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 microbial diagnosis of infective endocarditis

This review suggests an evidence-based algorithm for sequential testing in infective endocarditis. It discusses blood culture and the merits and drawbacks of serology in making the diagnosis. Newer techniques are briefly reviewed. The proposed algorithm will complement the Duke criteria in clinical practice. © 2003 The British Infection Society. Published by Elsevier Science Ltd. All rights reserved.

Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield:the use of a respiratory strain as a microbial cell factory

BACKGROUND: Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. RESULTS: Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. CONCLUSIONS: The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.

Inhibition of microbial colonization of shipboard fuel systems

The aim of the investigation was to study the problem of colonization of shipboard fuel systems and to examine the effect of a number of environmental factors on microbial growth and survival in order to find potential preservative treatments. A variety of microbial species were isolated from samples taken from fuel storage tanks. Bacteria were more numerous than yeasts or fungi and most microorganisms were found at the fuel/water interface. 1he salinity, pH and phosphate concentration of some water bottoms were characteristic of sea water. Others were brackish, acidic and varied in phosphate content. Microorganisms were cultured under a number of environmental conditions. After prolonged incubation, the inoculum size had no effect on the final biomass of Cladosporium resinae but the time required to achieve the final mass decreased with increasing spore number. Undecane supported better growth of the fungus than diesel fuel and of four types of diesel fuel, two allowed more profuse growth. With sea water as the aqueous phase, a number of isolates were inhibited but the addition of nutrients allowed the development of many of the organisms. Agitation increased the growth of C. resinae on glucose but inhibited it on hydrocarbons. The optimum temperature fgr growth of C. resinae on surface culture lay between 25º C and 30º C and growth was evident at 5º C but not at 45º C. In aqueous suspension, 90% of spores were inactivated in around 60 hours at 45ºC and the same proportion of spores of C. resinae and Penicillium corylophilum were destroyed after about 30 seconds at 65ºC. The majority of bacteria and all yeasts in a water bottom sample were killed within 10 seconds at this temperature. An increase in the concentration of an organo-boron compound caused more rapid inactivation of C. resinae spores and raising the temperature from 25ºC to 45°C significantly enhanced the potency of the biocide.

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.

Large scale organisational intervention to improve patient safety in four UK hospitals:mixed method evaluation

Objectives: To conduct an independent evaluation of the first phase of the Health Foundation's Safer Patients Initiative (SPI), and to identify the net additional effect of SPI and any differences in changes in participating and non-participating NHS hospitals. Design: Mixed method evaluation involving five substudies, before and after design. Setting: NHS hospitals in United Kingdom. Participants: Four hospitals (one in each country in the UK) participating in the first phase of the SPI (SPI1); 18 control hospitals. Intervention: The SPI1 was a compound (multicomponent) organisational intervention delivered over 18 months that focused on improving the reliability of specific frontline care processes in designated clinical specialties and promoting organisational and cultural change. Results: Senior staff members were knowledgeable and enthusiastic about SPI1. There was a small (0.08 points on a 5 point scale) but significant (P<0.01) effect in favour of the SPI1 hospitals in one of 11 dimensions of the staff questionnaire (organisational climate). Qualitative evidence showed only modest penetration of SPI1 at medical ward level. Although SPI1 was designed to engage staff from the bottom up, it did not usually feel like this to those working on the wards, and questions about legitimacy of some aspects of SPI1 were raised. Of the five components to identify patients at risk of deterioration - monitoring of vital signs (14 items); routine tests (three items); evidence based standards specific to certain diseases (three items); prescribing errors (multiple items from the British National Formulary); and medical history taking (11 items) - there was little net difference between control and SPI1 hospitals, except in relation to quality of monitoring of acute medical patients, which improved on average over time across all hospitals. Recording of respiratory rate increased to a greater degree in SPI1 than in control hospitals; in the second six hours after admission recording increased from 40% (93) to 69% (165) in control hospitals and from 37% (141) to 78% (296) in SPI1 hospitals (odds ratio for "difference in difference" 2.1, 99% confidence interval 1.0 to 4.3; P=0.008). Use of a formal scoring system for patients with pneumonia also increased over time (from 2% (102) to 23% (111) in control hospitals and from 2% (170) to 9% (189) in SPI1 hospitals), which favoured controls and was not significant (0.3, 0.02 to 3.4; P=0.173). There were no improvements in the proportion of prescription errors and no effects that could be attributed to SPI1 in non-targeted generic areas (such as enhanced safety culture). On some measures, the lack of effect could be because compliance was already high at baseline (such as use of steroids in over 85% of cases where indicated), but even when there was more room for improvement (such as in quality of medical history taking), there was no significant additional net effect of SPI1. There were no changes over time or between control and SPI1 hospitals in errors or rates of adverse events in patients in medical wards. Mortality increased from 11% (27) to 16% (39) among controls and decreased from17%(63) to13%(49) among SPI1 hospitals, but the risk adjusted difference was not significant (0.5, 0.2 to 1.4; P=0.085). Poor care was a contributing factor in four of the 178 deaths identified by review of case notes. The survey of patients showed no significant differences apart from an increase in perception of cleanliness in favour of SPI1 hospitals. Conclusions The introduction of SPI1 was associated with improvements in one of the types of clinical process studied (monitoring of vital signs) and one measure of staff perceptions of organisational climate. There was no additional effect of SPI1 on other targeted issues nor on other measures of generic organisational strengthening.

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo-reactivity

Modification of human islets prior to transplantation may improve long-term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo-rejection. Intragraft incorporation of stem cells secreting beta (β)-cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three-dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture-assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo-response in discrete T-cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo-response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T-cell subsets to AEC-bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre-transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo-reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long-term graft survival in islet transplantation.

Biodiversity of the surface microbial consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen cheeses

Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious "house" flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.