Efficient CRISPR/Cas9-Mediated BAX Gene Ablation in CHO Cells To Impair Apoptosis and Enhance Recombinant Protein Production.

Background: Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis. Objectives: Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster's ovary cells producing erythropoietin. Materials and Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein. Results: BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 µM.ml-1 Vs. 2505 µM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 µM oleuropein compared to the control cell line (p-value=0.0002). Conclusions: CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.

Exploring the experience of developing COVID-19 vaccines in Iran.

Widespread public vaccination is one of the effective mechanisms to ensure the health and prevent deaths in societies. The coronavirus disease 2019 (COVID-19) vaccine is a stark instance in this regard. Vaccine development is a complex process requiring firm-level capabilities, various infrastructures, long-term planning, and stable and efficient policies. Due to the global demand for vaccines during the pandemic, the national capability to produce vaccines is critical. To this end, the current paper investigates influential factors, at the firm- and policy-level, in the COVID-19 vaccine development process in Iran. By adopting a qualitative research method and conducting 17 semi-structured interviews and analyzing policy documents, news, and reports, we extracted internal and external factors affecting the success and failure of a vaccine development project. We also discuss the characteristics of the vaccine ecosystem and the gradual maturity of policies. This paper draws lessons for vaccine development in developing countries at both firm and policy levels.

Facile Capture of Recombinant Human Erythropoietin on Mesoporous Affinity Hydrogel Matrix Functionalized with Azoboronate.

Boronate affinity ligands (BALs) have gained attention for glycoproteins capture and recognition due to their unique affinity interaction with glycans. In this paper, the effect of azo immobilization of phenylboronic acid on the reduction of adsorption pH of a recombinant glycoprotein (i.e., rhEPO) on hydrogel microparticles was investigated. To evaluate the influence of intraparticle porosity on protein adsorption, microporous (MicroBead) and mesoporous (MesoBead) agarose beads carrying two levels of amine densities were functionalized with azoboronate ligand. Affinity adsorption of the glycoprotein during static and dynamic adsorptions at relatively low pHs of 8 and 7 was studied. Results revealed successful adsorption of rhEPO at pH = 8 through affinity capture of glycans by azoboronate ligands. Increased amine density provided 1.1 and 1.5 times higher static adsorption capacities and dynamic performance efficiencies, respectively. In addition, adsorption capacities and initial adsorption rates of rhEPO on MesoBeads were respectively 1.4 and 2.5-2.8 times of MicroBeads. Also, at pH = 8, MesoBeads recorded higher dynamic recoveries (59 and 91%) compared with microporous ones (46 and 69%) since mesoporosity facilitates intraparticle mass transfer. Reduction of binding pH from 8 to 7 resulted in a sharp decrease in dynamic recovery (14%), indicating the appropriate binding pH of azoPBA to be above 7. The azoboronate affinity ligand is a leading candidate for capturing glycoproteins at relatively low pH. Also, mesoporous microparticles are appropriate tools in more efficient medium-sized protein binding applications.

Targeting Caspase-3 Gene in rCHO Cell Line by CRISPR/Cas9 Editing Tool and Its Effect on Protein Production in Manipulated Cell Line.

Background: Chinese hamster ovary (CHO) cells are the widely used mammalian cell host for biopharmaceutical manufacturing. During cell cultures, CHO cells lose viability mainly from apoptosis. Inhibiting cell death is useful because prolonging cell lifespans can direct to more productive cell culture systems for biotechnology requests. Objectives: This study exploited a CRISPR/Cas9 technology to generate site-specific gene disruptions in the caspase-3 gene in the apoptosis pathway, which acts as an apoptotic regulator to extend cell viability in the CHO cell line. Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 system. The guide RNAs targeting the caspase-3 gene were designed, and vectors containing sgRNA and Cas9 were transfected into CHO cells that expressed erythropoietin as a heterologous protein. Indel formation was investigated by DNA sequencing. Caspase-3 expression was quantified by real-time PCR and western blot. The effect of editing the caspase-3 gene on the inhibition of apoptosis was also investigated by induction of apoptosis in manipulated cell lines by oleuropein. Finally, the erythropoietin production in the edited cells was compared to the control cells. Results: The caspase-3 manipulation significantly prolongation of the cell viability and decreased the caspase-3 expression level of protein in manipulated CHO cells (more than 6-fold, P-value < 0.0001). Manipulated cells displayed higher threshold tolerance to apoptosis compared to the control cells when they were induced by oleuropein. They show a higher IC50 than the control ones (7271 µM/mL Vs. 5741 µM/mL). They also show a higher proliferation rate than the control cells in the presence of an apoptosis inducer (P-value < 0.0001). Furthermore, manipulated cell lines significantly produce more recombinant protein in the presence of 2,000 µM oleuropein compared to the control ones (P-value = 0.0021). Conclusions: We understood that CRISPR/Cas9 could be effectively applied to suppress the expression of the caspase-3 gene and rescue CHO cells from apoptosis induced by cell stress and metabolites. The CRISPR/Cas9 system-assisted caspase-3 gene ablation can potentially increase erythropoietin yield in CHO cells.

Association between serum inflammatory parameters and the disease severity in COVID-19 patients.

OBJECTIVE: Most patients infected with the novel coronavirus (SARS-CoV-2), as the causative agent of COVID-19 disease, show mild symptoms, but some of them develop severe illness. The purpose of this study was to analyze the blood markers of COVID-19 patients and to investigate the correlation between serum inflammatory cytokines and the disease severity. METHODS: In this prospective cross-sectional study, 50 patients with COVID-19 and 20 patients without COVID-19 were enrolled. According to ICU admission criteria, patients were divided into two groups of non-severe and severe. Differences in the serum levels of C-reactive protein (CRP), IL-6, and TNF-α, as well as erythrocyte sedimentation rate (ESR), lymphocytes (LYM) count, and neutrophils (NEU) count between the two groups were determined and analyzed. RESULTS: Out of the 50 patients with COVID-19, 14 were diagnosed as severe cases. There was no significant difference between the two groups of COVID-19 patients in terms of gender and age. Blood tests of COVID-19 patients showed a significant decrease and increase in NEU and LYM counts, respectively. There were significant differences in the serum levels of IL-6, TNF-α, and CRP between the severe and non-severe groups, which were higher in the severe group. Also, there was a significant correlation between the disease severity and CRP with ESR (r = 0.79), CRP with IL-6 (r = 0.74), LYM with NEU (r = -0.97), and ESR with TNF-α (r = 0.7). CONCLUSION: The findings of this study, as the first study in Iran, suggest that the levels of IL-6, TNF-α, ESR, and CRP could be used to predict the severity of COVID-19 disease.

Computation screening and molecular docking of FDA approved viral protease inhibitors as a potential drug against COVID-19.

AIM: This study demonstrated potent inhibitors against COVID-19 using the molecular docking approach of FDA approved viral antiprotease drugs. BACKGROUND: COVID-19 has now spread throughout world. There is a serious need to find potential therapeutic agents. The 3C-like protease (Mpro/6LU7) is an attractive molecular target for rational anti-CoV drugs. METHODS: The tertiary structure of COVID-19 Mpro was obtained from a protein data bank repository, and molecular docking screening was performed by Molegro Virtual Docker, ver. 6, with a grid resolution of 0.30 Å. Docking scores (DOS) are representative of calculated ligand-receptor (protein) interaction energy; therefore, more negative scores mean better binding tendency. Another docking study was then applied on each of the selected drugs with the best ligands separately and using a more accurate RMSD algorithm. RESULTS: The docking of COVID-19 major protease (6LU7) with 17 selected drugs resulted in four FDA approved viral antiprotease drugs (Temoporfin, Simeprevir, Cobicistat, Ritonavir) showing the best docking scores. Among these 4 compounds, Temoporfin exhibited the best DOS (-202.88) and the best screened ligand with COVID-19 Mpro, followed by Simeprevir (-201.66), Cobicistat (-187.75), and Ritonavir (-186.66). As the best screened ligand, Temoporfin could target the Mpro with 20 different conformations, while Simeprevir, Cobicistat, and Ritonavir make 14, 10, and 10 potential conformations at the binding site, respectively. CONCLUSION: The findings showed that the four selected FDA approved drugs can be potent inhibitors against COVID-19; among them, Temoporfin may be more potent for the treatment of the disease. Based on the findings, it is recommended that in-vitro and in-vivo evaluations be conducted to determine the effectiveness of these drugs against COVID-19.

A Real-Time RT-PCR Assay for Genotyping of Rotavirus

Background: Human rotavirus (HRV) is the causative agent of severe gastroenteritis in children and responsible for two million hospitalizations and more than a half-million deaths annually. Sequence characteristics of the gene segments encoding the VP7 and VP4 proteins are used for the genotype classification of rotavirus. A wide variety of molecular methods are available, mainly based on reverse transcription PCR for rapid, specific and sensitive genotyping of rotaviruses. This study describes an alternative real-time PCR assay for genotyping of rotavirus. Methods: The samples of stools studied in this research have been collected from patients referred to Children's Medical Centers, Tehran, Iran. Rotavirus detection and genotyping were performed using the RT-PCR and semi-nested RT-PCR, respectively. Samples were then genotyped with a new real-time PCR. Results: The real-time PCR was able to genotype all positive samples with a mean Ct of 28.2. Besides, a concordance rate of 100% was detected between real-time PCR and semi-nested RT-PCR. Conclusion: In this study, the genotyping of rotavirus with real-time PCR showed that this method can provide several favorable features, including high sensitivity and specificity, and a wide dynamic range for rotavirus genotyping.

Laboratory Parameters in Detection of COVID-19 Patients with Positive RT-PCR; a Diagnostic Accuracy Study.

INTRODUCTION: The role of laboratory parameters in screening of COVID-19 cases has not been definitely established. This study aimed to evaluate the accuracy of laboratory parameters in predicting cases with positive RT-PCR for COVID-19. METHODS: This diagnostic accuracy study was conducted on suspected COVID-19 patients, who presented to Behpooyan Clinic Medical center in Tehran (Iran) from 22 February to 14 March, 2020. Patients were divided into two groups based on the results of real time reverse transcriptase-polymerase chain reaction (RT-PCR) for COVID-19, and the accuracy of different laboratory parameters in predicting cases with positive RT-PCR was evaluated using area under the ROC curve (AUC). RESULTS: Two hundred cases with the mean age of 41.3± 14.6 (range: 19-78) years were studied (0.53% male). The result of RT-PCR for COVID-19 was positive in 70 (35%) cases. Patients with positive RT-PCR had significantly higher neutrophil (NEU) count (p = 0.0001), and C-reactive protein (CRP) (p = 0.04), lactate dehydrogenase (LDH) (p = 0.0001), aspartate aminotransferase (AST) (p = 0.001), alanine aminotransferase (ALT) (p = 0.0001), and Urea (p = 0.001) levels in serum. In addition, patients with positive RT-PCR had lower white blood cell (WBC) count (p = 0.0001) and serum albumin level (p = 0.0001) compared to others. ALT (AUC = 0.879), CRP (AUC = 0.870), NEU (AUC = 0.858), LDH (AUC = 0.835), and Urea (AUC = 0.835) had very good accuracy in predicting cases with positive RT-PCR for COVID-19, respectively. CONCLUSION: Our findings suggest that level of LDH, CRP, ALT and NEU can be used to predict the result of COVID-19 test. They can help in detection of COVID-19 patients.

Optimization of parameters affecting on CHO cell culture producing recombinant erythropoietin.

Several factors may affect erythropoietin (EPO) sugar structures including designing cell culture procedure, pH, concentration of additives, dissolved oxygen, and other physicochemical parameters. In this study, we investigated the influence of changes in effective parameters and compounds on the growth rate of Chinese hamster ovary cell (CHO) cells producing recombinant EPO. Cell culture was performed at different temperature, buffering conditions, and varied concentrations of additives such as pyruvic acid, insulin, GlutaMAX, and sodium butyrate. Results indicated that the optimal temperature and pH were 37 °C and 7.2, respectively. Also, optimal concentrations for pyruvic acid, butyrate, glutamate, and insulin were obtained to be 20 mM, 1 mM, 2 mM, and 40 µg/mL, respectively. Then, cell culture was performed in microcarrier-coated spinner flasks under the optimized condition. The results showed recombinant human EPO (rhEPO) production with adequate purity. Optimization of physicochemical conditions and culture media are important factors to improve the quantity and quality of protein products. This study showed that cell growth and recombinant EPO protein production significantly increased under the optimized conditions. The results of this research can also be used in scale-up to increase the efficiency of EPO production. Abbreviations: EPO: erythropoietin; CHO cell: Chinese hamster ovary cell; rhEPO: recombinant human EPO; DMEM: modified eagle's medium; FBS: fetal bovine serum; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IGF-1: insulin-like growth factor 1.

Aggregate Forms of Recombinant Human Erythropoietin With Different Charge Profile Substantially Impact Biological Activities.

Recombinant human erythropoietin (rHuEPO) as a glycoprotein growth factor has been considered a biological drug for treatment of anemic patients with chronic renal failure or who receive cancer chemotherapy. Biological activity and circulation time are 2 parameters that are important to achieve EPO's efficacy. Previous efforts for increasing EPO's efficacy have focused on glycosylation modification via adding more sialic acid antenna and generates more negative charged protein. Evidences cleared that EPO's activity increased by numbers of N-glycan moieties with presence of sialic acids at their terminus. Correlation between bioactivity and glycosylation with terminal sialylation is theoretically achieved using the calculation of the amount of charge profile of the EPO variants called "I-number." Here, we studied and compared the relationship between bioactivities of different EPOs that contained various I-numbers and the effect of their secondary and tertiary protein structures on measured in vivo efficacy. Eight recombinant EPOs batches were produced under the same condition. I-numbers found out by EPO's charge profiles determination using capillary electrophoresis and activities were studied upon erythroid precursor cell stimulation in mice. Analyzing the bioactivity, I-number, and structural studies revealed that in spite of I-number, conformational changes in protein structure and presence of aggregated species impact bioactivity substantially.

Prevalence of astrovirus, adenovirus, and sapovirus infections among Iranian children with acute gastroenteritis.

AIM: This study aimed to determine the prevalence of Human Astroviruses (HAstVs), enteric Adenoviruses (HAdVs), and Sapoviruses (SaVs) in acute diarrhea patients, as well as their relation to age, sex, and season. BACKGROUND: Acute gastroenteritis is one of the most common diseases affecting children <5 years old and viral agents with approximately >75% are the major causative agent of acute infectious diarrhea. After Rotavirus and Norovirus, the greater viral agents of acute gastroenteritis include HAstVs, HAdVs, and SaVs. To the best of our knowledge, there are sparse studies in Iran detecting at least three enteric viruses as causative agents of diarrhea simultaneously. METHODS: The sample was collected from children referring to pediatric medical centers in Tehran, Iran; they were tested for Astrovirus, enteric Adenovirus, and Sapovirus by conventional PCR method. The association of incidence of viral enteric agents was evaluated with age, sex and seasonal pattern in children <5 years old. RESULTS: The positive case number among acute gastroenteritis patients was 17/120 (14.1%). Patients ranged in age within 1-60 months, but 52.9% were aged ≤ 12 months. Males comprised the majority (70.6), and the male: female ratio was 2.4. HAstV was the most frequently detected virus (6.7%), while SaVs were detected only in 2.5% of cases. Mixed infections were not detected in these samples. The highest rate of HAstV was identified in winter (66.7%), HAdV in fall (66.7%), and SaV in winter (33.3%). CONCLUSION: These findings underscore the importance of monitoring the epidemiology of HAstV, HAdV, and SaV as causative agents of viral diarrhea infections.

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.

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.

Erythropoietin-loaded solid lipid nanoparticles: Preparation, optimization, and in vivo evaluation.

Solid lipid nanoparticle (SLN) is a promising approach for delivery of various drugs including proteins and peptides. However, the loading of hydrophilic drugs into the lipoid matrix of SLNs is challenging. The statistical design is a potential method facilitating the optimization of nanoparticles characteristics. In this study, the Box-Behnken design was conducted to optimize the preparation of Erythropoietin (EPO) loaded SLNs. Circular dichroism, size exclusion chromatography, SDS-PAGE, and ELISA tests were used to prove the compatibility of the process with the stability of EPO. In the controlled situation, EPO preserved its conformation and activity during the SLN preparation. Regarding the particle size, entrapment efficiency, and polydispersity index, an optimum formulation was obtained with 130 mg Span®80, 152.5 µl EPO, and 1.9 min high-shear homogenization. Using the optimum condition, 280 nm sized SLNs with the narrow size distribution of 0.282 and entrapment efficiency of 43.4% were acquired. The in vitro cytotoxicity of optimum SLN formulation was conducted using MTT assay to show its safety on the evaluated cell line. The in vivo studies demonstrated that 2500 U EPO loaded SLN has similar or even better effects on elevating the RBC, hemoglobin, and hematocrit level compared to the 5000 U EPO solution. Generally, this study proposed a suitable EPO-loaded SLN preparation method as a potential drug delivery system for proteins.

Association of IL28B (IFNL3) rs12979860 mRNA levels, viral load, and liver function among HCV genotype 1a patients.

AIM: The present study was designed to evaluate the correlation of interleukin 28B (IL28B, IFNL3) rs12979860 mRNA levels, viral load, and liver function among hepatitis C virus (HCV) patients genotype 1a. BACKGROUND: HCV is considered essentially hepatotropic and is a major health problem around the world. METHODS: This study included 100 HCV-infected patients with HCV genotype1a (G1a) and rs12979860 CC genotype. These patients were divided into two groups according to HCV treatment. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and HCV Load were measured and recorded for each patient. IL28B mRNA levels were determined using real-time polymerase chain reaction assay, and their correlation with clinical data were analyzed. STRING was applied to construct a network and identify interactions between IL28B (IFNL3) and its significant neighbor proteins. RESULTS: The results revealed a significant relationship between the ALT as well as ALP levels with IL28B rs12979860 mRNA expression level in men, and also with age >50 years. In the treated group, AST level and HCV load had a significant relationship with IL28B mRNA expression level. The results showed that the level of ALP and AST decreased significantly with increased IL28B mRNA expression level in the treated and untreated group, respectively. STRING database showed that IL28B (IFNL3) interacted with ten important neighbor proteins with some of these proteins being involved in signal transduction pathway activating antiviral response. CONCLUSION: This study indicated that rs12979860CC genotype could predict IL28B mRNA expression level in HCV-infected patients with G1a. Furthermore, IL28B mRNA expression level may serve as a useful marker for the development of G1a HCV-associated outcomes.

Towards rational design of porous nanostructured biopolymeric microparticles for biomacromolecules separation: A case study of intraparticle diffusion facilitation and BSA adsorption on agarose microspheres.

Synthesis and employing advanced materials for emerging applications is of great challenge for the scientific community. Recombinant proteins production and purification is one of the fastest growing fields in the global economy. In this regard, it is essential to fabricate biocompatible low-cost materials with high specificity to enhance purification efficiency. This requires the regulation of mass transfer based on the protein molecular size and interactions with the matrix interface; thus, needs synthesizing novel materials with tuned porosity. In this study, we proposed rational alteration in porous structure of biopolymeric microspheres using appropriate-sized porogen to facilitate intraparticle molecular diffusion. The tailored porous nanostructures, which were generated by phase separation in the polymer blend of agarose and polyethylene glycol, were analyzed with optical and scanning electron microscopy, Fourier transform infrared spectroscopy, water diffusion, and albumin adsorption. The well-tuned beads possessed highly porous structures with dominant mesopores owing to PEG phase migration out of the network. The high speed homogenizer caused an uncommon dense morphology with interwoven two-type porosity. Optimally tuned mesoporous beads with considerably high specific surface area exhibited dramatically fast and enhanced intraparticle diffusion of both water and protein molecules. Thus, the introduced porosity modification is a promising design for enhancing mass transfer in the bio-separation process. Finally, useful insights for developing future smart hydrogel microparticles with tuned porous network for biomolecules purification are provided by the conducted experiments.

Physicochemical screening for chemical stabilizer of erythropoietin to prevent its aggregation.

Recombinant protein aggregation is a problematic issue and can provoke immunological response. The aim of this study was to analyze the stability of erythropoietin (EPO), as a therapeutic protein expressed in mammalian cells, in the presence of different chemicals and find a specific stabilizer for EPO. The effects of several chemicals, including mannitol, betaine, trehalose, taurine, linoleic acid, beta-cyclodextrin, copper sulfate, spermidine, maltose, maltodextrin, sucrose, dextran, beta-alanine, myo-inositol, and cysteine, on protein stabilization through the thermally induced aggregation of EPO were monitored. Based on the results of turbidity assay for thermal aggregation, three different patterns were observed for protein stability of active pharmaceutical ingredient of EPO, namely, accelerated, dose-dependent, and inhibitory behaviors for aggregate formation due to treatment with spermidine, mannitol, and betaine, respectively. According to circular dichroism outcomes, EPO treatment with betaine and spermidine resulted in different helical contents of the secondary structure. Dynamic light scattering experiments indicated that treating EPO with betaine resulted in less protein aggregation due to freeze and thaw stresses. Betaine was able to stabilize EPO and inhibit its aggregation, as opposed to spermidine that induced protein aggregation.

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (101 to 107 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Design and validation of a new method to detect and quantify residual host cell DNA in human recombinant erythropoietin (rEPO).

During the purification of human recombinant erythropoietin (rEPO) from host cells, residual DNA may remain in final products. This contamination is a risk factor for patients and may result in the inactivation of some tumor suppressor genes or activation of oncogenes if its concentration is more than the standard defined by WHO. Based on WHO's criteria, acceptable level of residual DNA in biopharmaceuticals is less than 10-100 pg/dose. In this study, we have designed a sensitive and specific quantitative real-time polymerase chain reaction (PCR) assay for the detection of residual DNA in human rEPO products. All reported sequences of CHO's GAPDH gene were retrieved from GenBank, and a multiple alignment was performed using Mega 6 software to find conserved regions of the gene. Primers and probe were designed by AlleleID7 software for the highly conserved region. Quantitative real-time PCR showed an R2 value more than 0.99 and the efficiency equal to 101% indicating a highly accurate and efficiency of the reaction, respectively. Based on the standard curve, the limit of detection of the assay was determined to be 10 copies/µL (0.00967 fg/µL). In addition, the inter- and intra-assay of the test were determined to be 1.14% and 0.65%, respectively, which are in acceptable range according to the WHO's guidelines.

Development of an in-House TaqMan Real-Time PCR-Based Method to Detect Residual Host Cell DNA in HBV Vaccine.

Biological therapeutic products such as recombinant hepatitis B virus (HBV) vaccine, produced by microbial fermentation in complex media, should be evaluated for host cell DNA contamination in purification steps. Eliminating these contaminations increases the efficacy of the vaccine and decreases its side effects. The objective of the present study is to trace the residual host cell DNA (HCD) in recombinant HBV vaccine by developing a TaqMan Real-Time PCR method which is more sensitive, specific, and reproducible than traditional methods such as Picogreen analysis and Threshold DNA assay. Primers and a probe were designed for the most highly conserved regions of Pichia pastoris genome. To determine the specificity of the assay, in addition to performing a BLAST for the primers and the probe in NCBI nucleotide database, 20 different human genomes and 8 bacterial and viral genomes were used. Moreover, serial dilutions of plasmids, from 10(2) to 10(7) copies/µL (from 0.00064 to 6.4 pg/µL), were prepared to find the sensitivity and the limit of detection (LOD) of the assay. Using 28 different genome samples, the specificity of the assay was determined to be 100 %. In addition, the sensitivity and LOD of the method was 0.39 × 10(-5) pg/µL. Moreover, the reproducibility of the assay based on intra- and inter-assay was 1.03 and 1.06 %, respectively. Considering the suitable specificity and sensitivity, ease of use, relatively low cost, and rapidity of the assay, it can be a reproducible and sensitive method to examine recombinant vaccines for P. pastoris residual DNA.