838 research outputs found
Reduced Temperature Production of Recombinant Proteins to Increase Productivity in Mammalian Cell Culture
The production of recombinant proteins from an industrial perspective has one of its main goals is to increase the product concentration whether in batch, fed-batch or continuous perfusion bioreactor systems. However, a major problem trying to achieve high product concentration over prolonged cultivation is the loss of cell viability leading to reduced production rate and lower product quality. One possible means to achieve high product concentration and main high cell viability is to perform the bioreactor operations at a reduced temperature than that traditional used for mammalian cell cultivation.
A collaborative research project between MIT and the Bioprocessing Technology Institute (BTI) was established where the MIT Ph.D. candidate (S.R. Fox) performed his research in Singapore with the assistances of BTI personnel. The goal of this project was the production of recombinant gamma interferon (γ -IFN) in Chinese Hamster Ovary (CHO) cells by operating the bioreactor at 32°C in contrast to cultivating the CHO cells at the traditional temperature of 37°C. By reducing the cultivation temperature to 32°C, we have found that the specific γ -IFN productivity can be increased to 400% as compared to the higher temperature (37°). This increase was the result of two factors. First the cell death was reduced at the lower temperature and second, the mRNA for the γ -IFN gene was greater (presumably through decreased mRNA degradation).
However, at the reduced temperature, the cellâs specific growth was also impaired. Mutation and selection for higher growth rate strain at the reduced temperature was successful but we are concerned with the genetic stability of such mutants. Therefore a new collaborative project has been initiated using molecular genetics to engineer new CHO strains with higher growth rate at the reduced temperatures. The preliminary findings from this new project will be presented as a poster in this Symposium by Mr. Hong Kiat Tan.Singapore-MIT Alliance (SMA
The Effect of Culture Temperature on Recombinant IFN-Îł Production and Quality
The goal of this research project is to analyze the effect of culture temperature on the production and quality of IFN-Îł produced and secreted by suspension culture CHO cells.The effect of low temperature on IFN-Îł glycosylation, which is under the control of a battery of enzymes whose activities will be influenced by temperature, is unknown. Work is focused on implementing a system for accurately monitoring the glycosylation of IFN-Îł and then using the system for quantifying the effect of culture temperature on glycosylation. The system consists of immunoaffinity purification of IFN-Îł , followed by capillary electrophoresis for determining glycosylation macroheterogeneity and MALDI-TOF MS and HPLC for determining glycosylation microheterogeneity. Initial results suggest that glycosylation macroheterogeneity is slightly decreased (~5%) at low temperature, thereby identifying a potential quality âbottleneckâ for the use of low temperature to increase IFN-Îł production.
Low temperature (32°C) shifts the cells towards the non-growth, G1 portion of the cell cycle. In batch culture, if cells are shifted to low temperature once a reasonably high cell density is reached, an approximately 4-fold improvement in total IFN-γ production compared to 37°C culture is achieved. Pseudo-continuous culture was used to show that IFN-γ production is statistically significantly higher at 32°C compared to 37°C even when nutrient depletion is not a concern (p < 0.5). In fed-batch bioreactor culture, cells grown at low temperature display a short period of growth followed by a prolonged stationary phase of high specific IFN-γ productivity (~4-fold higher than compared to 37°C) whereas cells at 37°C grow rapidly, reach a peak cell density and then begin to die immediately. The net result is a 2-fold increase in total IFN-γ production at low temperature. Real-time RT-PCR was used to show that the amount of IFN-γ mRNA present during the 32°C stationary production phase is approximately 4-fold higher than the amount present during the exponential growth phase of the 37°C culture.
To further explore the effect of low temperature on cell RNA levels, total RNA per cell was quantified during the course of batch cultures at 32°C and 37°C. Total RNA levels were found to be approximately 50% higher at 32°C than 37°C. The kinetics of the low temperature RNA concentration profile was modeled to obtain transcription (Ks) and degradation (Kd) rate constants and these were found to be consistent with literature values. This finding suggests that temperature shift may offer a novel approach for measuring RNA kinetic parameters in any cell system that can tolerate mild temperature changes.Singapore-MIT Alliance (SMA
Active Hypothermic Growth: A Novel Means For Increasing Total Interferon-Îł Production by Chinese Hamster Ovary Cells
When grown under hypothermic conditions, Chinese Hamster Ovary (CHO) cells become growth arrested in the Gâ/Gâ phase of the cell cycle and also often exhibit increased recombinant protein production. In this study, we have validated this hypothesis by stimulating hypothermic growth using basic fibroblast growth factor and fetal bovine serum supplementation. This method led to 7.7- and 4.9-fold increase in total production compared to the 37°C and 32°C control cultures, respectively. This proof-of-concept study will motivate the creation of cell lines capable of growing at low temperatures for use in industrial processes.Singapore-MIT Alliance (SMA
Improvement of Wear Performance of Nano-Multilayer PVD Coatings under Dry Hard End Milling Conditions Based on Their Architectural Development
The TiAlCrSiYN-based family of PVD (physical vapor deposition) hard coatings was specially designed for extreme conditions involving the dry ultra-performance machining of hardened tool steels. However, there is a strong potential for further advances in the wear performance of the coatings through improvements in their architecture. A few different coating architectures (monolayer, multilayer, bi-multilayer, bi-multilayer with increased number of alternating nano-layers) were studied in relation to cutting-tool life. Comprehensive characterization of the structure and properties of the coatings has been performed using XRD, SEM, TEM, micro-mechanical studies and tool-life evaluation. The wear performance was then related to the ability of the coating layer to exhibit minimal surface damage under operation, which is directly associated with the various micro-mechanical characteristics (such as hardness, elastic modulus and related characteristics; nano-impact; scratch test-based characteristics). The results presented exhibited that a substantial increase in tool life as well as improvement of the mechanical properties could be achieved through the architectural development of the coatings
Radiative Corrections to One-Photon Decays of Hydrogenic Ions
Radiative corrections to the decay rate of n=2 states of hydrogenic ions are
calculated. The transitions considered are the M1 decay of the 2s state to the
ground state and the E1(M2) decays of the and states to
the ground state. The radiative corrections start in order , but the method used sums all orders of . The leading
correction for the E1 decays is calculated and compared
with the exact result. The extension of the calculational method to parity
nonconserving transitions in neutral atoms is discussed.Comment: 22 pages, 2 figure
Scaling anomaly in cosmic string background
We show that the classical scale symmetry of a particle moving in cosmic
string background is broken upon inequivalent quantization of the classical
system, leading to anomaly. The consequence of this anomaly is the formation of
single bound state in the coupling interval \gamma\in(-1,1). The inequivalent
quantization is characterized by a 1-parameter family of self-adjoint extension
parameter \omega. It has been conjectured that the formation of loosely bound
state in cosmic string background may lead to the so called anomalous
scattering cross section for the particles, which is usually seen in molecular
physics.Comment: 4 pages,1 figur
Coherent states for exactly solvable potentials
A general algebraic procedure for constructing coherent states of a wide
class of exactly solvable potentials e.g., Morse and P{\"o}schl-Teller, is
given. The method, {\it a priori}, is potential independent and connects with
earlier developed ones, including the oscillator based approaches for coherent
states and their generalizations. This approach can be straightforwardly
extended to construct more general coherent states for the quantum mechanical
potential problems, like the nonlinear coherent states for the oscillators. The
time evolution properties of some of these coherent states, show revival and
fractional revival, as manifested in the autocorrelation functions, as well as,
in the quantum carpet structures.Comment: 11 pages, 4 eps figures, uses graphicx packag
Analysis of Residual Stresses in Laser-Shock-Peened and Shot-Peened Marine Steel Welds
Laser peening is now the preferred method of surface treatment in many applications. The magnitude and depth of the compressive residual stress induced by laser peening can be influenced strongly by the number of peen layers (the number of laser hits at each point) and by processing conditions including the use of a protective ablative layer. In this study, residual stresses have been characterized in laser and shot-peened marine butt welds with a particular focus at the fatigue crack initiation location at the weld toe. X-ray diffraction, synchrotron X-ray diffraction, incremental center-hole drilling, and the contour method were used for determination of residual stress. Results showed that the use of ablative tape increased the surface compressive stress, and the depth of compressive stress increased with an increase in number of peening layers. A key result is that variation of residual stress profile across laser peen spots was seen, and the residual stress magnitude varies between the center and edges of the spots
Calibration of the Transport Parameters of a Local Problem of Water Quality in Igap\'o I Lake
The calibration of a model refers to the process by which one can estimate
some parameters by comparisons with observed data. Due to the dynamical nature
of the environment, variations between predicted and observed values occur.
Thus, the environmental parameters may vary due to random temperature changes,
time of discharge flow, time of the day, and other conditions. Such variations
can be minimized by identifying and optimizing some parameters of the transport
model, like the values of diffusion coefficients in x and y directions and the
kinetic parameter that describes the process of removing pollutants. This paper
presents results concerning the calibration of transport parameters for
two-dimensional problems of water quality (fecal coliform control) at Igap\'o I
Lake, located in Londrina, Paran\'a, Brazil. Thus, the
convection-diffusion-reaction equation, which describes mathematically the
process studied in this work, is resolved by a semidiscrete finite element
method (SUPG) which combines finite differences in time and finite elements in
space.Comment: 8 Pages, 3 figures, 2 tables, XVIII International Conference on Water
Resources CMWR 2010 - J. Carrera (Ed) - CIMNE, Barcelona 201
Search for Colour Singlet and Colour Reconnection Effects in Hadronic Z Decays at LEP
A search is performed in symmetric 3-jet hadronic Z decay events for evidence
of colour singlet production or colour reconnection effects. Asymmetries in the
angular separation of particles are found to be sensitive indicators of such
effects. Upper limits on the level of colour singlet production and colour
reconnection effects are established for a variety of models
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