Influence of porosity and microstructure on compression behavior of methacrylate polymers in flow-through applications.

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.

Rapid, Direct, Noninvasive Method to Determine the Amount of Immobilized Protein.

Protein immobilization is of utmost importance in many areas, where various proteins are used for selective detection of target compounds. Despite the importance given to determine the amount of immobilized protein, there is no simple method that allows direct, noninvasive detection. In this work, a method based on pH transition, occurring during change of solution ionic strength, was developed. The method utilized the ionic character of the immobilized protein while implementing biologically compatible buffers. Five different proteins, namely, glucose oxidase, horseradish peroxidase, bovine serum albumin, lysozyme, and protein A, were immobilized in different amounts on a porous polymeric matrix, and their pH transition was measured using lactate buffer of various concentrations and pH values. A linear correlation was found between the amount of immobilized protein and the amplitude of the pH transition, allowing the detection down to 2 nmol of immobilized protein. By changing the buffer concentration and pH, the sensitivity of the method could be tailored. Criteria based on the symmetry of the pH transition peak have been developed to determine if a particular measurement is within a linear range. In addition, a mathematical model was developed enabling prediction of pH transition profiles based solely on the protein amino acid sequence, the buffer pKa value(s), and the amount of immobilized protein.Hence, it can be used to design pH transition method experiments to achieve the required sensitivity for a target sample. Since the proposed method is noninvasive, it can be routinely applied during optimization of the immobilization protocol, for quality control, and also as an in-process monitoring tool.

Effect of plasmid DNA isoforms on preparative anion exchange chromatography.

Increased need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA) in gene therapy and vaccination brings the need for its large-scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion-exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6 µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6 kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5 µm has been shown to increase yields and prevent irreversible pressure build-up and column clogging during purification of plasmids at least up to 16 kbp in size.

Complex Protein Retention Shifts with a Pressure Increase: An Indication of a Standard Partial Molar Volume Increase during Adsorption?

Studies of protein adsorption on reversed-phase and ion exchange stationary phases demonstrated an increase in retention with increasing pressure, which is interpreted as a standard partial molar volume decrease during the transition of the protein from a mobile to a stationary phase. Investigation of the pressure effect on the retention of lysozyme and IgG on a cation exchange column surprisingly revealed a negative retention trend with the increase of pressure. Further investigation of this phenomenon was performed with ß-lactoglobulin, which enabled adsorption to be studied on both cation and anion exchange columns using the same mobile phase with a pH of 5.2. The same surface charge and standard partial molar volume in the mobile phase allowed us to examine only the effect of adsorption. Interestingly, a negative retention trend with a pressure increase occurred on an anion exchange column while a positive trend was present on a cation exchange column. This indicates that the interaction type governs the change in the standard partial molar volume during adsorption, which is independent of the applied pressure. Increasing the protein charge by decreasing the pH of the mobile phase to 4 reversed the retention trend (into a negative) with a pressure increase on the cation exchange column. A further decrease of the pH value resulted in an even more pronounced negative trend. This counterintuitive behavior indicates an increase in the standard partial molar volume during adsorption with the protein charge, possibly due to intermolecular repulsion of adsorbed protein molecules. While a detailed mechanism remains to be elucidated, presented results demonstrate the complexity of ion exchange interactions that can be investigated simply by changing the column pressure.

Characterization of a convection-based support microstructure through a flow resistance parameter.

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.

Designing scalable ultrafiltration/diafiltration process of monoclonal antibodies via mathematical modeling by coupling mass balances and Poisson-Boltzmann equation.

Ultrafiltration/diafiltration (UF/DF) operations are employed for achieving the desired therapeutic monoclonal antibody (mAb) formulations. Due to electrostatic interactions between the charged proteins, solute ions, and uncharged excipients, the final pH and concentration values are not always equal to those in the DF buffer. At high protein concentrations, typical for industrial formulations, this effect becomes predominant. To account for challenges occurring in industrial environments, a robust mathematical framework enabling the prediction of pH and concentration profiles throughout the UF/DF process is provided. The proposed mechanistic model combines a macroscopic mass balance approach with a molecular approach based on a Poisson-Boltzmann equation dealing with electrostatic interactions and accounting for protein exclusion volume effect. The mathematical model was validated with experimental data of two commercially relevant mAbs obtained from an industrial UF/DF process using scalable laboratory equipment. The robustness and flexibility of the model were tested by using proteins with different isoelectric points and net charges. The latter was determined via a titration curve, enabling realistic protein charge-pH evaluation. In addition, the model was tested for different DF buffer types containing both monovalent and polyvalent ions, with various types of uncharged excipients. The model generality enables its implementation for the UF/DF processes of other protein varieties.

Effect of Pressure Increase on Macromolecules' Adsorption in Ion Exchange Chromatography.

In this study a new method for evaluating the pressure effect on separations of oligonucleotides and proteins on an anion exchange column was developed. The pressure rise of up to 500 bar was attained by coupling restriction capillaries to the column outlet to minimize differences in pressure over the column. Using pH transient measurements it was demonstrated that no shift in ion exchange equilibria occurs due to a pressure increase. Results from isocratic and gradient separations of oligonucleotides (model compounds) were evaluated by stoichiometric displacement and linear gradient elution model, respectively. Both elution modes demonstrated that for smaller oligonucleotides the number of binding sites remained unchanged with pressure rise while an increase for large oligonucleotides was observed, indicating their alignment over the stationary phase. From the obtained model parameters and their pressure dependencies, a thermodynamic description was made and compared between the elution modes. A complementary pattern of a linear increase of partial molar volume change with a pressure rise was established. Furthermore, estimation of the pressure effect was performed for bovine serum albumin and thyroglobulin that required gradient separations. Again, a raise in binding site number was found with pressure increase. The partial molar volume changes of BSA and Tg at the maximal investigated pressure and minimal salt concentration were -31.6 and -34.4 cm3/mol, respectively, indicating a higher rigidity of Tg. The proposed approach provides an insight into the molecule deformation over a surface at high pressures under nondenaturing conditions. The information enables a more comprehensive UHPLC method development.

Determination of bacteriophage growth parameters under cultivating conditions.

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.

An alternative molecular approach for rapid and specific detection of clinically relevant bacteria causing prosthetic joint infections with bacteriophage K.

Prosthetic joint infections (PJI) represent the most serious cause of prosthetic joint loosening, with high impact on patient life and health economics. Although not entirely reliable, the cultivation of intraoperative prosthetic tissue or synovial fluid remains the gold standard for determining the cause of PJI. Therefore, molecular methods are increasingly being introduced. The aim of this study was to optimize and assess an alternative molecular approach with the use of bacteriophage K for more rapid and specific detection of staphylococci in sonicate fluid (SF) of PJI. The best results with the method were obtained after 180 min of sample incubation with 104 PFU/mL of bacteriophage K. DNA isolation prior to qPCR analysis was confirmed unnecessary, while chloroform addition to samples after incubation with bacteriophage K improved bacterial detection by 100×. The method had a limit of detection of 6.8×102 CFU/mL and was found suitable for the detection of staphylococci in SF of removed prosthetic joints, giving results comparable to standard microbiological methods in just four hours. The optimized method was found fit for the purpose, offering potential advantages over the use of molecular detection methods to detect bacterial DNA.

Bacteriophage production processes.

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.

Pressure drop in liquid chromatography.

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.

Adsorbed Layer Thickness Determination for Convective-Based Media from Pressure Drop Data.

In the present work, we derive a theoretical framework to determine the adsorbed layer thickness from pressure drop measurements for convective-based media without any assumptions about the geometry of the pore structure of the stationary phase matrix. Equations are presented to calculate accuracy of the estimated adsorbed layer thickness as a consequence of measurement error and approximations of the mathematical model. We discovered that there is a minimum in the error for certain pressure drops that results in optimal experimental conditions for determining the adsorbed layer thickness. We demonstrate that the adsorbed layer thickness can be determined with less than 10% error using a wide range of experimental conditions simply from pressure drop data. By careful selection of porous bed dimensions and flow rates, the adsorbed layer thicknesses from subnanometer dimensions up to several hundred nanometers can be determined by measurement of the pressure drop in a range of several bars. The method was experimentally tested on methacrylate monolithic columns using monodisperse latex nanoparticles as a reference standard and two different proteins as unknowns demonstrating close agreement with calculations.

Effect of dilution rate on productivity of continuous bacteriophage production in cellstat.

Ability to efficiently propagate high quantities of bacteriophages (phages) is of great importance considering higher phage production needs in the future. Continuous production of phages could represent an interesting option. In our study, we tried to elucidate the effect of dilution rate on productivity of continuous production of phages in cellstat. As a model system, a well-studied phage T4 and Escherichia coli K-12 as a host were used. Experiments where physiology of bacteria was changing with dilution rate of cellstat and where bacterial physiology was kept constant were performed. For both setups there exists an optimal dilution rate when maximal productivity is achieved. Experimentally obtained values of phage concentration and corresponding productivity were compared with mathematical model predictions, and good agreement was obtained for both types of experiments. Analysis of mathematical model coefficients revealed that latent period and burst size to dilution rate coefficient mostly affect optimum dilution rate and productivity. Due to high sensitivity, it is important to evaluate phage growth parameters carefully, to run cellstat under optimal productivity.

Effect of pore size on performance of monolithic tube chromatography of large biomolecules.

Effect of pore size on the performance of ion-exchange monolith tube chromatography of large biomolecules was investigated. Radial flow 1 mL polymer based monolith tubes of different pore sizes (1.5, 2, and 6 µm) were tested with model samples such as 20 mer poly T-DNA, basic proteins, and acidic proteins (molecular weight 14 000-670 000). Pressure drop, pH transient, the number of binding site, dynamic binding capacity, and peak width were examined. Pressure drop-flow rate curves and dynamic binding capacity values were well correlated with the nominal pore size. While duration of the pH transient curves depends on the pore size, it was found that pH duration normalized on estimated surface area was constant, indicating that the ligand density is the same. This was also confirmed by the constant number of binding site values being independent of pore size. The peak width values were similar to those for axial flow monolith chromatography. These results showed that it is easy to scale up axial flow monolith chromatography to radial flow monolith tube chromatography by choosing the right pore size in terms of the pressure drop and capacity.

Quick bacteriophage-mediated bioluminescence assay for detecting Staphylococcus spp. in sonicate fluid of orthopaedic artificial joints.

Staphylococcus spp. accounts for up to two thirds of all microorganisms causing prosthetic joint infections, with Staphylococcus aureus and Staphylococcus epidermidis being the major cause. The present study describes a diagnostic model to detect staphylococci using a specific bacteriophage and bioluminescence detection, exploring the possibility of its use on sonicate fluid of orthopaedic artificial joints. Intracellular adenosine-5'-triphosphate release by bacteriophage mediated lysis of staphylococci was assessed to determine optimal parameters for detection. With the optimized method, a limit of detection of around 103 CFU/mL was obtained after incubation with bacteriophage for 2 h. Importantly, sonicate fluid did not prevent the ability of bacteriophage to infect bacteria and all simulated infected sonicate fluid as well as 6 clinical samples with microbiologically proven staphylococcal infection were detected as positive. The total assay took approximately 4 h. Collectively, the results indicate that the developed method promises a rapid, inexpensive and specific diagnostic detection of staphylococci in sonicate fluid of infected prosthetic joints. In addition, the unlimited pool of different existing bacteriophages, with different specificity for all kind of bacteria gives the opportunity for further investigations, improvements of the current model and implementation in other medical fields for the purpose of the establishment of a rapid diagnosis.

Surfaces energies of monoliths by inverse liquid chromatography and contact angles.

Seven porous chromatographic columns, termed monoliths, and seven nonporous sheets were produced from polymethacrylates. Their surfaces were activated by different densities of butyl and phenyl ligands. We determined the retention times of highly dilute molecular probes in monoliths and accessed contact angles of pure molecular probes of sheets. We calculated surface energies for both systems. We applied theories of Young, Dupré, and van Oss and compared the results of both types of experiments with respect to Lifshitz-van der Waals and Lewis acid and Lewis base contributions and find agreement but an additive constant.

Simple and Tailorable Synthesis of Silver Nanoplates in Gram Quantities.

Due to plasmonic and catalytic properties, silver nanoplates are of significant interest; therefore, their simple preparation in gram quantities is required. Preferably, the method is seedless, consists of few reagents, enables preparation of silver nanoplates with desired optical properties in high concentration, is scalable, and allows their long-term storage. The developed method is based on silver nitrate, sodium borohydride, polyvinylpyrrolidone, and H2O2 as the main reagents, while antifoam A204 is implemented to achieve better product quality on a larger scale. The effect of each component was evaluated and optimized. Solution volumes from 3 to 450 mL and concentrations of silver nanoplates from 0.88 to 4.8 g/L were tested. Their size was tailored from 25 nm to 8 µm simply by H2O2 addition, covering the entire visible plasmon spectra and beyond. They can be dried and spontaneously dispersed after at least one month of storage in the dark without any change in plasmonic properties. Their potential use in modern art was demonstrated by drying silver colloids on different surfaces in the presence of reagents or purified, resulting in a variety of colors but, more importantly, patterns of varying complexity, from simple multi-coffee-rings structures to dendritic forms and complex multilevel Sierpinski triangle fractals.

Application of monoliths for bioparticle isolation.

Monoliths are today probably the most studied chromatographic supports. There are plethora of publications dealing with different aspects of their preparation, characterization, and applications. The reason for this interest is their inherent properties related to their particular structure, like ease of preparation in various volumes, fast analytics at low pressure and room temperature, and high productivity as a consequence of flow-unaffected properties, especially important for isolation of large biological molecules. Because of that, structure of several monoliths was optimized for analytics and purification of biologic nanoparticles like viruses, virus-like particles (VLPs), cells structures, or even intact cells. In this review, some recent applications of monoliths in the field of bioparticle isolation are described and results are discussed in terms of particular monolith properties.

Noninvasive method for determination of immobilized protein A.

The pH transition method, developed for the determination of the ion-exchange group density on chromatographic stationary phase, was used for the quantification of immobilized protein A. Monolithic epoxy polyHIPE and particulate CNBr-Sepharose supports were used for immobilization. A lactate buffer was selected, having a buffer capacity peak approximately 0.5 pH units below the maximum buffer capacity of protein A. The pH transition measurements were performed at pH 4.3, where protein A exhibits maximum buffer capacity, with a lactate buffer concentration of 1 mM for protein A immobilized on polyHIPE monoliths and of 5 mM for protein A immobilized on CNBr-Sepharose. The pH transition height and full width at half maximum for the particulate support and the height for the polyHIPE matrix, showed a linear correlation with the amount of immobilized protein A determined from the absorbance difference before and after immobilization for both supports. The developed method allows a simple, non-invasive on-line determination of immobilized protein A using biological buffers, even for chromatographic columns with an amount of immobilized protein A as low as 0.25 mg. In addition, its sensitivity and duration can be easily adjusted by varying the buffer concentration and pH.

E. coli biofilm formation and its susceptibility towards T4 bacteriophages studied in a continuously operating mixing - tubular bioreactor system.

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.