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.


 

A New Assay to Study IgG Galactosylation in Serum

March 21, 2018 | Applications, Products |

IgGZERO-glycation

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.

 

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.

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.

Unique Enzymatic Digestions Allow Study of Antibody Disulphides

December 12, 2017 | Products, References |

Antikropp-klyvning-över-och-under-hinge

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). In the paper, 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, the Fab and the Fc/2 fragments. These fragments were separated using RP-HPLC on a diphenyl column and analyzed using mass spectrometry. This setup enabled analysis of both inter- and intrachain disulphide bonds as well as other quality attributes.

 

The disulphide bonds of a therapeutic antibody can serve as indicators for misfolded antibodies during antibody manufacturing and has also been directly linked to mAb stability and are considered a quality parameter during antibody manufacturing. Traditionally, the methods for investigating the disulphide bonds of antibodies include labelling free cysteine residues using Ellman’s reagent or fluorescent dyes, resulting in a measure of the overall free sulfhydryls. The new assay using FabALACTICA and FabRICATOR is based on the unique digestion sites of the enzymes and utilizes the fact that both enzymes works under non-reducing conditions. The authors conclude:

 

“This method is easy to use, generic for mAbs and presents the advantage to deal with the whole complexity of a single mAb molecule by generating three well separated smaller and less heterogeneous fragments. Besides a direct application to the detection of intra- and inter-chain free sulfhydryls, this combined digestion could be promising to investigate other mAbs quality attributes“ (Faid et al. 2017).


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Read more about FabALACTICA and FabRICATOR.

 

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.

 

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).

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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.

 

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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.