The Role of Technology in Transforming Bioanalysis

The emergence of novel therapeutic modalities has altered the bioanalytical landscape, resulting in the development and maturation of a diversified suite of technologies.

FREMONT, CA: With breakthroughs in genomics and a better understanding of the biological processes connected to human diseases, the framework of drug discovery and development is rapidly shifting away from traditional protein targets and toward those previously thought to be "undruggable," like specific genes, deoxyribonucleic acid (DNA)/ribonucleic acid (RNA), and protein-protein interactions. Novel target identification technologies, coupled with emerging new drug modalities other than small molecules, monoclonal antibodies, and antibody-drug conjugates, have widened the repertoire of possibilities for addressing unmet medical requirements.

As with traditional drugs, determining the safety and effectiveness of new therapeutic modalities involves a comprehensive understanding of their pharmacokinetic (PK) and toxicokinetic (TK) properties. Latest bioanalytical techniques and platforms are required to measure the parent drug and applicable metabolites and to assess the possible immunogenicity connected with novel therapies.

RNA, cell, and gene therapies are non-protein drugs in nature, thus, technology platforms other than LC-MS and LBA are developed to evaluate the molecular or cellular drug form, like quantitative polymerase chain reaction (qPCR), sequencing, hybrid LBA, or flow cytometry.

While regulatory guidelines for the latest drug modalities have yet to be developed, bioanalytical approaches based on an understanding of the drug's molecular structure, functionality, biotransformation,and immunogenicity have been implemented. 

The current review focuses on six types of new modalities' bioanalytical difficulties and factors: RNA-based drugs, bispecific antibodies and multi-domain protein therapies, prodrugs, therapeutic, cell therapies, and fusion proteins.

Bioanalytical Challenges and Considerations

The process for PK bioanalysis of TFPs must be developed based on the protein's nature, like size and structure, and accessibility of necessary reagents, interference from soluble proteins, and the essential assay sensitivity. LBA and LC-MS/MS are both frequently used. The primary difficulty for the LBA method is the presence of endogenous analytes, specific binding proteins, and nonspecific matrix elements in the circulation, which can create significant interference.

Strategies to enhance LBA assay sensitivity often involve using buffers with various blockers, like sheep and mouse sera, to decrease interference by human-anti-mouse-antibodies (HAMAs) and increase the incubation time from 2 to 3 hours to overnight. Washing steps following capture reagent incubation must be prevented to avoid signal reduction created by dissociating the weakly formed complex of capture reagents with the analyte.

A nano-surface and molecular-orientation restricted (nSMOL) proteolysis technology for etanercept in human serum can be used, which has an exceptional two-solid-surface supported Fab-selective proteolysis by trypsin immobilized on the surface of nanoparticles for protein bioanalysis, which is geared by the binding antibody Fc through Protein A/G in a pore. This method analyses the essential proteins of interest while preventing extra peptides produced by the drug or matrices carryover.