Multiangle Light Scattering as a Lentivirus Purification Process Analytical Technology.

The limited biomolecular and functional stability of lentiviral vectors (LVVs) for cell therapy poses the need for analytical tools that can monitor their titers and activity throughout the various steps of expression and purification. In this study, we describe a rapid (25 min) and reproducible (coefficient of variance ∼0.5-2%) method that leverages size exclusion chromatography coupled with multiangle light scattering detection (SEC-MALS) to determine size, purity, and particle count of LVVs purified from bioreactor harvests. The SEC-MALS data were corroborated by orthogonal methods, namely, dynamic light scattering (DLS) and transmission electron microscopy. The method was also evaluated for robustness in the range of 2.78 × 105-2.67 × 107 particles per sample. Notably, MALS-based particle counts correlated with the titer of infectious LVVs measured via transduction assays (R2 = 0.77). Using a combination of SEC-MALS and DLS, we discerned the effects of purification parameters on LVV quality, such as the separation between heterogeneous LV, which can facilitate critical decision-making in the biomanufacturing of gene and cell therapies.

Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing.

Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.

Identification and characterization of CHO host-cell proteins in monoclonal antibody bioprocessing.

Host-cell proteins (HCPs) are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.

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.

One-Step Quantification of anti-Covid-19 Antibodies via Dual Affinity Ratiometric Quenching Assays.

The global pandemic caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people and paralyzed healthcare systems worldwide. Developing rapid and accurate tests to detect and quantify anti-SARS-CoV-2 antibodies in complex fluids is critical to (i) track and address the spread of SARS-CoV-2 variants with different virulence and (ii) support the industrial manufacturing and clinical administration of anti-SARS-CoV-2 therapeutic antibodies. Conventional immunoassays, such as lateral flow, ELISA, and surface plasmon resonance (SPR), are either qualitative or, when quantitative, are laborious and expensive and suffer from high variability. Responding to these challenges, this study evaluates the performance of the Dual-Affinity Ratiometric Quenching (DARQ) assay for the quantification of anti-SARS-CoV-2 antibodies in bioprocess harvests and intermediate fractions (i.e., a Chinese hamster ovary (CHO) cell culture supernatant and a purified eluate) and human fluids (i.e., saliva and plasma). Monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid as well as the spike protein of the delta and omicron variants are adopted as model analytes. Additionally, conjugate pads loaded with dried protein were studied as an at-line quantification method that can be used in clinical or manufacturing laboratories. Our results indicate that the DARQ assay is a highly reproducible (coefficient of variation ∼0.5-3%) and rapid (<10 min) test, whose sensitivity (∼0.23-2.5 ng/mL), limit of detection (23-250 ng/mL), and dynamic range (70-1300 ng/mL) are independent of sample complexity, thus representing a valuable tool for monitoring anti-SARS-CoV-2 antibodies.

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.

Characterization and implications of host-cell protein aggregates in biopharmaceutical processing.

In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.

Towards continuous mAb purification: Clearance of host cell proteins from CHO cell culture harvests via "flow-through affinity chromatography" using peptide-based adsorbents.

The growth of advanced analytics in manufacturing monoclonal antibodies (mAbs) has highlighted the challenges associated with the clearance of host cell proteins (HCPs). Of special concern is the removal of "persistent" HCPs, including immunogenic and mAb-degrading proteins, that co-elute from the Protein A resin and can escape the polishing steps. Responding to this challenge, we introduced an ensemble of peptide ligands that target the HCPs in Chinese hamster ovary (CHO) cell culture fluids and enable mAb purification via flow-through affinity chromatography. This study describes their integration into LigaGuard™, an affinity adsorbent featuring an equilibrium binding capacity of ~30 mg of HCPs per mL of resin as well as dynamic capacities up to 16 and 22 mg/ml at 1- and 2-min residence times, respectively. When evaluated against cell culture harvests with different mAb and HCP titers and properties, LigaGuard™ afforded high HCP clearance, with logarithmic removal values (LRVs) up to 1.5, and mAb yield above 90%. Proteomic analysis of the effluents confirmed the removal of high-risk HCPs, including cathepsins, histones, glutathione-S transferase, and lipoprotein lipases. Finally, combining LigaGuard™ for HCP removal with affinity adsorbents for product capture afforded a global mAb yield of 85%, and HCP and DNA LRVs > 4.

Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands.

We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.75 µM for YAP and 0.68 µM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.

Dual-Affinity Ratiometric Quenching (DARQ) Assay for the Quantification of Therapeutic Antibodies in CHO-S Cell Culture Fluids.

More than 100 monoclonal antibodies (mAbs) are in industrial and clinical development to treat myriad diseases. Accurate quantification of mAbs in complex media, derived from industrial and patient samples, is vital to determine production efficiency or pharmacokinetic properties. To date, mAb quantification requires time and labor-intensive assays. Herein, we report a novel dual-affinity ratiometric quenching (DARQ) assay, which combines selective biorecognition and quenching of fluorescence signals for rapid and sensitive quantification of therapeutic monoclonal antibodies (mAbs). The reported assay relies on the affinity complexation of the target mAb by the corresponding antigens and Protein L (PrL, which targets the Fab region of the antibody), respectively, labeled with fluorescein and rhodamine. Within the affinity complex, the mAb acts as a scaffold framing the labeled affinity tags (PrL and antigen) in a molecular proximity that results in ratiometric quenching of their fluorescence emission. Notably, the decrease in fluorescence emission intensity is linearly dependent upon mAb concentration in solution. Control experiments conducted with one affinity tag only, two tags labeled with equal fluorophores, or two tags labeled with fluorophores of discrete absorbance and emission bands exhibited significantly reduced effect. The assay was evaluated in noncompetitive (pure mAb) and competitive conditions (mAb in a Chinese Hamster Ovary (CHO) cell culture harvest). The "DARQ" assay is highly reproducible (coefficient of variation ∼0.8-0.7%) and rapid (5 min), and its sensitivity (∼0.2-0.5 ng·mL-1), limit of detection (75-119 ng·mL-1), and dynamic range (300-1600 ng·mL-1) are independent of the presence of CHO host cell proteins.

Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.

Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis.

The clearance of host cell proteins (HCPs) is of crucial importance in biomanufacturing, given their diversity in composition, structure, abundance, and occasional structural homology with the product. The current approach to HCP clearance in the manufacturing of monoclonal antibodies (mAbs) relies on product capture with Protein A followed by removal of residual HCPs in flow-through mode using ion exchange or mixed-mode chromatography. Recent studies have highlighted the presence of "problematic HCP" species, which are either difficult to remove (Group I), can degrade the mAb product (Group II), or trigger immunogenic reactions (Group III). To improve the clearance of these species, we developed a family of synthetic peptides that target HCPs and exhibit low binding to IgG product. In this study, these peptides were conjugated onto chromatographic resins and evaluated in terms of HCP clearance and mAb yield, using an industrial mAb-producing CHO harvest as model supernatant. To gather detailed knowledge on the binding of individual HCPs, the unbound fractions were subjected to shotgun proteomic analysis by mass spectrometry. It was found that these peptide ligands exhibit superior HCP binding capability compared to those of the benchmark commercial resins commonly used in mAb purification. In addition, some peptide-based resins resulted in much lower losses of product yield compared to these commercial supports. The proteomic analysis showed effective capture of many "problematic HCPs" by the peptide ligands, especially some that are weakly bound by commercial media. Collectively, these results indicate that these peptides show great promise toward the development of next-generation adsorbents for safer and cost-effective manufacturing of biologics.

A multiscale coarse-grained model to predict the molecular architecture and drug transport properties of modified chitosan hydrogels.

Hydrogels constructed with functionalized polysaccharides are of interest in a multitude of applications, chiefly the design of therapeutic and regenerative formulations. Tailoring the chemical modification of polysaccharide-based hydrogels to achieve specific drug release properties involves the optimization of many tunable parameters, including (i) the type, degree (χ), and pattern of the functional groups, (ii) the water-polymer ratio, and (iii) the drug payload. To guide the design of modified polysaccharide hydrogels for drug release, we have developed a computational toolbox that predicts the structure and physicochemical properties of acylated chitosan chains, and their impact on the transport of drug molecules. Herein, we present a multiscale coarse-grained model to investigate the structure of networks of chitosan chains modified with acetyl, butanoyl, or heptanoyl moieties, as well as the diffusion of drugs doxorubicin (Dox) and gemcitabine (Gem) through the resulting networks. The model predicts the formation of different network structures, in particular the hydrophobically-driven transition from a uniform to a cluster/channel morphology and the formation of fibers of chitin chains. The model also describes the impact of structural and physicochemical properties on drug transport, which was confirmed experimentally by measuring Dox and Gem diffusion through an ensemble of modified chitosan hydrogels.

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.

Tailoring the Chemical Modification of Chitosan Hydrogels to Fine-Tune the Release of a Synergistic Combination of Chemotherapeutics.

Combination chemotherapy with a defined ratio and sequence of drug release is a clinically established and effective route to treat advanced solid tumors. In this context, a growing body of literature demonstrates the potential of hydrogels constructed with chemically modified polysaccharides as depots for controlled release of chemotherapeutics. Identifying the appropriate modification in terms of physicochemical properties of the functional group and its degree of substitution (χ) to achieve the desired release profile for multiple drugs is, however, a complex multivariate problem. To address this issue, we have developed a computational toolbox that models the migration of a drug pair through a hydrated network of polysaccharide chains modified with hydrophobic moieties. In this study, we chose doxorubicin (DOX) and Gemcitabine (GEM) as model drugs, as their synergistic effect against breast cancer has been thoroughly investigated, and chitosan as the model polymer. Our model describes how the modification of chitosan chains with acetyl, butanoyl, and heptanoyl moieties at different values χ governs both the structure of the hydrogel network and drug migration through it. Our experimental data confirm the in silico predictions for both single- and dual-drug release and, most notably, the counterintuitive inversion of release vs χ that occurs when switching from a single- to a dual-drug system. Consensus between predicted and experimental data indicates that acetyl modifications (χ = 32-42%) and butanoyl modifications (χ = 19-24%) provide synergistic GEM/DOX release molar ratios (i.e., 5-10). Collectively, these results demonstrate the potential of this model in guiding the design of chemotherapeutic hydrogels to combat cancer.

DendriPeps: Expanding Dendrimer Functionality by Hybridizing Poly(amidoamine) (PAMAM) Scaffolds with Peptide Segments.

In this work, the first synthesis of poly(amidoamine) (PAMAM) dendrimers whose branches are hybridized with peptide segments (DendriPeps) is reported. The intercalation of amino acids within the branches of PAMAMs provides supplementary internal functionalities to the coronal groups. Four DendriPep prototypes are synthesized with lysine or glutamic acid as "guest" amino acids, displaying, respectively, a primary amine or a carboxyl group, on generation (G)2 and G3 PAMAMs as host scaffolds. The precise control over the number, type, and topological placement of functional groups expands the functional behavior of DendriPeps beyond current PAMAM dendrimers toward new frontiers or colloids, drug delivery vectors, and catalysis.

Multiplexed Competitive Screening of One-Bead-One-Component Combinatorial Libraries Using a ClonePix 2 Colony Sorter.

Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies-beyond mere library blocking and competitive screening-to ensure binding selectivity of selected leads. This work presents a method for multiplexed solid-phase peptide library screening using a ClonePix 2 Colony Picker that integrates (i) orthogonal fluorescent labeling for positive selection against a target protein and negative selection against competitor species with (ii) semi-quantitative tracking of target vs. competitor binding for every library bead. The ClonePix 2 technology enables global at-a-glance evaluation and customization of the parameters for bead selection to ensure high affinity and selectivity of the isolated leads. A case study is presented by screening a peptide library against green-labeled human immunoglobulin G (IgG) and red-labeled host cell proteins (HCPs) using ClonePix 2 to select HCP-binding ligands for flow-through chromatography applications. Using this approach, 79 peptide ligand candidates (6.6% of the total number of ligands screened) were identified as potential HCP-selective ligands, enabling a potential rate of >3,000 library beads screened per hour.

Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality.

This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.