Articles by Maria Ekemohn

FabALACTICA Facilitates the Structural Insight into SARS-CoV-2 Neutralizing Antibodies


The first steps of infection with SARS-CoV-2 is binding of a viral Spike protein to a host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Antibodies that block this interaction are emerging as early COVID-19 therapies, however, the neutralization potencies of the antibodies are less studied.
Read more »

New Application Note on Antibody Oxidation Analysis


A Rapid LC-MS Assay for Monitoring of mAb Oxidation at the Subunit Level
 
Methionine oxidation is considered a critical quality attribute of therapeutic antibodies and may impact the clinical safety and efficacy. Therefore, monitoring of methionine oxidations is required during the discovery, development, and production of therapeutic antibodies. Traditional methods to characterize oxidation rely on tryptic peptide mapping and LC-MS, a labor intensive and time- consuming process that generates large data sets and requires trained and skilled manual interpretations.
Read more »

Launching New Anti-FabRICATOR Formats!

May 7, 2021 | New Product, News |

We are happy to announce two new affinity purified antibody formats for detecting the FabRICATOR enzyme!
 
The Anti-FabRICATOR Affinity Purified is an affinity purified goat polyclonal antibody and
the Anti-FabRICATOR Affinity Purified Biotin Conjugated is an affinity purified and biotin-conjugated goat polyclonal antibody.
 
Read more »

FabRICATOR in an Evaluation of Mobile-phase Additives for LC-MS Characterization of mAbs


Biopharmaceuticals, including monoclonal antibodies (mAbs), have become an important class of therapeutics. The manufacturing procedure of mAbs is complex, and many possible variants of a particular mAb can be generated as a result of enzymatical and chemical modifications. Some of these modifications are critical for the efficacy and safety of the therapeutic mAb and are known as critical quality attributes (CQAs). CQAs need to be thoroughly monitored to ensure the quality and safety of the therapeutic agent.

Read more »

Genovis Teams up with Waters to Deliver Enzymatic Workflows for the Biopharma Industry


The new collaboration between Genovis and Waters Corporation is intended to bring fast and easy analytical workflows for biopharmaceuticals by combining the SmartEnzymes™ portfolio from Genovis with the unique instrumentation from Waters™. The lab work has already started, and we got a quick word with Andreas Nägeli, one of the scientists at Genovis contributing to this collaboration.

Read more »

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.

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.

Characterizing ADCs using FabRICATOR and middle-down MS

September 24, 2019 | Applications, Products |

The process of characterizing an antibody drug conjugate (ADC) requires the evaluation of critical quality attributes including primary sequence analysis and drug conjugation assessment. Addressing glycoprofile determination as well as drug load distribution and drug-to-antibody ratio (DAR) is however challenging using peptide mapping. In addition, further challenges arise from the increased hydrophobicity of the ADC and the risk of drug-linker dissociation in an MS/MS experiment.

   

In an article by Hernandez-Alba et al. (2019) the authors characterized a site-specific ADC using middle-down MS. They combined three different fragmentation strategies for improved sequence coverage and drug conjugation assessment. The site-specific ADC (DAR=4) was digested using FabRICATOR and reduced to generate homogenous Fc/2, Fd’ and LC fragments for analysis by multiple ion activation techniques. By combining MS/MS data obtained with HDC (hydrodynamic chromatography), ETD (electron-transfer dissociation) and UVPD (ultraviolet photodissociation) fragmentation modes, the scientists obtained valuable information with the advantages of minimal sample preparation and analysis time using middle-down MS. 

   

The UVPD mode showed better performance compared to ETD and HDC. This indicated that the performance of this activation technique was unaffected by the hydrophobicity of the ADC. The complementarity between UVPD and ETD was further highlighted for drug conjugation assessment by allowing primary sequence validation and accurate identification of drug conjugation and glycosylation sites. These results highlight the potential of middle-down MS as a complement in next-generation strategies for the characterization of  mAb-based compounds including ADCs. 

 
 

Read more about FabRICATOR and Applications of FabRICATOR.

   

Hernandez-Alba, O. et al., 2019. A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate using Middle-Down Mass Spectrometry. American Society for Mass Spectrometry, 30(8). pp. 1-11.