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:

SmartEnzymes™ assist MALDI in-source decay FT-ICR Mass Spectrometry analysis

March 14, 2019 | References |

The Consortium for Top-down Proteomics is currently conducting a large inter lab study. They are developing methods for the analysis of intact mAbs and antibody subunits generated by digestion with either FabRICATOR or GingisKHAN. In this paper, van der Burgt et al. present a novel method for the analysis of mAbs based on MALDI in-source decay fragmentation coupled with high resolution FT-ICR mass spectrometry. The standard method for antibody sequence confirmation by MS is based on fragmentation using electron transfer dissociation (ETD). The MALDI-ISD based method yielded complementary fragments to those observed in ETD experiments, translating to increased sequence coverage. Using digestion with either FabRICATOR or GingisKHAN, a higher total sequence coverage could be achieved than for the intact mAbs. FabRICATOR digestion also allowed for direct analysis of Fc glycosylation by MALDI-FT-ICR without the need for LC separation.

 

Meet the Scientist

We got the opportunity to interview the last author of the paper, Dr Simone Nicolardi at Leiden University Medical Center.

 

Tell us about yourself?

DSC09608 (003)

I am a senior researcher at the Center for Proteomics and Metabolomics at Leiden University Medical Center (https://www.lumc.nl/org/proteomics-metabolomics/). My work is focused on the development of ultrahigh resolution mass spectrometry-based methods for the analysis of biomolecules such as glycans, peptides, and proteins. This includes the structural characterization of monoclonal antibodies for determination of primary amino acid sequence and post-translational modifications.

 

What is new with the MALDI in-source decay fragmentation you have published?

Our newly developed method combines the advantage of ultrahigh resolution mass measurements, obtained using high-end instrumentation and novel spectra processing software, with the efficient fragmentation provided by MALDI-in-source decay using 1,5-diaminonaphthalene as a reducing MALDI matrix. The main advantages are complementary sequence information compared to other mass spectrometric fragmentation techniques and the use of minimal sample preparation procedure that does not require separation techniques such as liquid chromatography.

 

What are the advantages of using FabRICATOR or GingisKHAN?

In MALDI-ISD experiments singly charged ions are generated from the protein backbone. Thus, a wide m/z-range is needed for the detection of all fragments generated from large compounds, such as monoclonal antibodies. Even in high-end MS instrumentation, this m/z-range is limited and sequence information is obtained from N- and C-terminal protein portions only. The use of FabRICATOR or GingisKHAN allows extending sequence information of heavy chains to more internal protein regions. In addition, after digestion mAb Fc portion can be analyzed directly by MALDI-MS allowing the detection of most abundant Fc glycoforms.

 

How do you view sample preparation prior to MS analysis of mAbs?

The structure characterization of mAbs is generally performed using a multi-level approach based on different analytical methods. Many of these methods are based on liquid chromatography (LC) electrospray ionization (ESI) MS. LC is used to separate mAb subunits and to remove ESI-non-compatible compounds such as salts. Our method is based on MALDI which is known to be more tolerant to salts in the sample. Thus, MALDI-ISD MS avoids laborious sample preparation steps that can lead to artificial modification of mAbs. Also, it comes with short analysis times in contrast to other chromatographic techniques.

 

What are you working on now?

Our attention is now on bispecific mAbs. We apply our MALDI-based method for the simultaneous analysis of all six different polypeptide chains generated after treatment with FabRICATOR and chemical reduction of disulfide bonds. Our aim is to develop a fast method for the monitoring of chemically induced mAb modifications.

 

Sequence Analysis

For more information on FabRICATOR or GingisKHAN please visit the following pages:

The full text paper is available online:

OpeRATOR Presentation at GlycoBioTec

February 13, 2019 | Uncategorized |

IMG_6888 2

Genovis was selected to present at the GlycoBioTec 2019 Conference in Berlin in January. Andreas Nägeli, Senior Scientist at Genovis, presented novel workflows based on the O-glycan specific OpeRATOR enzyme and GlycOCATCH coupled to LC-MS.

 

 

OpeRATOR_vit_Kant_500px
OpeRATOR-1200px

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.