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
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
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
*
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 *
****
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 *
***
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 *
*****
Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique
Yunxiao Zhu, Deepti Ahluwalia, Yingchen Chen, Madesh Belakavadi, Amit Katiyar, Tapan K. Das
Fragmentation in protein‐based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size‐exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE‐SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein‐based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab‐related low molecular weight fragments, a non‐reducible thio‐ether linked 75 kDa HL fragment was also identified.
https://onlinelibrary.wiley.com/doi/abs/10.1002/elps.201900119
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
FabALACTICA
*
Forced Degradation Testing as Complementary Tool for Biosimilarity Assessment
Yan Felix Karl Dyck, Daniel Rehm, Jan Felix Joseph, Karsten Winkler, Volker Sandig, Wolfgang Jabs, Maria Kristina Parr
Abstract: Oxidation of monoclonal antibodies (mAbs) can impact their efficacy and may therefore
represent critical quality attributes (CQA) that require evaluation. To complement classical CQA, bevacizumab and infliximab were subjected to oxidative stress by H2O2 for 24, 48, or 72 h to probe their oxidation susceptibility. For investigation, a middle-up approach was used utilizing liquid chromatography hyphenated with mass spectrometry (LC-QTOF-MS). In both mAbs, the Fc/2 subunit was completely oxidized. Additional oxidations were found in the light chain (LC) and in the Fd’ subunit of infliximab, but not in bevacizumab. By direct comparison of methionine positions, the oxidized residues in infliximab were assigned to M55 in LC and M18 in Fd’. The forced oxidation approach was further exploited for comparison of respective biosimilar products. Both for bevacizumab and infliximab, comparison of posttranslational modification profiles demonstrated high similarity of the unstressed reference product (RP) and the biosimilar (BS). However, for bevacizumab, comparison after forced oxidation revealed a higher susceptibility of the BS compared to the RP. It may thus be considered a useful tool for biopharmaceutical engineering, biosimilarity assessment, as well as for quality control of protein drugs.
http://scholar.google.se/scholar_url?url=https://www.mdpi.com/2306-5354/6/3/62/pdf&hl=en&sa=X&d=15749021992924168919&scisig=AAGBfm10AT-UuLS_ZyLOQP6ZKfFPgBcRKA&nossl=1&oi=scholaralrt&hist=lh8u15cAAAAJ:12383316450227222707:AAGBfm08V9WcS82pPkGWWTIHxUo3x9pMMA
https://www.genovis.com/products/igg-proteases/fabricator/
Oxidation
Monoclonal Ab *
LC/MS *
FabRICATOR *
*****
Utility of High Resolution NMR Methods to Probe the Impact of Chemical Modifications on Higher Order Structure of Monoclonal Antibodies in Relation to Antigen Binding
Subhabrata Majumder, Andrew Saati, Shibu Philip, Lucy L. Liu, Elaine Stephens, Jason C. Rouse, Arun Alphonse Ignatius
Purpose An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest.
Methods To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks. Samples were aliquoted at various time points and high reso- lution accurate mass liquid chromatography-mass spectrome- try (LC-MS/MS) was performed using low-artifact trypsin digestion (LATD) peptide mapping to identify and quantify chemical modifications. 2D backbone amide and sidechain methyl NMR spectra were acquired to gauge the effect of HOS changes upon chemical modification. Differential scan- ning calorimetry was also performed to assess the effect of thermal stability of mAbs upon modification. Finally, func- tional studies via target-binding based ELISA were performed to connect HOS changes to any loss of potency.
Results The extent of deamidation in the mAb domains were quantified by LC-MS/MS. The HOS changes as obtained from 2D NMR were mostly localized around the affected sites leaving the overall structure relatively unchanged. The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged.
https://link.springer.com/article/10.1007/s11095-019-2652-1
https://www.genovis.com/products/igg-proteases/fabricator/
Deamidation
Monoclonal Ab *
NMR;LC-MS/MS
FabRICATOR *
***
Site-specifically labeled 89Zr-DFO-trastuzumab improves immuno-reactivity and tumor uptake for immuno-PET in a subcutaneous HER2-positive xenograft mouse model
Lotte K. Kristensen, Camilla Christensen, Mette M. Jensen, Brian J. Agnew, Christina Schjöth-Frydendahl, Andreas Kjaer, Carsten H. Nielsen
Antibody-based PET tracers are exceptionally well-suited for determination of the in vivo biodistribution and quantification of therapeutic antibodies. The continued expansion in antibody-based therapeutics has accordingly driven the development towards more robust conjugation strategies in order to reliably predict the performance of such agents. We therefore aimed to evaluate the effect of site-specific labeling by enzymatic remodeling on the stability, immuno-reactivity and tumor-targeting properties of the monoclonal antibody (mAb) trastuzumab and compare it to conventional, random labeling in a HER2-positive xenograft mouse model.
Methods: Trastuzumab was conjugated with the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator randomly on lysine residues or site-specifically on enzymatically modified glycans using either β-galactosidase or endoglycosidase S2 prior to 89Zr radiolabeling. 89Zr-DFO-trastuzumab was injected into SK-OV-3 tumor-bearing NMRI nude mice. The antibody dose was titrated with either 100 µg or 500 µg of unlabeled trastuzumab. Mice underwent small animal PET/CT imaging 24, 70 and 120 hours post-injection for longitudinal assessment. Parallel experiments were conducted with an isotype control matched antibody. In vivo imaging was supported by conventional ex vivo biodistribution and HER2 immuno-histochemistry. Furthermore, site-specifically labeled 89Zr-DFO-trastuzumab was evaluated in a panel of subcutaneous patient-derived xenograft (PDX) models. Additionally, the affinity, in vitro stability and immuno-reactivity were assessed for all tracers.
Results: Site-specific labeling significantly increased PET tumor uptake (One-way ANOVA, p<0.0001) at all time-points when compared to random labeling. Mean tumor uptakes were 6.7 ± 1.7, 13.9 ± 3.3 and 15.3 ± 3.8 % injected dose per gram tissue (%ID/g) at 70 hours post-injection, for random, β-galactosidase or endoglycosidase S2 labeled probes, respectively. Co-injection with unlabeled trastuzumab increased the circulation time of tracers but did not alter tumor uptake notably. Site-specific probes presented with a superior in vitro stability and immuno-reactivity compared to the randomly labeled probe. Ex vivo biodistribution confirmed the data obtained by in vivo PET imaging, and site-specific 89Zr-DFO-trastuzumab successfully detected HER2-positive tumors in PDX mouse models.
Conclusion: 89Zr-DFO-trastuzumab is well-matched for specific immuno-PET imaging of HER2-positive tumors and site-specific labeling of trastuzumab by the SiteClickTM technology minimizes the impact of the DFO chelator on immuno-reactivity, stability and biodistribution. These findings support further development of site-specifically radiolabeled mAbs for immuno-PET.
http://www.thno.org/v09p4409.htm
https://www.genovis.com/products/glyclick/
ADC *
GlyCLICK
*****
Susceptibility of protein therapeutics to spontaneous chemical modifications by oxidation, cyclization, and elimination reactions
Luigi Grassi, Chiara Cabrele
Peptides and proteins are preponderantly emerging in the drug market, as shown by the increasing number of biopharmaceutics already approved or under development. Biomolecules like recombinant monoclonal antibodies have high therapeutic efficacy and offer a valuable alternative to small-molecule drugs. However, due to their complex three-dimensional structure and the presence of many functional groups, the occurrence of spontaneous conformational and chemical changes is much higher for peptides and proteins than for small molecules. The characterization of biotherapeutics with modern and sophisticated analytical methods has revealed the presence of contaminants that mainly arise from oxidation- and elimination-prone amino-acid side chains. This review focuses on protein chemical modifications that may take place during storage due to (1) oxidation (methionine, cysteine, histidine, tyrosine, tryptophan, and phenylalanine), (2) intra- and inter-residue cyclization (aspartic and glutamic acid, asparagine, glutamine, N-terminal dipeptidyl motifs), and (3) β-elimination (serine, threonine, cysteine, cystine) reactions. It also includes some examples of the impact of such modifications on protein structure and function.
https://link.springer.com/article/10.1007/s00726-019-02787-2
https://www.genovis.com/products/igg-proteases/gingiskhan/
FabRICATOR *;GingisKHAN *
****
Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has Mucin 2–processing properties
Elisabeth E.L. Nyström, Liisa Arike, Erik Ehrencrona, Gunnar C. Hansson, and Malin E.V. Johansson
Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major non-mucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1’s biochemical properties both in vitro and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that non-covalently interacts with the N terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.
http://www.jbc.org/content/early/2019/09/29/jbc.RA119.009940.full.pdf
https://www.genovis.com/products/enzymes-for-o-glycans/sialexo/
SialEXO
**
Unambiguous Sequence Characterization of a Monoclonal Antibody in a Single Analysis Using a Nonspecific Immobilized Enzyme Reactor
Joshua Hinkle, Robert Anthony D'Ippolito, Maria C. Panepinto, Weihan Wang, Dina L. Bai, Jeffrey Shabanowitz, and Donald F. Hunt
Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1-20 kDa. Utilizing the Thermo Orbitrap Fusion’s unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b02666
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
FabRICATOR *
***
On enzymatic remodeling of IgG glycosylation; unique tools with broad applications
Jonathan Sjögren, Rolf Lood, Andreas Nägeli
The importance of IgG glycosylation has been known for many years not only by scientists in glycobiology, but also by human pathogens that have evolved specific enzymes to modify these glycans with fundamental impact on IgG function. The rise of IgG as a major therapeutic scaffold for many cancer and immunological indications combined with the availability of unique enzymes acting specifically on IgG Fc-glycans have spurred a range of applications to study this important post-translational modification on IgG. This review article introduces why the IgG glycans are of distinguished interest, gives a background on the unique enzymatic tools available to study the IgG glycans, and finally presents an overview of applications utilizing these enzymes for various modifications of the IgG glycans. The applications covered include; site-specific glycan transglycosylation and conjugation, analytical workflows for monoclonal antibodies, serum diagnostics and many more. Additionally, the review looks ahead and discusses the importance of O- glycosylation for IgG3, Fc-fusion proteins, and other new formats of biopharmaceuticals.
https://watermark.silverchair.com/cwz085.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAnYwggJyBgkqhkiG9w0BBwagggJjMIICXwIBADCCAlgGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMli3plFt9NITZBzdkAgEQgIICKZnm2XXYCKXi2A_y9VhCacNNkNNZXUnWjKvR9xUhn-oeYzH2
https://www.genovis.com/products/glycinator/
https://www.genovis.com/products/glycinator/
Monoclonal Ab *;IgG *
IgGZERO *;GlycINATOR *
*****
Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh resolution MALDI FT-ICR mass spectrometry
Christoph Gstöttner, Dietmar Reusch, Markus Haberger, Irina Dragan, Peter van Veelen, David P. A. Kilgour, Yury O. Tsybin, Yuri E.M. van der Burgt, Manfred Wuhrer & Simone Nicolardi
Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The “bi-specificity” of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in formulation or in vivo after application of the drug. Glycation affects the structure, function and stability of monoclonal antibodies, and consequently, detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle- down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd’ regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168h forced- glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.
https://www.tandfonline.com/doi/full/10.1080/19420862.2019.1682403?scroll=top&needAccess=true
https://www.genovis.com/products/igg-proteases/fabricator/
Glycation
Monoclonal Ab *;Bispecific *
MALDI-TOF-MS *
FabRICATOR *
*****
A generic method for intact and subunit level characterization of mAb charge variants by native mass spectrometry
Y. Leblanc, V. Faid, M.A. Lauber, Q. Wang, N. Bihoreau, G. Chevreux
Monoclonal antibodies (mAbs) are heterogeneous macromolecules that display a complex isoform profile as a result of the large series of modifications they can undergo. Product-related charge variants that are associated with a loss of biological activity or affected half-life and immunogenicity are especially important. Consequently, they are often considered critical quality attributes such that acceptance criteria and controls should be established. The characterization of mAbs charge variants has long been a time and resource consuming task. Recent successes in the use of salt mediated pH gradient ion exchange chromatography with volatile mobile phases have shown there to be significant promise in using online mass spectrometric (MS) detection to facilitate peak detection.
In this study, a newly developed 3 μm non-porous cation exchange column technology was investigated for its capability to be hyphenated to MS for the purpose of characterizing mAb charge variants. A 2 mm ID format was selected for the ease of configuring it to classical MS ESI ion sources. A monoclonal antibody reference material from NIST (RM 8671; NISTmAb) was used in its intact and IdeS/IgdE-digested forms to test for column performance and MS sensitivity. Furthermore, three different mAbs with highly basic isoelectric points (pI) were analyzed in their native and proteolyzed forms to demonstrate the straightforward application of the developed technique even with mAbs having strong retention on cation exchange media. The MS detection of low-abundance charge variant species (<0.1%) demonstrated there to be acceptable sensitivity and dynamic range even from routine analyses. The capability of the column to separate different mAbs having high basic pI was demonstrated, and it was found that slight adjustment of ammonium acetate concentration in the eluent can be a convenient way to rapidly optimize a separation if necessary. Linearity was shown to exist between protein mass loads of 2.5 to 50 μg while an optimal balance between chromatographic resolution and MS sensitivity was observed between 5 and 10 μg. Excellent run-to-run and column-to-column repeatability was achieved in terms of retention times, resolution and recovery.
https://www.sciencedirect.com/science/article/abs/pii/S1570023219311171
https://www.genovis.com/products/igg-proteases/fabalactica/
Monoclonal Ab *
Native MS *;IEX chromatography
FabRICATOR *;FabALACTICA
****
Affinity-matured ‘aquaporumab’ anti-aquaporin-4 antibody for therapy of seropositive neuromyelitis optica spectrum disorders
Tianjiao Duan, Lukmanee Tradtrantip, Puay-Wah Phuan, Jeffrey L.Bennett, Alan S. Verkman
Pathogenesis in seropositive neuromyelitis optica spectrum disorders (herein called NMO) involves binding of IgG1 autoantibodies to aquaporin-4 (AQP4) on astrocytes in the central nervous system, which initiates complement and cellular injury. We previously developed an antibody blocking approach for potential therapy of NMO in which an engineered, monoclonal, anti-AQP4 antibody lacking cytotoxicity effector functions (called aquaporumab) blocked binding of NMO autoantibodies to astrocyte AQP4 (Tradtrantip et al. Ann. Neurol. 71, 314–322, 2012). Here, a high-affinity aquaporumab, which was generated by affinity maturation using saturation mutagenesis, was shown to block cellular injury caused by NMO patient sera. Anti-AQP4 antibody rAb-53, a fully human antibody with effector function neutralizing Fc mutations L234A/L235A and affinity-enhancing Fab mutations Y50R/S56R, called AQmabAM, bound to AQP4 in cell cultures with Kd ∼18 ng/ml (∼0.12 nM), ∼8-fold greater affinity than the original antibody. AQmabAM, but without L234A/L235A Fc mutations, produced complement-dependent cytotoxicity (CDC) with EC50 ∼ 82 ng/ml. AQmabAM prevented CDC produced by sera from eight NMO patients with IC50 ranging from 40 to 80 ng/ml, and similarly prevented antibody-dependent cellular cytotoxicity (ADCC). Mechanistic studies demonstrated that AQmabAM blocked binding of serum NMO autoantibodies to AQP4. AQmabAM offers a targeted, non-immunosuppressive approach for therapy of seropositive NMO. Autoantibody blocking may be a useful therapeutic strategy for other autoimmune diseases as well.
https://www.sciencedirect.com/science/article/pii/S0028390819303934
https://www.genovis.com/products/igg-proteases/fabricator/fragit/
ADCC
FragIT / FragIT Kit
***
Fc Receptors Contribute to the Antiviral Properties of Influenza Virus Neuraminidase-Specific Antibodies
E. R. Job, T. Ysenbaert, A. Smet, A. Van Hecke, L. Meuris, H. Kleanthous, X. Saelens, T. U. Vogeld
ABSTRACT Influenza virus neuraminidase (NA) has been under intense study re- cently as a vaccine antigen, yet there remain unanswered questions regarding the immune response directed toward NA. Antibodies (Abs) that can inhibit NA activity have been shown to aid in the control of disease caused by influenza virus infection in humans and animal models, yet how and if interactions between the Fc portion of anti-NA Abs and Fc receptors (Fc R) contribute to protection has not yet been extensively studied. Herein, we show that poly- and monoclonal anti-NA IgG anti- bodies with NA inhibitory activity can control A(H1N1)pdm09 infection in the ab- sence of Fc Rs, but Fc R interaction aided in viral clearance from the lungs. In con- trast, a mouse-human chimeric anti-NA IgG1 that was incapable of mediating NA inhibition (NI) solely relied on Fc R interaction to protect transgenic mice (with a humanized Fc R compartment) against A(H1N1)pdm09 infection. As such, this study suggests that NA-specific antibodies contribute to protection against influenza A vi- rus infection even in the absence of NI activity and supports protection through multiple effector mechanisms.
https://mbio.asm.org/content/mbio/10/5/e01667-19.full.pdf
https://www.genovis.com/products/igg-proteases/fabricator/fragit-kit/
ADCC
Monoclonal Ab *;IgG Polyclonal *
FragIT / FragIT Kit
***
TR1801-ADC: A highly potent cMet antibody-drug conjugate with high activity in patient-derived xenograft models of solid tumors
Marco Gymnopoulos, Oscar Betancourt, Vincent Blot, Ryo Fujita, Diana Galvan, Vincent Lieuw, Sophie Nguyen, Jeanette Snedden, Christine Stewart, Jose Villicana, Jon Wojciak, Eley Wong, Raul Pardo, Neki Patel, Francois D’Hooge, Balakumar Vijayakrishnan, Conor Barry, John A. Hartley, Philip W. Howard, Roland Newman, Julia Coronella
cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and more recently,antibody-drug conjugates (ADCs). However, the clinical benefit from cMet targeted therapy has been limited. We developed a novel cMet targeted “third generation” ADC, TR1801-ADC, that was optimized at different levels including specificity, stability, toxin linker, conjugation site, and in vivo efficacy.
Our non-agonistic cMet antibody was site-specifically conjugated to the PBD (pyrrolobenzodiazepine)-toxin linker tesirine and has picomolar activity in cancer cell lines derived from different solid tumors including lung, colorectal and gastric cancer. The potency of our cMet ADC is independent of MET gene copy number and its anti-tumor activity was high not only in high cMet expressing cell lines but also in medium to low cMet cell lines (40,000 to 90,000 cMet/cell) in which a cMet ADC with tubulin inhibitor payload was considerably less potent. In vivo xenografts with low-medium cMet expression were also very responsive to TR1801-ADC at a single dose while a cMet-ADC using a tubulin inhibitor showed a substantially reduced efficacy.
Furthermore, TR1801-ADC had excellent efficacy with significant anti-tumor activity in 90% of tested PDX models of gastric, colorectal and head & neck cancer: 7/10 gastric models, 4/10 colorectal cancer models and 3/10 head & neck cancer models showed complete tumor regression after a single dose administration.
Altogether, TR1801-ADC is a new generation cMet ADC with best in class preclinical efficacy and good tolerability in rats.
https://febs.onlinelibrary.wiley.com/doi/pdf/10.1002/1878-0261.12600
https://www.genovis.com/products/igg-proteases/fabricator/
ADC *
FabRICATOR *
***
Comparing different domains of analysis for the characterisation of N-Glycans on monoclonal antibodies
Sara Carillo, Raquel Pérez-Robles, CraigJakes, Meire Ribeiro da Silva, Silvia Millán Martín, Amy Farrell, Natalia Navas, Jonathan Bones
https://www.sciencedirect.com/science/article/pii/S2095177919306781
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
LC/MS *
FabRICATOR *;GingisKHAN *
***
Strong Cation Exchange Ultra-Performance Liquid Chromatography Coupled to UV and MS Detectors Allows Robust and Reproducible Characterization of mAb and ADC Charge Variants
Michal Kliman, Arnie Aistars, Yutaka Matsuda, Brian A. Mendelsohn
New methods for strong cation exchange (SCX) ultra-performance liquid chromatography (UPLC) have allowed effective resolution and characterization of charge variants throughout the ADC manufacturing process.
Retention time reproducibility after SCX UPLC column equilibration enabled monitoring of important changes in product quality.
SCX UPLC-MS performed with novel MS friendly mobile phases allowed for the confirmation of linker position, number of attachments, and glycoform heterogeneity. The method also provided high mass accuracy MS information for the verification of linker/drug incorporation.
https://www.waters.com/webassets/cms/library/docs/2019worldadc_kliman_uv-ms_of_adcs_bioaccord.pdf
https://www.genovis.com/products/igg-proteases/fabricator/
Charge variants
Monoclonal Ab *;ADC *
SCX-MS
FabRICATOR *
***
Analysis of cetuximab N-Glycosylation using multiple fractionation methods and capillary electrophoresis mass spectrometry
Csaba Váradi, Craig Jakes, Jonathan Bones
Site-specific glycosylation of Cetuximab was characterized in this study using multiple fractionation methods and capillary electrophoresis coupled to mass spectrometry (CE-MS) based glycomics. IdeS digested Cetuximab with subsequent reduction was fractionated using reversed-phase chromatography resulting in 3 fragments; Fd, Lc and Fc/2. Glycan release of the different fragments was performed in 18O enriched water providing the possible quantification of site occupancy. 2-AA labelled glycan structures were annotated by CE-MS profiling in combination with exoglycosidase sequencing, revealing potential structures with terminal α-galactose and N-glycolyl-neuraminic acid (NGNA) mainly originating from the Fd fragment. Glycosylation analysis was also performed on different charge variants of Cetuximab that were separated using pH gradient cation-exchange chromatography to investigate the impact of glycosylation on the net charge of the protein.
https://www.sciencedirect.com/science/article/abs/pii/S0731708519323581
https://www.genovis.com/products/igg-proteases/fabricator/
Fc-glycosylation;Charge variants
Monoclonal Ab *
CE-MS
FabRICATOR *
***
The impact of proline isomerization on antigen binding and the analytical profile of a trispecific anti-HIV antibody
Alessandro Masiero, Lechat Nelly, Gentric Marianne, Sourrouille Christophe, Laville Florian, Crépin Ronan, Borel Claire, Ziegler Cornelia, Bisch Grégoire, Leclerc Eric, Laurent Ludovic, Brault Dominique, Alexandre Sylvie, Gagnaire Marie, Duffieux Francis, Soubrier Fabienne, Capdevila Cécile, Arnould Isabelle, Dumas Jacques, Dabin Jérôme, Genet Bruno, Radošević Katarina, Menet Jean-Michel & Prades Catherine
Proline cis-trans conformational isomerization is a mechanism that affects different types of protein functions and behaviors. Using analytical characterization, structural analysis, and molecular dynamics simulations, we studied the causes of an aberrant two-peak size-exclusion chromatography profile observed for a trispecific anti-HIV antibody. We found that proline isomerization in the tyrosine-proline-proline (YPP) motif in the heavy chain complementarity-determining region (CDR)3 domain of one of the antibody arms (10e8v4) was a component of this profile. The pH effect on the conformational equilibrium that led to these two populations was presumably caused by a histidine residue (H147) in the light chain that is in direct contact with the YPP motif. Finally, we demonstrated that, due to chemical equilibrium between the cis and trans proline con- formers, the antigen-binding potency of the trispecific anti-HIV antibody was not significantly affected in spite of a potential structural clash of 10e8v4 YPtransPtrans conformers with the membrane-proximal ectodomain region epitope in the GP41 antigen. Altogether, these results reveal at mechanistic and molecular levels the effect of proline isomerization in the CDR on the antibody binding and analytical profiles, and support further development of the trispecific anti-HIV antibody.
https://www.tandfonline.com/doi/full/10.1080/19420862.2019.1698128
https://www.genovis.com/products/igg-proteases/fabalactica/
Structure
SEC *
FabALACTICA
****
Minimal B Cell Extrinsic IgG Glycan Modifications of Pro- and Anti-Inflammatory IgG Preparations in vivo
Anja Schaffert, Maja Hanic´, Mislav Novokmet, Olga Zaytseva, Jasminka Krištic´, Anja Lux, Lars Nitschke, Matthias Peipp, Marija Pezer, René Hennig, Erdmann Rapp, Gordan Lauc, Falk Nimmerjahn
Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo.
https://www.frontiersin.org/articles/10.3389/fimmu.2019.03024/full
https://www.genovis.com/products/igg-proteases/fabricator/
Fc-glycosylation
Monoclonal Ab *
HILIC
FabRICATOR *
***
Middle-level IM-MS and CIU experiments for improved therapeutic immunoglobulin isotype fingerprinting
Thomas Botzanowski, Oscar Hernandez-Alba, Martine Malissard, Elsa Wagner-Rousset, Evolène Deslignière, Olivier Colas, Jean-François Haeuw, Alain Beck, Sarah Cianférani
Currently approved therapeutic monoclonal antibodies (mAbs) are based on immunoglobulin G (IgG) 1, 2 or 4 isotypes, which differ in their specific inter-chains disulfide bridge connectivities. Different analytical techniques have been reported for mAb isotyping, among which native ion mobility mass spectrometry (IM-MS) and collision induced unfolding (CIU) experiments. However, mAb isotyping by these approaches is based on detection of subtle differences and thus remains challenging at the intactlevel. We report here on middle-level (after IdeS digestion) IM-MS and CIU approaches to afford better differentiation of mAb isotypes. Our method provides simultaneously CIU patterns of F(ab’)2 and Fc domains within a single run. Middle-level CIU patterns of F(ab’)2 domains enable more reliable classification of mAb isotypes compared to intact level CIU, while CIU fingerprints of Fc domains are overall less informative for mAb isotyping. F(ab’)2 regions can thus be considered as diagnostic domains providing specific CIU signatures for mAb isotyping. Benefits of middle-level IM-MS and CIU approaches are further illustrated on the hybrid IgG2/IgG4 eculizumab. While classical analytical techniques led to controversial results, middle-level CIU uniquely allowed to face the challenge of eculizumab « hybridicity », highlighting that its F(ab’)2 and Fc CIU patterns corresponds to an IgG2 and an IgG4, respectively. Altogether, the middle-level CIU approach is more clear-cut, accurate and straightforward for canonical but also more complex, engineered next generation mAb formats isotyping. Middle-level CIU thus constitutes a real breakthrough for therapeutic protein analysis, paving the way for its implementation in R&D laboratories.
https://www.biorxiv.org/content/10.1101/2020.01.20.911750v1
https://www.genovis.com/products/iggzero/
IdeS Characterization
Monoclonal Ab *;ADC *;IgG *
Native MS *;IM/MS *
FabRICATOR *
***
Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry
Jérémie Giorgetti, Alain Beck, Emmanuelle Leize-Wagner, Yannis-Nicolas François,
Significant growth of biopharmaceuticals requires powerful analytical methods to better understand their structure by establishing a complete characterization. To this end, a combination of bottom-up, middle-up and intact molecule levels with a capillary electrophoresis-mass spectrometry coupling has been performed to have a comprehensive picture of monoclonal antibodies. In this study, 7 worldwide health authorities approved mAbs have been analyzed to get information about their charge heterogeneity, the identification of post translational modifications (PTMs), their location and relative quantitation. Intact mAbs isoforms have been partially separated in less than 12 min and enabled to have a global illustration of mAbs heterogeneity and high masses PTMs characterization notably major N-glycosylation forms. Particularly, 2X-glycosylated and 1X-glycosylated forms have been partially separated. To deepen characterize PTMs carried by the backbone structure, advanced investigations at a middle-up level have been performed. Limited IdeS proteolysis allowed to study independently Fc/2 and F(ab)’2 fragments. Following the same separation conditions, isoforms of these fragments have been separated and data interpretation allowed to disclose additional PTMs as K-clip, oxidations or deamidations. A second intermediate level has been examined by adding a reduction step to establish a more precise assessment of PTMs and isoforms from the F(ab)’2 fragment. This reduction step released the light chains from the Fd fragment to get only 25 kDa fragments to analyze. CE-ESI-MS coupling allowed to get more information particularly about low masses PTMs. The precise location and relative quantitation of each PTM has been investigated at the peptidic level induced by a tryptic digestion of the studied mAbs. The concordance of the results shows the efficiency of the CE-ESI-MS coupling to characterize mAbs and highlight the need of the multi-level combination to get a comprehensive characterization of biotherapeutics.
https://www.sciencedirect.com/science/article/abs/pii/S0731708519328791
Deamidation;Oxidation;Structure;IdeS Characterization;Charge heterogeneity
Monoclonal Ab *
CE-MS
FabRICATOR *
***
Implementation of a High-Resolution Liquid Chromatography–Mass Spectrometry Method in Quality Control Laboratories for Release and Stability Testing of a Commercial Antibody Product
Izabela Sokolowska, Jingjie Mo,Fatie Rahimi Pirkolachahi, Carol McVean, Lars A. T. Meijer, Linda Switzar, Crina Balog, Michael J. Lewis, Ping Hu
Liquid chromatography–mass spectrometry (LC–MS) has been widely used throughout biotherapeutic development. However, its implementation in GMP-compliant commercial quality control (QC) laboratories remains a challenge. In this publication, we describe the covalidation and implementation of an automated, high-throughput, and GMP compliant subunit LC–MS method for monitoring antibody oxidation for commercial product release and stability testing. To our knowledge, this is the first report describing the implementation of a high-resolution LC–MS method in commercial QC laboratories for product release and stability testing in the biopharmaceutical industry. This work paves the road for implementing additional LC–MS methods to modernize testing in commercial QC with more targeted control of product quality.
https://pubs.acs.org/doi/full/10.1021/acs.analchem.9b05036
https://www.genovis.com/products/igg-proteases/fabricator/
Oxidation
Monoclonal Ab *;IgG *
LC/MS *
FabRICATOR *;IgGZERO *
***
Understanding how Fc-modification transforms a pathogenic HIT-like monoclonal antibody into a novel treatment for sepsis
Kandace Gollomp, Amrita Sarkar, Sanjiv Harikumar, Steven H Seeholzer, Gowthami M.
Arepally, Kristin Hudock, Lubica Rauova, M. Anna Kowalska, Mortimer Poncz
Sepsis is characterized by multi-organ system dysfunction that occurs due to infection. It is associated with high morbidity and mortality and in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multi-organ damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to deoxyribonuclease I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody, KKO, which binds to PF4-NET complexes, further enhances deoxyribonuclease resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination, and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis.
https://www.ncbi.nlm.nih.gov/pubmed/31722003
https://www.genovis.com/products/iggzero/
EndoS therapeutics
Monoclonal Ab *;IgG *
LC/MS *;LC-MS/MS
IgGZERO *
***
Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies
Srikanth Kotapati, David Passmore, Sayumi Yamazoe, Rajesh Kishore Kumar Sanku, Qiang Cong, Yam B. Poudel, Naidu S. Chowdari, Sanjeev Gangwar, Chetana Rao, Vangipuram S. Rangan, Pina M. Cardarelli, Shrikant Deshpande, Pavel Strop, Gavin Dollinger, and Arvind Rajpal
Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC–MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC–MS. To address these challenges, we developed a generic affinity capture LC–MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) “mono capture” or “dual capture” of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) “on-bead” digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC–HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.
https://pubs.acs.org/doi/10.1021/acs.analchem.9b04572
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Antibody Ratio;Disulfide reduction;ADCC
Monoclonal Ab *;ADC *
LC/MS *
FabRICATOR *
***
Quantitative PET imaging of PD-L1 expression in xenograft and syngeneic tumour models using a site-specifically labelled PD-L1 antibody
Camilla Christensen, Lotte K. Kristensen, Maria Z. Alfsen, Carsten H. Nielsen, Andreas Kjaer
Purpose
Despite remarkable clinical responses and prolonged survival across several cancers, not all patients benefit from PD-1/PD-L1 immune checkpoint blockade. Accordingly, assessment of tumour PD-L1 expression by immunohistochemistry (IHC) is increasingly applied to guide patient selection, therapeutic monitoring, and improve overall response rates. However, tissue-based methods are invasive and prone to sampling error. We therefore developed a PET radiotracer to specifically detect PD-L1 expression in a non-invasive manner, which could be of diagnostic and predictive value.
Methods
Anti-PD-L1 (clone 6E11, Genentech) was site-specifically conjugated with DIBO-DFO and radiolabelled with 89Zr (89Zr-DFO-6E11). 89Zr-DFO-6E11 was optimized in vivo by longitudinal PET imaging and dose escalation with excess unlabelled 6E11 in HCC827 tumour-bearing mice. Specificity of 89Zr-DFO-6E11 was evaluated in NSCLC xenografts and syngeneic tumour models with different levels of PD-L1 expression. In vivo imaging data was supported by ex vivo biodistribution, flow cytometry, and IHC. To evaluate the predictive value of 89Zr-DFO-6E11 PET imaging, CT26 tumour-bearing mice were subjected to external radiation therapy (XRT) in combination with PD-L1 blockade.
Results
89Zr-DFO-6E11 was successfully labelled with a high radiochemical purity. The HCC827 tumours and lymphoid tissue were identified by 89Zr-DFO-6E11 PET imaging, and co-injection with 6E11 increased the relative tumour uptake and decreased the splenic uptake. 89Zr-DFO-6E11 detected the differences in PD-L1 expression among tumour models as evaluated by ex vivo methods. 89Zr-DFO-6E11 quantified the increase in PD-L1 expression in tumours and spleens of irradiated mice. XRT and anti-PD-L1 therapy effectively inhibited tumour growth in CT26 tumour-bearing mice (p < 0.01), and the maximum 89Zr-DFO-6E11 tumour-to-muscle ratio correlated with response to therapy (p = 0.0252).
Conclusion
PET imaging with 89Zr-DFO-6E11 is an attractive approach for specific, non-invasive, whole-body visualization of PD-L1 expression. PD-L1 expression can be modulated by radiotherapy regimens and 89Zr-DFO-6E11 PET is able to monitor these changes and predict the response to therapy in an immunocompetent tumour model.
https://link.springer.com/article/10.1007%2Fs00259-019-04646-4#citeas
https://www.genovis.com/products/glyclick/
Imaging;EndoS therapeutics
IgG *
HPLC *
EndoS;GlyCLICK
***
Comparison of two glycoengineering strategies to control the fucosylation of a monoclonal antibody
Neha Mishraa, Maureen Spearman, Lynda Donald, Helene Perreault, Michael Butler
Core fucosylation of an Fc N-linked glycan affects antibody effector functions, as the absence of fucose increases the antibody dependent cell cytotoxicity (ADCC) response with increased binding to the Fcγ receptors. The work presented here compares two different approaches to incrementally reduce core fucosylation of a camelid heavy chain antibody, EG2-hFc expressed in CHO cells which targets the EGFR receptor. The first method uses a fucosyltransferase (FUT) inhibitor, 2- fluoro peracetylated fucose (2FF), which was added to cell cultures expressing the EG2-hFc antibody in increasing concentrations up to 50μM. At this concentration there was no observed effect on cell growth. Glycan analysis was performed on antibodies collected from culture samples using HILIC-HPLC. The inhibitor reduced total fucosylation from 80% to 17.5% at 20μM 2FF.
The second method involved transfecting the EG2-hFc producing cells with a prokaryotic GDP- 6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene in order to deflect the fucose de novo pathway into producing rhamnose which is not incorporated into a glycan. Stable clones from transfected pools were isolated following flow cytometry using the green fluorescent protein (GFP) marker which was co-expressed with the RMD gene. High expressing RMD clones reduced the fucosylation of the antibody glycan to as low as 16%. The addition of 2FF to cultures of these RMD clones reduced the fucosylation level even further to 3% of the antibody glycan. An incremental increase in fucosylation was obtained by step-wise addition of fucose (up to 1 mM) to the RMD cells, in which the fucosylation level increased to a maximum of 87%.
We also used ESI-MS to analyze the fucosylation pattern of EG2-hFc with addition of increasing concentrations of 2FF. This showed that 2FF inhibits the addition of fucose in a concentration- dependent and specific manner with the inhibition of fucose occurring one fucose at a time. Control cultures showed the presence of a predominant peak indicating two fucose moieties per antibody. As the 2FF inhibitor concentration was increased peaks corresponding to one fucose per antibody and non-fucosylated antibody predominated with a gradual decrease of the 2 fucose peak to insignificance at 15 μM 2FF.
https://www.sciencedirect.com/science/article/pii/S2590155920300020
https://www.genovis.com/products/iggzero/
Structure;EndoS therapeutics;Antigen Binding;ADCC
Monoclonal Ab *;IgG *
HILIC
IgGZERO *;EndoS
***
Monitoring CD8a+ T Cell Responses to Radiotherapy and CTLA-4 Blockade Using [64Cu]NOTA-CD8a PET Imaging
Lotte K. Kristensen, Camilla Christensen, Maria Z. Alfsen, Sigrid Cold, Carsten H. Nielsen & Andreas Kjaer
Current response assessment systems for cancer patients receiving immunotherapy are limited. This is due to the associated inflammatory response that may confound the conventional morphological response evaluation criteria in solid tumors and metabolic positron emission tomography (PET) response criteria in solid. Recently, novel PET imaging techniques using radiolabeled antibodies and fragments have emerged as a particularly sensitive and specific modality for quantitative tracking of immune cell dynamics. Therefore, we sought to investigate the utility of Cu-64 labeled F(ab)′2 fragments for in vivo detection of CD8a+ T cells as a prognostic imaging biomarker of response to immunotherapy in an immunocompetent mouse model of colorectal cancer.
https://link.springer.com/article/10.1007/s11307-020-01481-0
https://www.genovis.com/products/igg-proteases/fabricator/
Imaging
Monoclonal Ab *
HPLC *
FabRICATOR *
***
Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn
John S. Ruano-Salguero, Kelvin H. Lee
The blood-brain barrier (BBB) hinders the brain delivery of therapeutic immunoglobulin γ (IgG) antibodies. Evidence suggests that IgG-specific processing occurs within the endothelium of the BBB, but any influence on transcytosis remains unclear. Here, involvement of the neonatal Fc receptor (FcRn), which mediates IgG recycling and transcytosis in peripheral endothelium, was investigated by evaluating the transcytosis of IgGs with native or reduced FcRn engagement across human induced pluripotent stem cell-derived brain endothelial-like cells. Despite differential trafficking, the permeability of all tested IgGs were comparable and remained constant irrespective of concentration or competition with excess IgG, suggesting IgG transcytosis occurs nonspecifically and originates from fluid-phase endocytosis. Comparison with the receptor-enhanced permeability of transferrin indicates that the phenomena observed for IgG is ubiquitous for most macromolecules. However, increased permeability was observed for macromolecules with biophysical properties known to engage alternative endocytosis mechanisms, highlighting the importance of biophysical characterizations in assessing transcytosis mechanisms.
https://www.nature.com/articles/s41598-020-60438-z
https://www.genovis.com/products/glycinator/
Fc-glycosylation;Fab-glycosylation;Imaging
IgG *
LC-MS/MS
GlycINATOR *
***
Comprehensive N- and O-glycosylation Mapping of Human Coagulation Factor V
Cheng Ma, Ding Liu, Dong Li, Junping Zhang, Xiao‐Qian Xu, He Zhu, Xiu‐Feng Wan, Carol H. Miao, Barbara A. Konkle, Philip Onigman, Weidong Xiao, Lei Li
Background/Objective: Coagulation Factor V (FV), a multi-domain glycoprotein, is an essential
cofactor in the blood clotting cascade. FV deficiency is a rare bleeding disorder that results in poor clotting
after an injury or surgery. The only treatment for the disease is infusions of fresh frozen plasma and blood
platelets. Glycosylation affects the biological activity, pharmacokinetics, immunogenicity, and in vivo
clearance rate of proteins in the plasma. The glycan profile of FV, as well as how it affects the activity,
stability, and immunogenicity, remain unknown. Methods: In this study, we comprehensively mapped the
glycosylation patterns of human plasma-derived FV by combining multi-enzyme digestion, hydrophilic
interaction chromatography enrichment of glycopeptides, and alternated fragmentation mass spectrometry
analysis. Results/Conclusion: A total of 57 unique N-glycopeptides and 51 O-glycopeptides were identified,
which were categorized into 40 N-glycan and 17 O-glycan compositions. Such glycosylation details are
fundamental for future functional studies and therapeutics development. In addition, the established
methodology can be readily applied to analyze glycosylation patterns of proteins with over 2000 amino acids
https://onlinelibrary.wiley.com/doi/abs/10.1111/jth.14861
https://www.genovis.com/products/#o-glycans
Amino acid sequence;Structure
IgG *
HILIC
OpeRATOR;SialEXO
****
Generation and validation of structurally defined antibody–siRNA conjugates
Alex R Nanna, Alexander V Kel’in, Christopher Theile, Justin M Pierson, Zhi Xiang Voo, Ashish Garg, Jayaprakash K Nair, Martin A Maier, Kevin Fitzgerald, Christoph Rader
Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody–siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.
https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkaa286/5826805
https://www.genovis.com/products/igg-proteases/fabricator/
Amino acid sequence
ADC *;IgG *
HPLC *;LC/MS *;MALDI-TOF-MS *
FabRICATOR *
***
Glycan-mediated technology for obtaining homogeneous site-specific conjugated antibody-drug conjugates: synthesis and analytical characterization by using complementary middle-up LC/HRMS analysis
Bastiaan L. Duivelshof, Evolène Deslignière, Oscar Hernandez-Alba, Anthony Ehkirch, Hanna Toftevall, Jonathan Sjögren, Sarah Cianférani, Alain Beck, Davy Guillarme, and Valentina D'Atri
Conventional antibody-drug conjugate (ADC) manufacturing methods are based on the non-selective bioconjugation of cytotoxic drugs to lysine and cysteine residues. This results in highly heterogeneous mixtures of different drug-antibody ratios (DAR) that can significantly affect the safety and efficacy of the ADC product. Recently, an innovative procedure named GlyCLICK was suggested, consisting of a two-step enzymatic procedure to transform Fc-glycans present on IgG mAbs into two site-specific anchor points for the conjugation of any alkyne-containing payload of choice. Here, we evaluated the conjugation process by comparing trastuzumab and trastuzumab conjugated with DM1, following the GlyCLICK procedure. Complementary reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS) were used to analyze the protein subunits (ca. 25-100 kDa) obtained after different levels of enzymatic digestion and chemical reduction. Our results demonstrated that the hydrophobic character of the drug molecule allows to rapidly confirm the Fc-drug conjugation at the chromatographic level. Furthermore, the hyphenation to MS detection provided accurate mass information on the ADC subunits and facilitated the DAR determination of 2.0. Therefore, this work illustrates how middle-up analysis using LC/HRMS can provide accurate and complementary information on the critical quality attributes of these novel site-specific ADC products.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.0c00282
https://www.genovis.com/products/glyclick/
Drug Antibody Ratio;Amino acid sequence;Drug Load Distribution;Disulfide reduction
Monoclonal Ab *;ADC *
HILIC
GlycINATOR *;GlyCLICK
****
Distinctive binding properties of human monoclonal LGI1 autoantibodies determine pathogenic mechanisms
Melanie Ramberger, Antonio Berretta, Jeanne M M Tan, Bo Sun, Sophia Michael, Tianrong Yeo, Jakob Theorell, Rachael Bashford-Rogers, Sofija Paneva, Victoria O’Dowd, Neesha Dedi, Sarfaraj Topia, Robert Griffin, Jorge Ramirez-Franco, Oussama El Far, Stéphanie Baulac, Maria I Leite, Arjune Sen, Alexander Jeans, David McMillan, Diane Marshall, Daniel Anthony, Daniel Lightwood, Patrick Waters, Sarosh R Irani
Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.
https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awaa104/5841730
https://www.genovis.com/products/igg-proteases/fabricator/
Fab-glycosylation
Monoclonal Ab *;IgG *
Other
FabRICATOR *
***
IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies
Christian Leborgne, Elena Barbon, Jeffrey M. Alexander, Hayley Hanby, Sandrine Delignat, Daniel M. Cohen, Fanny Collaud, Saghana Muraleetharan, Dan Lupo, Joseph Silverberg, Karen Huang, Laetitia van Wittengerghe, Béatrice Marolleau, Adeline Miranda, Anna Fabiano, Victoria Daventure, Heena Beck, Xavier M. Anguela, Giuseppe Ronzitti, Sean M. Armour, Sebastien Lacroix-Desmazes & Federico Mingozzi
Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in humans1,2, and block liver transduction3,4,5 and vector readministration6; thus, they represent a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied7, which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients8. Here, we studied if IdeS could eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (intravenous Ig) in vitro upon endopeptidase treatment. In mice passively immunized with intravenous Ig, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.
https://www.nature.com/articles/s41591-020-0911-7
https://www.genovis.com/products/igg-proteases/fabricator/
IdeS Characterization
IgG *
Other
FabRICATOR *;IdeS
***
Middle Level IM–MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting
Thomas Botzanowski, Oscar Hernandez-Alba, Martine Malissard, Elsa Wagner-Rousset, Evolène Deslignière, Olivier Colas, Jean-François Haeuw, Alain Beck, and Sarah Cianférani
Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS). However, these two techniques exhibit significant limitations to differentiate mAb subclasses at the intact level. In the present work, we aimed at developing a unique IM–MS-based approach for the characterization of mAb subclasses at the middle level. Upon IdeS-digestion, the unfolding patterns of the F(ab′)2 and Fc domains were simultaneously analyzed in a single run to provide deeper structural insights of the mAb scaffold. The unfolding patterns associated with the F(ab′)2 domains are completely different in terms of unfolding energies and number of transitions. Thereby, F(ab′)2 regions are the diagnostic domain to provide specific unfolding signatures to differentiate IgG subclasses and provide more confident subclass categorization than CIU on intact mAbs. In addition, the potential of middle-level CIU was evaluated through the characterization of eculizumab, a hybrid therapeutic IgG2/4 mAb. The unfolding signatures of both domains were allowed to corroborate, within a single run, the hybrid nature of eculizumab as well as specific subclass domain assignments to the F(ab′)2 and Fc regions. Altogether, our results confirm the suitability of middle-level CIU of F(ab′)2 domains for subclass categorization of canonical and more complex new generation engineered antibodies and related products.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.0c00293
https://www.genovis.com/products/iggzero/
IdeS Characterization;Disulfide reduction
ADC *;IgG *
Native MS *;RP-HPLC *
FabRICATOR *;IgGZERO *
***
High microbiota reactivity of adult human intestinal IgA requires somatic mutations
Johanna Kabbert, Julia Benckert, Tim Rollenske, Thomas C.A. Hitch, Thomas Clavel, Vuk Cerovic, Hedda Wardemann, Oliver Pabst
The gut is home to the body’s largest population of plasma cells. In healthy individuals, IgA is the dominating isotype, whereas patients with inflammatory bowel disease also produce high concentrations of IgG. In the gut lumen, secretory IgA binds pathogens and toxins but also the microbiota. However, the antigen specificity of IgA and IgG for the microbiota and underlying mechanisms of antibody binding to bacteria are largely unknown. Here we show that microbiota binding is a defining property of human intestinal antibodies in both healthy and inflamed gut. Some bacterial taxa were commonly targeted by different monoclonal antibodies, whereas others selectively bound single antibodies. Interestingly, individual human monoclonal antibodies from both healthy and inflamed intestines bound phylogenetically unrelated bacterial species. This microbiota cross-species reactivity did not correlate with antibody polyreactivity but was crucially dependent on the accumulation of somatic mutations. Therefore, our data suggest that a system of affinity-matured, microbiota cross-species–reactive IgA is a common aspect of SIgA–microbiota interactions in the gut.
https://rupress.org/jem/article/217/11/e20200275/151927
https://www.genovis.com/products/glycinator/
Antigen Binding
Monoclonal Ab *;IgG *
GlycINATOR *
***
Large-scale site-specific mapping of the O-GalNAc glycoproteome
Weiming Yang, Angellina Song, Minghui Ao, Yuanwei Xu & Hui Zhang
Protein glycosylation is one of the most common protein modifications. A major type of protein glycosylation is O-GalNAcylation, in which GalNAc-type glycans are attached to protein Ser or Thr residues via an O-linked glycosidic bond. O-GalNAcylation is thought to play roles in protein folding, stability, trafficking and protein interactions, and identification of the site-specific O-GalNAc glycoproteome is a crucial step toward understanding the biological significance of the modification. However, lack of suitable methodology, absence of consensus sequon of O-GalNAcylation sites and complex O-GalNAc glycan structures pose analytical challenges. We recently developed a mass spectrometry-based method called extraction of O-linked glycopeptides (EXoO) that enables large-scale mapping of site-specific mucin-type O-GalNAcylation sites. Here we provide a detailed protocol for EXoO, which includes seven stages of: (1) extraction and proteolytic digestion of proteins to peptides, (2) sequential guanidination and de-salting of peptides, (3) enrichment of glycopeptides, (4) solid-phase peptide conjugation and release of O-GalNAc glycopeptides using the OpeRATOR protease, (5) liquid chromatography with tandem mass spectrometry analysis of O-GalNAc glycopeptides, (6) identification of O-GalNAc glycopeptides by database search and (7) quantification of O-GalNAc glycopeptides. Using this protocol, thousands of O-GalNAcylation sites from hundreds of glycoproteins with information regarding site-specific O-GalNAc glycan can be identified and quantified from complex samples. The protocol can be performed by a researcher with basic proteomics skills and takes about 4 d to complete.
https://www.nature.com/articles/s41596-020-0345-1
https://www.genovis.com/products/exoglycosidases/sialexo/
O-glycosylation
IgG *
LC-MS/MS
OpeRATOR;SialEXO
***
Inter-laboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry
Kristina Srzentic, Luca Fornelli, Yury O. Tsybin, Joseph A. Loo, Henrique Seckler, Jeffrey N. Agar, Lissa C. Anderson, Dina L. Bai, Alain Beck, Jennifer S. Brodbelt, Yuri E.M. van der Burgt, Julia Chamot-Rooke, Sneha Chatterjee, Yunqiu Chen, David James Clarke, Paul O. Danis, Jolene K. Diedrich, Robert Anthony D'Ippolito, Mathieu Dupré, Natalia Gasilova, Ying Ge, Young Ah Goo, David R. Goodlett, Sylvester M. Greer, Kim F. Haselmann, Lidong He, Christopher L. Hendrickson, Joshua D. Hinkle, Matthew Holt, Sam Hughes, Donald F. Hunt, Neil L Kelleher, Anton N. Kozhinov, Ziqing Lin, Christian Malosse, Alan G. Marshall, Laure Menin, Robert J. Millikin, Konstantin O. Nagornov, Simone Nicolardi, Ljiljana Paša-Tolić, Stuart Pengelley, Neil R. Quebbemann, Anja Resemann, Wendy Sandoval, Richa Sarin, Nicholas Daniel Schmitt, Jeffrey Shabanowitz, Jared B. Shaw, Michael R. Shortreed, Lloyd M. Smith, Frank Sobott, Detlev Suckau, Timothy Toby, Chad R. Weisbrod, Norelle C. Wildburger, John R. Yates, Sung Hwan Yoon, Nicolas L. Young, and Mowei Zhou
The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product’s primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to twenty laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide, and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.
https://pubs.acs.org/doi/abs/10.1021/jasms.0c00036
https://www.genovis.com/products/iggzero/
IdeS Characterization
Monoclonal Ab *
LC/MS *
IgGZERO *
***
Ligand binding to a humanized anti-cocaine mAb detected by non-reducing SDS-PAGE
Terence L. Kirley, Andrew B. Norman
Monoclonal antibodies are very useful tools in experimental biology, as well as being valuable and effective
therapeutic drugs. They can be targeted against proteins with varied functions, or against small molecules of
interest to both researchers and clinicians, such as drugs of abuse, including cocaine. Since there is no currently
FDA approved pharmacological treatment for cocaine abuse, our laboratory has developed an anti-cocaine mAb
for the treatment of cocaine use disorders. This humanized anti-cocaine antibody, named h2E2, has been
thoroughly characterized both functionally and structurally, in preparation for the start of clinical development.
We previously showed that this mAb could be characterized by sequential thermal unfolding of antibody domains using non-reducing SDS-PAGE. We also demonstrated that ligand-induced protein stabilization can be
used to quantitatively measure cocaine and cocaine metabolite binding to the h2E2 mAb, utilizing differential
scanning fluorimetry. Here, we demonstrate the utility of non-reducing SDS-PAGE for the qualitative assessment
of binding of cocaine and some of its metabolites, both to the intact mAb, as well as to fragments containing the antigen binding site (Fab and F(ab’)2 fragments). These results clearly show a ligand concentration dependence of the stabilization of the cocaine binding domain in non-reducing SDS-PAGE, as well as visually differentiating the relative binding affinities of various cocaine metabolites. Thus, non-reducing SDS-PAGE is a simple and widely available technique that is useful as a measure of binding of cocaine and its metabolites to the h2E2 mAb, and it is likely that this technique will also be applicable to other small molecule-directed mAbs.
https://www.sciencedirect.com/science/article/pii/S2405580820301047
https://www.genovis.com/products/igg-proteases/fabricator/fragit/
IdeS Characterization
Monoclonal Ab *
Other
FragIT / FragIT Kit
***
Biological and structural characterization of murine TRALI antibody reveals increased Fc-mediated complement activation
Eveline A. N. Zeeuw van der Laan, Saskia van der Velden, Arthur E. H. Bentlage, Mads D. Larsen, Thijs L. J. van Osch, Juk Yee Mok, Giso Brasser, Dionne M. Geerdes, Carolien A. M. Koeleman, Jan Nouta, John W. Semple, Leendert Porcelijn, Wim J. E. van Esch, Manfred Wuhrer, C. Ellen van der Schoot, Gestur Vidarsson, Rick Kapur
Transfusion-related acute lung injury (TRALI) remains a leading cause of transfusion-related deaths. In most cases, anti-leukocyte antibodies in the transfusion product trigger TRALI, but not all anti-leukocyte antibodies cause TRALI. It has been shown that the anti–major histocompatibility complex (MHC) class I antibody 34-1-2S (anti–H-2Kd) causes TRALI in BALB/c mice (MHC class I haplotype H-2Kd), whereas SF1.1.10 (anti–H-2Kd) does not. In C57BL/6 mice (MHC class I haplotype H-2Kb), TRALI only occurs when anti-MHC class I antibody AF6-88.5.5.3 (anti–H-2Kb) is administered together with a high dose of 34-1-2S. It remains unknown which specific antibody characteristics are responsible for eliciting TRALI. We therefore investigated several biological and structural features of 34-1-2S compared with other anti-MHC class I antibodies, which on their own do not cause TRALI: SF1.1.10 and AF6-88.5.5.3. No substantial differences were observed between the TRALI-causing 34-1-2S and the TRALI-resistant SF1.1.10 regarding binding affinity to H-2Kd. Regarding binding affinity to H-2Kb, only AF6-88.5.5.3 potently bound to H-2Kb, whereas 34-1-2S exhibited weak but significant cross-reactivity. Furthermore, the binding affinity to FcγRs as well as the Fc glycan composition seemed to be similar for all antibodies. Similar Fc glycosylation profiles were also observed for human TRALI-causing donor anti-HLA antibodies compared with human anti-HLA antibodies from control donors. 34-1-2S, however, displayed superior complement activation capacity, which was fully Fc dependent and not significantly dependent on Fc glycosylation. We conclude that TRALI induction is not correlated with Fab- and Fc-binding affinities for antigen and FcγRs, respectively, nor with the composition of Fc glycans; but increased Fc-mediated complement activation is correlated with TRALI induction.
https://ashpublications.org/bloodadvances/article/4/16/3875/463300
https://www.genovis.com/products/iggzero/
Fc-glycosylation
IgG *
LC/MS *
IgGZERO *;DeGlycIT;FabULOUS *
***
Improved N- and C-terminal sequencing of proteins by combining positive and negative ion MALDI in-source decay mass spectrometry
Simone Nicolardi, David Peter Alexander Kilgour, Yuri E.M. van der Burgt, Manfred Wuhrer
The development of various ionization and fragmentation techniques has been of key importance for establishing mass spectrometry (MS) as a powerful tool for protein characterization. One example of this is matrix-assisted laser desorption/ionization (MALDI) combined with in-source decay (ISD) fragmentation that allows mapping of N- and C-terminal regions of large proteins without the need for proteolysis. Positive ion mode ISD fragments are commonly assigned in the mass region above m/z 1000, while MALDI matrix ions generally hamper the detection of smaller singly-charged fragments. The ultrahigh resolving power provided by Fourier transform ion cyclotron resonance (FT-ICR) MS partially overcomes this limitation, but to further increase detection of smaller fragments we have revisited the application of negative ion mode MALDI-ISD and found a good coverage of the peptide chain termini starting from c’2 and z’2 fragment ions. For the first time, we demonstrate that the combination of negative and positive ion MALDI FT-ICR MS is a useful tool to improve the characterization of mAbs. The different specificities of the two ion modes allowed us to selectively cover the sequence of the light and heavy chains of mAbs at increased sensitivity. A comprehensive evaluation of positive and negative ion mode MALDI-ISD FT-ICR MS in the m/z-range 46-13,500 showed an increased sequence coverage for three standard proteins, namely myoglobin, SiLuLite mAb, and NIST mAb. The data obtained in the two ion modes were, in part, complementary.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.0c02198
https://www.genovis.com/products/igg-proteases/fabricator/
IdeS Characterization
Monoclonal Ab *
LC/MS *;MALDI-TOF-MS *
FabRICATOR *
***
Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS
Christoph Gstöttner, Simone Nicolardi, Markus Haberger, Dietmar Reusch, Manfred Wuhrer, Elena Domínguez-Vegaa
Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be observed contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new analytical strategies are needed for a more accurate and in-depth analysis.
Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a positively coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the separation permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond reduction. The six different subunits (Lc1, Lc2, Fd’1, Fd’2, (Fc/2)1 and (Fc/2)2) were separated using the same positively-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resolution (resolving power 140000 at m/z 1100) for a more confident analysis of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately determined in the subunits showing the complementarity of both approaches.
https://www.sciencedirect.com/science/article/abs/pii/S0003267020308072
https://www.genovis.com/products/igg-proteases/fabalactica/
Monoclonal Ab *
LC/MS *
FabALACTICA
****
Probing protein conformation destabilization in sterile liquid formulations through the formation of 3,4-dihydroxyphenylalanine
Olivier Mozziconacci, Natalia Subelzu, Christian Schoneich, Yong Liu, Andreas Abend, and W Peter Wuelfing
This work demonstrates the use of a fluorescent probe to screen protein conformational changes in mixtures of monoclonal antibodies and determine the region of where such changes may originate through a footprinting mass spectrometry approach. The oxidative stress of mixtures of two different immunoglobulins (IgG1, IgG2, or IgG4), performed in the presence of 2,2’-azobis(2-amidinopropane dihydrochloride) results in sequence-specific tyrosine oxidation reactions depending on the time of incubation of the IgG molecules and the nature of the excipients present in the formulation. The combination of a fluorescence assay, based on the detection of 3,4-dihydroxyphenylalanine (DOPA) and mass spectrometry analyses, permits the identification of protein conformation changes. In a mixture of IgG2 and IgG4, a destabilization of IgG4 in the presence of IgG2 is observed. The destabilized region involves the Fab region of IgG4 between Tyr63 and Tyr81, and potentially multiple regions of IgG2.
https://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.0c00554
https://www.genovis.com/products/gingisrex/
Oxidation;Amino acid sequence
Monoclonal Ab *
FRET;LC-MS/MS
GingisREX
***
Targeted bottom-up characterization of recombinant monoclonal antibodies by multi-dimensional LC/MS
Julien Camperi, Davy Guillarme, and Cinzia Stella
On-line bottom-up approaches have recently emerged as promising alternatives to standard off-line processes for char-acterizing post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs). The benefits of on-line processing include reductions in required sample amount and sample handling, as well as reducing the overall turnaround time. However, shortening digestion time for the on-line approach of an intact mAb can cause incomplete peptide cleavages, leading to low sequence coverage and poor repeatability of analyses. For the first time, we describe a novel, automated targeted bottom-up strategy consisting of reducing the complexity of intact mAb by digesting the product into small ~25 kDa fragments, followed by an on-line peptide mapping analysis of each fragment. For this pur-pose, a 4D-LC/MS method was developed using a immobilized IdeS-HPLC column as a first dimension (1D) for on-line digestion, followed by a (2D) on-column reversed-phase liquid chromatography (RPLC) for reduction and fragments separation. Then, only one fragment was selected for digestion using a (3D) immobilized trypsin cartridge and finally, the obtained peptides were analyzed by (4D) RPLC-MS. This strategy considerably improved the on-line digestion effi-ciency with higher sequence coverages (LC and HC > 97%), thus allowing various PTMs including oxidation, deami-dation, and isomerization located in the complementarity-determining regions (CDRs), as well as N-glycans present on the Fc/2 fragment, to be monitored with similar sensitivity to those obtained with standard off-line approaches. Additional investigations at a middle-up level were also performed via a 3D-LC/MS approach within the same system, demonstrating the feasibility to achieve a multi-level comprehensive characterization of mAbs.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.0c02780
https://www.genovis.com/products/igg-proteases/fabricator/fabricator-hplc/
Deamidation;IdeS Characterization
Monoclonal Ab *
LC/MS *
FabRICATOR-HPLC
***
Towards automation of Collision Induced Unfolding experiments through online Size Exclusion Chromatography coupled to native Mass Spectrometry
Evolène Deslignière, Anthony Ehkirch, Thomas Botzanowski, Alain Beck, Oscar Hernandez-Alba, and Sarah Cianférani
Ion mobility-based collision induced unfolding (CIU) has gained increasing attention to probe gas-phase unfolding of proteins and their noncovalent complexes, notably for biotherapeutics. CIU detects subtle conformational changes of proteins and emerges as an attractive alternative to circumvent poor IM resolution. However, CIU still lacks in automation for buffer exchange and data acquisition, precluding its wide adoption. We present here an automated workflow for CIU experiments, from sample preparation to data interpretation using online size exclusion chromatography coupled to native ion mobility mass spectometry (SEC-CIU). Online automated SEC-CIU experiments offer several benefits over nanoESI-CIU, among which i) improved and fast desalting compared to manual buffer exchange used for classical CIU experiments; ii) drastic reduction of the overall data collection time process along with iii) maintaining the number of unfolding transitions. We then evaluate the potential of SEC-CIU to distinguish monoclonal antibodies (mAbs) subclasses, illustrating the efficiency of our method for rapid mAb subclass identification at both intact and middle levels. Finally, we demonstrate that CIU data acquisition time can be further reduced either by setting up a scheduled CIU method relying on diagnostic trap collision voltages or by implementing mAbs-multiplexed SEC-CIU analyses to maximize information content in a single experiment. Altogether, our results confirm the suitability of SEC-CIU to automate CIU experiments, particularly for the fast characterization of next generation mAb-based products.
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.0c01426
https://www.genovis.com/products/iggzero/
Charge variants
Monoclonal Ab *;IgG *
LC/MS *
FabRICATOR *;IgGZERO *
***
Protein-Functionalized Gold Nanoparticles as Refractometric Nanoplasmonic Sensor for Detection of Proteolytic Activity of Porphyromonas gingivalis
Anna Svärd, Jessica Neilands, Eleonor Palm, Gunnel Svensäter, Torbjörn Bengtsson, and Daniel Aili
Periodontitis is an inflammatory oral disease that affects a large part of the adult population, causing significant costs and suffering. The key pathogen, Porphyromonas gingivalis, secretes gingipains, which are highly destructive proteases and the most important virulence factors in the pathogenesis of the disease. Currently, periodontitis is diagnosed mainly by mechanical manual probing and radiography, often when the disease has already progressed significantly. The possibilities of detecting gingipain activity in gingival fluid could enable early-stage diagnosis and facilitate treatment. Here, we describe a sensitive nanoparticle-based nanoplasmonic biosensor for the detection of the proteolytic activity of gingipains. Gold nanoparticles (AuNPs) were self-assembled as a submonolayer in multiwell plates and further modified with casein or IgG. The proteolytic degradation of the protein coating was tracked by monitoring the shift in the localized surface plasmon resonance (LSPR) peak position. The sensor performance was investigated using model systems with trypsin and purified gingipains (subtypes Kgp and RgpB) and further validated using supernatants from cultures of P. gingivalis. Proteolytic degradation by proteases in buffer results in a concentration- and time-dependent blueshift of the LSPR band of about 1−2 nm when using casein as a substrate. In bacterial supernatants, the degradation of the protein coating resulted in unspecific binding of proteins present in the complex sample matrix to the nanoparticles, which instead triggered a redshift of about 2 nm of the LSPR band. A significant LSPR shift was seen only in samples with gingipain activity. The sensor showed a limit of detection < 0.1 μg/mL (4.3 nM), which is well below gingipain concentrations detected in severe chronic periodontitis cases (∼50 μg/mL). This work shows the possibility of developing cost-effective nanoparticle-based biosensors for rapid detection of protease activity for chair-side periodontal diagnostics.
https://pubs.acs.org/doi/abs/10.1021/acsanm.0c01899
https://www.genovis.com/products/gingisrex/
IgG *
GingisKHAN *;GingisREX
****
Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila
Beatriz Trastoy, Andreas Naegeli, Itxaso Anso, Jonathan Sjögren, Marcelo E. Guerin
Akkermansia muciniphila is a mucin-degrading bacterium commonly found in the human gut that promotes a beneficial effect on health, likely based on the regulation of mucus thickness and gut barrier integrity, but also on the modulation of the immune system. In this work, we focus in OgpA from A. muciniphila, an O-glycopeptidase that exclusively hydrolyzes the peptide bond N-terminal to serine or threonine residues substituted with an O-glycan. We determine the high-resolution X-ray crystal structures of the unliganded form of OgpA, the complex with the glycodrosocin O-glycopeptide substrate and its product, providing a comprehensive set of snapshots of the enzyme along the catalytic cycle. In combination with O-glycopeptide chemistry, enzyme kinetics, and computational methods we unveil the molecular mechanism of O-glycan recognition and specificity for OgpA. The data also contribute to understanding how A. muciniphila processes mucins in the gut, as well as analysis of post-translational O-glycosylation events in proteins.
https://www.nature.com/articles/s41467-020-18696-y
https://www.genovis.com/products/enzymes-for-o-glycans/operator/
Structure;O-glycosylation
Other / not known
LC/MS *
OpeRATOR
****
Detecting molecular forms of antithrombin by LC-MRM-MS: defining the measurands
L. Renee Ruhaak, Fred P.H.T.M. Romijn, Nico P.M. Smit, Arnoud van der Laarse, Mervin M. Pieterse, Moniek P.M. de Maat, Fred J.L.M. Haas, Cornelis Kluft, Jean Amiral, Piet Meijer and Christa M. Cobbaert
Background: Antithrombin (AT) is a critical regulator of coagulation, and its overall activity is typically measured using functional tests. A large number of molecular forms of AT have been identified and each individual carries multiple molecular proteoforms representing variable activities. Conventional functional tests are completely blind for these proteoforms. A method that ensures properly defined measurands for AT is therefore needed. We here assess whether mass spectrometry technology, in particular multiple reaction monitoring (MRM), is suitable for the quantification of AT and the qualitative detection of its molecular proteoforms.
Methods: Plasma proteins were denatured, reduced and alkylated prior to enzymatic digestion. MRM transitions were developed towards tryptic peptides and glycopeptides using AT purified from human plasma. For each peptide, three transitions were measured, and stable isotope-labeled peptides were used for quantitation. Completeness of digestion was assessed using digestion time
curves.
Results: MRM transitions were developed for 19 tryptic peptides and 4 glycopeptides. Two peptides, FDTISEK and FATTFYQHLADSK, were used for quantitation, and using a calibration curve of isolated AT in 40 g/L human serum albumin, CVs below 3.5% were obtained for FDTISEK, whereas CVs below 8% were obtained for FATTFYQHLADSK. Of the 26 important AT mutations, 20 can be identified using this method, while altered glycosylation profiles can also be detected.
Conclusions: We here show the feasibility of the liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) technique for the quantitation of AT and the qualitative analysis of most of its molecular proteoforms. Knowing the measurands will enable standardization of AT tests by providing in-depth information on the molecular proteoforms of AT.
https://www.degruyter.com/view/journals/cclm/56/10/article-p1704.xml
https://www.genovis.com/products/gingisrex/
LC/MS *
GingisREX
Comprehensive histone epigenetics: A mass spectrometry based screening assay to measure epigenetic toxicity
Sigrid Verhelst, Laura De Clerck, Sander Willems, Bart Van Puyvelde, Simon Daled, Dieter Deforce, Maarten Dhaenens
Evidence of the involvement of epigenetics in pathologies such as cancer, diabetes, and neurodegeneration has increased global interest in epigenetic modifications. For nearly thirty years, it has been known that cancer cells exhibit abnormal DNA methylation patterns. In contrast, the large-scale analysis of histone post-translational modifications (hPTMs) has lagged behind because classically, histone modification analysis has relied on site specific antibody-based techniques. Mass spectrometry (MS) is a technique that holds the promise to picture the histone code comprehensively in a single experiment. Therefore, we developed an MS-based method that is capable of tracking all possible hPTMs in an untargeted approach. In this way, trends in single and combinatorial hPTMs can be reported and enable prediction of the epigenetic toxicity of compounds. Moreover, this method is based on the use of human cells to provide preliminary data, thereby omitting the need to sacrifice laboratory animals. Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening assay is an ongoing effort. Still, this novel toxicoepigenetic assay and the data it generates holds great potential for, among others, pharmaceutical industry, food science, clinical diagnostics and, environmental toxicity screening.
https://www.sciencedirect.com/science/article/pii/S2215016120302752
https://www.genovis.com/products/gingisrex/
LC/MS *
GingisREX
***
Towards middle‑up analysis of polyclonal antibodies: subclass‑specific N‑glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC‑MS
Constantin Blöchl, Christof Regl, Christian G. Huber, Petra Winter, Richard Weiss, Therese Wohlschlager
In recent years, advanced HPLC-MS strategies based on intact protein (“top-down”) or protein
subunit (“middle-up/middle-down”) analysis have been implemented for the characterization of
therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for
polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse,
representing an important model system in immunological investigations. To obtain Fc/2 portions as
conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially
determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different
subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to highresolution
mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses
and their N-glycosylation variants, both of which influence IgG effector functions. To assess method
capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies
obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a
shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different
mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications
including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for
quality control of functional antibodies.
https://www.nature.com/articles/s41598-020-75045-1.epdf
https://www.genovis.com/products/igg-proteases/fabulous/
Fc-glycosylation
IgG Polyclonal *
LC/MS *;RP-HPLC *
FabULOUS *
A highly potent CD73 biparatopic antibody blocks organization of the enzyme active site through dual mechanisms
James E. Stefano, Dana M. Lord , Yanfeng Zhou, Julie Jaworski, Joern Hopke, Tara Travaline, Ningning Zhang , Karen Wong, Amanda Lennon, Timothy He, Eva Bric-Furlong, Cornishia Cherrie, Tristan Magnay, Elisabeth Remy, William Brondyk, Huawei Qiu, Katarina Radošević
The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of eleven parental monoclonal antibodies against CD73wasgeneratedbyFab-armexchange.Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and sub-nanomolar EC50 values. Pairing the Fabs of parents with non-overlapping epitopes was both sufficient and necessary while monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a non- overlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.
https://www.jbc.org/cgi/doi/10.1074/jbc.RA120.012395
https://www.genovis.com/products/igg-proteases/fabricator-z/
Inhibitory antibodies
Bispecific *
LC/MS *;cIEF *
FabRICATOR Z
Integrating Intact Mass Analysis and Middle-Down Mass Spectrometry Approaches to Effectively Characterize Trastuzumab and Adalimumab Structural Heterogeneity
Wenwen Zhu, Menglin Li, Jinlan Zhang
Comprehensive characterization of therapeutic monoclonal antibody (mAb) structures is critical for drug development but remains challenging due to the inherent structural heterogeneity. In this study, an integrated strategy has been developed to characterize trastuzumab structural heterogeneity, which has prominent advantages in fast sample preparation with minimal artifacts, and complementary information obtained from intact mass and middle-down analyses. Our methods were all developed on an electron transfer dissociation (ETD)-enabled Q-TOF instrument. As a result, more than 13 structurally different proteoforms were easily identified and quantified through native and denatured intact mass analysis, which may result from the collective differences in glycosylation and C-terminal lysine clipping. Based on collision-induced dissociation and ETD-combined middle-down analysis, sequence coverage values of 28, 45, and 41% for trastuzumab Fc/2, Lc, and Fd subunits, respectively, were reached in a single LC run. The main glycan structure and relative abundance level were determined, and the glycosylation site was confirmed to be on the Fc fragment Asn 61. We finally integrated the native MS and middle-down results to have a more realistic detection of molecular weight, sequence variants, and glycosylation variants of trastuzumab. Applying the integrated strategy, we successfully completed the comprehensive characterization of adalimumab and found unexpected C-terminal lysine-modified variants (dataset identifier PXD021287). Overall, our integration strategy can be easily implemented for in-depth mAb structural heterogeneity characterization during pharmaceutical development and quality control.
https://dx.doi.org/10.1021/acs.jproteome.0c00373
https://www.genovis.com/products/igg-proteases/fabricator/
C-terminal Lysine;Amino acid sequence;Fc-glycosylation;Structure
Monoclonal Ab *
LC-MS/MS;CID ETD MS/MS
FabRICATOR *
Selectivity over coverage in de novo sequencing of IgGs
Maurits A. den Boer, Jean-Francois Greisch, Sem Tamara, Albert Bondt, Albert J. R. Heck
Although incredibly diverse in specificity, millions of unique Immunoglobulin G (IgG) molecules in the human antibody repertoire share most of their amino acid sequence. These constant parts of IgG do not yield any useful information in attempts to sequence antibodies de novo. Therefore, methods focusing solely on the variable regions and providing unambiguous sequence reads are strongly advantageous. We report a mass spectrometry-based method that uses electron capture dissociation (ECD) to provide straightforward-to-read sequence ladders for the variable parts of both the light and heavy chains, with a preference for the functionally important CDR3. We optimized this method on the therapeutic antibody Trastuzumab and demonstrate its applicability on two monoclonal quartets of the four IgG subclasses, IgG1, IgG2, IgG3 and IgG4. The method is based on proteolytically separating the variable F(ab0)2 part from the conserved Fc part, whereafter the F(ab0)2 portions are mass-analyzed and fragmented by ECD. Pure ECD, without additional collisional activation, leads to straightforward-to-read sequence tags covering the CDR3 of both the light and heavy chains. Using molecular modelling and structural analysis, we discuss and explain this selective fragmentation behavior and describe how structural features of the different IgG subclasses lead to distinct fragmentation patterns. Overall, we foresee that pure ECD on F(ab0)2 or Fab molecules can become a valuable tool for the de novo sequencing of serum antibodies.
https://pubs.rsc.org/en/content/articlehtml/2020/sc/d0sc03438j
https://www.genovis.com/products/igg-proteases/fabalactica/
Amino acid sequence;Structure;Comparability studies
Monoclonal Ab *
ECD MS/MS
FabRICATOR *;FabALACTICA
Cysteine in cell culture media induces acidic IgG1 species by disrupting the disulfide bond network
Elke Prade, Anne Zeck, Fabian Stiefel, Andreas Unsoeld, David Mentrup, Erik
Arango-Gutierrez, Ingo H. Gorr
A high degree of charge heterogeneity is an unfavorable phenomenon commonly observed for therapeutic monoclonal antibodies (mAb). Removal of these impurities during manufacturing often comes at the cost of impaired step yields. A wide spectrum of post-translational and chemical modifications is known to modify mAb
charge. However, a deeper understanding of underlying mechanisms triggeringcharged species would be beneficial for the control of mAb charge variants during bioprocessing. In this study, a comprehensive analytical investigation was carried out to define the root causes and mechanisms inducing acidic variants of an IgG1-derived
mAb. Characterization of differently charged species by LC-MS revealed the reduction of disulfide bonds in acidic variants, which is followed by cysteinylation and glutathionylation of cysteines. Importantly, biophysical stability and integrity of the mAb is not affected. By in vitro incubation of the mAb with the reducing agent cysteine, disulfide bond degradation was directly linked to an increase of numerous acidic species. Modifying the concentrations of cysteine during the fermentation of various mAbs illustrated that redox potential is a critical aspect to consider during bioprocess development with respect to charge variant control.
https://doi.org/10.1002/bit.27628
https://www.genovis.com/products/igg-proteases/fabricator/
Disulfide reduction;Charge variants
Monoclonal Ab *;IgG *
LC/MS *;SEC *;CE;LC-MS/MS;Cation-Exchange Chromatography (CEX)
FabRICATOR *
Using different proteolytic enzymes to digest antibody and its impact on stability of antibody mimetics
Hanieh Khalili
There are opportunities to formulate antibodies as solid-state depots for local therapy, which would minimise large systemic doses that are typically required. We have developed antibody mimetics known as Fab-PEG-Fab (FpF) that display similar binding affinity and functional activity as IgG antibodies. For head-to-head comparison between FpF and IgG, FpF is prepared from the Fabs obtained by enzymatic digestion of IgGs. Here, we report for the first time that using different enzymes to proteolytically digest IgG plays an important role in stability profile of the obtained Fabs leading in different stability profiles of the final conjugated product such as FpF. We prepared an anti-vascular endothelial growth factor (VEGF) FpF from either clinical Fabrani (ranibizumab) or Fabs obtained by enzymatic digestion of bevacizumab (IgG) using immobilised papain and gingisKHANTM (KGP) enzyme. The stability of FpFs was then studied after being lyophilised in comparison with both ranibizumab and bevacizumab. Lyophilisation is being evaluated to produce solid material that can be used for depot fabrication. We observed that using immobilised papain to digest IgG resulted in the heterogenous isomers Fab leading to the preparation of heterogenous FpFbeva-papain mimetic that underwent aggregation during lyophilisation. However, using KGP enzyme generated a homogenous intact Fabbeva-KGP as determined by mass spectral analysis. Interestingly, the FpF mimetics prepared from the homogenous Fabs (Fabrani and Fabbeva-KGP), displayed greater stability compared to their starting bevacizumab and ranibizumab after being lyophilised as determined by DLS analysis. There is a potential to lyophilize FpFs to be used to fabricate solid-state depots.
https://www.sciencedirect.com/science/article/abs/pii/S0022175920302271#!
https://www.genovis.com/products/igg-proteases/gingiskhan/
Other;Structure
Monoclonal Ab *
LC/MS *;Other
GingisKHAN *
In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo
N. N. Parayath, S. B. Stephan, A. L. Koehne, P. S. Nelson & M. T. Stephan
Engineering chimeric antigen receptors (CAR) or T cell receptors (TCR) helps create disease-specific T cells for targeted therapy, but the cost and rigor associated with manufacturing engineered T cells ex vivo can be prohibitive, so programing T cells in vivo may be a viable alternative. Here we report an injectable nanocarrier that delivers in vitro-transcribed (IVT) CAR or TCR mRNA for transiently reprograming of circulating T cells to recognize disease-relevant antigens. In mouse models of human leukemia, prostate cancer and hepatitis B-induced hepatocellular carcinoma, repeated infusions of these polymer nanocarriers induce sufficient host T cells expressing tumor-specific CARs or virus-specific TCRs to cause disease regression at levels similar to bolus infusions of ex vivo engineered lymphocytes. Given their ease of manufacturing, distribution and administration, these nanocarriers, and the associated platforms, could become a therapeutic for a wide range of diseases.
https://www.nature.com/articles/s41467-020-19486-2
https://www.genovis.com/products/iggzero/
Bioassays;Other;Imaging;Antigen Binding
Monoclonal Ab *;Other / not known
Other;Bioassays
IgGZERO *
Generating Informative Sequence Tags from Antigen-Binding Regions of Heavily Glycosylated IgA1 Antibodies by Native Top-Down Electron Capture Dissociation
Jean-Francois Greisch, Maurits A. den Boer, Frank Beurskens, Janine Schuurman, Sem Tamara, Albert Bondt, Albert J. R. Heck
Immunoglobulins A (IgA) include some of the most abundant human antibodies and play an important role in defending mucosal surfaces against pathogens. The unique structural features of the heavy chain of IgA subclasses (called IgA1 and IgA2) enable them to polymerize via the joining J-chain, resulting in IgA dimers but also higher oligomers. While secretory sIgA oligomers are dominant in milk and saliva, IgAs exist primarily as monomers in serum. No method currently allows disentangling the millions of unique IgAs potentially present in the human antibody repertoire. Obtaining unambiguous sequence reads of their hypervariable antigen-binding regions is a prerequisite for IgA identification. We here report a mass spectrometric method that uses electron capture dissociation (ECD) to produce straightforward-to-read sequence ladders of the variable parts of both the light and heavy chains of IgA1s, in particular, of the functionally critical CDR3 regions. We directly compare the native top-down ECD spectra of a heavily and heterogeneously N- and O-glycosylated anti-CD20 IgA1, the corresponding N-glycosylated anti-CD20 IgG1, and their Fab parts. We show that while featuring very different MS1 spectra, the native top-down ECD MS2 spectra of all four species are nearly identical, with cleavages occurring specifically within the CDR3 and FR4 regions of both the heavy and light chain. From the sequence-informative ECD data of an intact glycosylated IgA1, we foresee that native top-down ECD will become a valuable complementary tool for the de novo sequencing of IgA1s from milk, saliva, or serum.
https://pubs.acs.org/doi/full/10.1021/jasms.0c00461
https://www.genovis.com/products/enzymes-for-o-glycans/operator/
Amino acid sequence;Fab-glycosylation;Comparability studies;O-glycosylation
ECD MS/MS
FabALACTICA;OpeRATOR;SialEXO
Microdroplet Ultrafast Reactions Speed Antibody Characterization
Pengyi Zhao, Harsha P. Gunawardena, Xiaoqin Zhong, Richard N. Zare and Hao Chen
Recently, microdroplet reactions have aroused much interest because the microdroplet provides a unique medium where organic reactions could be accelerated by a factor of 103 or more. However, microdroplet reactions of proteins have been rarely studied. We report the occurrence of multiple-step reactions of a large protein, specifically, the digestion, reduction, and deglycosylation of an intact antibody, which can take place in microseconds with high reaction yields in aqueous microdroplets at room temperature. As a result, fast structural characterization of a monoclonal antibody, essential for assessing its quality as a therapeutic drug, can be enabled. We found that the IgG1 antibody can be digested completely by the IdeS protease in aqueous microdroplets in 250 microseconds, a 7.5 millionfold improvement in speed in comparison to traditional digestion in bulk solution (>30 min). Strikingly, inclusion of the reductant tris(2-carboxyethyl)phosphine in the spray solution caused simultaneous antibody digestion and disulfide bond reduction. Digested and reduced antibody fragments were either collected or analyzed online by mass spectrometry. Further addition of PNGase F glycosylase into the spray solution led to antibody deglycosylation, thereby producing reduced and deglycosylated fragments of analytical importance. In addition, glycated fragments of IgG1 derived from glucose modification were identified rapidly with this ultrafast digestion/reduction technique. We suggest that microdroplets can serve as powerful microreactors for both exploring largemolecule reactions and speeding their structural analyses.
https://dx.doi.org/10.1021/acs.analchem.0c04974?ref=pdf
https://www.genovis.com/products/igg-proteases/fabricator/
Glycation;Fc-glycosylation
Monoclonal Ab *;IgG *
nESI-MS
FabRICATOR *
Siplizumab Induces NK Cell Fratricide Through Antibody-Dependent Cell-Mediated Cytotoxicity
Christian Binder, Felix Sellberg, Filip Cvetkovski, Stefan Berg, Erik Berglund and David Berglund
The glycoprotein CD2 is expressed on T and NK cells and contributes to cell-cell conjugation, agonistic signaling and actin cytoskeleton rearrangement. CD2 has previously been shown to have an important function in natural NK cell cytotoxicity but to be expendable in antibody-mediated cytotoxicity. Siplizumab is a monoclonal anti-CD2 IgG1 antibody that is currently undergoing clinical trials in the field of transplantation. This study investigated the effect of CD2 binding and Fc γ receptor binding by siplizumab (Fc-active) and Fc-silent anti-CD2 monoclonal antibodies in allogeneic mixed lymphocyte reaction and autologous lymphocyte culture. Further, induction of NK cell fratricide and inhibition of natural cytotoxicity as well as antibody-dependent cytotoxicity by these agents were assessed. Blockade of CD2 via monoclonal antibodies in the absence of Fc γ receptor binding inhibited NK cell activation in allogeneic mixed lymphocyte reaction. In contrast, siplizumab increased NK cell activation in both mixed lymphocyte reaction and autologous lymphocyte culture due to FcγRIIIA binding. However, experiments using purified NK cells did not show an inhibitory effect of CD2 blockade on natural cytotoxicity or antibody-dependent cytotoxicity. Lastly, it was shown that siplizumab induces NK cell fratricide. Concluding, siplizumab is a promising biopharmaceutical drug candidate for depletion of T and NK cells with minimal off-target effects.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904868/
https://www.genovis.com/products/glycinator/glycinator-enzyme/
ADCC;Inhibitory antibodies
Monoclonal Ab *;IgG *
Other;Bioassays
GlycINATOR *
Electron Transfer Dissociation Parameter Optimization Using Design of Experiments Increases Sequence Coverage of Monoclonal Antibodies
Milos Cejkov, Tyler Greer, Reid O’Brien Johnson, Xiaojing Zheng and Ning Li
Middle-down analysis of monoclonal antibodies (mAbs) by tandem mass spectrometry (MS2) can provide detailed insight into their primary structure with minimal sample preparation. The middle-down approach uses an enzyme to cleave mAbs into Fc/2, LC, and Fd subunits that are then analyzed by reversed phase liquid chromatography tandem mass spectrometry (RPLC−MS2). As maximum sequence coverage is desired to obtain meaningful structural information at the subunit level, a host of dissociation methods have been developed, and sometimes combined, to bolster fragmentation and increase the number of identified fragments. Here, we present a design of experiments (DOE) approach to optimize MS2 parameters, in particular those that may influence electron transfer dissociation (ETD) efficiency to increase the sequence coverage of antibody subunits. Applying this approach to the NIST monoclonal antibody standard (NISTmAb) using three RPLC−MS2 runs resulted in high sequence coverages of 67%, 67%, and 52% for Fc/2, LC, and Fd subunits, respectively. In addition, we apply this DOE strategy to model the parameters required to maximize the number of fragments produced in “low”, “medium”, and “high” mass ranges, which ultimately resulted in even higher sequence coverages of NISTmAb subunits (75%, 78%, and 64% for Fc/2, LC, and Fd subunits, respectively). The DOE approach provides high sequence coverage percentages utilizing only one fragmentation method, ETD, and could be extended to other state-of-the-art techniques that combine multiple fragmentation mechanisms to increase coverage.
https://pubs.acs.org/doi/10.1021/jasms.0c00458?ref=pdf
https://www.genovis.com/products/igg-proteases/fabricator/
Amino acid sequence
Monoclonal Ab *;IgG *
LC-MS/MS
FabRICATOR *
Multiattribute Monitoring of Antibody Charge Variants by Cation- Exchange Chromatography Coupled to Native Mass Spectrometry
Markus Haberger, Anna-Katharina Heidenreich, Michaela Hook, Jürgen Fichtl, Rainer Lang, Florian Cymer, Mahdi Adibzadeh, Felix Kuhne, Harald Wegele, Dietmar Reusch, Lea Bonnington and Patrick Bulau
The aim of this study was to characterize the product variants of a therapeutic T-cell bispecific humanized monoclonal antibody (TCB Mab, ∼200 kDa, asymmetric) and to develop an online cation-exchange chromatography native electrospray mass spectrometry method (CEC-UV-MS) for direct TCB Mab charge variant monitoring during bioprocess and formulation development. For the identification and functional evaluation of the diverse and complex TCB Mab charge variants, offline fractionation combined with comprehensive analytical testing was applied. The offline fractionation of abundant product variant peaks enabled identification of coeluting acid charge variants such as asparagine deamidation, primary and secondary Fab glycosylation (with and without sialic acid), and the presence of O-glycosylation in the G4S-linker region. Consequently, a new nonconsensus N-glycosylation motif (N-338-FG) in the heavy chain CDR region was discovered. Functional evaluation by cellbased potency testing demonstrated a clear and negative impact of both asparagine deamidations, whereas the O-glycosylation did not affect the TCB Mab biological activity. We established an online native CEC-UV-MS method, with an ammonium acetate buffer and pH gradient, to directly monitor the TCB Mab charge variants. All abundant chemical degradations and post-translational amino acid modifications already identified by offline fraction experiments and liquid chromatography mass spectrometry peptide mapping could also be monitored by the online CEC-UV-MS method. The herein reported online native CEC-UV-MS methodology represents a complementary or even alternative approach for multiattribute monitoring of biologics, offering multiple benefits, including increased throughput and reduced sample handling and intact protein information in the near-native state.
https://doi.org/10.1021/jasms.0c00446
https://www.genovis.com/products/igg-proteases/fabalactica/
Oxidation;Amino acid sequence;Fc-glycosylation;Fab-glycosylation;Charge variants;O-glycosylation
Monoclonal Ab *;IgG *
HPLC *;Native MS *;Other;LC-MS/MS;Cation-Exchange Chromatography (CEX)
FabALACTICA
Application of middle-down approach in quantitation and catabolite identification of protein by LC–high-resolution mass spectrometry
Lijuan Kang, Shengsheng Xu, Yongle Pang, Tom Kirchner, Yue-mei Zhang, Wilson Edwards, Raul Camacho, Lisa Norquay, Naidong Weng & Wenying Jian
Aim: To further enhance the detection sensitivity and increase resolving power of top-down intact protein bioanalysis, middle-down approach was explored. Materials & methods: An monoclonal antibody (mAb) was used as a model protein to evaluate quantitative bioanalytical assay performance and a disulfide linked dimer protein was investigated for its pharmacokinetics properties and catabolism in vivo by middle-down approach. Results & Conclusion: For quantitation of the mAb, different subunits generated by middle-down approach provided different level of signal improvement in biological samples with Lc and half Fc giving five-times better sensitivity than intact mAb. For the dimer protein, middle-down analysis by reduction enabled effective differentiation of the unchanged protein and its oxidized form, and clearly elucidated their respective proteolytic catabolites.
https://www.future-science.com/doi/full/10.4155/bio-2020-0315
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Antibody Ratio;Oxidation;Other;Fc-glycosylation;Drug Load Distribution;Comparability studies;Charge variants
IgG *
LC/MS *
FabRICATOR *;GingisKHAN *
Automated and Faster Affinity Capture Method for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates
Aarti Jashnani, Srikanth Kotapati, Madhura Deshpande, Sayumi Yamazoe, Pavel Strop, Arvind Rajpal and Gavin Dollinger
Traditionally the biotransformation of antibody drug conjugates (ADCs) has been evaluated by affinity capture on streptavidin magnetic beads coated with a biotinylated capture reagent. To reduce the complexity of the analyte, the affinity captured ADCs are digested with enzymes (“on-bead” or after elution), and/or interchain disulfides are reduced to generate LC and HC fragments prior to mass spectrometry analysis. The “onbead” enzymatic digestion with IdeS and PNGase F is not efficient and requires longer incubation times to achieve complete Fc and N-glycan removal. This results in a prolonged sample preparation time (7−18 h) and is not suitable for labile ADCs due to the
possibility of assay-induced artifacts. To address these challenges, we developed an affinity capture method, where the ADCs are first captured onto streptavidin cartridges coated with a biotinylated generic capture reagent, followed by a 15 min “on-cartridge” digestion with IdeS or PNGase F. The ADCs are then eluted and directly analyzed by LC-HRMS. This method was successfully applied for the biotransformation assessment of site-specific ADCs with payload conjugated on the Fab or Fc. The reduced complexity of the analyte (Fc and N-glycan removal) combined with HRMS enabled sensitive and accurate identification of minor mass change catabolites and changes in the DAR distribution. This automated cartridge-based affinity capture method is fast with a total sample preparation time of less than 4 h (hands-on time of less than 1 h) and can be utilized for any human mAb/ADC independent of isotype (IgG1, IgG2, and IgG4).
https://pubs.acs.org/doi/full/10.1021/acs.analchem.0c04685
https://www.genovis.com/products/igg-proteases/fabricator/
Drug Antibody Ratio;Drug Load Distribution
ADC *
LC/MS *
FabRICATOR *
Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography – Mass spectrometry
Honorine Lardeux, Bastiaan L.Duivelshof, OlivierColas, AlainBeck, David V.McCalley, DavyGuillarme, ValentinaD’Atri
When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of the most effective additives in RPLC and HILIC, trifluoroacetic acid (TFA), is known as a strong suppressor of the MS signal and limits its use in hyphenated techniques. In this study, we evaluated a wide range of acidic additives to find alternatives to TFA that provided comparable chromatographic performance and improved MS sensitivity. It was observed that stronger acidic additives were required for intact level analysis compared to subunit level analysis and that the additive nature had a larger impact on the chromatographic performance in HILIC mode compared to RPLC. Therefore, four additives were identified as valuable alternatives to TFA in RPLC mode, namely, difluoroacetic acid (DFA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), and methanesulfonic acid (MSA). Only one of these additives provided acceptable performance in HILIC mode, namely, TClAA.
After evaluation of the MS performance, TClAA was discarded due to the apparent loss of intensity in both RPLC-MS and HILIC-MS mode. Together, these results demonstrate that for HILIC-MS analysis TFA remains the gold standard additive. However, DFA was found as promising alternative to TFA for RPLC-MS analysis and could play an important role in the development of methods for the characterization of the increasingly complex protein biopharmaceuticals.
https://www.sciencedirect.com/science/article/pii/S0003267021001732
https://www.genovis.com/products/igg-proteases/fabricator/
Monoclonal Ab *
LC/MS *;HILIC
FabRICATOR *
****
Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells
Christoph Gstöttner, Tao Zhang, Anja Resemann, Sophia Ruben, Stuart Pengelley, Detlev Suckau, Tim Welsink, Manfred Wuhrer and Elena Domínguez-Vega
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor-binding domain (RBD), mediates the interaction with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells and may be modulated by its structural features. Therefore, well-characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional post translational modifications), a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection, and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, a higher level of sialylation, and a combination of core 1 and core 2 type O-glycans. Additionally, using an alternative approach based on N-terminal cleavage of the O-glycosylation, the previously unknown O-glycosylation site was localized at T323. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to the ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides an analytical workflow for characterization of new RBDs and batch-to-batch comparison.
https://pubs.acs.org/doi/10.1021/acs.analchem.1c00893
https://www.genovis.com/products/exoglycosidases/fucosexo/
Bioassays;Other;O-glycosylation
Other / not known
Bioassays;CE-MS;LC-MS/MS
OpeRATOR;OglyZOR;SialEXO;FucosEXO;GalactEXO
Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia
Daniel Asarnow, Bei Wang, Wen-Hsin Lee, Yuanyu Hu, Ching-Wen Huang, Bryan Faust, Patricia Miang Lon Ng, Eve Zi Xian Ngoh, Markus Bohn, David Bulkley, Andrés Pizzorno, Beatrice Ary, Hwee Ching Tan, Chia Yin Lee, Rabiatul Adawiyah Minhat, Olivier Terrier, Mun Kuen Soh, Frannie Jiuyi Teo, Yvonne Yee Chin Yeap, Shirley Gek Kheng Seah, Conrad En Zuo Chan, Emily Connelly, Nicholas J. Young, Sebastian Maurer-Stroh, Laurent Renia, Brendon John Hanson, Manuel Rosa- Calatrava, Aashish Manglik, Yifan Cheng, Charles S. Craik, Cheng-I Wang
Infection by SARS-CoV-2 is initiated by binding of viral Spike protein to host receptor angiotensin-converting enzyme (ACE2), followed by fusion of viral and host membranes. While antibodies that block this interaction are in emergency use as early COVID-19 therapies, precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that all potently block ACE2 binding, yet exhibit divergent neutralization efficacy against live virus. Strikingly, these neutralizing antibodies can either inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in COVID-19 patients. Multiple cryogenic electron microscopy structures of Spike-antibody complexes reveal distinct binding modes that not only block ACE2 binding, but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion, with profound implications in COVID-19 pathology and immunity.
https://www.cell.com/cell/pdf/S0092-8674(21)00536-5.pdf
https://www.genovis.com/products/igg-proteases/fabalactica/immobilized-fabalactica/
Infection biology;Inhibitory antibodies
Monoclonal Ab *
SEC *;SEC-MS *
FabALACTICA
***
Chromatographic and Adsorptive Behavior of a Bivalent Bispecific Antibody and Associated Fragments
Lucas K. Kimerer, Ben Niu, Timothy M. Pabst, Weiguo Zhai, Alan K. Hunter, Giorgio Carta
The elution and adsorptive behavior of a bivalent bispecific antibody (BiSAb), comprising an IgG1 framework with a scFv domain genetically fused to each heavy chain C-terminus via flexible linkers, and of two associated fragments were studied on two cation exchange chromatography media – ProPac WCX-10, which is pellicular and suitable for analytical use, and Nuvia HR-S, which is macroporous and suitable for preparative and process scale uses. Both fragments were identified by MS as missing one of the two scFv domains and its flexible linker, but one of them also contains an additional C-terminal lysine. The separation of these fragments on both resins occurs as a result of differences in non-specific ligand-protein interactions that are modulated by the salt concentration. For the ProPac WCX-10 column, complex, multipeak elution behaviors are observed, since, as a result of the linker flexibility, both the intact molecule and the fragments appear to exist in multiple binding configurations with each scFv domains either collapsed onto the IgG framework or extended away from it. With a residence time of 2.5 min and at 21 °C, two peak elution is observed for the fragments which contain a single linked scFv and three peak elution for the intact molecule which contains two linked scFvs. This behavior is affected by residence time, temperature, and hold time. Increasing the residence time to 25 min or increasing temperature to 40°C results in elution of a single, merged peak for each of the protein species. For Nuvia HR-S, the broader peaks, obtained as a result of mass transfer limitations, tend to obscure the multipeak elution behavior. Nevertheless, even for this resin, the effects of configurational flexibility are still manifested at the single-particle scale and affect the evolution of the patterns of protein binding within individual resin particles as evident from confocal microscopy observations.
https://www.sciencedirect.com/science/article/abs/pii/S0021967321003058
https://www.genovis.com/products/igg-proteases/fabricator/
C-terminal Lysine
Bispecific *
Cation-Exchange Chromatography (CEX)
FabRICATOR *
***
Glycoproteomics: growing up fast
Thomas. D & Scott. N
Glycoproteomics is a rapidly growing field which seeks to identify and characterise glycosylation events at a proteome scale. Over the last few years considerable effort has been made in developing new technologies, enrichment systems, and analysis strategies to enhance the quality of glycoproteomic studies. Within this review we discuss the recent developments in glycoproteomics and the current state of the art approaches for analysing glycosylated substrates. We highlight key improvements in mass spectrometry instrumentation coupled with the advancements in enrichment approaches for key classes of glycosylation including mucin-O-glycosylation, O-GlcNAc glycosylation and N-linked glycosylation which now allow the identification/quantification of hundreds to thousands of glycosylation sites within individual experiments. Finally, we summarise the emerging trends within glycoproteomics to illustrate how the field is moving away from studies simply focused on identifying glycosylated substrates to studying specific mechanisms and disease states.
https://doi.org/10.1016/j.sbi.2020.10.028
https://www.genovis.com/products/enzymes-for-o-glycans/operator/
O-glycosylation
Other / not known
LC-MS/MS
OpeRATOR
Charge variant analysis of protein-based biopharmaceuticals using two-dimensional liquid chromatography hyphenated to mass spectrometry
Simon Jaag , Marina Shirokikh , Michael Lämmerhofer
The profile of charge variants represents an important critical quality attribute of protein-based biopharmaceuticals, in particular monoclonal antibodies and must therefore be closely controlled. In this work, 2D-LC methods for charge variant analysis were developed using a strong cation-exchange chromatography (SCX) as first dimension (1D) separation. Non-porous SCX (3 μm) particle columns and different mobile phases were evaluated using a test mixture of some standard proteins of different size and pI (comprising myoglobin, bovine serum albumin, cytochrome c, lysozyme and β-lactoglobulin) and two monoclonal IgG1 antibodies (NIST mAb and Secukinumab). The most promising 1D eluent for SCX was a salt-mediated pH-gradient system using a ternary mobile phase system with 2-(N-morpholino)ethanesulfonic acid, 1,3-diamino-2-propanol and sodium chloride. For the second dimension (2D), a desalting reversedphase liquid chromatography (RP-LC) was chosen to enable the hyphenation of the charge variant separation with mass spectrometric (MS) detection. While for intact mAbs the 2D just served for desalting without additional selectivity, the 2D contributed some orthogonal selectivity for the mAb fragment separation. Various core-shell and monolithic columns were tested and variables such as gradient time and flow rate systematically optimized. Unexpectedly, a C4 400 Å column (3.4 μm diameter with 0.2 μm porous shell) provided higher peak capacities compared to the same 1000 Å column (2.7 μm diameter with 0.5 μm porous shell). A thinner shell appeared to be more advantageous than wider pores under high flow regime. An ultra-fast RPLC method with a run time of one minute was developed first using trifluoroacetic acid and later with formic acid as additive for better MS compatibility. The successful hyphenation of the two orthogonal separation modes SCX and RP-LC could be demonstrated in the multiple heartcutting and the full comprehensive mode. MS analysis using a high-resolution quadrupole time-of-flight instrument enabled to identify different glycoforms and some major charge variants of the antibody at the intact protein level as well as on the subunit level (Fc/2, Lc, Fd’) in a middleup approach by 2D-LC-ESI-MS analysis.
https://www.sciencedirect.com/science/article/abs/pii/S0021967320310608
https://www.genovis.com/products/igg-proteases/fabricator/fragit/
Fc-glycosylation;Charge variants
Monoclonal Ab *
2D-LC;SCX-MS;Cation-Exchange Chromatography (CEX)
FragIT / FragIT Kit
Photoactivatable Fluorophore for Stimulated Emission Depletion(STED) Microscopy and Bioconjugation Technique forHydrophobic Labels
Michael Weber, Taukeer A. Khan, Lukas J. Patalag, Mariano Bossi, Marcel Leutenegger, Vladimir N. Belov and Stefan W. Hell
The use of photoactivatable dyes in STED micros-copy has so far been limited by two-photon activation through the STED beam and by the fact that photoactivatable dyes are poorly solvable in water. Herein, we report ONB-2SiR, a fluorophore that can be both photoactivated in the UV and specifically de-excited by STED at 775 nm. Like-wise, we introduce a conjugation and purification protocol to effectively label primary and secondary antibodies with moderately water-soluble dyes. Greatly reducing dye aggregation, our technique provides a defined and tunable degree of labeling, and improves the imaging performance of dye conjugates in general.
doi.org/10.1002/chem.202004645
https://www.genovis.com/products/glyclick/
Imaging
IgG *
STED
GlyCLICK
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