SmartEnzymes™ in the Manufacturing Process for a Biological Drug

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

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:

• FabRICATOR Product Page
• FabALACTICA Product Page
• Generation of Antibody Fragments

Genovis Certified to ISO 9001:2015

The Digested Guide to ASMS 2018

Interview with Valegh Faid at LFB Biotechnologies in France

Unique enzymatic digestions in study of antibody disulphides

Valegh Faid and colleagues at LFB Biotechnologies in France have developed and published an assay to study antibody disulphide bonds using middle-up LC-MS (Faid et al., 2017). The combination of FabRICATOR® for digestion below the hinge and FabALACTICA™ for digestion above the hinge, generated three fragments from a human IgG1 antibody; the hinge peptide, Fab and Fc/2 fragments. These fragments were resolved using RP-HPLC and mass spectrometry and enabled analysis of antibody disulphide bridges and other quality attributes.

Interview with Valegh Faid, Scientist at LFB and first author of the paper:

Why are antibody disulphide bonds important?

Disulphide bonds are highly important because of their critical role in the stabilization of protein conformations. Breaking and/or scrambling of disulphide bond occur during manufacturing and storage of biotherapeutics which is a concern in terms of safety and efficacy. The monitoring of these product-derived impurities is mandatory during development operations in order to minimize these forms.

How did you come up with the idea to combine FabRICATOR (IdeS) and FabALACTICA (IgdE)?

We have been using IdeS for many years in order to cleave IgG’s below the hinge; following DTT reduction, more amenable fragments for RP-HPLC/MS analysis are generated as previously published by our laboratory (Chevreux et al., 2011). This middle-up analysis is fast and very informative regarding the protein sequence integrity and post-translational modifications. However, investigating the oxidative state of disulfide bridges is tricky and often involved a time-consuming peptide mapping in non-reducing conditions.

In this context, IgdE is an interesting enzyme that cleaves specifically IgGs above the hinge and without requiring reducing conditions as papain do. The combination of IdeS and IgdE in non-reducing conditions presents the advantage to generate specifically three fragments i.e. hinge, Fc/2 and Fab that are both easily separated by RP-HPLC and analysed by MS.

How does the new enzymatic assay compare to previous methods to study antibody disulphide bonds?

Peptide mapping in non-reducing condition is the gold standard to investigate disulphide bonding of biotherapeutics. However, data interpretation is time consuming even if dedicated software to improve the treatment of data has largely improved. Although being slightly less informative than peptide mapping, this combined IdeS/IgdE middle-up approach increases the throughput for the investigation of free thiols and disulphide scrambling. Considering that other CQAs can also be monitored in the same experiment, it should be more applicable to routine use in process optimization, formulation screening and stability studies.

Would the assay be used in a QC setting relying solely on liquid chromatography separation?

The analytical workflow is robust and requires mere handlings of the antibody samples. Once the identification of each peak of the chromatogram is confirmed by MS, quantitation based on the UV detection is a current practice. Such analytical configuration involving an HPLC and a UV detection is actually common in most of QC labs and thus easily and robustly implementable.

How are you implementing this assay at LFB Biotechnologies?

This assay is integrated in our portfolio of analytical approaches for the analysis of mAbs currently in development, for process optimisation, batch characterization and stability studies.

Read more about FabALACTICA and FabRICATOR.

References:

Chevreux, G. et al., 2011. Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry. Anal Biochem. 15;415(2): pp. 212-4.

Faid, V. et al., 2017. Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls. Journal of Pharmaceutical and Biomedical Analysis, 149, pp.541–546.

Meet Our New Colleague Kevin Cook

We are happy and proud to have you on board, Kevin. Welcome to Genovis!

Tell us a bit about yourself.
Even though a Focused Generalist does not make perfect sense, I think it fits since I have a broad life sciences discovery background with a specificity towards LC-MS and orthogonal technology.

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

Curious.

Tell us a bit about your previous working life.

My on-the-job learning experiences, with small and large companies, started with DNA/RNA focused laboratory work, moved into LC-MS to support small molecule pharmacokinetics and drug metabolism, and has migrated into a focus on providing biologics sample preparation technology for LC-MS analysis.

What will your main focus be here at Genovis?

On behalf of the Genovis team, I have the pleasure of building relationships with scientists in the western US. I am excited to learn more about the challenges my colleagues in the industry are facing and how our team can help.

What do you believe will be the biggest opportunity in your new position as a Senior Application and Market Area Manager?

Genovis has built a customer centric reputation that shows in current products and formats, and how LC-MS scientists are utilizing these tools. Genovis has demonstrated the ability to rapidly respond to customer requests and I am looking forward to helping promote current products while building for the future through customer feedback and collaboratory efforts.

4 Quick Questions:

Coffee or tea? 

Coffee, lots of coffee…

Aerosmith or Depeche Mode?

At the moment, Eric Church.

Ice cream or candy?

Chocolate chip cookies.

Soccer or ice hockey?

American football though staying with the question, ice hockey – Chicago Blackhawks.

Study on Glycoform Heterogeneity using Enzymatic Digestion and Native Mass Spectrometry

In a study by Wohlschlager et al. (2018), FragIT™ kit was used to digest the Fc-fusion protein etanercept, and the resulting fragments were analyzed using high-resolution native mass spectrometry (MS). Native MS offers a higher spatial resolution at a lower charge state, enabling studies of glycan heterogeneity, and FragIT digestion reduces sample complexity, enabling a detailed annotation of glycoforms on complex compounds.

A detailed knowledge about structure and post-translational modifications (PTMs) is required for biopharmaceuticals to be approved for clinical use, and an important quality attribute that may affect both the efficacy and safety of biopharmaceuticals is glycosylation.

Etanercept is a highly glycosylated Fc-fusion protein that is used to treat autoimmune diseases such as rheumatoid arthritis, and it consists of the TNF-? receptor domain fused to the Fc domain of human IgG1. FragIT – an immobilized version of the FabRICATOR® (IdeS) enzyme – digests IgG from several species and subclasses at a specific site below the hinge region. The resulting fragments are easily purified using the Fc-specific affinity resin that, together with FragIT, comprises the FragIT kit.

In this study, the researchers analyzed the glycosylation of etanercept on both the intact level, and on the middle-down level after FragIT digestion. By combining native MS analysis with enzymatic remodelling of etanercept, a detailed annotation of glycoforms could be achieved and transferred from subunit to whole protein level.

The authors end by concluding:

“Comprehensive information on glycoform heterogeneity, fast analysis with minimal sample preparation and product-characteristic fingerprints render our method highly attractive for the quality control of biologics as well as for comparability studies following changes in the manufacturing process.”(Wohlschalger et al. 2018).

Read more about FragIT and FabRICATOR

References:

Genovis Launches GlycOCATCH™

Please welcome GlycOCATCH™ to the Genovis SmartEnzymes™ family!

GlycOCATCH is an enrichment resin for affinity purification of O-glycosylated proteins and peptides. The resin is designed to bind proteins and peptides carrying O-glycans with high affinity, and it is provided in convenient spin columns to allow easy-to-use O-glycoprotein enrichment.

The applications of GlycOCATCH involve glycomics, specific enrichment or removal of O-glycoproteins and peptides, studies of O-glycosylation in complex samples and characterization of biopharmaceuticals.

The GlycOCATCH product is available for purchasing today.

Read more about GlycOCATCH here

A New Assay to Study IgG Galactosylation in Serum

In a study by Vanderschaeghe et al. (2018), a new assay to measure IgG galactosylation in serum has been developed. The setup includes hydrolysis of IgG Fc glycans using the IgG-specific endoglycosidase IgGZERO^® (EndoS).

A reduced level of IgG galactosylation in serum is a promising biomarker to evaluate the severity, determine the treatment and assess the efficacy of the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Crohn’s disease.

Traditionally, it has been difficult to study IgG galactosylation in serum because of the requirement to purify the antibodies, a procedure that is both complex and time-consuming. However, Vanderschaeghe et al. demonstrate a new assay where IgGZERO is used to efficiently hydrolyze serum IgG Fc glycans before analyzing galactosylation on high-throughput DNA sequencers. IgGZERO works on natively folded IgG, meaning that the assay can be performed on complex serum without first needing to purify the IgG, a feature that renders the assay both fast and simple.

The authors conclude by describing their new IgGZERO-based assay:

“… an important breakthrough towards the clinical implementation of the proposed biomarker” (Vanderschaeghe et al. 2018).

Read more about IgGZERO

Find the full text of the paper here:

Vanderschaeghe et al., 2018. Clinical assay for direct assessment of IgG galactosylation in serum using endoglycosidase S. BioRxiv.