Calculated Conjugated Payload from Immunoassay and LC–MS Intact Protein Analysis Measurements of Antibody-Drug Conjugate

Rago, Brian; Clark, Tracey; King, Lindsay; Zhang, Jenny; Tumey, L. Nathan; Li, Fengping; Barletta, Frank; Wei, Cong; Leal, Mauricio; Hansel, Steve; Han, Xiaogang

doi:10.4155/bio-2016-0160

Cited by 20 publications

(21 citation statements)

References 33 publications

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“…If ADCs need to be enriched from biological samples such as plasma or serum, immunocapture is a powerful technique. 61,62 ADC samples could be desalted and separated by reversed-phase chromatography or size-exclusion chromatography (SEC), 41 or directly infused by nanospray. 63,64 Alternatively, capillary electrophoresis (CE) interfaced with MS provides an orthogonal option for separation.…”

Section: Major Quality Attributes Of Adcmentioning

confidence: 99%

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

Wagh

Song

Zeng

et al. 2018

mAbs

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Antibody-drug conjugates (ADCs) are a growing class of biotherapeutics in which a potent small molecule is linked to an antibody. ADCs are highly complex and structurally heterogeneous, typically containing numerous product-related species. One of the most impactful steps in ADC development is the identification of critical quality attributes to determine product characteristics that may affect safety and efficacy. However, due to the additional complexity of ADCs relative to the parent antibodies, establishing a solid understanding of the major quality attributes and determining their criticality are a major undertaking in ADC development. Here, we review the development challenges, especially for reliable detection of quality attributes, citing literature and new data from our laboratories, highlight recent improvements in major analytical techniques for ADC characterization and control, and discuss newer techniques, such as two-dimensional liquid chromatography, that have potential to be included in analytical control strategies.

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Section: Major Quality Attributes Of Adcmentioning

confidence: 99%

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

Wagh

Song

Zeng

et al. 2018

mAbs

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“…Since LC-MS is able to measure both small and large molecules, this enables analysis of complex biotherapeutics that require multiple assays including antibody-conjugated drug, total antibody and intact ADCs [60,78,79,80,81]. By employing tandem mass spectrometry, LC-MS/MS can identify specific proteins by directly sequencing surrogate peptides as well as detailed structural information beyond sequence such as post-translational modifications, macromolecular complexes characterization and analysis of DAR distribution and dynamics [60,82,83,84,85].…”

Section: Bioanalytical Platforms For Adcsmentioning

confidence: 99%

“…A better understanding of DAR distribution, fraction of unconjugated mAb in vitro and in vivo is needed especially for PK/PD studies [105]. A few novel methods with hybrid immuno-affinity LC-MS have demonstrated direct quantification of released toxin by quantifying DAR distributions of the ADC in circulation using high resolution accurate mass spectrometry (HRAM-MS) [60,81,82,84,85,106]. While this intact LC-MS analysis approach provides a new aspect of direct measurement and structural characterization of ADCs, there still remain challenges related to assay sensitivity and dynamic range, obtaining internal standards for intact ADCs, relatively poor chromatographic peak shapes and limited separation resolution of intact molecules with different DAR values and additional complexity from potential biotransformations.…”

Section: Hybrid Lba-lc-ms For Adc Analysismentioning

confidence: 99%

ADME Considerations and Bioanalytical Strategies for Pharmacokinetic Assessments of Antibody-Drug Conjugates

Mou¹,

Huang²,

Rosenbaum³

2018

Antibodies

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Antibody-drug conjugates (ADCs) are a unique class of biotherapeutics of inherent heterogeneity and correspondingly complex absorption, distribution, metabolism, and excretion (ADME) properties. Herein, we consider the contribution of various components of ADCs such as various classes of warheads, linkers, and conjugation strategies on ADME of ADCs. Understanding the metabolism and disposition of ADCs and interpreting exposure-efficacy and exposure-safety relationships of ADCs in the context of their various catabolites is critical for design and subsequent development of a clinically successful ADCs. Sophisticated bioanalytical assays are required for the assessments of intact ADC, total antibody, released warhead and relevant metabolites. Both ligand-binding assays (LBA) and hybrid LBA-liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) methods have been employed to assess pharmacokinetics (PK) of ADCs. Future advances in bioanalytical techniques will need to address the rising complexity of this biotherapeutic modality as more innovative conjugation strategies, antibody scaffolds and novel classes of warheads are employed for the next generation of ADCs. This review reflects our considerations on ADME of ADCs and provides a perspective on the current bioanalytical strategies for pharmacokinetic assessments of ADCs.

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“…Caution is needed about this comparison and correlation when the DAR characteristics of the ADC and total-antibody LBAs are unclear. One interesting article from Rago et al [15] is on calculated conjugated-drug from immunoassay and LC-MS intact protein measurements of ADC. Their method consisted of an anti-human Fc immuno-capture of inter-chain disulfide bonds conjugated ADC followed by LC-MS analysis of the light and heavy chains.…”

Section: Dar Distribution-intact Adc Measurementmentioning

confidence: 99%

Current status of antibody-drug conjugate bioanalysis

Wang¹

2017

J Appl Bioanal

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Keywords: antibody-drug conjugate bioanalysis, ligand-binding assay, hybrid ligand-binding/LC-MS assay, drug-toantibody ratio Antibody-drug conjugate (ADC) consists of a cytotoxic drug covalently bound to a monoclonal antibody via a linker. Because of the complexity of ADC structure unique bioanalytical strategies are needed to identify, characterize and quantify the ADC species most relevant to safety and efficacy. ADC bioanalysis need an integrated bioanalytical approach including ligand-binding assay (LBA) and LC-MS based assays to provide comprehensive characterization of the exposure/response relationship. This mini-review will summarize recent publications on ADC bioanalytical strategies and will focus on the publications within last three years on the advancement of hybrid ligand-binding/LC-MS methods for ADC PK and stability evaluation and for intact ADC-DAR distribution measurement to profile ADC biotransformation and catabolism. Special attention is paid to the publications on selection of ADC analytes, assay platforms and DAR characteristics of LBA as well as on the transition of the bioanalytical testing strategy at different phases of clinical development.

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Calculated Conjugated Payload from Immunoassay and LC–MS Intact Protein Analysis Measurements of Antibody-Drug Conjugate

Abstract: PK profiles from LBA, ADC and calculated conjugated payload (DAR × monoclonal antibody) were in good agreement. We present a new tool for PK assessment of ADCs while also exploring ADC metabolism and DAR sensitivity of LBA ADC assay.

Cited by 20 publications

References 33 publications

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

ADME Considerations and Bioanalytical Strategies for Pharmacokinetic Assessments of Antibody-Drug Conjugates

Current status of antibody-drug conjugate bioanalysis

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