Articles by Jonathan Sjögren

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FabALACTICA

Using FabALACTICA® to Elucidate Proline Trans-cis Isomerization on a Trispecific Antibody

January 30, 2020 | References |
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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

OpeRATOR Presentation at GlycoBioTec

February 13, 2019 | Uncategorized |

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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.

 

 

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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.

OpeRATOR Publication from Johns Hopkins University

Scientists from the prestigious Johns Hopkins University School of Medicine have used OpeRATOR to develop a workflow to map O-glycosylated sites on proteins in very complex samples. O-glycoproteins are notoriously difficult to study due to the low abundance, high structural heterogeneity and low stability. Previous approaches using affinity enrichment or engineered cell culture systems either lack efficiency or are ill-suited forO-glycoproteomic studies of complex samples.

In the workflow developed by Weiming Yang and colleagues, protein samples such as serum or kidney tissue were digested with trypsin, immobilized onto beads through the N-terminus and treated with OpeRATOR and SialEXO. OpeRATOR is an endoprotease and derived from the gut commensal bacteria Akkermansia muciniphila that specifically cleaves peptides and proteins N-terminally of O-glycosylated serine or threonine residues. Therefore, only O-glycopeptides are released from the solid support and were identified using ETD mass spectrometry.

Using this workflow, Yang et al. were able to map over 3000 O-glycosylation sites from human serum, T cells and kidney tissue, almost doubling the number of known O-glycosylation sites. They were also able to detect and quantify the aberrant O-glycosylation patterns in kidney tumors, showcasing the potential use of such methodologies for both basic research and diagnostic purposes.

 

Meet the Scientist

We got the opportunity to interview the first author of the paper, Weiming Yang at Johns Hopkins University.

 

Weiming Yang

Tell us about yourself?
I am a Research Associate in Mass Spectrometry Core Facility in the Center for Biomarker Discovery and Translation (www.biomarkercenter.org) of the Johns Hopkins University. The Mass Spectrometry Core Facility carries large-scale proteomics with particular emphasis on protein glycosylation on proteome scale to elucidate functions of glycoproteins on biology and disease. Before this position, I was a postdoc fellow in the same lab and worked on innovative glycoproteomics methods and HIV research. My interest in protein O-linked glycosylation started from every beginning at Hopkins that I was able to identify an O-linked glycosylation site in HIV gp120 from the infectious virion. Later on, I developed a series of glycoproteomic methods to study protein N- and O-linked glycosylation. The development of novel glycoproteomic methodologies led to new areas toward the discovery of the biomarker for HIV reservoir and new insight into cancer biology.

 

What is new with the ExoO method you have developed?
The major advantage of EXoO is its applicability to analyze clinical samples that is a breakthrough and central to reveal the significance of protein O-linked glycosylation in diseases. Using EXoO, now, scientists can start to gain new insight into their biological systems regarding O-linked glycoproteins. O-linked glycoproteins are ubiquitous on the cell surface and extracellular environment that is highly relevant to new treatment for diseases and diagnostics. Also, the EXoO is advantageous to analyze mucin-type O-linked glycoproteins that cannot be easily analyzed by conventional methods. The EXoO method identifies a large number of O-linked glycosylation sites in the sample that may be easily identified by using other methods such as various enrichments coupled with ETD-MS/MS.

 

How did you perform the analysis prior to this method?
We tried to use the same solid phase method to immobilize the peptides but released O-glycopeptides using beta-elimination to study site of protein O-linked glycosylation. Beta-elimination is a chemical reaction that can tag the site of protein O-linked glycosylation but give some background release of peptides from the solid support. We tried ETD-MS/MS for O-linked glycopeptide analysis but the number of identification is lower than the use of the current method using EXoO to release the O-glycopeptides.

 

What are the benefits of applying Operator in the workflow?
The OpeRATOR enzyme is a key component in the workflow. The high specificity of OpeRATOR enabled release of site-specific O-linked glycopeptides from solid phase support. Therefore, the resulting glycopeptides are relatively pure for improved identification.

 

What can you tell us about what you currently are working on?
Currently, we are applying the method to study different diseases including cancers and HIV reservoir.

 

How would you describe the impact of OpeRATOR on the O-glycan field?
The discovery of OpeRATOR changes of the game in the field of O-linked glycoproteomics. It makes the analysis of large-scale and site-specific O-linked glycoproteome in clinical samples feasible. For O-glycans, the specificity of OpeRATOR is not completely clear that will need further investigation. O-glycans have many different structures. The glycomic methods may still be the best way to go.

 

What are your thoughts on the future of O-glycan analysis?
EXoO and OpeRATOR provide unique research tools to identify the site of O-linked glycosylation. So far, the evidence supports that core 1 Gal-GalNAc structure can be studied by the use of OpeRATOR. Glycomic method focus on the identification of all different O-glycan structures with linkage and quantitative information. In the future, the structures of O-linked glycans on the specific sites on the proteins can be revealed in a single workflow.

 

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For more information on OpeRATOR go the the following pages:

 

The full text paper is available online:
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Poster Presentations at PEGS Europe 2018

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This week, scientists from Genovis are presenting two poster at the Protein Engineering Summit in Lisbon, Portugal. The posters cover our O-glycan specific endoprotease Operator and the recently launched FabRICATOR-HPLC column for automated antibidy subunit generation. Check out the poster abstracts below:

An O-glycan Specific Endoprotease with Applications in Glycoprotein Analysis using LC-MS

Helen Nyhlen, Maria Nordgren, Stephan Björk, Rolf Lood, Fredrik Leo, Fredrik Olsson
Genovis AB, Sweden

Changes in protein glycosylation may have an impact on the structure and function of a glycoprotein and O-glycosylation has drawn more and more attention for its roles in a wide range of biological processes. Characterization of glycosylation is of growing importance for the development and quality control of recombinant glycoprotein drugs and biosimilars. The study of O-linked glycosylation within the field of glycoproteomics is however challenging due to complicated sample preparation, difficult analytical procedures and the lack of O-glycan specific enzymes.

An O-glycan specific protease originating from the mucin degrading bacteria Akkermansia muciniphila has been described previously. The enzyme is dependent on the presence of O-glycans for digestion and hydrolyzes the peptide bond N-terminally to O-glycosylated serine and threonine residues. This feature can be used for the generation of intact O-glycopeptides to study site occupancy and composition of O-glycans in various biologic samples. We present here workflows that enabled determination of O-glycan sites and composition for O-glycosylated biopharmaceuticals and for proteins in human serum.

The O-linked glycosylation sites of biopharmaceuticals were assessed by treatment with PNGaseF, sialidases, O-protease and/or trypsin overnight prior LC/MS. The unique MS/MS peptides obtained revealed and defined the O-glycosylated threonine and serine residues. Enrichment of O-glycoproteins from human serum was achieved in native conditions using an affinity binding resin for O-glycan protein based on agarose beads with immobilized inactive O-protease. The complex protein sample was desialylated during the incubation step for binding. Bound proteins were then eluted by urea and treated with PNGaseF, active O-protease and/or trypsin followed by RP-C18 or HILIC separation and ESI-QTOF/MS analysis. The resin displayed high affinity for core 1 mucin-type glycans. With this workflow peptides and O-glycopeptides, with site-specific information, from several serum proteins were identified.

To summarize, using the characteristics of the O-protease and the O-glycoprotein affinity binding resin, strategies for the characterization of O-glycosylated proteins from pure and complex protein samples have been developed. The O-protease and the O-glycoprotein binding resin are potentially useful tools for deep characterization of O-glycoproteins.

 

Rapid On-column Digestion for Automated Monoclonal Antibody Analysis

Stephan Björk, Andreas Nägeli, Maria Nordgren, Linda Andersson, Helen Nyhlen, Jonathan Sjögren, Fredrik Olsson
Genovis AB, Lund, Sweden

Monoclonal antibodies (mAbs) and other IgG-based biopharmaceuticals are a fast-growing market. The inherent heterogeneity of such biologics necessitates detailed characterization by liquid chromatography and mass spectrometry (LC-MS) during development and production. While bottom-up peptide mapping is still the gold standard for analysis of critical quality attributes, such approaches are resource and time intensive in terms of both data acquisition and analysis. Top-down and middle-down approaches are therefore gaining in popularity. Antibody subunit analysis has become a widely accepted analytical strategy for rapid characterization of therapeutic antibodies and related products. The IdeS enzyme specifically digests IgG just below the hinge, generating F(ab’)2 and Fc/2 fragments. Reduction of disulfide bonds yields fragments of 23-25kDa in size which are amenable to high-resolution mass spectrometry. The IdeS based middle-level LC-MS workflow therefore enables the analysis of multiple antibody quality attributes such as glycosylation, oxidation, and C-terminal lysine clipping.

Here we present a rapid and automatable solution for antibody subunit generation in an HPLC column format. FabRICATOR (IdeS) enzyme was immobilized on the column to allow for automated middle-level analysis in a 2D-HPLC setup. The mAbs are digested on-column in the first dimension and the resulting subunits are separated and analyzed in the second dimension by RP-HPLC. This could be achieved with minor modifications to an HPLC-MS setup and potentially be connected directly to a bioreactor for automated monitoring of an on-going mAb production. The column tolerates continuous operation at 37°C for >10 days without a significant decrease in digestion performance and delivers consistent results for Fc glycan analysis during the entire period of operation. Additionally, other critical quality attributes such as Fab glycosylation and lysine clipping could be monitored. FabRICATOR-HPLC provides a fast solution for antibody subunit generation while reducing sample handling errors and increasing throughput.

Introducing Rob Horsefield

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Genovis is expanding its sales and marketing organization at its Lund headquarters and has hired Rob Horsefield as Sales & Business Development Manager. Rob has multiple years of experience in the industry from sales in analytical chemistry, pharmaceutical development and protein chemistry.

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

 

Tell us a bit about yourself.
I moved to Sweden in 2004 from the UK and live outside Lund with my Swedish wife and two children. I am now a Swedish citizen and fluent in Swedish. In my spare time I am a passionate road cyclist and enjoy long rides with my friends.

 

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

Trustworthy.

 

Tell us a bit about your previous working life.

I have a background in protein biochemistry and did my PhD in membrane protein crystallography. After that I worked at Gothenburg University for four years and then AstraZeneca for three and a half years. But most recently I was with Agilent Technologies for six years selling their analytical instruments in Southern Sweden and more recently liquid chromatography as their product specialist for the Nordics.

 

What will your main focus be here at Genovis?

My focus is to be the trusted associate at Genovis for our customers. I believe in always putting our customers first and securing their satisfaction. I like to work in a structured manner, pay attention to details and be on-time.

 

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

I hope we can leverage my experiences in protein biochemistry research, the biopharma market and LC/LC-MS instrument sales to grow Genovis’s business in Europe and Asia, and further expand our customer base.

 

Four Quick Questions:

Coffee or tea?

Tea, very strong with milk. But not fruit tea. And I never drink coffee, no, really.

 

Aerosmith or Depeche Mode?

Depeche Mode of course.

 

Ice cream or candy?

Both please, lots of.

 

Cricket or Rugby?

Rugby for sure, but there is still a small space left in my heart for cricket too.

OpeRATOR™ Decodes O-glycans; Publication by FDA and Genovis

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Scientist at the Center for Biologics Evaluation and Research, Food and Drug Administration, have, in collaboration with Genovis, developed a method for analyzing O-glycosylated proteins based on a solid phase chemical modification and followed by OpeRATOR digestion. Using this method, up to 8-fold more O-glycosites were discovered as compared to previously reported data.

 

The method uses an on-bead system to capture tryptic peptides deglycosylated using PNGaseF from a glycoprotein mixture. First, the tryptic peptides are bound via the N-terminus to the beads, and subsequent modifications to the sugars can be carried out. Secondly, the OpeRATOR enzyme is applied to digest the peptide bond, N-terminal of the O-glycosylated serine or threonine. In this way, only O-glycosylated peptides will be cleaved off and enriched. The OpeRATOR digested peptides were then analyzed using LC-MS/MS.

 

OpeRATOR was launched at the American Society for Mass Spectrometry 2017 and the FDA team quickly became interested in this novel tool. The enzyme originates from Akkermansia muciniphila and has been engineered by Genovis for biotech applications and analytical workflows and denoted OpeRATOR. The enzyme binds to musin type O-glycans and cuts the protein backbone, N-terminally of the O-glycosylated site. OpeRATOR can be used to study site occupancy and composition of O-glycans on biopharmaceuticals and for O-glycomic workflows.

 

We establish the method on standard glycoproteins, confirming known O- glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity” (Shuang et al 2018)

 

The paper has been selected Editor’s choice in Analytical Chemistry and is available using the link below:

 

Shuang Yang et al., “Deciphering Protein O‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment,” Analytical Chemistry, June 3, 2018, 1–9, doi:10.1021/acs.analchem.8b01834.

 

More information on OpeRATOR and its applications:

 

https://www.genovis.com/products/enzymes-for-o-glycans/operator/

 

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mAb Deamidation Study using FabRICATOR® Digestion and HIC Separation

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Hydrophobic interaction chromatography (HIC) is often used in characterization of therapeutic antibody products due to its ability to separate direct or indirect structural changes in the studied protein. Scientists at Alexion have published a study where FabRICATOR (IdeS) was used to generate Fc and F(ab’)2 fragments of an antibody to study conformational changes of a monoclonal antibody (King et al. 2018).

 

Separation of the intact antibody on HIC reveled two major peaks that were collected and subjected to FabRICATOR digestion. After digestion, the Fc and F(ab’)2 fragments separated well, and the heterogeneity was localized to the F(ab’)2 domain. Variations in the Fc were observed and attributed to oxidation modifications. Peptide mapping of the domains were carried out and a 1 Da difference was localized , indicating deamidation of Asn to either Asp or isoAsp in the complementarity-determining region (CDR) of the light chain. The observed difference in HIC separation pattern was also linked to changes in antigen binding, since the deamidation of the Asn residues reduced the binding of the antibody to its target antigen.

 

Taken together, this paper indicates that a single deamidation in the light chain changed the hydrophobicity profile of the antibody and impacted the antigen binding. The use of FabRICATOR (IdeS) digestion and HIC separation could serve as a quick screening assay to study deamidation changes in the F(ab’)2 domain.

 

 

King, C. et al., 2018. Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis. Journal of Chromatography B, 1085, pp.96–103.


 

Antibody Glycation Study using Intact LC-MS

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A new study from Janssen by Mo et al, demonstrates the use of intact mass spectrometry to determine the levels of glycation on therapeutic antibodies. To perform the assay, the authors used IgGZERO for rapid removal of the Fc-glycans.

 

Glycation occurs when reducing sugars such as glucose, galactose or fructose, reacts with protein amino acids through the Maillard reaction, and results in attachment of sugars to the protein. For therapeutic antibodies, glycation not only increases the heterogeneity of the drug but may also affect safety and efficacy.

 

To study the level of glycation on antibodies, the authors used both intact mass of the reduced antibody and peptide mapping to find the +162 Da mass shift indicating an addition of a hexose sugar. The Fc-glycan of an antibody contain 0, 1 or 2 galactose sugars that also gives a mass shift of 162 Da. To specifically remove the Fc-glycans, the scientist used IgGZERO (EndoS) from Genovis. Using this enzymatic pretreatment, the authors could determine glycation levels using intact mass spectrometry.

 

The authors found the peptide mapping and the intact LC-MS to give correlating results but conclude: “intact LC- MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing”(Mo et al. 2018).

 

 

Find the full text of the paper here:

Mo, J. et al., 2018. Quantitative analysis of glycation and its impact on antigen binding. mAbs, 154, pp.1–10.