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.

Progress in affinity ligand-functionalized bacterial magnetosome nanoparticles for bio-immunomagnetic separation of HBsAg protein.

Efficient Bio-immunomagnetic separation (BIMS) of recombinant hepatitis B surface antigen (rHBsAg) with high binding capacity was studied using affinity ligand immobilized bacterial magnetosome nanoparticles (Magnetospirillum gryphiswaldense strain MSR-1 bacteria) as an immunomagnetic sorbent. Our results showed immunomagnetic adsorption, acted by affinity interactions with the immobilized monoclonal antibody, offered higher antigen adsorption and desorption capacities as compared with the commercially available immunoaffinity sorbents. Four different ligand densities of the Hep-1 monoclonal antibody were examined during covalent immobilization on Pyridyl Disulfide-functionalized magnetosome nanoparticles for HBsAg immunomagnetic separation. The average of adsorption capacity was measured as 3 mg/ml in optimized immunomagnetic sorbent (1.056 mg rHBsAg/ml immunomagneticsorbent/5.5 mg of total purified protein) and 5mg/ml in immunoaffinity sorbent (0.876 mg rHBsAg/ml immunosorbent/5.5 mg total purified protein during 8 runs. Immunomagnetic sorbent demonstrated ligand leakage levels below 3 ng Mab/Ag rHBsAg during 12 consecutive cycles of immunomagnetic separation (IMS). The results suggest that an immunomagnetic sorbent with a lower ligand density (LD = 3 mg Mab/ml matrix) could be the best substitute for the immunosorbent used in affinity purification of r-HBsAg there are significant differences in the ligand density (98.59% (p-value = 0.0182)), adsorption capacity (97.051% (p-value = 0.01834)), desorption capacity (96.06% (p-value = 0.036)) and recovery (98.97% (p-value = 0.0231)). This study indicates that the immunosorbent approach reduces the cost of purification of Hep-1 protein up to 50% as compared with 5 mg Mab/ml immunoaffinity sorbent, which is currently used in large-scale production. As well, these results demonstrate that bacterial magnetosome nanoparticles (BMs) represent a promising alternative product for the economical and efficient immobilization of proteins and the immunomagnetic separation of Biomolecules, promoting innovation in downstream processing.

Lower frequency of T stem cell memory (TSCM) cells in hepatitis B vaccine nonresponders.

Despite the availability of an effective vaccine and antiviral treatments, hepatitis B is still a global public health problem. Hepatitis B vaccination can prevent the disease. Vaccination induces long-lasting protective immune memory, and the identification of memory cell subsets can indicate the effectiveness of vaccines. Here, we compared the frequency of CD4+ memory T cell subsets between responders and nonresponders to HB vaccination. Besides, the frequency of IFN-γ+ memory T cells was compared between studied groups. Study participants were grouped according to their anti-HBsAb titer. For restimulation of CD4+ memory T cells, peripheral blood mononuclear cells (PBMCs) were cultured in the presence of HBsAg and PHA for 48 h. Besides, PMA, ionomycin, and brefeldin were added during the last 5 h of incubation to induce IFN-γ production. Flow cytometry was used for analysis. There was a statistically significant difference in the frequency of CD4+CD95+, CD4+CD95Hi, and CD4+CD95low/med T stem cell memory (TSCM) cells between responder and nonresponder groups. However, the comparison of the frequency of memory T cells producing IFN-γ showed no differences. Our results identified a possible defect of immunological CD4+ memory T cell formation in nonresponders due to their lower frequency of CD4+ TSCM cells.

Fast antibody responses by immuno-targeting and nanotechnology strategies versus HBsAg vaccine.

OBJECTIVES: Though immunization with HBsAg has been routine since the 1980s, it has numerous limitations such as low or none humoral immune responses. Today, nanotechnology is used in vaccinology to achieve higher potency. The present study deals with the achievement of fast antibody response of humoral immune responses using immune-targeting through mannosylated nanocarriers of the vaccine. MATERIALS AND METHODS: Mannose sugar and HBsAg were attached to the surface of iron oxide nanoparticles. Mannosylated iron oxide nanoparticles conjugated HBsAg (HBsAg +MLCMNP), iron oxide nanoparticles conjugated HBsAg (HBsAg +LCMNP), hepatitis B vaccine, and mere HBsAg were injected twice to BALB/c mice subcutaneously, while suitable control groups were considered. Specific total IgG antibodies were evaluated on the 7th and 14th days after the final immunization. The avidity maturation of the humoral immune response was assessed with an optimized ELISA. Graph pad prism software was used to analyze statistical data. RESULTS: Results showed that on the seventh day of the final shooting, the mannosylated nano-vaccine caused higher antibody response induction than nano-vaccine without mannose and commercial vaccine groups. After 14 days of the second injection, a significant difference was seen versus the nano-vaccine without mannose but not the commercial vaccine group. In addition, the avidity index in mannosylated nano-vaccine showed a significant increase compared with the nano-vaccine without mannose and mere HBsAg group but not compared with the commercial vaccine. CONCLUSION: It seems that mannosylated nano-vaccine has more potency to achieve fast antibody responses and also higher quality of humoral immune response.

Application of beta and gamma carbonic anhydrase sequences as tools for identification of bacterial contamination in the whole genome sequence of inbred Wuzhishan minipig (Sus scrofa) annotated in databases.

Sus scrofa or pig was domesticated thousands of years ago. Through various indigenous breeds, different phenotypes were produced such as Chinese inbred miniature minipig or Wuzhishan pig (WZSP), which is broadly used in the life and medical sciences. The whole genome of WZSP was sequenced in 2012. Through a bioinformatics study of pig carbonic anhydrase (CA) sequences, we detected some ß- and γ-class CAs among the WZSP CAs annotated in databases, while ß- or γ-CAs had not previously been described in vertebrates. This finding urged us to analyze the quality of whole genome sequence of WZSP for the possible bacterial contamination. In this study, we used bioinformatics methods and web tools such as UniProt, European Bioinformatics Institute, National Center for Biotechnology Information, Ensembl Genome Browser, Ensembl Bacteria, RSCB PDB and Pseudomonas Genome Database. Our analysis defined that pig has 12 classical α-CAs and 3 CA-related proteins. Meanwhile, it was approved that the detected CAs in WZSP are categorized in the ß- and γ-CA families, which belong to Pseudomonas spp. and Acinetobacter spp. The protein structure study revealed that the identified ß-CA sequence from WZSP belongs to Pseudomonas aeruginosa with PDB ID: 5JJ8, and the identified γ-CA sequence from WZSP belongs to P. aeruginosa with PDB ID: 3PMO. Bioinformatics and computational methods accompanied with bacterial-specific markers, such as 16S rRNA and ß- and γ-class CA sequences, can be used to identify bacterial contamination in mammalian DNA samples.

Using Various Approaches of Design of Experiments for High Cell Density Production of the Functionally Probiotic Lactobacillus plantarum Strain RPR42 in a Cane Molasses-based Medium.

Considering the economic importance of the probiotics, industrial production of their biomass became important. Cane molasses, as an industrial byproduct, was used in this study to design a medium for biomass overproduction of a functionally probiotic strain, designated as Lactobacillus plantarum strain RPR42. The results showed that strain RPR42 can be best grown anaerobically in 22.5% cane molasses solution. Also, the findings of the single variable at a time experiments and either factorial design indicated that the optimal growth of strain RPR42 can be observed when beef extract, casein hydrolysate, and yeast extract were added into the medium. The central composite design experiments suggested a medium which was designated as cane molasses medium (CMM). Eventually, this medium contained 21.9% cane molasses, 30.72 g/L of a combined mixture of nitrogenous compounds: 0.0754% of a 1:1:1 mixture of polysorbates 20, 60, and 80, and 18.53 gr/L of the combined minerals. Such an optimized cane molasses-based medium supported a significant biomass production since a considerably high cell density, 13.8 g/L/24 h of dry biomass, of the strain was produced. Hence, cane molasses can be regarded as a promising substrate for industrial production purposes.

Screening for efficient nitrogen sources for overproduction of the biomass of the functionally probiotic L. plantarum strain RPR42 in a cane molasses-based medium.

Nitrogen source has a vital role for the efficient growth of lactobacilli. The effects of cheese whey, corn steep liquor, and wheat germ extract on the growth of L. plantarum strain RPR42 in cane molasses-based media was evaluated using various approaches of design of experiments. Our results showed that such protein-rich agricultural by-products significantly increase the biomass production of the strain RPR42 in cane molasses-based media. The most affecting nitrogenous material was cheese whey followed by CSL and the minor effect was reported for wheat germ extract as revealed in factorial and Box-Behnken design experiments. The replacement of costly beef extract and yeast extract with a defined mixtures of the above nitrogenous agricultural by-products in cane molasses-based medium led to production of up to 12.64 g/L/24 h of dry biomass of strain RPR42. A detectable cell density of strain RPR42 (~ 9.81 × 109 CFU/mL 24 h) which was observed in such an economic medium showed that the large-scale production of the strain RPR42 tend to be feasible at significantly low costs.

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.

Application of hydrocyclone for separation of Pichia pastoris produced r-HBsAg from fermentation culture: impact of concentration and pressure on hydrocyclone performance.

Separation of biomass from culture media by centrifugation and then washing the biomass are mandatory steps in the fermentation process of recombinant Pichia pastoris expressed HBsAg intracellularly. Biomass has to be washed many times to eliminate the culture media residues thoroughly. In this study, we tried to develop the hydrocyclone as an alternative method for separation of biomass from fermentation culture, an attractive replacement for centrifugation processes. The advantages of using hydrocyclone in biomass separation could be summarized in its suitability for continuous separation and its low risk of contamination. To evaluate the performance of hydrocyclone, concentration ratio in underflow to feed stream, capacity, and centrifugal force by considering three parameters of pressure drop, concentration, and the type of hydrocyclone were investigated. Using three level factorial design a concentration ratio equation was developed, with the correlation coefficient R2 = 0.977 ensured the good fitness of the predicted data with the experimental results. In optimal conditions, maximum concentration ratio was 1.246, for flow rate 13.5 LPM and C-force equal to 1276.11 at maximum pressure drop (3 bar) and minimum concentration (0.5% w/w) in hydrocyclone 1. Herein, two different hydrocyclones with the cylindrical diameters of 19 mm and 21 mm were used for separating the yeast cells.

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.

HBs antigen and mannose loading on the surface of iron oxide nanoparticles in order to immuno-targeting: fabrication, characterization, cellular and humoral immunoassay.

Mannosylation of nanovaccine is an appropriate strategy for targeting the mannose receptors on DCs. Here, HBsAg and mannose loaded on the surface of iron oxide nanoparticles to increases HBsAg vaccine potency. Nanoparticles are made by co-precipitation method and bonded to the HBsAg and mannose by chemical bonding. The physicochemical properties of nano-vaccines, their toxicity and antigenicity were determined. The synthesized nano-vaccine showed spherical shape with a mean particle size of 60 nm, a zeta potential of -44 mV, an antigen-binding efficiency of around 100% and for mannose 78%. In vitro release of nanoparticles exhibited about 30% at the first day and about 60% until the third day. SDSPAGE analysis confirmed structural integrity of HBsAg loaded on nanoparticles. The HBsAg-loaded LCMNP and MLCMNP nanoparticles had no toxic effects on HEK293 cell line. The quantification of the intracellular Fe by ICP-OES as a criterion of nano-vaccine uptake revealed mannose intensify uptake of MLCMNP. In addition, mannose in the structure of MLCMNP improved IL-6, TNF-α and IFN-γ (>16 fold) cytokines genes expression by macrophage/dendritic cells after exposure in 12 h. Immunization of experimental mice (subcutaneously, two times with 2-week intervals) with 5 µg of HBsAg loaded on MLCMNP nanoparticles increased specific total IgG and IgG2a/IgG1 ratio. In addition, TNF-α, IL-12, IL-2 and IL-4 cytokines in mannosylated nano-vaccine increased versus nano-vaccine group while lymphocyte proliferation and IFN-γ responses in the targeted nano-vaccine group show a tiny increase versus the nano-vaccine group. The results show that mannosylated nano-vaccine promotes higher level of cellular and humoural immune responses against HBsAg nano-vaccine.

Using chemical chaperones to increase recombinant human erythropoietin secretion in CHO cell line.

In recombinant protein production, over-expressed genes induce unfolded protein response (UPR), overloaded protein aggregation in endoplasmic reticulum and its expansion. In this study, we have used 16 chemicals to improve erythropoietin production in engineered CHO cells and tried to study the mechanism of reducing protein aggregation in each treatment. Endoplasmic reticulum expansion was studied through endoplasmic reticulum specific labeling with utilizing fluorescent glibenclamide and its molecular chaperones expression were studied by real-time polymerase chain reaction. The increase in the mRNA level of EPO and endoplasmic reticulum chaperones GRP78/BiP, XBP1, ATF6, and ATF4 in different chemical treatments were not related to ER expansion. On the other hand, ER expansion in beta alanine, beta cyclodextrin and taurine treatments resulted in increased EPO secretion. Dramatically increase in EPO expression in conjugated linoleic acid, spermidine, trehalose, and maltose (19, 20, 16, and 19-fold, respectively) did not increase erythropoietin productivity, but betaine which did not caused ER expansion, with minor increase in EPO gene expression increase EPO productivity. The results indicated that betaine increase EPO secretion in engineered CHO cell line without relation to ER expansion and molecular chaperones expression.

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.

Application of recurrent neural network for online prediction of cell density of recombinant Pichia pastoris producing HBsAg.

Artificial neural networking (ANN) seems to be a promising soft sensor for implementing current approaches of quality by design (QbD) and process analytical technologies (PAT) in the biopharmaceutical industry. In this study, we aimed to implement best-fitted ANN architecture for online prediction of the biomass amount of recombinant Pichia pastoris (P. pastoris) - expressing intracellular hepatitis B surface antigen (HBsAg) - during the fed-batch fermentation process using methanol as a sole carbon source. For this purpose, at the induction phase of methanol fed-batch fermentation, carbon evolution rate (CER), dissolved oxygen (DO), and methanol feed rate were selected as input vectors and total wet cell weight (WCW) was considered as output vector for the ANN. The obtained results indicated that after training recurrent ANN with data sets of four fed-batch runs, this toolbox could predict the WCW of the next fed-batch fermentation process at each specified time point with high accuracy. The R-squared and root-mean-square error between actual and predicted values were found to be 0.9985 and 13.73, respectively. This verified toolbox could have major importance in the biopharmaceutical industry since recombinant P. pastoris is widely used for the large-scale production of HBsAg.

Effects of Environmental Conditions on High-Yield Magnetosome Production by Magnetospirillum gryphiswaldense MSR-1

Background: Magnetotactic bacteria are a heterogeneous group of Gram-negative prokaryote cells that produce linear chains of magnetic particles called magnetosomes, intracellular organelles composed of magnetic iron particles. Many important applications have been defined for magnetic nanoparticles in biotechnology, such as cell separation applications, as well as acting as carriers of enzymes, antibodies, or anti-cancer drugs. Since the bacterial growth is difficult and the yield of magnetosome production is low, the application of magnetosome has not been developed on a commercial scale. Methods: Magnetospirillum gryphiswaldense strain MSR-1 was used in a modified current culture medium supplemented by different concentrations of oxygen, iron, carbon, and nitrogen, to increase the yield of magnetosomes. Results: Our improved MSR-1 culture medium increased magnetosome yield, magnetosome number per bacterial cell, magnetic response, and bacterial cell growth yield significantly. The yield of magnetosome increased approximately four times. The optimized culture medium containing 25 mM of Na-pyruvate, 40 mM of NaNO3, 200 µM of ferrous sulfate, and 5-10 ppm of dissolved oxygen (DO) resulted in 186.67 mg of magnetosome per liter of culture medium. The iron uptake increased significantly, and the magnetic response of the bacteria to the magnetic field was higher than threefold as compared to the previously reported procedures. Conclusion: This technique not only decreases the cultivation time but also reduces the production cost. In this modified method, the iron and DO are the major factors affecting the production of magnetosome by M. gryphiswaldense strain MSR-1. However, refining this technique will enable a further yield of magnetosome and cell density.

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.