GlyCLICK PET-tracers in Quantitative Imaging to Predict Immunotherapy Response

February 26, 2020 | References |

Scientists at Minerva Imaging demonstrate the potential of site-specific immuno-PET tracers as early identifiers of immune response activation using in vivo imaging. 

 

The programmed cell death protein (PD-1) on immune cells and its corresponding tumor-associated ligand (PD-L1) have emerged as effective targets for immunocheckpoint therapy. To date, the selection of patients eligible for PD-L1 blockade therapy and response rate monitoring is guided by immunohistochemistry of randomly sampled biopsies. This method is not only invasive and prone to errors, but also poorly reflects the heterogeneity and potential metastasis of the tumor.

 

Immunoimaging using PET-tracers is an attractive option to overcome these challenges since it provides a more comprehensive portrayal of the tumor and its temporal dynamics in vivo. In this study, Christensen et al. developed a site-specifically labeled immuno-PET tracer using a GlyCLICK-conjugated anti-PD-L1 antibody. The PET-tracer was used for quantitative detection of PD-L1 expression in order to non-invasively monitor radiotherapy-induced changes and demonstrate the predictive value of such tracers prior to PD-L1 blockade immunotherapy.

 

The anti-PD-L1 antibody was site-specifically conjugated with DIBO-functionalized DFO chelators using the GlyCLICK technology. Chelated conjugates were then radiolabeled with 89Zr to generate PET-tracers carrying two radioisotopes per antibody (DOL=2). Comparing conjugation strategies, the authors found that the GlyCLICK-conjugated antibodies displayed higher immuno-reactivity, stability and affinity compared to random conjugates. In vivo PET imaging and ex vivo biodistribution showed clear PD-L1-specificity of the GlyCLICK-tracers that allowed for the detection of different PD-L1 expression levels among mouse models of human and murine cancer. Importantly, the authors were also able to monitor therapy-induced changes of syngenetic mouse models in a combination study using XRT and anti-PD-L1 therapy. The tumor-to-muscle ratio of GlyCLICK-tracers enabled the scientists to obtain results predictive of response to PD-L1 immunocheckpoint inhibition.

 

Christensen et al., 2019. Quantitative PET imaging of PD-L1 expression in xenograft and syngeneic tumour models using a site-specifically labelled PD-L1 antibody. Journal of Nuclear Medicine and Molecular Imaging. doi: 10,1007/s00259-019-04646-4

 

Learn more about how GlyCLICK works by watching the GlyCLICK Movie.

 

Read more about GlyCLICK on the Product page or download our GlyCLICK Poster

 

 

 

 

 

SmartEnzymes Poster Highlights 2019

February 25, 2020 | Conferences, Products |

Which are the top five poster from Genovis in 2019? We have selected the posters that are both popular with our website visitors and that describe the most exciting new applications using SmartEnzymes. Click on the images to download the full posters.

 

1) ASMS Poster, 2019

1 – Analysis of O-glycosylated-Biopharmaceuticals using an O-glycan Dependent Endoprotease and LC-MS (ASMS, 2019)

In this collaborative work, we set out to combine our novel, specific enzymes with the latest LC-MS technologies from Thermo Fisher Scientific in order to improve and simplify analytical workflows for biotherapeutics. We demonstrate in-depth characterization of O-glycosylated biopharmaceuticals and quantitative comparison of O-glycosylation patterns. We also present a workflow for total deglycosylation of heavily glycosylated biopharmaceuticals, allowing for intact mass spectrometry analysis without interference from glycan heterogeneity.

 

2) World ADC Poster, 2019

2 – Robust Generation of Site-specific and Homogeneous Antibody Conjugates using GlyCLICK® (World ADC, 2019)

In this work, we present details on the enzymatic processing of the Fc-glycans that result in the homogenous conjugates, and confirmed it by mass spectrometry. The immunoreactivity of the conjugated antibodies was studied using surface plasmon resonance and toxicity by a dose-dependent response of a DM1 GlyCLICK conjugated trastuzumab (T-DM1).

 

CASSS AT Poster, 2019

3 – An Automated Workflow for Analysis of Monoclonal Antibody Subunits (CASSS AT 2019)

Here we present a rapid, automated solution for antibody subunit generation and analysis using a standard HPLC-MS setup with only minor modifications. FabRICATOR® (IdeS) enzyme was immobilized in an HPLC column format to allow for easy on-column digestion of IgG-based biologics followed by RP-HPLC-MS analysis. This facilitates a fully automated, completely hands-off workflow for analysis of several critical quality attributes.

 

4 – FabALACTICA® Generates Pure and Homogenous mAb Subunits that Facilitate 2D-NMR Spectroscopy Analysis (Festival of Biologics, 2019)

4) Festival of Biologics Poster, 2019

Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) allows for the precise atomic-level comparison of higher order structure (HOS) for IgG-based biopharmaceuticals. Since most of the approved therapeutic mAbs today have a human IgG1 backbone, the cysteine protease FabALACTICA (IgdE) can simplify the analysis. The enzyme digests human IgG1 at a specific site above the hinge, generating intact Fab and Fc fragments. In this work, we present a workflow for obtaining homogeneous Fab and Fc fragments that are ideal for evaluating HOS of the chimeric mAb infliximab using 2D-NMR.

 

5 – Complete and Rapid Desialylation of Therapeutic Glycoproteins using Immobilized SialEXO® (AET 2019)

The enzymatic performance of the Immobilized SialEXO column was tested on therapeutic glycoprotein substrates: human C1 inhibitor, etanercept, cetuximab, and human tissue-type plasminogen activator (tPA). Treated and native glycoproteins were then analyzed using released glycan analysis, antibody subunit LC-MS, and capillary iso-electric focusing. The sialidase column delivered complete de-sialylation of 0.5 mg glycoprotein after 30 min at room temperature. In summary, the new Immobilized SialEXO column provides robust and rapid desialylation ideal for routine analysis of biopharmaceuticals with a range of commonly used analytical techniques

 

AET Poster, 2019

 

 

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

First Paper on the FabRICATOR®-HPLC Column from Genentech

February 5, 2020 | References |

Scientist at Genentech and the University of Geneva have used FabRICATOR-HPLC to set up an automated workflow for the analysis of monoclonal antibodies by LC-MS.

 

Developing and manufacturing therapeutic antibodies requires the analysis of many product quality attributes. Robust and fast analytical methods are needed to support process development and quality control. In this new paper by Camperi et al., a novel workflow for automated subunit analysis of mAbs is described. Samples were digested on-column using FabRICATOR-HPLC and then transferred to a reverse phase HPLC column where the subunits could be separated and analyzed by mass spectrometry. This allowed for a completely hands-off analysis of critical quality attributes such as Fc glycosylation, glycation and C-terminal lysine clipping. More than 150 injections were performed on the same FabRICATOR-HPLC column with reliable results. Furthermore, analysis of a bispecific antibody revealed product related impurities due to mispairing of the chains.

 

FabRICATOR-HPLC enables on-line sample preparation for middle-level analysis and therefore reduces operator time and errors due to sample handling.

 

Camperi et al., 2020. Automated middle-up approach for the characterization of biotherapeutic products by combining on-line hinge-specific digestion with RPLC-HRMS analysis. Journal of Pharmaceutical and Biomedical analysis. doi: 10.1016/j.jpba.2020.113130

 

Link to FabRICATOR-HPLC Product page and Poster below

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FabALACTICA

Using FabALACTICA® to Elucidate Proline Trans-cis Isomerization on a Trispecific Antibody

January 30, 2020 | References |
FabALACTICA digestion

FabALACTICA digestion above hinge of a trispecific anti-HIV antibody (Masiero et al 2020).

Proline isomerization can occur in the antigen binding complementary determining regions (CDRs) of an antibody and impact the interaction with the antibody target. In this paper, scientists at Sanofi in Vitry-sur-Seine, France, found an unusual size exclusion chromatography profile of a trispecific anti-HIV antibody and determined that the heterogeneity originated from a proline isomerization.

 

Peptide bonds are planar due to the partial double bond character of the C-N bond and typically occur in a trans conformation since the cis confirmation is energetically unfavorable. However, proline with its ring structure, has a significantly lower energetic threshold and cis conformers occur more frequently as observed in crystal structures. The proline trans-cis isomerization plays various roles in biology where it can act as a molecular switch in immune function and cell signaling but it also plays a role in pathologies such as cancer and Alzheimer’s disease.

 

Scientists at Sanofi in Vitry-sur-Seine, France, found an unusual size exclusion profile of a trispecific anti-HIV antibody and determined that the heterogeneity originates from a proline isomerization.  In this paper,  Masiero et al., studied a trispecific antibody carrying three variable domains that displayed two non-resolved peaks in a UHPLC-SEC analysis. In combination with mass spectrometry, identical masses for the two peaks were observed. To dissect the origin of the heterogeneity, FabALACTICA was used to digest the antibody above the hinge and generate three fragments; an intact Fc fragment, a Fab fragment binding one antigen and a second Fab fragment with two antigen binding domains. Due to the specificity of FabALACTICA, the fragments could be analyzed using SEC-MS with high accuracy and the origin of the double peak was attributed to the domain with two antigen binding domains.

 

In summary, proline trans-cis isomerization can occur in the CDRs of antibodies and impact the analytical profile of the antibody. The complexity of multispecific antibodies can be reduced using specific enzymatic tools such as FabALACTICA for more detailed analysis.

 

  1. Masiero, A. et al., 2020. The impact of proline isomerization on antigen binding and the analytical profile of a trispecific anti-HIV antibody. mAbs, 12(1), p.1698128.

 

Link to FabALACTICA Product page and Poster below

SmartEnzymes™ in Antibody Subunit Analysis by Janssen and Celgene

January 23, 2020 | References |

 

Scientists at Janssen and Celgene use SmartEnzymes to analyze symmetric and asymmetric structure–function relationships of modified bispecific antibodies.

 

Forced degradation studies are useful for quickly evaluating critical quality attributes. Such modifications may have different biological impacts depending on if they occur symmetrically, in both chains of the antibody, or asymmetrically, in only one chain.

 

In the recently published article, Evans et al. first generated symmetrically and asymmetrically oxidized or deamidated samples. They then used two SmartEnzymes, IgGZERO® and FabRICATOR®, to do middle level mass spectrometry analysis, also referred to as antibody subunit analysis. They first employed IgGZERO to deglycosylated the N-glycans from the Fc region, FabRICATOR was used to digest the antibodies below the hinge and a reduction step resulted in subunits in the range of 25kDa – ideal for MS analysis.

 

Using this approach, the authors could probe the impact of symmetrical and asymmetrical oxidation or deamidation on IgG1 binding to the FcRn receptor or on protein A, respectively. The authors propose their technique, with subunit analysis, is ideal as a platform method for monitoring these or other modifications where symmetry might be crucial.

 

Read more about IgGZERO or FabRICATOR. 

 

Full article available here:
Evans, A. R. et al., 2019. Using bispecific antibodies in forced degradation studies to analyze the structure–function relationships of symmetrically and asymmetrically modified antibodies, mAbs, 11:6, 1101-1112, DOI: 10.1080/19420862.2019.1618675

 

Fluorescent Imaging using GlyCLICK®, an Interview with ImaGene-iT

January 14, 2020 | Interview |

Bo Holmqvist at ImaGene-iT gives us insight about microscopy imaging and how ImaGene-iT is using the site-specific GlyCLICK® technology to obtain high-quality fluorescence images.

 

Tell us about yourself and ImaGene-iT? 

 

I am CSO at ImaGene-iT, an independent contract research company supporting the life science industry and academia. Concurrently, as associate professor in experimental pathology, I have a research background in histology and neurobiology with extensive experience in microscope imaging and digital image analysis. As a principal investigator at ImaGene-iT, I perform most of the quality assessments using high-resolution confocal microscopy along with imaging for further digital analysis.

Our projects comprise a variety of questions and involve various types of biological samples on both the histological and cellular levels. At ImaGene-iT, we are engaged in finding suitable solutions and strategies for our customers and partners, concerning the choice of experimental labeling techniques combined with the optimal microscope and image analysis technique.

 

What are the key aspects of experimental design in imaging experiments? 

 

From the question at hand and to obtain the best end result, we want to participate in the whole chain of tailor-made procedures. This includes the initial handling of samples, the choice of labeling technology and the best suited detection and imaging equipment. This allows us to extract the most relevant data possible for high quality image analysis.

 

What are the advantages of using the GlyCLICK technology? 

 

In our tests, antibodies with GlyCLICK conjugated fluorophores work very well for imaging in both in vitro cell and tissue analyses. The conjugates provide an excellent signal-to-noise ratio for detection and imaging with fluorescence microscopy. For confocal microscope analysis, GlyCLICK conjugated antibodies give an optimal range of intensity levels. This allows us to obtain the ideal settings for detection of both the lower and higher intensity levels with a reduced loss of signal and saturation. The optimal range of detected intensity levels improves the quality of assessment as well as the digital imaging which in turn improves the image analyses.

 

What does the future of imaging look like? 

 

In our field, one of the major recent advances in imaging is the ability to extract a large amount of data from images, for example from multi-fluorescence labeled samples. The use of markers with a known number of conjugation sites per target molecule, such as the GlyCLICK technology offers, can significantly improve quantitation possibilities for all types of imaging-based analysis. This opportunity benefits the fields of basic research and drug development and may form the basis for exciting diagnostic tools in clinical histopathology. The combined use of GlyCLICK conjugated fluorescence with small animal in vivo imaging could further expand the quality of quantitative data that can be collected for our clients, from the cellular level to the whole animal.

 

Read more about the Genovis GlyCLICK technology and its applications.

 

FabRICATOR®-HPLC for Automated Antibody Subunit Analysis

December 17, 2019 | References |

Middle-level analysis is a universally accepted analytical strategy for rapid characterization of antibodies. The FabRICATOR® (IdeS)enzyme specifically digests IgG below the hinge, generating  fragments that are suitable for high-resolution mass spectrometry. By immobilizing the FabRICATOR enzyme in an HPLC column format, a fully automated on-column digestion of IgG is possible.
 

In a recent article written by the Genovis team and published in Chromatography Today, an LC-MS workflow for automated middle-level analysis of monoclonal antibodies (mAbs) and mAbs-based biologics is described and evaluated. FabRICATOR-HPLC was shown to facilitate easy on-line sample preparation for middle-level LC-MS. On-column digestion of mAbs followed by on-column reduction and analysis by RP-HPLC and MS yielded results indistinguishable from those produced using an off-line sample preparation protocol.
 

“With minimal carry-over, low sample requirements and a tolerance for a wide concentration range, this method is very versatile. It is well suited for both routine analysis as well as more advanced applications such as automatic monitoring of mAb CQAs during production in a bioreactor.”
 

The full article is available here:

Nägeli et al., 2020. On-column digestion of mAbs for automated middle-level analysis by LC-MS. Chromatograpy Today
 

Read more about FabRICATOR-HPLC and automated antibody subunit analysis

SmartEnzymes™ Simplify Characterization of Charge Variants by Native Mass Spectrometry

November 19, 2019 | References |

Monoclonal antibodies (mAbs) are complex macromolecules that undergo a wide range of post-translational modifications that can result in charge heterogeneity. Ion exchange (IEX) chromatography is considered the gold standard for characterization of charge variants. This approach separates charge variants and allows for the collection of isoforms for protein identification by mass spectrometry (MS). However, the collection of multiple fractions often needs to be combined with top-down and bottom-up characterization methods, which is both resource and time consuming.

 

In a recent study by LeBlanc et al., (2019) from LFB Biotechnologies, a newly developed cation exchange column technology was evaluated for its ability to separate charge variants under native conditions with direct coupling to MS. In total, three different mAbs with high basicisoelectric points (pI) and a monoclonal antibody reference material from NIST were analyzed in both their native and proteolyzed forms. The fragments were obtained using the IgG-specific proteases FabRICATOR®, which digests below the hinge, and FabALACTICA®, which digests above the hinge.

 

The column was shown to separate mAbs with high basicpI, demonstrating that mAbs having a strong retention on cation exchange media could be separated. A good repeatability was achieved in terms of resolution, recovery and retention times. Overall, the results demonstrate how SmartEnzymes have made mAb charge variant characterization, which was formerly challenging, more user-friendly with an easy-to-replicate protocol.

 

Read more about FabRICATOR and FabALACTICA.

 

Leblanc, Y. et al., 2019. A generic method for intact and subunit level characterization of mAb charge variants by native mass spectrometry, Journal of Chromatography B. doi: https://doi.org/10.1016/j.jchromb.2019.121814

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.