Deamidation
Asparagine deamidation of antibody products may occur during natural cell processing and degradation of the protein but also as a result of production and storage. Deamidation can be increased by high temperature and high pH. By utilizing FabRICATOR® and capillary electrophoresis, domain specific charge profiling has been achieved.
70.0
33.0
false
IsoAspartic acid
The isomerization of aspartic acid to isoaspartic acid (Iso-Asp) occurs spontaneously on proteins and if it occurs in the antigen binding part of an therapeutic antibody, it may have consequences for the clinical efficiency of the therapeutic agent.
FabRICATOR® has been employed in combination with hydrophobic interaction chromatography to separate the F(ab’)2 domains containging Iso-Asp residues.
FabRICATOR® has been employed in combination with hydrophobic interaction chromatography to separate the F(ab’)2 domains containging Iso-Asp residues.
68.0
21.0
true
Oxidation
Soluble antibody products are susceptible to oxidation. The oxidation reaction is increased when the product is exposed to light. Oxidation of therapeutic antibodies may have serious consequences for the functional properties of the antibody, including; decreased antigen binding, increased aggregation and immunogenicity.
By FabRICATOR fragmentation and chromatographic separation the oxidation levels of therapeutic antibodies can be estimated.
By FabRICATOR fragmentation and chromatographic separation the oxidation levels of therapeutic antibodies can be estimated.
60.0
44.0
false
Pyroglutamination
N-terminal glutamic acid and glutamine can spontaneously form a cyclic form of pyro-glutamic acid, a process known as pyro-glutamination (-17 Da). This can occur in the purification of the antibody, or as a result of pH and buffer composition. The formation of pyro-glutamic acid leads to increased hydrophobicity and can be monitored using antibody fragmentation by FabRICATOR®.
74.0
28.0
false
Glycation
Glycation is a non-enzymatic process where reducing sugars is added to lysine side chains or other free amines. Glycation occurs slowly at physiological levels of glucose but can be increased when sugar content is elevated such as in mAb formulation buffers. Major concerns of glycation of therapeutic mAbs is the development of immunogenicity, also reports have shown that antigen binding and complement activation may be negatively affected by glycation.
The following SmartEnzymes from Genovis has been used to study glycation:
• FabRICATOR® – The glycan on antibody fragments give better resolution
• IgGZERO® – by cleaving off the Fc glycans, sample complexity is dramatically reduced.
The following SmartEnzymes from Genovis has been used to study glycation:
• FabRICATOR® – The glycan on antibody fragments give better resolution
• IgGZERO® – by cleaving off the Fc glycans, sample complexity is dramatically reduced.
21.0
22.0
true
Multi-attribute
Methods of analyzing multiple quality attributes of biopharmaceuticals using SmartEnzymes.
46.0
65.0
true
Fab-glycosylation
Up to 30% of serum Fab fragments contain glycosylation sites and the glycans occupying these site have been shown to differ significantly from the Fc glycans on IgG. Typically, Fab glycans contains a higher degree of sialylated glycans as well as fewer fucosylated structures.
Genovis SmartEnzyme FabRICATOR® have been used to fragment antibodies to look at the Fc and Fab glycosylation separately both using LC/MS and capillary electrophoresis as analytical strategies.
Genovis SmartEnzyme FabRICATOR® have been used to fragment antibodies to look at the Fc and Fab glycosylation separately both using LC/MS and capillary electrophoresis as analytical strategies.
29.0
17.0
false
Amino acid sequence
To monitor the amino acid sequence of a theraputic antibody is crucial and is required by regulatory authorities. By fragmentation of the antobody into 25 kDa fragments using FabRICATOR® and subsequent middle-down mass spectrometry, variations in the amino acid sequence can be monitored.
37.0
27.0
false
Drug Load Distribution
Antibody drug conjugates (ADCs) carries drugs, normally attached to cysteine or lysine residues. The drug antibody ration (DAR) varies and as does the site of linkage. To determine the position of the linked drugs, ADCs have been fragmented using FabRICATOR® to generate Fd, Lc and Fc fragments and the drug load distribution could be determined using LC/MS analytical strategies.
40.0
43.0
true
Drug Antibody Ratio
For antibody drug conjugates (ADC) the number of drug per antibody, Drug Antibody Ratio (DAR) is an important quality parameter.
The following SmartEnzymes from Genovis have been used for DAR analysis:
• FabRICATOR® to generate Fd, Lc and Fc fragments
• IgGZERO® to reduce sample complexity and analyze the DAR on the intact antibody
The following SmartEnzymes from Genovis have been used for DAR analysis:
• FabRICATOR® to generate Fd, Lc and Fc fragments
• IgGZERO® to reduce sample complexity and analyze the DAR on the intact antibody
30.0
33.0
true
FcR inhibition
The Fc region of an antibody plays crucial roles in mediating the immune response directed by Fc-receptors on immune cells. The complement system is also dependent on the Fc domain of antibodies. Genovis SmartEnzymes specifically removes or inactivates (deglycosylates) the Fc domain and allows the Fab binding activity to be studied alone.
· FabRICATOR® - removes the Fc domain by specific cleavage below the hinge region and generates an intact F(ab’)2 domain
· IgGZERO® and GlycINATOR™ – specifically deglycosylates the Fc domain and renders the antibody unable to bind Fc receptors.
· FabRICATOR® - removes the Fc domain by specific cleavage below the hinge region and generates an intact F(ab’)2 domain
· IgGZERO® and GlycINATOR™ – specifically deglycosylates the Fc domain and renders the antibody unable to bind Fc receptors.
46.0
50.0
true
Aggregation
Aggregation may occur when monoclonal antibodies as formulated for therapeutic use. The susceptibility of different antibody candidates differs and the propensity to form aggregates can be monitored for quality by design approaches (QbD). The aggregates commonly occur via the Fab domains of the antibody and by digestion of the antibody by FabRICATOR®, the glycosylated Fc domain can be removed to reduce sample complexity and allow a more sensitive analytical approach.
46.0
75.0
true
C-terminal Lysine
Truncation of C-terminal lysine is commonly occurring on monoclonal antibodies (lysine clipping). The clipping of C-terminal lysines have recently been shown to maximize the antibody's complement dependent cytotoxicity (van den Bremer et al. 2015, mAbs).
By generating an Fc antibody fragment of 25 kDa by FabRICATOR® digestion, the degree of truncation of the Fc fragments can easily be monitored using liquid chromatography methods.
By generating an Fc antibody fragment of 25 kDa by FabRICATOR® digestion, the degree of truncation of the Fc fragments can easily be monitored using liquid chromatography methods.
54.0
75.0
false
Fc-glycosylation
Fc glycosylation affects the antibody’s ability to bind Fc receptor and have a major impact on the effector functions elicited by the antibody. The Fc glycosylation of therapeutic antibodies is a critical quality attribute and regulatory authorities require detailed glycan profiling on therapeutic mAbs.
The SmartEnzymes have been utilized to:
• Determine the Fc glycans on intact Fc fragments (25 kDa) using FabRICATOR
• Study the site specific glycosylation using FabRICATOR generated fragments
• Reduce sample complexity by deglycosylation using IgGZERO or GlycINATOR
The SmartEnzymes have been utilized to:
• Determine the Fc glycans on intact Fc fragments (25 kDa) using FabRICATOR
• Study the site specific glycosylation using FabRICATOR generated fragments
• Reduce sample complexity by deglycosylation using IgGZERO or GlycINATOR
54.0
66.0
false
Disulfide bridges
During cell harvesting of mammalian cells producing monoclonal antibodies, cell breakage may result in disulfide reduction of the interchain bonds of the antibody. Free thiols, pH, and solvent exposure can lead to disulfide rearrangements. The results of a rearrangement include decreased target affinity and loss of selectivity and specificity.
54.0
50.0
false
A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function
Wael Bahnan, Lotta Happonen , Hamed Khakzad, Vibha Kumra Ahnlide,
Therese de Neergaard , Sebastian Wrighton , Oscar Andre1, Eleni Bratanis , Di Tang , Thomas Hellmark , Lars Björck , Oonagh Shannon, Lars Malmström, Johan Malmström & Pontus Nordenfelt
Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vacci- nes or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had success- fully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M pro- tein. Such antibodies are typically non-opsonic. However, one anti- body could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody pri- marily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis-binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region cannot bypass the M protein’s virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings high- light the concept of dual-Fab cis binding as a means to access con- served, and normally non-opsonic regions, regions for protective antibody targeting.
https://www.embopress.org/doi/pdf/10.15252/emmm.202216208
https://www.genovis.com/products/antibody-digestion/fabalactica/
Antigen Binding
Monoclonal Ab *
LC/MS *
FabALACTICA
Development, validation, and implementation of a robust and quality control-friendly focused peptide mapping method for monitoring oxidation of co-formulated monoclonal antibodies
Chengdong Xu, Sumee Khanal, Nicholas A Pierson, Jorge Quiroz, Brent Kochert, Xiaoyu Yang, David Wylie, Christopher A Strulson
Monoclonal antibody (mAb) coformulation containing two therapeutic proteins provides benefits of improved therapeutic efficacy and better patient compliance. Monitoring of the individual mAb stability in the coformulation is critical to ensure its quality and safety. Among post-translational modifications (PTMs), oxidation is often considered as one of the critical quality attributes (CQAs) as it potentially affects the structure and potency. Although hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC) have been used to monitor overall protein oxidation, mass spectrometry of peptide digests resolved by LC methods can afford superior selectivity and sensitivity for specific PTMs. With the advent of the Quadrupole Dalton (QDa) mass spectrometer as an affordable add-on detector, implementation of targeted oxidation assays in development and quality control (QC) laboratories is now feasible. In this study, as the first effort to implement MS-based methods for antibody coformulation in QC laboratories, we developed and validated a high-throughput and robust focused peptide mapping method using QDa for simultaneous site-specific monitoring of oxidation of methionine and tryptophan residues in heavy-chain (HC) complementary determining regions (CDRs) of two co-formulated mAbs. The method was validated in terms of accuracy, precision, linearity, range, quantitation limit (QL), specificity, and solution stability per recommendations in ICH Q2. The method robustness was systematically assessed involving multiple sample preparation and instrument method parameters. The method met the validation criteria in GMP laboratories with excellent robustness and was implemented in both GMP and development environments.
https://link.springer.com/article/10.1007/s00216-022-04366-z
https://www.genovis.com/products/antibody-digestion/fabricator/
Oxidation
Monoclonal Ab *
LC/MS *
FabRICATOR *
Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions
Simon Daled, Sander Willems, Bart Van Puyvelde, Laura Corveleyn, Sigrid Verhelst, Laura De Clerck, Dieter Deforce and Maarten Dhaenens
Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment between runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX®, Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423.
https://www.mdpi.com/2227-7382/9/2/17/htm
https://www.genovis.com/products/gingisrex/
Other
Other / not known
LC-MS/MS
GingisREX
Evaluation of strategies for overcoming trifluoroacetic acid ionization suppression resulted in single-column intact level, middle-up, and bottom-up reversed-phase LC-MS analyses of antibody biopharmaceuticals
Maria A. Khalikova, Laimonas Skarbalius, Denis K. Naplekov, Siddharth Jadeja, František Švec, Juraj Lenčo
A wide range of strategies for efficient chromatography and high MS sensitivity in reversed-phase LC-MS analysis of antibody biopharmaceuticals and their large derivates has been evaluated. They included replacing trifluoroacetic acid with alternative acidifiers, relevancy of elevated column temperature, use of dedicated stationary phases, and counteraction of the suppression effect of trifluoroacetic acid in electrospray ionization. At the column temperature of 60 °C, which significantly reduces in-column protein degradation, the BioResolve RP mAb Polyphenyl, BioShell IgG C4 columns performed best using mobile phases with full or partial replacement of trifluoroacetic acid with difluoroacetic acid in the analysis of intact antibodies. Similarly, 0.03% trifluoroacetic acid in combination with 0.07% formic acid is a good alternative in analyzing antibody chains at 60 °C. Collectively, the addition of 3% 1-butanol to the mobile phase acidified with 0.1% formic acid was the most efficient approach to simultaneously achieving good chromatographic separation and MS sensitivity for intact and reduced antibody biopharmaceuticals. Moreover, this mobile phase combined with the BioResolve RP mAb Polyphenyl column was subsequently demonstrated to provide excellent results for peptide mapping of antibody biopharmaceuticals fully comparable with those obtained using a state-of-the-art column for peptide separation, thus opening an avenue for a single-column multilevel analysis of these biotherapeutics.
https://www.sciencedirect.com/science/article/abs/pii/S0039914021004331
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
LC/MS *
FabRICATOR *
In-capillary (electrophoretic) digestion-reduction-separation: A smart tool for middle-up analysis of mAb
Meriem Dadouch, Yoann Ladner, Claudia Bich, Jérôme Montels, Jacques Morel, Cherine Bechara, Catherine Perrin
Comprehensive characterization of physicochemical properties of monoclonal antibodies (mAbs) is a critical process to ensure their quality, efficacy, and safety. For this purpose, mAb analysis at different levels (bottom-up, middle-up) is a common approach that includes rather complex multistep sample preparation (reduction, digestion). To ensure high analysis performance, the development of fully integrated methodologies is highly valuable. Capillary zone electrophoresis is a particularly well-adapted technique for the multistep implementation of analytical strategies from sample preparation to detection. This feature was employed to develop novel integrated methodologies for the analysis of mAb at the middle-up level. Multiple in-line reactions (simultaneous reduction and digestion) were performed for the first time in the separation capillary. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used as an effective reducing agent under a broad pH range and IdeS (Immunoglobulin degrading enzyme from Streptococcus) as a highly specific enzyme for mAb digestion. Transverse diffusion of laminar flow profile (TDLFP) was applied for reactants mixing. Both in-line sample preparation and separation parameters were optimized under non-denaturing and denaturing conditions. The developed in-line methodologies provided good reproducibility and higher peak efficiencies comparing with off-line assays. They were successfully applied to different mAbs.
https://www.sciencedirect.com/science/article/abs/pii/S002196732100337X
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
CE
FabRICATOR *
State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis
Evolène Deslignière, Anthony Ehkirch, Bastiaan L. Duivelshof, Hanna Toftevall, Jonathan Sjögren, Davy Guillarme, Valentina D’Atri, Alain Beck, Oscar Hernandez-Alba, and Sarah Cianférani
Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.
https://www.mdpi.com/1424-8247/14/6/498
https://www.genovis.com/products/glyclick/
Monoclonal Ab *;ADC *
IM/MS *;SEC-MS *
FabRICATOR *;IgGZERO *;GlyCLICK
*****
Purification of antibody fragments via interaction with detergent micellar aggregates
Gunasekaran Dhandapani, Ellen Wachtel, Ishita Das, Mordechai Sheves & Guy Patchornik
The research described in this report seeks to present proof-of-concept for a novel and robust platform for purification of antibody fragments and to define and optimize the controlling parameters. Purification of antigen-binding F(ab′)2 fragments is achieved in the absence of chromatographic media or specific ligands, rather by using clusters of non-ionic detergent (e.g. Tween-60, Brij-O20) micelles chelated via Fe2+ ions and the hydrophobic chelator, bathophenanthroline (batho). These aggregates, quantitatively capture the F(ab′)2 fragment in the absence or presence of E. coli lysate and allow extraction of only the F(ab′)2 domain at pH 3.8 without concomitant aggregate dissolution or coextraction of bacterial impurities. Process yields range from 70 to 87% by densitometry. Recovered F(ab′)2 fragments are monomeric (by dynamic light scattering), preserve their secondary structure (by circular dichroism) and are as pure as those obtained via Protein A chromatography (from a mixture of F(ab′)2 and Fc fragments). The effect of process parameters on Ab binding and Ab extraction (e.g. temperature, pH, ionic strength, incubation time, composition of extraction buffer) are reported, using a monoclonal antibody (mAb) and polyclonal human IgG’s as test samples.
https://www.nature.com/articles/s41598-021-90966-1
https://www.genovis.com/products/igg-proteases/fabricator/fragit-kit/
Aggregation
Monoclonal Ab *
Other
FragIT / FragIT Kit
The monoclonal antibody combination REGEN-COV protects against SARS-CoV-2 mutational escape in preclinical and human studies
Richard Copin, Alina Baum, Elzbieta Wloga, Kristen E. Pascal, Stephanie Giordano, Benjamin O. Fulton, Anbo Zhou, Nicole Negron, Kathryn Lanza, Newton Chan, Angel Coppola, Joyce Chiu, Min Ni, Yi Wei, Gurinder S. Atwal, Annabel Romero Hernandez, Kei Saotome, Yi Zhou, Matthew C. Franklin, Andrea T. Hooper, Shane McCarthy, Sara Hamon, Jennifer D. Hamilton, Hilary M. Staples, Kendra Alfson, Ricardo Carrion, Jr., Shazia Ali, Thomas Norton, Selin Somersan-Karakaya, Sumathi Sivapalasingam, Gary A. Herman, David M. Weinreich, Leah Lipsich, Neil Stahl, Andrew J. Murphy, George D. Yancopoulos, Christos A. Kyratsous
Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. As rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment-induced emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of virus variants in SARS-COV-2 isolates found in COVID-19 patients treated with the two-antibody combination REGEN-COV, as well as in preclinical in vitro studies using single, dual, or triple antibody combinations, and in hamster in vivo studies using REGEN-COV or single monoclonal antibody treatments. Our study demonstrates that the combination of non-competing antibodies in REGEN-COV provides protection against all current SARS-CoV-2 variants of concern/interest and also protects against emergence of new variants and their potential seeding into the population in a clinical setting.
https://www.sciencedirect.com/science/article/pii/S0092867421007030
https://www.genovis.com/products/igg-proteases/fabalactica/fabalactica-fab-kit/
Monoclonal Ab *
SEC *
FabALACTICA
Characterization of charge variants of a monoclonal antibody using weak anion exchange chromatography at subunit levels
G. Ponniah, C. Nowak, A. Neill & H. Liu
An efficient strategy to characterize recombinant monoclonal antibody charge variants was established using weak anion exchange chromatography, LC-MS and IdeS digestion to allow subunit level characterization. Significantly higher resolution was achieved at subunit levels by weak anion exchange chromatography and LC-MS. In addition, subunit analysis localized potential modifications to either F(ab')2 or Fc fragments to facilitate further characterization. Peptide mapping of fractions from various charge variants after IdeS digestion identified aspartate isomerization, asparagine deamidation and glycation as the modifications. Although, aspartate isomerization does not generate net charge difference directly, it does generate antibody basic species. Antibodies with either isoaspartate or aspartate from deamidation showed different retention times by chromatography. Even more interestingly, the antibody contained succinimide as the isomerization intermediate, which though more basic compared to aspartate, eluted off the weak anion exchange column as an acidic species. The results demonstrated not only the utility of subunit level characterization but also the unpredictable chromatographic behavior of antibody charge variants.
https://www.ncbi.nlm.nih.gov/pubmed/28024755
https://www.genovis.com/products/fabricator/
Deamidation;Glycation;IsoAspartic acid
Monoclonal Ab *
LC/MS *
FabRICATOR *
Highly sensitive glycosylamine labelling of O-glycans using non-reductive β-elimination
Kenichiro Furuki, Toshimasa Toyo’oka & Kazutoshi Ban
When developing biopharmaceuticals, glycans are
the most important posttranslational protein modifications that
must be addressed because they affect the between-protein
interactions that maintain homeostasis. The glycan profile
may be defined as a critical quality attribute of a biopharmaceutical.
Comprehensive analysis of protein glycosylation
must overcome challenges such as the release, labelling, separation
and detection of O-glycans. In contrast, N-glycans can
be readily released non-reductively from peptide backbones
using an enzyme such as peptide N-glycosidase F. We developed
a highly sensitive protocol using RapiFluor-MS to label
glycosylamines for O-glycan analysis combined with a nonenzyme
treatment for efficient release of the reduced Oglycans
from the glycoproteins. Here we used the cytotoxic
T lymphocyte associated protein 4-immunoglobulin G (Ig)
fusion protein and fetuin as models for O-glycan analysis
and compared the analytical methods glycopeptide mapping,
2-AB labelling and RapiFluor-MS labelling. The structures of
major O-glycans and low-abundance O-glycans were successfully
identified using the third technique, which detected the
O-glycans with high sensitivity.
https://www.ncbi.nlm.nih.gov/pubmed/28091715
https://www.genovis.com/products/fabricator/
Fc-fusion protein *
LC/MS *
FabRICATOR *
Subunit mass analysis for monitoring antibody oxidation
Izabela Sokolowska, Jingjie Mo, Jia Dong, Michael J. Lewis, Ping Hu
Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.
https://www.ncbi.nlm.nih.gov/pubmed/28106519
https://www.genovis.com/products/fabricator/;https://www.genovis.com/products/iggzero/
Oxidation
Monoclonal Ab *
LC/MS *;RP-HPLC *
FabRICATOR *;IgGZERO *
****
Anti-Hinge Antibodies Recognize IgG Subclass- and Protease-Restricted Neoepitopes
Willem J. J. Falkenburg, Dirkjan van Schaardenburg, Pleuni Ooijevaar-de Heer, Michel W. P. Tsang-A-Sjoe,Irene E. M. Bultink, Alexandre E. Voskuyl,
Arthur E. H. Bentlage, Gestur Vidarsson, Gertjan Wolbink, and Theo Rispens
Anti-hinge Abs (AHAs) target neoepitopes exposed after proteolytic cleavage of IgG. In this study, we explored the diversity of
protease- and IgG subclass–restricted AHAs and their potential as immunological markers in healthy donors (HDs) and patients
with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). AHA reactivity against IgG-degrading enzyme of Streptococcus
pyogenes (IdeS)– or pepsin-generated F(ab9)2 fragments of all four human IgG subclasses was determined. AHA reactivity
against one or more out of eight F(ab9)2 targets was found in 68% (68 of 100) of HDs, 69% (68 of 99) of SLE patients, and
81% (79 of 97) of RA patients. Specific recognition of hinge epitopes was dependent on IgG subclass and protease used to create
the F(ab9)2 targets, as confirmed by inhibition experiments with F(ab9)2 fragments and hinge peptides. Reactivity against IdeSgenerated
F(ab9)2 targets was found most frequently, whereas reactivity against pepsin-generated F(ab9)2 targets better discriminated
between RA and HDs or SLE, with significantly higher AHA levels against IgG1/3/4. In contrast, AHA levels against
pepsin-cleaved IgG2 were comparable. No reactivity against IdeS-generated IgG2-F(ab9)2s was detected. The most discriminatory
AHA reactivity in RA was against pepsin-cleaved IgG4, with a 35% prevalence, ‡5.8-fold higher than in HDs/SLE, and significantly
higher levels (p < 0.0001). Cross-reactivity for F(ab9)2s generated from different IgG subclasses was only observed for
subclasses having homologous F(ab9)2 C termini (IgG1/3/4). For IgG2, two pepsin cleavage sites were identified; anti-hinge
reactivity was restricted to only one of these. In conclusion, AHAs specifically recognize IgG subclass– and protease-restricted
hinge neoepitopes. Their protease-restricted specificity suggests that different AHA responses developed under distinct inflammatory
or infectious conditions and may be markers of, and participants in, such processes.
https://www.ncbi.nlm.nih.gov/pubmed/27864476
https://www.genovis.com/products/fabricator/
Anti-hinge ab
IgG Polyclonal *
Native MS *;Other
FabRICATOR *
***
Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS
Yan Wang, Peng Feng, Zoran Sosic and Li Zang
Rapid and sensitive product quality analysis is important for real-time monitoring during biopharmaceutical development and manufacturing. However, low level of protein concentration and complex cell culture matrix pose challenges for product quality characterization at early stages of cell line selection and process development. Here, antibody protein directly from cell culture supernatant. Cell culture supernatant samples were characterized spectrometer. Under sample reducing conditions, multiple protein glycosylation attributes were determined on the heavy chain, whereas titer information was obtained from comparison of light chain signal intensity following sample spiking-in with heavy labeled mAb. Therefore, the protein expression and product quality can be monitored using is needed, whereas analysis time is within three minutes per sample. In addition, comparison of new method with traditional RP-LC-MS method using a set of time-course bioreactor cell culture samples has been performed. A good correlation of the levels of N-glycosylation attributes between ZipChip CE-MS of crude samples and RPLC-MS analysis following Protein A (ProA) purification step has been demonstrated.
http://908devices.com/wp-content/uploads/2017/06/ZipChip-CE_Publication_Anal-Bioanal-Tech-8-359-2017_Biogen.pdf
https://www.genovis.com/products/glycinator
Fc-glycosylation
Monoclonal Ab *
cIEF *
GlycINATOR *
****
Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach
Balázs Bobály, Valentina D’Atri, Alain Beck, Davy Guillarme, Szabolcs Fekete
Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.
http://www.sciencedirect.com/science/article/pii/S0731708517312098
https://www.genovis.com/products/fabricator/
Drug Antibody Ratio;Fc-glycosylation
Monoclonal Ab *;ADC *
HPLC *
FabRICATOR *
Antibody blood-brain barrier efflux is modulated by glycan modification
John M. Finke, Kari R. Ayres, Ryan P. Brisbin, Hali A. Hill, Emily E. Wing, William A. Banks
Background
Drug delivery to the brain is a major roadblock to treatment of Alzheimer's disease. Recent results of the PRIME study indicate that increasing brain penetration of antibody drugs improves Alzheimer's treatment outcomes. New approaches are needed to better accomplish this goal. Based on prior evidence, the hypothesis that glycan modification alters antibody blood-brain barrier permeability was tested here.
Methods
The blood-brain barrier permeability coefficient Pe of different glycosylated states of anti-amyloid IgG was measured using in vitro models of brain microvascular endothelial cells. Monoclonal antibodies 4G8, with sialic acid, and 6E10, lacking sialic acid, were studied. The amount of sialic acid was determined using quantitative and semi-quantitative surface plasmon resonance methods.
Results
Influx of IgG was not saturable and was largely insensitive to IgG species and glycosylation state. By contrast, efflux of 4G8 efflux was significantly lower than both albumin controls and 6E10. Removal of α2,6-linked sialic acid group present on 12% of 4G8 completely restored efflux to that of 6E10 but increasing the α2,6-sialylated fraction to 15% resulted in no change. Removal of the Fc glycan from 4G8 partially restored efflux. Alternate sialic acid groups with α2,3 and α2,8 linkages, nor on the Fc glycan, were not detected at significant levels on either 4G8 or 6E10.
Conclusions
These results support a model in which surface-sialylated 4G8 inhibits its own efflux and that of asialylated 4G8.
General significance
Glycan modification has the potential to increase antibody drug penetration into the brain through efflux inhibition.
Keywords
Sialic acidFab Fc antibody glycosylationAlzheimer's diseaseBlood brain barrierSurface plasmon resonance
http://www.sciencedirect.com/science/article/pii/S0304416517301940
https://www.genovis.com/products/iggzero/
Fc-glycosylation
Monoclonal Ab *
Bioassays
EndoS
*
Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates
Balázs Bobály, Sandrine Fleury-Souverain, Alain Beck, Jean-Luc Veuthey, Davy Guillarme, Szabolcs Fekete
Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics.
ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DARav), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.
http://www.sciencedirect.com/science/article/pii/S0731708517312116
https://www.genovis.com/products/fabricator/
Drug Antibody Ratio
ADC *
LC/MS *;RP-HPLC *
FabRICATOR *
****
Discovery and Characterization of Histidine Oxidation Initiated Cross-links in an IgG1 Monoclonal Antibody
Chong-Feng Xu, Yunqiu Chen, Linda Yi, Tim Brantley, Brad Stanley, Zoran Sosic, and Li Zang
Novel cross-links between an oxidized histidine
and intact histidine, lysine, or cysteine residues were discovered
and characterized from high-molecular weight (HMW) fractions
of an IgG1 monoclonal antibody (mAb). The mAb HMW
fractions were collected using preparative size-exclusion
chromatography (SEC) and extensively characterized to under-
stand the mechanism of formation of the nonreducible and
covalently linked portion of the HMWs. The HMW fractions
were IdeS digested, reduced, and analyzed by size-exclusion
chromatography coupled with mass spectrometry (SEC-MS).
The nonreducible cross-links were found to be enriched in the
fragment crystallizable (Fc) region of the heavy chain, with a net
mass increase of 14 Da. Detailed peptide mapping revealed as
many as seven covalent cross-links in the HMW fractions, where oxidized histidines react with intact histidine, lysine, and free cysteine to form cross-links. It is the first time that histidine−cysteine (His−Cys) and histidine−lysine (His−Lys) in addition to histidine−histidine (His−His) cross-links were discovered in monoclonal antibody HMW species. The histidine oxidation hot spots were identified, which include conserved histidine residues His292 and His440 in the Fc region and His231 in the hinge region of the IgG1 mAb heavy chain. Their cross-linking partners include His231, His292, His440, and Cys233 in the hinge region and Lys297 in the Fc region. A cross-linking mechanism has been proposed that involves nucleophilic addition by histidine, cysteine, or lysine residues to the carbonyl-containing histidine oxidation intermediates to form the cross-links.
http://pubs.acs.org/doi/pdf/10.1021/acs.analchem.7b00860
https://www.genovis.com/products/fabricator/
Oxidation
Monoclonal Ab *
LC/MS *;RP-HPLC *
FabRICATOR *
***
Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells
Janike Ehret Martina Zimmermann Thomas Eichhorn Aline Zimmer
Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half‐life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed‐batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high‐mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2‐F‐peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.
https://onlinelibrary.wiley.com/doi/full/10.1002/bit.26904
https://www.genovis.com/products/igg-proteases/fabricator/
Fc-glycosylation
Monoclonal Ab *
RP-HPLC *
FabRICATOR *
***
Hydrophilic Monomethyl Auristatin E Derivatives as Novel Candidates for the Design of Antibody-Drug Conjugates
Filip S. Ekholm, Suvi-Katriina Ruokonen, Marina Redón, Virve Pitkänen,
Anja Vilkman, Juhani Saarinen, Jari Helin, Tero Satomaa and Susanne K. Wiedmer
Antibody-drug conjugates (ADCs) are promising state-of-the-art biopharmaceutical drugs
for selective drug-delivery applications and the treatment of diseases such as cancer. The idea behind
the ADC technology is remarkable as it combines the highly selective targeting capacity of monoclonal
antibodies with the cancer-killing ability of potent cytotoxic agents. The continuous development
of improved ADCs requires systematic studies on the nature and effects of warhead modification.
Recently, we focused on the hydrophilic modification of monomethyl auristatin E (MMAE), the most
widely used cytotoxic agent in current clinical trial ADCs. Herein, we report on the use of micellar
electrokinetic chromatography (MEKC) for studying the hydrophobic character of modified MMAE
derivatives. Our data reveal a connection between the hydrophobicity of the modified warheads as
free molecules and their cytotoxic activity. In addition, MMAE-trastuzumab ADCs were constructed
and evaluated in preliminary cytotoxic assays.
https://www.mdpi.com/2297-8739/6/1/1
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Antibody Ratio
ADC *
MALDI-TOF-MS *
FabRICATOR *
****
A generic workflow for the characterization of therapeutic monoclonal antibodies—application to daratumumab
Bastiaan L. Duivelshof, Szabolcs Fekete, Davy Guillarme, Valentina D’Atri
In the present analytical workflow, chromatographic methods have been developed and hyphenated to mass spectrometry (MS) for the characterization of protein size, charge, hydrophobic, and hydrophilic variants of daratumumab. Multiple critical quality attributes (CQAs) were characterized in forced degraded daratumumab sample, using size exclusion, ion exchange (IEX), and hydrophobic interaction (HIC) chromatography coupled to fluorescence detection for relative quantification and fractionation. Mass assignment was performed by using a fast, non-denaturing and universal size exclusion chromatography (SEC) method prior to native MS analysis of the collected fractions (off-line approach). This allowed the identification of N-terminal lysine clipping, and the extent of glycation and oxidation at intact protein level. Finally, middle-up analysis of daratumumab was performed using reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography coupled to MS to obtain a comprehensive overview of all PTMs after the forced stressed conditions and a fine characterization of the glycosylation profile. Conveniently, the presented workflow maintains the established golden standard non-denaturing chromatography techniques and additionally introduces a straightforward and automated desalting procedure prior to MS analysis. Therefore, it is expected that the off-line coupling of SEC, IEX, and HIC to SEC-MS has great potential to be implemented in routine characterization of mAbs.
https://link.springer.com/article/10.1007/s00216-018-1561-1
https://www.genovis.com/products/igg-proteases/fabricator/
Aggregation;Glycation;Oxidation;Fc-glycosylation
Monoclonal Ab *
LC/MS *;RP-HPLC *;HILIC;LC-MS/MS
FabRICATOR *
****
Characterization of Whole and Fragmented Wild-Type Porcine IgG
Claudia Nelson, Raymond Bacala, Baylie Gigolyk, Evelyn Ang, Haley Neustaeter, Emy Komatsu, Oleg Krokhin, Dave Hatcher and Hélène Perreault
Glycoproteomic analyses of tryptic (glyco)peptides from wild-type (WT) porcine IgG were performed. In a first protocol, intact antibody was digested with trypsin, followed by glycopeptide enrichment and liquid chromatography-tandem MS (HPLC–MS/MS). This procedure allowed to detect N-glycopeptides observed previously (Lopez, P. G. et al., Glycoconj. J. 2016, 33 (1), 79), plus other non-reported N-glycopeptides. The method provided useful information but did not allow to discern between Fab (antigen-binding region) and Fc (constant region, fragment crystallizable) peptides/glycopeptides. In a second scheme, glycoproteomic analysis was attempted for Fab and Fc fragments obtained by papain and Fabulous™ hydrolysis. Usually employed for milligram amounts of antibodies, the papain and Fabulous™ protocols were adapted to 200 μg of WT IgG. Fab and Fc fragments were separated by size-exclusion (SEC) HPLC. Fractions collected were reanalyzed by gel electrophoresis (SDS-PAGE). Bands were excised, and fragments digested in-gel, followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and HPLC/MS–MS. In the protocol no glycopeptide enrichment was involved, that is, whole tryptic digests were analyzed. Fc N-glycopeptides were identified, and greater numbers of non-glycosylated peptides were tabulated. Very few peptides overlapped between Fc and Fab, as most peptides were clearly from Fc or Fab. HPLC-MS/MS detected more sialylated glycoforms than MALDI-TOF-MS. Sections of Fab and Fc were assigned de novo, through a database search or manually.
https://www.intechopen.com/books/recent-advances-in-analytical-chemistry/characterization-of-whole-and-fragmented-wild-type-porcine-igg
https://www.genovis.com/products/igg-proteases/fabulous/
Fc-glycosylation;Fab-glycosylation
IgG *
LC/MS *;MALDI-TOF-MS *
FabULOUS *
*
Fc Sialylation Prolongs Serum Half-Life of Therapeutic Antibodies
Mathilde Bas, Aurélie Terrier, Emilie Jacque, Aurélie Dehenne, Virginie Pochet-Béghin, Cécile Beghin, Anne-Sophie Dezetter, Gilles Dupont, Anaïs Engrand, Benjamin Beaufils, Philippe Mondon, Nathalie Fournier, Christophe de Romeuf, Sylvie Jorieux, Alexandre Fontayne, Lennart T. Mars and Céline Monnet
The long serum t1/2 of IgGs is ensured by their interaction with the neonatal Fc receptor (FcRn), which salvages IgG from intracellular degradation. Fc glycosylation is thought not to influence FcRn binding and IgG longevity in vivo. In this article, we demonstrate that hypersialylation of asparagine 297 (N297) enhances IgG serum persistence. This polarized glycosylation is achieved using a novel Fc mutation, a glutamate residue deletion at position 294 (Del) that endows IgGs with an up to 9-fold increase in serum lifespan. The strongest impact was observed when the Del was combined with Fc mutations improving FcRn binding (Del-FcRn+). Enzymatic desialylation of a Del-FcRn+ mutant or its production in a cell line unable to hypersialylate reduced the in vivo serum t1/2 of the desialylated mutants to that of native FcRn+ mutants. Consequently, our study proves that sialylation of the N297 sugar moiety has a direct impact on human IgG serum persistence.
http://www.jimmunol.org/content/early/2019/01/24/jimmunol.1800896.abstract
https://www.genovis.com/products/iggzero/
Fc-effector functions
Monoclonal Ab *
Bioassays
FabRICATOR *;IgGZERO *
**
Aptamers as quality control tool for production, storage and biosimilarity of the anti-CD20 biopharmaceutical rituximab
Sabrina Wildner, Sara Huber, Christof Regl, Christian G. Huber, Urs Lohrig & Gabriele Gadermaier
Detailed analysis of biopharmaceuticals is crucial for safety, efficacy and stability. Aptamers, which
are folded, single-stranded oligonucleotides, can be used as surrogate antibodies to detect subtle conformational changes. We aimed to generate and assess DNA aptamers against the therapeutic anti-CD20 antibody rituximab. Six rituximab-specific aptamers with Kd = 354–887 nM were obtained using the magnetic bead-based systematic evolution of ligands by exponential enrichment (seLeX) technology. Aptamer folds were analysed by online prediction tools and circular dichroism spectroscopy suggesting quadruplex structures for two aptamers while others present B-DNA helices. Aptamer binding and robustness with respect to minor differences in buffer composition or aptamer folding were verified in the enzyme-linked apta-sorbent assay. Five aptamers showed exclusive specificity to the Fab- fragment of rituximab while one aptamer revealed a broader recognition pattern to other monoclonal antibodies. Structural differences upon incubation at 40 °C for 72 h or UV exposure of rituximab were uncovered by four aptamers. High similarity between rituximab originator and biosimilar lots was demonstrated. The most sensitive aptamer (RA2) detected signal changes for all lots of a copy product suggesting conformational differences. For the first time, a panel of rituximab-specific aptamers was generated allowing the assessment of conformational coherence during production, storage, and biosimilarity of different products.
https://www.nature.com/articles/s41598-018-37624-1
https://www.genovis.com/products/igg-proteases/fabricator/fragit-kit/
Antigen Binding
Monoclonal Ab *
Other
FragIT / FragIT Kit
*
Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry
Stefan Mittermayr, Simone Albrecht, Csaba Váradi, Silvia Millán-Martín, and Jonathan Bones
Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein- derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statisti- cal analysis when optical detection methods are used. The approach outlined herein describes the utiliza- tion of stable isotope variants of commonly used uorescent tags that allow for mass-based glycan identi cation and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromato- gram/electropherogram ratios generated from the light and heavy isotope clusters.
https://link.springer.com/protocol/10.1007/978-1-4939-6990-6_23
https://www.genovis.com/products/fabricator/
Fc-glycosylation;Fab-glycosylation
Monoclonal Ab *
LC/MS *;cIEF *
FabRICATOR *
**
One-pot enzymatic glycan remodeling of a therapeutic monoclonal antibody by endoglycosidase S (Endo-S) from Streptococcus pyogenes
XinTong, Tiezheng Li, Jared Orwenyo, Christian Toonstra, Lai-Xi Wang
A facile, one-pot enzymatic glycan remodeling of antibody rituximab to produce homogeneous high-mannose and hybrid type antibody glycoforms is described. This method was based on the unique substrate specificity of the endoglycosidase S (Endo-S) from Streptococcus pyogenes. While Endo-S efficiently hydrolyzes the bi-antennary complex type IgG Fc N-glycans, we found that Endo-S did not hydrolyze the “ground state” high-mannose or hybrid glycoforms, and only slowly hydrolyzed the highly activated high-mannose or hybrid N-glycan oxazolines. Moreover, we found that wild-type Endo-S could efficiently use high-mannose or hybrid glycan oxazolines for transglycosylation without product hydrolysis. The combination of the remarkable difference in substrate specificity of Endo-S allows the deglycosylation of heterogeneous rituximab and the transglycosylation with glycan oxazoline to take place in one-pot without the need of isolating the deglycosylated intermediate or changing the enzyme to afford the high-mannose type, hybrid type, and some selectively modified truncated form of antibody glycoforms.
https://www.genovis.com/products/iggzero/
Fc-glycosylation
Monoclonal Ab *
LC/MS *
IgGZERO *
*
Protocols for the analytical characterization of therapeutic monoclonal antibodies. II – Enzymatic and chemical sample preparation
Balazs Bobaly, Valentina D’Atri, Alexandre Goyon, Olivier Colas, Alain Beck, Szabolcs Fekete, Davy Guillarme
The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g. N-glycans) and/or by decreasing the molecular size of the tested protein by enzymatic or chemical fragmentation. These approaches make the sample more suitable for chromatographic and mass spectrometric analysis. Structural elucidation and quality control (QC) analysis of biopharmaceutics are usually performed at intact, subunit and peptide levels. In this paper, general sample preparation approaches used to attain peptide, subunit and glycan level analysis are overviewed. Protocols are described to perform tryptic proteolysis, IdeS and papain digestion, reduction as well as deglycosylation by PNGase F and EndoS2 enzymes. Both historical and modern sample preparation methods were compared and evaluated using rituximab and trastuzumab, two reference therapeutic mAb products approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA). The described protocols may help analysts to develop sample preparation methods in the field of therapeutic protein analysis.
http://www.sciencedirect.com/science/article/pii/S1570023217306451
https://www.genovis.com/products/fabricator/
Fc-glycosylation
Monoclonal Ab *
LC/MS *;RP-HPLC *
FabRICATOR *;GlycINATOR *
*****
Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates
Nathan Tumey, Fengping Li, Brian Rago, Xiaogang Han, Frank Loganzo, Sylvia Musto, Edmund I. Graziani, Sujiet Puthenveetil, Jeffrey Casavant, Kimberly Marquette, Tracey Clark, Jack Bikker, Eric M. Bennett, Frank Barletta, Nicole Piche-Nicholas, Amy Tam, Christopher J. O’Donnell, Hans Peter Gerber, Lioudmila Tchistiakova
As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build “design rules” that will enable more efficient prosecution of next-generation ADC discovery programs.
https://link.springer.com/article/10.1208/s12248-017-0083-7
https://www.genois.com/products/fabricator/
Drug Antibody Ratio;Drug Load Distribution
ADC *
LC/MS *
***
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
Moelleken J, Endesfelder M, Gassner C, Lingke S, Tomaschek S, Tyshchuk O, Lorenz S, Reiff U, Mølhøj M.
The determination of the binding strength of immunoglobulins (IgGs) to targets can be influenced by avidity when the targets are soluble di- or multimeric proteins, or associated to cell surfaces, including surfaces introduced from heterogeneous assays. However, for the understanding of the contribution of a second drug-to-target binding site in molecular design, or for ranking of monovalent binders during lead identification, affinity-based assessment of the binding strength is required. Typically, monovalent binders like antigen-binding fragments (Fabs) are generated by proteolytic cleavage with papain, which often results in a combination of under- and over-digestion, and requires specific optimization and chromatographic purification of the desired Fabs. Alternatively, the Fabs are produced by recombinant approaches. Here, we report a lean approach for the functional assessment of human IgG1s during lead identification based on an in-solution digestion with the GingisKHAN™ protease, generating a homogenous pool of intact Fabs and Fcs and enabling direct assaying of the Fab in the digestion mixture. The digest with GingisKHAN™ is highly specific and quantitative, does not require much optimization, and the protease does not interfere with methods typically applied for lead identification, such as surface plasmon resonance or cell-based assays. GingisKHAN™ is highly suited to differentiate between affinity and avidity driven binding of human IgG1 monoclonal and bispecific antibodies during lead identification.
https://www.ncbi.nlm.nih.gov/pubmed/?term=gingiskhan
https://www.genois.com/products/gingiskhan/
Bioassays;Structure;Comparability studies
Monoclonal Ab *;Bispecific *
LC/MS *
GingisKHAN *
*****
A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection
Christof Regl, Therese Wohlschlager, Johann Holzmann and Christian G. Huber
Oxidation of biopharmaceuticals may affect their bioactivity, serum half-life, and (bio)chemical stability. The Fc domain of IgG monoclonal antibodies (mAbs) contains two methionine residues which are susceptible to oxidation. Here, we present a middle-down approach employing the cysteine protease IdeS under reducing conditions to obtain three mAb subunits of approximately 25 kDa: Fc/2, Fd′, and LC. These subunits were separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) and detected by UV spectroscopy as well as Orbitrap mass spectrometry (MS), as well as MS upon all-ion fragmentation (AIF-MS). We evaluated the feasibility of three strategies for absolute quantification of oxidation in the Fc region of hydrogen peroxide-stressed Rituximab, using a single, commercially available software platform both for data acquisition and evaluation: UV spectroscopy, full-scan MS, and monitoring of product ions obtained by AIF-MS. UV spectroscopy showed the lowest limits of quantification (LOQ) (0.96 ng μL–1) and featured the lowest relative process standard deviation (Vx0%) of 7.2% compared to MS and AIF-MS with LOQs of 1.24–4.32 ng μL–1 and relative process standard deviations of 9.0–14%, respectively. Our approach is generic in that it allows monitoring and quantification of oxidation in the Fc regions of fully human and humanized IgG1 mAbs as well as of Fc-fusion proteins. This is exemplified by limits of detection of 1.2%, 1.0%, and 1.2% of oxidation in drug products containing the biopharmaceuticals Rituximab, Adalimumab, and Etanercept, respectively. The presented method is an attractive alternative to conventional time-intensive peptide mapping which is prone to artificial oxidation due to extensive sample preparation.
http://pubs.acs.org/doi/pdf/10.1021/acs.analchem.7b01755
https://www.genois.com/products/fabricator/
Oxidation
Monoclonal Ab *
HPLC *;LC/MS *;RP-HPLC *
FabRICATOR *
*****
Preclinical Characterization of CTL-1, A Biosimilar Anti-EGFR Monoclonal Antibody for Cetuximab
Peisheng Hu, Ryan Gra, Long Zheng, Leslie A Khawli and Alan L Epstein
The monoclonal antibody (mAb) market has helped millions of patients across the globe in treating serious and chronic diseases. The expiry of patents for the rst generation of approved therapeutic mAbs has led to the development and authorization of biosimilar alternatives for which the market is of great interest due to the demand of affordable treatments. Here we report the preclinical development of a biosimilar mAb to cetuximab (Erbitux®) referred to as CTL-1. Analyses of the physicochemical properties and biological activities of both CTL- 1 and cetuximab were performed to allow for comparisons of the primary structure, glycoform heterogeneity, in vitro anti-tumor activity, and in vivo pharmacokinetics, tissue distribution, and ef cacy of the two antibodies. Our results con rm that CTL-1 has the same amino acid sequence and a similar glycosylation pro le as the cetuximab reference. In vitro bioassays indicate that CTL-1 and cetuximab achieve comparable dose-dependent inhibition of EGFR+ tumor cell proliferation, and maintain similar abilities to induce Antibody-Dependent Cell- Mediated Cytotoxicity (ADCC). Preclinical studies in mice con rm that the proposed biosimilar to cetuximab is comparable with regard to its pharmacokinetic and tissue distribution properties as well as its ability to reduce tumor growth in vivo. Based on these comprehensive similarity studies, CTL-1 was con rmed to be highly similar to the reference mAb cetuximab and is suggested as a viable biosimilar alternative to this important therapeutic mAb.
http://smjournals.com/bioequivalence-bioavailability/fulltext/smbb-v1-1003.pdf
https://www.genois.com/products/fabricator/
Bioassays;Fc-glycosylation
Biosimilar *
LC/MS *
FabRICATOR *
**
High resolution top-down experimental strategies on the Orbitrap platform
Kai Scheffler, Rosa Viner, Eugen Damoc
Top-down mass spectrometry (MS) strategies allow in-depth characterization of proteins by fragmentation of the entire molecule(s) inside a mass spectrometer without requiring prior proteolytic digestion. Importantly, the fragmentation techniques on commercially available mass spectrometers have become more versatile over the past decade, with different characteristics in regards to the type and wealth of fragment ions that can be ob- tained while preserving labile protein post-translational modifications. Due to these and other improvements, top-down MS has become of broader interest and has started to be applied in more disciplines, such as the quality control of recombinant proteins, analysis and characterization of biopharmaceuticals, and clinical biochemistry to probe protein forms as potential disease biomarkers. This article provides a technical overview and guidance for data acquisition strategies on the Orbitrap platform for single proteins and low complexity protein mixtures. A protein standard mixture composed of six recombinant proteins is also introduced and analysis strategies are discussed in detail.
Biological significance: The article provides a detailed overview and guidance on how to choose from the variety of available methods for protein characterization by top-down analysis on the Orbitrap platform. Technical details are provided explaining important observations and phenomena when working with intact proteins and data from a number of different samples should serve to provide a solid understanding on how experiments were and should be setup and to set the right expectations on the outcome of these types of experiments. Additionally, a new intact protein standard sample is introduced that will help as a QC sample to check the instrument's hard- ware and method setup conditions as a requirement for obtaining high quality data from biologically relevant samples.
http://www.sciencedirect.com/science/article/pii/S1874391917301197
https://www.genovis.com/products/fabricator/
Monoclonal Ab *
LC/MS *
FabRICATOR *
**
Orthogonal liquid chromatography–mass spectrometry methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level
Eric Largy, Fabrice Cantais, Géry Van Vyncht, Alain Beck, Arnaud Delobel
Proteins are increasingly used as therapeutics. Their characterization is challenging due to their size and inherent heterogeneity notably caused by post-translational modifications, among which glycosylation is probably the most prominent. The glycosylation profile of therapeutic proteins must therefore be thoroughly analyzed. Here, we illustrate how the use of a combination of various cutting-edge LC or LC/MS(/MS) methods, and operating at different levels of analysis allows the comprehensive characterization of both the N- and O-glycosylations of therapeutic proteins without the need for other approaches (capillary electrophoresis, MALDI-TOF). This workflow does not call for the use of highly specialized/custom hardware and software nor an extensive knowledge of glycan analysis. Most notably, we present the point of view of a contract research organization, with the constraints associated to the work in a regulated environment (GxP). Two salient points of this work are i) the use of mixed-mode chromatography as a fast and straightforward mean of profiling N-glycans sialylation as well as an orthogonal method to separate N-glycans co-eluting in the HILIC mode; and ii) the use of widepore HILIC/MS to analyze challenging N/O-glycosylation profiles at both the peptide and subunit levels. A particular attention was given to the sample preparations in terms of duration, specificity, versatility, and robustness, as well as the ease of data processing.
http://www.sciencedirect.com/science/article/pii/S002196731730331X
https://www.genovis.com/products/fabricator/
Fc-glycosylation
Monoclonal Ab *
LC/MS *
FabRICATOR *;GlycINATOR *
****
Direct quantitation of therapeutic antibodies for pharmacokinetic studies using immuno-purification and intact mass analysis
Lisa A Vasicek, Xin Zhu, Daniel S Spellman & Kevin P Bateman
Aim: The quantitation of therapeutic antibodies by MS often utilizes a surrogate peptide approach. Recent enhancements in instrumentation and sample preparation have enabled quantitation by detection of the intact molecule using MS. Methods & Results: A comparison of three methods for quantitative analysis of therapeutic monoclonal antibodies including analysis after deglycosylation, after hinge digestion and at the fully intact antibody level is reported. The optimized methodology provided sensitivity down to 0.1 μg/ml and a lower limit of quantitation of 0.5 ug/ml from a 30 μl sample volume. Conclusion: Application of this approach to a pharmacokinetic study compared with a conventional surrogate peptide and a ligand-binding assays provided consistent data with direct detection of the dosed molecule.
https://www.future-science.com/doi/abs/10.4155/bio-2018-0240
https://www.genovis.com/products/igg-proteases/fabricator/
Other
Monoclonal Ab *
LC/MS *;Bioassays
FabRICATOR *
**
Molecular Basis of Broad Spectrum N-Glycan Specificity and Processing of Therapeutic IgG Monoclonal Antibodies by Endoglycosidase S2
Erik H. Klontz, Beatriz Trastoy, Daniel Deredge, James K. Fields, Chao Li, Jared Orwenyo, Alberto Marina, Robert Beadenkop, Sebastian Günther, Jair Flores, Patrick L. Wintrod, Lai-Xi Wang, Marcelo E. Guerin, and Eric J. Sundberg
Immunoglobulin G (IgG) glycosylation critically modulates antibody effector functions. Streptococcus pyogenes secretes a unique endo-β-N-acetylglucosaminidase, EndoS2, which deglycosylates the conserved N-linked glycan at Asn297 on IgG Fc to eliminate its effector functions and evade the immune system. EndoS2 and specific point mutants have been used to chemoenzymatically synthesize antibodies with customizable glycosylation for gain of functions. EndoS2 is useful in these schemes because it accommodates a broad range of N-glycans, including high-mannose, complex, and hybrid types; however, its mechanism of substrate recognition is poorly understood. We present crystal structures of EndoS2 alone and bound to complex and high-mannose glycans; the broad N-glycan specificity is governed by critical loops that shape the binding site of EndoS2. Furthermore, hydrolytic experiments, domain-swap chimeras, and hydrogen–deuterium exchange mass spectrometry reveal the importance of the carbohydrate-binding module in the mechanism of IgG recognition by EndoS2, providing insights into engineering enzymes to catalyze customizable glycosylation reactions.
https://pubs.acs.org/doi/full/10.1021/acscentsci.8b00917
Structure
IgG *
Other
GlycINATOR *
**
Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 1: Alternative mobile phases and fine tuning of the separation
Evelin Farsang, Amarande Murisier, Krisztián Horvátha, Alain Beck, Róbert Kormányd, Davy Guillarme, Szabolcs Fekete
Cation exchange chromatography (CEX) of therapeutic monoclonal antibodies is generally performed with either salt gradient (MES buffer + NaCl) or using commercial pH gradient buffer. The goal of this study was to find out some alternative buffer systems for CEX separation of mAbs, which may offer alternative selectivity, while maintaining similar peak shapes. Among the new buffers that were tested, (N-morpholino)ethanesulfonic acid (MES) / 1,3-diamino-2-propanol (DAP), and citric acid / 2-(cyclohexylamino)ethanesulfonic acid (CHES) systems were particularly promising, especially when combining them with a moderate salt gradient of NaCl. This two buffer system provides an equivalent or slightly better separation than the standard, mobile phases for therapeutic mAbs.
It was also demonstrated that working with salt-mediated pH gradients, allows to extend the possibilities in method development, since the concentration of salt in the mobile phase has a significant impact on selectivity. Using HPLC modeling software (Drylab), it was possible to successfully develop CEX methods for authentic mAb samples within only 6 h, by optimizing the gradient steepness and salt concentration in the B eluent.
https://www.sciencedirect.com/science/article/abs/pii/S0731708518328553
https://www.genovis.com/products/igg-proteases/fabricator/
Charge variants
Monoclonal Ab *
Cation-Exchange Chromatography (CEX)
FabRICATOR *
****
Persistent Antibody Clonotypes Dominate the Serum Response to Influenza over Multiple Years and Repeated Vaccinations
Jiwon Lee, Philipp Paparoditis, Andrew P. Horton, Alexander Frühwirth, Jonathan R.McDaniel, Jiwon Jung, Daniel R. Boutz, Dania A. Hussein, Yuri Tanno, Leontios Pappas, Gregory C. Ippolito, Davide Corti, Antonio Lanzavecchia, GeorgeGeorgiou
Humans are repeatedly exposed to influenza virus via infections and vaccinations. Understanding how multiple exposures and pre-existing immunity impact antibody responses is essential for vaccine development. Given the recent prevalence of influenza H1N1 A/California/7/2009 (CA09), we examined the clonal composition and dynamics of CA09 hemagglutinin (HA)-reactive IgG repertoire over 5 years in a donor with multiple influenza exposures. The anti-CA09 HA polyclonal response in this donor comprised 24 persistent antibody clonotypes, accounting for 72.6% ± 10.0% of the anti-CA09 HA repertoire over 5 years. These persistent antibodies displayed higher somatic hypermutation relative to transient serum antibodies detected at one time point. Additionally, persistent antibodies predominantly demonstrated cross-reactivity and potent neutralization toward a phylogenetically distant H5N1 A/Vietnam/1203/2004 (VT04) strain, a feature correlated with HA stem recognition. This analysis reveals how “serological imprinting” impacts responses to influenza and suggests that once elicited, cross-reactive antibodies targeting the HA stem can persist for years.
https://www.sciencedirect.com/science/article/abs/pii/S1931312819300484
https://www.genovis.com/products/igg-proteases/fabricator/
IgG Polyclonal *;Bioassays
Bioassays
FabRICATOR *
*
MHC class II proteins mediate cross-species entry of bat influenza viruses
Umut Karakus, Thiprampai Thamamongood, Kevin Ciminski, Wei Ran, Sira C. Günther, Marie O. Pohl, Davide Eletto, Csaba Jeney, Donata Hoffmann, Sven Reiche, Jan Schinköthe, Reiner Ulrich, Julius Wiener, Michael G. B. Hayes, Max W. Chang, Annika Hunziker, Emilio Yángüez, Teresa Aydillo, Florian Krammer, Josua Oderbolz, Matthias Meier, Annette Oxenius, Anne Halenius, Gert Zimmer, Christopher Benner, Benjamin G. Hale, Adolfo García-Sastre, Martin Beer, Martin Schwemmle & Silke Stertz
Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR–Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.
https://www.genovis.com/products/igg-proteases/fabricator/
IgG Polyclonal *
Bioassays
FabRICATOR *
*
Sample Preparation for LC‐MS Bioanalysis of Antibody–Drug Conjugates
Cong Wei Ragu Ramanathan
Antibody‐drug conjugates (ADCs) have emerged as an important class of targeted biological therapeutics. ADCs are composed with monoclonal antibodies (mAb) and cytotoxic drugs (payloads) linked together through cysteine, lysine or other engineered residues via a variety of linkers. Bioanalysis plays a critical role in ADC drug development for the assessment of safety and efficacy. The unique and complex structural features of ADC require specific bioanalytical strategies that are different from ones applied to small‐molecule xenobiotics and large‐molecule biologics (e.g. peptide or protein therapeutics). Quantification of four major analytes, including unconjugated payload, conjugated payload, ADC (i.e., conjugated mAb) and total mAb, as well as characterization of changes in drug‐to‐antibody ratio (DAR) distribution in biological matrices, are integral components to the ADC bioanalysis. Due to the complexity of ADCs, sample preparation can be potentially challenging and should be carefully considered and executed in the bioanalytical workflows for various moieties of the ADC. This chapter describes different sample preparation techniques and their applications in quantitative analysis of four major analytes mentioned above. In addition, sample preparation for the determination of DAR from biological matrices is also discussed, which offers an indication of the ADC stability and possible biotransformations.
https://onlinelibrary.wiley.com/doi/pdf/10.1002/9781119274315.ch26
https://www.genovis.com/products/iggzero/
Drug Antibody Ratio
ADC *
LC/MS *;HIC
IgGZERO *
***
Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual
Shelly J. Krebs, Young D. Kwon,
Chaim A. Schramm, ..., Merlin L. Robb,
Peter D. Kwong, Nicole A. Doria-Rose
Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.
https://www.sciencedirect.com/science/article/abs/pii/S1074761319300743
https://www.genovis.com/products/igg-proteases/fabricator/
IgG Polyclonal *
Bioassays
FabRICATOR *
*
Advances toward mapping the full extent of protein site-specific O-GalNAc glycosylation that better reflects underlying glycomic complexity
Kay-Hooi Khoo
Recent advances in mass spectrometry has empowered unbiased global analysis of site-specific O-GalNAc glycosylation. Despite thousands of sites being identified, significant technical hurdles remain, particularly in the delineation of fully extended, larger O-GalNAc glycans on heavily O-glycosylated mucin domain. Current approaches require simplification of the O-GalNAc glycans either by genetic means or glycosidase treatments to allow unambiguous sequencing of the derived O-glycopeptides. In contrast, a full mapping of the O-GalNAc glycomic complexity still necessitates a detailed analysis of the released glycans. Chromatographic resolution and multistage fragmentation coupled with judicious choice of chemical derivatization are key to increase the analytical precision and glycomic coverage depth, which should be duly considered along with attainable sensitivity and throughput for meaningful glycobiology applications.
https://www.sciencedirect.com/science/article/abs/pii/S0959440X1830068X
https://www.genovis.com/products/enzymes-for-o-glycans/operator/
O-glycosylation
LC-MS/MS
OpeRATOR
**
IgG Charge: Practical and Biological Implications
Danlin Yang, Rachel Kroe-Barrett, Sanjaya Singh, Thomas Laue
Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to −13; (2) the F(ab’)2 fragments are nearly neutral for IgG1s and IgG2s, and about −5 for some of the IgG4s; (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; (4) the charge on the intact IgGs does not equal the sum of the F(ab’)2 and Fc charge. In no case is the calculated charge, based solely on H+ binding, remotely close to the measured charge. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. A thermodynamically rigorous, concentration-dependent protein–protein interaction parameter is introduced. Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. Example curves are provided.
https://www.mdpi.com/2073-4468/8/1/24/htm
https://www.genovis.com/products/igg-proteases/fabricator/
Charge variants
Monoclonal Ab *
Other
FabRICATOR *
****
Discriminating cross-reactivity in polyclonal IgG1 responses against SARS-CoV-2 variants of concern
Danique M. H. van Rijswijck, Albert Bondt, Max Hoek, Karlijn van der Straten, Tom G. Caniels, Meliawati Poniman, Dirk Eggink, Chantal Reusken, Godelieve J. de Bree, Rogier W. Sanders, Marit J. van Gils & Albert J. R. Heck
Existing assays to measure antibody cross-reactivity against different SARS-CoV-2 spike (S) protein variants lack the discriminatory power to provide insights at the level of individual clones. Using a mass spectrometry-based approach we are able to monitor individual donors’ IgG1 clonal responses following a SARS-CoV-2 infection. We monitor the plasma clonal IgG1 profiles of 8 donors who had experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we chart the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer VOC antigens. The plasma of each donor contains numerous anti-spike IgG1 antibodies, accounting for <0.1% up to almost 10% of all IgG1s. Some of these antibodies are VOC-specific whereas others do recognize multiple or even all VOCs. We show that in these polyclonal responses, each clone exhibits a distinct cross-reactivity and also distinct virus neutralization capacity. These observations support the need for a more personalized look at the antibody clonal responses to infectious diseases.
https://www.nature.com/articles/s41467-022-33899-1
https://www.genovis.com/products/igg-proteases/fabalactica/
Antigen Binding;Neutralization
Monoclonal Ab *
LC/MS *
FabALACTICA
A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans
Thapakorn Jaroentomeechai, Yong Hyun Kwon, Yiwen Liu, Olivia Young, Ruchika Bhawal, Joshua D. Wilson, Mingji Li, Digantkumar G. Chapla, Kelley W. Moremen, Michael C. Jewett, Dario Mizrachi & Matthew P. DeLisa
The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.
https://www.nature.com/articles/s41467-022-34029-7
https://www.genovis.com/products/glycinator/
Bioassays;Other;Structure
Monoclonal Ab *
Bioassays;HILIC;LC-MS/MS
GlycINATOR *
A Combination of Native LC-MS Approaches for the Comprehensive Characterization of the Antibody-Drug Conjugate Trastuzumab Deruxtecan
Evolène Deslignière, Hélène Diemer, Stéphane Erb, Pierre Coliat, Xavier Pivot, Alexandre Detappe, Oscar Hernandez-Alba, Sarah Cianférani
Background: Native mass spectrometry (nMS) approaches appear attractive to complement bottom-up strategies traditionally used in biopharmaceutical industries thanks to their quite straightforward and rapid workflows, especially through online hyphenation of non- denaturing liquid chromatography (LC) to nMS. The present work provides an overview of the state-of-the-art chromatographic tools available for the detailed characterization of monoclonal antibody (mAb) formats, exemplified on the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd). Methods: T-DXd was first characterized by conventional reversed phase LC (rpLC) and peptide mapping. Couplings of size exclusion chromatography (SEC), cation exchange chromatography (CEX), and hydrophobic interaction chromatography (HIC) to nMS were used to gain further insights into size, hydrophobic, and charge variants of T-DXd and its parental mAb trastuzumab, at intact and middle-up levels. Results: SEC-nMS first offered a direct snapshot of the homogeneous conjugation of T-DXd, with an average drug-to-antibody ratio (DAR) of 8 in agreement with a conjugation on cysteines after reduction of all interchain disulfide bonds. Moreover, SEC-nMS afforded precise identification and quantification of aggregates and fragments. Middle-up level experiments performed after IdeS digestion confirmed that drug conjugation occurs in the Fab region of the mAb, as seen with rpLC. HIC separated two DAR8 species that could not be differentiated by nMS. Although middle-up HIC-nMS proved to be more informative for oxidized forms, the identification of minor variants was still difficult because of poor MS signal quality, showing how the coupling of HIC to nMS remains challenging. Lastly, middle-up CEX-nMS provided accurate determination and localization of post-translational modifications, with several acidic/basic variants within Fab and Fc regions of T-DXd that were also identified by peptide mapping. Conclusions: This study illustrates the strengths and drawbacks of each LC-nMS coupling. By combining SEC-, HIC-, and CEX-nMS, we were able to achieve a comprehensive characterization of T-DXd without extensive sample preparation prior to MS analysis.
https://www.imrpress.com/journal/FBL/27/10/10.31083/j.fbl2710290/htm
https://www.genovis.com/products/igg-proteases/fabricator/
Aggregation;Drug Antibody Ratio;Drug Load Distribution;Characterization
Monoclonal Ab *;ADC *
LC/MS *;HIC;SEC-MS *;Cation-Exchange Chromatography (CEX);Peptide mapping
FabRICATOR *
Challenges and Strategies for a Thorough Characterization of Antibody Acidic Charge Variants
Y. Diana Liu , Lance Cadang, Karenna Bol, Xiao Pan, Katherine Tschudi, Mansour Jazayri, Julien Camperi , David Michels, John Stults, Reed J. Harris and Feng Yang
Heterogeneity of therapeutic Monoclonal antibody (mAb) drugs are due to protein variants generated during the manufacturing process. These protein variants can be critical quality attributes (CQAs) depending on their potential impact on drug safety and/or efficacy. To identify CQAs and ensure the drug product qualities, a thorough characterization is required but challenging due to the complex structure of biotherapeutics. Past characterization studies for basic and acidic variants revealed that full characterizations were limited to the basic charge variants, while the quantitative measurements of acidic variants left gaps. Consequently, the characterization and quantitation of acidic variants are more challenging. A case study of a therapeutic mAb1 accounted for two-thirds of the enriched acidic variants in the initial characterization study. This led to additional investigations, closing the quantification gaps of mAb1 acidic variants. This work demonstrates that a well-designed study with the right choices of analytical methods can play a key role in characterization studies. Thus, the updated strategies for more complete antibody charge variant characterization are recommended
https://www.mdpi.com/2306-5354/9/11/641
https://www.genovis.com/products/igg-proteases/fabricator/
Characterization
Monoclonal Ab *
LC/MS *;cIEF *;SEC *;HDX-MS
FabRICATOR *;FabALACTICA;SialEXO
Mucinomics as the Next Frontier of Mass Spectrometry
Valentina Rangel-Angarita and Stacy A. Malaker
Mucin-domain glycoproteins comprise a class of proteins whose densely O-glycosylated mucin domains adopt a secondary structure with unique biophysical and biochemical properties. The canonical family of mucins is well-known to be involved in various diseases, especially cancer. Despite this, very little is known about the site-specific molecular structures and biological activities of mucins, in part because they are extremely challenging to study by mass spectrometry (MS). Here, we summarize recent advancements toward this goal, with a particular focus on mucin-domain glycoproteins as opposed to general O-glycoproteins. We summarize proteolytic digestion techniques, enrichment strategies, MS fragmentation, and intact analysis, as well as new bioinformatic platforms. In particular, we highlight mucin directed technologies such as mucin-selective proteases, tunable mucin platforms, and a mucinomics strategy to enrich mucin-domain glycoproteins from complex samples. Finally, we provide examples of targeted mucin-domain glycoproteomics that combine these techniques in comprehensive site-specific analyses of proteins. Overall, this Review summarizes the methods, challenges, and new opportunities associated with studying enigmatic mucin domains.
https://pubs.acs.org/doi/abs/10.1021/acschembio.1c00384
https://www.genovis.com/products/enzymes-for-o-glycans/oglyzor/
O-glycosylation
LC/MS *
OglyZOR
Site-specific glycan-conjugated NISTmAb antibody drug conjugate mimetics: synthesis, characterization, and utility
Brian Agnew, Shanhua Lin, Terry Zhang, Robert Aggeler, Trina Mouchahoir & John Schiel
Antibody drug conjugates (ADCs) represent a rapidly growing modality for the treatment of numerous oncology indications. The complexity of analytical characterization method development is increased due to the potential for synthetic intermediates and process-related impurities. In addition, the cytotoxicity of such materials provides an additional challenge with regard to handling products and/or sharing materials with analytical collaborators and/or vendors for technology development. Herein, we have utilized a site-specific chemoenzymatic glycoconjugation strategy for preparing ADC mimetics composed of the NIST monoclonal antibody (NISTmAb) conjugated to non-cytotoxic payloads representing both small molecules and peptides. The materials were exhaustively characterized with high-resolution mass spectrometry-based approaches to demonstrate the utility of each analytical method for confirming the conjugation fidelity as well as deep characterization of low-abundance synthetic intermediates and impurities arising from payload raw material heterogeneity. These materials therefore represent a widely available test metric to develop novel ADC analytical methods as well as a platform to discuss best practices for extensive characterization.
https://link.springer.com/article/10.1007%2Fs00216-021-03460-y
https://www.genovis.com/products/glycinator/
ADC *
LC/MS *;Native MS *
FragIT / FragIT Kit;GlycINATOR *
Cysteine-SILAC Mass Spectrometry Enabling the Identification and Quantitation of Scrambled Interchain Disulfide Bonds: Preservation of Native Heavy-Light Chain Pairing in Bispecific IgGs Generated by Controlled Fab-arm Exchange
Ewald T. J. van den Bremer, Aran F. Labrijn, Ramon van den Boogaard, Patrick Priem, Kai Scheffler, Joost P. M. Melis, Janine Schuurman, Paul W. H. I. Parren and Rob N. de Jong
Bispecific antibodies (bsAbs) are one of the most versatile and promising pharmaceutical innovations for countering heterogeneous and refractory disease by virtue of their ability to bind two distinct antigens. One critical quality attribute of bsAb formation requiring investigation is the potential randomization of cognate heavy (H) chain/light (L) chain pairing, which could occur to a varying extent dependent on bsAb format and the production platform. To assess the content of such HL-chain swapped reaction products with high sensitivity, we developed cysteine-stable isotope labeling using amino acids in cell culture (SILAC), a method that facilitates the detailed characterization of disulfide-bridged peptides by mass spectrometry. For this analysis, an antibody was metabolically labeled with 13C3,15N-cysteine and incorporated into a comprehensive panel of distinct bispecific molecules by controlled Fab-arm exchange (DuoBody technology). This technology is a postproduction method for the generation of bispecific therapeutic IgGs of which several have progressed into the clinic. Herein, two parental antibodies, each containing a single heavy chain domain mutation, are mixed and subjected to controlled reducing conditions during which they exchange heavy–light (HL) chain pairs to form bsAbs. Subsequently, reductant is removed and all disulfide bridges are reoxidized to reform covalent inter- and intrachain bonds. We conducted a multilevel (Top-Middle-Bottom-Up) approach focusing on the characterization of both “left-arm” and “right-arm” HL interchain disulfide peptides and observed that native HL pairing was preserved in the whole panel of bsAbs produced by controlled Fab-arm exchange.
http://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b02543
https://www.genovis.com/products/igg-proteases/gingiskhan/
Structure;Disulfide reduction
Bispecific *
LC/MS *
GingisKHAN *
****
Identification and Quantification of Signal Peptide Variants in an IgG1 Monoclonal Antibody Produced in Mammalian Cell Lines
Yunping Huang, Jinmei Fu, Richard Ludwig, Li Tao, Jacob Bongers, Li Ma, Ming Yao, Mingshe Zhu, Tapan Das, Reb Russell
Sequence variants of a monoclonal antibody resulting from incomplete processing of signal peptide were identified and characterized using multiple mass spectrometry platforms and reverse phase chromatography. Detection and quantification of these variants by three LC/MS platforms were assessed. Quantification was also performed by mass spectrometric analysis of the subunits of the antibody generated by reduction and IdeS proteolysis. Peptide mapping with LC/MS/MS detection was used to quantify and confirm the identities of signal peptide sequence variants. Although quantification of the signal peptide variants thru mass spectrometry approaches is system dependent, our data revealed the results are close to the values determined by chromatographic separation with UV detection. Each of the methods have proven effective in demonstrating the consistency of signal peptide variants levels across the manufacture history of the antibody.
http://www.sciencedirect.com/science/article/pii/S1570023217314551
https://www.genovis.com/products/igg-proteases/fabricator/
Amino acid sequence
Monoclonal Ab *
LC/MS *
FabRICATOR *
****
Massive immune response against IVIg interferes with response against other antigens in mice: A new mode of action?
Laetitia Sordé, Sebastian Spindeldreher, Ed Palmer, Anette Karle
Administration of high dose intravenous immunoglobulin (IVIg) is widely used in the clinic to treat autoimmune and severe inflammatory diseases. However, its mechanisms of action remain poorly understood. We assessed the impact of IVIg on immune cell populations using an in vivo ovalbumin (Ova)-immunization mouse model. High dose IVIg significantly reduced the Ova-specific antibody response. Intriguingly, the results obtained indicate an immediate and massive immune reaction against IVIg, as shown by the activation and expansion of B cells and CD4+ T cells in the spleen and draining lymph nodes and the production of IVIg-specific antibodies. We propose that IVIg competes at the T-cell level with the response against Ova to explain the immunomodulatory properties of IVIg. Two monoclonal antibodies did not succeeded in reproducing the effects of IVIg. This suggests that in addition to the mouse response against human constant domains, the enormous sequence diversity of IVIg may significantly contribute to this massive immune response against IVIg. While correlation of these findings to IVIg-treated patients remains to be explored, our data demonstrate for the first time that IVIg re-directs the immune response towards IVIg and away from a specific antigen response.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186046
https://www.genovis.com/products/igg-proteases/fabricator/
IVIG *
Bioassays
FabRICATOR *
**
Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis
Cory King, Rekha Patel, Gomathinayagam Ponniah, Christine Nowak, Alyssa Neill, Zhenyu Gu, Hongcheng Liu
In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab′)2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen.
https://www.sciencedirect.com/science/article/pii/S1570023218301673
https://www.genovis.com/products/igg-proteases/fabricator/
Deamidation;IsoAspartic acid
Monoclonal Ab *
LC/MS *;cIEF *;HIC
FabRICATOR *
*****
Development of Comprehensive Online Two-Dimensional Liquid Chromatography/Mass Spectrometry Using Hydrophilic Interaction and Reversed-Phase Separations for Rapid and Deep Profiling of Therapeutic Antibodies
Dwight R Stoll, David Christopher Harmes, Gregory O. Staples, Oscar G. Potter, Carston T. Dammann, Davy Guillarme, and Alain Beck
Monoclonal antibodies (mAb) and related molecules are being developed at a remarkable pace as new therapeutics for the treatment of diseases ranging from cancer to inflammatory disorders. However, characterization of these molecules at all stages of development and manufacturing presents tremendous challenges to existing analytical technologies because of their large size (ca. 150 kDa) and inherent heterogeneity resulting from complex glycosylation patterns and other post-translational modifications. Multidimensional liquid chromatography is emerging as a powerful platform technology that can be used to both improve analysis speed for these molecules by combining existing one-dimensional separations into a single method (e.g., Protein A affinity separation and size-exclusion chromatography) and increasing the resolving power of separations by moving from one dimension of separation to two. In the current study, we have demonstrated the ability to combine hydrophilic interaction (HILIC) and RP separations in an online comprehensive 2D separation coupled with high resolution MS detection (HILIC × RP-HRMS). We find that active solvent modulation (ASM) is critical for coupling these two separation modes, because it mitigates the otherwise serious negative impact of the acetonitrile-rich HILIC mobile phase on the second dimension RP separation. The chromatograms obtained from these HILIC × RP-HRMS separations of mAbs at the subunit level reveal the extent of glycosylation on the Fc/2 and Fd subunits in analysis times on the order of 2 h. In comparison to previous CEX × RP separations of the same molecules, we find that chromatograms from the HILIC × RP separations are richer and reveal separation of some glycoforms that coelute in the CEX × RP separations.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.8b00776
https://www.genovis.com/products/igg-proteases/fabricator/
Fc-glycosylation
Monoclonal Ab *
LC/MS *;RP-HPLC *
FabRICATOR *
****
Targeting of embryonic annexin A2 expressed on ovarian and breast cancer by the novel monoclonal antibody 2448
Simeon Cua, Heng Liang Tan, Wey Jia Fong, Angela Chin, Ally Lau, Vanessa Ding, Zhiwei Song, Yuansheng Yang and Andre Choo
Monoclonal antibodies (mAbs) play an increasingly important role in cancer therapy. To address the wide heterogeneity of the disease, the identification of novel antigen targets and the development of mAbs against them are needed. Our lab previously generated a panel of mAbs against human embryonic stem cells (hESC) using a whole cell immunization approach in mice. These mAbs can potentially target oncofetal antigens and be repurposed for antibody or antibody drug conjugate (ADC) therapy. From this panel, the novel IgG1 2448 was found to bind surface antigens on hESC and multiple cancer cell lines. Here, we show 2448 targets a unique glycan epitope on annexin A2 (ANXA2) and can potentially monitor the Epithelial-Mesenchymal Transition (EMT) in ovarian and breast cancer. To evaluate 2448 as a potential drug, 2448 was engineered and expressed as a chimeric IgG1. Chimeric 2448 (ch2448) demonstrated efficient and specific killing when conjugated to cytotoxic payloads as an ADC. In addition, ch2448 elicited potent antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro and in vivo. Further engineering of ch2448 to remove fucose in the Fc domain enhanced ADCC. Overall, these findings indicate that embryonic ANXA2 is an attractive target and suggest that ch2448 is a promising candidate for further therapeutic development.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862572/
https://www.genovis.com/products/igg-proteases/fabricator/
Antigen Binding;ADCC
Monoclonal Ab *;ADC *
Bioassays
FabRICATOR *
***
Hydrophilic Auristatin Glycoside Payload Enables Improved Antibody-Drug Conjugate Efficacy and Biocompatibility
Tero Satomaa, Henna Pynnönen, Anja Vilkman, Titta Kotiranta, Virve Pitkänen, Annamari Heiskanen, Bram Herpers, Leo S. Price, Jari Helin and Juhani Saarinen
Antibody-drug conjugates (ADCs) offer a combination of antibody therapy and specific delivery of potent small-molecule payloads to target cells. The properties of the ADC molecule are determined by the balance of its components. The efficacy of the payload component increases with higher drug-to-antibody ratio (DAR), while homogeneous DAR = 8 ADCs are easily prepared by conjugation to the four accessible antibody hinge cystines. However, use of hydrophobic payloads has permitted only DAR = 2–4, due to poor pharmacokinetics and aggregation problems. Here, we describe generation and characterization of homogeneous DAR = 8 ADCs carrying a novel auristatin β-D-glucuronide, MMAU. The glycoside payload contributed to overall hydrophilicity of the ADC reducing aggregation. Compared to standard DAR = 2–4 ADCs, cytotoxicity of the homogeneous DAR = 8 ADCs was improved to low-picomolar IC50 values against cancer cells in vitro. Bystander efficacy was restored after ADC internalization and subsequent cleavage of the glycoside, although unconjugated MMAU was relatively non-toxic to cells. DAR = 8 MMAU ADCs were effective against target antigen-expressing xenograft tumors. The ADCs were also studied in 3D in vitro patient-derived xenograft (PDX) assays where they outperformed clinically used ADC. In conclusion, increased hydrophilicity of the payload contributed to the ADC’s hydrophilicity, stability and safety to non-target cells, while significantly improving cytotoxicity and enabling bystander efficacy.
http://resolver.ebscohost.com/openurl?sid=google&auinit=T&aulast=Satomaa&atitle=Hydrophilic+Auristatin+Glycoside+Payload+Enables+Improved+Antibody-Drug+Conjugate+Efficacy+and+Biocompatibility&id=doi%3a10.3390%2fantib7020015&title=Antibodies&volume=7&issue
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Antibody Ratio
ADC *
LC/MS *
FabRICATOR *
***
Glyco-engineered Long Acting FGF21 Variant with Optimal Pharmaceutical and Pharmacokinetic Properties to Enable Weekly to Twice Monthly Subcutaneous Dosing
Yan Weng, Tetsuya Ishino, Annette Sievers, Saswata Talukdar, Jeffrey R. Chabot, Amy Tam, Weili Duan, Kelvin Kerns, Eric Sousa, Tao He, Alison Logan, Darwin Lee, Dongmei Li, Yingjiang Zhou, Barbara Bernardo, Alison Joyce, Mania Kavosi,
Denise M. O’Hara, Tracey Clark, Jie Guo, Craig Giragossian, Mark Stahl, Roberto A. Calle, Ron Kriz, Will Somers & Laura Lin
Pharmacological administration of FGF21 analogues has shown robust body weight reduction and lipid pro le improvement in both dysmetabolic animal models and metabolic disease patients. Here we report the design, optimization, and characterization of a long acting glyco-variant of FGF21. Using
a combination of N-glycan engineering for enhanced protease resistance and improved solubility,
Fc fusion for further half-life extension, and a single point mutation for improving manufacturability
in Chinese Hamster Ovary cells, we created a novel FGF21 analogue, Fc-FGF21[R19V][N171] or PF-06645849, with substantially improved solubility and stability pro le that is compatible with subcutaneous (SC) administration. In particular, it showed a low systemic clearance (0.243 mL/hr/kg) and long terminal half-life (~200 hours for intact protein) in cynomolgus monkeys that approaches those of monoclonal antibodies. Furthermore, the superior PK properties translated into robust improvement in glucose tolerance and the e ects lasted 14 days post single SC dose in ob/ob mice. PF-06645849
also caused greater body weight loss in DIO mice at lower and less frequent SC doses, compared to previous FGF21 analogue PF-05231023. In summary, the overall PK/PD and pharmaceutical pro le of PF-06645849 o ers great potential for development as weekly to twice-monthly SC administered therapeutic for chronic treatment of metabolic diseases.
https://www.nature.com/articles/s41598-018-22456-w
https://www.genovis.com/products/iggzero/
Fab-glycosylation
Monoclonal Ab *
LC/MS *
IgGZERO *
****
N-glycans of complex glycosylated biopharmaceuticals and their impact on protein clearance
Fabian Higel, Theresa Sandl, Chi-Ya Kao, Nicole Pechinger, Fritz Sörgel, Wolfgang Friess, Florian Wolschina, Andreas Seidl
N-glycosylation is a common post-translational modification of biopharmaceutical products. Certain types of N-glycans have been shown to influence important properties of monoclonal antibody products including pharmacokinetics and effector functions. Complex biopharmaceuticals e.g. Fc fusion proteins may contain several N- and O-glycosylation sites. Domain specific characterization of two Fc fusion proteins showed an Fc N-glycosylation pattern comparable to IgG molecules. The receptor N-glycosylation was found to contain some larger and more complex N-glycans compared to the Fc part. Analyses of samples from non-clinical studies of the two studied fusion proteins indicate that their N-glycans impact pharmacokinetic properties. Interestingly, besides the type of N-glycan this influence on the pharmacokinetics depends also on the glycosylation site and thus the accessibility on the protein. The same type of N-glycan can influence the clearance of fusion proteins when located at the receptor part, but not if located at the Fc part. In this study, it is shown that N-glycans with terminal galactose or N-acetylglucosamine residues have a negative impact on serum half-life when located at the receptor part. Terminal sialylation of galactose residues prevents this faster clearance even when only one sialic acid is present. O-acetylation, a modification of sialic acids does not impact pharmacokinetics. Thus, type and accessibility of N-glycan moieties of fusion proteins both play an important role in pharmacokinetics. Finally, detailed site specific analysis is critical in the development of biopharmaceuticals.
https://www.sciencedirect.com/science/article/pii/S0939641118311688
https://www.genovis.com/products/igg-proteases/fabricator/
Fc-glycosylation;Fab-glycosylation
Monoclonal Ab *
Bioassays
FabRICATOR *
***
Application of a label-free and domain-specific free thiol method in monoclonal antibody characterization
Yi Pu, Yunqiu Chen, Tai Nguyen, Chong-Feng Xu, Li Zang, Zoran Sosic, Tyler Carlage
Characterization of free thiol variants in antibody therapeutics is important for biopharmaceutical development,
as the presence of free thiols may have an impact on aggregate formation, structural and thermal stability, as
well as antigen-binding potency of monoclonal antibodies. Most current methods for free thiol quantification
involve labeling of free thiol groups by different tagging molecules followed by UV, fluorescence or mass
spectrometry (MS) detection. Here, we optimized a label-free liquid chromatography (LC)-UV/MS method for
free thiol quantification at a subunit level and compared this method with two orthogonal and conventional
approaches, Ellman's assay and peptide mapping with differential alkylation. This subunit unit approach was
demonstrated to be able to provide domain-specific free thiol quantification and comparable results with labeling
approaches, using a relatively simple and efficient workflow.
https://www.sciencedirect.com/science/article/pii/S1570023219301928
https://www.genovis.com/products/igg-proteases/fabalactica/
Disulfide reduction
Monoclonal Ab *
LC/MS *
FabRICATOR *;FabALACTICA
*****
Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms
Guusje van Schaick Bob W.J. Pirok Rob Haselberg
Govert W. Somsen Andrea F.G. Gargano
Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glycoforms of glycoproteins. Recently, hydrophilic-interaction liquid chromatography (HILIC) -mass spectrometry has demonstrated to be a useful tool for the efficient separation and characterization of intact protein glycoforms. In particular, amide-based stationary phases in combination with acetonitrile-water gradients containing ion-pairing agents, have been used for the characterization of glycoproteins. However, finding the optimum gradient conditions for glycoform resolution can be quite tedious as shallow gradients (small decrease of acetonitrile percentage in the elution solvent over a long time) are required. In the present study, the retention mechanism and peak capacity of HILIC for non-glycosylated and glycosylated proteins were investigated and compared to reversed-phase liquid chromatography (RPLC). For both LC modes, ln k vs. φ plots of a series of test proteins were calculated using linear solvent strength (LSS) analysis. For RPLC, the plots were spread over a wider φ range than for HILIC, suggesting that HILIC methods require shallower gradients to resolve intact proteins. Next, the usefulness of computer-aided method development for the optimization of the separation of intact glycoform by HILIC was examined. Five retention models including LSS, adsorption, and mixed-mode, were tested to describe and predict glycoprotein retention under gradient conditions. The adsorption model appeared most suited and was applied to the gradient prediction for the separation of the glycoforms of six glycoproteins (Ides-digested trastuzumab, alpha-acid glycoprotein, ovalbumin, fetuin and thyroglobulin) employing the program PIOTR. Based on the results of three scouting gradients, conditions for high-efficiency separations of protein glycoforms varying in the degree and complexity of glycosylation was achieved, thereby significantly reducing the time needed for method optimization.
https://www.sciencedirect.com/science/article/pii/S0021967319302912
https://www.genovis.com/products/igg-proteases/fabricator/
Fc-glycosylation
Monoclonal Ab *
LC/MS *
FabRICATOR *
*
Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns. II. Biomolecular perspectives
Lucas K. Kimerer, Timothy M. Pabst, Alan K.
Hunter, Giorgio Carta
In Part I of this work we determined the experimental cation exchange behavior of bivalent bispecific antibodies (BiSAb) comprising a pair of single chain variable fragment (scFv) domains flexibly linked to a framework immunoglobulin G (IgG), which exhibit a complex, three-peak elution pattern dependent on the residence time. A phenomenological model was developed assuming that the BiSAb molecules exist in multiple configurations that interact differently with the resin surface and interconvert at finite rates. In Part II of this work we provide relevant biomolecular perspectives that shed light on the underlying mechanisms. Firstly, we show that crosslinking the BiSAb molecules with a bifunctional reagent, which limits conformational flexibility, suppresses multiple peak elution. Secondly, we show that of the fragments obtained by enzymatic digestion of the BiSAb molecules only those that exhibit a pair of scFv domains show three-peak elution, while only two peaks are observed if a single scFv is present. Thirdly, we analyze the roles of electrostatic and hydrophobic surface properties of the BiSAb domains, identifying regions that are likely responsible for inter-domain and protein-surface interactions. The results demonstrate that the complex elution behavior catalyzed by the combination of surface charge and hydrophobicity of the stationary phase is associated with outstretched and collapsed configurations of the scFv domains relative to the framework IgG.
https://www.sciencedirect.com/science/article/pii/S002196731930367X
https://www.genovis.com/products/igg-proteases/fabalactica/
Charge heterogeneity;Charge variants
Monoclonal Ab *
Cation-Exchange Chromatography (CEX)
FabRICATOR *;FabALACTICA
***
Capacitive Sensor to Monitor Enzyme Activity by Following Degradation of Macromolecules in Real Time
GE Bergdahl, M Hedström, B Mattiasson
A capacitive sensor was developed to analyze the presence and enzymatic activity of a model protease from standard solutions by following the degradation of the substrate in real time. The enzyme was chosen based on its specific digestion of the hinge region of immunoglobulin G (IgG). Real-time enzyme activity was monitored by measuring the change in capacitance (ΔC) based on the release of IgG fragments after enzymatic digestion by the enzyme. The results indicated that the developed capacitive system might be used successfully for label-free and real-time monitoring of enzymatic activity of different enzymes in a sensitive, rapid, and inexpensive manner in biotechnological, environmental, and clinical applications.
https://link.springer.com/article/10.1007/s12010-019-03006-0
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
FabRICATOR *
*
Mapping the O‐glycoproteome using site‐specific extraction of O‐linked glycopeptides (EXoO)
Weiming Yang, Minghui Ao, Yingwei Hu, Qing Kay Li, Hui Zhang
Protein glycosylation is one of the most abundant post‐translational modifications. However, detailed analysis of O‐linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, a chemoenzymatic method is introduced for the site‐specific extraction of O‐linked glycopeptides (EXoO), which enabled the mapping of over 3,000 O‐linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells, and serum. This large‐scale localization of O‐linked glycosylation sites demonstrated that EXoO is an effective method for defining the site‐specific O‐linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the Golgi, and the endoplasmic reticulum (ER). EXoO was also able to reveal significant differences in the O‐linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.
http://msb.embopress.org/content/14/11/e8486
https://www.genovis.com/products/enzymes-for-o-glycans/operator/
O-glycosylation
LC-MS/MS
OpeRATOR
*****
Mechanical strain determines the site-specific localization of inflammation and tissue damage in arthritis
Isabelle Cambré, Djoere Gaublomme, Arne Burssens, Peggy Jacques, Nadia Schryvers,
Amélie De Muynck, Leander Meuris, Stijn Lambrecht, Shea Carter, Pieter de Bleser, Yvan Saeys, Luc Van Hoorebeke, George Kollias, Matthias Mack, Paul Simoens, Rik Lories, Nico Callewaert, Georg Schett & Dirk Elewaut
Many pro-inflammatory pathways leading to arthritis have global effects on the immune system rather than only acting locally in joints. The reason behind the regional and patchy distribution of arthritis represents a longstanding paradox. Here we show that biomechanical loading acts as a decisive factor in the transition from systemic autoimmunity to joint inflammation. Distribution of inflammation and erosive disease is confined to mechano-sensitive regions with a unique microanatomy. Curiously, this pathway relies on stromal cells but not adaptive immunity. Mechano-stimulation of mesenchymal cells induces CXCL1 and CCL2 for the recruitment of classical monocytes, which can differentiate into bone-resorbing osteoclasts. Genetic ablation of CCL2 or pharmacologic targeting of its receptor CCR2 abates mechanically-induced exacerbation of arthritis, indicating that stress-induced chemokine release by mesenchymal cells and chemo-attraction of monocytes determines preferential homing of arthritis to certain hot spots. Thus, mechanical strain controls the site-specific localisation of inflammation and tissue damage in arthritis.
https://www.nature.com/articles/s41467-018-06933-4
https://www.genovis.com/products/iggzero/
Fc-glycosylation
IgG Polyclonal *
IgGZERO *
*
Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE
Terence L. Kirley, Kenneth D. Greis, Andrew B. Norman
Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically.
Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage
stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We
recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG
domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and
analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the
observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE.
Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb
structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of
SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)2, Fab,
and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain
unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to
generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented
should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of
conformational stabilities between chemically or genetically modified versions of a given antibody. Such
modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and
therapeutic applications.
https://www.sciencedirect.com/science/article/pii/S2405580818301882
https://www.genovis.com/products/igg-proteases/fabricator/fragit-kit/
Structure
Monoclonal Ab *
Other
FragIT / FragIT Kit
**
Fast and Simple Qualitative/Semi‐Quantitative Analysis of Monoclonal Antibody Mixtures Using Liquid Chromatography–Electrospray Triple Time‐of‐Flight Mass Spectrometry
Jin‐Ju Byeon Min‐Ho Park Seok‐Ho Shin Young G. Shin
Bispecific antibodies are generally prepared by co-expression of mixtures of recombinant monoclonal
antibodies. Because of increased molecular complexity, characterization of a desired bispecific antibody
or a mixture of monoclonal antibodies is more challenging than characterization of a conventional single
monoclonal antibody. The purpose of this study is to develop a fast and simple method for qualitative/
semi-quantitative analysis of antibody mixtures using liquid chromatography–electrospray triple time-offlight
mass spectrometry (LC-ESI-TOF/MS) to complement the enzyme-linked immunosorbent assay. To
demonstrate the proof of concept for the analysis of antibody mixtures, three different tool monoclonal
antibodies (trastuzumab, rituximab, and cetuximab) with various mixture ratios were treated by either
PNGase or Fabricator to simplify the antibody structures without glycans. After deglycosylation, the mixtures
of antibodies were analyzed by LC-ESI-TOF/MS in the positive ion mode. The m/z scan range of
2000–4000 was used for the deconvolution of each peak from antibodies. Because each antibody could
show different ionization efficiency in TOF MS, the peak intensities obtained from various mixture antibodies
(1:6:3 or 3:1:6 or 6:3:1) were normalized by the peak intensities of 1:1:1 mixture of three antibodies.
Overall, two different methods treated by either PNGase or Fabricator were comparable in
estimating the mixture ratios; however, the accuracy and precision data from the Fabricator group were
slightly better than PNGase group possibly due to the generation of smaller fragments by Fabricator.
https://onlinelibrary.wiley.com/doi/pdf/10.1002/bkcs.11610
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
LC/MS *
FabRICATOR *
***
Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography
Mingyan Cao, Niluka De Mel, Jihong Wang, Conner Parthemore, Yang Jiao, Weimin Chen, Shihua Lin, Dengfeng Liu, Greg Kilby, Xiaoyu Chen
N-terminal glutamate (E) cyclization to form pyroglutamate (pE) generates charge heterogeneities for mAbs and proteins. Thus far, pE formation rate in lyophilized formulation as compared to in liquid formulation has not been reported. Impact of pE on antibody biological activity has only been predicted or assessed using stressed samples that may contain other confounding degradations besides pE. Additionally, application of hydrophobic interaction chromatography (HIC) to separate pE has not been reported. In our study, N-terminal E cyclization was identified as the major degradation pathway in lyophilized formulation at elevated temperature for both monoclonal antibody (mAb-A) and IgG-like bispecific antibody (bsAb-A). pE was enriched in salt-gradient ion exchange chromatography (IEC) as pre-peak and in HIC as post-peak for both mAb-A and bsAb-A. Structure-function studies with pE-enriched IEC and HIC fractions confirmed that pE did not affect binding activities for mAb-A and bsAb-A. In vitro incubation of bsAb-A in serum and PBS revealed that the serum matrix may play a role in pE conversion in human serum, in contrast to the chemical reaction mechanism reported. These techniques can help in characterization of N-terminal E-to-pE cyclization and quality attribute severity assessment during therapeutic protein product development.
https://www.sciencedirect.com/science/article/pii/S0022354921004767
https://www.genovis.com/products/igg-proteases/fabricator/
Pyroglutamination;Structure
Monoclonal Ab *;Bispecific *
HIC;IEX chromatography
FabRICATOR *
Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody–Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry
Konstantin O. Nagornov, Natalia Gasilova, Anton N. Kozhinov, Pasi Virta, Patrik Holm, Laure Menin, Victor J. Nesatyy, and Yury O. Tsybin
The therapeutic efficacy and pharmacokinetics of antibody–drug conjugates (ADCs) in general, and antibody–oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap’s unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.
https://pubs.acs.org/doi/10.1021/acs.analchem.1c02247
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Antibody Ratio
Monoclonal Ab *;ADC *
HPLC *;LC/MS *;Antibody-Oligonucleotide Conjugates (AOC)
FabRICATOR *
*****
EGFR binding Fc domain-drug conjugates: stable and highly potent cytotoxic molecules mediate selective cell killing
Sebastian Jäger, Stephan Dickgiesser, Jason Tonillo, Stefan Hecht, Harald Kolmar and Christian Schröter
The exposition of cancer cells to cytotoxic doses of payload is fundamental for the therapeutic efficacy of antibody drug conjugates (ADCs) in solid cancers. To maximize payload exposure, tissue penetration can be increased by utilizing smaller-sized drug conjugates which distribute deeper into the tumor. Our group recently explored small human epidermal growth factor receptor 2 (HER2) targeting Fc antigen binding fragments (Fcabs) for ADC applications in a feasibility study. Here, we expand this concept using epidermal growth factor receptor (EGFR) targeting Fcabs for the generation of site-specific auristatin-based drug conjugates. In contrast to HER2-targeting Fcabs, we identified novel conjugation sites in the EGFR-targeting Fcab scaffold that allowed for higher DAR enzymatic conjugation. We demonstrate feasibility of resultant EGFR-targeting Fcab-drug conjugates that retain binding to half-life prolonging neonatal Fc receptor (FcRn) and EGFR and show high serum stability as well as target receptor mediated cell killing at sub-nanomolar concentrations. Our results emphasize the applicability of the Fcab format for the generation of drug conjugates designed for increased penetration of solid tumors and potential FcRn-driven antibody-like pharmacokinetics.
https://www.degruyter.com/document/doi/10.1515/hsz-2021-0321/html
https://www.genovis.com/products/glycinator/
Drug Antibody Ratio
ADC *
GlycINATOR *
****
Human plasma IgG1 repertoires are simple, unique, and dynamic
Albert Bondt, Max Hoek, Sem Tamara, Olaf L. Cremer, Marc J.M. Bonten, Albert J.R. Heck
Although humans can produce billions of IgG1 variants through recombination and hypermutation, the diversity of IgG1 clones circulating in human blood plasma has largely eluded direct characterization. Here, we combined several mass-spectrometry-based approaches to reveal that the circulating IgG1 repertoire in human plasma is dominated by a limited number of clones in healthy donors and septic patients. We observe that each individual donor exhibits a unique serological IgG1 repertoire, which remains stable over time but can adapt rapidly to changes in physiology. We introduce an integrative protein- and peptide-centric approach to obtain and validate a full sequence of an individual plasma IgG1 clone de novo. This IgG1 clone emerged at the onset of a septic episode and exhibited a high mutation rate (13%) compared with the closest matching germline DNA sequence, highlighting the importance of de novo sequencing at the protein level.
https://www.cell.com/cell-systems/fulltext/S2405-4712(21)00331-8
https://www.genovis.com/products/igg-proteases/fabalactica/
Other
Monoclonal Ab *;IgG *
LC/MS *
FabALACTICA
*****
Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins
Jose Antonio Navarro-Huerta, Amarande Murisier, Jennifer M. Nguyen, Matthew A. Lauber, Alain Beck, Davy Guillarme, Szabolcs Fekete
The purpose of this work was to study the potential of recently developed ultra-short column hardware for ion exchange chromatography (IEX). Various prototype and commercial columns having lengths of 5, 10, 15, 20 and 50 mm and packed with non-porous 3 µm particles were systematically compared. Both pH and salt gradient modes of elution were evaluated. Similarly, what has been previously reported for reversed phase liquid chromatography (RPLC) mode, an “on-off” retention mechanism was observed in IEX for therapeutic proteins and their fragments (25–150 kDa range).
Because of the non-porous nature of the IEX packing material, the column porosity was relatively low (ε = 0.42) and therefore the volumes of ultra-short columns were very small. Based on this observation, it was important to reduce as much as possible all the sources of extra-column volumes (i.e. injection volume, extra-bed volume, detector cell volume and connector tubing volume), to limit peak broadening. With a fully optimized UHPLC system, very fast separations of intact and IdeS digested mAb products were successfully performed in about 1 min using an IEX column with dimensions of 15 × 2.1 mm. This column was selected for high-throughput separations, since it probably offers the best compromise between efficiency and analysis time. For such ultra-fast separations, PEEK tubing was applied to bypass the column oven (column directly connected) to the optical detector via a zero dead volume connection.
https://www.sciencedirect.com/science/article/abs/pii/S0021967321006920
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
IEX chromatography
FabRICATOR *
Glycoform analysis of intact erythropoietin by MALDI FT-ICR mass spectrometry
Steffen Lippold, Raashina Thavarajah, Dietmar Reusch, Manfred Wuhrer, Simone Nicolardi
Recombinant human erythropoietin (EPO) is a complex therapeutic glycoprotein with three N- and one O-glycosylation sites. Glycosylation of EPO influences its safety and efficacy and is defined as a critical quality attribute. Thus, analytical methods for profiling EPO glycosylation are highly demanded. Owing to the complexity of the intact protein, information about EPO glycosylation is commonly derived from released glycan and glycopeptide analysis using mass spectrometry (MS). Alternatively, comprehensive insights into the glycoform heterogeneity of intact EPO are obtained using ESI MS-based methods with or without upfront separation of EPO glycoforms. MALDI MS, typically performed with TOF mass analyzers, has been also used for the analysis of intact EPO but, due to the poor glycoform resolution, has only provided limited glycoform information. Here, we present a MALDI FT-ICR MS method for the glycosylation profiling of intact EPO with improved glycoform resolution and without loss of sialic acid residues commonly observed in MALDI analysis. Three EPO variants were characterized in-depth and up to 199 glycoform compositions were assigned from the evaluation of doubly-charged ions, without any deconvolution of the mass spectra. Key glycosylation features such as sialylation, acetylation, and N-acetyllactosamine repeats were determined and found to agree with previously reported data obtained from orthogonal analyses. The developed method allowed for a fast and straightforward data acquisition and evaluation and can be potentially used for the high-throughput comparison of EPO samples throughout its manufacturing process.
https://www.sciencedirect.com/science/article/pii/S0003267021009107
https://www.genovis.com/products/exoglycosidases/sialexo/
O-glycosylation
SialEXO
Inline electrochemical reduction of NISTmAb for middle-up subunit liquid chromatography-mass spectrometry analysis
Tomos E. Morgan, Craig Jakes, Hendrik-Jan Brouwer, Silvia Millán-Martín, Jean-Pierre Chervet, Ken Cook, Sara Carillo and Jonathan Bones
to produce antibody subunits for middle-down and middle-up analysis. In this contribution we offer an online electrochemical reduction method for the reduction of antibodies coupled with liquid chromatography (LC) and mass spectrometry (MS), reducing the disulfide bonds present in the antibody without the need for chemical reducing agents. An electrochemical cell placed before the analytical column and mass spectrometer facilitated complete reduction of NISTmAb inter- and intrachain disulfide bonds. Reduction and analysis were carried out under optimal solvent conditions using a trapping column and switching valve to facilitate solvent exchange during analysis. The level of reduction was shown to be affected by electrochemical potential, temperature and solvent organic content, but with optimization, complete disulfide bond cleavage was achieved. The use of an inline electrochemical cell offers a simple, rapid, workflow solution for liquid chromatography mass spectrometry analysis of antibody subunits.
https://pubs.rsc.org/en/content/articlehtml/2021/an/d1an01184g
https://www.genovis.com/products/igg-proteases/fabricator/
Disulfide reduction
Monoclonal Ab *
LC/MS *
FabRICATOR *
A three-point identity criteria tool for establishing product identity using icIEF method
Deepti Ahluwalia⁎, Madesh Belakavadi, Tapan K. Das, Amit Katiyar
Product identity is one of the release testing requirements that needs to be established to ensure that there is no misidentification of drugs. Here, we demonstrated the challenges that can come across while establishing a product identity method for monoclonal antibody (mAb) and mAb-related products using icIEF method. A unique three-point identity criteria tool (visual comparison, pI of individual peaks and ΔpIs) was applied to distinguish mAb1 from the other in-house mAbs. A reduction approach followed by icIEF showed higher potential for establishing identity for mAb1 product as compared to native and enzymatic digestion approach. In general, icIEF method lacks specificity required to unequivocally establish the identity for mAbs, therefore, risk analysis is recommended before implementing icIEF as a stand-alone identity method for monoclonal antibodies.
https://www.sciencedirect.com/science/article/pii/S1570023218300916
https://www.genovis.com/products/igg-proteases/fabalactica/
Amino acid sequence;Other
Monoclonal Ab *
LC/MS *
FabULOUS *;FabALACTICA
*
Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: implications for B-cell selection
Katy A. Lloyd, Johanna Steen, Khaled Amara, Philip J. Titcombe, Lena Israelsson, Susanna L. Lundström, Diana Zhou, Roman A. Zubarev, Evan Reed, Luca Piccoli, Cem Gabay, Antonio Lanzavecchia, Dominique Baeten, Karin Lundberg, Daniel L. Mueller, Lars Klareskog, Vivianne Malmström, and Caroline Grönwall
Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico and compared to 452 highly- mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly-mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA- mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decreased in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly also through non-antigen driven mechanisms.
https://arxiv.org/pdf/1802.10401.pdf
https://www.genovis.com/products/iggzero/
Fab-glycosylation
Monoclonal Ab *
Other
IgGZERO *
***
Protein de novo sequencing by top-down and middle-down MS/MS: Limitations imposed by mass measurement accuracy and gaps in sequence coverage
Lidong He, Chad R. Weisbrod, Alan G. Marshall
With the increasing accessibility of Fourier transform (FT) mass spectrometers, top-down/middle-down MS/MS characterization of protein sequences is rapidly gaining popularity. Compared to conventional bottom-up sequencing, the top-down/middle-down approach offers the advantages of fast sample preparation and unambiguous characterization of proteoforms in a mixture. If the modified or mutated peptide segment of interest is not found or recovered with the bottom-up approaches, top-down becomes attractive relative to spending time seeking the right enzyme or chromatographic approach. Here, we discuss the potential and limitations of protein sequence analysis by top-down/middle-down MS/MS alone. Even if 100% protein sequence coverage is achieved by MS/MS, fragment mass error tolerance as low as 1 ppm or 0.5 ppm is needed to differentiate glutamine from lysine at positions not exceeding 330 amino acids (AAs) or 660 AAs from the N-/C-terminus for a protein with 660 AAs (72,760 Da) or 1320 AAs (145,520 Da). To characterize the “AA sequence gap” between two adjacent fragments, we show that the number of gap AA sequences with identical masses for di-, tri-, and tetra-AA gaps grows exponentially with increasing number of gap amino acids. If peptide fragment mass could be measured exactly (in practice, to 0.00001 Da), it would then be possible to define the overall atomic composition for the group of amino acids spanning a product ion gap 3–4 amino acids long. However, when we consider any 3–4 amino acid gap, we find that 50–75% of the possible compositions describe at least two sets of amino acids. Moreover, a next-generation protein fragment deconvolution algorithm is critical to exploit the experimentally observed high mass accuracy generated from the 21 T FT-ICR MS/MS for high confidence and high throughput top-down/middle-down analysis of proteins with unknown sequences. Finally, we show that de novo top-down/middle-down MS/MS can determine the germline sequence category for a given monoclonal antibody (mAb) and further serve to identify its novel mutations.
http://www.sciencedirect.com/science/article/pii/S1387380617302543
https://www.genovis.com/products/igg-proteases/fabricator/
Amino acid sequence
Monoclonal Ab *
LC/MS *
FabRICATOR *
****
Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls
Valegh Faid, Yann Leblanc, Nicolas Bihoreau, Guillaume Chevreux
Despite significant analytical improvements during this last decade, characterizing the whole integrity of monoclonal antibodies during their bioproduction remains a challenge. In this study, we report a new analytical approach to evaluate the overall heterogeneity/integrity of mAbs by LC-MS after combined proteolysis at their lower- and upper-hinge sites using the immunoglobulin-degrading enzymes IdeS and IgdE respectively. The whole sample preparation did not use any harsh conditions such as low pH, high temperature or reductive conditions and enables the splitting of mAbs structure into three fragments, namely the hinge dimer, Fab and Fc/2. Using the NIST mAb reference material, this method was demonstrated to be particularly suited for the analysis of mAbs disulfide bridges. The three fragments as well as their corresponding free sulfhydryl forms were well separated by chromatography and identified online by mass spectrometry. The method was then successfully applied to several mAbs of variable hydrophobicities.
https://www.sciencedirect.com/science/article/pii/S0731708517319775
https://www.genovis.com/products/igg-proteases/fabalactica/
Disulfide reduction
Monoclonal Ab *
LC/MS *
FabRICATOR *;FabALACTICA
*****
Parsimonious Charge Deconvolution for Native Mass Spectrometry
Marshall Bern, Tomislav Caval, Yong J Kil, Wilfred Tang, Christopher Becker, Eric Carlson, Doron Kletter, K. Ilker Sen, Nicolas Galy, Dominique Hagemans, Vojtech Franc, and Albert J.R. Heck
Charge deconvolution infers the mass from mass over charge (m/z) measurements in electrospray ionization mass spectra. When applied over a wide input m/z or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new “parsimonious” charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously, and when focused on the m/z range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using m/z peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies.
https://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.7b00839
https://www.genovis.com/products/igg-proteases/fabricator/
Fab-glycosylation
Monoclonal Ab *
LC/MS *;Native MS *
FabRICATOR *
*****
Practical approaches for overcoming challenges in heightened characterization of antibody-drug conjugates with new methodologies and ultrahigh-resolution mass spectrometry
Olga V. Friese, Jacquelynn N. Smith, Paul W. Brown & Jason C. Rouse
Antibody-drug conjugation strategies are continuously evolving as researchers work to improve the safety and efficacy of the molecules. However, as a part of process and product development, confirmation of the resulting innovative structures requires new, specialized mass spectrometry (MS) approaches and methods, as compared to those already established for antibody-drug conjugates (ADCs) and the heightened characterization practices used for monoclonal antibodies (mAbs), in order to accurately elucidate the resulting conjugate forms, which can sometimes have labile chemical bonds and more extreme chemical properties like hydrophobic patches. Here, we discuss practical approaches for characterization of ADCs using new methodologies and ultrahigh-resolution MS, and provide specific examples of these approaches. Denaturing conditions of typical liquid chromatography (LC)/MS analyses impede the successful detection of intact, 4-chain ADCs generated via cysteine site-directed chemistry approaches where hinge region disulfide bonds are partially reduced. However, this class of ADCs is detected intact reliably under non-denaturing size-exclusion chromatography/MS conditions, also referred to as native MS. For ADCs with acid labile linkers such as one used for conjugation of calicheamicin, careful selection of mobile phase composition is critical to the retention of intact linker-payload during LC/MS analysis. Increasing the pH of the mobile phase prevented cleavage of a labile bond in the linker moiety, and resulted in retention of the intact linker-payload. In-source fragmentation also was observed with typical electrospray ionization (ESI) source parameters during intact ADC mass analysis for a particular surface-accessible linker-payload moiety conjugated to the heavy chain C-terminal tag, LLQGA (via transglutaminase chemistry). Optimization of additional ESI source parameters such as cone voltages, gas pressures and ion transfer parameters led to minimal fragmentation and optimal sensitivity. Ultrahigh-resolution (UHR) MS, combined with reversed phase-ultrahigh performance (RP-UHP)LC and use of the FabRICATOR® enzyme, provides a highly resolving, antibody subunit-domain mapping method that allows rapid confirmation of integrity and the extent of conjugation. For some ADCs, the hydrophobic nature of the linker-payload hinders chromatographic separation of the modified subunit/domains or causes very late elution/poor recovery. As an alternative to the traditionally used C4 UHPLC column chemistry, a diphenyl column resulted in the complete recovery of modified subunit/domains. For ADCs based on maleimide chemistry, control of pH during proteolytic digestion is critical to minimize ring-opening. The optimum pH to balance digestion efficiency and one that does not cause ring opening needed to be established for successful peptide mapping.
http://www.tandfonline.com/doi/abs/10.1080/19420862.2018.1433973
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Antibody Ratio;Drug Load Distribution
ADC *
LC/MS *;Native MS *
FabRICATOR *
****
Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry
Valentina D’Atri, Szabolcs Fekete, Dwight Stoll, Matthew Lauber, Alain Beck, Davy Guillarme
Brentuximab vedotin (Adcetris) is a cysteine-linked antibody-drug conjugate (ADC) used in the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). In this study, the drug payload and glycan modifications of this ADC were simultaneously characterized using a unique LC-MS middle-up analysis, involving hydrophilic interaction chromatography (HILIC). This work demonstrates that HILIC is an effective and complementary analytical technique to reversed phase liquid chromatography (RPLC) for subunit-level characterization of immuno-conjugates.
https://www.sciencedirect.com/science/article/pii/S1570023218300771
https://www.genovis.com/products/igg-proteases/fabricator/
Fc-glycosylation;Drug Load Distribution
ADC *
LC/MS *
FabRICATOR *
*****
A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody–Drug Conjugates
Jorge M. M. Verkade, Marloes A. Wijdeven, Remon van Geel, Brian M. G. Janssen, Sander S. van Berkel and Floris L. van Delft
Despite tremendous efforts in the field of targeted cancer therapy with antibody–drug conjugates (ADCs), attrition rates have been high. Historically, the priority in ADC development has been the selection of target, antibody, and toxin, with little focus on the nature of the linker. We show here that a short and polar sulfamide spacer (HydraSpace™, AE Oss, The Netherland) positively impacts ADC properties in various ways: (a) efficiency of conjugation; (b) stability; and (c) therapeutic index. Different ADC formats are explored in terms of drug-to-antibody ratios (DAR2, DAR4) and we describe the generation of a DAR4 ADC by site-specific attachment of a bivalent linker–payload construct to a single conjugation site in the antibody. A head-to-head comparison of HydraSpace™-containing DAR4 ADCs to marketed drugs, derived from the same antibody and toxic payload components, indicated a significant improvement in both the efficacy and safety of several vivo models, corroborated by in-depth pharmacokinetic analysis. Taken together, HydraSpace™ technology based on a polar sulfamide spacer provides significant improvement in manufacturability, stability, and ADC design, and is a powerful platform to enable next-generation ADCs with enhanced therapeutic index.
http://www.mdpi.com/2073-4468/7/1/12
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Load Distribution
ADC *
LC/MS *
FabRICATOR *
**
Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies
Subhabrata Majumder, Michael T. Jones, Michael Kimmel, Arun Alphonse Ignatius
Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs.
Methods
Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [13C-1H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments.
Results
Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution.
Conclusion
The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.
https://link.springer.com/article/10.1007/s11095-018-2500-8
https://www.genovis.com/products/igg-proteases/fabricator/fragit/
Aggregation;Fc-glycosylation
Monoclonal Ab *
NMR
FragIT / FragIT Kit
**
Tuning selectivity in cation-exchange chromatography applied for monoclonal antibody separations, part 2: evaluation of recent stationary phases
Amarande Murisiera, Evelin Farsang, Krisztián Horváthb, Matthew Lauberc, Alain Beck, Davy Guillarmea, Szabolcs Feketea
In this second part of the series, recently commercialized cation exchanger stationary phases were systematically investigated for their capabilities to separate therapeutic monoclonal antibodies. It was demonstrated that the different combinations of stationary and mobile phases result in diverse retention, selectivity and efficiency. Hence, the whole phase system (combination of stationary and mobile phase) should be considered when developing a method. In addition, retention behavior is mAb dependent and should be individually optimized.
Another interesting observation was that in cation exchange chromatographic separations of large proteins, the particle size of the columns probably impacts retention rather than efficiency, due to the non-porous particle structure - and therefore the higher specific surface area of smaller particles -. Particle size influences the specific surface area and total porosity. Therefore, columns packed with larger particles showed lower retention (when the ion exchanger group was the same e.g. strong exchanger sulfonic group) while no link was observed between efficiency and particle size.
The retention, efficiency and selectivity of the studied columns were quite different and strongly dependent on the elution mode (i.e. salt gradient, pH gradient or combined salt/pH gradient mode). The columns can be considered to be complementary, suggesting that it is useful to have more than one type of column on hand while developing new charge variant assays. Moreover, this work shows that it is especially attractive to make use of short, narrow bore ion exchange columns that offer the possibility to perform 4-6 minutes long separations of both intact and partially digested antibodies.
https://www.sciencedirect.com/science/article/abs/pii/S0731708519305242
https://www.genovis.com/products/igg-proteases/fabricator/
Charge variants
Monoclonal Ab *
Cation-Exchange Chromatography (CEX)
FabRICATOR *
****
Two sequential layers of antibody‐mediated control of Legionella pneumophila infection
Stefan S. Weber, Diana Stoycheva, Falk Nimmerjahn, Annette Oxenius
Protective immunity against intracellular pathogens, including bacteria, usually relies on cellular immunity. However, antibodies are also implicated in mediating protection against intracellular bacteria. In case of airway infection with Legionella pneumophila (Lpn), the causative agent of Legionnaires' disease, pre‐existing Lpn‐specific antibodies were shown to afford protection within two days of infection. Here we dissected the early kinetics of Ab‐mediated protection against airway Lpn infection and observed two kinetically and mechanistically distinct phases of protection by passively administered antibodies. Within the first hour of infection, Lpn‐opsonizing antibodies provided almost 10 fold protection in an antibody Fc‐dependent, but FcR‐independent manner. Later on, by two days post infection, Lpn‐specific Ab‐mediated protection strictly involved FcγR, Syk kinase activity in alveolar macrophages and induction of reactive oxygen species (ROS). The findings presented here contribute to the understanding of the mechanisms of Ab‐mediated control of Lpn infection in actively or passively immunized individuals.
https://onlinelibrary.wiley.com/doi/abs/10.1002/eji.201948106
Fc-glycosylation
Monoclonal Ab *;IgG *
FabRICATOR *;IgGZERO *
**
Confident monoclonal antibody sequence verification by complementary LC-MS techniques
Amy Farrell, Sara Carillo, Jonathan Bones, Kai Scheffler, Ken Cook
To highlight the possibility of errors originating from using only a single technique for primary sequence identi cation and to show the bene ts of
the application of multiple, orthogonal techniques to address this problem. Discussing the importance of investigation of protein primary sequence at different domains together with a high level of certainty on the data generated thanks to high-resolution accurate mass MS techniques.
https://www.separatedbyexperience.com/documents/an-21919-lc-ms-monoclonal-antibody-sequence-verification-an21919-en.pdf
https://www.separatedbyexperience.com/documents/an-21919-lc-ms-monoclonal-antibody-sequence-verifica
Monoclonal Ab *
LC/MS *
FabRICATOR *
****
Using bispecific antibodies in forced degradation studies to analyze the structure–function relationships of symmetrically and asymmetrically modified antibodies
Adam R. Evans, Michael T. Capaldi, Geetha Goparaju, David Colter, Frank F. Shi, Sarah Aubert, Lian-Chao Li, Jingjie Mo, Michael J. Lewis, Ping Hu, Pedro Alfonso & Promod Mehndiratta
Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.
https://www.tandfonline.com/doi/full/10.1080/19420862.2019.1618675
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
HPLC *;HIC;SEC *
FabRICATOR *
*****
A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry
Oscar Hernandez-Alba, Stéphane Houel, Steve Hessmann, Stéphane Erb, David Rabuka, Romain Huguet, Jonathan Josephs, Alain Beck, Penelope M. Drake, Sarah Cianférani
Middle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. Middle-level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds, providing information on drug load distribution and average drug-to-antibody ratio (DAR). However, peptide mapping is still the gold standard for primary amino acid sequence assessment, post-translational modifications (PTM), and drug conjugation identification and localization. However, peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats, like antibody-drug conjugates (ADCs). We report here, for the first time, MD MS analysis of a third-generation site-specific DAR4 ADC using different fragmentation techniques, including higher-energy collisional- (HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photodissociation (UVPD). UVPD used as a standalone technique for ADC subunit analysis afforded, within the same liquid chromatography-MS/MS run, enhanced performance in terms of primary sequence coverage compared to HCD- or ETD-based MD approaches, and generated substantially more MS/MS fragments containing either drug conjugation or glycosylation site information, leading to confident drug/glycosylation site identification. In addition, our results highlight the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment. Altogether, our results highlight the potential of UVPD for ADC MD MS analysis for drug conjugation/glycosylation site assessment, and indicate that MD MS strategies can improve structural characterization of empowered next-generation mAb-based formats, especially for PTMs and drug conjugation sites validation.
https://link.springer.com/article/10.1007/s13361-019-02296-2
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Load Distribution
Monoclonal Ab *;ADC *
LC/MS *
FabRICATOR *
*****
In Vivo Surveillance and Elimination of Teratoma-forming Human Embryonic Stem Cells with Monoclonal Antibody 2448 targeting Annexin A2
Heng Liang Tan, Bao Zhu Tan, Winfred Xi Tai Goh, Simeon Cua, Andre Choo
This study describes the use of a previously reported chimerised monoclonal antibody (mAb), ch2448, to kill human embryonic stem cells (hESCs) in vivo and prevent or delay the formation of teratomas. ch2448 was raised against hESCs and was previously shown to effectively kill ovarian and breast cancer cells in vitro and in vivo. The antigen target was subsequently found to be Annexin A2, an oncofetal antigen expressed on both embryonic cells and cancer cells. Against cancer cells, ch2448 binds and kills via antibody‐dependent cell‐mediated cytotoxicity (ADCC) and/or antibody‐drug conjugate (ADC) routes. Here, we investigate if the use of ch2448 can be extended to hESC. ch2448 was found to bind specifically to undifferentiated hESC but not differentiated progenitors. Similar to previous study using cancer cells, ch2448 kills hESC in vivo either indirectly by eliciting ADCC or directly as an ADC. The treatment with ch2448 post‐transplantation eliminated the in vivo circulating undifferentiated cells and prevented or delayed the formation of teratomas. This surveillance role of ch2448 adds an additional layer of safeguard to enhance the safety and efficacious use of pluripotent stem cell‐derived products in regenerative medicine. Thereby, translating the use of ch2448 in the treatment of cancers to a proof of concept study in hESC (or pluripotent stem cell [PSC]), we show that mAbs can also be used to eliminate teratoma forming cells in vivo during PSC‐derived cell therapies. We propose to use this strategy to complement existing methods to eliminate teratoma‐forming cells in vitro. Residual undifferentiated cells may escape in vitro removal methods and be introduced into patients together with the differentiated cells.
https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.27135
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
FabRICATOR *
*
Middle-Down Multi-Attribute Analysis of Antibody- Drug Conjugates with Electron Transfer Dissociation
Bifan Chen, Ziqing Lin, Yanlong Zhu, Yutong Jin, Eli Larson, Qingge Xu, Cexiong Fu, Zhaorui Zhang, Qunying Zhang, Wayne Pritts, and Ying Ge
Antibody-drug conjugates (ADCs) are designed to combine the target specificity of monoclonal antibodies and potent cytotoxin drugs to achieve better therapeutic outcome. Comprehensive evaluation of the quality attributes of ADCs is critical for drug development but remains challenging due to heterogeneity of the construct. Currently, peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the predominant approach to characterize ADCs. However, it is suboptimal for sequence characterization and quantification of ADCs because it lacks a comprehensive view of co-existing variants and suffers from varying ionization effects of drug-conjugated peptides compared to unconjugated counterparts. Here, we present the first middle-down RPLC-MS analysis of both cysteine (Adcetris®; BV) and lysine (Kadcyla®; T-DM1) conjugated ADCs at the subunit level (~25 kDa) with electron transfer dissociation (ETD). We successfully achieved high-resolution separation of subunit isomers arising from different drug conjugation and subsequently localized the conjugation sites. Moreover, we obtained a comprehensive overview of the micro-variants associated with each subunits and characterized them such as oxidized variants with different sites. Furthermore, we observed relatively high levels of conjugation near complementarity-determining regions (CDRs) from the heavy chain but no drug conjugation near CDRs of light chain (Lc) from lysine conjugated T-DM1. Based on the extracted ion chromatograms, we measured accurate average drug to antibody ratio (DAR) values and relative occupancy of drug-conjugated subunits. Overall, the middle-down MS approach enables the evaluation of multiple quality attributes including DAR, positional isomers, conjugation sites, occupancy, and micro-variants, which potentially open up a new avenue to characterize ADCs.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b02194
https://www.genovis.com/products/igg-proteases/fabricator/
ADC *
LC/MS *
FabRICATOR *;GlycINATOR *;GingisKHAN *
*****
Side-by-Side Comparison of uPAR-Targeting Optical Imaging Antibodies and Antibody Fragments for Fluorescence-Guided Surgery of Solid Tumors
Victor M. Baart, Labrinus van Manen, Shadhvi S. Bhairosingh, Floris A. Vuijk, Luisa Iamele, Hugo de Jonge, Claudia Scotti, Massimo Resnati, Robert A. Cordfunke, Peter J. K. Kuppen, Andrew P. Mazar, Jacobus Burggraaf, Alexander L. Vahrmeijer & Cornelis F. M. Sier
Purpose
Radical resection is paramount for curative oncological surgery. Fluorescence-guided surgery (FGS) aids in intraoperative identification of tumor-positive resection margins. This study aims to assess the feasibility of urokinase plasminogen activator receptor (uPAR) targeting antibody fragments for FGS in a direct comparison with their parent IgG in various relevant in vivo models.
Procedures
Humanized anti-uPAR monoclonal antibody MNPR-101 (uIgG) was proteolytically digested into F(ab’)2 and Fab fragments named uFab2 and uFab. Surface plasmon resonance (SPR) and cell assays were used to determine in vitro binding before and after fluorescent labeling with IRDye800CW. Mice bearing subcutaneous HT-29 human colonic cancer cells were imaged serially for up to 120 h after fluorescent tracer administration. Imaging characteristics and ex vivo organ biodistribution were further compared in orthotopic pancreatic ductal adenocarcinoma (BxPc-3-luc2), head-and-neck squamous cell carcinoma (OSC-19-luc2-GFP), and peritoneal carcinomatosis (HT29-luc2) models using the clinical Artemis fluorescence imaging system.
Results
Unconjugated and conjugated uIgG, uFab2, and uFab specifically recognized uPAR in the nanomolar range as determined by SPR and cell assays. Subcutaneous tumors were clearly identifiable with tumor-to-background ratios (TBRs) > 2 after 72 h for uIgG-800F and 24 h for uFab2-800F and uFab-800F. For the latter two, mean fluorescence intensities (MFIs) dipped below predetermined threshold after 72 h and 36 h, respectively. Tumors were easily identified in the orthotopic models with uIgG-800F consistently having the highest MFIs and uFab2-800F and uFab-800F having similar values. In biodistribution studies, kidney and liver fluorescence approached tumor fluorescence after uIgG-800F administration and surpassed tumor fluorescence after uFab2-800F or uFab-800F administration, resulting in interference in the abdominal orthotopic mouse models.
Conclusions
In a side-by-side comparison, FGS with uPAR-targeting antibody fragments compared with the parent IgG resulted in earlier tumor visualization at the expense of peak fluorescence intensity.
https://link.springer.com/article/10.1007/s11307-021-01657-2
https://www.genovis.com/products/igg-proteases/gingiskhan/
Imaging
Monoclonal Ab *
Other
FabRICATOR *;GingisKHAN *
Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis
Bastiaan L. Duivelshof, Steffy Denorme, Koen Sandra, Xiaoxiao Liu, Alain Beck, Matthew A. Lauber, Davy Guillarme and Valentina D’Atri
The identification and accurate quantitation of the various glycoforms contained in ther- apeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to var- ious glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Fi- nally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute mon- itoring method.
https://pubmed.ncbi.nlm.nih.gov/34834160/
https://www.genovis.com/products/igg-proteases/fabricator/
Glycation
Monoclonal Ab *
HILIC
FabRICATOR *
Mechanistic insights into the rational design of masked antibodies
Carolina T Orozco, Manuela Bersellini, Lorraine M Irving, Wesley W Howard, David Hargreaves, Paul W A Devine, Elise Siouve, Gareth J Browne, Nicholas J Bond, Jonathan J Phillips,, Peter Ravn, Sophie E Jackson
Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.
https://www.tandfonline.com/doi/full/10.1080/19420862.2022.2095701
https://www.genovis.com/products/igg-proteases/fabalactica/
Structure;Antigen Binding
Monoclonal Ab *
HDX-MS
FabALACTICA
Kinetic performance comparison of superficially porous, fully porous and monolithic reversed-phase columns by gradient kinetic plots for the separation of protein biopharmaceuticals
Simon Jaag, Chunmei Wen, Benjamin Peters, Michael Lämmerhofer
To find the best performing column for the analysis of protein-based biopharmaceuticals is a significant challenge as meanwhile numerous modern columns with distinct stationary phase morphologies are available for reversed-phase liquid chromatography. Especially when besides morphology also several other column factors are different, it is hard to decide about the best performing column a priori. To cope with this problem, in the present work 13 different reversed-phase columns dedicated for protein separations were systematically tested by the gradient kinetic plot method. A comprehensive comparison of columns with different morphologies (monolithic, fully porous and superficially porous particle columns), particle sizes and pore diameters as well as column length was performed. Specific consideration was also given to various monolithic columns which recently shifted a bit out of the prime focus in the scientific literature. The test proteins ranged from small proteins starting from 12 kDa, to medium sized proteins (antibody subunits obtained after IdeS-digestion and disulphide reduction) and an intact antibody. The small proteins cytochrome c, lysozyme and β-lactoglobulin could be analysed with similar performance by the best columns of all three column morphologies while for the antibody fragments specific fully porous and superficially porous particle columns were superior. A 450 Å 3,5 µm superficially porous particle column showed the best performance for the intact antibody while a 1.7 µm fully porous particle column with 300 Å showed equivalent performance to the best superficially porous column with thin shell and 400 Å pore size for proteins between 12 and 25 kDa. While the majority of the columns had C4 bonding chemistry, the silica monolith with C18 bonding and 300 Å mesopore size approximated the best performing particle columns and outperformed a C4 300 Å wide-pore monolith. The current work can support the preferred choice for the most suitable reversed-phase column for protein separations.
https://www.sciencedirect.com/science/article/abs/pii/S0021967322004447
https://www.genovis.com/products/igg-proteases/fabricator/fragit/
Pharmacokinetic assessment
Monoclonal Ab *
HPLC *
FragIT / FragIT Kit
Capillary (Gel) Electrophoresis-Based Methods for Immunoglobulin (G) Glycosylation Analysis
Samanta Cajic, René Hennig, Robert Burock, Erdmann Rapp
The in-depth characterization of protein glycosylation has become indispensable in many research fields and in the biopharmaceutical industry. Especially knowledge about modulations in immunoglobulin G (IgG) N-glycosylation and their effect on immunity enabled a better understanding of human diseases and the development of new, more effective drugs for their treatment. This chapter provides a deeper insight into capillary (gel) electrophoresis-based (C(G)E) glycan analysis, addressing its impressive performance and possibilities, its great potential regarding real high-throughput for large cohort studies, as well as its challenges and limitations. We focus on the latest developments with respect to miniaturization and mass spectrometry coupling, as well as data analysis and interpretation. The use of exoglycosidase sequencing in combination with current C(G)E technology is discussed, highlighting possible difficulties and pitfalls. The application section describes the detailed characterization of N-glycosylation, utilizing multiplexed CGE with laser-induced fluorescence detection (xCGE-LIF). Besides a comprehensive overview on antibody glycosylation by comparing species-specific IgGs and human immunoglobulins A, D, E, G, and M, the chapter comprises a comparison of therapeutic monoclonal antibodies from different production cell lines, as well as a detailed characterization of Fab and Fc glycosylation. These examples illustrate the full potential of C(G)E, resolving the smallest differences in sugar composition and structure.
https://link.springer.com/chapter/10.1007/978-3-030-76912-3_4
https://www.genovis.com/products/igg-proteases/fabricator-z/
Fc-glycosylation
Monoclonal Ab *
CE;CE-MS
FabRICATOR Z
Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography
Siddharth S. Parasnavis, Ben Niu, Matthew Aspelund, Wai K. Chung, Mark Snyder, Steven M. Cramer
In this article, a systematic workflow was formulated and implemented to understand selectivity differences and preferred binding patches for bispecific monoclonal antibodies (mAbs) and their parental mAbs on three multimodal cation exchange resin systems. This workflow incorporates chromatographic screening of the parent mAbs and their fragments at various pH followed by surface property mapping and protein footprinting using covalent labeling followed by liquid chromatography–mass spectrometry analysis. The chromatography screens on multimodal resins with the intact mAbs indicated enhanced selectivity as compared to single-mode interaction systems. While the bispecific antibody (bsAb) eluted between the two parental mAbs on most of the resins, the retention of the bispecific transitioned from co-eluting with one parental mAb to the other parental mAb on Capto MMC. To investigate the contribution of different domains, mAb fragments were evaluated and the results indicated that the interactions were likely dominated by the Fab domain at higher pH. Protein surface property maps were then employed to hypothesize the potential preferred binding patches in the solvent-exposed regions of the parental Fabs. Finally, protein footprinting was carried out with the parental mAbs and the bsAb in the bound and unbound states at pH 7.5 to identify the preferred binding patches. Results with the intact mAb analysis supported the hypothesis that interactions with the resins were primarily driven by the residues in the Fab fragments and not the Fc. Furthermore, peptide mapping data indicated that the light chain may be playing a more important role in the higher binding of Parent A as compared with Parent B in these resin systems. Finally, results with the bsAb indicated that both halves of the molecule contributed to binding with the resins, albeit with subtle differences as compared to the parental mAbs. The workflow presented in this paper lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules which can provide insights into improved biomanufacturability and expedited downstream bioprocess development.
https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.27967
https://www.genovis.com/products/igg-proteases/fabricator/
Structure
Monoclonal Ab *;Bispecific *
LC/MS *;SEC *
FabRICATOR *
Elevated Fab glycosylation of anti-hinge antibodies
J Koers, NIL Derksen, WJJ Falkenburg, P Ooijevaar-de Heer, MT Nurmohamed, GJ Wolbink & T Rispens
Abstract
Objective
Rheumatoid arthritis (RA) is characterized by systemic inflammation and the presence of anti-citrullinated protein antibodies (ACPAs), which contain remarkably high levels of Fab glycosylation. Anti-hinge antibodies (AHAs) recognize immunoglobulin G (IgG) hinge neoepitopes exposed following cleavage by inflammation-associated proteases, and are also frequently observed in RA, and at higher levels compared to healthy controls (HCs). Here, we investigated AHA specificity and levels of Fab glycosylation as potential immunological markers for RA.
Method
AHA serum levels, specificity, and Fab glycosylation were determined for the IgG1/4-hinge cleaved by matrix metalloproteinase-3, cathepsin G, pepsin, or IdeS, using enzyme-linked immunosorbent assay and lectin affinity chromatography, in patients with early active RA (n = 69) and HCs (n = 97).
Results
AHA reactivity was detected for all hinge neoepitopes in both RA patients and HCs. Reactivity against CatG-IgG1-F(ab´)2s and pepsin-IgG4-F(ab´)2s was more prevalent in RA. Moreover, all AHA responses showed increased Fab glycosylation levels in both RA patients and HCs.
Conclusions
AHA responses are characterized by elevated levels of Fab glycosylation and highly specific neoepitope recognition, not just in RA patients but also in HCs. These results suggest that extensive Fab glycosylation may develop in response to an inflammatory proteolytic microenvironment, but is not restricted to RA.
https://www.tandfonline.com/doi/abs/10.1080/03009742.2021.1986959
https://www.genovis.com/products/igg-proteases/fabricator/
Fab-glycosylation
Monoclonal Ab *
Bioassays
FabRICATOR *
Next generation Fc scaffold for multispecific antibodies
Bram Estes, Athena Sudom, Danyang Gong, Douglas A. Whittington, Vivian Li, Christopher Mohr, Danqing Li, Timothy P. Riley, Stone D.-H. Shi, Jun Zhang, Fernando Garces, and Zhulun Wang
Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH30 interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8633962/
https://www.genovis.com/products/igg-proteases/fabricator/
Structure;Chain pairing
Bispecific *
SEC *;HDX-MS;DSC (differential scanning calorimetry)
FabRICATOR *
Clinical assay for direct assessment of IgG galactosylation in serum using endoglycosidase S
Dieter Vanderschaeghe, Leander Meuris, Hendrik Grootaert, Tom Raes, Annelies Van Hecke, Xavier Verhelst, Frederique Van de Velde, Bruno Lapauw, Hans Van Vlierberghe, and Nico Callewaert
A wide spectrum of (autoimmune) diseases such as rheumatoid arthritis (RA), Crohn’s disease, systemic lupus erythematosus, but also other pathologies such as alcoholic and non-alcoholic steatohepatitis (ASH and NASH) are driven by chronic inflammation and hallmarked by a reduced level of serum Immunoglobulin G (IgG) galactosylation. IgG undergalactosylation is a promising biomarker to assess the severity of disease, monitor therapy efficacy and adjust therapy accordingly. The main hurdle for clinical implementation is the necessity for purifying the antibodies in order to specifically determine IgG Fc glycan galactosylation. We addressed this issue by using endo-- N-acetylglucosaminidase from Streptococcus pyogenes (endoS) and optimized the reaction conditions in which the enzyme specifically releases the IgG Fc N-glycans in serum. The sample preparation takes two hours and only entails the addition of endoS to serum and subsequent labeling with the fluorophore 8-aminopyrene-1,3,6- trisulphonic acid (APTS). Samples are then readily analyzed on high-throughput DNA sequencers, which are able to analyze up to 96 samples in one hour. We demonstrate in two separate patient cohorts that the degree of IgG undergalactosylation from our assay correlates very well with undergalactosylation scores calculated from N-glycan profiles derived from antibodies that have been purified from serum and deglycosylated with peptide N-glycosidase from Flavobacterium meningosepticum (PNGaseF). The presented new assay thus allows to directly and specifically measure the degree of IgG galactosylation in serum without the requirement of isolating the antibodies, and should help in advancing this biomarker towards clinical implementation.
https://www.biorxiv.org/content/biorxiv/early/2018/02/22/269795.full.pdf
https://www.genovis.com/products/iggzero/
IgG Polyclonal *
IgGZERO *
****
Quantitative analysis of glycation and its impact on antigen binding
Jingjie Mo, Renzhe Jin, Qingrong Yan, Izabela Sokolowska, Michael J. Lewis & Ping Hu
Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.
https://www.tandfonline.com/doi/full/10.1080/19420862.2018.1438796
https://www.genovis.com/products/iggzero/
Glycation
Monoclonal Ab *
LC/MS *;cIEF *
IgGZERO *
*****
Deciphering Protein O‑Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment
Yang S., Onigman P., Wu WW., Sjogren J., Nyhlen H., Shen RF. and Cipollo J.
Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. N-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on O-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of O-glycans. Although O-glycosidase can catalyze the removal of core 1 and core 3 O-linked disaccharides from glycoproteins, analysis of other types of O-glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available O-protease and solid phase platform. This method enables the assignment of O-glycosylated peptides, N-glycan profile, sialyl O-glycopeptides linkage, and mapping of heterogeneous O-glycosylation. For the first time, we can analyze intact O-glycopeptides generated by O-protease and enriched using a solid-phase platform. 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. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant O-glycosites and setting a basis set of O-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein O-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where O-glycosylation is central.
https://www.ncbi.nlm.nih.gov/pubmed/29936827
https://www.genovis.com/products/enzymes-for-o-glycans/operator/
LC/MS *
OpeRATOR
****
Multiplexed Middle-Down Mass Spectrometry Reveals Light and Heavy Chain Connectivity in a Monoclonal Antibody
Kristina Srzentic, Konstantin O. Nagornov, Luca Fornelli, Anna A. Lobas, Daniel Ayoub, Anton Kozhinov, Natalia Gasilova, Laure Menin, Alain Beck, Mikhail Gorshkov, Konstantin Aizikov, and Yury O. Tsybin
Pairing light and heavy chains in monoclonal antibodies (mAbs) using top
down (TD) or middle-down (MD) mass spectrometry (MS) may complement the
sequence information on single chains provided by high-throughput genomic
sequencing and bottom-up proteomics, favoring the rational selection of drug
candidates. The 50 kDa F(ab) subunits of mAbs are the smallest structural
units that contain the required information on chain pairing. These subunits
can be enzymatically produced from whole mAbs and interrogated in their
intact form by TD/MD MS approaches. However, the high structural
complexity of F(ab) subunits requires increased sensitivity of the modern
TD/MD MS for a comprehensive structural analysis. To address this and
similar challenges, we developed and applied a multiplexed TD/MD MS
workflow based on spectral averaging of tandem mass spectra (MS/MS) across
multiple liquid chromatography (LC)-MS/MS runs acquired in reduced or full
profile mode using an Orbitrap Fourier transform mass spectrometer (FTMS).
We first benchmark the workflow using myoglobin as a reference protein and
then validate it for the analysis of the 50 kDa F(ab) subunit of a therapeutic
mAb, trastuzumab. Obtained results confirm the envisioned benefits in terms
of increased signal-to-noise ratio of product ions from utilizing multiple LC
MS/MS runs for TD/MD protein analysis using mass spectral averaging. The
workflow performance is compared with the earlier introduced multiplexed
TD/MD MS workflow based on transient averaging in Orbitrap FTMS. For the
latter, we also report on enabling absorption mode FT processing and
demonstrate its comparable performance to the enhanced FT (eFT) spectral
representation.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.8b02398
https://www.genovis.com/products/igg-proteases/gingiskhan/
Structure;Chain pairing
Monoclonal Ab *;IgG *
LC-MS/MS
GingisKHAN *
Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry
Yutong Jin, Ziqing Lin, Qingge Xu, Cexiong Fu, Zhaorui Zhang, Qunying Zhang, Wayne A. Pritts, Ying Ge
The pharmaceutical industry’s interest in monoclonal antibodies (mAbs) and their derivatives
has spurred rapid growth in the commercial clinical pipeline of these effective therapeutics. The
complex micro-heterogeneity of mAbs requires in-depth structural characterization for critical
quality attribute assessment and quality assurance. Currently, mass spectrometry (MS)-based
methods are the gold standard in mAb analysis, primarily with a bottom-up approach in which
immunoglobulins G (IgGs) and their variants are digested into peptides to facilitate the analysis.
Comprehensive characterization of IgGs and the micro-variants remains challenging at the
proteoform level. Here, we used both top-down and middle-down MS for in-depth
characterization of a human IgG1 using ultra-high resolution Fourier transform MS. Our top
down MS analysis provided characteristic fingerprinting of the IgG1 proteoforms at unit mass
resolution. Subsequently, the tandem MS analysis of intact IgG1 enabled the detailed sequence
characterization of a representative IgG1 proteoform at the intact protein level. Moreover, we
used the middle-down MS analysis to characterize the primary glycoforms and micro-variants.
Micro-variants such as low-abundance glycoforms, C-terminal glycine clipping, and C-terminal
proline amidation were characterized with bond cleavages higher than 44% at the subunit level.
By combining top-down and middle-down analysis, 76% of bond cleavage (509/666 amino acid
bond cleaved) of IgG1 was achieved. Taken together, we demonstrated the combination of top
down and middle-down MS as powerful tools in the comprehensive characterization of mAbs.
https://doi.org/10.1080/19420862.2018.1525253
https://www.genovis.com/products/igg-proteases/fabricator/
Fc-glycosylation;C-terminal processing
Monoclonal Ab *;IgG *
LC/MS *;LC-MS/MS
FabRICATOR *;IgGZERO *
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