Optimization and application of caprylic acid precipitation in the purification of monoclonal antibody / 生物工程学报

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.

Research on the orientedly immobilized urease via concanavalin A / 生物工程学报

Concanavalin A (ConA) is immobilized on a pre-activated chitosan microspheres, and then oriented immobilization of urease is carried out based on the strong interaction between ConA and glycoprotein. The optimum immobilization conditions are as follows: glutaraldehyde concentration is 3.5%, ConA concentration 1 mg/mL, ConA pH 7.0 and urease concentration 0.4 mg/mL. For orientedly immobilized urease, the highest activity was allowed at pH 5.0-6.0 and temperature 77 degrees C, and the Michaelis constant (Km) was disclosed to be 11.76 mmol/L by Lineweaver-Burk plot. Compared with the free urease and the randomly immobilized urease, the optimum pH of the orientedly immobilized urease becomes smaller and the pH domain wider. Orientedly immobilized urease presents higher temperature resistance, higher affinity to the substrate, and higher stability of operation.

Study on blood supply of lung metastasis with trans-pulmonary arterial lipiodol infusion / 介入放射学杂志

Objectvive To evaluate the blood supply of pulmonary metastases using small volume of lipiodol through pulmonary arterial infusion. Methods 10 cases of lung metastasis were enroled including the primary tumors of liver cancer(n = 5), renal carcinoma(n = 3), chordoma(n = 1) and malignant neurofibroma (n = 1). Plain CT scan was performed to exclude calcification or ossification within metastasis and then pulmonary arterial DSA was undertaken to evaluate tumor vessels or staining. After pulmonary arteriovenous fistula or other anomalous circulation was excluded by lobar arterial DSA, small volume of lipiodol was infused under fluoroscopy (0.5 -1.5 ml for each lobar artery, total volume less than 3.0 ml). CT scan was immediately performed. Blood supply of the pulmonary metastases was assessed according to the accumulation of lipiodol on CT scans. Results No cases but one experienced cough, expectoration, suffocating or dyspnea. No complication of cerebral or visceral embolism occurred. Totally 27 nodules were studied including 6 nodules with cloudy lipiodol accumulation and 6 nodules with tiny granules of lipiodol accumulation. No enlarged tumor vessel or tumor stain was observed within all 27 nodules on pulmonary arterial DSA. Conclusions Pulmonary artery supplys only parts of pulmonary metastases, especially those sited at the peripheral region of the lung. Infusion of small volume of lipiodol through pulmonary artery is safe, and the increased density of lung field could return normal after several days.

Total flavonoid extracted from Sophara japonica buds with assistant of laccase from endophytic Fusarium sp. C-8 / 中草药

Objective To study the application of extracellular laccase from endophytic fungi to total flavonoid extraction. Methods The extracellular laccase vitality of an endophytic Fusarium sp.C-8 from Artemisa annua reached 36.1 U/mL when the fungus was cultivated in revised PDA medium with initial pH 7.0 at 20℃ on a rotary shaker incubator for 6 d. The crude laccase from the cultural medium was applied for extraction of flavonoid from the buds of Sophara japonica. The optimization on the ratio of dry buds to crude enzyme liquid,temperature,time,and pH value was carried out during the process of laccase-assistant treatment. Results After incubation with 40∶1 of crude enzyme (pH 7.0) at 20℃ to dry buds for 1 h,the extraction rate of total flavonoids was 11.4 %,a more increase of 28.7 % than that in regular extraction process. Conclusion The results present a practical method of laccase-assistant extraction process on total flavones in S. japonica buds.