Computational modelling of the therapeutic outputs of photodynamic therapy on spheroid-on-chip models.

Photodynamic therapy (PDT) is a medical radio chemotherapeutic method that uses light, photosensitizing agents, and oxygen to produce cytotoxic compounds, which eliminate malignant cells. Recently, Microfluidic systems have been used to analyse photosensitizers (PSs) due to their potential to replicate in vivo environments. While prior studies have established a strong correlation between reacted singlet oxygen concentration and PDT-induced cellular death, the effects that the ambient fluid flow might have on the concentration of oxygen and PS have been disregarded in many, which limits the reliability of the results. Herein, we coupled the transport of oxygen and PS throughout the ambient medium and within the spheroidal multicellular aggregate to initially study the profiles of oxygen and PS concentration alongside PDT-induced cellular death throughout the spheroid before and after radiation. The attained results indicate that the PDT-induced cellular death initiates on the surface of the spheroids and subsequently spreads to the neighbouring regions, which is in great accordance with experimental results. Afterward, the effects that drug-light interval (DLI), fluence rate, PS composition, microchannel height, and inlet flow rate have on the therapeutic outcomes are studied. The findings show that adequate DLI is critical to ensure uniform distribution of PS throughout the medium, and a value of 5 h was found to be sufficient. The composition of PS is critical, as ALA-PpIX induces earlier cell death but accelerates oxygen consumption, especially in the outer layers, depriving the inner layers of oxygen necessary for PDT, which in turn disrupts and prolongs the exposure time compared to mTHPC and Photofrin. Despite the fluence rate directly influencing the singlet oxygen generation rate, increasing the fluence rate by 189 mW/cm2 would not significantly benefit us. Microwell height and inlet flow rate involve competing phenomena-increasing height or decreasing flow reduces oxygen supply and increases PS "washout" and its concentration.

Highly efficient strategy of lipopolysaccharide (LPS) decontamination from rHBsAg: synergistic effect of enhanced magnetic nanoparticles (MNPs) as an LPS affinity adsorbent (LAA) and surfactant as a dissociation factor.

The interaction of lipopolysaccharide with a recombinant protein is a serious bottleneck, particularly in the purification step of bioprocessing. Recombinant hepatitis B surface antigen (rHBsAg), the active ingredient of the hepatitis B vaccine, is probably contaminated by extrinsic LPS like other biopharmaceuticals. This research intends to eliminate LPS from its mixture with rHBsAg efficiently. Immobilized polymyxin B on magnetic nanoparticles (PMB-MNPs) was synthesized and implemented as an enhanced LPS affinity adsorbent (LAA). The 20-80 EU/dose binary samples with and without surfactant were applied to PMB-MNPs. Formerly, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were examined on the samples to qualitatively show the dissociation effect of the surfactant. Considering the high potential interaction of LPS with HBsAg, the dissociation effects of 0.5 and 1.5% Tween 20 on the binary samples were assessed using immunoaffinity chromatography (IAC) as a quantification tool. The dissociation effect of Tween 20 substantially diminished the interaction, leading to a proportional increase of free LPS up to 66%. The synergetic effect of Tween 20 and privileged LAA was highly effective in eliminating more than 80% of LPS with a remarkable LPS clearance factor of 5.8 and a substantial protein recovery rate of 97%.

Application of a 3D printed miniaturized hydrocyclone in biopharmaceutical industry-numerical and experimental studies of yeast separation from fermentation culture media.

Various industries ranging from water purification to pharmaceutical production have experienced multi separation steps that impose more process time and contamination possibility by batch operation. We propose a developed microfluidic particle sorter (miniaturized hydrocyclone) that adopts centrifugal force as it has ability to decline the number of separation steps and the risk of extrinsic contamination in continuous process. While biological industries have not relied on mini hydrocyclones considerably because of low efficiency and microfabrication difficulties, current work has been planned to conquer these obstacles. In this research, biomass separation from fermentation broth by 3 mm hydrocyclones was investigated. The effect of apex size, feed flow rate, hydrocyclone geometry were analyzed numerically in four mini-hydrocyclones. The most efficient mini-hydrocyclone was chosen to be made by elegant additive manufacturing technology and studied experimentally. The separation efficiency was achieved up to 90% while the concentration ratio of heavy stream (apex) to dilute stream (vortex finder) was reached more than twofold. The mini hydrocyclone performance in view of energy target was studied by Euler-Reynolds-Efficiency plots. The 4 µm cut size was achieved that is promising high throughput separation for biological particles.

Quantitative assessment of LPS-HBsAg interaction by introducing a novel application of immunoaffinity chromatography.

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.

Assessing virus like particles formation and r-HBsAg aggregation during large scale production of recombinant hepatitis B surface antigen from Pichia pastoris.

The aggregation of recombinant proteins in the different stages of purification leads to the loss of a considerable portion of target protein and reduction in the process efficiency. As the active HBsAg used in Hepatitis B vaccine production is in the form of virus-like particle (VLP), therefore the time and stages at which the VLP assembling happened through the process would be important. The aim of this study was to explore the product aggregation during different stages of large scale production of rHBsAg in Pichia pastoris at production unit of the Pasteur Institute of Iran. Dynamic light scattering (DLS) and transmission electron microscopy (TEM), and also size exclusion-high-performance liquid chromatography (SE-HPLC) were carried out on samples taken from each downstream processes steps to determine the rate of VLPs formation as the desired product and the aggregated form at each stage of the purification. Based on the results, it was found that VLPs formation started at the acid precipitation stage and reached up to 80% at the thermal treatment stage. The ultrafiltration, ion exchange chromatography and immunoaffinity chromatography stages were disclosed to have the highest contribution in the formation of VLP (virus like particle) 22 nm.

Accurate and cost-effective prediction of HBsAg titer in industrial scale fermentation process of recombinant Pichia pastoris by using neural network based soft sensor.

In the current work, the attempt was made to apply best-fitted artificial neural network (ANN) architecture and the respective training process for predicting final titer of hepatitis B surface antigen (HBsAg), produced intracellularly by recombinant Pichia pastoris Mut+ in the commercial scale. For this purpose, in large-scale fed-batch fermentation, using methanol for HBsAg induction and cell growth, three parameters of average specific growth rate, biomass yield, and dry biomass concentration-in the definite integral form with respect to fermentation time-were selected as input vectors; the final concentration of HBsAg was selected for the ANN output. Used dataset consists of 38 runs from previous batches; feed-forward ANN 3:5:1 with training algorithm of backpropagation based on a Bayesian regularization was trained and tested with a high degree of accuracy. Implementing the verified ANN for predicting the HBsAg titer of the five new fermentation runs, excluded from the dataset, in the full-scale production, the coefficient of regression and root-mean-square error were found to be 0.969299 and 2.716774, respectively. These results suggest that this verified soft sensor could be an excellent alternative for the current relatively expensive and time-intensive analytical techniques such as enzyme-linked immunosorbent assay in the biopharmaceutical industry.

Optimization of non-detergent treatment for enveloped virus inactivation using the Taguchi design of experimental methodology (DOE).

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness-in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120 min), and temperature (25 °C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.

A novel application of ion exchange chromatography in recombinant hepatitis B vaccine downstream processing: Improving recombinant HBsAg homogeneity by removing associated aggregates.

Production of recombinant HBsAg as a main component of the hepatitis B vaccine has already been established in commercial scale. So far, many studies have been performed to optimize the production process of this recombinant vaccine. However, still aggregation and dissociation of rHBsAg virus-like particles (VLPs) are major challenges in downstream processing of this biomedicine. The structural diversity of rHBsAg is dependent on many factors including cell types, molecular characteristics of the expressed recombinant rHBsAg, buffer composition as well as operation condition and specific characteristics of each downstream processing unit. Hence, it is not relatively easy to implement a single strategy to prevent aggregation formation in already established rHBsAg production processes. In this study, we examined the efficacy of weak anion exchange chromatography (IEC)- packed with DEAE Sepharose Fast Flow medium- on isolation of rHBsAg VLPs from aggregated structures. For this purpose, the influence of ionic strength of elution buffer as a key factor was investigated in isolation and recovery of rHBsAg VLPs. The elution buffer with electrical conductivity between 27 and 31 mS/cm showed the best results for removing aggregated rHBsAg based on SEC-HPLC analysis. The results showed that in the selected conductivity range, about 79% of rHBsAg was recovered with purity above 95%. The percentage of rHBsAg VLPs in the recovered sample was between 94% and 97.5% indicating that we could obtain highly homogeneous rHBsAg within the acceptable quality level. The TEM, SDS-PAGE and western blot analysis were also in agreement with our quantitative measurements.

Integration of size-exclusion chromatography and ultracentrifugation for purification of recombinant hepatitis B surface antigen: An alternative method for immunoaffinity chromatography.

In purification process of recombinant hepatitis B surface antigen (rHBsAg), immunoaffinity chromatography (IAF) is one of the most important and effective steps in rHBsAg purification. However, the buffer composition and the interaction of ligands-rHBsAg often lead to disassembly, deformation, and clumping of a portion of these virus-like particles (VLPs). Besides, the expensive media, variable biospecific ligand density and the possibility of product contamination are other reported drawbacks of using IAF which makes the production process of rHBsAg more challenging. This study investigated the possibility of substituting IAF with purification methods of size-exclusion chromatography (SEC) and ultracentrifugation. In the SEC, the efficacy of rHBsAg purification was examined by four different media in which Toyopearl HW 65S resin demonstrated the best results. By integrating Toyopearl HW 65S resin - with a bed height of 51 cm - and ultracentrifugation process at 47,000 rpm for 48 hr, 95% of protein impurities were removed. Compared to the IAF in rHBsAg production, the purified sample contained a higher percentage of multimeric rHBsAg particles without any noticeable monomer and aggregate forms. The result of this study indicates that the proposed integrated system could be an efficient mild purification alternative for conventional IAF.

High recovery of intracellular recombinant HBsAg from Pichia pastoris via continuous pulsed laser cell disruption system optimized by response surface methodology.

In our previous study, we demonstrated that continuous power laser could be a clean, rapid, and convenient alternative to the other conventional disruption techniques for the release of recombinant hepatitis B surface antigen (rHBsAg) from Pichia pastoris. In the current work, we examined the effect of pulsed laser in the continuous laboratory-scale process on cell disruption. Design-of-experiments (DOE) methodology was used for optimization of cell disruption process to obtain the highest protein concentration in the disruption buffer. Our investigations for the pulsed laser at wavelength of 1,064 nm demonstrated that for disrupting P. pastoris cell and releasing rHBsAg, the laser power was the most influential factor, and laser pulse duration and cycle number were in the second and third places. According to the results, the effect of laser power and pulse duration (time) had a direct relationship with protein concentration. For the number of cycles, however, increasing the value from the lowest point at first led to the enhancement and then reduction of protein concentration. The maximum cell disruption and rHBsAg release were recorded for the laser system in the energy input of 284 mW and the pulse duration of 100 mSec after four complete rounds of circulation.

Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography.

The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400 SEC, we obtained highly pure, i.e., > 90%, rHBsAg virus-like particles (VLP).

Improving the recovery of clarification process of recombinant hepatitis B surface antigen in large-scale by optimizing adsorption-desorption parameters on Aerosil-380.

In the downstream process of recombinant hepatitis B surface antigen (rHBsAg), nano-colloidal silica adsorbent (Aerosil-380) is one of the possible methods to separate the antigen from other main impurities partially. The current study aimed to maximize the adsorptive capacity of Aerosil-380 as well as rHBsAg recovery for large-scale production of recombinant hepatitis B vaccine. The experimental design methodology was used to optimize the eight critical parameters influencing the efficiency, rHBsAg recovery, of the adsorption-desorption process in the lab-scale. These examined parameters were the adsorption-desorption temperature, pH, contact time, agitation speed, antigen concentration, and desorption buffer. Under optimal condition, the maximum adsorption capacity of Aerosil-380 was equal to 3333 µg.g-1 (rHBsAg/adsorbent), and we could recover about 95% of rHBsAg with purity of 54% (rHBsAg/total protein) in the lab scale. Using the optimum parameters for rHBsAg clarification process in large-scale by Aerosil-380, we recovered about 78% of rHBsAg with 43% purity. Based on the obtained experimental data, Langmuir adsorption isotherm and pseudo-first-order kinetic model provide the best correlations of experimental data for the adsorbent. Findings of this study significantly increase the recovery of clarification process of rHBsAg in large-scale compared to previous reports.

Label-free and simple detection of endotoxins using a sensitive LSPR biosensor based on silver nanocolumns.

This paper describes the construction of a silver-based LSPR biosensor for endotoxin detection. We used GLAD method to procure reproducible silver nanocolumns. In this work, the silver nanostructures were considerably stabilized by a SAM of MPA, and the limit of detection of biosensor was measured to be 340 pg/ml for endotoxin E. coli. Considering endotoxin B. abortus as the second type of endotoxin contamination in our target samples (HBs-ag produced in Institute Pasteur, Iran), we investigated selectivity of the biosensor in various experiments. We showed that this biosensor can selectively detect both types of endotoxins compared to other biological species. Overall, this study proposes that LSPR biosensing can be considered as a sensitive, simple, and label-free method for endotoxin detection in the quality control laboratories.

Isolation of recombinant Hepatitis B surface antigen with antibody-conjugated superparamagnetic Fe3O4/SiO2 core-shell nanoparticles.

In the production process of recombinant Hepatitis B surface antigen (rHBsAg) various separation techniques are used to purify this virus-like particle (VLP). In this study, we developed antibody-conjugated super-paramagnetic Fe3O4/SiO2 core-shell nanoparticles as a highly selective method for isolation of expressed rHBsAg in yeast Pichia pastoris. For this purpose, first, iron oxide magnetic nanoparticles (MNPs) were prepared by co-precipitation method in alkali media and coated with silica. Then the surface was activated by amine groups and conjugated with oxidized antibodies. X-ray diffraction (XRD), transmission electron microscopy (TEM), and vibrating sample magnetometer (VSM) were used to study the physical properties of MNPs. To evaluate the efficacy of these MNPs as a purification technique successfully synthesized MNPs were added to the rHBsAg sample to couple with the antigen and then be isolated based on their magnetic property. In the present research, in the optimum condition, we could isolate 65% of total rHBsAg from the final vaccine sample with purity above 95%. In this procedure, the maximum obtained specific yield (mg HBsAg/mg MNPs) was equal to 37.6. These results underline the potential application of the immune-magnetic separation (IMS) in the future bioseparation systems.

Co-expression of alpha-1 antitrypsin with cytoplasmic domain of v-SNARE in Pichia pastoris: Preserving biological activity of alpha-1 antitrypsin.

Alpha-1-antitrypsin (A1AT) is a major serum protein in human with protease inhibitory activity. Because of its extensive application in medicine, recombinant DNA technology has been considered for its production. The current study examines coexpression of A1AT and soluble domain of v-SNARE in Pichia pastoris, which can prevent the secretion of A1AT after thoroughly passing the secretory pathway. This was done mainly to preserve the biological activity of A1AT, which in the secretory mode might be impaired in the fermentation and early clarification conditions. SNARE proteins are the driving force for vesicle docking and membrane fusion in the exocytosis. Intracellular expression of the cytoplasmic domain of v-SNARE and its subsequent interaction to form SNARE complex can intensify the competition for A1AT secretory vesicles to be fused and released to the media. Our investigation shows successful coexpression of A1AT in the form of post-Golgi vesicles and the cytoplasmic domain of v-SNARE. Our findings confirmed the reduction of A1AT secretion by 45% caused accumulation of post-Golgi secretory vesicles filled with A1AT inside the yeast cell. A1AT trapped in secretory vesicles were biologically more active than secretory A1AT. These results indicate that the inhibition of A1AT secretion can protect its biological activity in fermentation and clarification processes.

Enhancing recovery of recombinant hepatitis B surface antigen in lab-scale and large-scale anion-exchange chromatography by optimizing the conductivity of buffers.

In biopharmaceutical science, ion-exchange chromatography (IEC) is a well-known purification technique to separate the impurities such as host cell proteins from recombinant proteins. However, IEC is one of the limiting steps in the purification process of recombinant hepatitis B surface antigen (rHBsAg), due to its low recovery rate (<50%). In the current study, we hypothesized that ionic strengths of IEC buffers are easy-to-control parameters which can play a major role in optimizing the process and increasing the recovery. Thus, we investigated the effects of ionic strengths of buffers on rHBsAg recovery via adjusting Tris-HCl and NaCl concentrations. Increasing the conductivity of equilibration (Eq.), washing (Wash.) and elution (Elut.) buffers from their initial values of 1.6 mS/cm, 1.6 mS/cm, and 7.0 mS/cm to 1.6 mS/cm, 7 mS/cm and 50 mS/cm, respectively yielded an average recovery rate of 82% in both lab-scale and large-scale weak anion-exchange chromatography without any harsh effect on the purity percentage of rHBsAg. The recovery enhancement via increasing the conductivity of Eq. and Wash. buffers can be explained by their roles in reducing the binding strength and aggregation of retained particles in the column. Moreover, further increase in the salt concentration of Elut. Buffer could substantially promote the ion exchange process and the elution of retained rHBsAg.

Active Brownian particles and run-and-tumble particles separate inside a maze.

A diverse range of natural and artificial self-propelled particles are known and are used nowadays. Among them, active Brownian particles (ABPs) and run-and-tumble particles (RTPs) are two important classes. We numerically study non-interacting ABPs and RTPs strongly confined to different maze geometries in two dimensions. We demonstrate that by means of geometrical confinement alone, ABPs are separable from RTPs. By investigating Matryoshka-like mazes with nested shells, we show that a circular maze has the best filtration efficiency. Results on the mean first-passage time reveal that ABPs escape faster from the center of the maze, while RTPs reach the center from the rim more easily. According to our simulations and a rate theory, which we developed, ABPs in steady state accumulate in the outermost region of the Matryoshka-like mazes, while RTPs occupy all locations within the maze with nearly equal probability. These results suggest a novel technique for separating different types of self-propelled particles by designing appropriate confining geometries without using chemical or biological agents.

An effective DNA priming-protein boosting approach for the cervical cancer vaccination.

Considerable advances have been made in developing human papillomaviruses (HPV) prophylactic vaccines based on L1 virus-like particles (VLPs). However, there are limitations in the availability of these vaccines in developing countries, where most cases of cervical cancer occur. In the current study, the prime-boost immunization strategies were studied using a DNA vaccine carrying HPV-16 L1 gene (pcDNA/L1) and insect cell baculovirus-derived HPV-16 L1 VLP. The humoral immunity was evaluated by measuring the specific IgG levels, and the T-cell immune response was assessed by measuring different cytokines such as IFN-γ, TNF-α and IL-10. Results showed that although immunization with pcDNA/L1 alone could induce strong cellular immune responses, higher immunogenicity especially antibody response was achieved in pcDNA/L1 priming-VLP boosting regimen. Therefore, we suggest that prime-boost regimen can be considered as an efficient prophylactic and therapeutic vaccine.