Engineering Next Generation Cyclized Peptide Ligands for Light‐Controlled Capture and Release of Therapeutic Proteins

Abstract

Photo‐affinity adsorbents (i.e., translucent matrices functionalized with ligands featuring light‐controlled biorecognition) represent a futuristic technology for purifying labile biologics. In this study, a framework for prototyping photo‐affinity adsorbents comprising azobenzene‐cyclized peptides (ACPs) conjugated to translucent porous beads (ChemMatrix) is presented. This approach combines computational and experimental tools for designing ACPs and investigating their light‐controlled isomerization kinetics and protein biorecognition. First, a modular design for tailoring ACP's conformation, facilitating sequencing, and streamlining the in silico modeling of cis/trans isomers and their differential protein binding is introduced. Then, a spectroscopic system for measuring the photo‐isomerization kinetics of ACPs on ChemMatrix beads is reported; using this device, it is demonstrated that the isomerization at different light intensities is correlated to the cyclization geometry, specifically the energy difference of trans versus cis isomers as calculated in silico. Also, a microfluidic device for sorting ACP‐ChemMatrix beads to select and validate photo‐affinity ligands using Vascular Cell Adhesion Molecule 1 (VCAM‐1) as target protein and cycloAZOB[GVHAKQHRN‐K*]‐G‐ChemMatrix as model photo‐affinity adsorbent is presented. The proposed ACPs exhibit rapid and defined light‐controlled isomerization and biorecognition. Controlling the adsorption and release of VCAM‐1 using light demonstrates the potential of photo‐affinity adsorbents for targets whose biochemical liability poses challenges to its purification.

A Novel Electrochemically Switchable Conductive Polymer Interface for Controlled Capture and Release of Chemical and Biological Entities

“…A significant example of such need is in realizing the promise of liquid biopsies for precise and personal diagnostics. [1] Research efforts have been directed towards the capture and release of a wide range of clinically relevant targets, including small drug molecules, [2] proteins, [3] oligonucleotides, [4] extracellular vesicles, [5] and various types of cells, including circulating epithelial cells (CECs) in preneoplastic or benign disease [6] and circulating tumor cells (CTCs) in cancers. [7] Controlled release of small molecules offers opportunities for a number of applications, including in drug delivery [2,8] and for cell stimulation.…”

Towards continuous mAb purification: Clearance of host cell proteins from CHO cell culture harvests via “flow‐through affinity chromatography” using peptide‐based adsorbents

“…The future manufacturing of therapeutic mAb will rely on novel chromatographic adsorbents that enable continuous and more affordable processes, and have an improved ability to remove the high‐risk HCPs that have been identified as persistent in current bioprocesses (Jones et al, 2021). In this context, our group has developed a downstream toolbox of peptide‐based chromatographic adsorbents that purify therapeutic proteins either in bind‐and‐elute mode (Barozzi et al, 2020; Chu et al, 2021; Day et al, 2019; Kish et al, 2017, 2018; Menegatti et al, 2016; Prodromou et al, 2021) or flow‐through mode (Lavoie, Chu, et al, 2021; Lavoie et al, 2020). The latter, named LigaGuard™, operates by capturing CHO HCPs while allowing the mAb product to flow through unbound as the clarified cell culture harvest is continuously fed to the adsorbent.…”

Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates

“…sate. The screening process of AAV-targeting peptides was performed as reported in the previous studies 50,55 . A screening mix was initially prepared by spiking AF594-labeled AAV2 and AAV9 in AF488-labeled HEK 293 cell culture lysate to obtain a final AAV titer of 5•10 11 vp/mL and a HEK 293 HCP titer of ~0.5 mg/mL.…”