Standardized approach for accurate and reliable model development of ion-exchange chromatography based on parameter-by-parameter method and consideration of extra-column effects.

Developing an accurate and reliable model for chromatographic separation that meets regulatory requirements and ensures consistency in model development remains challenging. In order to address this challenge, a standardized approach was proposed in this study with ion-exchange chromatography (IEC). The approach includes the following steps: liquid flow identification, system and column-specific parameters determination and validation, multi-component system identification, protein amount validation, steric mass action parameters determination and evaluation, and validation of the calibrated model's generalization ability. The parameter-by-parameter (PbP) calibration method and the consideration of extra-column effects were integrated to enhance the accuracy of the developed models. The experiments designed for implementing the PbP method (five gradient experiments for model calibration and one stepwise experiment for model validation) not only streamline the experimental workload but also ensure the extrapolation abilities of the model. The effectiveness of the standardized approach is successfully validated through an application about the IEC separation of industrial antibody variants, and satisfactory results were observed with R2 ≈ 0.9 for the majority of calibration and validation experiments. The standardized approach proposed in this work contributes significantly to improve the accuracy and reliability of the developed IEC models. Models developed using this standardized approach are ready to be applied to a broader range of industrial separation systems, and are likely find further applications in model-assisted decision-making of process development.

DFT and microkinetic study of acetylene transformation on Pd(111), M(111) and PdM(111) surfaces (M = Cu, Ag, Au).

Density functional calculations and microkinetic simulations were performed on the transformation network of acetylene on Pd(111), M(111) and PdM(111) (M = Cu, Ag, Au) surfaces. It is demonstrated that the adsorption energies on alloy surfaces linearly correlate with the values on the pure metal surfaces. A good linear relationship between the co-adsorption energies of initial states and transition states is revealed with which the barriers of most elementary steps in the reaction network were estimated. To shed light on the transformation of acetylene, microkinetic simulations were conducted on the network. The results show that CHCH and H are dominant species on the surfaces and CCH, CCH2 and CCH3 are the main intermediates. Analysis indicates that introduction of coinage metals into Pd reduces the activity, but promotes the selectivity by lowering the barrier of CHCH2 → CH2CH2. The present work provides a comprehensive overview of acetylene transformation on palladium, coinage metals and their alloy surfaces. The linear relationship of adsorption energies between the component metal and alloy surfaces and usage of the TSS relationship to evaluate barriers for microkinetic simulations are worthy of being further studied and extended to other systems.

Optimization of moxifloxacin hydrochloride tablets by formulationscreening and preparation process / 国际药学研究杂志

Objective To screen prescriptions for moxifloxacin hydrochloride tablets and optimize its preparation technology. Methods Taking the angle of repose,tap density,hardness,friability,disintegration time,tablet weight difference,and dissolution rate as indexes,the amount of each component,binder solvent,amount of binder,size of the mesh for granulation and particle drying process were investigated. The optimal formulation and process were determined based on the above results. Results With water as the binder solvent,binder volume of 6 ml,screen mesh number of 26 mesh,and finally drying 1 h at 50℃,the indicators of the tablet prepared met the quality requirements of tablet in the second part of the Pharmacopoeia of People′s Republic of China the 2015 ver-sion. And the dissolution profile was in good agreement with the commercially available preparation. Conclusion The quality of moxi-floxacin hydrochloride tablets prepared by the optimal formulation and process in this study is in accordance with the standard. The pre-scription and process can be used for the preparation of generic drugs of moxifloxacin hydrochloride tablets.

Suppression of phospho-p85α-GTP-Rac1 lipid raft interaction by bichalcone analog attenuates cancer cell invasion.

The p85α subunit of phosphatidylinositol 3-kinase (PI3K) acts as a key regulator of cell proliferation and motility, which mediates signals that confer chemoresistance to many human cancer cells. Using small interfering RNAs against matrix metalloproteinase-2 (MMP-2) and the MMP-2 promoter-driven luciferase assay, we showed that the new synthetic bichalcone analog TSWU-CD4 inhibits the invasion of human cancer cells by down-regulating MMP-2 expression. Treatment with TSWU-CD4 inhibited MMP-2 expression and cell invasion, which were restored by ectopic wild type (wt) p85α or a constitutively active form of MAPK kinase 3 (CA MKK3), CA MKK6, or CA p38α mitogen-activated protein kinase (MAPK). The attenuated formation of lipid raft-associated phospho (p)-p85α-GTP-Rac1 complexes, protein kinase B (Akt) Ser 473 phosphorylation, and cell invasion by TSWU-CD4 was reversed by overexpression of wt p85α or the p85α Brc-homology (BH) domain. The ectopic expression of CA Rac1L61 (but not wt Rac1) could overcome the suppression of Ser 473 phosphorylation, lipid raft association of Akt, the interaction between GTP-bound Rac1 and p85α in lipid rafts, and cell invasion by TSWU-CD4. The involvement of Akt activity in the functions of NF-κB-mediated MMP-2 was further confirmed through the attenuation of Akt phosphorylation signaling using the Akt-specific inhibitor MK-2206 and ectopic expression of NF-κB p65. Collectively, the inhibitory effect of TSWU-CD4 on cancer cell invasion was likely to suppress the p-p85α-GTP-Rac1 interaction in lipid rafts by targeting the p85α BH domain, which resulted in the suppression of MMP-2 expression via the PI3K-Akt-mediated ERK-MKK3/MKK6-p38 MAPK-NF-κB signaling pathway. © 2016 Wiley Periodicals, Inc.

Preparation and evaluation of dextran-grafted agarose resin for hydrophobic charge-induction chromatography.

Hydrophobic charge-induction chromatography (HCIC) is a new and effective technology for antibody separation. In the present work, HCIC resin MMI-B-XL was prepared with dextran-grafted agarose gel as the matrix and 2-mercapto-1-methyl-imidazole (MMI) as the functional ligand. The preparation procedures were optimized, and the maximum ligand density could reach as high as 200 µmol/g gel. The adsorption isotherms and kinetics on new resins were investigated with human immunoglobulin G (hIgG) as the model protein, which were compared with non-grafted HCIC resin MMI-B-6FF. It was found that the saturated adsorption capacity (Qm) increased with the increase of ligand density for MMI-B-XL. Moreover, the effective diffusivity (De) could be dramatically enhanced with the increase of ligand density for MMI-B-XL, and the De for MMI-B-XL with the ligand density of 200 µmol/g gel was 18-40 times higher than that for MMI-B-6FF. The breakthrough experiments indicated that new resins with the ligand density of 200 µmol/g gel could be used for high superficial velocity and high dynamic adsorption could be obtained. The results indicated that dextran-grafted layer on the resin could increase the ligand density, enhance the mass transport in the pore, and improve the dynamic adsorption at high velocity, which showed a potential application for large-scale antibody purification.

Development of ß-amino-carbonyl compounds as androgen receptor antagonists.

AIM: Androgen receptor (AR) antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel ß-amino-carbonyl-based androgen receptor antagonist. METHODS: Different isomers of the ß-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SCID mice bearing LNCaP xenografts. RESULTS: Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 21-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist (compound 6012), which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 (1R, 3S). CONCLUSION: Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.

Evaluation of mixed-mode chromatographic resins for separating IgG from serum albumin containing feedstock.

Mixed-mode chromatography has been focused as a cost-effective new technique for antibody purification. In this study, four mixed-mode resins with N-benzyl-N-methyl ethanol amine, 2-benzamido-4-mercaptobutanoic acide, 4-mercapto-ethyl-pyridine and phenylpropylamine as the ligands were tested and the multi-functional interactions between ligand and protein were discussed. Immunoglobulin G (IgG), bovine serum albumin (BSA) and the binary mixture of BSA and IgG were used as the model feedstock to compare the separation behaviors by pH gradient elution. The comparison analysis showed mixed-mode resin with N-benzyl-N-methyl ethanol amine as the ligand had the best ability to separate IgG and BSA. The results indicated that for four resins tested ionic interaction might play the dominant role in the separation of IgG and BSA while the hydrophobic interactions and hydrogen bonding have some subsidiary effects. The pH stepwise elution and sample loading were optimized to improve the IgG purification from serum albumin containing feedstock. High purity (92.3%) and high recovery (95.6%) of IgG were obtained. The results indicated that mixed-mode chromatography would be a potential option for antibody purification with the control of loading and elution conditions.

Evaluation of immunoglobulin adsorption on the hydrophobic charge-induction resins with different ligand densities and pore sizes.

Hydrophobic charge-induction chromatography (HCIC) is a novel technology for antibody purification. The ligand densities and pore properties of HCIC resins have significant effects on the separation behavior of protein, however, the understandings are quite limited. In the present work, new HCIC ligand, 2-mercapto-1-methylimidazole (MMI) was coupled to three agarose matrices with different pore sizes. A series of MMI resins with different ligand density and pore size was prepared by the control of ligand coupling. The adsorption isotherms and kinetics on the series of MMI resins were investigated with bovine serum immunoglobulin as the model IgG, and the effects of salt addition were studied. The Langmuir equation and pore diffusion model were used to fit the experimental data, and the influences of ligand density, pore size and salt addition on the saturated adsorption capacity, the dissociation constant and the effective diffusivity were discussed. It was found that the adsorption capacities and the effective pore diffusion coefficient increased with the increase of ligand density and pore size. The effects of salt addition on the adsorption behaviors were dependent on the ligand density. For low ligand density the IgG adsorption was salt-promoted, while the resins with high ligand density showed a salt-independent property. The results indicated that for a given protein the ligand density and pore size of HCIC resins should be optimized for improving the protein adsorption.

Effects of ligand density and pore size on the adsorption of bovine IgG with DEAE ion-exchange resins.

Immunoglobulin G is an important plasma protein with many applications in therapeutics and diagnostics, which can be purified effectively by ion exchange chromatography. The ligand densities and pore properties of ion-exchange resins have significant effects on the separation behaviors of protein, however, the understandings are quite limited. In this work, with bovine immunoglobulin as the model IgG, the adsorption isotherms and adsorption kinetics were investigated systematically with series of diethylaminoethyl ion-exchange resins with different ligand densities and pore sizes. The Langmuir equation and pore diffusion model were used to fit the experimental data. The influences of ligand density and pore size on the saturated adsorption capacity, the dissociation constant and the effective diffusivity were discussed. The adsorption capacities increased with the increase of ligand density and the decrease of pore size, and an integrative parameter was proposed to describe the combined effects of ligand density and pore size. It was also found that the effective pore diffusion coefficient of the adsorption kinetics was influenced by pore sizes of resins, but was relatively independent on the ligand densities of resins. For a given protein, the ligand density and pore size should be optimized for improving the protein adsorption.

Protein adsorption on DEAE ion-exchange resins with different ligand densities and pore sizes.

Ion exchange chromatography (IEC) is a common and powerful technique for the purification of proteins. The ligand density and pore properties of ion-exchange resins have significant effects on the separation behaviors of protein, however, the understandings are quite limited. In the present work, the adsorption isotherms of bovine serum albumin (BSA) and human serum albumin (HSA) were investigated systematically with series of diethylaminoethyl (DEAE) ion-exchange resins, which have different ligand densities and pore sizes. The Langmuir equation was used to fit the experimental data and the influences of ligand density and pore size on the saturated adsorption capacity and the dissociation constant were discussed. The zeta potentials and hydrodynamic diameters of proteins at different pHs were also measured, and the surface charge characteristics of proteins and the adsorption mechanism were discussed. The results demonstrated that the ligand density, pore size, and protein properties affect the protein adsorption capacities in an integrative way. An integrative parameter was introduced to describe the complicated effects of ligand density and pore size on the protein adsorption. For a given protein, the ligand density and pore size should be optimized for improving the protein adsorption.

Expression and purification of bioactive high-purity human midkine in Escherichia coli.

Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation. The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor. In the present work, rhMK was expressed in Escherichia coli (E. coli) BL21 (DE3). The expression of midkine was efficiently induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). After sonication, midkine was recovered in an insoluble form, and was dissolved in guanidine hydrochloride buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose ion-exchange chromatography. The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC). The purified rhMK enhanced the proliferation of NIH3T3 cells.

Rhodanine derivatives as novel peroxisome proliferator-activated receptor gamma agonists.

AIM: To characterize the in vitro bioactivities of rhodanine derivatives as novel peroxisome proliferator-activated receptor (PPAR) gamma modulators, based on a hit (SH00012671) identified during high-throughput screening (HTS) of a diverse synthetic compound library, and to preliminarily elucidate the structure-activity relationship of this class of PPARgamma agonists. METHODS: Full-length PPARgamma and retinoid X receptor alpha (RXRalpha), biotinylated PPAR response element (PPRE), [3H]BRL49653 (rosiglitazone), and streptavidin-coated FlashPlate or microbeads were used to measure the receptor-binding properties of various compounds based on the scintillation proximity assay (SPA) technology. A recombinant PPRE vector was transiently cotransfected with PPARgamma and RXRalpha plasmids into the African green monkey kidney (CV-1) cells, and the effects of BRL49653 and test compounds on transcription mediated by PPARgamma were determined by examining luciferase (reporter) responses. 3T3-L1 cells were employed to determine whether the compounds facilitated adipogenesis upon PPARgamma activation. RESULTS: Of the 16,000 samples screened with the SPA method, only 1 compound (SH00012671) displayed a similar binding affinity (Ki=186.7 nmol/L) to PPARgamma as BRL49653, but it was inactive in the cell-based assays. A series of rhodanine derivatives were synthesized based on the core structure of SH00012671 and 8 of them showed agonist activities in both cotransfection and pre-adipocyte differentiation assays. To reduce intrinsic cytotoxicities, the sulphur on the rhodanine was changed to oxygen. This alteration led to a decrease in receptor-binding affinities while modified analogues generally maintained agonist efficacies in the cell-based assays. Of the analogues studied, compound 31 exhibited about 70% the efficacy exerted by BRL49653 in both cotransfection and pre-adipocyte differentiation assays. CONCLUSION: Through minor chemical modifications on the core structure of the initial HTS hit, SH00012671 was transformed to possess both molecular (PPARgamma binding) and cellular (adipogenesis) activities. The rhodanine derivatives reported here may represent a new scaffold in further understanding the molecular mechanism of agonism at PPARgamma.

Inhibitory effect of Ca2+ on in vivo gene transfer by electroporation.

AIM: To investigate the specific effects of Ca2+ on transgene expression during electroporation-mediated gene transfer in mice. METHODS: Skeletal muscle and skin were subjected to in vivo electroporation with a luciferase reporter plasmid, with or without Ca2+ and various other ions. RESULTS: For in vivo electroporation, the presence of just 10 mmol/L Ca2+ in the DNA solution drastically reduced the resulting transgene expression, to less than 5% of control values. Only Ca2+, not other ions, caused inhibition, and the effect was not tissue specific. More surprisingly, even when Ca2+ ions were delivered by electroporation before or after DNA administration, similar effects were still observed. CONCLUSION: The inhibitory effect of Ca2+ on in vivo gene transfer by electroporation is specific, ie, the inhibitory effect may be related to the cell membrane properties after electroporation and the subsequent resealing event.