LRRC1 knockdown downregulates MACF1 to inhibit the malignant progression of acute myeloid leukemia by inactivating ß-catenin/c-Myc signaling.

Acute myeloid leukemia (AML) is a hematologic disease associated with genetic abnormalities. This study aimed to explore the role of leucine-rich repeat-containing protein 1 (LRRC1) in the malignant activities of AML and to reveal the molecular mechanism related to microtubule actin cross-linking factor 1 (MACF1). GEPIA database was used to analyze the expression of LRRC1 in bone marrow tissues of AML patients and the correlation between LRRC1 expression and survival analysis. LRRC1 was knocked down to assess the change of AML cell proliferation, cell cycle and apoptosis using CCK-8 assay and flow cytometry. Besides, the contents of extracellular acidification and oxygen consumption rates were measured to evaluate the glycolysis. Additionally, the interaction between LRRC1 and MACF1 predicted by MEM database and was verified by co-immunoprecipitation (Co-IP) assay. Then, MACF1 was overexpressed to conduct the rescue experiments. Expression of proteins in ß-catenin/c-Myc signaling was detected by western blot. Finally, AML xenograft mouse model was established to observe the impacts of LRRC1 silencing on the tumor development. Notably upregulated LRRC1 expression was observed in bone marrow tissues of AML patients and AML cells, and patients with the higher LRRC1 expression displayed the lower overall survival. LRRC1 depletion promoted cell cycle arrest and apoptosis and inhibited the glycolysis. Co-IP confirmed the interaction between LRRC1 and MACF1. MACF1 upregulation relieved the impacts of LRRC1 knockdown on the malignant activities of AML cells. Moreover, LRRC1 silencing inhibited the development of xenograft tumor growth of HL-60 cells in nude mice, suppressed MACF1 expression and inactivated the ß-catenin/c-Myc signaling. Collectively, LRRC1 knockdown suppressed proliferation, glycolysis and promoted apoptosis in AML cells by downregulating MACF1 expression to inactivate ß-catenin/c-Myc signaling.

ADSC-Exosomes Alleviate MTX-induced Rat Neuronal Damage by Activating Nrf2-ARE Pathway.

The aim of this study was to analyze the efficacy and underlying mechanism of adipose-derived mesenchymal stem cell exosome (ADSC-exosomes)-mediated protection on methotrexate (MTX)-induced neuronal damage. We established a H2O2-induced oxidative stress model in vitro, as well as an MTX-induced neuronal damage rat model in vivo. We analyzed the effects of ADSC-exosomes on neuronal damage and Nrf2-ARE signaling pathway in rats and related mechanisms. The morphological and functional recovery of rat hippocampal neurons by ADSC-exosomes was examined by Nissl staining and modified neurological severity score (mNSS) score. The activation of Nrf2-ARE pathway effectively inhibited H2O2-induced oxidative stress. ADSC-exosomes treatment restored the activity of hippocampal neuronal cells, reduced ROS production, and inhibited hippocampal neuronal cells apoptosis. In in vivo experiments, ADSC-exosomes ameliorates MTX-induced hippocampal neuron damage by triggering Nrf2-ARE pathway, decreasing IL-6, IFN-, and TNF-a levels and TUNEL positive cells in hippocampus, and repairing hippocampal neuronal cell damage. ADSC-exosomes ameliorated MTX-induced neuronal damage and suppressed oxidative stress induced by neuronal damage through the activation of Nrf2-ARE signaling pathway.

Establishment of an induced pluripotent stem cell line (WMUi016-A) from a patient with X-linked Dent disease (X-Dent) carrying the hemizygote mutation p.R718* (c.2152C > T) in the CLCN5 gene.

The gene mutations of the chloride channel gene (CLCN5) can lead to the inherited X-linked Dent disease (X-Dent). The urine cells of a 4-year-old male X-Dent patient with the hemizygous CLCN5 gene mutation p.R718* (c.2152C > T) were reprogrammed into induced pluripotent stem cells (iPSCs) using integration free Sendai virus reprogramming system. The generated iPSCs stably expressed pluripotent stem cell markers and can be induced to differentiate into three germ layers in vitro. The karyotype of the generated iPSCs was normal (46, XY).

Generation of a human induced pluripotent stem cell line with Cas9 driven by Tet-on operator via AAVS1 safe harbor gene-editing.

Human induced pluripotent stem (iPS) cells expressing Cas9 protein are valuable for the pathogenic mechanism study and drug discovery. These cells can be efficiently induced to differentiate into disease cell models with specific mutations through adding designed sgRNAs. Here, we generated a human gene-editable iPS cell line by gene editing method that Cas9 gene driven by Tet-on operator was perfectly integrated into the human AAVS1 safe harbor locus. The established Cas9 expression iPS cell line named as WMUi013-A can express endogenous pluripotent markers, has the ability to differentiate into the three germ layers, and possesses a normal karyotype.

Generation of an urine-derived induced pluripotent stem cell line from a 5-year old X-linked Alport syndrome (X-LAS) patient.

The gene mutations of the collagen type IV alpha 5 chain (COL4A5) can lead to the inherited haematuria to end-stage renal disease X-linked Alport syndrome (X-LAS). The urine cells of a 5-year-old male X-LAS patient carrying a hemizygous COL4A5 gene mutation p.G1433V (c.4298G>T) were reprogrammed to induced pluripotent stem cells (iPSCs) with Sendai virus reprogramming kit containing OCT4, SOX2, c-MYC, and KLF4 Yamanaka factors. The generated iPSC line WMUi015-A stably expressed pluripotent markers, maintained a normal karyotype (46, XY), and had differentiation potential into three germ layers in vitro.

Emodin Reverses Gemcitabine Resistance of Pancreatic Cancer Cell Lines Through Inhibition of IKKß/NF-κB Signaling Pathway.

BACKGROUND: Pancreatic cancer is one of the most malignant tumors, and gemcitabine has been considered as the standard treatment and been widely utilized as a first-line drug for advanced pancreatic cancer, but gemcitabine-resistance always occurs after a short period of treatment. METHODS: Two pancreatic cancer cell lines Panc-1 and MIA-PaCa-2 were used as the study subject and their gemcitabine-resistant cells were established. Both drug-resistant cells were divided into four groups: blank, emodin, gemcitabine, and emodin+gemcitabine. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis rate and P-gp function. Quantitative real-time polymerase chain reaction and Western blotting were used to detect Survivin, XIAP, Caspase-9/3, NF-κB p65, IKKß and IκB-α mRNA/protein expressions, respectively. Electrophoretic mobility shift assay (EMSA) was performed to detect NF-κB binding activity. Rhodamine 123 efflux assay was used to detect P-gp function. RESULTS: Emodin could inhibit cell activity in all cell lines. Both emodin and gemcitabine can significantly increase the apoptosis rate, and the combination of the two drugs can further significantly increase the apoptosis rate in normal pancreatic cancer cell lines. In both drug-resistant pancreatic cancer cell lines, it can be observed that although gemcitabine can increase the apoptosis rate, the effect of promoting apoptosis is significantly lower than that of emodin; the drug combination can still significantly increase the apoptosis rate on the basis of emodin alone. Emodin can significantly reduce the mRNA and protein expression levels of Survivin, XIAP, NF-κB, and IKKß, and significantly increase the mRNA and protein expression levels of Caspase-3/9 and IκB-α. Emodin significantly reduced NF-κB activity and emodin significantly promoted P-gp fluorescence intensity from Rhodamine 123 efflux assay. CONCLUSION: Emodin inhibits the expression of IKKß, thereby inhibiting the expression and activity of downstream NF-κB, and inhibits P-gp function at the same time, ultimately achieving the purpose of reversing the drug-resistance of pancreatic cancer cell lines.

Effects of evodiamine on PI3K/Akt and MAPK/ERK signaling pathways in pancreatic cancer cells.

The effective antitumor drug evodiamine (EVO) is attracting increased attention. Therefore, the present study aimed to investigate the effects of EVO on the proliferation, apoptosis and autophagy of human pancreatic cancer (PC) cell lines in vitro and in vivo. Human PANC­1 and SW1990 PC cell lines were treated with different concentrations of EVO and proliferation was detected using a Cell Counting Kit (CCK)­8 assay. Colony formation and wound­healing assays showed that EVO inhibited PC cell viability and migration, and apoptosis was detected using flow cytometry. Western blotting and immunofluorescence detected the expression of proteins in PANC­1 and SW1990 cells. The PANC­1 cells were used to establish an orthotopic pancreatic tumor model in nude mice. Tumor­bearing nude mice were administered with different concentrations of EVO, and growth was monitored. High­resolution positron emission tomography and fluorine­18­labeled fluorodeoxyglucose were used to monitor the tumor/non­tumor (T/NT) ratio and standard uptake value (SUV) of the mice, which were subsequently sacrificed to measure the transplanted tumor weight. Apoptosis increased with increasing EVO concentration. The EVO­treated PC cells exhibited significantly higher expression of LC3II than the controls cells. EVO decreased LC3II, enhanced P62 and inhibited the expression of Akt, extracellular­signal­regulated protein kinase (ERK)1/2 and p38. Compared with the control group, the T/NT ratio, SUV and tumor weight decreased more markedly in the EVO­treated group. The tumor expression of phosphorylated AKT, detected using immunohistochemistry, decreased with increasing EVO doses in vivo. EVO induced PC cell apoptosis by inhibiting phosphoinositide 3­kinase/AKT and mitogen­activated protein kinase/ERK and inhibiting the phosphorylation of signal transducer and activator of transcription activator 3 in PC cells to inhibit autophagy, suggesting that EVO may be considered as a novel PC treatment.

Emodin sensitizes human pancreatic cancer cells to EGFR inhibitor through suppressing Stat3 signaling pathway.

BACKGROUND: Excessive expression of EGFR is closely related to tumor formation, transfer and deterioration, which has attracted much attention. EGFR overexpression may be detected in up to 90% of pancreatic tumors. However, drug resistance of EGFR inhibitors targeting treatment severely limits its clinical application. METHODS: In this study, Western blotting was used to detect the expression of p-Stat3, EGFR, Bcl-2, cleaved-caspase3 and Bax. Cell apoptosis was evaluated via flow cytometry. The colon assay and MTT assay were applied for detecting the cell proliferation in vitro. The xenograft mouse model was used to examine the cell proliferation in vivo. RESULTS: Emodin remarkably enhanced the anti-cancer effect of EGFR inhibitor on pancreatic cancer cells. In addition, emodin promoted afatinib-induced apoptosis by inhibiting the Stat3 signaling pathway. Meanwhile, siRNAs against Stat3 significantly increased the apoptosis of pancreatic cancer cells. EGFR inhibitor promoted phosphorylation of Stat3 in pancreatic cancer cells. Interestingly, emodin combined with EGFR inhibitor inhibited the proliferation of pancreatic cancer cells in vitro. The tumor xenograft mice model was further confirmed that emodin possessed a synergy anticancer effect with afatinib on pancreatic cancer cells by regulating the Stat3 expression. CONCLUSION: These results indicate that the combination of emodin with EGFR inhibitor is an effective therapeutic strategy to sensitize human pancreatic cancer.

Bioinformatics analysis of molecular genetic targets and key pathways for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second leading cause of death among cancers worldwide. In this study, we aimed to identify the molecular target genes and detect the key mechanisms of HCC. Three gene expression profiles (GSE84006, GSE14323, GSE14811) and two miRNA expression profiles (GSE40744, GSE36915) were analyzed to determine the molecular target genes, microRNAs (miRNAs) and the potential molecular mechanisms in HCC. METHODS: All profiles were extracted from the Gene Expression Omnibus database. The identification of the differentially expressed genes (DEGs) was analyzed by the GEO2R method. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology (GO) enrichment analysis performed database for Integrated Discovery, Visualization and Annotation. The miRNA-gene network and protein-protein interaction (PPI) network were correlated by the Cytoscape software. The key target genes were identified by the CytoHubba plugin, Molecular Complex Detection (MCODE) plugin and miRNA-gene network. The identified hub genes were testified for survival curve using the Kaplan-Meier plotter database. RESULTS: Expression profiles had 592 overlapped DEGs. The majority of the DEGs were enriched in membrane-bounded organelles and intracellular membrane-bounded organelles. These DEGs were significantly enriched in metabolic, protein processing in the endoplasmic reticulum and thyroid cancer pathways. PPI network analysis showed these genes were mostly involved in the pathogenic Escherichia coli infection and the regulation of actin cytoskeleton pathways. Combining these results, we identified 10 key genes involving in the progression of HCC. Finally, PLK1, PRCC, PRPF4 and PSMA7 exhibited higher expression levels in HCC patients with poor prognosis than those for lower expression via Kaplan-Meier plotter database. CONCLUSION: PLK1, PRCC, PRPF4 and PSMA7 could be potential biomarkers or therapeutic targets for HCC. Meanwhile, the metabolic pathway, protein processing in the endoplasmic reticulum and the thyroid cancer pathway may play vital roles in the progression of HCC.

Preparation and evaluation of dextran-grafted mixed-mode chromatography adsorbents.

In the present work, mixed-mode chromatography adsorbents grafted with dextran of different molecular weight were prepared. Compared to non-grafted adsorbent (BA-6B), the morphology of dextran-grafted adsorbents (BA-6B-Ts) did not change significantly, while the water content (ω), porosity (P) and pore volume (V) decreased. Static adsorption, salt tolerance and dynamic adsorption behaviors of adsorbents BA-6B-Ts against BSA/IgG were studied. Compared to adsorbent BA-6B, adsorbents BA-6B-Ts had higher saturated adsorption capacity (Qm) toward BSA/IgG, especially for BA-6B-T20, the Qm values toward BSA and IgG increased by 32.8% and 39.5%, respectively. Adsorbents BA-6B-Ts showed enhanced salt tolerance toward BSA, and relatively weaker salt tolerance toward IgG. The relative adsorption capacity of BA-6B-T20 was 66.7% at 0.1 mol/L NaCl and 39.2% at 0.5 mol/L NaCl, higher than those of BA-6B. Moreover, for both BSA and IgG, the effective pore diffusivity (De) of BA-6B-Ts was higher than that of BA-6B. De values of BA-6B-T20 toward BSA and IgG increased by 77% and 139% compared to BA-6B, respectively. Dynamic binding capacities (DBCs) of BA-6B-Ts were also improved. DBC of BSA and IgG on BA-6B-T20 were 25.6% and 46.5% higher than those on BA-6B. The possible mechanisms for the above difference in adsorption behavior of dextran-grafted adsorbents toward BSA/IgG were discussed, providing some insights into protein adsorption and mass transport for dextran-grafted MMC resins.

Molecular dynamics perspective on the thermal stability of mandelate racemase.

Mandelate racemase from Pseudomonas putida is a promising candidate for the dynamic kinetic resolution of α-hydroxy carboxylic acids. In the present study, the thermal stability of mandelate racemase was investigated through molecular dynamics simulations in the temperature range of 303-363 K, which can guide the design of mandelate racemase with higher stability. The basic features such as radius of gyration, surface accessibility, and secondary structure content suggested the instability of mandelate racemase at high temperatures. With increase in temperature, α-helix content reduced significantly, especially the α-helices exposed to the environment. At the simulation time scale considered, intra-protein hydrogen bonds, hydrogen bonds between protein and water decreased at 363 K, while the number of salt-bridges increased. The long-distance networks remarkably changed at 363 K. A considerable number of long-lived (percentage existence time higher than 90%) hydrogen bonds and Cα contacts were lost. Root mean square fluctuation analysis revealed regions with high fluctuation, which should be helpful in the reengineering of mandelate racemase for enhanced thermal stability.

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.

Effects of cytochrome P450 family 3 subfamily A member 5 gene polymorphisms on daunorubicin metabolism and adverse reactions in patients with acute leukemia.

The present study aimed to investigate the association between the genetic polymorphism of cytochrome P450 family 3 subfamily A member 5 (CYP3A5) and the activity of CYP3A and plasma concentrations of daunorubicin (DNR) in patients with acute leukemia. A total of 36 children with newly diagnosed acute lymphoblastic leukemia were enrolled in the study. Polymerase chain reaction (PCR)­restriction fragment length polymorphism and PCR product sequencing were used to detect the genotype of CYP3A5*3. PCR was then used to express the mRNA expression of CYP3A5. A midazolam probe method was used to detect CYP3A enzyme activity, and DNR concentrations were measured using high performance liquid chromatography. Children with different genotypes had different mRNA expression levels of CYP3A5, and CYP3A enzyme activity in children with the CYP3A5*1 allele was higher, compared with that in children with the CYP3A5*3 allele. In addition, the area under the curve (AUC)0­24 h and AUC0­∞ of DNR were significantly different in children with different genotypes, however, no statistically significant differences were found in half­life or maximum concentration. The AUC of DNR was increased in children with acute lymphatic leukemia who suffered from cardiotoxicity, compared with those in the normal group. The CYP3A5*3 gene polymorphism was closely associated with the mRNA expression of CYP3A5, CYP3A enzyme activity and DNR plasma drug concentration, and exhibited different drug adverse reactions.

Quantitative analysis of gadoxetic acid-enhanced magnetic resonance imaging predicts histological grade of hepatocellular carcinoma.

OBJECTIVE: To confirm the histological grade of hepatocellular carcinoma (HCC) by gadoxetic acid-enhanced MRI. METHODS: Ninety-five HCC patients underwent gadoxetic acid-enhanced MRI before surgical intervention. The correlations among the signal absolute enhancement, contrast enhancement ratio (CER) and tumor histological grade were analyzed. RESULTS: The correlation between CER of tumor-to-liver and the grades of tumor differentiation is the most significant negative. The k-value for the CER of tumor-to-liver and histopathologic analysis is 0.62, which gives evidence of good agreement. CONCLUSION: The quantitative analysis of gadoxetic acid-enhanced MRI can predict the histological grades of small HCCs.

Post-therapeutic recovery of serum interleukin-35 level might predict positive response to immunosuppressive therapy in pediatric aplastic anemia.

BACKGROUND: The predictive value of interleukin-35 (IL-35) on efficacy of immunosuppressive therapy (IST) in aplastic anemia (AA) has not been well investigated. The aim of the study was to evaluate the association between serum IL-35 level and response to IST in pediatric AA. METHODS: A total of 154 children with AA and 154 controls were included between January 2012 and December 2013. Blood and bone marrow fluid specimens were collected. Serum level of IL-35 was determined by enzyme-linked immunosorbent assay. Patients were treated with IST, and response to therapy was evaluated during 180-day follow-up period after starting therapy. RESULTS: Serum levels of IL-35 at admission decreased significantly in patients compared with that in controls (10.9 ± 5.5 pg ml-1 and 45.3 ± 8.8 pg ml-1, p < 0.001). After starting IST, serum levels of IL-35 in patients recovered 30.7 ± 9.7 pg ml-1 in the first 28 days (p < 0.001). During the follow-up period, increased range of serum IL-35 level ≥30.7 pg ml-1 in the first 28 days was associated with effective response to therapy (odds ratio 7.97, 95% confidence interval 3.82-16.79). In addition, Fas/FasL protein expression in bone marrow mononuclear cells dropped significantly in the same group of patients in the first 28 days (p < 0.05). CONCLUSION: The study revealed that post-therapeutic recovery of circulating IL-35 concentration might be an independent predictor for effective response to IST in pediatric AA. Moreover, apoptosis might be involved in such a forecasting process.

Transcriptomic analysis of gene expression profiles of stomach carcinoma reveal abnormal expression of mitotic components.

AIMS: In order to explore the etiology of gastric cancer on global gene expression level, we developed advanced bioinformatic analysis to investigate the variations of global gene expression and the interactions among them. MAIN METHODS: We downloaded the dataset GSE63288 from Gene Expression Omnibus (GEO) database which included 22 human gastric cancer and 22 healthy control samples. We identified the differential expression genes, and explored the Gene ontology (GO) and pathways of the differentially expressed genes. Furthermore, integrative interaction network and co-expression network were employed to identify the key genes which may contribute to gastric cancer progression. KEY FINDINGS: The results indicated that 5 kinases including BUB1, TTK protein kinase, Citron Rho-interacting kinase (CIT), ZAK and NEK2 were upregulated in gastric cancer. Interestingly, BUB1, TTK, CIT and NEK2 have shown high expression similarities and bound with each other, and participated in multiple phases of mitosis. Moreover, a subnet of co-expression genes e.g. KIF14, PRC1, CENPF and CENPI was also involved in mitosis which was functionally coupled with the kinases above. By validation assays, the results indicated that CIT, PRC1, TTK and KIF14 were significantly upregulated in gastric cancer. SIGNIFICANCE: These evidences have suggested that aberrant expression of these genes may drive gastric cancer including progression, invasion and metastasis. Although the causal relationships between gastric cancer and the genes are still lacking, it was reasonable to take them as biomarkers for diagnosis of gastric cancer.

Effect of the Polymorphism of Folylpolyglutamate Synthetase on Treatment of High-Dose Methotrexate in Pediatric Patients with Acute Lymphocytic Leukemia.

BACKGROUND The aim of this study was to investigate the association of the polymorphism of folylpolyglutamate synthetase (FPGS) with the dynamic plasma concentration of methotrexate (MTX) in pediatric patients with acute lymphocytic leukemia (ALL), as well as the prognosis. MATERIAL AND METHODS 57 ALL patients and 31 age and sex-matched children (control) were included in this study. Polymerase chain reaction-restriction fragment length polymorphism was performed for the analysis of the genotype of FPGS rs1544105 and high-performance liquid chromatography for measurement of MTX plasma concentration after 24-h and 44-h treatment. Overall survival was analyzed by Kaplan-Meier method. RESULTS No differences were observed between patients and controls regarding the distribution frequency of genotype and alleles of rs1544105. Patients carrying AA genotype had a significantly higher plasma concentration of MTX after 24 h than those carrying GG or GA (P<0.05) and no differences were found after 44 h. Kaplan-Meier survival analysis showed a longer median survival time in patients with AA than other genotypes with significant difference in overall survival. CONCLUSIONS Polymorphism of FPGS rs1544105 might be used as an effective approach for prediction of the treatment outcome of MTX.

Evaluation of magnetic particles modified with a hydrophobic charge-induction ligand for antibody capture.

Magnetic particles modified with 5-amino-benzimidazole (ABI), a ligand for hydrophobic charge-induction chromatography, were prepared and used for antibody capture. In this study, with IgG as the model target, and bovine serum albumin (BSA) as the model impurity, the separation mechanism and process of IgG was investigated. The adsorption isotherms of IgG and BSA were measured, and the effects of pH were investigated in the range of pH 4.0-8.0. The maximum adsorption capacity of IgG on the particles was 180mg/ml at pH 7.0, while low adsorption capacity of BSA (64mg/ml) was found at pH 7.0, resulting in good selectivity. The protein-ligand interactions were elucidated by adding NaCl and glycerol. The results indicated the hydrophobic interactions were the main forces for IgG-ligand association. Moreover, the batch uptake and desorption experiments demonstrated the fast adsorption and desorption processes for IgG separation. The purity of IgG separated from mimetic serum could reach 98.6%, and the purity of monoclonal antibody (mAb) from a cell culture supernatant was 97.1%. Magnetic particles with hydrophobic charge-induction ligands showed a robust performance and could purify antibody directly from the complicated feedstock without clarification, which would improve the efficiency of antibody purification.

Multimodal charge-induction chromatography for antibody purification.

Hydrophobic charge-induction chromatography (HCIC) has advantages of high capacity, salt-tolerance and convenient pH-controlled elution. However, the binding specificity might be improved with multimodal molecular interactions. New ligand W-ABI that combining tryptophan and 5-amino-benzimidazole was designed with the concept of mutimodal charge-induction chromatography (MCIC). The indole and benzimidazole groups of the ligand could provide orientated mutimodal binding to target IgG under neutral pH, while the imidazole groups could induce the electrostatic repulsion forces for efficient elution under acidic pH. W-ABI ligand was coupled successfully onto agarose gel, and IgG adsorption behaviors were investigated. High affinity to IgG was found with the saturated adsorption capacity of 70.4 mg/ml at pH 7, and the flow rate of mobile phase showed little impact on the dynamic binding capacity. In addition, efficient elution could be achieved at mild acidic pH with high recovery. Two separation cases (IgG separation from albumin containing feedstock and monoclonal antibody purification from cell culture supernatant) were verified with high purity and recovery. In general, MCIC with the specially-designed ligand is an expanding of HCIC with improved adsorption selectivity, which would be a potential alternative to Protein A-based capture for the cost-effective purification of antibodies.

5-Aminobenzimidazole as new hydrophobic charge-induction ligand for expanded bed adsorption of bovine IgG.

Expanded bed adsorption (EBA) can capture target proteins directly from unclarified feedstock without prior solid-liquid separation. Hydrophobic charge-induction chromatography (HCIC) is a promising technology for biomolecule separation with high capacity, good selectivity and relatively low cost without the pretreatment of dilution or salt addition. In this work, EBA and HCIC were combined to develop a new separation technology, hydrophobic charge-induction EBA. Two HCIC ligands, 4-mercapto-ethyl-pyridine (MEP) and 5-aminobenzimidazole (ABI), were coupled onto agarose beads containing tungsten carbide to prepare the resins for EBA, named T-MEP and T-ABI, respectively. The static adsorption and dynamic binding behaviors of bovine IgG (bIgG) were investigated. Two resins had similar saturated adsorption capacities and salt-tolerant properties, but T-ABI showed higher dynamic binding capacity than T-MEP, indicating that ABI ligand was more suitable for EBA. The performances in expanded bed were verified. With the protein mixture (2mg/ml bIgG and 10mg/ml bovine serum albumin) as the model feedstock, the effects of loading and elution pH, expansion factor and loading volume on the separation performance of bIgG were evaluated. Finally, T-ABI EBA was used to separate bIgG directly from bovine whey with optimized operation conditions. The purity and recovery of bIgG reached 90.6% and 78.2%, respectively. The purification factor was about 19.3. The results demonstrated that the combination of HCIC and EBA would be a potential platform for antibody capture with less feedstock pretreatments, high efficiency and relatively low cost.