Use of Fondaparinux Off-Label or Approved Anticoagulants for Management of Heparin-Induced Thrombocytopenia.

BACKGROUND: Life-threatening heparin-induced thrombocytopenia (HIT) is treated with the alternative nonheparin anticoagulants argatroban, lepirudin, or danaparoid. Frequently, the pentasaccharide fondaparinux is used off-label. OBJECTIVES: The authors sought to investigate the safety and efficacy of the different anticoagulants for treating HIT. METHODS: In a national, multicenter registry study, hospitalized patients who were diagnosed with HIT, an at least intermediate clinical HIT-risk (4Ts score ≥4 points), and received treatment with ≥1 dose of the aforementioned anticoagulants were included. Main outcome measures were the incidences of HIT-specific complications (thromboembolic venous/arterial events, amputations, recurrent/persistent thrombocytopenia, skin lesions) and bleedings. RESULTS: Of 195 patients, 46 (23.6%), 4 (2.1%), 61 (31.3%), and 84 (43.1%) had been treated first-line with argatroban, lepirudin, danaparoid, and fondaparinux, respectively. The composite endpoint of HIT-specific complications (thromboembolic events, amputation, skin necrosis) occurred in 11.7% of patients treated with approved alternative anticoagulation and in 0.0% of fondaparinux-treated patients. The all-cause in-hospital mortality rates were 14.4% during approved alternative anticoagulation and 0.0% during fondaparinux treatment. Bleeding complications occurred in alternatively anticoagulated patients and in fondaparinux-treated patients in 6.3% and 4.8%, respectively. Post hoc analysis of clinical and laboratory features confirmed "true" HIT in at least 74 of 195 (38.0%) patients; 35 of 74 (47.3%) were treated with fondaparinux. CONCLUSIONS: Fondaparinux is effective and safe in suspected acute HIT; no HIT-specific complications occurred in the fondaparinux-treated patients, even among those with a high clinical HIT probability. Further data from randomized controlled trials are urgently needed because lepirudin was recalled from the market; danaparoid access has been limited and is not approved in the United States; and argatroban is contraindicated in patients with impaired liver function, and activated partial thromboplastin time confounding may interfere with monitoring. (Retrospective Registry of Patients With Acute Heparin-induced Thrombocytopenia Type II; NCT01304238).

A robust feeding strategy to maintain set-point glucose in mammalian fed-batch cultures when input parameters have a large error.

Industrial CHO cell cultures run under fed-batch conditions are required to be controlled in particular ranges of glucose, while glucose is constantly consumed and must be replenished by a feed. The most appropriate feeding rate is ideally stoichiometric and adaptive in nature to balance the dynamically changing rate of glucose consumption. However, high errors in biomass and glucose estimation as well as limited knowledge of the true metabolic state challenge the control strategy. In this contribution, we take these errors into account and simulate the output with uncertainty trajectories in silico in order to control glucose concentration. Other than many control strategies, which require parameter estimation, our assumptions are founded on two pillars: (i) first principles and (ii) prior knowledge about the variability of fed-batch CHO cell culture. The algorithm was exposed to an in-silico Design of Experiments (DoE), in which variations of parameters were changed simultaneously, such as clone-specific behavior, precision of equipment and desired control range used. The results demonstrate that our method achieved the target of holding the glucose concentration within an acceptable range. A robust and sufficient level of control could be demonstrated even with high errors for biomass or metabolic state estimation. In a time where blockbuster drugs are queuing up for time slots of their production, this transferable control strategy that is independent of tedious establishment runs may be a decisive advantage for rapid implementation during technology transfer and scale up and decrease in campaign change over time. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:317-336, 2017.

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors.

The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-rhamnolipids are converted to di-rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical rhamnolipids containing two fatty acids, valuable for the production of designer rhamnolipids with desired physicochemical properties.

Metabolic Control in Mammalian Fed-Batch Cell Cultures for Reduced Lactic Acid Accumulation and Improved Process Robustness.

Biomass and cell-specific metabolic rates usually change dynamically over time, making the "feed according to need" strategy difficult to realize in a commercial fed-batch process. We here demonstrate a novel feeding strategy which is designed to hold a particular metabolic state in a fed-batch process by adaptive feeding in real time. The feed rate is calculated with a transferable biomass model based on capacitance, which changes the nutrient flow stoichiometrically in real time. A limited glucose environment was used to confine the cell in a particular metabolic state. In order to cope with uncertainty, two strategies were tested to change the adaptive feed rate and prevent starvation while in limitation: (i) inline pH and online glucose concentration measurement or (ii) inline pH alone, which was shown to be sufficient for the problem statement. In this contribution, we achieved metabolic control within a defined target range. The direct benefit was two-fold: the lactic acid profile was improved and pH could be kept stable. Multivariate Data Analysis (MVDA) has shown that pH influenced lactic acid production or consumption in historical data sets. We demonstrate that a low pH (around 6.8) is not required for our strategy, as glucose availability is already limiting the flux. On the contrary, we boosted glycolytic flux in glucose limitation by setting the pH to 7.4. This new approach led to a yield of lactic acid/glucose (Y L/G) around zero for the whole process time and high titers in our labs. We hypothesize that a higher carbon flux, resulting from a higher pH, may lead to more cells which produce more product. The relevance of this work aims at feeding mammalian cell cultures safely in limitation with a desired metabolic flux range. This resulted in extremely stable, low glucose levels, very robust pH profiles without acid/base interventions and a metabolic state in which lactic acid was consumed instead of being produced from day 1. With this contribution, we wish to extend the basic repertoire of available process control strategies, which will open up new avenues in automation technology and radically improve process robustness in both process development and manufacturing.

Universal Capacitance Model for Real-Time Biomass in Cell Culture.

: Capacitance probes have the potential to revolutionize bioprocess control due to their safe and robust use and ability to detect even the smallest capacitors in the form of biological cells. Several techniques have evolved to model biomass statistically, however, there are problems with model transfer between cell lines and process conditions. Errors of transferred models in the declining phase of the culture range for linear models around +100% or worse, causing unnecessary delays with test runs during bioprocess development. The goal of this work was to develop one single universal model which can be adapted by considering a potentially mechanistic factor to estimate biomass in yet untested clones and scales. The novelty of this work is a methodology to select sensitive frequencies to build a statistical model which can be shared among fermentations with an error between 9% and 38% (mean error around 20%) for the whole process, including the declining phase. A simple linear factor was found to be responsible for the transferability of biomass models between cell lines, indicating a link to their phenotype or physiology.

Frequent off-label use of fondaparinux in patients with suspected acute heparin-induced thrombocytopenia (HIT)--findings from the GerHIT multi-centre registry study.

INTRODUCTION: In life-threatening immune heparin-induced thrombocytopenia (HIT), treatment with an approved non-heparin anticoagulant is essential. However, off-label use with fondaparinux has been reported in the literature. The study aim was to collect data on "real-life" management of patients with suspected acute HIT regarding diagnostic and therapeutic strategies. PATIENTS AND METHODS: In a national multi-centre registry study, patients with a 4T's HIT-probability score of ≥ 4 points and treatment with at least one dose of (A)rgatroban, (L)epirudin, (D)anaparoid, or (F)ondaparinux were retrospectively evaluated. RESULTS: Of 195 patients, the 4T's scores were 4/5/6/7/8 points in 46 (23.6%)/50 (25.6%)/74 (38.0%)/13 (6.7%)/7 (3.6%) patients, respectively. During heparin therapy, 47 (24.1%) thromboembolic events, 5 (2.6%) skin lesions, 1 (0.5%) amputation, 24 (12.3%) Hb-relevant bleedings, and 2 (1.0%) fatalities occurred. A functional heparin-induced platelet activation assay was performed in 96.9%, a platelet factor 4/heparin-dependent enzyme immunoassay in 89.2%, a particle gel immunoassay in 12.3%, and a serotonin-release assay in none of the patients. Argatroban was used in 16.4%, lepirudin in 2.1%, danaparoid in 23.6%, fondaparinux in 40.0% of the patients; the sequential therapy strata were: AF (5.6%), DA (5.6%), DF (2.6%), DL (2.1%), ADF (1.5%), and DFL (0.5%). CONCLUSIONS: The current diagnostic laboratory strategy for suspected HIT is mostly (>96%) based on the recommended 2-step strategy (immunoassay plus functional assay). However, there is a wide fondaparinux off-label use (up to 50.3%) for suspected HIT, even in those patients with a high clinical pretest probability. Efficacy and safety of fondaparinux for HIT-treatment require further evaluation.

Production of microbial rhamnolipid by Pseudomonas aeruginosa MM1011 for ex situ enhanced oil recovery.

Recently, several investigations have been carried out on the in situ bacteria flooding, but the ex situ biosurfactant production and addition to the sand pack as agents for microbial enhanced oil recovery (MEOR) has little been studied. In order to develop suitable technology for ex situ MEOR processes, it is essential to carry out tests about it. Therefore, this work tries to fill the gap. The intention of this study was to investigate whether the rhamnolipid mix could be produced in high enough quantities for enhanced oil recovery in the laboratory scale and prove its potential use as an effective material for field application. In this work, the ability of Pseudomonas aeruginosa MM1011 to grow and produce rhamnolipid on sunflower as sole carbon source under nitrogen limitation was shown. The production of Rha-C10-C10 and Rha2-C10-C10 was confirmed by thin-layer chromatography and high-performance liquid chromatography analysis. The rhamnolipid mixture obtained was able to reduce the surface and interfacial tension of water to 26 and 2 mN/m, respectively. The critical micelle concentration was 120 mg/L. Maximum rhamnolipid production reached to about 0.7 g/L in a shake flask. The yield of rhamnolipid per biomass (Y RL/x ), rhamnolipid per sunflower oil (Y RL/s ), and the biomass per sunflower oil (Y x/s ) for shake flask were obtained about 0.01, 0.0035, and 0.035 g g(-1), respectively. The stability of the rhamnolipid at different salinities, pH and temperature, and also, its emulsifying activity has been investigated. It is an effective surfactant at very low concentrations over a wide range of temperatures, pHs, and salt concentrations, and it also has the ability to emulsify oil, which is essential for enhanced oil recovery. With 120 mg/L rhamnolipid, 27 % of original oil in place was recovered after water flooding from a sand pack. This result not only suggests rhamnolipids as appropriate model biosurfactants for MEOR, but it even shows the potential as a biosurfactant of choice for actual MEOR applications.

Rhamnolipids--next generation surfactants?

The demand for bio-based processes and materials in the petrochemical industry has significantly increased during the last decade because of the expected running out of petroleum. This trend can be ascribed to three main causes: (1) the increased use of renewable resources for chemical synthesis of already established product classes, (2) the replacement of chemical synthesis of already established product classes by new biotechnological processes based on renewable resources, and (3) the biotechnological production of new molecules with new features or better performances than already established comparable chemically synthesized products. All three approaches are currently being pursued for surfactant production. Biosurfactants are a very promising and interesting substance class because they are based on renewable resources, sustainable, and biologically degradable. Alkyl polyglycosides are chemically synthesized biosurfactants established on the surfactant market. The first microbiological biosurfactants on the market were sophorolipids. Of all currently known biosurfactants, rhamnolipids have the highest potential for becoming the next generation of biosurfactants introduced on the market. Although the metabolic pathways and genetic regulation of biosynthesis are known qualitatively, the quantitative understanding relevant for bioreactor cultivation is still missing. Additionally, high product titers have been exclusively described with vegetable oil as sole carbon source in combination with Pseudomonas aeruginosa strains. Competitive productivity is still out of reach for heterologous hosts or non-pathogenic natural producer strains. Thus, on the one hand there is a need to gain a deeper understanding of the regulation of rhamnolipid production on process and cellular level during bioreactor cultivations. On the other hand, there is a need for metabolizable renewable substrates, which do not compete with food and feed. A sustainable bioeconomy approach should combine a holistic X-omics strategy with metabolic engineering to achieve the next step in rhamnolipid production based on non-food renewable resources. This review discusses different approaches towards optimization of rhamnolipid production and enhancement of product spectra. The optimization of rhamnolipid production with P. aeruginosa strains, screening methods for new non-pathogenic natural rhamnolipid producers and recombinant rhamnolipid production are examined. Finally, biocatalysis with rhamnolipids for the synthesis of l-rhamnose, ß-hydroxyfatty acids, and tailor-made surfactants is discussed. Biosurfactants are still in the phase of initial commercialization. However, for next generation development of rhamnolipid production processes and next generation biosurfactants there are still considerable obstacles to be surmounted, which are discussed here.

Regulatory and metabolic network of rhamnolipid biosynthesis: traditional and advanced engineering towards biotechnological production.

During the last decade, the demand for economical and sustainable bioprocesses replacing petrochemical-derived products has significantly increased. Rhamnolipids are interesting biosurfactants that might possess a broad industrial application range. However, despite of 60 years of research in the area of rhamnolipid production, the economic feasibility of these glycolipids is pending. Although the biosynthesis and regulatory network are in a big part known, the actual incidents on the cellular and process level during bioreactor cultivation are not mastered. Traditional engineering by random and targeted genetic alteration, process design, and recombinant strategies did not succeed by now. For enhanced process development, there is an urgent need of in-depth information about the rhamnolipid production regulation during bioreactor cultivation to design knowledge-based genetic and process engineering strategies. Rhamnolipids are structurally comparable, simple secondary metabolites and thus have the potential to become instrumental in future secondary metabolite engineering by systems biotechnology. This review summarizes current knowledge about the regulatory and metabolic network of rhamnolipid synthesis and discusses traditional and advanced engineering strategies performed for rhamnolipid production improvement focusing on Pseudomonas aeruginosa. Finally, the opportunities of applying the systems biotechnology toolbox on the whole-cell biocatalyst and bioprocess level for further rhamnolipid production optimization are discussed.

Evaluation of rhamnolipid production capacity of Pseudomonas aeruginosa PAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874.

A lack of understanding of the quantitative rhamnolipid production regulation in bioreactor cultivations of Pseudomonas aeruginosa and the absence of respective comparative studies are important reasons for achieving insufficient productivities for an economic production of these biosurfactants. The Pseudomonas strains DSM 7108 and DSM 2874 are described to be good rhamnolipid over-producers. The strain PAO1 on the other hand is the best analyzed type strain for genetic regulation mechanisms in the species P. aeruginosa. These three strains were cultivated in a 30-L bioreactor with a medium containing nitrate and sunflower oil as sole C-source at 30 and 37 °C. The achieved maximum rhamnolipid concentrations varied from 7 to 38 g/L, the volumetric productivities from 0.16 to 0.43 g/(L·h), and the cellular yield from 0.67 to 3.15 g/g, with PAO1 showing the highest results for all of these variables. The molar di- to mono-rhamnolipid ratio changed during the cultivations; it was strain dependent but not significantly influenced by the temperature. This study explicitly shows that the specific rhamnolipid synthesis rate per cell follows secondary metabolite-like courses coinciding with the transition to the stationary phase of typical logistic growth behavior. However, the rhamnolipid synthesis was already induced before N-limitation occurred.

Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production in bioreactor systems.

Rhamnolipids are biosurfactants with interesting physico-chemical properties. However, the main obstacles towards an economic production are low productivity, high raw-material costs, relatively expensive downstream processing, and a lack of understanding the rhamnolipid production regulation in bioreactor systems. This study shows that the sequenced Pseudomonas aeruginosa strain PAO1 is able to produce high quantities of rhamnolipid during 30 L batch bioreactor cultivations with sunflower oil as sole carbon source and nitrogen limiting conditions. Thus PAO1 could be an appropriate model for rhamnolipid production in pilot plant bioreactor systems. In contrast to well-established production strains, PAO1 allows knowledge-based systems biotechnological process development combined with the frequently used heuristic bioengineering approach. The maximum rhamnolipid concentration obtained was 39 g/L after 90 h of cultivation. The volumetric productivity of 0.43 g/Lh was comparable with previous described production strains. The specific rhamnolipid productivity showed a maximum between 40 and 70 h of process time of 0.088 g(RL)/g(BDM)h. At the same time interval, a shift of the molar di- to mono-rhamnolipid ratio from 1:1 to about 2:1 was observed. PAO1 not only seems to be an appropriate model, but surprisingly has the potential as a strain of choice for actual biotechnological rhamnolipid production.