Abstract
In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been carefully designed to characterize the intact drug and each functional domain in terms of quantity, functionality, biotransformation, and immunogenicity. The present review focuses on the bioanalytical challenges and considerations for RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene and cell therapies, and fusion proteins. Methods ranging from the conventional ligand binding assays and liquid chromatography-mass spectrometry assays to quantitative polymerase chain reaction or flow cytometry often used for oligonucleotides and cell and gene therapies are discussed. Best practices for method selection and validation are proposed as well as a future perspective to address the bioanalytical needs of complex modalities.
Highlights
With the advances in genomics and a deeper understanding of the biological pathways linked to human diseases, the paradigm of discovery and development of novel pharmaceutical therapies is quickly shifting from intervention of conventional protein targets to those previously considered “undruggable” such as specific genes, deoxyribonucleic acid (DNA)/ribonucleic acid (RNA), and protein-protein interactions [1]
Among the new drug modalities, RNA, cell, and gene therapies are nonprotein drugs in nature; technology platforms other than liquid chromatography-mass spectrometry (LC-MS) and ligand binding assays (LBA), such as quantitative polymerase chain reaction, sequencing, hybrid LBA, or flow cytometry, are necessary to measure the molecular or cellular drug form
In a LC-MS/MS method for ethanercept in human serum, Iwamoto et al [86] used a nano-surface and molecular-orientation limited proteolysis technology which has a unique two-solidsurface assisted Fab-selective proteolysis by trypsin immobilized on the surface of nanoparticles (200-nm diameter) for protein bioanalysis which is oriented by the binding antibody Fc via Protein A/G in a pore (100nm diameter)
Summary
With the advances in genomics and a deeper understanding of the biological pathways linked to human diseases, the paradigm of discovery and development of novel pharmaceutical therapies is quickly shifting from intervention of conventional protein targets to those previously considered “undruggable” such as specific genes, deoxyribonucleic acid (DNA)/ribonucleic acid (RNA), and protein-protein interactions [1]. The assessment of safety and efficacy of the new therapeutic modalities requires a thorough understanding of their pharmacokinetic (PK) and toxicokinetic (TK) characteristics. New bioanalytical strategies and platforms are needed to measure the parent drug and relevant metabolites [4, 5] and to evaluate potential immunogenicity which is often associated with the novel therapies. Whereas regulatory guidelines for the new drug modalities are yet to be developed, bioanalytical strategies have been implemented based on understanding of the drug’s molecular structure, functionality, biotransformation, and immunogenicity. The present review highlights the various bioanalytical challenges and considerations for six types of new modalities: RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene therapies, cell therapies, and fusion proteins.
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