Identification of a broad-spectrum high-affinity peptide ligand for the purification of spike proteins.

Since the outbreak of coronavirus disease 2019, the global demand for vaccines has increased rapidly to prevent infection and protect high-risk populations. However, identifying viral mutations poses an additional challenge for chromatographic purification of vaccines and subunit vaccines. In this study, a new affinity peptide model, X1VX2GLNX3WX4RYSK, was established, and a library of 612 peptides was generated for ligand screening. Based on a multistep strategy of ligand screening, 18 candidate peptides were obtained. The top ranking peptide, LP14 (YVYGLNIWLRYSK), and two other representative peptides, LP02 and LP06, with lower rankings were compared via molecular dynamics simulation. The results revealed that peptide binding to the receptor binding domain (RBD) was driven by hydrophobic interactions and the key residues involved in the binding were identified. Surface plasmon resonance analysis further confirmed that LP14 had the highest affinity for the wild RBD (Kd=0.520 µmol/L), and viral mutation had little influence on the affinity of LP14, demonstrating its great potential as a broad-spectrum ligand for RBD purification. Finally, chromatographic performance of LP14-coupled gel-packed column verified that both wild and omicron RBDs could be purified and were eluted by 0.1 mol/L Gly-HCl buffer (pH 3.0). This research identified a broad-spectrum peptide for RBD purification based on rational design and demonstrated its potential application in the purification of RBDs from complex feedstock.

Affinity chromatography for virus-like particle manufacturing: Challenges, solutions, and perspectives.

The increasing medical application of virus-like particles (VLPs), notably vaccines and viral vectors, has increased the demand for commercial VLP production. However, VLP manufacturing has not yet reached the efficiency level achieved for recombinant protein therapeutics, especially in downstream processing. This review provides a comprehensive analysis of the challenges associated with affinity chromatography for VLP purification with respect to the diversity and complexity of VLPs and the associated upstream and downstream processes. The use of engineered affinity ligands and matrices for affinity chromatography is first discussed. Although several representative affinity ligands are currently available for VLP purification, most of them have difficulty in balancing ligand universality, ligand selectivity and mild operation conditions. Then, phage display technology and computer-assisted design are discussed as efficient methods for the rapid discovery of high-affinity peptide ligands. Finally, the VLP purification by affinity chromatography is analyzed. The process is significantly influenced by virus size and variation, ligand type and chromatographic mode. To address the updated regulatory requirements and epidemic outbreaks, technical innovations in affinity chromatography and process intensification and standardization in VLP purification should be promoted to achieve rapid process development and highly efficient VLP manufacturing, and emphasis is given to the discovery of universal ligands, applications of gigaporous matrices and platform technology. It is expected that the information in this review can provide a better understanding of the affinity chromatography methods available for VLP purification and offer useful guidance for the development of affinity chromatography for VLP manufacturing in the decades to come.

Co-Immobilization of Laccase and Mediator into Fe-Doped ZIF-8 Significantly Enhances the Degradation of Organic Pollutants.

Co-immobilization of laccase and mediator 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) for wastewater treatment could simultaneously achieve the reusability of laccase and avoid secondary pollution caused by the toxic ABTS. Herein, Fe-induced mineralization was proposed to co-immobilize laccase and ABTS into a metal-organic framework (ZIF-8) within 30 min. Immobilized laccase (Lac@ZIF-8-Fe) prepared at a 1:1 mass ratio of Fe2+ to Zn2+ exhibited enhanced catalytic efficiency (2.6 times), thermal stability, acid tolerance, and reusability compared to free laccase. ABTS was then co-immobilized to form Lac+ABTS@ZIF-8-Fe (ABTS = 261.7 mg/g). Lac@ZIF-8-Fe exhibited significantly enhanced bisphenol A (BPA) removal performance over free laccase due to the local substrate enrichment effect and improved enzyme stability. Moreover, the Lac+ABTS@ZIF-8-Fe exhibited higher BPA removal efficiency than the free laccase+ABTS system, implying the presence of a proximity effect in Lac+ABTS@ZIF-8-Fe. In the successive malachite green (MG) removal, the MG degradation efficiency by Lac@ZIF-8-Fe was maintained at 96.6% at the fifth reuse with only an extra addition of 0.09 mM ABTS in each cycle. As for Lac+ABTS@ZIF-8-Fe, 58.5% of MG was degraded at the fifth cycle without an extra addition of ABTS. Taken together, this research has provided a novel strategy for the design of a co-immobilized laccase and ABTS system for the degradation of organic pollutants.

Interface-Based Design of High-Affinity Affibody Ligands for the Purification of RBD from Spike Proteins.

The outbreak of coronavirus disease 2019 (COVID-19) has sparked an urgent demand for advanced diagnosis and vaccination worldwide. The discovery of high-affinity ligands is of great significance for vaccine and diagnostic reagent manufacturing. Targeting the receptor binding domain (RBD) from the spike protein of severe acute respiratory syndrome-coronavirus 2, an interface at the outer surface of helices on the Z domain from protein A was introduced to construct a virtual library for the screening of ZRBD affibody ligands. Molecular docking was performed using HADDOCK software, and three potential ZRBD affibodies, ZRBD-02, ZRBD-04, and ZRBD-07, were obtained. Molecular dynamics (MD) simulation verified that the binding of ZRBD affibodies to RBD was driven by electrostatic interactions. Per-residue free energy decomposition analysis further substantiated that four residues with negative-charge characteristics on helix α1 of the Z domain participated in this process. Binding affinity analysis by microscale thermophoresis showed that ZRBD affibodies had high affinity for RBD binding, and the lowest dissociation constant was 36.3 nmol/L for ZRBD-07 among the three potential ZRBD affibodies. Herein, ZRBD-02 and ZRBD-07 affibodies were selected for chromatographic verifications after being coupled to thiol-activated Sepharose 6 Fast Flow (SepFF) gel. Chromatographic experiments showed that RBD could bind on both ZRBD SepFF gels and was eluted by 0.1 mol/L NaOH. Moreover, the ZRBD-07 SepFF gel had a higher affinity for RBD. This research provided a new idea for the design of affibody ligands and validated the potential of affibody ligands in the application of RBD purification from complex feedstock.

Biomimetic affinity chromatography for antibody purification: Host cell protein binding and impurity removal.

Peptide affinity chromatography has received increasing attention as an alternative to protein A chromatography in antibody purification. However, its lower selectivity than protein A chromatography has impeded its success in practical applications. In particular, efficient removal of contaminants, including host cell proteins (HCPs) and DNA, is a great challenge for peptide affinity chromatography in monoclonal antibody (mAb) manufacturing. In this work, a biomimetic peptide ligand (bPL), FYWHCLDE, was coupled onto Sepharose 6 Fast Flow (SepFF) to synthesize a peptide affinity gel, SepFF-bPL, for the investigation of the binding mechanism of HCP as well as the feasibility of antibody capture. The results showed that the SepFF-bPL column exhibited effective removal of mAb aggregates as well as mAb capture from feedstocks of various origins, whereas poor removal of HCP and DNA was found. Mechanistic studies of HCP binding indicated that electrostatic interactions dominated HCP binding on the SepFF-bPL gel and that ionic conductivity had a significant influence on HCP binding at low salt concentrations. Thus, combined chromatin extraction and anion exchange adsorption were introduced prior to SepFF-bPL chromatography for initial contaminant removal to reduce mAb aggregation induced by HCP and the loading burden of contaminants in SepFF-bPL chromatography. A proof-of-concept study of the purification train demonstrated a high recovery of mAb (68.7%) and low levels of HCP (23 ppm) and DNA (below the limit of detection) in the final product, which were acceptable for the mandatory requirements in clinical applications. This research provided a deep understanding of HCP binding on the peptide affinity column and led to the development of an effective purification train.

Investigation on Flexural Fracture Behaviour of Bolted Spherical Joints with Crack Propagation in Screw Threads.

Bolted spherical joints, due to their prominent merits in installation, have been widely used in modern spatial structures. Despite significant research, there is a lack of understanding of their flexural fracture behaviour, which is important for the catastrophe prevention of the whole structure. Given the recent development to fill this knowledge gap, it is the objective of this paper to experimentally investigate the flexural bending capacity of the overall fracture section featured by a heightened neutral axis and fracture behaviour related to variable crack depth in screw threads. Accordingly, two full-scale bolted spherical joints with different bolt diameters were evaluated under three-point bending. The fracture behaviour of bolted spherical joints is first revealed with respect to typical stress distribution and fracture mode. A new theoretical flexural bending capacity expression for the fracture section with a heightened neutral axis is proposed and validated. A numerical model is then developed to estimate the stress amplification and stress intensity factors related to the crack opening (mode-I) fracture for the screw threads of these joints. The model is validated against the theoretical solutions of the thread-tooth-root model. The maximum stress of the screw thread is shown to take place at the same location as the test bolted sphere, while its magnitude can be greatly reduced with an increased thread root radius and flank angle. Finally, different design variants related to threads that have influences on the SIFs are compared, and the moderate steepness of the flank thread has been found to be efficient in reducing the joint fracture. The research findings could thus be beneficial for further improving the fracture resistance of bolted spherical joints.

Kinetic and molecular insight into immunoglobulin G binding to immobilized recombinant protein A of different orientations.

Mechanistic understanding of immunoglobulin G (IgG) binding to protein A is crucial for the design and development of high-performance protein A chromatography. In this work, the IgG binding domain (Z) of protein A from Staphylococcus aureus was genetically modified by introducing a cysteine residue at the N-terminus (Cys-Z) or a cysteine-lysine dipeptide at the C-terminus (Z-Cys), and the two ligands were used to unravel the IgG binding mechanism by means of binding kinetics and different single molecule measurements. Surface plasma resonance (SPR) measurement of the binding kinetics of mouse myeloma IgG2a (mIgG2a) to the two ligands indicated that oriented ligand immobilization significantly increased the association rate constant of mIgG2a, and Z-Cys had the highest binding affinity to mIgG2a among the three ligands (Cys-Z, Z-Cys and Z). This was attributed to the synergistic contribution of the high association rate constant and low dissociation rate constant to mIgG2a. Furthermore, quartz crystal microbalance with energy dissipation monitoring (QCM-D) measurement provided the maximum adsorption densities of IgGs on the Z-Cys-immobilized chip as zeta potentials of IgGs were nearly zero. The QCM-D investigation revealed that the adsorbed layer was dependent on ligand type and density, and IgG. Moreover, Z-Cys and Cys-Z induced IgG binding in flipped orientations, as evidenced by the antigen-antibody reaction. Finally, rectangular DNA origami tiles were introduced to analyze the molecular orientation of adsorbed IgG. Single-molecule imaging showed that mIgG2a was associated with flexible Z-Cys on the tiles predominantly in side-on and end-on orientations. The research has provided molecular insight into the binding mechanism of IgG molecules at liquid-solid interfaces and would help design new protein A-based ligands and high-capacity adsorbents.

Kinetic investigation of protein adsorption into polyelectrolyte brushes by quartz crystal microbalance with dissipation: The implication of the chromatographic mechanism.

With the growing concerns of polymer-grafted ion-exchange chromatography, the importance of protein adsorption on charged polymer-grafted surfaces cannot be stressed enough. However, a full understanding in adsorption in polymer brushes is still a great challenge due to the lack of in situ characterization technique. In this work, we use quartz crystal microbalance with dissipation to in situ investigate adsorption kinetics of γ-globulin and recombinant human lactoferrin on poly(3-sulfopropyl methacrylate) (pSPM) sensors prepared via atom transfer radical polymerization. With an increase of chain length and grafting density, great increasing amounts of proteins on pSPM-grafted sensors revealed that protein underwent a transition from monolayer to multilayer adsorption. It was attributed to direct protein binding into charged brushes, in which more binding sites involved and more coupled water lost. However, such a strong binding and rigid structure of proteins limited the protein transport in pSPM brushes and "chain delivery" effect. With an increase in grafting density, moreover, denser brushes hindered adjustment in protein conformation in pSPM brushes and further exacerbated protein transport in pSPM brushes. Furthermore, the influence of buffer pH and salt concentration further validated the ion exchange characteristics of protein adsorption into pSPM brushes. The research provided a variety of in situ evidence of protein binding and conformation evolution in pSPM brushes and elucidated mechanism of protein adsorption in pSPM brushes.

Increasing immunoglobulin G adsorption in dextran-grafted protein A gels.

The formation of a stable spatial arrangement of protein A ligands is a great challenge for the development of high-capacity polymer-grafted protein A adsorbents due to the complexity in interplay between coupled ligands and polymer chain. In this work, carboxymethyl dextrans (CMDs) with different molecular weight were introduced to provide stable spatial ligand arrangement in CMD-grafted protein A gels to improve IgG adsorption. The result showed that coupling of protein A ligand in CMD-grafted layer had no marked influence on pore size and dextran layers coupled with the ligands were stable in experimental range of salt concentrations. The result of IgG adsorption revealed that carboxymethyl dextran T10, a short CMD, was more suitable as a scaffold for the synthesis of high-capacity protein A gels. Moreover, the maximal adsorption capacity for IgG was obtained to be 96.4 mg/g gel at ionic capacities of 300-350 mmol/L and a ligand density of 15.2 mg/g gel. Dynamic binding capacity for IgG exhibited a higher capacity utilization in CMD-grafted protein A gels than non-grafted protein A gel. The research presented a tactics to establish a stable dextran layer coupled with protein A ligands and demonstrated its importance to improve binding capacity for IgG.

Signature RNAS and related regulatory roles in type 1 diabetes mellitus based on competing endogenous RNA regulatory network analysis.

BACKGROUND: Our study aimed to investigate signature RNAs and their potential roles in type 1 diabetes mellitus (T1DM) using a competing endogenous RNA regulatory network analysis. METHODS: Expression profiles of GSE55100, deposited from peripheral blood mononuclear cells of 12 T1DM patients and 10 normal controls, were downloaded from the Gene Expression Omnibus to uncover differentially expressed long non-coding RNAs (lncRNAs), mRNAs, and microRNAs (miRNAs). The ceRNA regulatory network was constructed, then functional and pathway enrichment analysis was conducted. AT1DM-related ceRNA regulatory network was established based on the Human microRNA Disease Database to carry out pathway enrichment analysis. Meanwhile, the T1DM-related pathways were retrieved from the Comparative Toxicogenomics Database (CTD). RESULTS: In total, 847 mRNAs, 41 lncRNAs, and 38 miRNAs were significantly differentially expressed. The ceRNA regulatory network consisted of 12 lncRNAs, 10 miRNAs, and 24 mRNAs. Two miRNAs (hsa-miR-181a and hsa-miR-1275) were screened as T1DM-related miRNAs to build the T1DM-related ceRNA regulatory network, in which genes were considerably enriched in seven pathways. Moreover, three overlapping pathways, including the phosphatidylinositol signaling system (involving PIP4K2A, INPP4A, PIP4K2C, and CALM1); dopaminergic synapse (involving CALM1 and PPP2R5C); and the insulin signaling pathway (involving CBLB and CALM1) were revealed by comparing with T1DM-related pathways in the CTD, which involved four lncRNAs (LINC01278, TRG-AS1, MIAT, and GAS5-AS1). CONCLUSION: The identified signature RNAs may serve as important regulators in the pathogenesis of T1DM.

PET/CT in a patient with cardiac paraganglioma.

A 22-year-old female with SDHB-positive who presented with palpitation and hypertension after adrenalectomy was performed 18F-FDG PET/CT to detect the primary ectopic pheochromocytoma (PCC) and rule out metastasis. PET/CT is useful for detecting and localizing the primary ectopic PCC.

The incidence risk of programmed cell death-1/programmed cell death ligand 1 inhibitor-related alopecia for cancer patients: A systematic review and meta-analysis.

PURPOSE: To evaluate the incidence risk of programmed cell death-1/programmed cell death ligand 1 (PD-1/PD-L1) inhibitor-related alopecia for cancer patients, the meta-analysis was put into practice. METHOD: The meta-analysis was designed and put into practice according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. RESULTS: After rigorous screening and verification, 22 clinical trials involving PD-1/PD-L1 inhibitors were collected for the final comprehensive analysis. The incidence risk of alopecia for all-grade in the PD-1/PD-L1 group was significantly lower than that in the control chemotherapy group (odds ratio [OR] = 0.01, 95% confidence interval [CI]: [0.01, 0.04], I = 86%, Z = 8.73 [P < .00001]). Similar to the above, the incidence risk of alopecia for grade 3-5 related to PD-1/PD-L1 was obvious lower than the control group (OR = 0.17, 95% CI:[0.05, 0.55], I = 0%, Z = 2.97 [P = .003]). When 7 clinical trials (PD-1/PD-L1 + Chemotherapy vs Chemotherapy) were taken to evaluate the risk of alopecia for all-grade and grade 3-5, no statistically significant results were found. CONCLUSION: The incidence risk of alopecia caused by PD-1/PD-L1 is significantly lower than chemotherapy, and there is no statistical significant evidence that PD-1/PD-L1 combined with chemotherapy would increase the incidence risk of alopecia.

CircAGFG1modulates autophagy and apoptosis of macrophages infected by Mycobacterium tuberculosis via the Notch signaling pathway.

BACKGROUND: Recent studies have revealed the involvement of circular RNAs (circRNAs) in the control and elimination of invading Mycobacterium tuberculosis (Mtb) by macrophages. However, the regulatory mechanism of circAGFG1 in macrophages infected by Mtb has not been fully explored. In this study, we sought to investigate the role of circAGFG1 on autophagy and apoptosis of Mtb-infected macrophages and reveal its the molecular mechanism. METHODS: The expression of circAGFG1 in macrophages from patients with active tuberculosis and in Mtb-treated macrophages in vitro was explored. Then, the effect of circAGFG1 on autophagy and apoptosis of Mtb-infected macrophages was evaluated by flow cytometry, electron microscope, immunofluorescence, and Western blotting. Bioinformatics analysis was used to identify and validate the downstream regulatory pathway of circAGFG1, miRNA-1257/Notch. For further analysis, the role of miRNA-1257 on autophagy and apoptosis was assessed. RESULTS: In vitro, ectopic expression of circAGFG1 upregulated autophagy and reduced apoptosis significantly in Mtb-infected cells. Notch levels were discovered to be increased by the silencing effect of circAGFG1 on miRNA-1257 expression. miRNA-1257 was found to noticeably reduce autophagy and promote macrophage apoptosis. Increased circAGFG1 expression, decreased monocyte apoptosis, and enhanced autophagy were found in macrophages from patients with active tuberculosis. CONCLUSIONS: In active tuberculosis, circAGFG1 enhances autophagy and reduces apoptosis via the miRNA-1257/Notch axis; this provides new therapeutic targets for tuberculosis patients.

Characterization of the metabolites of gigantol in rat, dog, monkey, and human hepatocytes using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

RATIONALE: Gigantol (3',4-dihydroxy-3,5'-dimethoxybibenzyl) is a bibenzyl compound isolated from Dendrobii Caulis that has been widely used as a medicinal herb in China. To fully understand the mechanism of action of gigantol, it is necessary to determine its metabolic profile. METHODS: Gigantol at a concentration of 20 µM was incubated with hepatocytes (rat, dog, monkey, and human) at 37°C. After 120 min incubation, the samples were analyzed using liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The structures of the metabolites were characterized by their molecular masses, product ions, and retention times. RESULTS: A total of 17 metabolites were detected and structurally identified. The metabolism involved the following pathways: (a) oxidation to form quinone-methide species and subsequently conjugation with glutathione (GSH); (b) demethylation to form demethylated gigantol, which was further conjugated with GSH; (c) hydroxylation to yield hydroxyl-gigantol followed by glucuronidation or GSH conjugation; and (d) glucuronidation to form glucuronide conjugates. Glucuronidation was the primary metabolic pathway in all tested species. CONCLUSIONS: Hydroxylation, demethylation, glucuronidation, and GSH conjugation were the major metabolic pathways of gigantol. This study provides new information on the metabolic profiles of gigantol and helps us understand the disposition of the compound.

Characterization of the metabolites of TUG-891 in rat, dog, and human hepatocytes using ultra-high-performance liquid chromatography tandem mass spectrometry and nuclear magnetic resonance spectroscopy.

RATIONALE: TUG-891 is a potent and selective agonist of the long chain free fatty acid receptor 4. However, its metabolic profiles have not been revealed. The aim of this study was to investigate the in vitro metabolism of TUG-891 in hepatocytes. METHODS: TUG-891 at a concentration of 20 µM was incubated with rat, dog, and human hepatocytes at 37°C for 120 min. The samples were analyzed using ultra-high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry. The structures of the metabolites were proposed according to their MS/MS product ions. Furthermore, M4 and M5 were biosynthesized using human liver microsomes, and their structures were characterized using 13 C-NMR spectroscopy. RESULTS: Under the current conditions, eight metabolites were detected and structurally identified using high-resolution LC/MS and MS/MS spectra. The metabolites M4 and M5 were unambiguously confirmed to be TUG-891 alcohol and TUG-891 acid, respectively, using 13 C-NMR spectroscopy. Our results revealed that hydroxylation of methyl group at C-21 position to form TUG-891 alcohol (M5) followed by oxidation to yield TUG-891 aldehyde (M7) and carboxylic acid (M4) were the major metabolism processes. Phase II metabolism processes included glucuronidation and sulphation. CONCLUSIONS: Hydroxylation at the C-21 position was the primary metabolic site of TUG-891. This study provided an overview of the metabolic profiles of TUG-891 in hepatocytes.

Expression of protein disulfide isomerase A3 precursor in colorectal cancer.

INTRODUCTION: Over 20% of colorectal cancer (CRC) patients seek medical attention for the first time when they are in the advanced stages of CRC. Thus, early and reliable detection of CRC is critical to early diagnosis of CRC. Protein disulfide isomerase A3 precursor (PDIA3) has been implicated in various types of cancers. However, little is known about PDIA3 in CRC. METHODS: In this study, we screened PDIA3 expression in CRC tissues and cell lines. Small interfering RNA (siRNA) was introduced into SW480 cells to knockdown PDIA3 expression. The effect of PDIA3 in cell growth was evaluated. RESULTS: Significant upregulation of PDIA3 expression was found in CRC tissues as compared with adjacent non-cancer tissues, and was found in established CRC cell lines (SW480, HCT116, CACO2, NCM460 and HT-29). In SW480 cells, knockdown of PDIA3 expression with siRNA resulted in subcellular morphological change, reduced cell proliferation and increased apoptosis. CONCLUSION: PDIA3 inhibition could suppress CRC, likely through inducing apoptosis. PDIA3 could be a potential therapeutic target for CRC.

[Structural regulation by calcium ion in preparing cross-linked enzyme aggregates].

We studied the effect of calcium ion on particle size and pore structure of cross-linked enzyme aggregates (CLEAs) of glucose oxidase, with activity and stability of the enzyme as evaluation criteria. With calcium ion to prepare CLEA significantly decreased particle sizes of CLEAs whilst the pore structures of CLEAs gradually disappeared with the increase of calcium concentration. When glucose oxidase was precipitated at 0.1 mmol/L Ca²âº, glucose oxidase in CLEA showed the definitive pore structure. Moreover, glucose oxidase activity in CLEA with Ca²âº was 1.69 times higher than that without Ca²âº. Even at Ca²âº as high as 1.0 mmol/L, glucose oxidase activity in CLEA was 42% higher than that of CLEA without Ca²âº. Furthermore, CLEA prepared with 0.1 mmol/L Ca²âº not only exhibited higher substrate conversion and operational stability, but also increased the maximum reaction speed. Therefore, calcium ion improved the performance of glucose oxidase in CLEAs.

Dual-ligand affinity systems with octapeptide ligands for affinity chromatography of hIgG and monoclonal antibody.

This work reports the development of affinity systems with dual octapeptide ligands for affinity adsorption and purification of human IgG (hIgG) and monoclonal antibody (mAb). The three octapeptide ligands, FYWHCLDE (1), FYCHWALE (2), and FYCHTIDE (3), identified earlier by the biomimetic design strategy were used; any two of the three were mixed and coupled to Sepharose gel, leading to the formation of three dual-ligand affinity systems. Research emphasis was first placed on hIgG adsorption isotherms and the results were compared to the three single-ligand affinity systems. It was found that there was synergistic effect of the two peptide ligands in a dual-ligand system, so the affinity of a dual-ligand resin for hIgG was higher than those of its counterparts, single-ligand resins. Of the three dual-ligand systems, the FYWHCLDE (1)-FYCHTIDE (3) resin showed the highest affinity, so it was selected for investigating the effects of ligand density and molar ratio on hIgG adsorption equilibrium. It was found that the synergistic effect increased with increasing the total ligand density of the two peptides in the dual-ligand affinity system. Moreover, the FYWHCLDE (1)-FYCHTIDE (3) system at a molar ratio of 2:1 displayed the highest affinity for hIgG (0.69 µM at a total ligand density of 31.1 µmol/mL), indicating that the synergistic effect reached the maximum at this ratio. This dual-ligand affinity column was then used for the purification of hIgG and mAb by affinity chromatography, resulting in over 95% pure hIgG and mAb at recovery yield over 90%. Molecular docking of the two peptides to the Fc fragment simultaneously showed that FYWHCLDE (1) stood still but FYCHTIDE (3) shifted aside the CH2CH3 inter-domain. Molecular dynamics simulation of the binding process of the two octapeptides to Fc revealed that both the peptide ligands kept stable interactions with Fc. The synergistic effect of the dual-ligand affinity system was thus elucidated by the molecular simulations.

Improved purification of immunoglobulin G from plasma by mixed-mode chromatography.

Efficient loading of immunoglobulin G in mixed-mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed-mode ligand, 4-(1H-imidazol-1-yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15-64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification.

Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations.