Antibody-drug conjugate characterization by chromatographic and electrophoretic techniques

Chen, Tao; Chen, Yan; Stella, Cinzia; Medley, Colin D.; Gruenhagen, Jason; Zhang, Kelly

doi:10.1016/j.jchromb.2016.07.023

Cited by 57 publications

(60 citation statements)

References 97 publications

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“…Two levels of heterogeneity are characterized: (1) The antibody contains species conjugated with different number of payloads, or (2) an antibody with the same number of payloads may have different conjugation sites. 9 Conventional conjugation strategies include conjugation to surface lysine residues or the interchain cysteines ( Fig. 1).…”

Section: Heterogeneitymentioning

confidence: 99%

“…7 In addition, the analytical characterization to evaluate physical and chemical integrity can be of increased complexity, 8 particularly in quantification of free drug, total drug, drug-to-antibody ratio (DAR), and drug load distribution. 9 Chemical and physical degradation pathways of biologics and chemical entities have been extensively investigated. Antibody-drug conjugates bear degradation pathways of their 3 components.…”

Section: Introductionmentioning

confidence: 99%

“…A wide range of excellent reviews cover the different linker chemistries, 3 the toxins used for ADCs, 14,15 the variety of conjugation strategies and techniques, [16][17][18] analytical techniques, 8,9,19 and ADC PKs. 20,21 This review pursues the path of Ross and Wolfe 4 with a focus on physical and chemical stabilities of ADCs but sets the mAb component and its structure and biophysical properties in the primary focus, supported by currently available literature.…”

Section: Introductionmentioning

confidence: 99%

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Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

Buecheler

Winzer

Weber

et al. 2020

Journal of Pharmaceutical Sciences

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a b s t r a c tAntibody conjugates, in particular antibody-drug conjugates (ADCs), are a fast-growing area in research and in the pharmaceutical industry. The covalent attachment of an antibody to a chemical moiety can be an effective measure for drug targeting or can also positively impact pharmacokinetics of small molecular compounds by serum half-life extension. Stability, physicochemical properties, and degradation pathways of biotherapeutics or small molecule therapeutics are often not totally known and understood. However, ADCs represent a hybrid of small molecular and macromolecular components, and their properties are still not fully understood and described. This review discusses the alteration of the physicochemical properties of antibodies upon conjugation of chemical moieties to its surface and the resulting impact on ADC stability.

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Section: Heterogeneitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

Buecheler

Winzer

Weber

et al. 2020

Journal of Pharmaceutical Sciences

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“…3A, second panel), but a lower recovery of the LC and the Fd glycovariants to 39% and 62% was observed at 40 • C. Brentuximab vedotin has several highly hydrophobic loaded fragments due to cytotoxic drugs attached to them (LC I, Fd I, Fd II and Fd III fragments, with I, II and III corresponding to the number of bound payloads). The proper recovery of these loaded subunits is important for the estimation of the average drug-to-antibody ratio (DAR), which is a critical quality attribute (CQA) of the antibody drug conjugates [31,[42][43][44]. Average DAR can be calculated based on the formula reported by Wagner-Rousset et al [45] and it is generally close to 4.0 [46,47].…”

Section: Recovery Of Mab Species In Hilic Conditionsmentioning

confidence: 99%

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Bobály

D’Atri

Beck³

et al. 2017

Journal of Pharmaceutical and Biomedical Analysis

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a b s t r a c tHydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

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“…Whilst much work has been done to control the regioselectivity of the covalent attachment of drug molecules onto the antibodies and new modes of drugrelease, [12][13][14][15] little attention has been paid to ADC-analyses until recently. Recent publications described both novel techniques for the assessment of the drug-to-antibody ratio (DAR), [16][17][18][19][20][21] ADC stability, [22][23][24][25] aggregation degree assessment, [26][27][28] bioanalysis, 29 the quantification of unconjugated drug molecules 22 and PTMs. 8,[30][31][32] There exist numerous reviews discussing ADC analysis techniques by comparing data from different studies; 27,[33][34][35] however, to our knowledge comprehensive studies that have experimentally compared the performance of multiple techniques for ADC DAR analysis based on a single sample batch are scarce.…”

mentioning

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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Antibody-drug conjugate characterization by chromatographic and electrophoretic techniques

Cited by 57 publications

References 97 publications

Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

Alteration of Physicochemical Properties for Antibody-Drug Conjugates and Their Impact on Stability

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

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

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