Articles in the Category ”References”

Fully automated 3D LC-MS workflow for the characterization of antibody glycosylation

March 19, 2020 | References |

Genentech used FabRICATOR®-HPLC to develop a fast and automated 3D LC-MS workflow for the characterization of glycosylation on therapeutic antibodies.

 

The glycosylation profile is a critical quality attribute of biopharmaceuticals including antibodies. This heterogenous quality attribute can be tackled using a range of different analytical technologies including separation of released glycans , intact glycoprotein mass analysis, subunit mass analysis and many more. An antibody subunit approach using FabRICATOR (IdeS) combined with separation on hydrophilic interaction chromatography (HILIC) offers a promising strategy for characterization of IgG glycosylation. Typically, the FabRICATOR digestion is done manually, offline, prior to LC-MS. In a growing number of cases, like online monitoring of glycosylation, it is desirable to automate such a workflow.

 

Researchers at Genentech in collaboration with University of Geneva recently published an automated workflow using three dimensional separation coupled to mass spectrometry. To automate the workflow, they employed the FabRICATOR-HPLC column to deliver online antibody subunit generation. In their setup, this was followed by on column reduction and separation of the subunits using a reversed phase column, coupled to HILIC column to separate the Fc/2 glycoforms. In summary, the system incorporated three columns controlled by two valves: FabRICATOR-HPLC x Reduction/RPLC x HILIC/MS (Fig. 1 below).

 

Camperi et al. compared their automated workflow to the standard manual digestion by testing two of their candidate mAbs. The authors concluded that the 3D automated workflow, which took 95 minutes per sample, could deliver comparable data for all N-glycan variants for the Fc/2 subunit and therefore could be applied to the analysis of not only mAbs but also antibody drug conjugated (ADCs) and bispecific antibodies. Finally, the scientists emphasize that the key strength of the 3D approach was not just the automation aspect but also the fact that no tedious sample preparation was required, and that it is applicable to routine analysis tasks.
 


Fig 1. 3D-LC Workflow for automated middle-up analysis of mAbs (Camperi et al., 2020.)
 

Camperi et al., 2020. Development of a 3D–LC/MS workflow for fast, automated and effective characterization of glycosylation patterns of biotherapeutic products. Anal. Chem. 2020, 92, 6, 4357-4363. doi:10.1021/acs.analchem.9b05193

FabRICATOR® presents F(ab’)2 PET-tracers for Prognostic Imaging

March 18, 2020 | References |

Scientists at Minerva Imaging demonstrate the use of FabRICATOR® to generate a 64Cu-labeled F(ab’)2 PET-tracer for the detection of changes in T Cell response to combined radiation and immunoblockade therapy.
 

Current immunotherapy response-evaluation criteria are limited in discriminating responsive patients eligible for immunotherapy from non-responders. Accurate evaluations of progression are limited by pseudo-progression, a phenomenon where therapy-induced inflammatory events temporarily increase tumor volume, thereby delaying evidence of response. Routine and standardized clinical evaluations are needed that require predictive biomarkers combined with reproducible methods for monitoring biomarker expression and dynamics.
 

Targeting T cells as essential players in the anti-tumor immune response, Kristensen et al. developed a 64Cu-labeled F(ab’)2 against murine CD8a+ T cells. The scientists further demonstrate its potential as a prognostic PET-imaging biomarker

for immunotherapy response in mouse models of colorectal cancer. Rat anti-mouse CD8a antibodies were digested using the FabRICATOR enzyme to generate homogenous F(ab’)2 and Fc/2 subunits. Purification by preparative HPLC yielded isolated F(ab’)2 subunits for random chelation with p-SCN-Bn-NOTA and radiolabeling using the 64Cu isotope. Treatment response assessment was conducted using in vivo PET-imaging of tumor-bearing mice subjected to combined radiation and anti-CTLA-4 therapy.
 

The scientists found that the tumor-to-heart ratio of the PET-tracer increased with combined therapy, this was also confirmed by flow cytometry and IHC analysis showing increased tumor infiltration by CD8+ T cells. The prognostic value of the PET-tracer was further demonstrated using in vivo imaging as the scientists were able to distinguish responsive mice from non-responders prior to treatment-induced variations in tumor volume.
 

Kristensen et al., 2020. Monitoring CD8+ T Cell Responses to Radiotherapy and CTLA-4 Blockade Using [64Cu]NOTA-CD8a PET imaging. Mol Imaging Biol (2020).

 

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

 

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™ 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 |

Scientists 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

 

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