Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates (original) (raw)
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Journal of Chromatography B, 2018
Brentuximab vedotin (Adcetris) is a cysteine-linked antibody-drug conjugate (ADC) used in the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). In this study, the drug payload and glycan modifications of this ADC were simultaneously characterized using a unique LC-MS middle-up analysis, involving hydrophilic interaction chromatography (HILIC). This work demonstrates that HILIC is an effective and complementary analytical technique to reversed phase liquid chromatography (RPLC) for subunit-level characterization of immuno-conjugates.
BioDrugs, 2014
Currently, the most bioanalytically challenging drugs are antibody-drug conjugates (ADCs), constructs comprising a monoclonal antibody and a cytotoxic drug connected by a linker. The bioanalytical challenges arise from the heterogeneous nature of ADCs and their complex in vivo behavior, resulting in a high number of analytes to be measured. Measuring the concentration of biologics in blood/plasma/serum is a necessity to properly assess their pharmacokinetic (PK)/pharmacodynamic behaviors in vivo. An additional bioanalytical challenge is to monitor the stability of the ADCs, as cytotoxic drugs released from the ADC in blood circulation may pose a potential safety risk because of their high cytotoxic potency. The nature of ADCs does not only complicate bioanalysis, but also immunogenicity assessment. Questions, such as 'Which part of the ADCs is the anti-drug antibodies directed against?' may arise, and their answer normally includes several immunogenicity risk assessment strategies. This review will focus on the bioanalytical challenges of ADCs, current approaches involving ligand-binding assays (LBAs), liquid chromatography and mass spectrometry platforms, and recommendations on which approach to use for which stage of drug development, and will close with immunogenicity assessment. In order to appropriately tackle the bioanalytical and immunogenic challenges of ADCs and consider every angle, the authors of this review have expertise in ligand binding and liquid chromatography-mass spectrometry.
Journal of Separation Science
Antibody drug conjugates are cytotoxic pharmaceuticals, designed to destroy malignant cells. A cytotoxic molecule is attached to an antibody that binds specific to a cancer-cell surface. Given the high toxicity of the drugs, strict safety standards have to be kept. For this reason, an antibody drug conjugates model was developed with fluorescein 5-isothiocyanate as the nontoxic payload surrogate. Due to the similar hydrophobicity, this model is used to establish a suitable purification process and characterization method for antibody drug conjugates. Because of the pH dependent solubility of fluorescein, the hydrophobicity of conjugates can be modulated by the pH value. Based on the complex heterogeneity and hydrophobicity of the conjugates a chromatographic purification is challenging. Hydrophobic interaction chromatography is used for analytical as well as for preparative separation. Because of the increased hydrophobicity of the conjugates compared to native antibody, hydrophobic interaction chromatography often suffer from resolution and recovery problems. Conjugates were separated differing on the number of payloads attached to the antibody.
Journal of pharmaceutical and biomedical analysis, 2016
Hydrophobic interaction chromatography (HIC) is a historical strategy used for the analytical purification and characterization of proteins. Similarly to what can be done in reversed-phase liquid chromatography (RPLC), HIC is able to separate protein species based on their hydrophobicity, but using different conditions. Compared to RPLC, the main benefit of HIC is its ability to perform separations under non denaturing conditions (i.e. physiological pH conditions, ambient mobile phase temperature and no need for organic solvents) and so an orthogonal method. The goal of this review is to provide a general overview of theoretical and practical aspects of modern HIC applied for the characterization of therapeutic protein biopharmaceuticals including monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) and bispecific antibodies (bsAbs). Therefore, method development approaches, state-of-the-art column technology, applications and future perspectives are described and criticall...
Journal of Clinical Medicine
Membrane chromatography is routinely used to remove host cell proteins, viral particles, and aggregates during antibody downstream processing. The application of membrane chromatography to the field of antibody-drug conjugates (ADCs) has been applied in a limited capacity and in only specialized scenarios. Here, we utilized the characteristics of the membrane adsorbers, Sartobind® S and Phenyl, for aggregate and payload clearance while polishing the ADC in a single chromatographic run. The Sartobind® S membrane was used in the removal of excess payload, while the Sartobind® Phenyl was used to polish the ADC by clearance of unwanted drug-to-antibody ratio (DAR) species and aggregates. The Sartobind® S membrane reproducibly achieved log-fold clearance of free payload with a 10 membrane-volume wash. Application of the Sartobind® Phenyl decreased aggregates and higher DAR species while increasing DAR homogeneity. The Sartobind® S and Phenyl membranes were placed in tandem to simplify th...
2020
An antibody-drug conjugate (ADC) is a unique therapeutic modality composed of a highly potent drug molecule conjugated to a monoclonal antibody. As the number of ADCs in various stages of nonclinical and clinical development has been increasing, pharmaceutical companies have been exploring diverse approaches to understanding the disposition of ADCs. To identify the key absorption, distribution, metabolism, and excretion (ADME) issues worth examining when developing an ADC and to find optimal scientifically based approaches to evaluate ADC ADME, the International Consortium for Innovation and Quality in Pharmaceutical Development launched an ADC ADME working group in early 2014. This white paper contains observations from the working group and provides an initial framework on issues and approaches to consider when evaluating the ADME of ADCs.
2017
Antibody-Drug Conjugates (ADCs) are important biotherapeutic candidates that combine highly potent cytoxic drugs with monoclonal antibodies (mAb) for targeted drug delivery in the treatment of cancer or neurodegenerative disorders. However, while the underlying mAb may be a relatively stable molecule, the addition of the drug and linker often destabilizes the protein or adds undesirable intermolecular interactions, so that ADC biotherapeutics are heavily prone to aggregation. Uncontrolled aggregation can lead to a loss in clinical efficacy in vivo or, in extreme cases, invoke a serious immunogenic response. Monitoring stability during formulation is therefore essential to ensure that ADC compounds meet commercial, performance and safety targets.