Articles by Jonathan Sjögren

ADC Subunit Characterization of Drug Load and Glycosylation using HILIC-MS

FabRICATOR-HILIC-MS

In a collaboration headed by Davy Guillarme at University of Geneva, scientists have explored the characterization of subunits derived from antibody drug conjugates (ADCs) using hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (D’Atri et al. 2018).
The scientists used brentuximab vedotin (BV, Adcetris®), an approved ADC for treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). The BV consists of an antibody directed towards CD30, coupled to the vedotin toxin using cysteine conjugation chemistry. The random cysteine conjugation method results in a heterogeneous attachment of the drug, with differences in efficacy depending on the drug load. For this reason, the amount of conjugated toxins requires careful characterization. A key quality attribute of both antibodies and ADCs is the glycosylation profile, that may affect the stability, efficacy and safety. In this paper, a method to study ADC drug load and glycan profiling in a single experiment was demonstrated.

 

The intact ADC is around 150 kDa, which makes it very complicated to study details with high resolution. For this reason, D’Atri and colleagues used FabRICATOR digestion and reduction to generate specific antibody subunits of around 25 kDa, with increased resolution in both separation and mass determination. New wide-pore HILIC phase has enabled separation of larger molecules such as antibody subunits, and the team has already published a glycoprofiling strategy using HILIC on naked antibodies (Periat et al. 2016).

 

The coupling of HILIC separation to MS of subunits resulted in more detailed characterization of the subunits as compared to reverse phase separation (RP-HPLC). The relative percentage of each subunit aligned well with both methods of separation. However, additional positional isomers of the Fd’ fragment were observed using HILIC separation. Also, the glycoforms of the Fc/2 fragments were chromatographically separated, making mass deconvolution and determination easier. The authors conclude the middle-up HILIC-MS method to be orthogonal to RP-MS with the benefit that the methodology allows simultaneous characterization of drug load and glycosylation of the antibody drug conjugate.

 

FabRICATOR is a protease with a single digestion site below the hinge of IgG. The enzyme is widely used in middle-level analytical workflows for characterization of antibody based biopharmaceuticals. Learn more about FabRICATOR.

 

References

D’Atri, V. et al., 2018. Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry. Journal of Chromatography B, 1080, pp.37–41.

Periat, A. et al., 2016. Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals. Journal of chromatography. A, 1448, pp.81–92.

Subunit Comparability Analysis of Etanercept and Biosimilar

February 16, 2018 | References |

FabRICATOR+Enbrel-rakResearchers at the Free University of Berlin have performed a comparability study of the Fc-fusion protein etanercept and a biosimilar using FabRICATOR® and subunit analysis. The etanercept molecule consists of an IgG1 Fc domain fused to a tumor necrosis factor alpha receptor (TNFaR) and is used for autoimmune diseases such as rheumatoid arthritis. The originator etanercept (Enbrel®) was compared to its biosimilar Altebrel™ (AryoGen Pharmed), that has been launched in Iran.

The scientists used FabRICATOR to digest the Fc-fusion protein and studied the subunits, TNFaR and Fc/2 separately using middle-up mass spectrometry. Interestingly, differences in the glycosylation pattern,  the level of C-terminal lysine clipping and oxidation status of the two biopharmaceuticals were observed. The c-terminal lysine clipping was only observed in the originator molecule whereas the biosimilar showed no lysine clipping. Looking at the Fc/2 glycosylation profile using middle-up is a rapid way of determining the glycan content and the relative abundance of the species. In this case, the pattern was similar although the peak intensities differed, indicating a variation between the originator and the biosimilar.

Taken together, this paper highlights the use of FabRICATOR for comparability assessment of Fc-fusion proteins and shows that the middle-level approach can be used for fingerprinting of originator and biosimilar biopharmaceuticals.

Find the article using this link:

Montacir, O. et al., 2018. Physicochemical Characterization, Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept. The protein journal, 8(6), pp.1136–16.

Consortium for Top Down Proteomics uses FabRICATOR and GingisKHAN

October 13, 2017 | Applications |

FabRICATOR and GingisKHAN

The Monoclonal Antibody Project initiated by The Consortium for Top Down Proteomics (CTDP) aims to assess top-down mass spectrometry approaches for antibody characterization. It is a multicentre study, where the participants use several workflows (for example top-down sequencing, middle-down sequencing and intact mass analysis) to investigate the detailed structure and modifications of three monoclonal antibodies. Both intact antibodies and antibody subunits are thoroughly investigated in the study, and Genovis FabRICATOR and GingisKHAN enzymes are used to produce the subunits. FabRICATOR digests antibodies into F(ab’)2 and Fc fragments, and GingisKHAN into intact Fab and Fc fragments, and detailed protocols on how to use these enzymes are available from our website (Genovis IgG Proteases) and also from CTDP’s newly launched website (Top Down Proteomics Methods). The project is coordinated by Yury Tsybin from Spectroswiss Sarl, Joe Loo from UCLA and Kristina Srzentic from Northwestern University, and preliminary results are planned to be presented this fall! For updates about the study results, visit Top Down Proteomics.

 

GingisKHAN Used in Antibody Lead Identification – Publication by Roche

September 11, 2017 | Products, References |

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“GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies.”

 

In this paper, the scientists at Roche Innovation Center Munich, Moelleken et al, discuss the use of GingisKHAN in antibody lead selection screening and affinity measurements. The binding strength of an antibody is called affinity and when developing an antibody based drug, the selection of the best lead candidate involves measuring the affinity to its target antigen. A traditional antibody has two binding regions and the characteristics of the binding involves either one or two binding sites, depending on the target molecule. For this reason, the binding of a single Fab fragment is important to study during lead selection. Traditional methods of generating Fab fragments includes unspecific digestion with enzymes such as papain or LysC, mild reduction, or recombinant expression of the Fab fragment. Methods involving unspecific proteases suffer from low reproducibility, unspecific and incomplete digestion, and requires optimization for each IgG molecule to obtain a homogenous pool of Fab fragments. Another drawback is that none of the traditional methods allow high-throughput generation of Fabs.

 

In this setting Moelleken et al turned to GingisKHAN and studied the digestion efficiency, specificity, and ease of use for antibodies, bispecific antibodies, and new molecules with more than two antigen binding domains. The conclusions from the paper indicate that GingisKHAN has a high degree of specificity above the hinge of human IgG1 with no overdigestion or incomplete digestion. The protocol using GingisKHAN can be used as a platform method since there is very limited optimization needed. Due to the specificity of GingisKHAN, Moelleken et al were able to study the binding of Fab fragments directly from the digestion mixture without the need for purification. This feature shortens the analysis time and allows higher throughput.

 

The authors conclude the following:

“In summary, we have shown that the GingisKHAN protease is highly suited to differentiate between affinity- and avidity-driven binding of human IgG1s monoclonal antibodies and bispecific antibody formats in a lean and efficient manner”. (Moelleken et al, 2017)

 

GingisKHAN is an enzyme from Genovis for specific digestion above the hinge of human IgG1 and generation of homogenous Fab and Fc fragments.

 

Read more about GingisKHAN

  • Specific digestion of human IgG1
  • 1 h incubation at 37°C
  • Available in 2000 units or as a Fab preparation kit

 

 

Moelleken, J. et al., 2017. GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies. mAbs, pp.1–12.

Middle level analysis of site-specific ADC

May 29, 2017 | References |

In a collaboration between Universite de Strasbourg, Pierre-Fabre, and Catalent Biologics a multilevel mass spectrometry analysis of a site-specific conjugated ADC was recently published in the journal mAbs. As a first step in the analysis, FabRICATOR (IdeS) was used to perform middle-up analysis and confirm the drug to antibody ratio, DAR. The scientists write on p.7: “Middle level analysis, which is now the first line strategy used in most laboratories for mAb and ADC analytical characterization, revealed a highly homogeneous sample in terms of drug load, with mainly DAR4 detected and low amounts of DAR3” (Botzanowski et al. 2017).

For intact mass analysis using top-down mass spectrometry, the ADC was deglycosylated using IgGZERO for rapid removal of the glycan heterogeniety on the intact ADC. For this approach the newer and improved GlycINATOR enzyme could have been used. Studies have shown that GlycINATOR (EndoS2) removes all glycans from IgG whereas IgGZERO (EndoS) leaves the high-mannose glycans (Sjögren et al. 2015).

FabRICATOR_Logo_GubbeIgGZERO_Logo_Gubbe


 

Botzanowski, T. et al., 2017. Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates. mAbs, ePublished ahead of print.

Sjögren, J. et al., 2015. EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans. Glycobiology, 25(10), pp.1053–1063.

Glycan analysis by LC/MS in regulated environments

April 28, 2017 | References |

A team of scientist from Quality Assistance in collaboration with Alain Beck from Pierre Fabre, have published a detailed article highlighting the analytical strategies for both N- and O-linked glycan analysis of biotherapeutics using LC/MS. The workflows for glycan analysis on antibodies includes FabRICATOR digestion and study of the Fc/2 fragment for identification of the Fc glycoforms. The researchers used widepore HILIC-MS to separate the individual glycoforms of the Fc/2 subunit of adalimumab and obtained the glycoprofile with increased chromatography and MS resolution as compared to intact analysis. When applying the same workflow to cetuximab, site specific glycan profiles of both the Fc and Fd glycosylation sites were obtained.

 

In the same paper, a workflow combining GlycINATOR (EndoS2) and FabRICATOR (IdeS) was applied to study the level of core afucosylation. This digestions, that can be carried out simultaneously in 30 min, results in a dramatic reduction of the complexity of the Fc/2 subunit. After this simple sample processing the level of GlcNAc, GlcNAc + Fucose, or aglycosylated Fc/2 fragments can be quantified using LC/MS.

 

FabRICATOR_Logo_Gubbe GlycINATOR_Logo_Gubbe

Find the paper available in open access here:

Largy, E. et al., 2017. Orthogonal liquid chromatography-mass spectrometry methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level. Journal of chromatography. A, 1498, pp.128–146.

Biosimilar Comparability Assesment using FabRICATOR

March 31, 2017 | References |

Biosimilars are gaining in popularity as patents of innovator drugs are expiring. The analytical strategies to characterize and assess the similarity between the innovator product and the biosimilar often involve liquid chromatography and mass spectrometry (LC/MS). In a recent paper from the Freie Universität Berlin, Montacir et al. studied Rituximab and follow-on molecules using FabRICATOR digestion, reduction, and middle-down LC/MS. Using this approach the researchers found differences in the level of c-terminal lysine clipping of the Fc/2 fragment, a sequence error in the Fd fragment (as previously reported by Beck et al 2014), and a pyro-glutamic acid formation in the light chain.

The comparability assessment of biosimilars and innovator drugs using middle-down LC/MS with FabRICATOR digestion have also been published by the FDA a couple of years ago (Wang et al. 2013). Wang et al. argues that the FabRICATOR middle-down approach is very suitable for rapid fingerprinting of complex molecules, due to the high robustness and specificity of the enzyme.

Fabricator_Text_Logo_110pxfabricator_30min


 

Montacir, O. et al., 2017. Comparability study of Rituximab originator and follow-on biopharmaceutical. Journal of Pharmaceutical and Biomedical Analysis. E-published ahead of print.

Wang, B. et al., 2013. Structural comparison of two anti-CD20 monoclonal antibody drug products using middle-down mass spectrometry. The Analyst, 138(10), pp.3058–3065.

 

Assessment of Oxidation using FabRICATOR and LC/MS


Oxidation of methionine residues in the Fc region of a therapeutic antibody may affect the binding of the antibody to Protein A and FcRn leading to difficulties in purification or increased clearence in vivo. For the variable regions of the antibody, oxidation may affect antigen binding or lead to increased immunogenicity. For these reasons, the propensity of an IgG molecule to become oxidized is a critical quality attribute to consider early in the selection of therapeutic antibody candidates. The team at Adimab have developed an high-throughput assay based on FabRICATOR digestion and LC/MS analysis to evaluate the oxidation levels of 121 clinical stage antibodies. The antibodies were digested with FabRICATOR for 30 min at 37°C and reduced with DTT to obtain Fc, Fd and LC, prior to analysis on LC/MS, an approach called middle-down. The scientists correlated the observed oxidations with a model of predicted solvent-exposed methionine residues. They authors discovered oxidation at antibodies experimentally that were not predicted in the model,  probably due to inaccurate crystal structures or differences in expression host.

 

Taken together, the paper by Yang et al demonstrates the robustness of the oxidation assay based on FabRICATOR digestion and subunit analysis. The 121 antibodies analyzed in the paper indicated this method applicable to early clone selection for evaluation of antibody oxidation at the subunit level.

 

 

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Generate and purify Fab fragments from human and mouse IgG using Genovis SmartEnzymes

Antibodies are important tools in several scientific research areas, and sometimes it is necessary to cleave antibodies to generate antigen binding (Fab) fragments. The Fab fragments can be used within e.g. imaging, removal of effector functions, infection biology, binding studies, and mass spectrometry.

In a recently published book (“Bacterial Pathogenesis, Methods and Protocols”, Humana Press, 2017), the Genovis Team has written a chapter of how to generate and purify Fab fragments from human and mouse IgG by using the bacterial proteases IdeS (FabRICATOR), SpeB (FabULOUS) and Kgp (GingisKHAN).

 

  • The FabRICATOR enzyme is derived from Streptococcus pyogenes, and generates F(ab’)2 fragments in all human IgG subclasses. A homogenous pool of Fab’ fragments can be generated under mild reducing conditions. Read more about FabRICATOR here.
  • The FabULOUS enzyme is also derived from Streptococcus pyogenes, and can be used to digest mouse IgG. Using light chain affinity resins, Fab fragments can be purified. Read more about FabULOUS here.
  • The GingisKHAN enzyme is derived from Porphyromonas gingivalis, digests the upper hinge and generates intact Fab fragments from human IgG1 in one step. Using a CH1-specific affinity resin, the Fab fragments can be purified. Read more about GingisKHAN here.

Pathogenesis Book
Find the full text paper here:
Sjögren, J. et al., 2017. Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp. Methods in Molecular Biology, 1535, pp.319–329.

First GingisKHAN Publication

February 27, 2017 | References |

Researchers at LFB Biotechnologies have published the first paper using the Genovis SmartEnzyme GingisKHAN. GingisKHAN digests human IgG1 antibodies in the upper hinge, resulting in intact Fab and Fc fragments. The paper published in Journal of Chromatography describes how charge variants of therapeutic antibodies are characterized in a long term stability study using ion exchange chromatography coupled to native mass spectrometry. FabRICATOR was applied to digest antibodies below the hinge region and generated good separation of the Fc fragments. To specifically investigate any variations in the Fab domain, GingisKHAN was applied to digest the antibodies in the upper hinge and generate Fab fragments of about 50 kDa. The specific digestion site in combination with native MS allowed both oxidations (+16 Da) and deamidations (+1 Da) to be characterized.

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Leblanc, Y. et al., 2017. Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: Case study after a long-term storage at +5°C. Journal of Chromatography B, 1048, pp.130–139.