Differences in accelerated tooth movement promoted by recombinant human parathyroid hormone after mandibular ramus osteotomy.

INTRODUCTION: This study investigated the effects of different doses of parathyroid hormone (PTH) on orthodontic tooth movement after mandibular ramus osteotomy and the associated dose-response relationship. METHODS: One-hundred twenty rabbits were divided into 2 experimental groups (A and B) and 2 control groups (control group and negative control group). An experimental model of mandibular ramus osteotomy with installation of an orthodontic tooth movement device was established in groups A and B and the control group. After surgery, groups A and B received intermittent subcutaneous injections of PTH, 20 and 40 µg/kg, respectively, and the control group received injections of normal saline solution. The negative control group underwent installation of the orthodontic tooth movement device without mandibular ramus osteotomy and received normal saline solution after surgery. Changes in expression of RANKL and RUNX2 in the periodontal tissues of the first molars were evaluated by means of immunohistochemical analysis and quantitative fluorescence polymerase chain reaction. RESULTS: Movement of the first molars was more rapid in group B than in group A in the 21 days after surgery. Significantly higher RANKL mRNA levels and lower RUNX2 mRNA levels were detected on the compression side of the periodontal tissues in groups A and B than in the control groups. There was a significant difference in RANKL and RUNX2 expression levels between group B and the control groups at all time points. CONCLUSIONS: Mandibular ramus osteotomy combined with high-dose PTH can increase catabolism on the compressed periodontal tissues, thereby accelerating remodeling of periodontal bone and promoting orthodontic tooth movement after surgery.

Poly(glycidyl methacrylate)-grafted hydrophobic charge-induction agarose resins with 5-aminobenzimidazole as a functional ligand.

Hydrophobic charge-induction chromatography is a new technology for antibody purification. To improve antibody adsorption capacity of hydrophobic charge-induction resins, new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins with 5-aminobenzimidazole as a functional ligand were prepared. Adsorption isotherms, kinetics, and dynamic binding behaviors of the poly(glycidyl methacrylate)-grafted resins prepared were investigated using human immunoglobulin G as a model protein, and the effects of ligand density were discussed. At the moderate ligand density of 330 µmol/g, the saturated adsorption capacity and equilibrium constant reached the maximum of 140 mg/g and 25 mL/mg, respectively, which were both much higher than that of non-grafted resin with same ligand. In addition, effective pore diffusivity and dynamic binding capacity of human immunoglobulin G onto the poly(glycidyl methacrylate)-grafted resins also reached the maximum at the moderate ligand density of 330 µmol/g. Dynamic binding capacity at 10% breakthrough was as high as 76.3 mg/g when the linear velocity was 300 cm/h. The results indicated that the suitable polymer grafting combined with the control of ligand density would be a powerful tool to improve protein adsorption of resins, and new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins have a promising potential for antibody purification applications.

Evaluation of a PEG/hydroxypropyl starch aqueous two-phase system for the separation of monoclonal antibodies from cell culture supernatant.

In this study, an aqueous two-phase system (ATPS) with PEG and hydroxypropyl starch (HPS) was used to separate monoclonal antibody (mAb) from Chinese hamster ovary cell culture supernatant. The phase diagram of the PEG/HPS ATPS was determined, and the effects of NaCl addition were investigated. The results showed that NaCl addition could lead to a shift of the binodal curve and that phase separation would occur at higher PEG and HPS concentrations. The effects of NaCl addition, pH, and the load of cell supernatant on the partitioning of mAb in a PEG/HPS ATPS were investigated. It was found that with 6% cell supernatant and 15% NaCl addition at pH 6.0, the yield of mAb in the upper phase was 96.7% with a purity of 96.0%. The back-extraction of mAb with a PEG/phosphate ATPS were also studied, and the results showed that after the two-step extraction with ATPSs the purity of mAb could reach 97.6 ± 0.5% with a yield of 86.8 ± 1.0%, which was comparable to the purification with Protein A chromatography. These results indicate that the two-step extraction with PEG/HPS and PEG/phosphate ATPSs might be a promising alternative for the separation of mAb from cell culture supernatant.

Design of chitosan and its water soluble derivatives-based drug carriers with polyelectrolyte complexes.

Chitosan, the cationic polysaccharide derived from the natural polysaccharide chitin, has been studied as a biomaterial for more than two decades. As a polycationic polymer with favorable properties, it has been widely used to form polyelectrolyte complexes with polyanions for various applications in drug delivery fields. In recent years, a growing number of studies have been focused on the preparation of polyelectrolyte complexes based on chitosan and its water soluble derivatives. They have been considered well-suited as biomaterials for a number of vital drug carriers with targeted/controlled release profiles, e.g., films, capsules, microcapsules. In this work, an overview highlights not only the favorable properties of chitosan and its water soluble derivatives but also the good performance of the polyelectrolyte complexes produced based on chitosan. Their various types of applications as drug carriers are reviewed in detail.

Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)-based anion-exchange cryogel.

Bovine milk whey contains several bioactive proteins such as α-lactalbumin, ß-lactoglobulin, and immunoglobulin G (IgG). Chromatographic separation of these proteins has received much attention in the past few years. In this work, we provide a chromatographic method for the efficient isolation of IgG from bovine milk whey using a poly(2-hydroxyethyl methacrylate)-based anion-exchange cryogel. The monolithic cryogel was prepared by grafting 2-(dimethylamino) ethyl methacrylate onto the poly(2-hydroxyethyl methacrylate)-based cryogel matrix and then employed to separate IgG under various buffer pH and salt elution conditions. The results showed that the buffer pH and the salt concentration in the step elution have remarkable influences on the purity of IgG, while the IgG recovery depended mainly on the loading volume of whey for a given cryogel bed. High purity IgG (more than 95%) was obtained using the phosphate buffer with pH of 5.8 as the running buffer and the salt solution in as the elution liquid. With suitable loading volume of whey, the maximum IgG recovery of about 94% was observed. The present separation method is thus a potential choice for the isolation of high-purity IgG from bovine milk whey.

Poly(hydroxyethyl methacrylate)-based composite cryogel with embedded macroporous cellulose beads for the separation of human serum immunoglobulin and albumin.

A novel super-macroporous monolithic composite cryogel was prepared by embedding macroporous cellulose beads into poly(hydroxyethyl methacrylate) cryogel. The cellulose beads were fabricated by using a microchannel liquid-flow focusing and cryopolymerization method, while the composite cryogel was prepared by cryogenic radical polymerization of the hydroxyethyl methacrylate monomer with poly(ethylene glycol) diacrylate as cross-linker together with the cellulose beads. After graft polymerization with (vinylbenzyl)trimethylammonium chloride, the composite cryogel was applied to separate immunoglobulin-G and albumin from human serum. Immunoglobulin-G with a mean purity of 83.2% and albumin with a purity of 98% were obtained, indicating the composite cryogel as a promising chromatographic medium in bioseparation for the isolation of important bioactive proteins like immunoglobulins and albumins.

Parameter-by-parameter method for steric mass action model of ion exchange chromatography: Simplified estimation for steric shielding factor.

Mechanistic models play a crucial role in the process development and optimization of ion-exchange chromatography (IEC). Recent researches in steric mass action (SMA) model have heightened the need for better estimation of nonlinear parameter, steric shielding factor σ. In this work, a straightforward approach combination of simplified linear approximation (SLA) and inverse method (IM) was proposed to initialize and further determine σ, respectively. An existed, unique, and positive σ can be derived from SLA. Compared with linear approximation (LA) developed in our previous study, σ of the multi-component system can be calculated easily without solving the complex system of linear equations, leading to a time complexity reduction from O(n3) to O(n). The proposed method was verified first in numerical experiments about the separation of three charge variants. The calculated σ was more reasonable than that of LA, and the error of elution profiles with the parameters estimated by SLA+IM was only one-sixth of that by LA in numerical experiments. Moreover, the error accumulation effect could also be reduced. The proposed method was further confirmed in real-world experiments about the separation of monomer-dimer mixtures of monoclonal antibody. The results gave a lower error and better physical understanding compared to LA. In conclusion, SLA+IM developed in the present work provides a novel and straightforward way to determine σ. This simplification would help to save the effort of calibration experiments and accelerate the process development for the multi-component IEC separation.

The Promoting Effect of a Local Pulsatile Parathyroid Hormone Delivery on Healing of the Mandibular Fracture in Rats.

Parathyroid hormone (PTH) can promote bone formation and mineralization in mandibular fractures, and is systemically administered through daily injections. In this study, the local delivery of PTH using carboxymethyl chitosan/polyvinyl alcohol and alginate was investigated. Bovine serum albumin was used as a drug substitute, and the delivery system was verified to release drugs in a pulsed rhythm. After the delivery system was subcutaneously implanted in Sprague-Dawley (SD) rats, no rejection reaction was detected, indicating that it has good biocompatibility and biodegradability in vivo. Then, an SD rat model of mandibular fracture was established, and 24 rats were randomly divided into two groups. The control group was reduced and fixed with screws and a microplate, and the experimental group received pulsatile PTH release system (14 µg PTH) + screws and microplate fixation. The animals were euthanized on postoperative weeks 1-4. Observation of gross specimens, digital radiography, and hematoxylin and eosin showed that the local PTH pulsatile release system promoted osteogenesis and accelerated fracture healing. In summary, PTH can be loaded by biomaterials to locally target the fracture and stimulate bone formation. Moreover, the pulsatile PTH release system provides a potential therapeutic protocol for mandibular fracture. Impact statement Our study prepares a drug release system that could impulsively release parathyroid hormone. The system could enhance bone regeneration in rats with mandibular fracture. These data provide a foundation for future studies aimed to understand and optimize the use of bioactive molecule pulsatile delivery for bone regeneration and tissue engineering applications.

Dietary sugar consumption and health: umbrella review.

OBJECTIVE: To evaluate the quality of evidence, potential biases, and validity of all available studies on dietary sugar consumption and health outcomes. DESIGN: Umbrella review of existing meta-analyses. DATA SOURCES: PubMed, Embase, Web of Science, Cochrane Database of Systematic Reviews, and hand searching of reference lists. INCLUSION CRITERIA: Systematic reviews and meta-analyses of randomised controlled trials, cohort studies, case-control studies, or cross sectional studies that evaluated the effect of dietary sugar consumption on any health outcomes in humans free from acute or chronic diseases. RESULTS: The search identified 73 meta-analyses and 83 health outcomes from 8601 unique articles, including 74 unique outcomes in meta-analyses of observational studies and nine unique outcomes in meta-analyses of randomised controlled trials. Significant harmful associations between dietary sugar consumption and 18 endocrine/metabolic outcomes, 10 cardiovascular outcomes, seven cancer outcomes, and 10 other outcomes (neuropsychiatric, dental, hepatic, osteal, and allergic) were detected. Moderate quality evidence suggested that the highest versus lowest dietary sugar consumption was associated with increased body weight (sugar sweetened beverages) (class IV evidence) and ectopic fatty accumulation (added sugars) (class IV evidence). Low quality evidence indicated that each serving/week increment of sugar sweetened beverage consumption was associated with a 4% higher risk of gout (class III evidence) and each 250 mL/day increment of sugar sweetened beverage consumption was associated with a 17% and 4% higher risk of coronary heart disease (class II evidence) and all cause mortality (class III evidence), respectively. In addition, low quality evidence suggested that every 25 g/day increment of fructose consumption was associated with a 22% higher risk of pancreatic cancer (class III evidence). CONCLUSIONS: High dietary sugar consumption is generally more harmful than beneficial for health, especially in cardiometabolic disease. Reducing the consumption of free sugars or added sugars to below 25 g/day (approximately 6 teaspoons/day) and limiting the consumption of sugar sweetened beverages to less than one serving/week (approximately 200-355 mL/week) are recommended to reduce the adverse effect of sugars on health. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42022300982.

Poor Oral Health and Risk of Incident Dementia: A Prospective Cohort Study of 425,183 Participants.

BACKGROUND: The association between poor oral health and the risk of incident dementia remains unclear. OBJECTIVE: To investigate the associations of poor oral health with incident dementia, cognitive decline, and brain structure in a large population-based cohort study. METHODS: A total of 425,183 participants free of dementia at baseline were included from the UK Biobank study. The associations between oral health problems (mouth ulcers, painful gums, bleeding gums, loose teeth, toothaches, and dentures) and incident dementia were examined using Cox proportional hazards models. Mixed linear models were used to investigate whether oral health problems were associated with prospective cognitive decline. We examined the associations between oral health problems and regional cortical surface area using linear regression models. We further explored the potential mediating effects underlying the relationships between oral health problems and dementia. RESULTS: Painful gums (HR = 1.47, 95% CI [1.317-1.647], p < 0.001), toothaches (HR = 1.38, 95% CI [1.244-1.538], p < 0.001), and dentures (HR = 1.28, 95% CI [1.223-1.349], p < 0.001) were associated with increased risk of incident dementia. Dentures were associated with a faster decline in cognitive functions, including longer reaction time, worse numeric memory, and worse prospective memory. Participants with dentures had smaller surface areas of the inferior temporal cortex, inferior parietal cortex, and middle temporal cortex. Brain structural changes, smoking, alcohol drinking, and diabetes may mediate the associations between oral health problems and incident dementia. CONCLUSION: Poor oral health is associated with a higher risk of incident dementia. Dentures may predict accelerated cognitive decline and are associated with regional cortical surface area changes. Improvement of oral health care could be beneficial for the prevention of dementia.

Proteomic analysis of mitochondria from infantile hemangioma endothelial cells treated with sodium morrhuate and its liposomal formulation.

Hemangioma is the most common benign tumor of infancy. The aim of this study is to evaluate the biological effects of sodium morrhuate (SM) and its liposomal formulation on infantile hemangioma endothelial cells (IHECs). Morphological analysis revealed that exposure to liposomal sodium morrhuate (LSM) preferentially caused apoptotic death in IHECs, manifested as shrunken configuration and formation of apoptotic bodies. In contrast, necrotic death was prominent in IHECs treated with an equal concentration of SM. By means of proteomic analysis and confirmation experiments, we revealed that the apoptosis-inducing effects of LSM were associated with an upregulation of a set of genes involved in mitochondrial death pathway, including apoptosis-inducing factor, cytochrome c1, caspase-8, and lamin B1. In conclusion, our data highlight the proapoptotic activity of LSM in IHECs through the mitochondrial apoptotic pathway and may provide a promising avenue to treat hemangiomas of infancy.

Sulfate-dependant microbially induced corrosion of mild steel in the deep sea: a 10-year microbiome study.

BACKGROUND: Metal corrosion in seawater has been extensively studied in surface and shallow waters. However, infrastructure is increasingly being installed in deep-sea environments, where extremes of temperature, salinity, and high hydrostatic pressure increase the costs and logistical challenges associated with monitoring corrosion. Moreover, there is currently only a rudimentary understanding of the role of microbially induced corrosion, which has rarely been studied in the deep-sea. We report here an integrative study of the biofilms growing on the surface of corroding mooring chain links that had been deployed for 10 years at ~2 km depth and developed a model of microbially induced corrosion based on flux-balance analysis. METHODS: We used optical emission spectrometry to analyze the chemical composition of the mooring chain and energy-dispersive X-ray spectrometry coupled with scanning electron microscopy to identify corrosion products and ultrastructural features. The taxonomic structure of the microbiome was determined using shotgun metagenomics and was confirmed by 16S amplicon analysis and quantitative PCR of the dsrB gene. The functional capacity was further analyzed by generating binned, genomic assemblies and performing flux-balance analysis on the metabolism of the dominant taxa. RESULTS: The surface of the chain links showed intensive and localized corrosion with structural features typical of microbially induced corrosion. The microbiome on the links differed considerably from that of the surrounding sediment, suggesting selection for specific metal-corroding biofilms dominated by sulfur-cycling bacteria. The core metabolism of the microbiome was reconstructed to generate a mechanistic model that combines biotic and abiotic corrosion. Based on this metabolic model, we propose that sulfate reduction and sulfur disproportionation might play key roles in deep-sea corrosion. CONCLUSIONS: The corrosion rate observed was higher than what could be expected from abiotic corrosion mechanisms under these environmental conditions. High corrosion rate and the form of corrosion (deep pitting) suggest that the corrosion of the chain links was driven by both abiotic and biotic processes. We posit that the corrosion is driven by deep-sea sulfur-cycling microorganisms which may gain energy by accelerating the reaction between metallic iron and elemental sulfur. The results of this field study provide important new insights on the ecophysiology of the corrosion process in the deep sea.

Analysis of Three-Dimensional Morphological Differences in the Mandible between Skeletal Class I and Class II with CBCT Fixed-Point Measurement Method.

This study was aimed at determining the three-dimensional differences in the mandible morphology between skeletal class I and II patients, at exploring the pathogenic mechanisms and morphological characteristics of skeletal class II, and at providing clinical references. The subjects were assigned to two groups according to the size of ANB angle: skeletal class I (2° < ANB angle < 5°) and skeletal class II (5° < ANB angle < 8°). After cone-beam computed tomography (CBCT) scanning, 31 landmarks and 25 measurement items were determined by In Vivo Dental 5.1 software (Anatomage, CA) for statistical analysis. The results were as follows: Co-Go, Go-Me, and CdM-CdD in skeletal class II cases were smaller than those in skeletal class I, and GoR-Me-GoL, GoR-Me-CoL, and, Ig-Men were larger than those in skeletal class I cases. In conclusion, there were significant differences in the three-dimensional morphology of the mandible between skeletal class I and class II patients. The vertical growth of the ramus, the horizontal growth of the mandibular body, and the condyle in skeletal class II patients were smaller than those in skeletal class I cases. In skeletal class II, the growth of the anterior part of the mandible in the vertical direction was larger than that in skeletal class I, and the shape of the mandible was more extended.

Enamel matrix derivative (EMD) enhances the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

To investigate the EMD's capacity in BMSCs osteogenic differentiation. In vivo and in vitro, BMSCs were treated with EMD, scanning electron microscopy, and Alizarin Red staining were used to detect the changes in the osteogenic ability of BMSCs, and the proliferation ability of BMSCs was evaluated by CCK8. In addition, by adding xav939, a typical inhibitor of Wnt/ß-catenin signaling pathway, the regulatory function of Wnt/ß-catenin signaling was clarified. The results showed that EMD promote cell proliferation and 25 µg/ml EMD had the most significant effect. Cells inducing osteogenesis for 2 and 3 even 4 weeks, the cell staining is deeper in EMD treated group than that of the control (P < 0.05) by alizarin Red staining, suggesting more mineralization of BMSCs. In vivo implanting the titanium plate wrapped with 25 µg/ml EMD treated-BMSC film into nude mice for 8 weeks, more nodules were formed on the surface of the titanium plate than that the control (P < 0.05). HE showed that there is a little blue-violet immature bone-like tissue block. Besides, the expression of RUNX Family Transcription Factor 2 (Runx2), Osterix, Osteocalcin (OCN), collagen I (COLI), alkaline phosphatase (ALP) and ß-catenin were inhibited in xav939 group (P < 0.05); Inversely, all were activated in EMD group (P < 0.05). In conclusion, EMD promoted the proliferation and osteogenic differentiation of BMSCs. EMD's function on BMSCs might be associated with the Wnt/ß-catenin signaling pathway.

[Study on the correlation between the mandibular masticatory muscle movement and sleep tooth wear].

OBJECTIVE: To investigate the correlation between the clinical diagnostic criteria of sleep bruxism and the frequency of mandibular movements during sleep. METHODS: Video polysomnography was used to record 20 healthy adults with at least one of the following clinical symptoms and signs: 1) report of frequent tooth grinding; 2) tooth wear and dentin exposure with at least three occlusal surfaces; 3) masticatory muscle symptoms in the morning; 4) masseter muscle hypertrophy. The rhythmic masticatory muscle activity (RMMA) and isolated tonic activity were scored to compare the correlations with clinical symptoms and signs. Finally, the incidence of temporomandibular disorders (TMD) was investigated in patients with isolated tonic and RMMA subjects. RESULTS: Among the 20 subjects, RMMA events were observed (5.8±3.1) times·h⁻¹ and isolated tonic episodes were observed (2.1±0.9) times·h⁻¹. The frequency of RMMA events was significantly greater in the patients with acoustic molars than in those without (P<0.05). Similarly, the frequency of RMMA events was significantly greater in the patients with tooth attrition than in those without (P<0.05). However, no difference was observed between the occurrence of RMMA and the symptoms of masticatory muscles or masseter hypertrophy in the morning. The incidence of TMD was significantly higher in the patients with RMMA than in the isolated tonic patients. CONCLUSIONS: The clinical symptoms and signs often used to diagnose sleep bruxism are different clinical and physiological mandibular movements during sleep. RMMA during sleep can reflect the occurrence of tooth attrition and the high risk of TMD.

Model-based process development and evaluation of twin-column continuous capture processes with Protein A affinity resin.

Multi-column continuous chromatography has advantages of high resin capacity utilization and productivity, low buffer consumption and small footprint. Experimental optimization is often time-consuming and inefficient due to the complexity of continuous processes. In this study, a model-based approach was investigated to improve process development of twin-column continuous capture with Protein A affinity resin MabSelect PrismA. Breakthrough curves under various conditions, productivity and capacity utilization (CU) of the continuous processes under varying operating conditions were predicted. Effects of three key operating parameters (feed concentration (c0), interconnected feed residence time (RT) and breakthrough percentage control of the first column during interconnected feeding (s)) on the productivity and CU were evaluated. A recommended working window can be determined directly from contour maps to balance the trade-off between productivity and CU. The model-optimized operating conditions at varying feed concentrations were verified by experiments, which indicated that the model-based approach was feasible and reliable. The results showed that the suitable RT was 1~2 min and suitable s was 0.6~0.75 for the continuous IgG capture with MabSelect PrismA. The maximum productivity varied from 14 to 47 g/L/h with the feed IgG concentrations at the range of 1 to 10 mg/mL. The results indicated that model-based approach could assist process development efficiently and promote target-orientated process design for continuous processes.

Evaluation of adsorption selectivity of immunoglobulins M, A and G and purification of immunoglobulin M with mixed-mode resins.

This study investigated adsorption selectivity of immunoglobulin M (IgM), immunoglobulin A (IgA) and immunoglobulin (IgG) on four mixed-mode resins with the functional ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. IgM purification processes with mixed-mode resins were also proposed. All resins showed typical pH-dependent adsorption, and high adsorption capacity was found at pH 5.0-8.0 with low adsorption capacity under acidic conditions. Meanwhile, high selectivity of IgM/IgA and IgM/IgG was obtained with ABI-4FF and MMI-4FF resins at pH 4.0-5.0, which was used to develop a method for IgM, IgA and IgG separation by controlling loading and elution pH. Capture of monoclonal IgM from cell culture supernatant with ABI-4FF resins was studied and high purity (∼99%) and good recovery (80.8%) were obtained. Moreover, IgM direct separation from human serum with combined two-step chromatography (ABI-4FF and MMI-4FF) was investigated, and IgM purity of 65.2% and a purification factor of 28.3 were obtained after optimization. The antibody activity of IgM was maintained after purification. The results demonstrated that mixed-mode chromatography with specially-designed ligands is a promising way to improve adsorption selectivity and process efficiency of IgM purification from complex feedstock.

Preparation and characterization of macroporous cellulose-tungsten carbide composite beads for expanded bed applications.

The specially designed adsorbent is one of the most important bases to achieve the expanded bed adsorption (EBA) process. The perfect adsorbents tend to be of small size, large pore and high density. In the present work a new kind of macroporous cellulose beads densified by high-density tungsten carbide were prepared through the method of water-in-oil suspension thermal regeneration. The cassava starch was used as porogenic agent during the preparation. Firstly the gelatinized starch solution was added into the cellulose viscose. After the formation of cellulose beads, the composite starch could be eliminated with boiling water wash and enzymatic hydrolyzation. The sphericity of cellulose beads was not influenced by starch addition and some amount of large pores with the diameter of about 1-3 microm could be formed in the beads. Varieties of physical properties, such as wet density, water content, porosity, specific surface area and mean pore diameter of the composite beads, as the function of starch addition were investigated. The analysis of elution peaks with model proteins (packed bed mode) indicated that the mass transfer kinetic inside the macroporous beads prepared was enhanced, which might certainly benefit the chromatographic operation under high flow rate. In addition, the prepared beads showed good expansion and stability in expanded bed, and are thus suitable for expanded bed applications.

Molecular insight into protein binding orientations and interaction modes on hydrophobic charge-induction resin.

Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the functional ligand has been developed as a new technology for antibody purification. In the present work, molecular simulation methods were developed to investigate the interactions between the Fc fragment of IgG and a MEP ligand net. The MM/PBSA method was used to evaluate the binding energy for the MEP ligand net at different densities. It was found that ligand density had significant influence on the binding of Fc. Potential binding conformations were further analyzed by molecular dynamics simulation. It was found that the interaction between Fc and MEP ligand net is driven by self-adaptive conformation adjustment and multiple-site binding. Hydrophobic forces dominate the binding interaction, which appeared as the results of synergistic actions of binding sites located on CH2, CH3, and the consensus binding site (CBS) of the Fc fragment. At acidic pH, the electrostatic repulsion between the basic residues and the protonated pyridine ring group on MEP ligands is the main driving force for the detachment of the Fc fragment from the MEP net.

Selectivity evaluation and separation of human immunoglobulin G, Fab and Fc fragments with mixed-mode resins.

Adsorption selectivity is critical important for mixed-mode chromatography with specially-designed ligands. Human immunoglobulin G (hIgG), Fc and Fab fragments were used in the present work to evaluate adsorption behavior and binding selectivity of four mixed-mode resins with the ligands of 4-mercatoethyl-pyridine (MEP), 2-mercapto-1-methylimidazole (MMI), 5-aminobenzimidazole (ABI) and tryptophan-5-aminobenzimidazole (W-ABI), respectively. The resins showed an obvious pH-dependent adsorption behavior. High adsorption capacities were found at neutral pH for hIgG, Fc and Fab, and almost no adsorption happened under acidic conditions. An adsorption selectivity index was proposed to evaluate separation efficiency. High specificity of hIgG/Fc was found at pH 8.9 for MEP resin, and for W-ABI resin at pH 8.0 and 8.9. In addition, isothermal titration calorimetry was used to evaluate ligand-protein interactions. Finally, the separation of hIgG and Fc (1:1) was optimized with mixed-mode resins, and the best separation performance was obtained with W-ABI-based resin. Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4% and recovery of 98.8%, while elution at pH 3.6 provided hIgG with purity of 99.7% and recovery of 86.5%.