SmartEnzymes™ in a new approach to characterize ADCs

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. 

SmartEnzymes at BGI, an interview with Aaron Bailey

September 24, 2019 | Interview |

We got the opportunity to talk to Aaron Bailey at BGI Americas. From their newly setup MS labs, they use SmartEnzymes to help provide analytical services to their customers globally. Read on to find out more.

 
 Tell us about yourself and BGI?Aaron_Bailey5

Currently I work at BGI Americas as the Associate Director of Product Management for Mass Spectrometry Services. I am part of the scientific team based in San Jose, California, at the newly built Mass Spectrometry Center.

 
 How has the MS team set up the industrial platform there at BGI in San Jose?

Our mass spec center is a service and R&D laboratory focused on providing LC-MS solutions for proteomic research and biologic drug characterization. Our current LC-MS service platform plays to our core strengths in automated sample prep, multi-mode HPLC separations, high resolution Orbitrap MS, and advanced bioinformatic analysis. We have built a versatile system which can cater to a wide scope of proteomics and biologic drug characterization needs. In San Jose our Q Exactive HF-X BioPharma can be coupled to either nano-flow or analytical flow UHPLC, which essentially affords us the ability to provide LC-MS services for multi-omic researchers and pharmaceutical research and development.

 
 How does the MS team do sample preparation prior to MS analysis?

For emerging formats, including some Fc-fusion protein drugs which are already established in global markets, glycosylation can be both highly heterogeneous and often involve both N- and O-linkages and is often distributed on potentially many sites throughout the amino acid sequence. These projects require that glycosylations can be selectively removed in order to validate the identity of each identified PTM. We have had success using several deglycosidases including SmartEnzymes from Genovis. In a sort of combinatorial sample prep matrix these enzymes help objectively determine for each therapeutic protein how highly complex glycoform patterns obseved at the (completely unprocessed) intact mass level are specifically derived from the contributions of possibly several types of glycosylation events (i.e., N-linked vs. O-linked vs. sialylated). Each may individually involve heterogeneous distibution of the specific sugar types found at any particular amino acid site. While protein isoform distributions must be measured at the “intact” or “subunit” level, for example using FabRICATOR, site specific measurements must be performed using peptide mapping approaches. Most importantly, the two types of datasets must essentially agree on the findings, and the sample prep strategy described here is a powerful way to navigate this challenge.

 
 What are the advantages of using SmartEnzymes like FragIT, OglyZOR and SialEXO?

We found that the activity and purity of these SmartEnzymes was a good fit for our native LC-MS platform in the sense that we are able to efficiently deglycosylate our therapeutic proteins using very little amount enzyme which remains near or below the actual level of detection in our experiments. This means that in normal usage we are not seeing these enzymes (FragIT, OglyZOR and SialEXO) in our datasets, which is particularly important for our native LC-MS intact mass analyses. This is a critical point for us as it means that we can keep our analyses streamlined by avoiding the need to address any artifact data which are not specifically related to any therapeutic protein isoforms.

 
 

FcFusionSamplePrepOverview_BaileyBGIAmericas

 
 

What are the next steps for you?

The San Jose Mass Spectrometry Center uniquely allows BGI to offer state-of-the-art genomic and proteomic analytical services globally. In the coming year we plan to grow our team and add new LC and MS instrumentation to further increase our ability to support this market. We expect to see exciting new developments in integrating NGS and LC-MS datasets. We also plan to introduce additional mass spectrometry services which will allow us to provide even deeper support in our main focus areas of proteomics and biologics characterization.

 
 Download Aaron’s ASMS poster here.

 
 For more information on FragIT, OglyZOR and SialEXO please visit the following pages:

 Prodults

Introducing our new CFO, Johny Humaloja

September 9, 2019 | Genovis Team, Interview |

 

Johny joined the Genovis team in August, and he brings more than 25 years of financial experience to the company. We are very excited to have you in the team, Johny! Welcome!

 

Tell us a bit about yourself

I live in Malmö, in the southern part of Sweden and I have two daughters. I have a cat named Zelda. On my free time I like to play hockey with my friends and go out for a ride on my road bike.

 

If you were to describe yourself using only one word – what word would that be?

Committed

 

Tell us about your previous working life

I have more than 18 years of finance leadership experience in the life science industry, mainly from working in US public listed companies such as Biogen and Boston Scientific. Focus has been on the Nordic region, to develop businesses and execute operational improvements. At Biogen I got the opportunity to work as Plant Controller and run all the financial activities at their large-scale manufacturing plant in North Carolina.

 

What will your main focus be here at Genovis?

Day-to-day business and make sure to enable Genovis to continue to grow and expand the business model. It will be key for me to understand the different parts of the organization and give adequate financial information and support. Financial areas to focus on will be;internal control, project management, statutory accounting, taxes, manage outsourced services, and external reporting.

 

What do you believe will be the biggest opportunity in your new position as CFO?

I believe I can utilize skills and experiences in sales/commercial and production area to find new ways to do business and provide financial and strategic leadership.

 

4 quick questions:

Coffee or tea?

Coffee – Zoegas

Aerosmith or Depeche Mode?

Depeche Mode

Ice cream or candy?

Chocolate Ice cream

Soccer or ice hockey?

Hockey, Carolina Hurricanes

FragIT™ kit in Study Evaluating IgG Charge

September 2, 2019 | References |

 

Protein charge is a fundamental property that influences both the ability to interact with other molecules and the structure, solubility and stability of the protein.

In this collaborative work by Boehringer Ingelheim Pharmaceuticals, Janssen BioTherapeutics and University of New Hampshire, the charge measurements of twelve monoclonal IgG and their F(ab’)2 and Fc fragments are presented. Besides other interesting findings, it is suggested that mAb charge measurements is valuable when it comes to selecting candidate molecules for development.

FragIT kit was used to achieve the IgG subunits before charge evaluation. This kit consists of spin columns of an immobilized version of the FabRICATOR (IdeS) enzyme for antibody digestion and spin columns for affinity binding of the Fc fragments. Using this kit, the Fc and F(ab’)2 fragments were easily separated from each other, with no enzyme in the final preparation.

 

For more information about FragIT kit, please visit the following page:

FragIT kit

 

The full-text paper is available online:

Yang et al., 2019. IgG Charge: Practical and Biological Implications. Antibodies, 8(1), 24

 

Improved antibody-PET tracers for in vivo imaging with GlyCLICK®

Radioactively labelled antibodies are excellent immuno-PET tracers for evaluating in vivo distribution and performance of therapuetic agents. Site-specific conjugation at the antibody Fc glycan site by enzymatic remodeling allows for a uniform label distribution of such PET-tracers, compared to conjugates generated with conventional random labelling strategies.

In an article by Kristensen et al. (2019), the authors evaluated the stability, immunoreactivity and in vivo biodistribution of the radioactively labelled mAb Trastuzumab (Herceptin). Using GlyCLICK, the antibody was enzymatically modified with GlycINATOR (EndoS2) and conjugated with a DIBO-DFO chelator prior to 89Zr radioactive labelling. Comparing the GlyCLICK technology with ß-galactosidase remodelled conjugates and two random labelling techniques, the authors obtained valuable data on the overall performance of the various PET-tracers.

Antibodies subjected to site-specific labelling showed significantly increased in vitro stability and immunoreactivity compared to randomly labeled Trastzumab. Furthermore, using in vivo immuno-PET imaging, these conjugates also displayed superior tumor-targeting properties based on the successful detection of HER2-positive tumors in mouse models. These results highlight the advantages of site-specific antibody conjugation.
For more information on GlyCLICK please visit

Reference:
Kristensen, L. et al., 2019. Site-specifically labeled 89Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake for immuno-PET in a subcutaneous HER2-positive xenograft mouse model. Theranostics, 9(15). pp.4409-4420.

FabRICATOR® in capillary zone electrophoresis-native mass spectrometry

June 19, 2019 | References |

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.

 

 

product-box-fabricator

For more information on FabRICATOR please visit the following pages:

The full text paper is available online:

The Digested Guide to ASMS 2019

Many of Genovis’ customers have submitted abstracts for ASMS 2019 in Atlanta, in which the SmartEnzymes have been used. Below is a digested guide to these abstracts so that you can plan your days at ASMS.

To read the full poster abstracts, visit the Online Planner for ASMS, and paste the abstract titel in the search field.

 

Sunday, June 2

New Enzymatic Workflows for Analysis of O-Glycosylated Biopharmaceuticals

Andreas Nägeli

Thermo Fisher Scientific User Meeting

Pharma/BioPharma Breakout Session 9:30 AM – 12.15 PM

Marquis Ballroom C

Thermo-user-meeting-blog

Monday, June 3

Improved middle-down characterization of antibodies using multiple ion activation techniques and Proton Transfer Reaction on a modified Orbitrap mass spectrometer 

Romain Huguet

MP 676, Poster Session 10.30 AM – 2.30 PM

FabRICATOR® and GingisKHAN®

 

Direct Determination of Antibody Chain Pairing by Top-Down Mass Spectrometry using Electron Capture Dissociation and Ultraviolet Photodissociation 

Jared Shaw

Room B302-305, Oral Presentation 3.50 PM – 4.10 PM

FabRICATOR® and GingisKHAN®

 

Unraveling a complex immunoprotein profile in multiple myeloma with middle-down de novo sequencing and native mass spectrometry 

Valerie J Winton

Room A411-412, Oral Presentation 9.30 AM – 9.50 AM

FabRICATOR®

 

Middle Down Approach for the Characterization of Monoclonal Antibodies After Ides Digestion and ETD Fragmentation

Colin Wynne

MP 782, Poster Session 10.30 AM – 2.30 PM

FabRICATOR®

 

Tuesday, June 4

Analysis of Zika Viral Polyprotein N- and O-glycosylation Using a Novel Lectin-chemoenzymatic Enrichment 

Shuang Yang

TP 655, Poster Session 10.30 AM – 2.30 PM

OpeRATOR® and GlycOCATCH™

 

Comprehensive Characterization of Antibody Drug Conjugates Enabled by Top-down and Middle-down Mass Spectrometry Strategies

Eli J Larson

TP 601, Poster Session 10.30 AM – 2.30 PM

GlycINATOR®FabRICATOR® and GingisKHAN®

 

Wednesday, June 5

Analysis of O-glycosylated Biopharmaceuticals using an O-glycan dependent Endoprotease and LC-MS

Andreas Nägeli

WP 334, Poster Session 10.30 AM – 2.30 PM

OpeRATOR® and OglyZOR®

 

MALDI-in-source decay FT-ICR MS for top-down and middle-down characterization of monoclonal antibodies 

Simone Nicolardi

WP 032, Poster Session 10.30 AM – 2.30 PM

FabRICATOR® and GingisKHAN®

 

Application of a label-free and domain-specific free thiol method in monoclonal antibody characterization

Yi Pu

WP 040, Poster Session 10.30 AM – 2.30 PM

FabALACTICA® and FabRICATOR®

 

Collision induced unfolding experiments to decipher the structural regions of a hybrid monoclonal antibody

Thomas Botzanowski

WP 481, Poster Session 10.30 AM – 2.30 PM

IgGZERO®

 

Thursday, June 6

Intact and Subunit Mass Analysis Using Native Ion Exchange Chromatography Coupled to an Orbitrap Mass Spectrometer 

Qian Liu

ThP 663, Poster Session 10.30 AM – 2.30 PM

FabRICATOR®

 

Ultra-Fast Analysis of Intact Proteins Using SPE- TOF 

Kevin McCann

ThP 149, Poster Session 10.30 AM – 2.30 PM

FabRICATOR®

 

Isomeric linkage determination of Sialic acid on O-glycopeptides using O-protease and LC-MS/MS 

Jieqiang Zhong

ThP 071, Poster Session 10.30 AM – 2.30 PM

OpeRATOR®

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® driven middle-down glycan analysis using NMR

March 15, 2019 | References |

Blog 2

 

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.

 

product-box-fabricator

For more information on FabRICATOR please visit the following pages:

The full text paper is available online: