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

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.

The downstream bioprocess toolbox for therapeutic viral vectors.

Viral vectors are poised to acquire a prominent position in modern medicine and biotechnology owing to their role as delivery agents for gene therapies, oncolytic agents, vaccine platforms, and a gateway to engineer cell therapies as well as plants and animals for sustainable agriculture. The success of viral vectors will critically depend on the availability of flexible and affordable biomanufacturing strategies that can meet the growing demand by clinics and biotech companies worldwide. In this context, a key role will be played by downstream process technology: while initially adapted from protein purification media, the purification toolbox for viral vectors is currently undergoing a rapid expansion to fit the unique biomolecular characteristics of these products. Innovation efforts are articulated on two fronts, namely (i) the discovery of affinity ligands that target adeno-associated virus, lentivirus, adenovirus, etc.; (ii) the development of adsorbents with innovative morphologies, such as membranes and 3D printed monoliths, that fit the size of viral vectors. Complementing these efforts are the design of novel process layouts that capitalize on novel ligands and adsorbents to ensure high yield and purity of the product while safeguarding its therapeutic efficacy and safety; and a growing panel of analytical methods that monitor the complex array of critical quality attributes of viral vectors and correlate them to the purification strategies. To help explore this complex and evolving environment, this study presents a comprehensive overview of the downstream bioprocess toolbox for viral vectors established in the last decade, and discusses present efforts and future directions contributing to the success of this promising class of biological medicines.

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.

Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids.

Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the CH1-3 and CL chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Qmax ∼ 20 mg of Fab per mL of resin) and dissociation constant (KD = 2.16·10-6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93-96%) and purity (89-96%) comparable to those provided by Protein L resins.

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.

De novo discovery of peptide-based affinity ligands for the fab fragment of human immunoglobulin G.

Antibody fragments and their engineered variants show true potential as next-generation therapeutics as they combine excellent targeting with superior biodistribution and blood clearance. Unlike full antibodies, however, antibody fragments do not yet have a standard platform purification process for large-scale production. Short peptide ligands are viable alternatives to protein ligands in affinity chromatography. In this work, an integrated computational and experimental scheme is described to de novo design 9-mer peptides that bind to Fab fragments. The first cohort of designed sequences was tested experimentally using human polyclonal Fab, and the top performing sequence was selected as a prototype for a subsequent round of ligand refinement in silico. The resulting peptides were conjugated to chromatographic resins and evaluated via equilibrium and dynamic binding studies using human Fab-κ and Fab-λ. The equilibrium studies returned values of binding capacities up to 32 mg of Fab per mL of resin with mild affinity (KD ∼ 10-5 M) that are conducive to high product capture and recovery. Dynamic studies returned values of product yield up to ∼90%. Preliminary purification studies provided purities of 83-93% and yields of 11-89%. These results lay the groundwork for future development of these ligands towards biomanufacturing translation.

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.

A technique for lyopreservation of Clostridium ljungdahlii in a biocomposite matrix for CO absorption.

A system capable of biocatalytic conversion of distributed sources of single carbon gases such as carbon monoxide into hydrocarbons can be highly beneficial for developing commercially viable biotechnology applications in alternative energy. Several anaerobic bacterial strains can be used for such conversion. The anaerobic carbon monoxide-fixing bacteria Clostridium ljungdahlii OTA1 is a model CO assimilating microorganism that currently requires cryogenic temperature for storage of the viable strains. If these organisms can be stabilized and concentrated in thin films in advanced porous materials, it will enable development of high gas fraction, biocomposite absorbers with elevated carbon monoxide (CO) mass transfer rate, that require minimal power input and liquid, and demonstrate elevated substrate consumption rate compared to conventional suspended cell bioreactors. We report development of a technique for dry-stabilization of C. ljungdahlii OTA1 on a paper biocomposite. Bacterial samples coated onto paper were desiccated in the presence of trehalose using convective drying and stored at 4°C. Optimal dryness was ~1g H2O per gram of dry weight (gDW). CO uptake directly following biocomposite rehydration steadily increases over time indicating immediate cellular metabolic recovery. A high-resolution Raman microspectroscopic hyperspectral imaging technique was employed to spatially quantify the residual moisture content. We have demonstrated for the first time that convectively dried and stored C. ljungdahlii strains were stabilized in a desiccated state for over 38 days without a loss in CO absorbing reactivity. The Raman hyperspectral imaging technique described here is a non-invasive characterization tool to support development of dry-stabilization techniques for microorganisms on inexpensive porous support materials. The present study successfully extends and implements the principles of dry-stabilization for preservation of strictly anaerobic bacteria as an alternative to lyophilization or spray drying that could enable centralized biocomposite biocatalyst fabrication and decentralized bioprocessing of CO to liquid fuels or chemicals.

Simple displays of talker location improve voice identification performance in multitalker, spatialized audio environments.

OBJECTIVE: The aim of this study was to assess the voice identification benefits of visual depictions of the relative locations of spatialized talkers in a serial listening task. BACKGROUND: Although spatialized audio is known to improve speech intelligibility and voice identification accuracy within multitalker environments, prior studies have not found any additional benefit for augmenting spatialized audio with visual depictions of relative voice locations. These studies, however, were restricted to small audio environments (four voices), potentially limiting the ability of simple talker location displays to provide additional identification benefit. METHOD: In the first experiment, 18 participants performed a voice identification task for four- and eight-voice environments under three display conditions: (a) nonspatialized voices with an audio-only display, (b) spatialized voices with an audio-only display, and (c) spatialized voices augmented by a visual display of relative talker locations. In the second experiment, 32 participants performed the same voice identification task within a spatialized eight-voice environment but with audio and visual displays of differing angular scale. RESULTS: Visually depicting relative talker locations improved voice identification performance in terms of both accuracy and response time, particularly for more populous auditory spaces. Both auditory and visual display scale affected these benefits, with large-angle displays performing the best for both modalities. CONCLUSION: Results indicate that simple visual representations of spatialized audio environments help listeners identify voices and that these representations are more effective when the angular spacing (auditory and visual) between talker locations is increased. APPLICATION: These results have important implications for the design and implementation of collaborative audio environments for shared, desktop, and portable communication devices.

Effects of single versus multiple warnings on driver performance.

OBJECTIVE: To explore how a single master alarm system affects drivers' responses when compared with multiple, distinct warnings. BACKGROUND: Advanced driver warning systems are intended to improve safety, yet inappropriate integration may increase the complexity of driving, especially in high workload situations. This study investigated the effects of auditory alarm scheme, reliability, and collision event type on driver performance. METHOD: Using a 2 x 2 x 4 mixed factorial design, we investigated the impact of two alarm schemes (master vs. individual) and two levels of alarm reliability (high and low) on distracted drivers' performance across four collision event types (frontal collision warnings, left and right lane departure warnings, and warnings for a fast-approaching following vehicle). RESULTS: Participants' reaction times and accuracy rates were significantly affected by the type of collision event and alarm reliability. The use of individual alarms, rather than a single master alarm, did not significantly affect driving performance in terms of reaction time or response accuracy. CONCLUSION: Even though a master alarm is a relatively uninformative warning, it produced statistically no different reaction times or accuracy results when compared with information-rich auditory icons, some of which were spatially located. In addition, unreliable alarms negatively impacted driver performance, regardless of event type or alarm scheme. APPLICATION: These results have important implications for the development and implementation of multiple driver warning systems.