Evaluation of dynamic control of continuous capture with periodic counter-current chromatography under feedstock variations.

Multi-column periodic counter-current chromatography is a promising technology for continuous antibody capture. However, dynamic changes due to disturbances and drifts pose some potential risks for continuous processes during long-term operation. In this study, a model-based approach was used to describe the changes in breakthrough curves with feedstock variations in target proteins and impurities. The performances of continuous capture of three-column periodic counter-current chromatography under ΔUV dynamic control were systematically evaluated with modeling to assess the risks under different feedstock variations. As the concentration of target protein decreased rapidly, the protein might not breakthrough from the first column, resulting in the failure of ΔUV control. Small reductions in the concentrations of target proteins or impurities would cause protein losses, which could be predicted by the modeling. The combination of target protein and impurity variations showed complicated effects on the process performance of continuous capture. A contour map was proposed to describe the comprehensive impacts under different situations, and nonoperation areas could be identified due to control failure or protein loss. With the model-based approach, after the model parameters are estimated from the breakthrough curves, it can rapidly predict the process stability under dynamic control and assess the risks under feedstock variations or UV signal drifts. In conclusion, the model-based approach is a powerful tool for continuous process evaluation under dynamic changes and would be useful for establishing a new real-time dynamic control strategy.

Model-assisted process development, characterization and design of continuous chromatography for antibody separation.

Continuous manufacturing in monoclonal antibody production has generated increased interest due to its consistent quality, high productivity, high equipment utilization, and low cost. One of the major challenges in realizing continuous biological manufacturing lies in implementing continuous chromatography. Given the complex operation mode and various operation parameters, it is challenging to develop a continuous process. Due to the process parameters being mainly determined by the breakthrough curves and elution behaviors, chromatographic modeling has gradually been used to assist in process development and characterization. Model-assisted approaches could realize multi-parameter interaction investigation and multi-objective optimization by integrating continuous process models. These approaches could reduce time and resource consumption while achieving a comprehensive and systematic understanding of the process. This paper reviews the application of modeling tools in continuous chromatography process development, characterization and design. Model-assisted process development approaches for continuous capture and polishing steps are introduced and summarized. The challenges and potential of model-assisted process characterization are discussed, emphasizing the need for further research on the design space determination strategy and parameter robustness analysis method. Additionally, some model applications for process design were highlighted to promote the establishment of the process optimization and process simulation platform.

Model-based evaluation and model-free strategy for process development of three-column periodic counter-current chromatography.

Multi-column counter-current chromatography is an advanced technology used for continuous capture processes to improve process productivity, resin capacity utilization and product consistency. However, process development is difficult due to process complexity. In this work, some general and convenient guidances for three-column periodic counter-current chromatography (3C-PCC) were developed. Boundaries and distributions of operating windows of 3C-PCC processes were clarified by model-based predictions. Interactive effects of feed concentration (c0), resin properties (qmax and De), recovery and regeneration times (tRR) were evaluated over a wide range for maximum productivity (Pmax). Furthermore, variation of Pmax was analyzed considering the constraint factors (capacity utilization target and flow rate limitation). The plateau value of Pmax was determined by qmax and tRR. The operating conditions for Pmax were controlled by qmax, tRR and c0 interactively, and a critical concentration existed to judge whether the operating conditions of Pmax under constraints. Based on the comprehensive understanding on 3C-PCC processes, a model-free strategy was proposed for process development. The optimal operating conditions could be determined based on a set of breakthrough curves, which was used to optimize process performance and screen resins. The approach proposed was validated using monoclonal antibody (mAb) capture with a 3C-PCC system under various mAb and feed concentrations. The results demonstrated that a thorough model-based process understanding on multi-column counter-current chromatography is important and could improve process development and establish a model-free strategy for more convenient applications.

Age-Stratified Cut-Off Values for Serum Levels of N-Terminal ProB-Type Natriuretic Peptide and Mortality from Sepsis in Children Under Age 18 Years: A Retrospective Study from a Single Center.

BACKGROUND The N-terminal fragment of proB-type natriuretic peptide (NT-proBNP) is an established predictive marker for sepsis-related mortality in adult. This retrospective study aimed to determine age-stratified cut-off values for serum levels of NT-proBNP and mortality from sepsis in children under 18 years. MATERIAL AND METHODS Patients were stratified by age as follows: <1 year, 1-3 years, 4-6 years, and 7-18 years (age groups). The control group consisted of age- and sex-matched healthy children. Serum NT-proBNP levels were detected by laboratory assays in the participants. The appropriate serum NT-proBNP cut-off values for predicting short-term mortality of the sepsis patients were calculated via receiver operating characteristic (ROC) curve analyses. RESULTS Among 327 pediatric patients with sepsis, the serum NT-proBNP cut-off concentrations for predicting sepsis-related mortality in the <1 year, 1-3 years, 4-6 years, and 7-18 years age groups were 5000 ng/L, 4500 ng/L, 4100 ng/L, and 3800 ng/L, respectively (P<0.001). The area under the ROC curve (AUC) values for these were 0.815, 0.812, 0.806 and 0.725, respectively (P<0.001). CONCLUSIONS This retrospective study provided the age range-specific serum NT-proBNP cut- off concentrations for predicting short-term mortality in children. In children <1 year, 1-3 years, 4-6 years, and 7-18 years, age-stratified cut-off values that predicted sepsis-associated mortality were 5000 ng/L, 4500 ng/L, 4100 ng/L, and 3800 ng/L, respectively.

Comparison of Protein A affinity resins for twin-column continuous capture processes: Process performance and resin characteristics.

Continuous chromatography is a promising technology for downstream processing of biopharmaceuticals. The operation of continuous processes is significantly different to batch-mode chromatography and needs comprehensive evaluation. In this work, the performances of four Protein A affinity resins were studied systematically for twin-column continuous capture processes. A model-based approach was used to evaluate the process performance (productivity and capacity utilization) under varying operation conditions, and the objective was to reveal the crucial resin properties for continuous capture. The trade-off between productivity and capacity utilization was found, and it is necessary to select appropriate resins for different feedstock and operation conditions. The capacity utilization heavily depends on mass transfer, and steep breakthrough curves are favorable for high capacity utilization. The productivity is determined by both equilibrium binding capacity and mass transfer, and the balance of feed amount and feed time is critical. Moreover, the influence of binding capacity and mass transfer on process productivity and parameter sensitivity with two important resin properties (equilibrium binding capacity qmax and effective pore diffusion coefficient De) were assessed by the model, and suitable resin parameter ranges for twin-column continuous capture were determined. The model-based approach is an effective and useful tool to evaluate the complex performance of different resins and guide the design of next-generation resins for continuous processes.

MiR-27a-3p enhances the cisplatin sensitivity in hepatocellular carcinoma cells through inhibiting PI3K/Akt pathway.

MicroRNAs (miRNAs) play an important role in drug resistance, and it is reported that miR-27a-3p regulated the sensitivity of cisplatin in breast cancer, lung cancer and ovarian cancer. However, the relationship between miR-27a-3p and chemosensitivity of cisplatin in hepatocellular carcinoma (HCC) was unclear, especially the underlying mechanism was unknown. In the present study, we analyzed miR-27a-3p expression levels in 372 tumor tissues and 49 adjacent tissues in HCC samples from TCGA database, and found that the miR-27a-3p was down-regulated in HCC tissues. The level of miR-27a-3p was associated with metastasis, Child-Pugh grade and race. MiR-27a-3p was regarded as a favorable prognosis indicator for HCC patients. Then, miR-27a-3p was overexpressed in HepG2 cell, and was knocked down in PLC cell. Next, we conducted a series of in vitro assays, including MTT, apoptosis and cell cycle assays to observe the biological changes. Further, inhibitor rate and apoptosis rate were detected with pre- and post-cisplatin treatment in HCC. The results showed that overexpression of miR-27a-3p repressed the cell viability, promoted apoptosis and increased the percentage of cells in G0/G1 phase. Importantly, overexpression of miR-27a-3p significantly increased the inhibitor rate and apoptosis rate with cisplatin intervention. Besides, we found that miR-27a-3p added cisplatin sensitivity potentially through regulating PI3K/Akt signaling pathway. Taken together, miR-27a-3p acted as a tumor suppressor gene in HCC cells, and it could be useful for modulating cisplatin sensitivity in chemotherapy.

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.

Role of E-selectin for diagnosing myocardial injury in paediatric patients with mycoplasma pneumoniae pneumonia.

Backgrounds Effects of myocardial injury on E-selectin remain unclear. Thus, we investigated the diagnostic value of E-selectin for myocardial injury in paediatric patients with mycoplasma pneumoniae pneumonia. Methods In this prospective and blinded clinical study, plasma E-selectin, cardiac troponin I, creatine kinase isoenzyme MB, interleukin-6 and tumor necrosis factor alpha concentrations were measured in paediatric patients with mycoplasma pneumoniae pneumonia (MPP group, n = 138). The control group comprised 120 healthy children. The definition of cardiac injury was based on cardiac troponin I or CK-MB (with or possibly without abnormal electrocardiogram evidence). Diagnostic value of E-selectin for myocardial injury was determined by analysing receiver operating characteristic curves. Results Among the 138 mycoplasma pneumoniae pneumonia patients, 40 patients were identified with myocardial injury, while 98 patients were identified without myocardial injury. Plasma E-selectin concentrations were: 40.22 ± 4.80 ng/mL, in patients with myocardial injury; 18.55 ± 2.16 ng/mL, in patients without myocardial injury and 12.39 ± 3.27 ng/mL, in healthy children. For the 40 patients identified with myocardial injury, area under the receiver operating characteristic curve value for plasma E-selectin concentrations was 0.945 (95% CI: 0.899-0.991), and optimal diagnostic cut-off value was 29.93 ng/mL (positive likelihood ratio = 72.5). Conclusion E-selectin was shown to be an effective index for myocardial injury in paediatric patients with mycoplasma pneumoniae pneumonia, and its role in other causes of myocardial injury warrants further investigation.

Role of E-selectin for diagnosis of myocardial injury in children of age up to 14 years.

BACKGROUND: Effects of myocardial injury on E-selectin remain unclear. Thus, we investigated the diagnostic value of E-selectin for myocardial injury in children of no more than 14 years of age, which determined the scoring method of myocardial injury. METHODS: In this prospective study, plasma E-selectin, cardiac troponin I (cTnI) and creatine kinase isoenzyme MB (CK-MB) concentrations in pediatric patients with myocardial injury (myocardial injury group, n=85) were measured. The control group comprised 80 patients without myocardial injury, and the case-control study method was selected at the same time. The definition of cardiac injury was based on cTnI and CK-MB (with or possibly without abnormal ECG evidence). Diagnostic value of E-selectin for myocardial injury was determined by analyzing receiver operating characteristic (ROC) curves. RESULTS: The differences between the two groups were of statistical significance (P<0.001). For the 85 patients with myocardial injury, the area under the ROC curve (AUC) value for plasma E-selectin levels was 0.945 with a 95% CI of 0.899-0.991 and the optimal diagnostic cut-off value 29.67 ng/ml (positive likelihood ratio (positive LR=72.5); AUC value for plasma cTnI level was 0.848 with a 95% CI: 0.737-0.960 and the optimal diagnostic cut-off value was 0.155 µg/L (positive LR=12.3); AUC value for plasma CK-MB levels was 0.946 with a 95% CI: 0.903-0.989 and the optimal diagnostic cut-off value 24.26 IU/L (positive LR=72.5). CONCLUSIONS: E-selectin is more effective than cTnI in diagnosing myocardial injury as an important biological marker of myocardial injury- an important index of pediatric cardiac injury score.

Effects of insulin combined with idebenone on blood-brain barrier permeability in diabetic rats.

This study investigates the effect of insulin combined with idebenone on blood-brain barrier (BBB) permeability in experimental streptozotocin-induced diabetic rats as well as the underlying mechanisms. With a diabetic rat model, we show that insulin and idebenone normalize body weight and water intake and restore BBB permeability and that their combination displays a synergistic effect. The results from transmission electron microscopy show that the combination of insulin and idebenone significantly closed the tight junction (TJ) in diabetic rats. The results from Western blotting in diabetic rats show that the upregulation of TJ-associated proteins occludin, and zonula occludens (ZO)-1 caused by the combination of insulin and idebenone is more remarkable than that with either agent alone. In addition, the activations of reactive oxygen species (ROS) and advanced glycation end products (AGEs) and the expression levels of receptors for advanced glycation end-products (RAGE) and nuclear factor-κB (NF-κB) were significantly decreased after treatment with insulin and idebenone in diabetic rats. These results suggest that the combination of insulin and idebenone could decrease the BBB permeability in diabetic rats by upregulating the expression of occludin, claudin-5, and ZO-1 and that the ROS/AGE/RAGE/NF-κB signal pathway might be involved in the process.