Molecular Engineering of Cyclic Azobenzene‐Peptide Hybrid Ligands for the Purification of Human Blood Factor VIII via Photo‐Affinity Chromatography

Abstract

The use of benign stimuli to control the binding and release of labile biologics for their isolation from complex feedstocks is a key goal of modern biopharmaceutical technology. This study introduces cyclic azobenzene‐peptide (CAP) ligands for the rapid and discrete photo‐responsive capture and release of blood coagulation factor VIII (FVIII). A predictive method—based on amino acid sequence and molecular architecture of CAPs—is developed to correlate the conformation of cis/trans‐CAP photo‐isomers to FVIII binding and release. Combined in silico ‐ in vitro analysis of FVIII:peptide interactions guide the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G‐cycloAZOB[Lys‐YYKHLYN‐Lys]‐G on translucent chromatographic beads, features high binding capacity (>6 mg of FVIII per mL of resin) and rapid photo‐isomerization kinetics (τ < 30 s) when exposed to 420–450 nm light at the intensity of 0.1 W cm−2. The adsorbent purifies FVIII from a recombinant harvest using a single mobile phase, affording high product yield (>90%), purity (>95%), and blood clotting activity. The CAPs introduced in this report demonstrate a novel route integrating gentle operational conditions in a rapid and efficient bioprocess for the purification of life‐saving biotherapeutics.

Peptide ligands targeting the vesicular stomatitis virus G (VSV‐G) protein for the affinity purification of lentivirus particles

“…The selection of candidate peptide ligands was initially performed by screening a solid‐phase peptide library using a device for ligand development introduced and demonstrated by our team in prior work (Chu et al, 2022; Kilgore et al, 2023; Prodromou et al, 2023; Sripada et al, 2022). Our technology relies on orthogonal fluorescence labeling to ensure the selection of ligands that possess strong and selective binding, but can also release the target when exposed to mild elution conditions.…”

“…Individual beads were imaged, and the values of green (AF488) and red (AF594) fluorescence emission were recorded; the bead was then washed for 5 min with 20 mM citrate buffer with 0.5 M MgCl 2 , and imaged to record the new values of green and red fluorescence emission. All values of fluorescence emission, emission ratio, and emission reduction were determined in real time via image analysis using a custom MATLAB code (MathWorks) (Prodromou et al, 2023). The beads that exhibited (i) high green fluorescence emission and red‐to‐green emission ratio before washing and (ii) >75% reduction of green fluorescence emission after washing were isolated, while all other beads were discarded.…”

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

“…The library was incubated with a model feedstock containing red‐fluorescently labeled AAV2 and green‐labeled HEK 293 HCPs, and screened using a bead sorting device developed by our team for the rapid selection of peptide ligands (Day et al, 2019; Saberi‐Bosari et al, 2019): AAV2 was utilized as a model target for being the most studied and utilized of the currently known human and nonhuman primate AAV serotypes (Schmidt et al, 2008); the formulation of the feedstock—namely AAV2 at 5×10 11 vp/mL and HEK 293 HCPs at 0.5 mg/mL—mimics industrial cell culture lysates and was adopted to identify peptide ligands capable of isolating AAV from complex sources in bind‐and‐elute mode. The device comprises a microfluidic chamber, where each bead is imaged using a multiple‐wavelength fluorescence microscope, and is controlled by software performing real‐time monitoring, image processing, and selection of the beads (Figure 1) (Chu et al, 2022; Kilgore et al, 2023; Prodromou et al, 2023; Sripada et al, 2022). Beads with high binding strength (i.e., ratio of the bead's vs. standard red fluorescence intensity >0.9) and selectivity (i.e., red vs. green intensity ratio >100) are retained in the device, while all other beads are discarded.…”

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

“…The library was incubated with a model feedstock containing red-fluorescently labeled AAV2 and green-labeled HEK 293 host cell proteins (HCPs), and screened using a bead sorting device developed by our team for the rapid selection of peptide ligands 56,57 : AAV2 was utilized as model target for being the most studied and utilized of the currently known human and nonhuman primate AAV serotypes 23 ; the formulation of the feedstock -namely AAV2 at 5 •10 11 vp/mL and HEK 293 HCPs at 0.5 mg/mL -mimics industrial cell culture lysates and was adopted to identify peptide ligands capable of isolating AAV from complex sources in bind-and-elute mode. The device comprises a microfluidic chamber, where each bead is imaged using a multiple wavelength fluorescence microscope, and is controlled by a software performing real-time monitoring, image processing, and selection of the beads (Figure 1A) 50,51,86,87 . Beads with high binding strength (i.e., ratio of the bead's vs. standard red fluorescence intensity > 0.9) and selectivity (i.e., red vs. green intensity ratio > 100) are retained in the device, while all other beads are discarded.…”