Universal Automated Immunoaffinity Purification-CE−MS Platform for Accelerating Next Generation Biologic Design

Abstract

As the pharmaceutical industry places greater emphasis on pairing biological pathways with appropriate therapeutic intervention, an increase in the use of biologic drugs has emerged. With increasing complexity of biotherapeutics, absorption, distribution, metabolism, and excretion (ADME) studies have also become increasingly complex. The characterization of ADME properties is critical to tuning the pharmacokinetic profiles of next generation biologics (NGBs). The knowledge of the fate of a drug is essential for the enhancement of the design processes, elongation of exposure at the desired site of action, and achieving efficacy with minimum toxicity. In vivo proteolytic cleavage of biotherapeutics may lead to undesirable in vivo properties, such as rapid clearance, low bioavailability, and loss of pharmacodynamic effect. All of these may affect drug efficacy and/or generate safety concerns through increases in immunogenicity or off-target toxicity. The work herein describes the development of a robust, fully automated immunoaffinity purification (IA)− capillary electrophoresis−mass spectrometry (CE−MS) workflow. The reagents were carefully optimized to maximize isolation yields while minimizing the number of experimental steps to analytical results. The result is the development of a comprehensive integrated platform for the characterization of a wide range of biotherapeutics, including peptibodies, monoclonal antibodies, and bispecific antibodies. Empowered by this automated IA−CE−MS approach, implementing biotransformation studies at an early drug discovery stage can speed up the drug development process.

Peptide mapping of proteins by capillary electromigration methods

“…Efficient reconcentration of diluted proteins can be achieved by SPE or SPME with RP-based chromatographic sorbents or hydrophobic membranes fixed in different types of microcartridges [39], see Figure 1. High enrichment of diluted proteins is obtained using specific immunoaffinity sorbents with immobilized whole antibodies or their antigen-binding fragments [40,41] or with aptamers [42]. For additional LC-and CE-based concentration techniques, see the recent reviews [39,43,44].…”

Future of Biotransformation Science in the Pharmaceutical Industry

“…Protein precipitation and solid phase extraction are often used for peptides and oligonucleotides while immunoaffinity purification is favored for proteins, antibodies, and ADCs (Fung et al, 2016;Donnelly et al, 2019). Having an expertise in separation science using HPLC or capillary electrophoresis, which can be coupled with mass spectrometry for biotransformation work, is also beneficial in this field (Han et al, 2021). High resolution MS is highly recommended for elucidating complex biotransformation and sites of metabolism of large molecules (Wagner-Rousset et al, 2014;Su et al, 2016;.…”

“Lab of the Future”─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics

“…The diversity and constant evolution in the MS field requires many state-of-the-art devices, most of which lack convenient application programming interfaces (APIs) and often were not designed to integrate into larger automation systems. Individual MS analysis steps have been automated thus far in clinical proteomics, − immunopeptidomics, bioanalysis, , and bioprocess monitoring, including automated and integrated sampling, dilutions, and even automated proteolytic digestion, − but none of these applications has demonstrated true end-to-end hands-free automation. Dynamic requirements for sample quantity, multistep sample preparations and variants, data acquisition, and data processing have traditionally limited the degree of automation; however, advances in automation technology have now made possible the automation of complex, multibranched MS-based processes.…”