Therapeutic protein expression platform of microbial system

A number of expression systems have been developed for the production of pharmaceutical products. Pichia pastoris and Escherichia coli expression system operate in our lab and express antibody fragment (scFv), cytokine, protein base adjuvant and vaccine and process enzyme. The expression platform are consisted of three part, first is strain generation , the second is fermentation process development in 250 ml fermentor and the last is process scale-up to 5 litter fermentor. Please click Additional Files below to see the full abstract

Gram Level scFv expression platform of Pichia pastoris

The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform. Please click Additional Files below to see the full abstract

Gram level scFv expression platform of Phichi pastoris

The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform Please click Additional Files below to see the full abstract

Engineering of Escherichia coli protein expression process development

It almost 30% protein drugs are expression by Escherichia coli, because of rapid growth and high production yield. We have developed E.coli base system for recombinant protein expression, scFv, Fab and vaccine. In this study we introduce example about process development for nutrient components selection. Shaker flasks were used for different nitrogen and carbon components screening by DoE. Seven media formulations for E. coli fermentation were used in this study. By changing nitrogen and carbon source ratio, product titer of target protein could be optimized, at least 1.4 folds increased. The best result from shaker flask was used in 250 mL parallel fermenter and pH, dissolved oxygen, feeding/induction strategy were evaluated. The processes from seed culture to harvest only require 64 hours. The optimized time was reduced to 32 hours. The result showed that both target protein expression and cell density value were comparable, but the total process time was significantly reduced by half Please click Additional Files below to see the full abstract

The microbial antibodies secretion expression platform with scale down fermentors

Therapeutic antibodies have become one of the most effective therapeutics for human diseases such as cancer, inflammation and viral infection. The production of antibody-based drugs using microbial expression systems is more cost effective with ease of gene manipulation compared to mammalian expression systems. In our team, antibody fragments (ex: BsAb, scFv and Fab) were produced from methylotrophic yeast Pichia pastoris secretion expression system with the AOX1 as driven promoter or E. coli secretion expression system. To achieve high production yield for both system, we investigated fermentation parameter such as base medium, induction medium, induction condition, feeding strategy and pH. For the 250 ml fermentor Pichia system, the nitrogen have been add into glycerol fed medium and/or methanol induction medium and also compared base-medium, buffered glycerol-complex medium (BMGY) and basal salt medium (BS). The highest scFv production was yielded from the basal salt medium as base medium, glycerol fed medium plus nitrogen and multiple carbon source methanol induction medium. This process can yielded over 500 mg/L scFv. After scale-up from 250 ml fermentor to 5L fermentor, the methanol fed-back control system also applied on the 5 L fermentor, can achieve 1.7 g/L scFv in 5 days. The E. coli expression process has passed through screening for high production yield clones in 2 ml deep-well then confirmed by using 250 ml flask scale. Feeding medium, DO, pH etc, parameters were investigated by parallel 250 ml-fermenter. The parameters from 250 ml fermentor were validated by using 5 L fermenter. Under this scale-up procedure, the antibody Fab was 100 folds production yield, production deep well stage at 1 mg/L, production from 250 ml fermentor stage is 50-100 mg/L and production 5 L fermentor stage is over 35-90 mg/L. Although different antibodies will result in different production yield, building a reliable platform to predict production yield from antibody cell clones under deep well and shake flask stage serves a good scale-down model for future scale-up prediction

A preliminary study on the immune responses of HPV16-E7 by combined intranasal immunization with lymphotoxin

Objectives: Human papillomavirus (HPV) ranks the first cause of cervical cancer. Cervical cancer has high prevalence ratesin women around the world. The HPV-E7 oncoprotein is expressed in cervical cancer and is a target of developing immunotherapiesagainst HPV-associated tumors. However, the antigenicity of this protein is low. Due to this reason, potentadjuvants are required to enhance its therapeutic efficacy. This preliminary study aims to evaluate whether lymphotoxin(LT) could act as an effective immune adjuvant for HPV infection in mice models.Material and methods: Intranasal immunization was used to explore the effect of HPV-E7 and/or LT immune response.After the third intranasal immunization, the titer for the HPV-E7 antibody was detected in serum and vaginal washing fluid.Also, we assessed the expression of chemokine ligand 13 (CXCL13) and Peripheral Node Addressin (PNAd) in the lymphnodes after intranasal immunization with immunohistochemical analysis.Results: compared to HPV-E7 immunization, intranasal immunization with HPV-E7 plus LT significantly increased HPV-E7-specificserum IgG and vaginal IgA titers. Furthermore, the combined use of HPV-E7 and LT strongly induced E7-specific CTLresponses.Conclusions: LT can be effective for intranasal immunized HPV-E7 to improve E7-specific immune responses to HPV infection.It is new approach to eradicate chronic HPV infection capable of inducing an effective anti-infection method

Lasing on nonlinear localized waves in curved geometry

The use of geometrical constraints opens many new perspectives in photonics and in fundamental studies of nonlinear waves. By implementing surface structures in vertical cavity surface emitting lasers as manifolds for curved space, we experimentally study the impacts of geometrical constraints on nonlinear wave localization. We observe localized waves pinned to the maximal curvature in an elliptical-ring, and confirm the reduction in the localization length of waves by measuring near and far field patterns, as well as the corresponding dispersion relation. Theoretically, analyses based on a dissipative model with a parabola curve give good agreement remarkably to experimental measurement on the transition from delocalized to localized waves. The introduction of curved geometry allows to control and design lasing modes in the nonlinear regime.Comment: 6 pages, 6 figure

The efficiency of endothelin receptor antagonist bosentan for pulmonary arterial hypertension associated with congenital heart disease: A systematic review and meta-analysis

BACKGROUND: Oral bosentan has been widely applied in pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD). A systemic review and meta-analysis was conducted for a therapeutic evaluation of oral bosentan in both adult and pediatric patients with PAH-CHD. The acute responses and a long-term effect were respectively assessed in a comparison with baseline characteristics, and the improvement of exercise tolerance was analyzed. METHODS: PubMed, Medline, Embase, and Cochrane Central Register of clinical controlled trails or observational studies have been searched for a recording of bosentan effects on the PAH-CHD participants. For mortality and rate of adverse events (AEs), it was described in detail. Randomized-effects model or fixed-effects model was used to calculate different effective values with a sensitivity analysis. RESULTS: Seventeen studies were pooled in this review, and 3 studies enrolled the pediatric patients. Among all studies, 456 patients were diagnosed with PAH-CHD, and 91.7% were treated with oral bosentan. With a term less than 6 months of bosentan therapy, there existed a significant improvement in 6-minute walk distance (6MWD) and the World Health Organization functional class (WHO-FC), but no such differences in Borg dyspnea index scores (BDIs) and the resting oxygen saturation (SpO2). Although with a prolonged treatment, not only 6MWD and FC, but also the resting SpO2 and heart rate were changed for a better exercise capability. Additionally, compared with the basic cardiopulmonary hemodynamics, it showed a statistically significant difference in mean pulmonary arterial pressure (mPAP) and pulmonary vascular resistance index (PVRi). Although a limitation of pooled studies with comparative outcomes of different terms, outcomes presented a lower WHO-FC which contributes to a success in a prolonged treatment. CONCLUSIONS: Bosentan in PAH-CHD is well established and still requires clinical trials for an identification of its efficiency on CHD patients for an optimized period lessening a serious complication and the common AEs

Shape Effects of Iron Nanowires on Hyperthermia Treatment

This research discusses the influence of morphology of nanomagnetic materials (one-dimensional iron nanowires and zero-dimensional iron nanoparticles) on heating efficiency of the hyperthermia treatment. One-dimensional iron nanowires, synthesized by reducing method in external magnetic field, are explored in terms of their material properties, magnetic anisotropy, and cytotoxicity of EMT-6 cells. The magnetic anisotropy of an array of nanowires is examined in parallel and perpendicular magnetic fields by VSM. For the magnetic hyperthermia treatment tests, iron nanowires and nanoparticles with different concentrations are heated in alternating magnetic field to measure their actual heating efficiency and SLP heating properties. The shape effects of iron nanomaterials can be revealed from their heating properties. The cytotoxicity of nanowires with different concentrations is measured by its survival rate in EMT-6 with the cells cultivated for 6 and 24 hours