Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity

A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day-1) for 4 days, increasing gradually to 2 day-1 at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line. © 2004 Kluwer Academic Publishers

Fluorescent quantitation method for differentiating the nativity of green fluorescent protein

A gel imaging method was employed to quantitate the GFP that had been subjected to denaturation and degradation treatments. This method is able to differentiate the nativity of GFP by relating the observed changes in the position of fluorescent bands which is unable to be detected using the spectrofluorometric method

Effect of Bcl-2 overexpression on cell cycle and antibody productivity in chemostat cultures of myeloma NS0 cells

Chemostat cultures of NS0 cell lines were carried out at dilution rates ranging from 0.8 d-1 to 0.2 d-1. Compared with the control, the viable cell density of the Bcl-2 cell line was approximately 10% higher at 0.8 d-1 and increased to 55% when the dilution rate was reduced to 0.2 d-1. As the dilution rate was reduced, the viability of the two cultures diverged reaching a difference of 43% at 0.2 d-1. The specific growth rate of the control cells was the same as the dilution rate down to a value of 0.6 d-1. By contrast, the specific growth rate of Bcl-2 cells was parallel to the dilution rate down to a value as low as 0.3 d-1. For both NS0 cell lines, the G1 cell population decreased, while the S and G2/M cell populations increased as the dilution rate was reduced. The antibody titer of the control cells increased from 7 to 21 μg·ml-1 as the dilution rate was reduced from 0.8 to 0.2 d-1. With an initial increase from 2 to 15 μg·ml-1 as the dilution rate was reduced from 0.8 to 0.4 d-1, the antibody titer of the Bcl-2 cells remained constant as the dilution rate was further reduced to 0.2 d-1. A good correlation between specific antibody production rate and the percentage of G2/M cells was observed. © 2005, The Society for Biotechnology

Onset of natural convection in gas-gas system induced by bottom-up transient mass diffusion

The onset of convection induced by transient mass diffusion in a stationary gas was succesfully predicted with transient instability theory and simulated using a computational fluid dynamics (CFD) scheme. 2D time-dependent simulations were conducted for bottom-up diffusion of a light gas in a stagnant heavy gas. The results of simulations were used to calculate the transient Rayleigh number adopted from the theory of Tan and Thorpe (1996 and 1999). The average transient maximum Rayleigh number from simulations is 707, which is close to the theoretical value of 817 for analogous bottom heating with constant heat flux. The simulated critical times of the onset of convection were in reasonably good agreement with the predicted values from theory

Core-shell versus inert polymer grafted adsorbents for the negative chromatography of virus-like particle

Core-shell and polymer grafted adsorbents are new generation media developed for the separation of virus-like particle (VLP) in a negative chromatography. The inert shell and grafted polymer chain are designed to exclude the big biomolecules such as VLP from adsorbing onto the ligands situated on the surface of the adsorbents. Meanwhile, these exclusion layers should be permeable for the smaller impurities which will be adsorbed by the ligands to prevent its presence in the flowthrough fractions. In this study, the performance of these negative chromatography media were compared in the purification of recombinant hepatitis B VLPs (HB-VLPs) from clarified E. coli feedstock. The core-shell adsorbents with different shell thickness (InertShell and InertLayer 1000) and poly[(ethylene glycol) methacrylate] grafted adsorbents (SQ) were studied in a packed bed mode. SQ adsorbed more impurities, thus achieving a higher purity in flowthrough while core-shell adsorbents recovered more HB-VLPs and recorded nearly 100% recovery in InertShell. This suggests the shielding effect of the core shell layer is higher than the inert polymer chain. For core-shell adsorbents, there was a trade-off between the purity and recovery of flow-through HB-VLPs due to the shell thickness. A thicker shell allows more HB-VLP exclusion but less impurities adsorption. Prolonging the residence time of the negative chromatography only resulted in a slight improvement in the impurities adsorption in all adsorbents, but the recovery of HB-VLPs in InertShell was reduced substantially. Atomic force microscopic (AFM) analysis revealed funnel-shaped pore channels on the shell layer which may contribute to the entrapment of HB-VLPs on core-shell adsorbents, thus decreasing the HB-VLP recovery. Overall, SQ performed better than the core-shell adsorbents in handling feedstock with high concentration

Regions on Nucleocapsid Protein of Newcastle Disease Virus that Interact with its Phosphoprotein

Summary. The nucloecapsid (NP) and phosphor-(P) proteins of paramyxoviruses are involved in transcription and replication of the viral genome. An in vitro protein binding assay was used to investigate the regions on NP protein that interact with the P protein of Newcastle disease virus (NDV). Truncated NP mutants were first immobilized on a solid phase and then interacted with radio-labelled [35S]-P protein synthesised in rabbit reticulocyte. The interaction affinity was quantitated by measuring the radioactivity that was retained on the solid phase. Using the approach, a highly interactive region was identified to be resided at the first 25 amino acids of NPN-terminal. The interaction between these two proteins remained strong even with the removal of 114 amino acids from the C-terminal end might have another contact region for P protein, which is not as critical as the N_terminal end. The interaction regions mapped in this study are significantly different from the other two paramyxoviruses: Sendai and measles viruses in which the C-termini of their NP proteins play an important role in binding to the P

Production and purification of the phosphoprotein of Nipah virus in Escherichia coli for use in diagnostic assays.

ABSTRACT Nipah Virus (NiV) is an emerging zoonotic paramyxovirus that can be fatal in humans and various types of animals. The phospho (P) protein of NiV plays an important role in RNA synthesis, replication, and genome synthesis. In this study, the NiV P gene was cloned into a pTrcHis2-TOPO vector and the recombinant protein containing a His-tag was produced in Escherichia coli. SDS-PAGE and Western blot analysis using the anti-His antibody confirmed the protein expression. An optimization study of E. coli fermentation showed that the optimal cultivation temperature was 37°C, while the optimal induction time for P protein expression was at 9 h with 1 mM IPTG. Solubility analysis showed that E. coli cultivated at 37°C produced the highest fraction (70%) of soluble P protein. The recombinant P protein was purified from clarified E. coli lysate using an immobilized metal affinity chromatography (IMAC) technique to a purity of 92.67%, with a purification factor of 11.58. The purified P protein strongly reacted with the anti-NiV swine sera collected during a NiV outbreak, suggesting its potential as a diagnostic reagent

Comparison of alginate and pectin based beads for production of poultry probiotic cells.

A comparative study on the stability and potential of alginate and pectin based beads for production of poultry probiotic cells using MRS medium in repeated batch fermentation was conducted. The bead cores, made of three types of materials, i.e., ca-alginate, ca-pectinate and ca-alginate/pectinate, were compared. The effect of single and double layer coatings using chitosan and core material, respectively, on the bead stability and cell production were also studied. The pectin based beads were found to be more stable than that of the alginate beads and their stability was further improved by coating with chitosan. The cell concentration in pectin based beads was comparable to that in the alginate beads. On the other hand, pectin based beads gave significantly lower cell concentration in the growth medium for the initial fermentation cycles when compared to the alginate beads. In conclusion, pectin was found to be potential encapsulation material for probiotic cell production owing to its stability and favourable microenvironment for cell growth

Purification of bacteriophage M13 by anion exchange chromatography

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious

Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography

Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8 mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500 mM imidazole and 1.5 M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP)