Bioanalytical Methods and Strategic Perspectives Addressing the Rising Complexity of Novel Bioconjugates and Delivery Routes for Biotherapeutics

Abstract

In recent years, an increase in the discovery and development of biotherapeutics employing new modalities, such as bioconjugates or novel routes of delivery, has created bioanalytical challenges. The inherent complexity of conjugated molecular structures means that quantification of the bioconjugate and its multiple components is critical for preclinical/clinical studies to inform drug discovery and development. Moreover, bioconjugates involve additional multifactorial complexity because of the potential for in vivo catabolism and biotransformation, which may require thorough investigations in multiple biological matrices. Furthermore, excipients that enhance absorption are frequently evaluated and employed for the development of oral and inhaled biotherapeutics. Risk-benefit assessments are required for novel or existing excipients that utilize dosages above previously approved levels. Bioanalytical methods that can measure both excipients and potential drug metabolites in biological matrices are highly relevant to these emerging bioanalysis challenges. We discuss the bioanalytical strategies for analyzing bioconjugates such as antibody-drug conjugates and antibody-oligonucleotide conjugates and review recent advances in bioanalytical methods for the quantification and characterization of novel bioconjugates. We also discuss bioanalytical considerations for both biotherapeutics and excipients through novel administration routes and review analyses in various biological matrices, from the extensively studied serum or plasma to tissue biopsy in the context of preclinical and clinical studies from both technical and regulatory perspectives.Ruipeng Mu and Jiaqi Yuan have contributed equally to this work.

Matrix effects demystified: Strategies for resolving challenges in analytical separations of complex samples

“…Methods to evaluate this phenomenon in complex matrices are dependent on the instrument as well as the sample preparation technique used. Correction of the ME can be achieved by combining several techniques such as post-extraction spike, post-column infusion method, and a quantitative structure-property relationship model to determine the retention time-dependent and structure-specific ME as shown by Tisler et al [3,11]. ME can also be quantitatively determined by the Matuszewski method, where the ME, recovery of the extraction procedure (RE), and -assuming exhaustive extraction-the overall processes efficiency (PE) can be calculated as:…”

“…Correction of the ME can be achieved by combining several techniques such as post‐extraction spike, post‐column infusion method, and a quantitative structure‐property relationship model to determine the retention time‐dependent and structure‐specific ME as shown by Tisler et al. [3, 11]. ME can also be quantitatively determined by the Matuszewski method, where the ME, recovery of the extraction procedure (RE), and ‐ assuming exhaustive extraction‐ the overall processes efficiency (PE) can be calculated as: $$$$\begin{equation}{\mathrm{ME}}\left( {\mathrm{\% }} \right) = \frac{{After\ Extraction\ Peak\ Area}}{{Peak\ Area\ in\ Neat\ Solution\ Standard}} \bullet 100{\mathrm{\% }}\end{equation}$$$$ $$$$\begin{equation}{\mathrm{RE}}\left( {\mathrm{\% }} \right) = \frac{{Before\ extraction\ Peak\ Area}}{{\ After\ Extraction\ Peak\ Area}}\ \bullet 100{\mathrm{\% }}\end{equation}$$$$ $$$$\begin{equation}{\mathrm{PE}}\left( {\mathrm{\% }} \right) = \frac{{ME}}{{RE}}\ \bullet \ 100{\mathrm{\% }}\end{equation}$$$$ Thus the ratio of the peak area of an analyte standard before and after extraction is used to assess associated ion suppression or enhancement [12].…”

“…The demand for reliable trace analysis in complex matrices has grown significantly throughout multiple sectors encompassing pharmacology, biology, and environmental sciences. Matrices like blood plasma, biological tissues, urine, and intricate environmental samples have proven to pose many challenges in modern analytical method development [1][2][3][4][5]. A considerable portion of these challenges arise from the presence of matrix effects (MEs), which refer to any alterations in analytical performance caused by components within the matrix.…”

Current Analytical Strategies for Antibody–Drug Conjugates in Biomatrices

“…Furthermore, owing to their high specificity of molecular-resolution, LC-MS-based methods are able to determine DARs in biological samples directly, facilitating the characterization of ADC forms with different DAR values. However, it should be noted that no regulatory guidance is currently available for LBA-LC–MS based assays for ADCs [ 42 ].…”

Implementation of Systematic Bioanalysis of Antibody–Drug Conjugates for Preclinical Pharmacokinetic Study of Ado-Trastuzumab Emtansine (T-DM1) in Rats

“…Multiple and diverse analytical methods contribute to the challenges of ADC bioanalysis to understand the overall PK and toxicokinetics (TK) of an ADC [ 5 , 24 ]. Three fundamental assays used to assess the exposure and catabolism of most FDA-approved ADCs are total antibody, conjugated antibody, and unconjugated drugs [ 25 ]. As they have the molecular characteristics of both small- and large-molecule therapeutics, typical bioanalytical methods for both therapeutics are needed.…”