Bacteriophage encapsulation using spray drying for phage therapy

Exploiting the potential of bacteriophages for phage therapy is an exciting future prospect. However, in order to be successful, there is a pressing need for the manufacture of safe and efficacious phage drug products to treat patients. Scalable manufacture of phage biologics as a stable solid dry powder form is highly desirable and achievable using the process of spray drying. Spray drying of purified phage suspensions formulated with suitable excipients can be carried out in a single step with high process throughput and at relatively low cost. The resulting phage-containing powders can possess good storage shelf-life. The process allows control over the final phage dose in the powder and production of microparticles suitable for a variety of therapeutic uses. Spray dried powders may include different polymer formulations employing a multitude of different triggers for phage release at the target site including pH, enzymes, virulence factors etc. The activity of the phages in spray dried powders is adversely affected during spray drying due to dessication and thermal stresses which need to be controlled. The choice of polymers, excipients and moisture content of the dry powders affects the material glass transition temperature and the stability of the phages during storage. The storage temperature and storage humidty are important factors affecting the stability of the phages in the dry powders. A quality by design (QbD) approach for phage drug product development needs to identify drug product characteristics that are critical to quality from the patient's perspective and translates them into the critical quality attributes (CQA) of the drug product. The relationship between the phage drug product CQAs and formulation development and spray drying process conditions are discussed in this article

The uses and abuses of rapid bioluminescence-based ATP assays

Bioluminescence-based ATP testing of solid surfaces has become well established in the food processing industry as part of general hazard analysis and critical control points (HACCP) measures. The rise in healthcare associated infections (HAIs) at the turn of the century focussed attention on the environment as a potential reservoir of the agents responsible for such infections. In response to the need for objective methods of assessing the efficiency of cleaning in healthcare establishments and for rapid methods for detecting the presence of the pathogens responsible for HAIs, it was proposed that ATP testing of environmental surfaces be introduced. We examine the basis behind the assumptions inherent in these proposals. Intracellular ATP levels are shown to vary between microbial taxa and according to environmental conditions. Good correlations between microbial numbers and ATP levels have been obtained under certain specific conditions, but never within healthcare settings. Notwithstanding, ATP testing may still have a role in providing reassurance that cleaning regimes are being carried out satisfactorily. However, ATP results should not be interpreted as surrogate indicators for the presence of microbial pathogens

Estimating bacterial surface contamination by means of ATP determinations: 20 pence short of a pound

Estimating bacterial surface contamination by means of ATP determinations: 20 pence short of a poun

High precision microfluidic microencapsulation of bacteriophages for enteric delivery

A Salmonella specific bacteriophage Felix O1 (Myoviridae) was microencapsulated in a pH responsive polymer formulation. The formulation incorporated a pH responsive methacrylic acid copolymer Eudragit® S100 (10% (w/v)) with the addition of the biopolymer sodium alginate, the composition of which was varied in the range (0.5% (w/v)e2% (w/v)). The microencapsulation process employed commercially available microfluidic droplet generation devices. We have used readily available low cost microfluidic chips instead of bespoke in-house fabricated glass capillary devices which are accessible only in specialist research facilities. We show that these co-flow microfluidic devices can easily be used to prepare phage encapsulated microparticles making them suitable for use by both the phage research community and industry in order to evaluate and optimise phage compatible formulations for microencapsulation. A novelty of the work reported here is that the size of the generated monodispersed droplets could be precisely controlled in the range 50 mme200 mm by varying the flow rates of the dispersed and continuous phases. Consequently, alginate concentration and microparticle size were shown to influence the phage release profile and the degree of acid protection afforded to phages upon exposure to simulated gastric fluid (SGF). Bigger microparticles (~100 mm) showed better acid protection compared with smaller beads (~50 mm) made from the same formulation. Increasing the alginate composition resulted in improved acid protection of phages for similar particle sizes. The high viscosity formulations containing higher amounts of alginate (e.g. 2% (w/v)) negatively affected ease of droplet generation in the microfluidic device thereby posing a limitation in terms of process scale-up. Felix O1 encapsulated in the formulation containing 10% (w/v) ES100 and 1% (w/v) alginate showed excellent protection upon exposure of the gelled microparticles to SGF (pH 1 for 2 h) without the use of any antacids in the encapsulation matrix. Encapsulated phages previously exposed to SGF (pH 1 for 2 h) were released at elevated pH in simulated intestinal fluid (SIF) and were shown to arrest bacterial growth in the log growth phase. We have therefore demonstrated the microencapsulation of phages using readily available microfluidic chips to produce solid dosage microcapsule forms with a rapid pH triggered release profile suitable for targeted delivery and controlled release in the gastrointestinal tract

Questioning the catalytic effect of Ni nanoparticles on CO2 hydration and the very need of such catalysis for CO2 capture by mineralization from aqueous solution

© 2017 Elsevier Ltd Recent publications claimed a significant catalytic effect of nickel nanoparticles on the hydration of CO 2 to carbonic acid. Others have claimed that such catalysis can significantly accelerate the overall process of CO 2 capture by mineralization to CaCO 3 from aqueous solution. Having repeated the experiments as closely as possible, we observed no catalytic effect of Ni nanoparticles. Numerical modelling revealed that hydration is not the slowest reaction in the chain ending with mineralization; hence its catalysis cannot have a significant effect on CaCO 3 formation

Evaluation and comparison of protein ultrafiltration test results: dead-end stirred cell compared with a cross-flow system

Dead-end stirred cell devices are commonly used in laboratories to characterise ultrafiltration membranes and their separation behaviour. Additionally, protein separation data from such systems are used for process scale-up. Such devices are operated under conditions that are inherently different from those used during the continuous or semi-continuous processing of industrial feed streams. The work presented in this paper compares the rejection behaviour of single protein solutions in both a dead-end stirred cell (SC) device with that for a crossflow system (CF). The effect of ionic strength (20 mM and 100 mM) and solution pH (4.9, 6.0, 7.1, 8.4 and 11.0) on protein filtration (bovine serum albumin (BSA) and lysozyme (LYZ) from buffered aqueous solutions) behaviour has been investigated using polyethersulfone (PES) membranes with a manufacturer specified molecular weight cut-off (MWCO) of 50 kDa. PES membranes were characterised in terms of dextran MWCO using both the SC and the CF systems. The mode of operation resulted in significant observed differences in the resulting dextran solute rejection curves for the two systems. The observed rejection (Robs) values for a series of dextran standards were consistently found to be lower for the CF system compared with the SC unit suggesting higher wall concentrations (Cw) due to concentration polarisation effects in the CF unit. Protein ultrafiltration studies with the 50 kDa PES membranes highlighted important differences in observed protein rejection behaviour despite operation of the two systems at the same transmembrane pressures (25 kPa). Solution pH was found to have little effect on the rejection of both BSA and LYZ. The solute rejection was found to be more sensitive to ionic strength effects for the SC device both during BSA and LYZ filtration. Convective mass transfer coefficients and hence the true rejection coefficients (Rtr) were calculated for both systems using the stagnant film model to understand the influence of hydrodynamic effects on the ultrafiltration behaviour of the two systems. The magnitude of the Peclet number (Pe) provides a means of comparing hydrodynamic conditions for the two systems and thereby allows differences in observed solute rejection to be better understood

Hydroxylation of benzene to phenol using nitrous oxide

There is an increasing commercial interest in finding alternative ways to produce phenol that overcome the disadvantages of the current cumene process used to synthesise phenol. The drivers for the change are both economical and environmental. A direct oxidation route for producing phenol from benzene is based on using N2O as an oxidizing agent in the gas phase in the presence of modified Fe-ZSM5 zeolite. A series of selective Fe-ZSM5 catalysts with different Si/Al ratios have been prepared and evaluated for selective formation of phenol. Catalysts synthesized with high Si/Al ratios (80) and with low iron content (35mg/g) showed good long term stability (reduced deactivation rates) and demonstrated good phenol selectivity and reaction rates (6 mmol/g.h). Catalysts with high amounts of iron (~500mg/g) showed considerable deactivation particularly at high reaction temperatures (450C). High reaction temperatures (450C) in comparison with 350C were found to favour higher reaction rates however, catalysts with high iron content were particularly prone to deactivation at this temperature

Microencapsulation of Clostridium difficile specific bacteriophages using microfluidic glass capillary devices for colon delivery using pH triggered release

The prevalence of pathogenic bacteria acquiring multidrug antibiotic resistance is a global health threat to mankind. This has motivated a renewed interest in developing alternatives to conventional antibiotics including bacteriophages (viruses) as therapeutic agents. The bacterium Clostridium difficile causes colon infection and is particularly difficult to treat with existing antibiotics; phage therapy may offer a viable alternative. The punitive environment within the gastrointestinal tract can inactivate orally delivered phages. C. difficile specific bacteriophage, myovirus CDKM9 was encapsulated in a pH responsive polymer (Eudragit® S100 with and without alginate) using a flow focussing glass microcapillary device. Highly monodispersed core-shell microparticles containing phages trapped within the particle core were produced by in situ polymer curing using 4-aminobenzoic acid dissolved in the oil phase. The size of the generated microparticles could be precisely controlled in the range 80 μm to 160 μm through design of the microfluidic device geometry and by varying flow rates of the dispersed and continuous phase. In contrast to free `naked' phages, those encapsulated within the microparticles could withstand a 3 h exposure to simulated gastric fluid at pH 2 and then underwent a subsequent pH triggered burst release at pH 7. The significance of our research is in demonstrating that C. difficile specific phage can be formulated and encapsulated in highly uniform pH responsive microparticles using a microfluidic system. The microparticles were shown to afford significant protection to the encapsulated phage upon prolonged exposure to an acid solution mimicking the human stomach environment. Phage encapsulation and subsequent release kinetics revealed that the microparticles prepared using Eudragit® S100 formulations possess pH responsive characteristics with phage release triggered in an intestinal pH range suitable for therapeutic purposes. The results reported here provide proof-of-concept data supporting the suitability of our approach for colon targeted delivery of phages for therapeutic purposes

The inactivation of bacillus subtilis spores at low concentrations of hydrogen peroxide vapour

Spores of the bacterium Bacillussubtilis were deposited onto the surface of membranes by a process of filtration and exposed to concentrations of hydrogen peroxide vapour between 10 and 90 mg/m3 (ppm) for times ranging from 1.5 to 48 h. The inactivation data obtained in this way was modelled using the Weibull, Series-Event and Baranyi inactivation models. The Weibull model provided the best fit, and its use was extended to previously published literature obtained at higher hydrogen peroxide concentrations to produce a correlation yielding D (decimal reduction value) values over a range from 10 to almost 4000 ppm

Microencapsulation of Clostridium difficile specific bacteriophage using glass microcapillary devices and pH dependent controlled release for colon targeted delivery [Abstract]

Microencapsulation of Clostridium difficile specific bacteriophage using glass microcapillary devices and pH dependent controlled release for colon targeted delivery [Abstract