Molecular engineering of cyclic azobenzene-peptide hybrid ligands for the purification of human blood Factor VIII via photo-affinity chromatography.

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) hybrid 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 - was developed to correlate the conformation of cis/trans CAP photo-isomers to FVIII binding and release. The combined in silico and in vitro analysis of FVIII:peptide interactions guided 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, featured high binding capacity (> 6 mg of FVIII per mL of resin) and rapid photo-isomerization kinetics (τ < 30s) when exposed to 420-450 nm light at the intensity of 0.1 W·cm-2. The adsorbent purified 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 for the affinity purification of adeno-associated viruses from HEK 293 cell lysates.

Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands-typically camelid antibodies-that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH = 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (KD ~ 10-5 -10- 6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC10% > 1013 vp/mL of resin) and product yields (~50%-80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50%-80%), 80- to 400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.

Affibody-Binding Ligands.

While antibodies remain established therapeutic and diagnostic tools, other protein scaffolds are emerging as effective and safer alternatives. Affibodies in particular are a new class of small proteins marketed as bio-analytic reagents. They feature tailorable binding affinity, low immunogenicity, high tissue permeation, and high expression titer in bacterial hosts. This work presents the development of affibody-binding peptides to be utilized as ligands for their purification from bacterial lysates. Affibody-binding candidates were identified by screening a peptide library simultaneously against two model affibodies (anti-immunoglobulin G (IgG) and anti-albumin) with the aim of selecting peptides targeting the conserved domain of affibodies. An ensemble of homologous sequences identified from screening was synthesized on Toyopearl® resin and evaluated via binding studies to select sequences that afford high product binding and recovery. The affibody-peptide interaction was also evaluated by in silico docking, which corroborated the targeting of the conserved domain. Ligand IGKQRI was validated through purification of an anti-ErbB2 affibody from an Escherichia coli lysate. The values of binding capacity (~5 mg affibody per mL of resin), affinity (KD ~1 µM), recovery and purity (64-71% and 86-91%), and resin lifetime (100 cycles) demonstrate that IGKQRI can be employed as ligand in affibody purification processes.

Discovery and Evaluation of Peptide Ligands for Selective Adsorption and Release of Cas9 Nuclease on Solid Substrates.

The rapid expansion of CRISPR in biotechnology, medicine, and bioprocessing poses an urgent need for advanced manufacturing of Cas nucleases. The lack of Cas-targeting ligands, however, prevents the development of platform processes for purifying this class of molecules. This work represents the first effort at developing short synthetic Cas9-binding peptides and demonstrates their applicability as affinity ligands for the purification of a Cas nuclease. Candidate Cas9-targeting peptides were initially identified by screening a solid-phase peptide library against a model mixture of Streptococcus pyogenes Cas9 spiked in Escherichia coli cell lysate. An ensemble of homologous sequences was identified, conjugated on Toyopearl resin, and evaluated by Cas9 binding studies to identify sequences providing selective Cas9 capture and efficient release. In silico docking studies were also performed to evaluate the binding energy and site of the various peptides on Cas9. Notably, sequences GYYRYSEY and YYHRHGLQ were shown to target the RecII domain of Cas9, which is not involved in nuclease activity and was targeted as an ideal binding site. The peptide ligands were validated by purifying Cas9 from the E. coli lysate in dynamic conditions and through measurements of binding capacity and strength (Qmax and KD). The resulting values of Qmax = 4-5 mg Cas9 per mL of resin and KD ∼ 0.1-0.3 µM, product recovery (86-89%), and purity (91%-93%) indicate that both peptides, and YYHRHGLQ in particular, can serve as capture ligands in a platform purification process of Cas9.

Development of peptide ligands for the purification of α-1 antitrypsin from cell culture fluids.

α-1 antitrypsin (AAT) deficiency, a major risk factor for chronic obstructive pulmonary disease, is one of the most prevalent and fatal hereditary diseases. The rising demand of AAT poses a defined need for new processes of AAT manufacturing from recombinant sources. Commercial affinity adsorbents for AAT purification present the intrinsic limitations of protein ligands - chiefly, the high cost and the lability towards the proteases in the feedstocks and the cleaning-in-place utilized in biomanufacturing - which limit their application despite their high capacity and selectivity. This work presents the development of small peptide affinity ligands for the purification of AAT from Chinese hamster ovary (CHO) cell culture harvests. An ensemble of ligand candidates identified via library screening were conjugated on Toyopearl resin and evaluated via experimental and in silico AAT-binding studies. Initial ranking based on equilibrium binding capacity indicated WHAKKSKFG- (12.9 mg of AAT per mL of resin), WHAKKSHFG- (16.3 mg/mL), and KWKHSHKWG- (15.8 mg/mL) Toyopearl resins as top performing adsorbents. Notably, the fitting of adsorption data to Langmuir isotherms concurred with molecular docking and dynamics in returning values of dissociation constant (KD) between 1 - 10 µM. These peptide-based adsorbents were thus selected for AAT purification from CHO fluids, affording values of AAT binding capacity up to 13 gram per liter of resin, and product yield and purity up to 77% and 97%. WHAKKSHFG-Toyopearl resin maintained its purification activity upon 20 consecutive uses, demonstrating its potential for AAT manufacturing from recombinant sources.

Peptides and pseudopeptide ligands: a powerful toolbox for the affinity purification of current and next-generation biotherapeutics.

Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as ß-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.

Photoinduced reconfiguration to control the protein-binding affinity of azobenzene-cyclized peptides.

The impact of next-generation biorecognition elements (ligands) will be determined by the ability to remotely control their binding activity for a target biomolecule in complex environments. Compared to conventional mechanisms for regulating binding affinity (pH, ionic strength, or chaotropic agents), light provides higher accuracy and rapidity, and is particularly suited for labile targets. In this study, we demonstrate a general method to develop azobenzene-cyclized peptide ligands with light-controlled affinity for target proteins. Light triggers a cis/trans isomerization of the azobenzene, which results in a major structural rearrangement of the cyclic peptide from a non-binding to a binding configuration. Critical to this goal are the ability to achieve efficient photo-isomerization under low light dosage and the temporal stability of both cis and trans isomers. We demonstrated our method by designing photo-switchable peptides targeting vascular cell adhesion marker 1 (VCAM1), a cell marker implicated in stem cell function. Starting from a known VCAM1-binding linear peptide, an ensemble of azobenzene-cyclized variants with selective light-controlled binding were identified by combining in silico design with experimental characterization via spectroscopy and surface plasmon resonance. Variant cycloAZOB[G-VHAKQHRN-K] featured rapid, light-controlled binding of VCAM1 (KD,trans/KD,cis ∼ 130). Biotin-cycloAZOB[G-VHAKQHRN-K] was utilized to label brain microvascular endothelial cells (BMECs), showing co-localization with anti-VCAM1 antibodies in cis configuration and negligible binding in trans configuration.

Affordable Microfluidic Bead-Sorting Platform for Automated Selection of Porous Particles Functionalized with Bioactive Compounds.

The ability to rapidly and accurately evaluate bioactive compounds immobilized on porous particles is crucial in the discovery of drugs, diagnostic reagents, ligands, and catalysts. Existing options for solid phase screening of bioactive compounds, while highly effective and well established, can be cost-prohibitive for proof-of-concept and early stage work, limiting its applicability and flexibility in new research areas. Here, we present a low-cost microfluidics-based platform enabling automated screening of small porous beads from solid-phase peptide libraries with high sensitivity and specificity, to identify leads with high binding affinity for a biological target. The integration of unbiased computer assisted image processing and analysis tools, provided the platform with the flexibility of sorting through beads with distinct fluorescence patterns. The customized design of the microfluidic device helped with handling beads with different diameters (~100-300 µm). As a microfluidic device, this portable novel platform can be integrated with a variety of analytical instruments to perform screening. In this study, the system utilizes fluorescence microscopy and unsupervised image analysis, and can operate at a sorting speed of up to 125 beads/hr (~3.5 times faster than a trained operator) providing >90% yield and >90% bead sorting accuracy. Notably, the device has proven successful in screening a model solid-phase peptide library by showing the ability to select beads carrying peptides binding a target protein (human IgG).