Developing a scalable model of recombinant protein yield from Pichia pastoris:the influence of culture conditions, biomass and induction regime

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

The cellular response to transglutaminase-cross-linked collagen

Collagen, type I, is a highly abundant natural protein material which has been cross-linked by a variety of methods including chemical agents, physical heating and UV irradiation with the aim of enhancing its physical characteristics such as mechanical strength, thermal stability, resistance to proteolytic breakdown, thus increasing its overall biocompatibility. However, in view of the toxicity of residual cross-linking agents, or impracticability at large scales, it would be more useful if the collagen could be cross-linked by a milder, efficient and more practical means by using enzymes as biological catalysts. We demonstrate that on treating native collagen type I (from bovine skin) with both tissue transglutaminase (TG2; tTG) and microbial transglutaminase (mTG; Streptoverticillium mobaraense) leads to an enhancement in cell attachment, spreading and proliferation of human osteoblasts (HOB) and human foreskin dermal fibroblasts (HFDF) when compared to culture on native collagen. The transglutaminase-treated collagen substrates also showed a greater resistance to cell-mediated endogenous protease degradation than the native collagen. In addition, the HOB cells were shown to differentiate at a faster rate than on native collagen when assessed by measurement of alkaline phosphatase activity and osteopontin expression. © 2005 Elsevier Ltd. All rights reserved.

Culture and behaviour in the English National Health Service:overview of lessons from a large multimethod study

Background - Problems of quality and safety persist in health systems worldwide. We conducted a large research programme to examine culture and behaviour in the English National Health Service (NHS). Methods - Mixed-methods study involving collection and triangulation of data from multiple sources, including interviews, surveys, ethnographic case studies, board minutes and publicly available datasets. We narratively synthesised data across the studies to produce a holistic picture and in this paper present a highlevel summary. Results - We found an almost universal desire to provide the best quality of care. We identified many 'bright spots' of excellent caring and practice and high-quality innovation across the NHS, but also considerable inconsistency. Consistent achievement of high-quality care was challenged by unclear goals, overlapping priorities that distracted attention, and compliance-oriented bureaucratised management. The institutional and regulatory environment was populated by multiple external bodies serving different but overlapping functions. Some organisations found it difficult to obtain valid insights into the quality of the care they provided. Poor organisational and information systems sometimes left staff struggling to deliver care effectively and disempowered them from initiating improvement. Good staff support and management were also highly variable, though they were fundamental to culture and were directly related to patient experience, safety and quality of care. Conclusions - Our results highlight the importance of clear, challenging goals for high-quality care. Organisations need to put the patient at the centre of all they do, get smart intelligence, focus on improving organisational systems, and nurture caring cultures by ensuring that staff feel valued, respected, engaged and supported.

Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation.

Economic life in the modern age

Werner Sombart (1863-1941) may well have been the most famous and controversial social scientist in Germany during the early twentieth century. Highly influential, his work and reputation have been indelibly tainted by his embrace of National Socialism in the last decade of his life. Although Sombart left an enormous opus spanning disciplinary boundaries, intellectual reaction to his work inside and outside of Germany is divided and ambivalent. Sombart consistently responded to the social and political developments that have shaped the twentieth century. Economic Life in the Modern Age provides a representative sampling of those portions of Sombart's work that have stood the test of time. The volume opens with a substantial introduction reviewing Sombart's life and career, the evolution of his major intellectual concerns, his relation to Marx and Weber, and his political affiliation with the Nazis. The editors' selection of texts emphasizes areas of Sombart's economic and cultural thought that remain relevant, particularly to those intellectual trends that seek a more broadly based, cross-disciplinary approach to culture and economics. Sombart's writings on capitalism are represented by essays on the nature and origin of the market system and the diversity of motives among the bourgeoisie and the proletariat. Also included is an excerpt from Sombart's controversial The Jews and Modern Capitalism, exploring the widely perceived relation between economic life and Judaism as a religion. In essays on the economics of cultural processes, Sombart's comprehensive and expansive idea of cultural science yields prophetic insights into the nature of urbanism, luxury consumption, fashion, and the cultural secularization of love. The volume's final section consists of Sombart's reflections on the social influences of technology, the economic life of the future, and on socialism, including the influential essay "Why is there no Socialism in the United States." Encapsulating the most valuable aspects of his work, Economic Life in the Modern Age provides clear demonstration of Sombart's sense for fine cultural distinctions and broad cultural developments and the predictive power of his analyses. It will be of interest to sociologists, economists, political scientists, and specialists in cultural studies.

Challenges and limitations of intercultural training for inpatriates in German multinationals:a case study

Despite the increasing popularity of research on intercultural preparation and its effectiveness, research on training for inpatriates has not been developed with the same level of rigour as research on training for expatriates. Furthermore, research on intercultural training hardly ever includes the aspect of preparing for the corporate culture of a company. For expatriates coming from headquarters’ national culture and equipped with a good knowledge of headquarters’ corporate culture, it might be sufficient to address only the national culture of the location abroad. But can the same be said for inpatriates coming from a foreign subsidiary? Therefore the qualitative research of my thesis was aimed at finding out if intercultural training programmes that address only the national culture of the host country are sufficient to prepare inpatriates for working at headquarters. A case study using a German multinational company has been conducted in order to find out what kind of problems and irritations inpatriates at the company’s headquarters perceive at work. In order to determine whether the findings are related to the national or the corporate culture, Hall’s and Hofstede’s approaches to culture were used. The interview analysis produced the following conclusion: Although the researched company promotes standardised worldwide corporate guidelines, there are many differences between headquarters and subsidiaries regarding the interpretation and realisation of these guidelines. These differences cause irritation, confusion and problems for the inpatriates. Therefore an effective intercultural preparation for inpatriates should be tailor-made and take into account the aspect of corporate culture, as well as the specific roles and functions of inpatriates.

The embryo and its future

The preimplantation mammalian embryo from different species appears sensitive to the environment in which it develops, either in vitro or in vivo, for example, in response to culture conditions or maternal diet. This sensitivity may lead to long-term alterations in the characteristics of fetal and/or postnatal growth and phenotype, which have implications for clinical health and biotechnological applications. We review the breadth of environmental influences that may affect early embryos and their responses to such conditions along epigenetic, metabolic, cellular, and physiological directions. In addition, we evaluate how embryo environmental responses may influence developmental potential and phenotype during later gestation. We conclude that a complex of different mechanisms may operate to associate early embryo environment with future health.

Membrane protein production in the Yeast, S. cerevisiae

The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca2+-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes.

A study of the morphological development of some filamentous fungi in tower fermenter culture, with particular reference to Aspergillus niger

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The role of national culture in advertising's sensitivity to business cycles:an investigation across continents

The authors conduct a systematic investigation into the cyclical sensitivity of advertising expenditures in 37 countries, covering four key media: magazines, newspapers, radio, and television. They show that advertising is considerably more sensitive to business-cycle fluctuations than the economy as a whole. Advertising behaves less cyclically in countries high in long-term orientation and power distance, but it is more cyclical in countries high in uncertainty avoidance. Furthermore, advertising is more sensitive to the business cycle in countries characterized by significant stock market pressure and few foreign-owned multinational corporations. The authors provide initial evidence on the long-term social and managerial losses incurred when companies tie ad spending too tightly to business cycles. Countries in which advertising behaves more cyclically exhibit slower growth of the advertising industry. Moreover, private-label growth is higher in countries characterized by more cyclical advertising spending, implying significant losses for brand manufacturers. Finally, an examination of 26 global companies shows that stock price performance is lower for companies that exhibit stronger procyclical advertising spending patterns.

Diversity, culture, leadership, performance:a performance-oriented model to leading across differences and managing internationalization processes

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Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate

Background aims: The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. Methods: In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based microcarrier process. Results: The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from microcarrier expansion. BM-hMSCs cultured on microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. Conclusions: This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and microcarrier culture, which will have key implications for process transfer during scale-up.