Flow-velocity programmed chromatography as an alternative method for increasing the efficiency of continuous- or integrated-chromatography processes

Solvent (mobile phase) programming is most commonly employed for controlling adsorption/desorption in chromatography (linear gradient elution or stepwise elution). For gas separation, temperature- or pressure-swing adsorption is frequently used. Although flow-velocity is another important parameter, which affects both the dynamic adsorption capacity (DBC) and the resolution, it is seldom used as a programmed operating variable. The one exception is the standard 4-zone simulated moving bed (SMB) chromatography, in which the flow-velocities of the 4-zones are different. Several researchers have already shown that DBC can be increased by using two different flow velocities. However, a rational method for determining the optimum flow velocity program has not been established. Moreover, application of this method to periodic counter-current (PCC) chromatography or connected flow-through chromatography (FTC) has not been attempted yet. In this study, we have developed a flow-velocity gradient method for analyzing the breakthrough curves of proteins in ion-exchange or protein A chromatography (Figure 1). The data were obtained at various different gradient slopes. The obtained curves were analyzed based on a model considering mass transfer (pore diffusion) and non-linear isotherm. Then, numerical simulations were carried out in order to find the optimum flow-velocity program for improving the efficiency. This method was further applied to PCC and FTC (Figure 2). The effect of flow programming on productivity and cost reduction has also been examined in both batch and continuous configuration in capture chromatography of mAbs by simulation of the process models. Experimental verification was also carried out using monoclonal antibody samples in the filtered cell culture liquid. Please click Additional Files below to see the full abstract

Flow-through chromatography as a continuous and integrated purification method

Continuous manufacturing is expected to improve the efficiency and economics of protein and other bio-product production processes. However, it is not easy to design and operate the continuous process especially for downstream processing as many unit operations (chromatography and membrane filtration) are involved. An operation method known as flow-through chromatography (FTC) is considered to be an efficient purification method as the flow is continuous. In FTC, a target bio-product is eluted from the chromatography column without adsorption whereas contaminants are strongly bound. Usually two different modes of chromatography are needed in order to remove various kinds of contaminants. Two FTC columns have to be connected in order to build the integrated continuous process. This is not an easy task since the mobile phase properties (pH, salt, buffer ions) are different for the two columns. Please click Additional Files below to see the full abstract

Increasing Ceftriaxone Resistance in Salmonellae, Taiwan

In Taiwan, despite a substantial decline of Salmonella enterica serotype Choleraesuis infections, strains resistant to ciprofloxacin and ceftriaxone persist. A self-transferable blaCMY-2-harboring IncI1 plasmid was identified in S. enterica serotypes Choleraesuis, Typhimurium, Agona, and Enteritidis and contributed to the overall increase of ceftriaxone resistance in salmonellae

MARCH1 protects the lipid raft and tetraspanin web from MHCII proteotoxicity in dendritic cells

Dendritic cells (DCs) produce major histocompatibility complex II (MHCII) in large amounts to function as professional antigen presenting cells. Paradoxically, DCs also ubiquitinate and degrade MHCII in a constitutive manner. Mice deficient in the MHCII-ubiquitinating enzyme membrane-anchored RING-CH1, or the ubiquitin-acceptor lysine of MHCII, exhibit a substantial reduction in the number of regulatory T (Treg) cells, but the underlying mechanism was unclear. Here we report that ubiquitin-dependent MHCII turnover is critical to maintain homeostasis of lipid rafts and the tetraspanin web in DCs. Lack of MHCII ubiquitination results in the accumulation of excessive quantities of MHCII in the plasma membrane, and the resulting disruption to lipid rafts and the tetraspanin web leads to significant impairment in the ability of DCs to engage and activate thymocytes for Treg cell differentiation. Thus, ubiquitin-dependent MHCII turnover represents a novel quality-control mechanism by which DCs maintain homeostasis of membrane domains that support DC's Treg cell-selecting function

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

[[abstract]]Background: Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. Principal Finding: In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10?5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). Conclusion:These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification

Pilot Scale Production of Highly Efficacious and Stable Enterovirus 71 Vaccine Candidates

BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials

Subalpine Loamy Spodosols in Taiwan: Characteristics, Micromorphology, and Genesis

Four subalpine loamy Spodosols with illuvial clay in the spodic horizons were selected for study to understand their characteristics, micromorphology, and genesis. These Spodosols, located in central Taiwan, are covered by coniferous vegetation, receive high levels of precipitation, and occupy gentle slope positions at elevations >2400 m. Soil textures, which range from loam to clay, are finer than those of Spodosols formed in temperate regions. The pedogenic products in the soils of the study area are mainly organo-metallic complexes or illuvial clay mixed with organo-metallic complexes in the spodic horizons. Typical micromorphological features in the spodic horizons of these Spodosols revealed infillings of organo-Fe complexes, argillans, or argiferrans along the irregular voids. The four selected pedons have loose spodic horizons where illuvial clay also has accumulated. The large amounts of spodic materials and illuvial clays in the spodic horizons indicate that podzolization and clay accumulation were the two dominant pedogenic processes. Some of the illuvial clay translocated into the spodic horizons is mixed with organo-Fe (or -Al) complexes along the irregular voids of the spodic horizons. This may also have resulted in a lack of well-oriented clay in these horizons despite the occurrence of large amounts of illuviated clays