Pore types, genesis, and evolution model of lacustrine oil-prone shale: a case study of the Cretaceous Qingshankou Formation, Songliao Basin, NE China.

A comprehensive characterisation of the pore structure in shale oil reservoirs is essential for forecasting oil production and exploration risks. This study forecasted these risks in the oil-rich Songliao Basin using combination of high-resolution field emission scanning electron microscopy and quantitative X-ray diffraction to analyze the pore genesis and evolution mode within the first member of the Cretaceous Qingshankou Formation (K2qn1). The results showed the dominance of inorganic pores over organic pores, wherein diagenetic processes, such as compaction, pressure solution, and cementation, were responsible for the destruction of pore structure in the formation. Notably, the pores formed by dissolution and shrinkage cracks resulting from clay mineral transformation improved the oil storage space. Furthermore, according to the geochemical data and clay composition, the K2qn1 shale is in the middle diagenetic stage A, which can be further subdivided into A1 and A2 stages from top to bottom. The porosity slowly decreased in both sub-stages A1 and A2, wherein the decrease was stable in the latter. The diagenetic observations in this study are significant for the exploration of unconventional shale oil in petroliferous basins worldwide.

A novel function for selenium in biological system: selenite as a highly effective iron carrier for Chinese hamster ovary cell growth and monoclonal antibody production.

As the market for biopharmaceuticals especially monoclonal antibodies (MAbs) rapidly grows, their manufacturing methods are coming under increasing regulatory scrutiny, particularly due to concerns about the potential introduction of adventitious agents from animal-sourced components in the media used for their production in mammalian cell culture. Chinese hamster ovary (CHO) cells are by far the most commonly used production vehicles for these recombinant glycoproteins. In developing animal-component free media for CHO and other mammalian cell lines, the iron-transporter function of serum or human/bovine transferrin is usually replaced by certain organic or inorganic chelators capable of delivering iron for cell respiration and metabolism, but few of them are sufficiently effective. Selenium is a well-known essential trace element (TE) for cell growth and development, and its positive role in biological system includes detoxification of free radicals by activating glutathione peroxidase. In cell culture, selenium in the form of selenite can help cells to detoxify the medium thus protect them from oxidative damage. In this presentation, we describe the discovery and application of a novel function of selenite, that is, as a highly effective carrier to deliver iron for cell growth and function. In our in-house-developed animal protein-free (APF) medium for CHO cells, using an iron-selenite compound to replace the well-established tropolone delivery system for iron led to comparable or better cell growth and antibody production. A high cell density of >10 x 10(6) viable cells/mL and excellent antibody titer of approximately 3 g/L were achieved in 14-day fed-batch cultures in shake flasks, followed by successful scale-up to stirred bioreactors. The preparation of the commercially unavailable iron-selenite compound from respective ions, and its effectiveness in cell-culture performance, were dependent on reaction time, substrates, and other conditions.

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture.

There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serum-free media into ones that do not contain any components of animal origin, or 'animal-free media'. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animal-sourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1~ 3x10(6) viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.

Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process.

Yeast extract (YE) is commonly used as a key component in the complex media for industrial fermentations. However, the lot-to-lot variation of this raw material frequently requires extensive "use testing" of many lots to identify only the few that support desired fermentation performance. Through extensive fermentation studies and chemical analyses, we have identified adenine and two metabolizable carbon sources, trehalose and lactate, as the principle components in YE that affect the production of a recombinant protein antigen by a yeast strain. Adenine is required for culture growth and the relationship between biomass and measured adenine can be expressed by a Michaelis-Menten model, while the slowly metabolized trehalose serves to maintain the energy supply to the continued antigen synthesis. The rapidly utilized lactate exerts an indirect positive effect by sparing some of the accumulated ethanol from being consumed for growth to being utilized in the product formation. The effects of these YE components are mutually dependent. Based on the database generated from 40 lots at laboratory scale, a relatively high level of carbon sources in YE (trehalose plus lactate, >9.5% w/w) and an intermediate level of adenine (0.14-0.24% w/w) appear to be the minimal requirement of a good lot for this recombinant yeast fermentation. Many poor lots were improved in lab fermenters by rational supplementation of trehalose, lactate, or adenine to compensate for their insufficiencies. At the large production scale, predictions based on adenine and trehalose/lactate contents in various YE lots used correlated reasonably well with culture growth and antigen yield, illustrating the feasibility of such a simple chemical/biochemical analysis as a rapid and reliable initial screening tool. Without incurring any compositional change to an established manufacturing medium, this study demonstrates an effective approach to achieve consistency in fermentations employing complex nutrients and to improve fermentation productivities supported by suboptimal lots of raw material.