Purification optimization for a recombinant single-chain variable fragment against type 1 insulin-like growth factor receptor (IGF-1R) by using design of experiment (DoE).

Design of experiment (DoE) is a statistics-based technique for experimental design that could overcome the shortcomings of traditional one-factor-at-a-time (OFAT) approach for protein purification optimization. In this study, a DoE approach was applied for optimizing purification of a recombinant single-chain variable fragment (scFv) against type 1 insulin-like growth factor receptor (IGF-1R) expressed in Escherichia coli. In first capture step using Capto L, a 2-level fractional factorial analysis and successively a central composite circumscribed (CCC) design were used to identify the optimal elution conditions. Two main effects, pH and trehalose, were identified, and high recovery (above 95%) and low aggregates ratio (below 10%) were achieved at the pH range from 2.9 to 3.0 with 32-35% (w/v) trehalose added. In the second step using cation exchange chromatography, an initial screening of media and elution pH and a following CCC design were performed, whereby the optimal selectivity of the scFv was obtained on Capto S at pH near 6.0, and the optimal conditions for fulfilling high DBC and purity were identified as pH range of 5.9-6.1 and loading conductivity range of 5-12.5 mS/cm. Upon a further gel filtration, the final purified scFv with a purity of 98% was obtained. Finally, the optimized conditions were verified by a 20-fold scale-up experiment. The purities and yields of intermediate and final products all fell within the regions predicted by DoE approach, suggesting the robustness of the optimized conditions. We proposed that the DoE approach described here is also applicable in production of other recombinant antibody constructs.

Microcatheter looping facilitates access to both the acutely angled parent artery and cerebral aneurysms for effective embolization.

Aneurysms with an acutely angled parent artery are difficult to access for coiling. This study aimed to investigate the safety and effectiveness of microcatheter looping for embolization of cerebral aneurysms with access difficulty. Ten patients (male:female=5:5) with cerebral aneurysms treated with the microcatheter looping technique were analyzed retrospectively. The parent artery formed an acute angle with the major artery in five aneurysms. The microcatheter was looped into a "α" loop for treatment in the anterior temporal artery aneurysm and a "U" loop in the remaining nine aneurysms. All ten aneurysms were successfully treated with the microcatheter looping technique. The microcatheter tip was successfully navigated into the aneurysm sac and remained stable throughout the embolization process. All aneurysms were occluded with total occlusion in five and near-total occlusion in five, and the parent artery remained patent in all cases. No complications occurred peri-procedurally. The Glasgow Outcome Scale was 5 in all patients before discharge. Follow-up angiography six to 12 months later revealed a good occlusion status of the aneurysms. The microcatheter looping technique is effective when the conventional embolization technique fails to treat cerebral aneurysms with difficult access especially when the parent artery forming an acute angle with the major artery exacerbates difficult access to the aneurysms.

The stent-assisted coil-jailing technique facilitates efficient embolization of tiny cerebral aneurysms.

OBJECTIVE: Tiny cerebral aneurysms are difficult to embolize because the aneurysm's sac is too small for a single small coil, and coils within the aneurysm may escape from the confinement of a stent. This study was performed to introduce the stent-assisted coil-jailing technique and to investigate its effect on the coil embolization of tiny intracranial aneurysms. MATERIALS AND METHODS: Sixteen patients with tiny intracranial aneurysms treated with the stent-assisted coil-jailing technique between January 2011 and December 2013 were retrospectively reviewed and followed-up. RESULTS: All aneurysms were successfully treated with the coil-jailing technique, and at the end of embolization, complete occlusion of the aneurysm was achieved in 9 cases (56.3%), incomplete occlusion in 6 (37.5%), and partial occlusion in 1 (6.3%). Intraprocedural complications included acute thrombosis in one case (6.3%) and re-rupture in another (6.3%). Both complications were managed appropriately with no sequela. Follow-up was performed in all patients for 3-24 months (mean, 7.7 months) after embolization. Complete occlusion was sustained in the 9 aneurysms with initial complete occlusion, progressive thrombosis to complete occlusion occurred in the 6 aneurysms with initial near-complete occlusion, and one aneurysm resulted in progressive thrombosis to complete occlusion after initial partial occlusion. No migration of stents or coils occurred at follow-up as compared with their positions immediately after embolization. At follow-up, all patients had recovered with no sequela. CONCLUSION: The stent-assisted coil-jailing technique can be an efficient approach for tiny intracranial aneurysms, even though no definite conclusion regarding its safety can be drawn from the current data.

Effects of (-)doxazosin on histomorphologic and cell apoptotic changes of the hyperplastic prostate in castrated rats.

BACKGROUND: (-)Doxazosin is one of the enantiomers of (+/-)doxazosin, and it was reported that (-)doxazosin possessed higher selectivity for lower urinary tract between the cardiovascular system and the urinary system in the animal experiments in comparison with that of (+/-)doxazosin and (+)doxazosin. Therefore, it is important to know whether (-)doxazosin has a therapeutic effect on the hyperplastic prostate. METHODS: (-)Doxazosin and (+/-)doxazosin were administered intragastrically to prostatic hyperplasia rats, induced by testosterone propionate for 4 weeks, and each experimental group contained 8 animals. The histomorphologic changes of the prostate were observed under light microscope, the quantitative analysis of the prostatic glandular cavity was performed using an image analysis system, and the cell apoptosis was detected by using flow cytometry. RESULTS: In comparison with model-control group, the volume index of the prostate in (-)doxazosin 3.0 mg/kg group became significantly smaller. The maximal diameter, perimeter, and area of the hyperplastic prostate glandular cavity, and the glandular epithelial cell height in (-)doxazosin (0.3, 1.0, and 3.0 mg/kg) groups and (+/-)doxazosin group were significantly reduced. (-)Doxazosin and (+/-)doxazosin did not significantly affect cell cycle distribution and cell proliferation index of the hyperplastic prostate. However, apoptotic rates of the prostatic cells in (-)doxazosin (0.3, 1.0, and 3.0 mg/kg) groups and (+/-)doxazosin group were significantly increased in comparison with those of model-control group. CONCLUSIONS: Both (-)doxazosin and (+/-)doxazosin inhibit the prostatic hyperplasia induced by testosterone propionate in castrated rats. The induction of prostate cell apoptosis might be one of the mechanisms underlying the therapeutic role of (-)doxazosin.