptide mapping in non-reducing condition is the gold standard to investigate disulphide bonding of biotherapeutics. However, data interpretation is time consuming even if dedicated software to improve the treatment of data has largely improved. Although being slightly less informative than peptide mapping, this combined IdeS/IgdE middle-up approach increases the throughput for the investigation of free thiols and disulphide scrambling. Considering that other CQAs can also be monitored in the same experiment, it should be more applicable to routine use in process optimization, formulation screening and stability studies.

 

Would the assay be used in a QC setting relying solely on liquid chromatography separation?

 

The analytical workflow is robust and requires mere handlings of the antibody samples. Once the identification of each peak of the chromatogram is confirmed by MS, quantitation based on the UV detection is a current practice. Such analytical configuration involving an HPLC and a UV detection is actually common in most of QC labs and thus easily and robustly implementable.

 

How are you implementing this assay at LFB Biotechnologies?

 

This assay is integrated in our portfolio of analytical approaches for the analysis of mAbs currently in development, for process optimisation, batch characterization and stability studies.

 

 

Read more about FabALACTICA and FabRICATOR.

 

References:

 

Chevreux, G. et al., 2011. Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry. Anal Biochem. 15;415(2): pp. 212-4.

 

Faid, V. et al., 2017. Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls. Journal of Pharmaceutical and Biomedical Analysis, 149, pp.541–546.