Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography

α-Fetoprotein (AFP) is a prospective biopharmaceutical candidate currently undergoing advanced-stage clinical trials for autoimmune indications. The high AFP expression yields in the form of inclusion bodies in Escherichia coli renders the inclusion body route potentially advantageous for process scale commercial manufacture, if high-throughput refolding can be achieved. This study reports the successful development of an ‘anion-exchange chromatography’-based refolding process for recombinant human AFP (rhAFP), which carries the challenges of contaminant spectrum and molecule complexity. rhAFP was readily refolded on-column at rhAFP concentrations unachievable with dilution refolding due to viscosity and solubility constraints. DEAE-FF functioned as a refolding enhancer to achieve rhAFP refolding yield of 28% and product purity of 95% in 3 h, at 1 mg/ml protein refolding concentration. Optimization of both refolding and chromatography column operation parameters (i.e. resin chemistry, column geometry, redox potential and feed conditioning) significantly improved rhAFP refolding efficiency. Compared to dilution refolding, on-column rhAFP refolding productivity was 9-fold higher, while that of off-column refolding was more than an order of magnitude higher. Successful demonstration that a simple anion-exchange column can, in a single step, readily refold and purify semi-crude rhAFP comprising 16 disulfide bonds, will certainly extend the application of column refolding to a myriad of complex industrial inclusion body proteins.     

Periodic orbits around the collinear libration points in the restricted three body problem when the smaller primary is a triaxial rigid body and bigger primary is a source of radiation pressure

Periodic orbits belonging to the stromgren families A, B and C around the collinear libration points in the restricted three body problem have been studied when the smaller primary is a triaxial rigid body and more massive body is a source of radiation pressure. These families are determined in three different cases: (i) classical case, (ii) when bigger primary is a source of radiation pressure, (iii) when smaller primary is a triaxial rigid body and bigger primary is a source of radiation pressure. The Liapunov stability of each periodic solution has also been examined.      

Radiation dose measurements for staff members involved in holmium-166 preclinical trial

AimNeutron-activated holmium-166 (¹⁶⁶Ho) is an excellent radionuclide for internal radiation therapy (Eβmax = 1.84 MeV) with an appropriate half-life (26.8 h), which emits photons (81 keV, 6.2%) suitable to be detected by gamma cameras. Preparing and injecting radiopharmaceuticals containing beta/gamma emitting holmium-166 implies a risk of exceeding the upper limit for skin and hand radiation equivalent doses (500 mSv/an). This study was aimed to estimate the whole body and finger exposure for staff responsible for dose preparation, dose dispensing, and dose injection of holmium-166 therapy.MethodsTo measure the finger dose from external exposure, all staff members wore TLD dosimeters. Personal dose equivalents Hp(10) were measured using electronic personal dosimeters (EPD MK2, Thermo Fischer Scientific) placed on the left side of the chest. During our study, staff members administered more than 40 ¹⁶⁶Ho-based therapies for preclinical trial. Appropriate radiation safety procedures and shielding were applied at each stage.ResultsIn this study, the whole body doses were 2.80 ± 1.56 nSv MBq⁻¹ for one ¹⁶⁶Ho-therapy preparation/formulation, and 2.68 ± 1.70 nSv MBq⁻¹ for one intravenous injection. Maximum finger doses were 2.9 ± 0.2 μSv MBq⁻¹ and 2.5 ± 0.3 μSv MBq⁻¹ for preparation and injection, respectively (activities injected: 72 ± 3 MBq).ConclusionExtrapolated annual doses from 300 ¹⁶⁶Ho radionuclide therapies were lower than the annual limit doses for skin and the whole body, 500 mSv and 20 mSv, respectively, reported in the European Directive EURATOM 96/29 when applying appropriate radiation protection standards. However, these doses have to be added to other diagnostic or therapeutic protocols, performed in preclinical facilities.     

PIV measurement of the flow past a generic car body with wheels at LES applicable Reynolds number

Experiments by using 2D–2C Particle Image Velocimetry (PIV) were carried out and reported concerning the flow field past a generic car body (modified Ahmed body) which is equipped with wheels and wheel-arches. The Reynolds number was chosen to not exceed 2E+5 based on the height of the Ahmed body which makes it possible to investigate the same configuration by means of Large Eddy Simulation (LES). The wheels were rotating but the ground was stationary. The wheel-ground contact was realized by means of small rectangular openings below the wheels in the ground plane in which the wheels were immersed. The transition contour of the immersed wheels and the ground, as well as the rectangular openings below the wheels were properly sealed to prevent parasite flow and to provide well defined boundary conditions for an upcoming LES investigation.The flow field was measured in several planes with normal vectors pointing towards the directions normal to the free stream. Statistical characteristics of the flow are provided and discussed.     

Application of nanodiamonds in human body fluid analysis by matrix-assisted laser desorption/ionization mass spectrometry

Direct mass spectrometric analysis of complex biological samples is very important and challenging. In this paper, nanodiamonds have been successfully used in matrix-assisted laser desorption/ionization mass spectrometric analysis of human serum and urine. As a practical tool and platform, it can be widely used in the field of humoral proteomics, and it plays a very promising role in clinical diagnosis, including identification of novel disease-associated biomarkers.     

Stability analyses on dynamic control for a moving body levitated magnetically over elastic guideways

Based on the Floquet theory on ordinary differential equations with periodically variable coefficients and the bifurcation approach to nonlinear equations, a numerical approach to determining the stability region of control parameters is established for a dynamic control system composed of a moving body levitated magnetically over flexible guideways. The system is nonlinearly coupled among the elastic deformation of guideways, disturbance the levitation position of the body and electromagnetic control forces. The numerical simulation is given for the system in the case in which the control parameters are taken within and out of the stability region respectively. Results show that this approach is reliable. This project is supported by NSFC(No. 19725207), the Pre-research Project of the Committee of Science and Technology for Defence of China and the Science Foundation of Education Ministry of China for Ph. D. Programmes.     

刚体位移对应变测试的影响

通过运用波前干涉理论,得到了贴片云纹干涉法测圆柱壳的位移与应变公式,分析了刚体位移及曲率对测试结果的影响情况.指出了在对压力容器及管道进行测试时应注意的事项.     

混合新几何体Γ_(-p，i)K的不等式

近来,Lutwak,Yang和Zhang提出了新几何体Γ_(-p)K的概念,获得一些结论.本文引入混合新几何体Γ_(-p,i)K的概念,即新几何体是它的特殊类,得到了混合新几何体的一些性质,并建立了相关的不等式.     

Exact solution of external tangential contact problem for a transversely isotropic elastic half-space

Summary  The following mixed boundary-value problem for a transversely isotropic elastic half-space is considered. Arbitrary tangential displacements are prescribed at the exterior of a circle, while the interior of the circle is free of tangential stress, and the normal stress vanishes all over the boundary. The governing integral equation is solved exactly, in closed form, and in terms of elementary functions. The method of continuation of solutions previously published by the author has been used here. Several examples are considered. No similar results has been reported before, even in the case of an isotropic body. Received 8 May 2000; accepted for publication 20 July 2000