Lipid reduction to improve clarification and filterability during primary recovery of intracellular products in yeast lysates using exogenous lipase

BACKGROUND: The yeast Pichia pastoris is a popular host organism for production of a range of biological products, several of which are intracellular. The disruption of yeast cells by homogenisation also releases large quantities of lipids, which can foul the downstream membranes and chromatography matrices used for purification. This work examines lipid removal from yeast cells following homogenisation by enzymatic degradation and its impact on the performance of the subsequent centrifugation and filtration. RESULTS: Lipase treatment of cell homogenate at 37°C for 2 hours, followed by clarification using a scale-down mimic of disc stack centrifugation, resulted in a 6.5-fold improvement in solids removal when compared to untreated feed material. The lipase treated and untreated materials that had undergone initial centrifugation were then tested for filtration performance by passing the material through a 0.45 μm polyethylene sulfone membrane under constant flux. A 50% increase in throughput was observed in comparison to the untreated material. CONCLUSION: This proof-of-concept data suggests enzymatic digestion of lipids, analogous to the widely performed DNA reduction using nucleases, could be a valuable process improvement strategy

Predicting free-space occupancy on novel artificial structures by an invasive intertidal barnacle using a removal experiment.

Artificial structures can create novel habitat in the marine environment that has been associated with the spread of invasive species. They are often located in areas of high disturbance and can vary significantly in the area of free space provided for settlement of marine organisms. Whilst correlation between the amount of free space available and recruitment success has been shown in populations of several marine benthic organisms, there has been relatively little focus on invasive species, a group with the potential to reproduce in vast numbers and colonise habitats rapidly. Invasion success following different scales of disturbance was examined in the invasive acorn barnacle, Austrominiusmodestus, on a unique art installation located in Liverpool Bay. Population growth and recruitment success were examined by comparing recruitment rates within disturbance clearings of 4 different sizes and by contrasting population development with early recruitment rates over a 10 week period. Disturbed areas were rapidly recolonised and monocultures of A. modestus formed within 6 weeks. The size of patch created during disturbance had no effect on the rate of recruitment, while a linear relationship between recruit density and patch size was observed. Density-dependent processes mediated initial high recruitment resulting in population stability after 8-10 weeks, but densities continued to greatly exceed those reported in natural habitats. Given that artificial structures are likely to continue to proliferate in light of climate change projections, free-space is likely to become more available more frequently in the future supporting the expansion of fast-colonising species

Holistic process development to mitigate proteolysis of a subunit rotavirus vaccine candidate produced in Pichia pastoris by means of an acid pH pulse during fed‐batch fermentation

To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein‐based rotavirus vaccine candidate derived from non‐replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small‐scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full‐length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full‐length material (60–75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield)

Extraction of pure components from overlapped signals in gas chromatography-mass spectrometry (GC-MS)

Gas chromatography-mass spectrometry (GC-MS) is a widely used analytical technique for the identification and quantification of trace chemicals in complex mixtures. When complex samples are analyzed by GC-MS it is common to observe co-elution of two or more components, resulting in an overlap of signal peaks observed in the total ion chromatogram. In such situations manual signal analysis is often the most reliable means for the extraction of pure component signals; however, a systematic manual analysis over a number of samples is both tedious and prone to error. In the past 30 years a number of computational approaches were proposed to assist in the process of the extraction of pure signals from co-eluting GC-MS components. This includes empirical methods, comparison with library spectra, eigenvalue analysis, regression and others. However, to date no approach has been recognized as best, nor accepted as standard. This situation hampers general GC-MS capabilities, and in particular has implications for the development of robust, high-throughput GC-MS analytical protocols required in metabolic profiling and biomarker discovery. Here we first discuss the nature of GC-MS data, and then review some of the approaches proposed for the extraction of pure signals from co-eluting components. We summarize and classify different approaches to this problem, and examine why so many approaches proposed in the past have failed to live up to their full promise. Finally, we give some thoughts on the future developments in this field, and suggest that the progress in general computing capabilities attained in the past two decades has opened new horizons for tackling this important problem

On the relevance of preprocessing in predictive maintenance for dynamic systems

The complexity involved in the process of real-time data-driven monitoring dynamic systems for predicted maintenance is usually huge. With more or less in-depth any data-driven approach is sensitive to data preprocessing, understood as any data treatment prior to the application of the monitoring model, being sometimes crucial for the final development of the employed monitoring technique. The aim of this work is to quantify the sensitiveness of data-driven predictive maintenance models in dynamic systems in an exhaustive way. We consider a couple of predictive maintenance scenarios, each of them defined by some public available data. For each scenario, we consider its properties and apply several techniques for each of the successive preprocessing steps, e.g. data cleaning, missing values treatment, outlier detection, feature selection, or imbalance compensation. The pretreatment configurations, i.e. sequential combinations of techniques from different preprocessing steps, are considered together with different monitoring approaches, in order to determine the relevance of data preprocessing for predictive maintenance in dynamical systems

Habitat complexity effects on diversity and abundance differ with latitude: an experimental study over 20 degrees

Habitat complexity is accepted as a general mechanism for increasing the abundance and diversity of communities. However, the circumstances under which complexity has the strongest effects are not clear. Over 20 degrees of Australia's east coast, we tested whether the effects of within-site structural habitat complexity on the diversity and community structure of sessile marine invertebrates was consistent over a latitudinal gradient where environmental conditions and species composition vary. We used experimental arrays with varied structural treatments to detect whether community cover, species richness, diversity and community composition (β-diversity) changed with increasing complexity. Community response to complexity varied over latitude due to differences in species richness and community development. Increased complexity had the greatest positive effects on community cover and species richness at higher latitudes where recruitment and growth were low. At lower latitudes, community cover and species richness were higher overall and did not vary substantially between complexity treatments. Latitudinal variation in within-treatment β-diversity relative to complexity further suggest divergent community responses. At higher latitudes, increased similarity in more complex treatments suggests community dominance of successful taxonomic groups. Despite limited effects on species richness and community cover at lower latitudes, β-diversity was higher in more complex treatments, signifying potential positive effects of increased complexity at these sites. These results demonstrate the context-dependency of complexity effects in response to variation in species richness and community development and should be taken into consideration to help direct conservation and restoration efforts

Latitudinal variation in the competition-colonisation trade-off reveals rate-mediated mechanisms of coexistence

Theories of species coexistence often describe a trade-off between colonising and competitive abilities. In sessile marine invertebrates, this trade-off can manifest as trends in species distributions relative to the size of isolated patches of substrate. Based on their abilities to find available substrate and competitively exclude neighbours, good colonisers tend to dominate smaller patches, whereas better competitors tend to monopolise larger patches. In theory, species with equivalent colonising and competitive abilities should display similar distributions across patch sizes. We used patch size to observe this manifestation of the competition-colonisation trade-off over 20° of latitude. The trade-off was more readily observed at lower latitudes and was proportional to the ‘ecological age’ of communities (i.e. the degree of resource acquisition and likelihood of species interactions). Results suggest that ecological age may mediate the prominence of stochastic or deterministic coexistence mechanisms and will depend on the rate of ecological processes

Rapid Developability Assessments to Formulate Recombinant Protein Antigens as Stable, Low-Cost, Multi-Dose Vaccine Candidates: Case-Study With Non-Replicating Rotavirus (NRRV) Vaccine Antigens

A two-step developability assessment workflow is described to screen variants of recombinant protein antigens under various formulation conditions to rapidly identify stable, aluminum-adjuvanted, multi-dose vaccine candidates. For proof-of-concept, a series of sequence variants of the recombinant non-replicating rotavirus (NRRV) P[8] protein antigen (produced in Komagataella phaffii) were compared in terms of primary structure, post-translational modifications, antibody binding, conformational stability, relative solubility and preservative compatibility. Based on these results, promising P[8] variants were down-selected and the impact of key formulation conditions on storage stability was examined (e.g., presence or absence of the aluminum-adjuvant Alhydrogel and the preservative thimerosal) as measured by differential scanning calorimetry (DSC) and antibody binding assays. Good correlations between rapidly-generated developability screening data and storage stability profiles (12 weeks at various temperatures) were observed for aluminum-adsorbed P[8] antigens. These findings were extended and confirmed using variants of a second NRRV antigen, P[4]. These case-study results with P[8] and P[4] NRRV variants are discussed in terms of using this vaccine formulation developability workflow to better inform and optimize formulation design with a wide variety of recombinant protein antigens, with the long-term goal of rapidly and cost-efficiently identifying low-cost vaccine formulations for use in low and middle income countries