Modern convection‐based supports differ substantially in pore size, porosity, and microstructure topology. Due to such variability, it is challenging to evaluate the contribution of a particular ...microstructure topology on flow resistance. We demonstrated that the flow resistance parameter (ϕ$\phi $) introduced decades ago can be used as a criterion to evaluate the effect of microstructure topology on a pressure drop when the pore size is used as a characteristic support dimension. Furthermore, the ϕ$\phi $ value of simple cubic packing was calculated over the entire range of open porosity and compared to the ϕ$\phi $ values determined for pressure drop models derived for particular convection‐based supports and experimental values of various convection‐based supports from the literature. It was shown that different convection‐based supports become clustered into distinct groups when plotted according to their ϕ$\phi $ and open porosity values, allowing their discrimination.
Pressure drop in liquid chromatography Podgornik, Aleš
Journal of separation science,
January 2019, 2019-Jan, 2019-01-00, 20190101, Letnik:
42, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Measurement of pressure drop is routinely performed during chromatographic runs. In many cases this information is only used for regulation of mobile phase flow rate to keep pressure drop below ...defined limit. However, pressure drop can provide additional important information about performance of chromatographic process. In this review different parameters affecting pressure drop such as compressibility of chromatographic media, nature of applied sample and mobile phase flow regime are discussed. Detailed analysis correlating organization of particle based chromatographic media and pressure drop is presented together with its extension to convective media such as membranes and monoliths but also novel 3D printed media. Finally, estimation of layer thickness formed by adsorbed molecules based on a pressure drop data is presented and its applicability is discussed.
Bacteriophage production processes Jurač, Katja; Nabergoj, Dominik; Podgornik, Aleš
Applied microbiology and biotechnology,
01/2019, Letnik:
103, Številka:
2
Journal Article
Recenzirano
High quantities of bacteriophages are currently used in the food industry and agriculture. However, growing antibiotic resistance of bacteria has recently awakened the interest to use bacteriophages ...for the treatment of bacterial infections in humans indicating that even higher quantities will be required in the future. High demand combined with a wide range of applications requires also efficient bacteriophage production processes operating at low production costs and with high productivity. To achieve this goal, different approaches were introduced and extensive studies of various parameters affecting bacteriophage formation were investigated. In this mini-review, we provide a short overview about different operation modes of bacteriophage production such as batch, semi-continuous and especially continuous with the pros and cons of each. We present factors affecting bacterial physiological state, its effect on phage formation and provide a description of methods for determination of bacteriophage growth parameters, through which bacteriophage formation is obtained. Understanding of described phenomena and inclusion of potential occurrence of mutations and selection in continuous systems enables evaluation of continuous process productivity and its optimization.
Mechanical properties of a material play a pivotal role in its performance when such porous material is used in a flow‐through mode. This study delves into the effect of porosity and microstructure ...on the compressibility of methacrylate polymer, focusing on two distinct microstructures: cauliflower and high internal phase emulsion. Samples with various porosities yet identical chemical composition were prepared and their Young's modulus was measured. The effect of porosity on Young's modulus was described by an exponential law model with the cauliflower microstructure exhibiting an exponent of 3.61, while the high internal phase emulsion of only 1.86. A mathematical analysis of the compression caused by a liquid flow unveiled significant disparities in the porosity threshold where minimal compression is observed, being around 0.45 for the cauliflower while there is monotone decrease in compression with porosity increase for the high internal phase emulsion microstructure. Evaluating exponent integer values between 1 and 5 over entire porosity range reveals that the porosity where the minimal compression occurs increases with a decrease in exponent value, being approximately 0.33 for n = 5, 0.4 for n = 4, 0.55 for n = 3, 0.65 for n = 2 while no minimum occurs for n = 1. These findings indicate that lower exponent value results in lower compression under identical experimental conditions.
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•Human insulin and six therapeutic analogues are resolved on a mixed mode column.•Development of two separation methods: for binary and quaternary pump delivery system.•SPE cleanup of ...formulations with insulin recoveries from 80 to 100% is optional.•Identification and quantitation of insulins is possible using simple HPLC-UV method.•Methods were validated and can be used for screening of pharmaceutical raw materials.
Human insulin and six most used therapeutic analogues are very similar in terms of retention on a reversed-phase column. Thus, the LC methods prescribed in the European Pharmacopoeia monographs for insulin and insulin analogues include many similar separation methods, which tend to be time consuming when separating individual products of insulins or are inadequate when handling a mixture. In this study, we present a simple, robust, versatile and accessible HPLC-UV separation method for identification and quantification of human insulin and its analogues in one run. The simultaneous separation and detection is possible by fine-tuning the mobile phase properties that affect the separation mechanism on a mixed mode column combining anion exchange and reversed-phase characteristics. Also developed was a simple and effective SPE sample cleaning procedure with insulin recoveries ranging from 80 to 100% for all analogues. On the other hand, the concentration of major excipients such as phenol and m-cresol fall below 1%. The two developed and validated separation methods differ in their compatibility with the use of a quaternary or binary pump, thus enabling sample characterisation independent of the HPLC solvent delivery system. The methods are compatible with the use of a mass spectrometric detector for an indisputable identification.
Several polymerized high internal phase emulsions (polyHIPE) of various porosity but the same pore diameter were prepared. It was demonstrated that the pore size distribution of void pores and ...interconnecting pores is similar and it can be described by a log-normal distribution. Flow-through porosity matches closely static porosity and the non-polymerizable fraction of initial emulsion, demonstrating an open structure of polyHIPE that allows flow through the entire porous polymer. Pulse response experiments demonstrated flow uniformity. Pressure drop analysis allowed estimation of porosity effect on permeability. Fitting of experimental data with different pressure drop models revealed that interconnecting pore diameter should be used as a characteristic dimension to describe pressure drop on polyHIPE accurately. Linear correlation between permeability and flow-through porosity demonstrated that the effect of the porous polymer structure on the flow properties is independent of porosity. This finding suggests that polyHIPE hydrodynamics does not change with porosity, facilitating prediction of their performance for various applications where flow-through mode is important.
•Porosity change of polyHIPE with the same pore sizes correlated with pressure drop.•Photocurable poly(GMA–EGDMA) columns with 75, 80, 85 and 90% porosities prepared.•Void and interconnected/window pore size measured with ellipses from SEM in ImageJ.•Pulse experiments for convective flow-through polymer performed for hydrodynamics.•Pressure drop data was evaluated with Happel, modified Happel, TSC and RUC model.
Summary
A system consisting of a connected mixed and tubular bioreactor was designed to study bacterial biofilm formation and the effect of its exposure to bacteriophages under different experimental ...conditions. The bacterial biofilm inside silicone tubular bioreactor was formed during the continuous pumping of bacterial cells at a constant physiological state for 2 h and subsequent washing with a buffer for 24 h. Monitoring bacterial and bacteriophage concentration along the tubular bioreactor was performed via a piercing method. The presence of biofilm and planktonic cells was demonstrated by combining the piercing method, measurement of planktonic cell concentration at the tubular bioreactor outlet, and optical microscopy. The planktonic cell formation rate was found to be 8.95 × 10−3 h−1 and increased approximately four‐fold (4×) after biofilm exposure to an LB medium. Exposure of bacterial biofilm to bacteriophages in the LB medium resulted in a rapid decrease of biofilm and planktonic cell concentration, to below the detection limit within < 2 h. When bacteriophages were supplied in the buffer, only a moderate decrease in the concentration of both bacterial cell types was observed. After biofilm washing with buffer to remove unadsorbed bacteriophages, its exposure to the LB medium (without bacteriophages) resulted in a rapid decrease in bacterial concentration: again below the detection limit in < 2 h.
In this article we present flow‐through system allowing formation and characterization of bacterial biofilms together with their susceptibly toward bacteriophages. We were able to determine generation rate of planktonic cells released by formed biofilm as well as bacteriophages efficiency in biofilm eradication in the presence of nutrients and under substrate limitation. It was demonstrated that bacteriophages infect biofilm in both environments but they are efficient in biofilm lysis only in the presence of bacteria nutrients
The determination of bacteriophage growth parameters, such as adsorption constant, latent period, and burst size, is essential for the proper design of bacteriophage production and the estimation of ...the efficacy of bacteriophage therapy. As they are dependent on the physiological state and cultivation conditions bacteria, they should be preferably determined in a non-invasive way. We propose a method that allows their determination under cultivation conditions. It is based on the cultivation of bacteria in a chemostat, the injection of bacteriophages, and monitoring of their concentration over a certain period. Phage growth parameters are determined by fitting a mathematical model to experimental data.
E. coli
–T4 system was investigated for various dilution rates covering a broad range of bacteria physiological states. Results were used for a prediction of bacteriophages and bacteria steady-state concentrations in a cellstat. A close match was found when adsorption of bacteriophages to the lysed cells was considered in the cellstat, while this mechanism can be neglected in the chemostat. Trends and values for burst size and latent period were consistent with literature data, demonstrating an increase in the burst size and decrease of the latent period with an increase of bacteria-specific growth rate (from 19 to 81 bacteriophage particles per cell and 89 to 29.8 min for a specific growth rate between 0.072 and 0.96 h
−1
, respectively). Adsorption constant also showed an increase with a specific growth rate increase (from 2.8E-10 to 4.0E-09 mL min
−1
), in contrast to chemostat literature data, probably due to its determination within the bioreactor. The proposed method also allowed estimation of latent period distribution. While its value for high-specific growth rates was determined to be constant of around 6 min, an increase of over an order of magnitude was found for the lowest specific growth rate, probably as a consequence of higher variability within bacteria population.
Key Points
• A method for determination of phage growth parameters under cultivating conditions was developed.
• The method was successfully tested on E. coli and T4 bacteriophage system comparing chemostat and cellstat values.
• Adsorption to lysed cells was found to be important for cellstat experiment but can be neglected in the chemostat.
• The determined burst size and latent period dependence on the bacterial physiological state was consistent with literature data, while differences were found for adsorption constant.
• Latent period distribution significantly increases for low bacteria–specific growth rates.
► Application of short monolithic columns for analytics of macromolecules and nanoparticles – recent overview. ► Impact of macromolecule interactions on peak spreading and resolution in IEX. ► ...Overview of binding sites number for different class of macromolecules and nanoparticles.
Chromatographic monoliths have already penetrated in many different areas of separation sciences. This is due to their properties, especially advantageous for fast separation and purification of large biologic macromolecules, even at low pressure drop. Probably the most outstanding features are flow unaffected binding capacity and resolution, later resulting in very short analysis times. Furthermore, since large biomolecules interact with the matrix via many binding sites, efficient separation can be achieved with the monolithic columns of a very short length, further reducing pressure drop over matrix. In this review brief introduction to the monoliths is given with the emphasize on the theory of separation of large molecules, particularly on a linear gradient elution and estimation of peak broadening. As an outcome of this analysis the most efficient separation is expected when short monolithic column with accordingly adjusted gradient is implemented, especially for macromolecules interacting with the monolith functionalities via over 10 binding sites. This is experimentally demonstrated by several recent examples of short monolithic column applications for analysis of antibodies, viruses, virus like particles (VLPs) and polynucleotides like plasmid DNA (pDNA) and RNA, indicating their potential for process monitoring, control and optimization but also for product final formulation and quality control.
It is important to understand how physiological state of the host influence propagation of bacteriophages (phages), due to the potential higher phage production needs in the future. In our study, we ...tried to elucidate the effect of bacterial growth rate on adsorption constant (δ), latent period (L), burst size (b), and bacteriophage population growth rate (λ). As a model system, a well‐studied phage T4 and Escherichia coli K‐12 as a host was used. Bacteria were grown in a continuous culture operating at dilution rates in the range between 0.06 and 0.98 hr−1. It was found that the burst size increases linearly from 8 PFU·cell−1 to 89 PFU·cell−1 with increase in bacteria growth rate. On the other hand, adsorption constant and latent period were both decreasing from 2.6∙10‐9 ml·min−1 and 80 min to reach limiting values of 0.5 × 10‐9 ml·min−1 and 27 min at higher growth rates, respectively. Both trends were mathematically described with Michaelis–Menten based type of equation and reasons for such form are discussed. By applying selected equations, a mathematical equation for prediction of bacteriophage population growth rate as a function of dilution rate was derived, reaching values around 8 hr−1 at highest dilution rate. Interestingly, almost identical description can be obtained using much simpler Monod type equation and possible reasons for this finding are discussed.
Due to versatility of applications of bacteriophages and consequently potential higher phage production needs in the future, it is important to evaluate the effect of physiological state of the host on propagation of phages. In our study, we demonstrated on system phage T4 and Escherichia coli K‐12 growing in a continuous culture, that all phage growth parameters and consequently also bacteriophage population growth rate depends significantly on bacterial growth rate. The trends of each phage growth parameter and bacteriophage population growth rate were mathematically described enabling accurate prediction of bacteriophage propagation for specific bacterial growth rate and by this design of phage production process as well.
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