Influence of cultivation parameters or supplement on product qualities and culture performances during perfusion

Perfusion processes have been developed with technological advances in single-use bioreactor and cell retention device. Perfusion has advantages such as high cell density culture in compact facilities and media change or culture parameter shifts for protein production with desired qualities. Single use bioreactor (200L or 1000L) with ATF System was used for the clinical production. For the process development, the scale-down model was established with lab-scale (2L) bioreactor with ATF2. A recombinant CHO cell line producing a fusion protein was cultivated using in-house serum-free media. Influence of insulin (0~3mg/L) on qualities was investigated in the established perfusion process using in-house serum-free medium and 2L scale-down model. The results showed that low concentration of insulin enhanced O-glycosylation and -2 charged N-glycan of fusion protein. Temperature (30~34oC) conditions, under no addition of insulin, were investigated to evaluate effect on qualities. High temperature enhanced O-glycosylation and -2 charged N-glycan of fusion protein. To evaluate interaction among culture parameters (Temperature, pH, and Dissolved Oxygen) in the perfusion process, Central Composite Inscribed (CCI) was selected as design of experiment. 20 perfusion cultures were carried out in the 2L scale-down model. The results showed each parameter and interactions among parameters had an effect on qualities and culture performances

Possible Novel Therapy for Malignant Gliomas with Secretable Trimeric TRAIL

Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and many novel therapeutic approaches, average survival for the patients with malignant gliomas is only about 1 year. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancers, but concerns over delivery and toxicity have limited progress. We have developed a secretable trimeric TRAIL (stTRAIL) and here evaluated the therapeutic potential of this stTRAIL-based gene therapy in brain tumors. An adenovirus (Ad-stTRAIL) delivering stTRAIL was injected into intra-cranial human glioma tumors established in nude mice and tumor growth monitored using the magnetic resonance imaging (MRI). Ad-stTRAIL gene therapy showed potent tumor suppressor activity with no toxic side effects at therapeutically effective doses. When compared with 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a conventional therapy for malignant gliomas, Ad-stTRAIL suppressed tumor growth more potently. The combination of Ad-stTRAIL and BCNU significantly increased survival compared to the control mice or mice receiving Ad-stTRAIL alone. Our data indicate that Ad-stTRAIL, either alone or combined with BCNU, has promise as a novel therapy for malignant gliomas

Structure and Dynamics of Theophylline in D2O Investigated by 2D IR

Theophylline is one of the xanthine derivatives. The molecular structure of theophylline is like purine (i.e., adenine and guanine), so it acts like a competitive and nonspecific ribonucleoside inhibitor. Because of this character, theophylline has been widely used as tablet pills to help asthma patients. The crystal structure of theophylline monohydride and the intermolecular interaction between theophylline and DNA/RNA sequences has been reported to improve its pharmaceutical usage. We investigated the structure and dynamics of theophylline using Fourier transform infrared (FT IR) and two-dimensional infrared (2D IR) spectroscopy to understand molecular couplings and vibrational dynamics of two carbonyl groups, which have not been studied so far. We obtained time-resolved 2D IR spectra of theophylline in D2O solution from T = 0 to T = 10 ps, with four different polarization sets. We observed cross peaks between the two C=O modes of theophylline in the 2D IR spectra, where the magnitude and sign of the cross peaks depend on the polarization of the involved IR pulses. The two modes were determined to be almost orthogonal to each other due to strong coupling. Time-dependent 2D IR spectra revealed energy transfer between the two modes and H-bond exchange between the C=O and water occurring predominantly in one of the C=O groups

2D IR Study of the Aggregation Pathway of hIAPP oligomers

Type 2 diabetes (T2D) is a disease in which human islet amyloid polypeptide (hIAPP) amyloid deposits attack ??-cells, causing insulin production problems and the loss of blood sugar control. Although the formation process of hIAPP deposits has been studied for several decades, the early stage of the aggregation mechanism is not fully understood due to the transient nature of intermediate oligomers. In this regard, we slowed down the aggregation process by lowering the sample temperature and observed the aggregation process in detail. The aggregation process was clearly observed by 2D IR to be reversible at the early stage aggregation

Direct Evidence of Channel-Water Dynamics Related with the Structure Changes in a Transmembrane Ion Channel

Knowledge of both structural and dynamical characteristics are essential to understand the function of membrane proteins. Two-dimensional infrared (2D IR) spectroscopy can detect small changes in secondary structure as well as measure fast structure dynamics on the picosecond timescale. Here, we report a 2D IR spectroscopic investigation to probe the backbone configurations of the transmembrane domain of M2 proton channel in a solid-supported membrane environment. Our results show that a helix kink at higher pH (7.5) occludes the passage of water molecules into the channel, making the channel inactive. A conformational change upon lowering the pH (5.5), shows ultrafast hydrogen-bond dynamics between the amide carbonyls of the transmembrane protein and the water molecules, confirming the presence of water inside the channel in the active state. This result suggests that the rate of proton diffusion is closely related to the water dynamics induced by the structural change in the protein

Application of 2D IR to Study h-IAPP Aggregation and Perovskite Films

Since two-dimensional infrared (2D IR) spectroscopy was introduced in 1998, 2D IR has rapidly developed and is being applied to various fields. A variety of experimental methods are used nowadays. The pump???probe (PP) and the heterodyned photon echo (HPE) methods are the most widely used among them. In terms of experimental difficulty, HPE has an advantage over PP. However, PP has a relatively lower signal-to-noise ratio than HPE. When both techniques are applied to samples producing strong scatterings, such as metal???organic frameworks (MOFs), protein aggregates, and perovskite films, the spectra are severely contaminated by the scattered light. In the PP method, this problem can be circumvented by phase cycling. However, in the HPE method, phase cycling does not function as effectively as in the PP method. This presentation demonstrates that the scattering problem in the HPE approach can be solved mechanically by introducing choppers or shutters without moving any translation stages.[1] This scatter-removing technique was applied to investigate a perovskite film with a very rough surface and hIAPP protein aggregation. The perovskite film was found to contain a tiny amount of dimethylformamide and H2O. Temperature-dependent study on hIAPP shows that the proteins are denatured at 4 ??C from ??-sheets to random coils and ??-turns