Characterization of Cysteine-Linked Conjugation Profiles of Immunoglobulin G1 and Immunoglobulin G2 Antibody–Drug Conjugates

Wiggins, B C; Liu-Shin, Lily; Yamaguchi, Hideto; Ratnaswamy, Gayathri

doi:10.1002/jps.24338

Cited by 67 publications

(59 citation statements)

References 27 publications

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“…51 Cysteine conjugation commonly generates a mixture of intact ADC with an increasing portion of non-covalently linked light and heavy chains for increasing average DAR. 52,53 Most publications depict light chain (LC) and heavy chains (HC) in separate spectra. However, by opting to display both LCs and HCs in the same spectrum, we could clearly observe a pronounced bias for LC in all QToF spectra (see Fig.…”

Section: Rplc-msmentioning

confidence: 99%

Qualitative analysis of antibody–drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs

Källsten

Hartmann

Artemenko

et al. 2018

Analyst

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Antibody-drug conjugates (ADCs) are an emerging type of biotherapeutics that utilize multiple tissuespecific antibodies combined with a range of linker designs to enable the transportation and selective release of cytotoxic drugs in close proximity to tumours. Consisting of antibodies conjugated to small drug molecules through a variety of linkers, ADCs are chemically complex analytes. Here we present a unique experimental comparison of four techniques for ADC analysis: hydrophobic interaction chromatography (HIC-UV/Vis), reversed phase liquid chromatography mass spectrometry (RPLC-MS), using either a QToF or an Orbitrap analyser, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Four different ADCs consisting of Trastuzumab, monomethyl auristatin E (MMAE) and a peptidic linker moiety differing in their respective stoichiometric ratios in regard to drugto-antibody ratio (DAR) were used for the comparison. We found that the determined DAR from all techniques was comparable, while the accuracy of the molecular weights for the conjugated light and heavy chain differed more extensively. This indicates that the choice of a mass analyser is more crucial for determining the accurate weights of the light and heavy chains than to evaluate the DAR of a given batch.However, ambiguous DAR assignment in HIC-UV/Vis or bias for either the light or heavy chain fragments in the mass spectrometry-based techniques can influence the obtained average DAR value and the use of complementary techniques is advisable. Out of the four techniques evaluated, HIC-UV/Vis and MALDI required less time to obtain an average DAR value and would therefore be good for initial screenings in the early stages of the discovery phase of new ADCs. † Electronic supplementary information (ESI) available: S-1. LC-chromatograms for HIC-UV/Vis and both RPLC-MS with consecutive blank runs depicting carry over effects, further information on MALDI samples and further comparison of mass accuracy of the different techniques (PDF). See

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Section: Rplc-msmentioning

confidence: 99%

Qualitative analysis of antibody–drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs

Källsten

Hartmann

Artemenko

et al. 2018

Analyst

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“…It is well-known that the semi-hydrophobic nature of cysteine allows for marked differences in solvent accessibility depending on the folded state of the protein [112,113,114,115]. The more solvent that has access to these residues, the faster the reaction rate [112].…”

Section: Probing Protein Conformation Via Differential Modificationmentioning

confidence: 99%

Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins

Irvine

Stillman

2017

IJMS

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Structural information regarding metallothioneins (MTs) has been hard to come by due to its highly dynamic nature in the absence of metal-thiolate cluster formation and crystallization difficulties. Thus, typical spectroscopic methods for structural determination are limited in their usefulness when applied to MTs. Mass spectrometric methods have revolutionized our understanding of protein dynamics, structure, and folding. Recently, advances have been made in residue modification mass spectrometry in order to probe the hard-to-characterize structure of apo- and partially metalated MTs. By using different cysteine specific alkylation reagents, time dependent electrospray ionization mass spectrometry (ESI-MS), and step-wise “snapshot” ESI-MS, we are beginning to understand the dynamics of the conformers of apo-MT and related species. In this review we highlight recent papers that use these and similar techniques for structure elucidation and attempt to explain in a concise manner the data interpretations of these complex methods. We expect increasing resolution in our picture of the structural conformations of metal-free MTs as these techniques are more widely adopted and combined with other promising tools for structural elucidation.

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“…In particular, the reduction of antibody interchain disulfide bridges is essential to enable attachment of cytotoxic payloads in the synthesis of ADC's such as brentuximab vedotin (Adcetris™). 11 Water soluble alkylphosphines have established themselves as popular reducing reagents for effecting the liberation of peptidyl thiols, as they provide numerous advantages over the traditional thiol based reagents such as dithiothreitol (DTT), β-mercaptoethanol (BME) and β-mercaptoethylamine (2-MEA). [12][13][14] The phosphorus atom of commercial watersoluble alkylphosphines have pKa's of 7-8 which is a common pH range for performing bioconjugations and as such, the trialkylphosphines are more effective nucleophiles than thiol-based reducing agents to effect reduction within this pH range.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Reactions between Water-Soluble Trialkylphosphines and Thiol Alkylating Reagents: Implications for Protein-Conjugation Reactions

Kantner

Watts

2016

Bioconjugate Chem.

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Water-soluble trialkylphosphines such as tris(carboxyethyl)phosphine (TCEP) and trishydroxypropyl phosphine (THPP) are effective agents for reducing disulfide bonds in proteins and are increasingly becoming the reagents of choice for bioconjugation strategies that modify cysteine (thiol containing) amino acids. These reducing agents are often considered as being chemically compatible with Michael acceptors such as maleimides and, as such, are often not removed prior to performing protein conjugation reactions. Here, we demonstrate the rapid and irreversible reaction of both TCEP and THPP with derivatives of the commonly employed thiol alkylating groups, maleimide and vinyl sulfone. Mechanistic investigations revealed distinct differences between the reactions of TCEP and THPP with maleimide, leading to the production of either nonproductive ylenes or succidimidyl derivatives, respectively. Importantly, we also demonstrate the incorporation of nonproductive ylenes formed between maleimide and TCEP into the Pneumococcal capsular polysaccharide Pn6b following strategies employed toward the production of conjugate vaccines.

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Characterization of Cysteine-Linked Conjugation Profiles of Immunoglobulin G1 and Immunoglobulin G2 Antibody–Drug Conjugates

Cited by 67 publications

References 27 publications

Qualitative analysis of antibody–drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs

Qualitative analysis of antibody–drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs

Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins

Characterization of Reactions between Water-Soluble Trialkylphosphines and Thiol Alkylating Reagents: Implications for Protein-Conjugation Reactions

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